POLYPEPTIDE, COMPOSITION, NUCLEIC ACID MOLECULE, VECTOR, TRANSGENIC MICRO-ORGANISM, POLYPEPTIDE PROD

专利摘要:
polypeptide, composition, nucleic acid molecule, vector, host cell, process for producing a polypeptide, combined vaccine, and, use of a composition or a combined vaccine.. the invention relates to the development of chimeric ospa molecules for use in a new lyme vaccine. more specifically, chimeric ospa molecules comprise the proximal portion of one ospa serotype, together with the distal portion of another ospa serotype, while retaining the antigenic properties of both parent polypeptides. the chimeric ospa molecules are delivered alone or in combination to provide protection against a variety of borrelia genospecies. the invention also provides methods for administering the chimeric ospa molecules to a subject in the prevention and treatment of lyme disease or borreliosis.
公开号:BR112012027315B1
申请号:R112012027315-9
申请日:2011-05-13
公开日:2021-08-17
发明作者:Brian A. Crowe；Ian Livey；Maria O'Rourke；Michael Schwendinger
申请人:Baxalta Incorporated；Baxalta GmbH；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The invention generally relates to chimeric OspA polypeptides, nucleic acids encoding the polypeptides, compositions comprising these molecules and methods of using them. FUNDAMENTALS OF THE INVENTION
[002] Lyme disease is a tick-borne disease caused by Borrelia burgdorferi sensu lato (s.l.). The disease is typically characterized by the development of an expanding red rash at the tick bite site, which may be followed by systemic complications including meningitis, carditis, or arthritis. Almost all cases of Lyme disease are caused by one of three genospecies, Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto (s.s.). In Europe, all three species that infect humans are found. However, in North America only a single species, Borrelia burgdorferi stricto sensu, is found. Borrelia burgdorferi is a species of Gram-negative bacteria of the spirochete class of the genus Borrelia. Antibiotic treatment of Lyme disease is generally effective, but some patients develop a chronic disabling form of the disease, involving the joints or nervous system, that does not substantially improve even after parenteral antibiotic therapy, thus underscoring the need for a vaccine for high-risk populations.
[003] The outer surface protein A (OspA) is a 31 kDa antigen expressed by Borrelia burgdorferi s.l. present in the midgut of Ixodes tick. OspA has been proven effective in preventing Lyme disease in North America (Steere et al., N. Engl. J. Med. 339: 209-15,1998; Sigal et al., N. Engl. J. Med. 339:216-22, 1998; erratum in: N. Engl. J. Med. 339:571, 1998). The amino terminus of fully processed OspA is a cysteine residue that is post-translationally modified, with three fatty acyl chains that anchor the protein to the outer surface of the bacterial membrane ( Bouchon et al., Anal. Biochem. 246: 52-61 : 52-61 , 1997). Lipidation of OspA is reported to stabilize the molecule (Luft, personal communication) and is essential for protection in the absence of a strong adjuvant (Erdile et al., Infect. Immun. 61:81-90, 1993). A recombinant soluble form of the protein lacking the amino terminal lipid membrane anchor was cocrystallized with the Fab fragment of an agglutinating mouse monoclonal antibody to determine the structure of OspA, which was shown to comprise 21 antiparallel β-strands followed by a single α-helix (Li et al., Proc. Natl. Acad. Sci. USA 94:3584-9, 1997).
[004] Monovalent OspA-based vaccine (LYMErix®) has been marketed in the US for the prevention of Lyme disease. However, in Europe heterogeneity in OspA sequences among the three genospecies precludes broad protection with a vaccine based on OspA from a single strain (Gern et al., Vaccine 15:1551-7, 1997). Seven major OspA serotypes have been recognized among European isolates (designated serotypes 1 to 7, Wilske et al., J. Clin. Microbiol. 31:340-50, 1993). OspA serotypes correlate with species; serotype 1 corresponds to B. burgdorferi s.s., serotype 2 corresponds to B. afzelii, and serotypes 3 to 7 correspond to B. garinii.
[005] Protective immunity acquired through immunization with OspA is unusual since the interaction between the host and pathogen immune response does not occur in the host, but in the midgut of the tick vector. In the case of Lyme disease, a tick acts as a vector or carrier for the transmission of Lyme disease to humans from animals. OspA-specific antibody acquired during feeding by an infected tick prevents transmission of B. burgdorferi s.l. for the immunized host mammal (de Silva et al., J. Exp. Med. 183: 271-5, 1996). Protection is mediated by antibodies and is influenced primarily through bactericidal antibody although an antibody that blocks the binding of the spirochete to a receptor on the lining of the tick's intestinal epithelium may also be effective (Pal et al., J. Immunol. 166: 7398-403, 2001).
The rational development of effective OspA vaccines requires the identification of protective epitopes, such as that defined by the protective monoclonal antibody LA-2 (Golde et al., Infect. Immun. 65:882-9, 1997). X-ray crystallography and NMR analysis were used to identify immunologically important hypervariable domains in OspA and mapped the LA-2 epitope to amino acids 203-257 ( Ding et al., J. Mol. Biol. 302: 1153- 64, 2000; Luft et al. J Infect Dis. 185 (Suppl. 1): S46-51, 2002).
[007] There is a need in the art for the development of an OspA vaccine that can provide broad protection against a variety of Borrelia species that are present in the United States, Europe and elsewhere. The following disclosure describes the details of such a vaccine. SUMMARY OF THE INVENTION
[008] The present invention addresses one or more of the needs in the art relating to the prevention and treatment of Lyme disease or Lyme borreliosis.
The invention includes a chimeric polypeptide comprising a first polypeptide fragment of a Borrelia garinii outer surface protein A (OspA) serotype 3 protein and a second polypeptide fragment of a Borrelia garinii OspA serotype 5 protein, the polypeptide having the property of inducing an immune response against OspA serotype 3 protein and OspA serotype 5 protein. In some aspects, the chimeric polypeptide comprises an N-terminal polypeptide fragment of OspA serotype 5 protein and a C-terminal polypeptide fragment of OspA serotype 3 protein. In other aspects, the chimeric polypeptide comprises an N-terminal polypeptide fragment of OspA serotype 3 protein and a C-terminal polypeptide fragment of OspA serotype 5 protein. In certain aspects, the chimeric polypeptide further comprises an N-terminal Borrelia outer surface protein B (OspB) polypeptide fragment, wherein the OspB polypeptide fragment comprises an OspB leader sequence. In particular aspects, the chimeric polypeptide comprises an amino acid sequence having at least 200 amino acid residues with at least about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91. 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the amino acid sequence defined in SEQ ID NO: 173. In various aspects, the chimeric polypeptide comprises the amino acid sequence defined in SEQ. ID NO: 173. In other aspects, the chimeric polypeptide consists of the amino acid sequence defined in SEQ ID NO: 173.
[0010] The invention includes compositions comprising a chimeric polypeptide of the invention and a pharmaceutically acceptable carrier. In some aspects, such compositions further comprise an additional polypeptide from a Borrelia outer surface protein A (OspA) protein. In various aspects, such compositions further comprise an additional polypeptide from an outer surface protein B (OspB) protein of Borrelia. In particular aspects, the additional polypeptide comprises a polypeptide fragment of an N-terminal outer surface protein B (OspB) of Borrelia, where the OspB polypeptide fragment comprises an OspB leader sequence. In various respects, it is Borrelia Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisiana, Borrelia lusitaniam, Borrelia andersonii, Borrelia spielman.
In some aspects, the additional polypeptide is a chimeric polypeptide comprising a first polypeptide fragment of a Borrelia garinii outer surface protein A (OspA) serotype 4 protein and a second polypeptide fragment of a Borrelia garinii serotype 6 protein Borrelia garinii OspA, the polypeptide having the property of inducing an immune response against OspA serotype 4 protein and OspA serotype 6 protein. In a particular aspect, the additional polypeptide comprises an N-terminal polypeptide fragment of OspA serotype 6 protein and a C-terminal polypeptide fragment of OspA serotype 4 protein. In other aspects, the additional polypeptide comprises an N-terminal OspA serotype 4 protein polypeptide fragment and a OspA serotype 6 protein C-terminal polypeptide fragment. In certain aspects, the additional polypeptide comprises an amino acid sequence having at least 200 amino acid residues with at least about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91. 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the amino acid sequence defined in SEQ ID NO: 171. In a particular aspect, the additional polypeptide comprises the amino acid sequence defined in SEQ ID NO: 171. In further aspects, the additional polypeptide consists of the amino acid sequence defined in SEQ ID NO: 171.
[0012] In some aspects, the additional polypeptide is a chimeric polypeptide comprising a first polypeptide fragment of a Borrelia burgdorferi sensu stricto outer surface protein A (OspA) serotype 1 protein and a second polypeptide fragment of a serotype protein Borrelia afzelii OspA 2, the polypeptide having the property of inducing an immune response against OspA serotype 1 protein and OspA serotype 2 protein. In a particular aspect, the additional polypeptide comprises an N-terminal polypeptide fragment of an OspA serotype 1 protein and a C-terminal polypeptide fragment of an OspA serotype 2 protein. In other aspects, the additional polypeptide comprises an N-terminal polypeptide fragment of OspA serotype 2 protein and a C-terminal polypeptide fragment of OspA serotype 1 protein. In a particular aspect, the additional polypeptide further comprises an N-terminal Borrelia outer surface protein B (OspB) polypeptide fragment, wherein the OspB polypeptide fragment comprises an OspB leader sequence. In certain aspects, the additional polypeptide comprises an amino acid sequence having at least 200 amino acid residues with at least about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 90, 91. 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the amino acid sequence defined in SEQ ID NO: 169. In a particular aspect, the additional polypeptide comprises the amino acid sequence defined in SEQ ID NO: 169. In further aspects, the additional polypeptide consists of the amino acid sequence defined in SEQ ID NO: 169.
[0013] The invention includes compositions comprising at least three OspA chimeric polypeptides, wherein the polypeptides have different sequences. In some aspects, the OspA chimeric polypeptides individually comprise the amino acid sequence defined in SEQ ID NOS: 169, 171, and 173. In other aspects, the OspA chimeric polypeptides include an immune response against at least OspA 1 serotype proteins. 2, 3, 4, 5, and 6.
The invention includes a chimeric nucleic acid molecule comprising a first nucleotide sequence fragment of an outer surface protein A (OspA) serotype 3 protein coding region of Borrelia garinii and a second nucleotide sequence fragment of Borrelia garinii a Borrelia garinii OspA serotype 5 protein coding region, the nucleic acid molecule encoding a polypeptide having the property of inducing an immune response against OspA serotype 3 protein and OspA serotype 5 protein. In some aspects, the chimeric nucleic acid molecule comprises a 5'-terminal nucleotide sequence encoding a fragment of the OspA serotype 5 protein coding region and a 3'-terminal nucleotide sequence encoding a fragment of a region of OspA serotype 3 protein coding. In other aspects, the chimeric nucleic acid molecule comprises a 3'-terminal nucleotide sequence encoding a fragment of the OspA serotype 5 protein coding region and a 5'-terminal nucleotide sequence encoding a fragment of a coding region of OspA serotype 3 protein. In various aspects, the chimeric nucleic acid molecule further comprises a Borrelia 5'-terminal outer surface protein B (OspB) nucleotide sequence fragment, wherein the OspB nucleotide sequence fragment comprises an OspB leader sequence. In certain aspects, the chimeric nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of at least about 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the nucleic acid sequence defined in SEQ ID NO: 172; and (b) a nucleotide sequence complementary to (a). In other aspects, the chimeric nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a polypeptide of at least about 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the amino acid sequence defined in SEQ ID NO: 173; and (b) a nucleotide sequence complementary to (a). In a particular aspect of the invention, the chimeric nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 173, the polypeptide having a substitution of one to 25 conservative amino acids; (b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 173, the polypeptide having an insertion of one to 25 conservative amino acids; (c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 173, the polypeptide having an internal deletion of one to 25 conservative amino acids; (d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 173, the polypeptide having a C and/or N-terminal truncation of one to 25 amino acids; (e) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 173, the polypeptide having a modification of one to 25 amino acids selected from amino acid substitutions, amino acid insertions, amino acid deletions, a truncation C-terminal, or an N-terminal truncation; and (f) a nucleotide sequence complementary to any one of (a)-(e). In various aspects, such substitutions, insertions, deletions or modifications occur at any of amino acid positions 1-4, 6, 8, 9, 11, 16, 18, 20-28, 47, 49, 50, 81, 82, 83, 100 139, 155, 160, 176, 189, 190, and 250 of SEQ ID NO: 173. In some aspects, the chimeric nucleic acid molecule comprises the nucleotide sequence defined in SEQ ID NO: 172. In other aspects , the chimeric nucleic acid molecule consists of the nucleotide sequence defined in SEQ ID NO:172.
The invention includes vectors, host cells, and processes for producing polypeptides by culturing the host cells discussed herein. In some aspects, the invention includes a vector comprising any of the nucleic acid molecules described herein. In other aspects, the invention includes a host cell that comprises such vectors. In some aspects, the host cell is a eukaryotic cell. In other aspects, the host cell is a prokaryotic cell. In various aspects, the process for producing a polypeptide comprises culturing the host cells described herein under conditions suitable to express the polypeptide and, optionally, isolating the polypeptide from the culture. In various aspects, the invention includes compositions comprising any of these chimeric nucleic acid molecules or any vectors comprising such nucleic acid molecules and a pharmaceutically acceptable carrier, or carriers.
As noted above, the present invention includes a composition comprising a chimeric nucleic acid molecule comprising a first nucleotide sequence fragment of an outer surface protein A (OspA) serotype 3 protein coding region of Borrelia garinii, and a second nucleotide sequence fragment of a Borrelia garinii OspA serotype 5 protein coding region, the nucleic acid molecule encoding a polypeptide having the property of inducing an immune response against the OspA serotype 3 protein, and the OsPA serotype 5 protein. In some aspects, the composition further comprises an additional nucleic acid molecule encoding a Borrelia outer surface protein A (OspA) protein. In other aspects, the composition further comprises an additional nucleic acid molecule encoding Borrelia outer surface protein B (OspB) protein. In particular aspects, the additional nucleic acid molecule further comprises a Borrelia 5'-terminal outer surface protein B (OspB) fragment nucleotide sequence, wherein the OspB nucleotide sequence fragment comprises an OspB leader sequence. In various respects, Borrelia is Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisiana, Borrelia lusitalonieli, Borrelia andersonii, Borrelia sp.
In some aspects, the additional nucleic acid molecule is a chimeric nucleic acid molecule comprising a first nucleotide sequence fragment of a Borrelia garinii outer surface protein A (OspA) serotype 6 protein coding region and a second nucleotide sequence fragment of a Borrelia garinii OspA serotype 4 protein coding region, the nucleic acid molecule encoding a polypeptide having the property of inducing an immune response against OspA serotype 6 protein and serotype protein 4 of OspA. In other aspects, the additional nucleic acid molecule comprises a 5'-terminal nucleotide sequence encoding a fragment of the OspA serotype 6 protein coding region and a 3'-terminal nucleotide sequence encoding a fragment of the coding region of OspA serotype 4 protein. In various aspects, the additional nucleic acid molecule comprises a 5'-terminal nucleotide sequence encoding a serotype 4 OspA protein fragment and a 3'-terminal nucleotide sequence encoding a serotype 6 OspA protein fragment. In some aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of at least about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the nucleotide sequence defined in SEQ ID NO: 170, and (b) a nucleotide sequence complementary to (a). In other aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a polypeptide of at least or about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171; and (b) a nucleotide sequence complementary to (a). In particular aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171, the polypeptide having a substitution from one to 25 conservative amino acids, (b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171, the polypeptide having an insertion of one to 25 conservative amino acids, (c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171, the polypeptide having an internal deletion of one to 25 conservative amino acids, (d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171, the polypeptide having a C- and/or N-terminal truncation of one to 25 amino acids; (e) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 171, the polypeptide having a one to 25 amino acid modification selected from amino acid substitutions, amino acid insertions, amino acid deletions, a truncation C-terminal, or an N-terminal truncation, and (f) a nucleotide sequence complementary to any one of (a) - (e). In various aspects, substitutions, insertions, deletions or modifications occur at any of amino acid positions 1-4, 6, 8, 9, 11, 16, 18, 20-28, 47, 49, 50, 81, 82. 83, 100 139, 155, 160, 176, 189, 190, and 250 of SEQ ID NO: 171. In some aspects, the additional nucleic acid molecule comprises a nucleotide sequence defined in SEQ ID NO: 170. In other aspects , the additional nucleic acid molecule consists of the nucleotide sequence defined in SEQ ID NO: 170.
In other aspects, the additional nucleic acid molecule is a chimeric nucleic acid molecule comprising a first nucleotide sequence fragment of a Borrelia burgdorferi outer surface protein A (OspA) serotype 1 protein coding region sensu stricto, and a second fragment of the nucleotide sequence a Borrelia afzelii OspA serotype 2 protein coding region, the nucleic acid molecule encoding a polypeptide having the property of inducing an immune response against the OspA serotype 1 protein and the OspA serotype 2 protein. In certain aspects, the additional nucleic acid molecule comprises a 5'-terminal nucleotide sequence encoding an OspA serotype 1 protein coding region and a 3'-terminal nucleotide sequence encoding a fragment of the OspA coding region. OspA serotype 2 protein. In other aspects, the additional nucleic acid molecule comprises a 5'-terminal nucleotide sequence encoding a fragment of the OspA serotype 2 protein coding region and a 3'-terminal nucleotide sequence encoding a protein coding region OspA serotype 1. In various aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence of at least about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with the nucleotide sequence defined in SEQ ID NO: 168, and (b) a nucleotide sequence complementary to (a). In additional aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a polypeptide of at least or about 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity with a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, and (b) a nucleotide sequence complementary to (a). In some aspects, the additional nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, the polypeptide having a substitution from one to 25 conservative amino acids, (b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, the polypeptide having an insertion of one to 25 conservative amino acids; (c) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, the polypeptide having an internal deletion of one to 25 conservative amino acids, (d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, the polypeptide having a C- and/or N-terminal truncation of one to 25 amino acids, (e) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence defined in SEQ ID NO: 169, the polypeptide having a one to 25 amino acid modification selected from amino acid substitutions, amino acid insertions, amino acid deletions, a C-terminal truncation, or an N-terminal truncation, and (f) a complementary nucleotide sequence to any one of (a) - (e). In various aspects, substitutions, insertions, deletions or modifications occur at any of amino acid positions 1-4, 6, 8, 9, 11, 16, 18, 20-28, 47, 49, 50, 81, 82. 83, 100 139, 155, 160, 176, 189, 190, and 250 of SEQ ID NO: 169. In some aspects, the additional nucleic acid molecule comprises the nucleotide sequence defined in SEQ ID NO: 168. In other aspects , the additional nucleic acid molecule consists of the nucleotide sequence defined in SEQ ID NO: 168.
As noted above, the present invention includes a composition comprising a chimeric nucleic acid molecule comprising a first nucleotide sequence fragment of an outer surface protein A (OspA) serotype 3 protein coding region of Borrelia garinii, and a second nucleotide sequence fragment of a Borrelia garinii OspA serotype 5 protein coding region, the nucleic acid molecule encoding a polypeptide having the property of inducing an immune response against the OspA serotype 3 protein, and the OsPA serotype 5 protein. In some aspects, the composition further comprises at least two other nucleic acid molecules that encode a Borrelia outer surface protein A (OspA) protein. In various aspects, such complementary nucleic acid molecules have different nucleotide sequences. In certain aspects, a composition of the invention comprises at least three nucleic acid molecules that encode a Borrelia outer surface protein A (OspA) protein, wherein the nucleic acid molecules have different nucleotide sequences. In particular aspects, a composition of the present invention comprises nucleic acid molecules, wherein the nucleic acid molecules individually comprise the nucleotide sequences defined in SEQ ID NOS: 168, 170 and 172. In some aspects, a composition of the present invention comprises chimeric nucleic acid molecules, wherein the nucleic acid molecules encode polypeptides that induce an immune response against at least the OspA proteins of serotype 1, 2, 3, 4, 5, and 6.
[0020] The invention also includes immunogenic compositions. In some aspects, an immunogenic composition of the invention comprises any of the compositions discussed herein and a pharmaceutically acceptable carrier. In several aspects, the immunogenic composition has the property of inducing the production of an antibody that specifically binds to an outer surface protein A (OspA) protein. In certain aspects, the immunogenic composition has the property of inducing the production of an antibody that specifically binds to Borrelia. In particular aspects, the immunogenic composition has the property of inducing the production of an antibody that neutralizes Borrelia. In some aspects, the antibody is produced by an animal. In additional aspects, the animal is a mammal. In still further aspects, the mammal is human.
[0021] The invention further includes vaccine compositions. In some aspects, a vaccine composition of the present invention comprises any immunogenic composition discussed herein and a pharmaceutically acceptable carrier. In various aspects, the invention includes a combination vaccine. In certain aspects, the combination vaccine of the present invention comprises any vaccine composition discussed herein in combination with at least one second vaccine composition. In some aspects, the second vaccine composition protects against a tick-borne disease. In many ways, the tick-borne disease is Rock Mountain spotted fever, babesiosis, recurrent fever, Colorado tick fever, human monocytic ehrlichiosis (HME), human granulocytic ehrlichiosis (HGE), Southern tick-associated rash disease (STARI). ), tularemia, tick paralysis, Powassan's encephalitis, Q fever, Crimean-Congo hemorrhagic fever, Citauxzoonosis, scaronodular fever or tick-borne encephalitis. In other aspects, the second vaccine composition is a vaccine selected from the group consisting of: a tick-borne encephalitis vaccine, a Japanese encephalitis vaccine, and a Rocky Mountain spotted fever vaccine. In various aspects, the second vaccine composition has a seasonal immunization schedule compatible with immunization against Borrelia infection or Lyme disease.
[0022] The invention also includes methods for inducing an immune response in an individual. In various aspects, such methods comprise the step of administering any of the immunogenic compositions or vaccine compositions discussed herein to the subject, in an amount effective to induce an immune response. In certain aspects, the immune response comprises the production of an anti-OspA antibody.
The invention includes methods for preventing or treating a Borrelia infection or Lyme disease in a subject. In various aspects, such methods comprise the step of administering any of the vaccine compositions discussed herein or any of the combination vaccines discussed herein to the subject, in an amount effective for preventing or treating Borrelia infection or Lyme disease.
[0024] The invention includes uses of compositions of the present invention for the preparation of medicaments. Other related aspects are also provided in the present invention.
[0025] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the detailed description that follows. The entire document is intended to be listed as a unified disclosure, and it is to be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence, paragraph, or section of this document. Other features and advantages of the invention will be apparent from the detailed description which follows. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. in the art of this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 is a schematic overview for the preparation of lipidated OspA chimera constructs.
Figure 2 is the amino acid sequence of lipB sOspA 1/2251 (SEQ ID NO: 2).
Figure 3 shows nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2) sequences of lipB sOspA 1/2251.
Figure 4 is the amino acid sequence of lipB sOspA 6/4 (SEQ ID NO: 4).
Figure 5 shows nucleotide sequences (SEQ ID NO: 3) and deduced amino acid sequences (SEQ ID NO: 4) of lipB sOspA 6/4.
Figure 6 is the amino acid sequence of lipB sOspA 5/3 (SEQ ID NO: 6).
[0032] Figure 7 shows nucleotide (SEQ ID NO: 5) and deduced amino acid sequences (SEQ ID NO: 6) of lipB sOspA 5/3.
[0033] Figure 8 represents optimization of the use of codons for high-level expression.
[0034] Figure 9 shows the sequence differences between lipidated and non-lipidated constructs.
[0035] Figure 10 is a description of the T7 expression system.
Figure 11 is an SDS-PAGE gel showing the expression of novel recombinant OspA proteins from induced and non-induced cultures.
[0037] Figure 12 is a map of plasmid pUC18.
[0038] Figure 13 is a map of plasmid pET30a.
[0039] Figure 14 shows the strategy for the creation of the lipB sOspA 5/3 Kpn I - Bam HI fragment.
[0040] Figure 15 is an alignment highlighting the amino acid change (SEQ ID NO: 39) in lipB sOspA 1/2251 and the PCR primer sequences (SEQ ID NOS: 21 and 41) used to introduce the change (lipB OspA 1/2 mod (SEQ ID NO:38)); consensus sequence (SEQ ID NO:40)).
Figure 16 is an OspA sequence alignment of Blip OspA BPBP/A1 with the modified molecule lipB sOspA 1/2251. The upper strand is the original sequence (SEQ ID NO: 42) and the lower strand is the optimized sequence (SEQ ID NO: 43). Note: Three bases (CAT) at the beginning of the sequence are not shown, they are part of the Nde I CATTG site.
Figure 17 is an OspA sequence alignment of Blip OspA KT with the modified molecule lipB sOspA 6/4. The upper strand is the original sequence (SEQ ID NO: 44) and the lower strand is the optimized sequence (SEQ ID NO: 45). Note: The single base (C) at the beginning of the sequence is not shown, they are part of the Ndel CATATG site.
Figure 18 is an OspA sequence alignment of Blip OspA 5/3 of the modified molecule lipB sOspA 5/3. The upper strand is the original sequence (SEQ ID NO: 46) and the lower strand is the optimized sequence (SEQ ID NO: 47).
Figure 19 shows the distribution of functional anti-OspA responses in growth inhibition and antibody surface binding assays between protected and infected animals immunized with 3 ng of OspA 1/2 prior to challenge with strain B. burgdorferi SS B31. Mann-Whitney p values demonstrated a highly significant difference in functional antibody content between protected and infected animals.
Figure 20 shows the distribution of functional anti-OspA responses in growth inhibition and antibody surface binding assays between protected and infected animals immunized with 3 ng OspA 1/2 prior to wild tick challenge. Mann-Whitney p values demonstrated a highly significant difference in functional antibody content between protected and infected animals.
[0046] Figure 21 shows surface binding (mean fluorescence intensities (MFI)) and growth inhibition (GI-50 titers) in pooled mouse sera after immunization with three doses of the chimeric OspA vaccine of 3 components. Efficient surface binding and growth inhibition were detected against all six strains of Borrelia that express homologues of the OspA types to the OspA types in the vaccine (types 1-6).
[0047] Figure 22 shows the mean fluorescence intensity (MFI) titrations that were obtained using 42-day sera from individual mice immunized with rOspA vaccine combinations in a surface binding assay (SBA). The results showed that all three components of rOspA (1/2, 6/4, and 5/3) are required in a multivalent vaccine to induce high titers of IgG binding surface antibodies against all six strains in C3H mice . Two-component vaccines do not completely cover the two missing strains.
Figure 23 shows growth inhibition of Borreliae using 42-day sera from individual mice (in groups of 10) immunized with combinations of rOspA vaccines. Only the multivalent vaccine (the vaccine comprising the three strains) gave > 50% growth inhibition in > 90% of animals (n = 10). Black bars (solid bars) indicate strains homologous to the vaccine used.
[0049] Figure 24 shows the coverage of Borreliae expressing intratype variants of OspA. Surface binding was categorized into strong (>10-fold increased fluorescence) or weak (2-10-fold increased fluorescence). DETAILED DESCRIPTION OF THE INVENTION
The present invention provides chimeric OspA molecules that are useful as antigens that can be provided as an immunogenic composition or vaccine composition for Lyme disease, or a Borrelia infection. Before any of the embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and arrangement of components defined in the following description or illustrated in the figures and examples. The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited in this patent application are expressly incorporated herein by reference.
[0051] The invention encompasses other embodiments and is practiced or carried out in various ways. Also, it should be understood that the phraseology and terminology used herein is for the purpose of description and should not be considered limiting. The terms "including", "comprising" or "possessing" and their variations are intended to cover the elements indicated below and their equivalents, as well as the additional items.
[0052] The embodiments of the invention are exemplified in the design and synthesis of three chimeric OspA coding sequences encoding three different lipidated OspA molecules, which share some common features. Each chimeric coding sequence represents two OspA serotypes and the chimeric coding sequences were designed to encode stable chimeric OspA molecules that are safe and highly immunogenic, and provide protection to the subject against infection with B. burgdorferi sensu lato (sl) .
[0053] In one aspect, the chimeric OspA molecules comprise the proximal portion of one OspA serotype, together with the distal portion of another OspA serotype, maintaining the protective properties of both source polypeptides. Chimeric OspA nucleic acid molecules were expressed in Escherichia coli (E. coli) to provide antigens that can be formulated as a combination vaccine to provide protection against all six prevalent serotypes (serotypes 1-6) associated with the disease of Lyme or Borrelia infection in Europe and against the unique OspA serotype associated with Lyme disease or Borrelia infection in North America. Because the vaccine comprising serotypes 1-6 provides protection against B. afzelii, B. garinii and B. burgdorferi, the vaccine is designed for global use.
The invention also includes the preparation of a second set of chimeric OspA coding sequences which is, in one aspect, derived from the first set of three genes, by removing nucleic acid sequences encoding a leader sequence necessary for the production of a lipidated OspA molecule. The two sets of constructs (giving rise to lipidated and non-lipidated polypeptides) were needed to assess their ease of production in the fermenter (biomass, stability, product yield, etc.), to assess how easily different types of antigen can be purified and to compare their biological characteristics (security profile and protection potency).
The present invention includes immunogenic compositions comprising the chimeric OspA molecules of the invention. The invention also includes vaccines and vaccine kits comprising such OspA molecules, the processes for preparing the immunogenic compositions and vaccines, and the use of the immunogenic compositions and vaccines in human and veterinary medical therapy and prevention. The invention further includes methods of immunizing against Lyme disease or Borrelia infection using the OspA compositions described herein and the use of the OspA compositions in the manufacture of a medicament for the prevention of Lyme disease or Borrelia infection. Definitions
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide a person skilled in the art with a general definition of many of the terms used in the present invention Singleton, et al., DICTIONARY OF MICROBIOLOGY and MOLECULAR BIOLOGY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE and TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY(1991).
[0057] The following abbreviations are used throughout the document.
[0058]


[0059] It should be noted that, as used in this specification and the accompanying claims, the singular forms "a", "an", and "o, a" include plural reference unless the context clearly indicates the contrary.
[0060] As used herein, the following terms have the meanings ascribed to them, unless otherwise specified.
[0061] The term "gene" refers to a DNA sequence that encodes an amino acid sequence that comprises all or part of one or more polypeptides, proteins or enzymes, and may or may not include introns, and DNA sequences regulatory, such as promoter or enhancer sequences, 5' untranslated region, or 3' untranslated region that affect, for example, the conditions under which the gene is expressed. In the present disclosure, the OspA gene is bacterial and therefore there are no introns. A "coding sequence" refers to a DNA sequence that encodes an amino acid sequence but does not contain introns or regulatory sequences. Likewise, in the present disclosure the coding sequence of OspA does not contain regulatory sequences.
The "nucleic acid" or "nucleic acid sequence" or "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides and polymers thereof either in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogues or modified scaffold residues or linkages, which are synthetic, naturally occurring and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized from a similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). The term encompasses molecules formed from any of the known base analogues of DNA and RNA such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5 -fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2.2 -dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylkeosine, 5 '-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-methylester of oxyacetic acid, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid N-methylester, uracil-5- oxyacetic, pseudouracil, keosine, 2-thiocytosine, and 2,6-diaminopurine.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions, in some respects, are achieved by generation sequences in which the third position of one or more selected codons (or all) is replaced with mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:26052608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0064] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues linked by peptide bonds. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers of. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
The term "Osp A molecule" or "chimeric OspA molecule" refers, in one aspect, to an "OspA nucleic acid" comprising the nucleotide sequence of SEQ ID NO: 1 (lipB sOspA 1/ 2251), SEQ ID NO: 3 (lipB sOspA 6/4), SEQ ID NO: 5 (lipB sOspA 5/3), SEQ ID NO: 7 (sOspA 1/2251), SEQ ID NO: 9 (sOspA 6/ 4), SEQ ID NO: 11 (sOspA 5/3), SEQ ID NO: 168 (origin sOspA 1/2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3), or, in another aspect to an "OspA Polypeptide" comprising the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3).
The term "lipB sOspA molecule" refers, in one aspect, to an "OspA nucleic acid" comprising the nucleotide sequence of SEQ ID NO: 1 (lipB sOspA 1/2251), SEQ ID NO: 3 (lipB sOspA 6/4), or SEQ ID NO: 5 (lipB sOspA 5/3) or, in another aspect to an "OspA Polypeptide" comprising the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1 /2251), SEQ ID NO: 4 (lipB sOspA 6/4), or SEQ ID NO: 6 (lipB sOspA 5/3). The nucleic acid sequences of SEQ ID NOS: 7, 9, and 11 do not show the nucleic acid sequence encoding the leader sequence lipB (MRLLIGFALALALIG (SEQ ID NO: 13) Furthermore, the nucleic acid sequences of SEQ ID NOS: 7, 9, and 11 encode a methionine residue at the amino terminus of SEQ ID NOS: 8, 10, and 12 in place of the cysteine residue present at the carboxy terminus of a lipB leader sequence in SEQ ID NOS: 2, 4 , and 6.
The term "sOspA source molecule" or "original sOspA molecule" refers, in one aspect, to an "OspA nucleic acid" comprising the nucleotide sequence of SEQ ID NO: 168 (sOspA source 1/ 2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3) or, in another aspect to an "OspA Polypeptide" comprising the amino acid sequence of SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3). These “original” molecules are chimeric constructs with no mutations and no codon optimization.
The invention includes chimeric molecules of "lipidated OspA" and "non-lipidated OspA". In several aspects, lipidation confers adjuvant properties on OspA. In some aspects of the invention, lipidated OspA molecules comprise an OspB leader sequence. In some aspects of the invention, the OspB leader sequence comprises amino acids MRLLIGFALALALIG (SEQ ID NO: 13). In other aspects, the OspB leader sequence includes other amino acids.
[0069] The terms "identical" and percent "identity" as known in the art refer to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences . In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by matching between strands of two or more nucleotides or two or more amino acid sequences. "Identity" measures the percentage of identical matches between the smallest of two or more gap-aligned sequences (if any) addressed by a given mathematical model or computer program (ie, "algorithms"). "Substantial identity" refers to sequences of at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77% , about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87% , about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97% , about 98%, or about 99% sequence identity over a specified sequence. In some aspects, identity exists over a region that is at least about 50-100 amino acids or nucleotides in length. In other aspects, identity exists over a region that is at least about 100200 amino acids or nucleotides in length. In other aspects, identity exists over a region that is at least about 200,500 amino acids or nucleotides in length. In certain aspects, percent sequence identity is determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, Blasta, BLASTX, BestFit and the SmithWaterman algorithm.
[0070] It is also specifically understood that any numeric value recited here includes all values from the lowest value to the highest value, that is, all possible combinations of numeric values between the lowest value and the highest value enumerated shall be deemed to be expressly referred to in this application. For example, if a concentration range is indicated as about 1% to 50%, values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are intended to be expressly listed in this descriptive report. The values listed above are just examples of what is specifically intended.
[0071] Ranges, in various respects, are expressed herein as "about" or "approximately" a particular value and/or "about" or "approximately" another particular value. When values are expressed as approximations, using the background "about", it is understood that a certain amount of variation is included in the range.
[0072] The term "similarity" is a related concept, but in contrast to "identity", it refers to a measure of similarity, which includes both identical matches and conservative substitution matches. If two polypeptide sequences are, for example, 10/20 amino acids identical and the rest are all non-conservative substitutions, then the percentage identity and similarity would be 50%. If, in the same example, there are five more positions where conservative substitutions exist, then the identity percentage remains 50%, but the similarity percentage would be 75% (15/20). Therefore, in cases where conservative substitutions exist, the degree of percentage similarity between two polypeptides will be greater than the percentage identity between these two polypeptides.
The term "isolated nucleic acid molecule" refers to a nucleic acid molecule of the present invention that (1) has been separated to any degree from proteins, lipids, carbohydrates or other materials with which it is naturally found when total DNA is isolated from the cells of origin, (2) it is not linked to all or a portion of a polynucleotide to which the "isolated nucleic acid molecule" is linked in nature, (3) it is linked operatively to a polynucleotide that is not linked in nature or (4) does not occur in nature as part of a larger polynucleotide sequence. Substantially free, as used herein, indicates that the nucleic acid molecule is free of any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that may interfere with its use in polypeptide production. or its therapeutic, diagnostic, prophylactic or research use.
[0074] The term "isolated polypeptide" refers to a polypeptide of the present invention that (1) has been separated to any degree from polynucleotides, lipids, carbohydrates or other materials with which it is naturally found when isolated from the cell of origin , (2) is not linked (by covalent or non-covalent interaction) to all or a portion of a polypeptide to which the "isolated polypeptide" is linked in nature, (3) is operatively linked (by covalent or non-covalent interaction ) to a polypeptide with which it is not linked in nature, or (4) does not occur in nature. In one aspect, the isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.
As used herein, a "fragment" of a polypeptide refers to any portion of the polypeptide smaller than the full-length protein or polypeptide expression product. Fragments are typically deletion analogs of the full-length polypeptide in which one or more amino acid residues have been removed from the amino terminus and/or the carboxyl terminus of the full-length polypeptide. Therefore, "fragments" are a subset of the deletion analogues described below.
As used herein an "analog" refers to a polypeptide of substantially similar structure and which has the same biological activity, albeit in certain cases, to a different degree, to a naturally occurring molecule. Analogs differ in the composition of their amino acid sequences compared to the naturally occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini. of the polypeptide (including fragments as described above) and/or one or more internal regions of the naturally occurring polypeptide sequence, (ii) insertion or addition of one or more amino acids at one or more termini (usually an analogous "addition") of the polypeptide and/or one or more internal regions (typically an analogous "insert") of the naturally-occurring polypeptide sequence, or (iii) the substitution of one or more amino acids for other amino acids in the naturally-occurring sequence of the polypeptide. Substitutions are conservative or non-conservative based on the physicochemical or functional relationship of the amino acid being replaced and the amino acid replacing it.
"Conservatively modified analogs" apply to both amino acid and nucleic acid sequences. With respect to certain nucleic acid sequences, conservatively modified nucleic acids refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence for the essentially identical sequences. . Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the GCA, GCC, GCG and GCU codons all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding described codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a kind of conservatively modified analogs. Each nucleic acid sequence herein which encodes a polypeptide also describes any possible silent variation of the nucleic acid. A skilled person will recognize that every codon in a nucleic acid (except AUG, which is normally just the codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.
As for amino acid sequences, one skilled in the art will recognize that individual substitutions, insertions, deletions, additions or truncations to a nucleic acid, peptide, polypeptide or protein sequence that alters, adds or deletes a single amino acid or a small percentage An amino acid in the coding sequence is a "conservatively modified analog", where the changes result from the replacement of one amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude polymorphic variants, cross-species homologs, and alleles of the invention.
[0079] The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)).
[0080] As used herein a "variant" refers to a polypeptide, protein, or analog that comprises at least one amino acid substitution, deletion, insertion, or modification, so long as the variant retains the biological activity of the native polypeptide.
[0081] As used herein an "allelic variant" refers to any of two or more polymorphic forms of a gene that occupy the same genetic locus. Allelic variations arise naturally by mutation and, in some respects, result in phenotypic polymorphism in populations. In certain aspects, genetic mutations are silent (no change in the encoded polypeptide) or, in other aspects, encode polypeptides that have altered amino acid sequences. "Allelic variants" also refers to cDNAs derived from mRNA transcripts of genetic allelic variants, as well as the proteins encoded by them.
[0082] The term "derivative" refers to polypeptides that are covalently modified by conjugation to therapeutic or diagnostic agents, labels (for example, with radionuclides or different enzymes), the covalent attachment of polymers such as pegylation (derivatization with polyethylene glycol ) and insertion or substitution by chemical synthesis of unnatural amino acids. In some aspects, derivatives are modified to comprise additional chemical fractions that are not normally part of the molecule. Such fractions, in several aspects, modulate the solubility, absorption and/or biological half-life of the molecule. Fractions, in various other respects, alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side-effects of the molecule, etc. Portions capable of mediating these effects are described in Remington's Pharmaceutical Sciences (1980). The procedure for coupling such moieties to a molecule is well known in the art. For example, in some aspects, an OspA derivative is an OspA molecule that has a chemical modification that confers a longer in vivo half-life for the protein. In one embodiment, polypeptides are modified by the addition of a water-soluble polymer known in the art. In a related embodiment, polypeptides are modified by glycosylation, PEGylation, and/or polysialylation.
The term "recombinant" when used with reference, for example, to a cell or nucleic acid, protein or vector indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or altering a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the cell's native (non-recombinant) form or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.
As used herein, "selectable marker" refers to a gene that encodes an enzyme or other protein that confers on the cell or organism in which it is expressed an identifiable phenotypic change such as resistance to a drug, a antibiotic or another agent, such that the expression or activity of the marker is selected for (eg, but not limited to, a positive marker, such as the neogene) or against (eg, and without limitation, a negative marker , such as the diphtheria gene). A "heterologous selectable marker" refers to a selectable marker gene that has been inserted into the genome of an animal where it would not normally be found.
[0085] Examples of selectable markers include, but are not limited to, an antibiotic resistance gene such as neomycin (neo), puromycin (Puro), diphtheria toxin, phosphotransferase, hygromycin phosphotransferase, xanthineguanine, phosphoribosyl transferase, thymidine kinase of Herpes simplex virus type 1, adenine phosphonbosyltransferase and hypoxanthine phosphoribosyltransferase. One of ordinary skill in the art will understand that any selectable marker known in the art is useful in the methods described herein.
The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences which are not in the same relationship to each other in nature. For example, the nucleic acid is typically produced recombinantly, having two or more unrelated gene sequences arranged to make a new nucleic acid functional, for example, a promoter from one source and a coding region from another source. Likewise, a heterologous protein indicates that the protein includes two or more subsequences that are not in the same relationship to each other in nature (for example, a fusion protein).
As used herein, the term "homologous" refers to the relationship between proteins that have a "common evolutionary origin", including proteins from superfamilies (for example, the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., Cell 50:667, 1987). These proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity, either in terms of percentage similarity or the presence of specific residues or motifs at conserved positions.
[0088] Optimal alignment of sequences for comparison is conducted, for example, and without limitation, through the local homology algorithm of Smith et al., Adv. Appl. Math. 2:482, 1981; by the homology alignment algorithm of Needleman et al., J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson et al., Proc. Natl. Academic Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see in general, Ausubel et al., supra ). Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyzes is publicly available through the National Biotechnology Information Center. In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993 ).
[0089] The term "vector" is used to refer to any molecule (eg nucleic acid, plasmid or virus) used to transfer coding information to a host cell.
[0090] A "cloning vector" is a small piece of DNA into which a foreign DNA fragment can be inserted. Insertion of the fragment into the cloning vector is accomplished by treating the vehicle and foreign DNA with the same restriction enzyme and then ligating the fragments together. There are many types of cloning vectors and all types of cloning vectors are used in the invention. Genetically modified plasmids and bacteriophages (such as phage X) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that allows the transcription of a particular nucleic acid of a host cell. The expression vector can be part of a virus, plasmid or nucleic acid fragment. In certain aspects, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
[0092] The term "coding sequence" is defined herein as a nucleic acid sequence that is transcribed into mRNA, which is translated into a polypeptide, when placed under the control of the appropriate control sequences. The boundaries of the coding sequence are generally determined by the ATG initiation codon, which is usually the beginning of the open reading frame at the 5' end of the mRNA and a transcription termination sequence located immediately downstream of the 3' end open reading frame. ' of the mRNA. A coding sequence can include, but is not limited to, genomic DNA, cDNA, and semi-synthetic, synthetic, and recombinant nucleic acid sequences. In one aspect, the promoter DNA sequence is defined as being the DNA sequence located upstream of an associated coding sequence and being capable of controlling the expression of that coding sequence.
[0093] A "promoter" is defined as an array of nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the start of the transcription site, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the beginning of the transcription site. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulations.
The term "operably linked" refers to a functional link between a nucleic acid expression control sequence (such as matrix transcription factor binding sites or a promoter) and a second nucleic acid sequence , wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
"Transduction" is used to refer to the transfer of nucleic acids from one bacterium to another, usually by a phage. "Transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses.
[0096] The term "transfection" is used to refer to the incorporation of foreign or exogenous DNA by a cell and a cell that has been "transfected" when the exogenous DNA has been introduced into the cell membrane. A number of transfection techniques are well known in the art and are described herein. See, for example, Graham et al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York, (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene, 13:197 (1981). Such techniques can be used to introduce one or more exogenous DNA fractions into suitable host cells.
[0097] The term "transformation" as used herein refers to a change in the genetic characteristics of cells, and a cell that was transformed when it was modified to contain the new DNA. For example, a cell is transformed when it is genetically modified from its native state. After transfection or transduction, the transforming DNA can recombine with that of the cell by physical integration into a chromosome in the cell. In some cases, DNA is transiently maintained as an unreplicated episomal element or independently replicates as a plasmid. A cell is considered to have been stably transformed when the DNA is replicated as the cell divides.
[0098] The term "endogenous" refers to a polypeptide or polynucleotide or other compound, which is naturally expressed in the host organism, or originates within a cell, tissue or organism. "Exogenous" refers to a polypeptide, polynucleotide or other compound that originates outside a cell, tissue or organism.
[0099] The term "agent" or "compound" describes any molecule, eg protein or pharmaceutical, with the ability to affect a biological parameter in the invention.
[00100] A "control", as used herein, may refer to an active, positive, negative, or vehicle control. As will be understood by those skilled in the art, controls are used to establish the relevance of the experimental results, and to provide a comparison for the condition being tested.
The term "reduces severity", when referring to a symptom of Lyme or Lyme disease, means that the symptom delayed onset, reduced severity, or caused less harm to the subject. Generally, the severity of a symptom is compared to a control, for example, that does not receive an active prophylactic or therapeutic composition. In that case, the composition can be said to reduce the severity of a Lyme disease symptom, if the symptom is reduced by 10%, 25%, 30%, 50%, 80%, or 100% (i.e. essentially eliminated ), while compared to the level of symptom control.
[00102] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and, additionally, capable of being used in a subject to produce antibodies able to bind to an epitope of each antigen. An antigen, in many ways, has one or more epitopes.
[00103] The term "antibody" refers to a molecule or molecules that have specificity for an OspA polypeptide. As used herein, the terms "specific", "specificity" and "specifically binds" refer to the ability of the antibody to bind to OspA polypeptides and not to bind to non-OspA polypeptides. In certain aspects, the antibody is a "neutralizing antibody", where the antibody reacts with an infectious agent and destroys or inhibits its infectivity or virulence. The invention includes immunogenic compositions comprising antibodies that "neutralize" Borrelia.
The terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refer to one or more formulation materials suitable for effecting or enhancing delivery of the OspA polypeptide, OspA nucleic acid molecule or antibody of OspA as a pharmaceutical composition.
[00105] The term "stabilizer" refers to a vaccine excipient or substance that protects the immunogenic composition of the vaccine from adverse conditions, such as those that occur during heating or freezing, and/or prolongs stability or shelf life of the immunogenic composition and in a stable immunogenic state or condition. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, mannose; sugar alcohols such as mannitol, amino acids such as glycine or glutamic acid; and proteins such as gelatin or human serum albumin.
[00106] The term "antimicrobial preservative" refers to any substance that is added to the immunogenic composition or vaccine, which inhibits the growth of microorganisms that can be introduced by repeated puncture of multiple-dose vials, if these containers are used . Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.
[00107] The term "immunogenic composition" refers to a composition comprising an antigen (eg, chimeric OspA molecules) against which antigen-specific antibodies are raised, an adjuvant that stimulates the host's immune response. subject, and a suitable, immunologically inert, pharmaceutically acceptable carrier. Optionally, an immunogenic compound comprises one or more stabilizers. Optionally, an immunogenic compound comprises one or more antimicrobial preservatives.
[00108] The term "vaccine" or "vaccine composition" refers to a biological preparation, which improves immunity to a particular disease (eg Lyme disease or Borrelia infection). The vaccine typically contains an agent that resembles a disease-causing microorganism (eg, the chimeric OspA molecules (antigen) of Borrelia). The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. Vaccines, in many ways, are prophylactic (prevent or ameliorate the effects of future infection by any natural or "wild" pathogen) or therapeutic (vaccines against the present infection). As set out above, vaccine compositions include formulations which comprise such pharmaceutically acceptable carriers. Optionally, the vaccine also comprises one or more stabilizers and/or one or more antimicrobial preservatives.
The terms "effective amount" and "therapeutically effective amount" each refer to the amount of nucleic acid molecule, composition, polypeptide, or antibody used to support an observable level of one or more of the biological activities of the OspA polypeptides as set forth herein. For example, an effective amount, in some aspects of the present invention, would be the amount needed to prevent, neutralize or reduce Borrelia infection.
The term "combination" refers to two or more nucleic acid molecules of the present invention, or two or more polypeptides of the invention. In some aspects, the combinations of molecules of the present invention are administered to provide immunity to or combat infection of at least four of the six Borrelia serotypes (1-6) described herein. In various aspects, combinations of two or three molecules or polypeptides of the present invention are used. In certain aspects, the combinations of molecules of the present invention are administered to a subject to provide immunity against all six Borrelia serotypes (1-6) described herein. The latter combination has been shown to confer immunity to heterologous strains of OspA types expressing Borrelia not present in the combination of nucleic acid molecules or polypeptides.
The term "combination vaccine" refers to a vaccine formulation containing more than one vaccine composition, or more than one protective antigen for one or more diseases. The invention includes a combined vaccine comprising the chimeric OspA antigens against Lyme or Borrelia disease in addition to an antigen against one or more other diseases. In many ways, one or more other diseases is a tick-borne disease. In certain respects, the other tick-borne disease is Rock Mountain spotted fever, babesiosis, recurrent fever, Colorado tick fever, human monocytic ehrlichiosis (HME), human granulocytic ehrlichiosis (HGE), Southern tick-associated rash disease ( STARI), tularemia, tick paralysis, Powassan's encephalitis, Q fever, Crimean-Congo hemorrhagic fever, Citauxzoonosis, scaronodular fever or tick-borne encephalitis. In particular aspects, the invention includes a combination vaccine comprising one or more vaccines, including a tick-borne encephalitis vaccine, the Japanese encephalitis vaccine, and a Rocky Mountain spotted fever vaccine. In some aspects, the combination vaccine comprises vaccine compositions that have a seasonal immunization schedule compatible with immunization against Borrelia infection or Lyme disease. In more specific respects, combination vaccines are useful in preventing multiple diseases for use in geographic locations where these diseases are prevalent.
[00112] The term "Borrelia" refers to a species of Gram-negative bacteria of the spirochete class of the genus Borrelia. In one respect, "Borrelia burgdorferi sensu lato (l.l.)" refers to Borrelia burgdorferi in the broadest sense. Almost all cases of Lyme disease or Borreliosis are caused by one of the three genospecies, Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto (s.s.), which refers to B. burgdorferi in the strict sense. Borrelia OspA serotypes correlate with species; serotype 1 corresponds to B. burgdorferi s.s., serotype 2 corresponds to B. afzelii and serotypes 3-7 corresponds to B. garinii. In various aspects, the immunogenic or vaccine compositions of the present invention also provide protection against other species of Borrelia, including, but not limited to, Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii, Borrelia miyamotoi or Borrelia lonestar.
[00113] A "subject" is given in its conventional sense of a non-plant, non-protist living being. In most respects, the subject is an animal. In particular aspects, the animal is a mammal. In more specific ways, the mammal is a human. In other aspects, the mammal is a pet or companion animal, a domesticated farm animal, or a zoo animal. In certain respects, the mammal is a cat, dog, horse, or cow. In many other respects, the mammal is a deer, mouse, chipmunk, chipmunk, opossum, or raccoon. Lyme Disease (Borreliosis or Lyme Borreliosis)
In some aspects, the present invention includes chimeric OspA molecules and compositions comprising these molecules in preventing Lyme disease or Borrelia infection. Lyme disease is also known in the art as Borreliosis or Lyme Borreliosis and therefore all these terms are included in the invention. Likewise, the invention includes methods for preventing or treating Lyme disease which comprise administering the chimeric OspA molecules described herein. Lyme disease, or borreliosis, is an infectious disease caused by at least three species of Gram-negative spirochete bacteria belonging to the genus Borrelia. There are at least 13 species of Borrelia that have been discovered, three of which are known to be related to Lyme. The species of Borrelia that cause Lyme disease are collectively known as Borrelia burgdorferi sensu lato, and show great genetic diversity. The group of Borrelia burgdorferi sensu lato is composed of three closely related species that are probably responsible for the vast majority of cases. Borrelia burgdorferi sensu stricto is the main cause of Lyme disease in the United States (but is also present in Europe), while Borrelia afzelii and Borrelia garinii cause more cases in Europe. Some studies have also proposed that species (eg, Borrelia bissettii, Borellia spielmanii, Borrellia lusitaniae, and Borrelia valaisiana) can sometimes infect humans. Although these species do not appear to be major causes of disease, immunogenic protection against these species is also included in the invention.
[00115] Lyme disease is the most common tick-borne disease in the Northern Hemisphere. The disease is named after the village of Lyme, Connecticut, where a series of cases was identified in 1975. Borrelia is transmitted to man by the bite of infected ticks belonging to some species of the genus Ixodes ("hard ticks"). Early symptoms in some cases include headache, fever, fatigue, depression, and a characteristic circular rash called erythema migrans. Left untreated, later symptoms can often involve the joints, heart and central nervous system. In most cases, the infection and its symptoms are eliminated by conventional treatment, especially when the disease is treated early. However, late, delayed, or inadequate treatment can lead to more severe symptoms, which can be disabling and difficult to treat. Occasionally, symptoms such as arthritis persist after the infection has been cleared by antibiotics.
[00116] Some groups argue that "chronic" Lyme disease is responsible for a range of medically unexplained symptoms beyond the recognized symptoms of late Lyme disease, and that additional, long-term antibiotic treatments are needed. However, in the long term, treatment is controversial and the dispute over this treatment has led to legal action over treatment guidelines.
[00117] Lyme disease is classified as a zoonosis as it is transmitted to humans from a natural reservoir that includes rodents and birds through ticks that feed on both sets of hosts. Hard-bodied ticks of the genus Ixodes are the main vectors of Lyme disease. Most human infections are caused by ticks at the nymph stage, as nymphal ticks are very small and can feed for long periods of time without being detected. Tick bites often go unnoticed because of the tick's small size in its nymph stage, as well as tick secretions that prevent the host from experiencing any itchiness or pain due to the bite.
[00118] Lyme disease is diagnosed clinically based on symptoms, objective physical findings (such as erythema migrans, facial paralysis, or arthritis), a history of possible exposure to infected ticks, as well as serologic blood tests. Approximately half of Lyme disease patients will develop the characteristic bulls-eye rash, but many may not recall a tick bite. Laboratory tests are not recommended for people who do not have Lyme disease symptoms.
[00119] Due to the difficulty in culturing Borrelia bacteria in the laboratory, the diagnosis of Lyme disease is typically based on clinical examination results and a history of exposure to Lyme endemic areas. Erythema migratory (EM) eruption, which only occurs in about 50% of all cases, is considered sufficient to establish a diagnosis of Lyme disease, even when serologic blood tests are negative. Serologic tests can be used to support a clinically suspected case, but it is not diagnostic in and of itself. Diagnosing end-stage Lyme disease is often difficult due to the multifaceted appearance that can mimic the symptoms of many other diseases. For this reason, one reviewer called Lyme the "great impersonator". New Lyme disease, in some cases, is diagnosed as multiple sclerosis, rheumatoid arthritis, fibromyalgia, chronic fatigue syndrome (CFS), lupus, or other autoimmune and neurodegenerative diseases. Thus, there is a great need in the art for a vaccine to prevent or treat Lyme disease. Outer surface protein A (OspA) of BORRELIA
In various aspects, the invention includes Borrelia OspA chimeric molecules and compositions comprising these molecules in the prevention and treatment of Lyme disease or Borrelia infection. Several Borrelia outer surface proteins have been identified in the past decade as being upregulated by mammalian host-specific and/or temperature signals as this spirochete is transmitted from ticks to mammals.
The major outer surface protein, OspA, from Borrelia burgdorferi is a lipoprotein of particular interest due to its potential as a vaccine candidate. Serotypic and genetic analysis of OspA from both European and North American strains of Borrelia demonstrated antigenic and structural heterogeneities. OspA is described in published PCT patent application WO 92/14488, in Jiang et al. (Clin. Diagn. Lab. Immunol. 1: 406-12, 1994) and is known in the art. OspA has been shown to induce protective immunity in challenge studies with mice, hamsters and dogs. Clinical trials in humans have demonstrated OspA formulations to be safe and immunogenic in humans (Keller et al., JAMA (1994) 271:17641768).
[00122] While OspA is expressed in the vast majority of clinical isolates of Borrelia burgdorferi from North America, a different picture has emerged from the examination of clinical isolates of Borrelia in Europe. In Europe, Lyme disease is mainly caused by three Borrelia genospecies, namely B. burgdorferi, B. garinii and B. afzelii. The invention is directed to chimeric OspA molecules that provide protective immunity against all Borrelia genospecies. The invention describes the design and synthesis of three chimeric OspA genes that encode three different lipidated OspA molecules that share common characteristics. Each gene represents two OspA serotypes and the genes were designed to encode stable OspA molecules that are safe and highly immunogenic, and provide subjective protection against infection with the subject B. burgdorferi sensu lato (s.l.). The invention also describes the three original chimeric OspA genes without mutations and without codon optimization that encode three different lipidated OspA molecules that share common characteristics. Each gene represents two OspA serotypes and encodes molecules that provide subjective protection against infection with B. burgdorferi sensu lato (s.l.).
Seven major OspA serotypes have been recognized among European isolates (designated serotypes 1 to 7, Wilske et al., J. Clin. Microbiol. 31:340-50, 1993). OspA serotypes are correlated between species; serotype 1 corresponds to B. burgdorferi s.s., serotype 2 corresponds to B. afzelii and serotypes 3 to 7 correspond to B. garinii. Epidemiological studies of European Borrelia isolates indicate that an OspA-based vaccine of types 1, 2, 3, 4, 5 and 6 would provide theoretical coverage in Europe of 98.1% of Lyme disease and 96.7% coverage of invasive disease isolates. The invention provides six chimeric nucleic acid OspA molecules (SEQ ID NOS: 1, 3, and 5, and SEQ ID NOS: 168, 170, and 172) and six chimeric polypeptide OspA molecules (SEQ ID NOS: 2, 4, and 6, and SEQ ID NOS: 169, 171, and 173) that can provide protective immunity against all six serotypes 1-6. Six synthetic OspA genes were designed to encode OspA molecules with the protective epitopes of OspA serotypes 1 and 2 (lipB sOspA 1/2251 (SEQ ID NOS: 1 (nucleic acid) and 2 (amino acid) and orig sOspA 1/2 ( SEQ ID NOS: 168 (nucleic acid) and 169 (amino acid)); OspA serotypes 6 and 4 (lipB sOspA 6/4 (SEQ ID NOS: 3 (nucleic acid) and 4 (amino acid) and orig sOspA 6/4 ( SEQ ID NOS: 170 (nucleic acid) and 171 (amino acid)); and OspA serotypes 5 and 3 (lipB sOspA 5/3 (SEQ ID NOS: 5 (nucleic acid) and 6 (amino acid) and orig sOspA 5/3 ( SEQ ID NOS: 172 (nucleic acid) and 173 (amino acid).) The OspA chimeric genes were made using the overlapping synthetic oligonucleotides.These recombinant proteins are, in certain respects, produced in high yield and purity, and in several respects , manipulated to maximize desired activities and minimize unwanted ones. Polypeptide Molecules and Chimeric OspA Nucleic Acid Molecules
In various aspects, the present invention includes Borrelia chimeric OspA polypeptide and nucleic acid molecules. The OspA nucleic acids of the invention include a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleotide sequence defined in SEQ ID NO: 1 (lipB sOspA 1/2251), SEQ ID NO: 3 (lipB sOspA 6 /4), SEQ ID NO: 5 (lipB sOspA 5/3), SEQ ID NO: 7 (sOspA 1/2251), SEQ ID NO: 9 (sOspA 6/4), SEQ ID NO: 11 (sOspA 5/ 3), SEQ ID NO: 168 (origin sOspA 1/2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3), or a nucleotide sequence that encodes the polypeptide as defined in SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 ( sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3).
[00125] The nucleic acid sequences of SEQ ID NOS: 7, 9, and 11 do not show the nucleic acid sequence encoding the lipB leader sequence (MRLLIGFALALALIG (SEQ ID NO: 13). Furthermore, the nucleic acid sequences of SEQ ID NOS: 7, 9, and 11 encode a methionine residue at the amino terminus of SEQ ID NOS: 8, 10, and 12 in place of the cysteine residue present at the carboxy terminus of a lipB leader sequence in SEQ ID NOS: 2, 4, and 6. SEQ ID NOS: 1, 3, and 5 are lipB sOspA polypeptides, and SEQ ID NOS: 2, 4, and 6 are lipB sPolypeptide from OspAs.
[00126] In some aspects, the invention includes Borrelia chimeric OspA polypeptide and nucleic acid molecules without mutations and with codon optimization. The OspAs nucleic acid of the invention, therefore, includes a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleotide sequence as defined in SEQ ID NO: 168 (origin sOspA 1/2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3), or a nucleotide sequence encoding the polypeptide as defined in SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO : 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3).
[00127] The DNA Sequence numbers and amino acid sequence identification for the chimeric OspA molecules are shown in Table 1 below. Table 1. Amino acid and DNA sequences of Chimeric OspA
LipB sOspA 1/2251 amino acid sequence (SEQ ID NO: 2) MRLLIGFALALALIGCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLEL KGTSDKNNGS GVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSE KIITMADGT RLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSMNISKSGEVSVELND TDSSAATKKT AAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 1) catatgcgtctgttgatcggctttgctctggcgctggctctgatcggctgcgcacagaaaggtgctgagt ctattggttccgtttctgtagatctgcccggtgaaatgaaggttctggtgagcaaagaaaaagacaagaa cggcaagtacgatctcatcgcaaccgtcgacaagctggagctgaaaggtacttctgataaaaacaacggc tctggtgtgctggagggcgtcaaaactaacaagagcaaagtaaagcttacgatctctgacgatctcggtc agaccacgctggaagttttcaaagaggatggcaagaccctcgtgtccaaaaaagtaacttccaaagacaa gtcctctacggaagaaaaattcaacgaaaaaggtgaggtgtctgaaaagatcatcaccatggcagacggc acccgtcttgaatacaccggtattaaaagcgatggtaccggtaaagcgaaatatgttctgaaaaacttca ctctggaaggcaaagtggctaatgataaaaccaccttggaagtcaaggaaggcaccgttactctgagcat gaatatctccaaatctggtgaagtttccgttgaactgaacgacactgacagcagcgctgcgacta aaaaa actgcagcgtggaattccaaaacttctactttaaccattagcgttaacagcaaaaaaactacccagctgg tgttcactaaacaagacacgatcactgtgcagaaatacgactccgcaggcaccaacttagaaggcacggc agtcgaaattaaaacccttgatgaactgaaaaacgcgctgaaataagctgagcggatcc complementary strand (SEQ ID NO: 48) catatgcgtctgttgatcggctttgctttggcgctggctttaatcggctgtgcacagaaaggtgctgagt ctattggttccgtttctgtagatctgcccgggggtatgaaagttctggtaagcaaagaaaaagacaaaaa cggtaaatacagcctgatggcaaccgtagaaaagctggagcttaaaggcacttctgataaaaacaacggt tctggcaccctggaaggtgaaaaaactaacaaaagcaaagtaaagcttactattgctgaggatctgagca aaaccacctttgaaatcttcaaagaagatggcaaaactctggtatctaaaaaagtaaccctgaaagacaa gtcttctaccgaagaaaaattcaacgaaaagggtgaaatctctgaaaaaactatcgtaatggcaaatggt acccgtctggaatacaccgacatcaaaagcgataaaaccggcaaagctaaatacgttctgaaagacttta ctctggaaggcactctggctgctgacggcaaaaccactctgaaagttaccgaaggcactgttactctgag catgaacatttctaaatccggcgaaatcaccgttgcactggatgacactgactctagcggcaataaaaaa tccggcacctgggattctgatacttctactttaaccattagcaaaaacagccagaaaactaaacagctgg tattcaccaaagaaaacactatcaccgtacagaactataaccgtgcag gcaatgcgctggaaggcagccc ggctgaaattaaagatctggcagagctgaaagccgctttgaaataagctgagcggatcc LipB 6/4 sOspA amino acid sequence (SEQ ID NO: 4) MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMTVLVSKEKDKNGKYSLEATVDKLEL KGTSDKNNGS GTLEGEKTNKSKVKLTIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTEEKFNEKGETSE KTIVMANGT RLEYTDIKSDGSGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVVLSMNILKSGEITVALDD SDTTQATKK TGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 3) catatgcgtctgttgatcggctttgctctggcgctggctctgatcggctgcgcacagaaaggtgctgagt ctattggttccgtttctgtagatctgcccggtggcatgaccgttctggtcagcaaagaaaaagacaaaaa cggtaaatacagcctcgaggcgaccgtcgacaagcttgagctgaaaggcacctctgataaaaacaacggt tccggcaccctggaaggtgaaaaaactaacaaaagcaaagtgaaactgaccattgctgatgacctcagcc agaccaaattcgaaattttcaaagaagatgccaaaaccttagtatccaaaaaagtgaccctgaaagacaa gtcctctaccgaagaaaaattcaacgaaaagggtgaaacctctgaaaaaaccatcgtaatggcaaatggt acccgtctggaatacaccgacatcaaaagcgatggctccggcaaagccaaatacgttctgaaagacttca ccctggaaggcaccctcgctgccgacggcaaaaccaccttgaaagttaccgaagg cactgttgttttaag catgaacatcttaaaatccggtgaaatcaccgttgcgctggatgactctgacaccactcaggccactaaa aaaaccggcaaatgggattctaacacttccactctgaccatcagcgtgaattccaaaaaaactaaaaaca tcgtgttcaccaaagaagacaccatcaccgtccagaaatacgactctgcgggcaccaacctcgaaggcaa cgcagtcgaaatcaaaaccctggatgaactgaaaaacgctctgaaataagctgagcggatcc additional tape (SEQ ID NO: 49) ggatccgctcagcttatttcagcgcgtttttcagttcatcaagggttttaatttcgactgccgtgccttc taagttggtgcctgcggagtcgtatttctgcacagtgatcgtgtcttgtttagtgaacaccagctgggta gtttttttgctgttaacgctaatggttaaagtagaagttttggaattccacgctgcagtttttttagtcg cagcgctgctgtcagtgtcgttcagttcaacggaaacttcaccagatttggagatattcatgctcagagt aacggtgccttccttgacttccaaggtggttttatcattagccactttgccttccagagtgaagtttttc agaacatatttcgctttaccggtaccatcgcttttaataccggtgtattcaagacgggtgccgtctgcca tggtgatgatcttttcagacacctcacctttttcgttgaatttttcttccgtagaggacttgtctttgga agttacttttttggacacgagggtcttgccatcctctttgaaaacttccagcgtggtctgaccgagatcg tcagagatcgtaagctttactttgctcttgttagttttgacgccctccagcacaccagagccgttgtttt tatcagaagtacctttcagctccagcttgtcgacg gttgcgatgagatcgtacttgccgttcttgtcttt ttctttgctcaccagaaccttcatttcaccgggcagatctacagaaacggaaccaatagactcagcacct ttctgtgcgcagccgatcagagccagcgccagagcaaagccgatcaacagacgcatatg LipB 5/3 sOspA amino acid sequence (SEQ ID NO: 6) MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMKVLVSKEKDKNGKYSLMATVEKLEL KGTSDKNNGS GTLEGEKTNKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEK TIVMANGT RLEYTDIKSDKTGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVTLSMNISKSGEITVALDD TDSSGNKKS GTWDSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK DNA sequence (SEQ ID NO: 5) catatgcgtctgttgatcggctttgctttggcgctggctttaatcggctgtgcacagaaaggtgctgagt ctattggttccgtttctgtagatctgcccgggggtatgaaagttctggtaagcaaagaaaaagacaaaaa cggtaaatacagcctgatggcaaccgtagaaaagctggagcttaaaggcacttctgataaaaacaacggt tctggcaccctggaaggtgaaaaaactaacaaaagcaaagtaaagcttactattgctgaggatctgagca aaaccacctttgaaatcttcaaagaagatggcaaaactctggtatctaaaaaagtaaccctgaaagacaa gtcttctaccgaagaaaaattcaacgaaaagggtgaaatctctgaaaaaactatcgtaatggcaaatggt acccgtctggaatacaccgacatcaaaagcgataaaaccggca aagctaaatacgttctgaaagacttta ctctggaaggcactctggctgctgacggcaaaaccactctgaaagttaccgaaggcactgttactctgag catgaacatttctaaatccggcgaaatcaccgttgcactggatgacactgactctagcggcaataaaaaa tccggcacctgggattctgatacttctactttaaccattagcaaaaacagccagaaaactaaacagctgg tattcaccaaagaaaacactatcaccgtacagaactataaccgtgcaggcaatgcgctggaaggcagccc ggctgaaattaaagatctggcagagctgaaagccgctttgaaataagctgagcggatcc complementary strand (SEQ ID NO: 50) ggatccgctcagcttatttcagagcgtttttcagttcatccagggttttgatttcgactgcgttgccttc gaggttggtgcccgcagagtcgtatttctggacggtgatggtgtcttctttggtgaacacgatgttttta gtttttttggaattcacgctgatggtcagagtggaagtgttagaatcccatttgccggtttttttagtgg cctgagtggtgtcagagtcatccagcgcaacggtgatttcaccggattttaagatgttcatgcttaaaac aacagtgccttcggtaactttcaaggtggttttgccgtcggcagcgagggtgccttccagggtgaagtct ttcagaacgtatttggctttgccggagccatcgcttttgatgtcggtgtattccagacgggtaccatttg ccattacgatggttttttcagaggtttcacccttttcgttgaatttttcttcggtagaggacttgtcttt cagggtcacttttttggatactaaggttttggcatcttctttgaaaatttcgaatttggtctggctgagg tcatcagcaatggtcagtttcacttt gcttttgttagttttttcaccttccagggtgccggaaccgttgt ttttatcagaggtgcctttcagctcaagcttgtcgacggtcgcctcgaggctgtatttaccgtttttgtc tttttctttgctgaccagaacggtcatgccaccgggcagatctacagaaacggaaccaatagactcagca cctttctgtgcgcagccgatcagagccagcgccagagcaaagccgatcaacagacgcatatg sOspA 1/2251 amino acid sequence (SEQ ID NO: 8) MAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKGTSDKNNGSGVL EGVKTNKSKVKL TISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITMADGTRLEYTG IKSDGTGKA KYVLKNFTLEGKVANDKTTLEVKEGTVTLSMNISKSGEVSVELNDTDSSAATKKTAAWN SKTSTLTISVN SKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 7) catatggcacagaaaggtgctgagtctattggttccgtttctgtagatctgcccggtgaaatgaaggttc tggtgagcaaagaaaaagacaagaacggcaagtacgatctcatcgcaaccgtcgacaagctggagctgaa aggtacttctgataaaaacaacggctctggtgtgctggagggcgtcaaaactaacaagagcaaagtaaag cttacgatctctgacgatctcggtcagaccacgctggaagttttcaaagaggatggcaagaccctcgtgt ccaaaaaagtaacttccaaagacaagtcctctacggaagaaaaattcaacgaaaaaggtgaggtgtctga aaagatcatcaccatggcagacggcacccgtcttgaatacaccggtat taaaagcgatggtaccggtaaa gcgaaatatgttctgaaaaacttcactctggaaggcaaagtggctaatgataaaaccaccttggaagtca aggaaggcaccgttactctgagcatgaatatctccaaatctggtgaagtttccgttgaactgaacgacac tgacagcagcgctgcgactaaaaaaactgcagcgtggaattccaaaacttctactttaaccattagcgtt aacagcaaaaaaactacccagctggtgttcactaaacaagacacgatcactgtgcagaaatacgactcca acggcaccaacttagaaggcacggcagtcgaaattaaaacccttgatgaactgaaaaacgcgctgaaata agctgagcggatcc complementary strand (SEQ ID NO: 56) gtataccgtgtctttccacgactcagataaccaaggcaaagacatctagacgggccactttacttccaag accactcgtttctttttctgttcttgccgttcatgctagagtagcgttggcagctgttcgacctcgactt tccatgaagactatttttgttgccgagaccacacgacctcccgcagttttgattgttctcgtttcatttc gaatgctagagactgctagagccagtctggtgcgaccttcaaaagtttctcctaccgttctgggagcaca ggttttttcattgaaggtttctgttcaggagatgccttctttttaagttgctttttccactccacagact tttctagtagtggtaccgtctgccgtgggcagaacttatgtggccataattttcgctaccatggccattt cgctttatacaagactttttgaagtgagaccttccgtttcaccgattactattttggtggaaccttcagt tccttccgtggcaatgagactcgtacttatagaggtttagaccacttcaaaggcaacttgacttgctgtg actgt cgtcgcgacgctgatttttttgacgtcgcaccttaaggttttgaagatgaaattggtaatcgcaa ttgtcgtttttttgatgggtcgaccacaagtgatttgttctgtgctagtgacacgtctttatgctgaggt tgccgtggttgaatcttccgtgccgtcagctttaattttgggaactacttgactttttgcgcgactttat tcgactcgcctagg 6/4 sOspA amino acid sequence (SEQ ID NO: 10) MAQKGAESIGSVSVDLPGGMTVLVSKEKDKNGKYSLEATVDKLELKGTSDKNNGSGTLE GEKTNKSKVKL TIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTEEKFNEKGETSEKTIVMANGTRLEYTDI KSDGSGKA KYVLKDFTLEGTLAADGKTTLKVTEGTVVLSMNILKSGEITVALDDSDTTQATKKTGKWD SNTSTLTISV NSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 9) catatggcacagaaaggtgctgagtctattggttccgtttctgtagatctgcccggtggcatgaccgttc tggtcagcaaagaaaaagacaaaaacggtaaatacagcctcgaggcgaccgtcgacaagcttgagctgaa aggcacctctgataaaaacaacggttccggcaccctggaaggtgaaaaaactaacaaaagcaaagtgaaa ctgaccattgctgatgacctcagccagaccaaattcgaaattttcaaagaagatgccaaaaccttagtat ccaaaaaagtgaccctgaaagacaagtcctctaccgaagaaaaattcaacgaaaagggtgaaacctctga aaaaaccatcgtaatggcaaatggtacccgtctggaatacaccgacatcaaaagcgatggctccggcaaa gccaa atacgttctgaaagacttcaccctggaaggcaccctcgctgccgacggcaaaaccaccttgaaag ctctgacaccactcaggccactaaaaaaaccggcaaatgggattctaacacttccactctgaccatcagc gtgaattccaaaaaaactaaaaacatcgtgttcaccaaagaagacaccatcaccgtccagaaatacgact ctgcgggcaccaacctcgaaggcaacgcagtcgaaatcaaaaccctggatgaactgaaaaacgctctgaa ttaccgaaggcactgttgttttaagcatgaacatcttaaaatccggtgaaatcaccgttgcgctggatga ataagctgagcggatcc complementary strand (SEQ ID NO: 57) gtataccgtgtctttccacgactcagataaccaaggcaaagacatctagacgggccaccgtactggcaag accagtcgtttctttttctgtttttgccatttatgtcggagctccgctggcagctgttcgaactcgactt tccgtggagactatttttgttgccaaggccgtgggaccttccacttttttgattgttttcgtttcacttt gactggtaacgactactggagtcggtctggtttaagctttaaaagtttcttctacggttttggaatcata ggttttttcactgggactttctgttcaggagatggcttctttttaagttgcttttcccactttggagact tttttggtagcattaccgtttaccatgggcagaccttatgtggctgtagttttcgctaccgaggccgttt cggtttatgcaagactttctgaagtgggaccttccgtgggagcgacggctgccgttttggtggaactttc aatggcttccgtgacaacaaaattcgtacttgtagaattttaggccactttagtggcaacgcgacctact gagactgtggtgagtccggtgatttttttg gccgtttaccctaagattgtgaaggtgagactggtagtcg cacttaaggtttttttgatttttgtagcacaagtggtttcttctgtggtagtggcaggtctttatgctga gacgcccgtggttggagcttccgttgcgtcagctttagttttgggacctacttgactttttgcgagactt tattcgactcgcctagg 5/3 sOspA amino acid sequence (SEQ ID NO: 12) MAQKGAESIGSVSVDLPGGMKVLVSKEKDKNGKYSLMATVEKLELKGTSDKNNGSGTLE GEKTNKSKVKL TIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVMANGTRLEYTDIK SDKTGKA KYVLKDFTLEGTLAADGKTTLKVTEGTVTLSMNISKSGEITVALDDTDSSGNKKSGTWDS DTSTLTISKN SQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK DNA sequence (SEQ ID NO: 11) catatggcacagaaaggtgctgagtctattggttccgtttctgtagatctgcccgggggtatgaaagttc tggtaagcaaagaaaaagacaaaaacggtaaatacagcctgatggcaaccgtagaaaagctggagcttaa aggcacttctgataaaaacaacggttctggcaccctggaaggtgaaaaaactaacaaaagcaaagtaaag cttactattgctgaggatctgagcaaaaccacctttgaaatcttcaaagaagatggcaaaactctggtat ctaaaaaagtaaccctgaaagacaagtcttctaccgaagaaaaattcaacgaaaagggtgaaatctctga aaaaactatcgtaatggcaaatggtacccgtctggaatacaccgacatcaaaagcgataaaaccggcaaa gctaaatacgttctgaaagactttact ctggaaggcactctggctgctgacggcaaaaccactctgaaag cactgactctagcggcaataaaaaatccggcacctgggattctgatacttctactttaaccattagcaaa aacagccagaaaactaaacagctggtattcaccaaagaaaacactatcaccgtacagaactataaccgtg caggcaatgcgctggaaggcagcccggctgaaattaaagatctggcagagctgaaagccgctttgaaata ttaccgaaggcactgttactctgagcatgaacatttctaaatccggcgaaatcaccgttgcactggatga agctgagcggatcc additional tape (SEQ ID NO: 58) gtataccgtgtctttccacgactcagataaccaaggcaaagacatctagacgggcccccatactttcaag accattcgtttctttttctgtttttgccatttatgtcggactaccgttggcatcttttcgacctcgaatt tccgtgaagactatttttgttgccaagaccgtgggaccttccacttttttgattgttttcgtttcatttc gaatgataacgactcctagactcgttttggtggaaactttagaagtttcttctaccgttttgagaccata gattttttcattgggactttctgttcagaagatggcttctttttaagttgcttttcccactttagagact tttttgatagcattaccgtttaccatgggcagaccttatgtggctgtagttttcgctattttggccgttt cgatttatgcaagactttctgaaatgagaccttccgtgagaccgacgactgccgttttggtgagactttc aatggcttccgtgacaatgagactcgtacttgtaaagatttaggccgctttagtggcaacgtgacctact gtgactgagatcgccgttattttttaggccgtggaccctaagactatgaagatga aattggtaatcgttt ttgtcggtcttttgatttgtcgaccataagtggtttcttttgtgatagtggcatgtcttgatattggcac gtccgttacgcgaccttccgtcgggccgactttaatttctagaccgtctcgactttcggcgaaactttat tcgactcgcctagg Orig 1/2 sOspA amino acid sequence (SEQ ID NO: 169) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDK LEL KGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTE EKF NEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTL SK NISKSGEVSVELNDTDSSAATKKTAAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDS AG TNLEGTAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 168) atgaaaaaatatttattgggaataggtctaatattagccttaatagcatgtaagcaaaatgt tagcagccttgacgagaaaaacagcgtttcagtagatttgcctggtgaaatgaaagttcttg taagcaaagaaaaaaacaaagacggcaagtacgatctaattgcaacagtagacaagcttgag cttaaaggaacttctgataaaaacaatggatctggagtacttgaaggcgtaaaagctgacaa aagtaaagtaaaattaacaatttctgacgatctaggtcaaaccacacttgaagttttcaaag aagatggcaaaacactagtatcaaaaaaagtaacttccaaagacaagtcatcaacagaagaa aaattcaatgaaaaaggtgaagtatctgaaaaaataataacaagagcagacggaaccagact tgaatacacagga attaaaagcgatggatctggaaaagctaaagaggttttaaaaaacttta ctcttgaaggaaaagtagctaatgataaagtaacattggaagtaaaagaaggaaccgttact ttaagtaaaaatatttcaaaatctggggaagtttcagttgaacttaatgacactgacagtag tgctgctactaaaaaaactgcagcttggaattcaaaaacttctactttaacaattagtgtta acagcaaaaaaactacacaacttgtgtttactaaacaagacacaataactgtacaaaaatac gactccgcaggtaccaatttagaaggcacagcagtcgaaattaaaacacttgatgaacttaa aaacgctttaaaatag Orig 6/4 sOspA amino acid sequence (SEQ ID NO: 171) MKKYLLGIGLILALIACKQNVSTLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDK LKGTSDKNNGSGTLEGEKTDKSKVKLTIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTE LE V E KFNEKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGT VLSKNILKSGEITVALDDSDTTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQK YDSAGTNLEGNAVEIKTLDELKNALK DNA sequence (SEQ ID NO: 170) atgaaaaaatatttattgggaataggtctaatattagccttaatagcatgtaagcaaaatgt tagcacgcttgatgaaaaaaatagcgtttcagtagatttacctggtggaatgacagttcttg taagtaaagaaaaagacaaagacggtaaatacagtctagaggcaacagtagacaagcttgag cttaaaggaacttctgataaaaacaacggttctggaacacttgaaggtgaaaaaac tgacaa aagtaaagtaaaattaacaattgctgatgacctaagtcaaactaaatttgaaattttcaaag aagatgccaaaacattagtatcaaaaaaagtaacccttaaagacaagtcatcaacagaagaa aaattcaacgaaaagggtgaaacatctgaaaaaacaatagtaagagcaaatggaaccagact tgaatacacagacataaaaagcgatggatccggaaaagctaaagaagttttaaaagacttta ctcttgaaggaactctagctgctgacggcaaaacaacattgaaagttacagaaggcactgtt gttttaagcaagaacattttaaaatccggagaaataacagttgcacttgatgactctgacac tactcaggctactaaaaaaactggaaaatgggattcaaatacttccactttaacaattagtg tgaatagcaaaaaaactaaaaacattgtatttacaaaagaagacacaataacagtacaaaaa tacgactcagcaggcaccaatctagaaggcaacgcagtcgaaattaaaacacttgatgaact taaaaacgctttaaaataa Orig 5/3 sOspA amino acid sequence (SEQ ID NO: 173) and MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVE KLE LKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTE KFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGT V TLSKNISKSGEITVALDDTDSSGNKKSGTWDSDTSTLTISKNSQKTKQLVFTKENTITVQNY NRAGNALEGSPAEIKDLAELKAALK DNA sequence (SEQ ID NO: 172) atgaaaaaatatttattgggaataggtctaatat tagccttaatagcatgtaagcaaaatgt tagcagccttgatgaaaaaaatagcgtttcagtagatttacctggtggaatgaaagttcttg taagtaaagaaaaagacaaagatggtaaatacagtctaatggcaacagtagaaaagcttgag cttaaaggaacttctgataaaaacaacggttctggaacacttgaaggtgaaaaaactgacaa aagtaaagtaaaattaacaattgctgaggatctaagtaaaaccacatttgaaatcttcaaag aagatggcaaaacattagtatcaaaaaaagtaacccttaaagacaagtcatcaacagaagaa aaattcaacgaaaagggtgaaatatctgaaaaaacaatagtaagagcaaatggaaccagact tgaatacacagacataaaaagcgataaaaccggaaaagctaaagaagttttaaaagacttta ctcttgaaggaactctagctgctgacggcaaaacaacattgaaagttacagaaggcactgtt actttaagcaagaacatttcaaaatccggagaaataacagttgcacttgatgacactgactc tagcggcaataaaaaatccggaacatgggattcagatacttctactttaacaattagtaaaa acagtcaaaaaactaaacaacttgtattcacaaaagaaaacacaataacagtacaaaactat aacagagcaggcaatgcgcttgaaggcagcccagctgaaattaaagatcttgcagagcttaa agccgctttaaaataa
[00128] OspA polypeptides of the invention include a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4 ), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3) , SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3) and related polypeptides. Related polypeptides include OspA polypeptide analogs, OspA polypeptide variants, and OspA polypeptide derivatives. In some aspects, an OspA polypeptide has an amino terminal methionine residue, depending on the method by which they are prepared. In related aspects, the OspA polypeptide of the invention comprises OspA activity.
[00129] In one embodiment, the nucleic acid molecules comprise or consist of a nucleotide sequence that is about 70 percent (70%) identical or similar to the nucleotide sequence shown in SEQ ID NO: 1 (lipB sOspA 1/ 2251), SEQ ID NO: 3 (lipB sOspA 6/4), SEQ ID NO: 5 (lipB sOspA 5/3), SEQ ID NO: 7 (sOspA 1/2251), SEQ ID NO: 9 (sOspA 6/ 4), SEQ ID NO: 11 (sOspA 5/3), SEQ ID NO: 168 (origin sOspA 1/2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3), in certain respects comprise, consist essentially of, or consist of a nucleotide sequence encoding a polypeptide that is about 70 percent (70%) identical to the polypeptide as defined in SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 ( sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sospA 5/3). In various embodiments, the nucleotide sequences are about 70 percent, or about 71, 72, 73, 74, 75, 76, 77, 78, or 79 percent, or about 80 percent, or about 81 , 82, 83, 84, 85, 86, 87, 88, or 89 percent, or about 90 percent, or about 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence shown in SEQ ID NO: 1 (lipB sOspA 1/2251), SEQ ID NO: 3 (lipB sOspA 6/4), SEQ ID NO: 5 (lipB sOspA 5/3), SEQ ID NO: 7 (sOspA 1/2251), SEQ ID NO: 9 (sOspA 6/4), SEQ ID NO: 11 (sOspA 5/3), SEQ ID NO: 168 (origin sOspA 1/2), SEQ ID NO: 170 (origin sOspA 6/4), or SEQ ID NO: 172 (origin sOspA 5/3), or the nucleotide sequences that condition a polypeptide that is about 70 percent, or about 71, 72, 73, 74 , 75, 76, 77, 78, or 79 percent, or about 80 percent, or about 81, 82, 83, 84, 85, 86, 87, 88, or 89 percent, or about 90 percent percent, or about 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to the pol sequence. peptides as defined in SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3).
[00130] In some embodiments, the methods for determining sequence identity and/or similarity are designed to give the highest match between the sequences tested. Methods for determining identity and similarity are described in publicly available computer programs. In some aspects, computer program methods for determining identity and similarity between two sequences include, but are not limited to, packaged GCG programs, including GAP (Devereux et al., Nucl. Acid. Res., 12: 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI, BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra (1990)) The well-known Smith Waterman algorithm is also used to determine identity.
Certain alignment schemes for aligning two amino acid sequences, in some respects, result in only a small region of the two sequences being matched and this small aligned region may have very high sequence identity even if there is no significant relationship between the two full-length sequences. Consequently, in one embodiment, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide. For example, using the GAP (Genetics Computer Group, University of Wisconsin, Madison, WI) computer algorithm, two polypeptides for which percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "span" matched", as determined by the algorithm). Gap opening penalty (which is calculated as the mean of 3x the diagonal; "mean diagonal" is the mean of the diagonal of the comparison matrix being used, "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix like PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. A standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250 matrix comparison; Henikoff et al., Proc. Natl. Acad. Sci USA, 89: 10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.
[00132] In various aspects, parameters for comparing a polypeptide sequence include the following: Algorithm: Needleman et al., J. Mol. Biol., 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., supra (1992); Gap Penalty: 12 Gap Length Penalty: 4 Similarity Threshold: 0
[00133] The GAP program is useful with the above parameters. The above parameters are the default parameters for polypeptide comparisons (along with no penalty for final gaps) using the GAP algorithm.
In some aspects, parameters for nucleic acid molecule sequence comparisons include the following: Algorithm: Needleman et al., supra (1970); Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3
[00135] The GAP program is also useful with the above parameters. The above parameters are the standard parameters for nucleic acid molecule comparisons. Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices and similarity thresholds, and the like, are used by those skilled in the art, including those described in the Program Manual, Wisconsin Package, Version 9, September 1997 The particular choices to be made will be evident to those skilled in the art and will depend on the specific comparison being made, such as DNA-to-DNA, protein-to-protein, protein-to-DNA, and additionally whether the comparison is between even data of sequences (in which case GAP or BestFit are generally preferred) or between a sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).
Differences in nucleic acid sequence, in some aspects, result in conservative and/or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO : 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO : 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3).
Conservative modifications to the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4), SEQ ID NO: 6 (lipB sOspA 5/3) , SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3), SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3) (and corresponding modifications to the coding nucleotides) will produce OspAs polypeptides having functional and chemical characteristics similar to those of a polypeptide of naturally occurring OspA. In contrast, substantial modifications in the functional and/or chemical characteristics of the OspA polypeptides are made by selecting substitutions in the amino acid sequence of SEQ ID NO: 2 (lipB sOspA 1/2251), SEQ ID NO: 4 (lipB sOspA 6/4 ), SEQ ID NO: 6 (lipB sOspA 5/3), SEQ ID NO: 8 (sOspA 1/2251), SEQ ID NO: 10 (sOspA 6/4), SEQ ID NO: 12 (sOspA 5/3) , SEQ ID NO: 169 (origin sOspA 1/2), SEQ ID NO: 171 (origin sOspA 6/4), or SEQ ID NO: 173 (origin sOspA 5/3) which differ significantly in maintenance (a) of structure of the molecular scaffold in the replacement area, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
[00138] For example, a "conservative amino acid substitution", in some aspects, involves replacing a native amino acid residue with a non-native residue in such a way that there is little or no effect on the polarity or charge of the residue. amino acid at that position. Furthermore, any native residue on the polypeptide, in some respects, is also replaced by alanine, as previously described for "alanine scanning mutagenesis".
Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptide mimetics and other reversed or inverted forms of amino acid moieties.
[00140] Naturally occurring residues, in various aspects, are divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acid: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe.
[00141] For example, non-conservative substitutions, in some aspects, involve exchanging a member of one of these classes for a member of another class. Such substituted residues, in various aspects, are introduced into regions of the OspA polypeptide that are homologous, or similar, to OspA polypeptide orthologs, or into non-homologous regions of the molecule.
[00142] When making such changes, the hydropathic index of the amino acids is often considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); Threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
[00143] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and yet retain a similar biological activity. When making changes based on the hydropathic index, the replacement of amino acids whose hydropathic indices are within ± 2 in certain respects is preferred, those which are within ± 1 are in other respects particularly preferred, and those within ± 0.5 are, in various aspects, more particularly preferred.
[00144] It is also understood in the art that amino acid substitution can be made effectively on the basis of hydrophilicity, particularly where the equivalent biologically functional protein or peptide thus created is intended, in part, for use in immunological modalities, as in the case gift. The greater local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, that is, with a biological property of the protein.
[00145] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); Threonine (-0.4); proline (-0.5 ± 1); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2,3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ± 2 is in certain respects preferred, those which are within ± 1 are in other respects particularly preferred, and those within ± 0.5 are in various respects more particularly preferred. A skilled person also identifies epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as "epitopic core regions".
Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the OspA polypeptide, or to increase or decrease the affinity of the OspA polypeptides for their substrates, described herein.
In some aspects, nucleotide substitutions in nucleotide sequences and amino acid in amino acid sequences are included in the invention. Substitutions include one through 5, one through 10, one through 15, one through 20, one through 25, one through 30, one through 35, one through 40, one through 45, one through 50, one through 55, one through 60, one through 65, one through 70, one through 75, one through 80, one through 85, one through 90, one through 95, one through 100, one through 150, and one through 200 nucleotides. Likewise, substitutions include one through 5, one through 10, one through 15, one through 20, one through 25, one through 30, one through 35, one through 40, one through 45, one through 50, one through 55 , one through 60, one through 65, one through 70, one through 75, one through 80, one through 85, one through 90, one through 95, and one through 100 amino acids. Substitutions, in many respects, are either conservative or non-conservative. Exemplary amino acid substitutions are defined in Table 2. Table 2. Amino Acid Substitutions

[00148] One of skill in the art can determine the appropriate analogues or variants of the polypeptide as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173 using well known techniques. To identify suitable areas of the molecule that can be altered without destroying the activity, those skilled in the art can target areas not believed to be important for the activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art can compare the amino acid sequence of an OspA polypeptide to such similar polypeptides. With this comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of an OspA polypeptide that are not conserved relative to those similar polypeptides would be less likely to adversely affect the biological activity and/or structure of the OspA polypeptide. One skilled in the art would also know that, even in relatively conserved regions, it is possible to chemically replace amino acids similar to naturally occurring residues during activity retention (conservative amino acid residue substitutions).
In some embodiments, OspA polypeptide variants include glycosylation variants, in which the number and/or type of glycosylation sites has been altered compared to the amino acid sequence defined in SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173. In one embodiment, OspA polypeptide variants comprise a greater or lesser number of N-linked glycosylation sites than the amino acid sequence defined in SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue is designated as X can be any amino acid residue except proline. Substituting amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that delete that sequence will remove an existing N-linked carbohydrate chain. A rearrangement of N-linked carbohydrate chains is also provided, in which one or more N-linked glycosylation sites (typically those that occur naturally) are deleted and one or more new N-linked sites are created. Additional variants of OspA include cysteine variants, in which one or more cysteine residues are deleted or replaced by another amino acid (eg serine) compared to the amino acid sequence defined in SEQ ID NO: 2, 4, 6, 8 , 10, 12, 169, 171, or 173. Cysteine variants are useful when OspA polypeptides must be refolded into a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically they have a uniform number to minimize interactions resulting from mismatched cysteines.
The invention also provides polypeptides which comprise an epitope-bearing portion of a protein as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173. The term "epitope" refers to a region of a protein to which an antibody can bind. See, for example, Geysen et al., Proc. Natl. Academic Sci. USA 81:3998-4002 (1984). Epitopes can be linear or conformational, the latter being composed of discontinuous regions of protein that form an epitope upon protein folding. Linear epitopes are generally at least 6 amino acid residues in length. Relatively short synthetic peptides that mimic part of a protein sequence are routinely able to induce an antiserum that reacts with the partially mimicked protein. See, Sutcliffe et al., Science 219:660-666 (1983). Antibodies that recognize short linear epitopes are particularly useful in analytical and diagnostic applications that use denatured protein, such as in Western blotting. See Tobin, Proc. Natl. Academic Sci. USA, 76:4350-4356 (1979). Antibodies to the short peptides, in certain cases, also recognize the proteins in the native conformation and thus will be useful for monitoring protein expression and protein isolation, and for detecting OspA proteins in solution, such as by ELISA or in immunoprecipitation studies. Synthesis of Chimeric OspA Nucleic Acid Molecules and Polypeptide Molecules
Nucleic acid molecules encode a polypeptide comprising the amino acid sequence of an OspA polypeptide and can be readily obtained in a variety of ways, including, without limitation, recombinant DNA and chemical synthesis methods.
Recombinant DNA methods are generally those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and/or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994). Recombinant expression techniques conducted in accordance with the descriptions set forth below, in various aspects, are followed to produce such polynucleotides and to express the encoded polypeptides. For example, by inserting a nucleic acid sequence encoding the amino acid sequence of an OspA polypeptide into an appropriate vector, one skilled in the art can easily produce large amounts of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding the amino acid sequence of an OspA polypeptide can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the OspA polypeptide or encoded OspA polypeptides are, in some aspects, produced in large quantities.
Likewise, chemical syntheses of nucleic acids and polypeptides are well known in the art, such as those described by Engels et al., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate method for nucleic acid synthesis. In one aspect, one method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry. Typically, the DNA encoding the amino acid sequence of an OspA polypeptide will be several hundred nucleotides in length. Nucleic acids longer than about 100 nucleotides are synthesized as multiple fragments using these methods. The fragments are then ligated together to form the full-length nucleotide sequences of the present invention. In particular aspects, the DNA fragment encoding the amino terminus of the polypeptide has an ATG, which encodes a methionine residue.
In a particular aspect of the invention, the chimeric OspA coding sequences are made using the overlapping synthetic oligonucleotides. Because Borrelia cell DNA is not used, an additional benefit of the synthetic approach is the prevention of contamination with adventitious agents contained in the animal source material (ie, serum or serum albumin) present in the Borrelia culture medium . This strategy also substantially reduces the number of manipulations needed to make the genes chimeric, as it allows sequence changes to be made in a single step, such as modifications to optimize expression (OspB leader sequence), to introduce restriction sites to facilitate cloning, or to avoid potential intellectual property problems. This also allows the use of codons that are optimized for an E. coli host, since the presence of codons that are rarely used in E. coli is known to present a potential impediment to high level expression of foreign genes (Makoff et al. al., Nucleic Acids Res. 17:10191-202, 1989; Lakey et al., Infect. Immun. 68:233-8, 2000). Other methods known to those skilled in the art are also used.
[00155] In certain embodiments, nucleic acid variants contain codons that have been altered for optimal expression of an OspA polypeptide from a particular host cell. Specific codon changes will depend on the host cell(s) and OspA polypeptide(s) selected for expression. This "codon optimization" can be accomplished by a variety of methods, for example, by selecting the codons that are preferred for use in highly expressed genes in a given host cell. Computer algorithms that incorporate codon frequency tables such as "Ecohigh.cod" for the codon preference of highly expressed bacterial genes are used in some cases and are provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group , Madison, WI. Other useful codon frequency tables include "Celegans_high.cod", "Celegans_low.cod", "Drosophila_high.cod", "Human_high.cod", "Maize_high.cod", and "Yeast_high.cod".
A nucleic acid molecule encoding the amino acid sequence of an OspA polypeptide, in certain aspects, is inserted into an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that gene amplification and/or gene expression can occur). A nucleic acid molecule encoding the amino acid sequence of an OspA polypeptide, in various aspects, is amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Host cell selection will depend, in part, on whether an OspA polypeptide must be post-transductionally modified (eg, glycosylated and/or phosphorylated). If so, yeast, insect or mammalian host cells are preferred. For a review of expression vectors, see Meth. Enz., vol.185, D.V. Goeddel, ed., Academic Press Inc., San Diego, CA (1990).
[00157] Cloning vectors include all known in the art. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989. In one aspect, pUC18 is used as a cloning vector for all intermediate steps, because manipulations and genetic sequencing are easier with this plasmid than with the pET30a vector . The main features are namely, the lacZ gene fragment encoding the 149-469 base pair LacZ alpha peptide (lac promoter at 507 base pairs), the bla gene encoding the base pair ampicillin resistance determinant 1629 to 2486 (bla promoter at base pairs 2521), origin of replication at 867 base pairs and multiple cloning sites from base pairs 185 to 451 (Fig. 12).
[00158] Expression vectors include all known in the art, including, without limitation, cosmids, plasmids (e.g., naked, or contained in liposomes) and viruses that incorporate the recombinant polynucleotide. The expression vector is inserted (eg, by transformation or transduction) into an appropriate host cell for polynucleotide and polypeptide expression by transformation or transfection by techniques known in the art. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989. In one aspect, pET30a (Novagen) is used as an expression vector for the insertion of the final full-length OspA gene. In pET vectors, genes have been cloned under the control of a T7 promoter and expression is induced by providing a source of T7 RNA polymerase in the host cell (no expression occurs until a source of T7 RNA polymerase is provided) . The main features are the gene encoding kanamycin resistance (kan) at base pairs 4048-4860, the lacI gene base pairs 826 to 1905, the F1 origin of replication at base pairs 4956 to 5411 and multiple sites from cloning base pairs 158 to 346 (Fig. 13.).
After the vector has been constructed and a nucleic acid molecule encoding an OspA polypeptide has been inserted into the proper site of the vector, the complete vector is inserted into a suitable host cell for polypeptide amplification and/or expression. Transformation of an expression vector for an OspA polypeptide in a selected host cell is, in many respects, accomplished by well-known methods such as transfection, infection, calcium chloride-mediated transformation, electroporation, microinjection, lipofection or the DEAE-dextran method or other known techniques. The method selected will, in part, be a function of the type of host cell to be used. These methods and other suitable methods are well known to those skilled in the art and are set out, for example, in Sambrook et al., Supra.
Host cells, in some aspects, are prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as yeast, insect or vertebrate cells). The host cell, when cultivated under suitable conditions, synthesizes an OspA polypeptide which can be subsequently harvested from the culture medium (if the host cell secretes the same into the medium) or directly from the host cell producing it (if not is secreted). Selection of an appropriate host cell will depend on several factors, such as the desired expression levels, the polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and the ease of folding into a biologically active molecule. These host cells include, but are not limited to, yeast, bacterial, fungal, invertebrate, viral, and mammalian host cell sources. For examples of such host cells, see Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). In additional aspects, host cells used in the art since the publication of the Maniatis manual (supra) are also used in the present invention.
[00161] In one aspect, the host cell is an E. coli cell. Suitable E. coli strains include, but are not limited to, BL21, DH5D, HMS174(DE3), DH10B, or E. CLONI 10G (Lucigen, Middleton, Wis.). In some embodiments, host cells are designed to increase the efficiency of vector transformation and/or maintenance.
[00162] In one aspect, the E. coli strain DH5α [genotype: end A1 hsdR17 (rK-mK+) supE44 thi-1 recA1 gyrA (Nalr) relA1 D(lacZYA-argF)U169 deoR (F80dlacD(lacZ)M15] (Gibco BRL) is used for all intermediate cloning steps. This strain is derived from the E. coli K12 strain, one of the most widely used hosts in genetic engineering. The strain is amplifying to allow selection of transformants with vectors containing the ampicillin (amp) gene resistance.
[00163] In another aspect, the E. coli strain HMS174 (DE3) is used as a host for expression. E. coli host cells HMS174 (DE3) [genotype: F-recA1 hsdR (rk12-MK12 +) RifR (DE3)] (Novagen) are used in several examples described in this document for the final cloning steps. The strain is kan- to allow selection of transformants with vectors containing the kanamycin resistance gene (kan).
Host cells comprising an OspA polypeptide expression vector are cultured using standard media well known to those skilled in the art. The media will normally contain all the nutrients needed for cell growth and survival. Suitable media for culturing E. coli cells include, for example, Luria broth (LB) and/or Terrific broth (TB). Suitable media for culturing eukaryotic cells include Roswell Park Memorial Institute's 1640 medium (RPMI 1640), Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle's Medium (DMEM), all of which, in some cases, being supplemented with serum and/or growth factors, as indicated by the particular cell line being cultured. A suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolyzate, or fetal calf serum, if necessary.
Typically, an antibiotic or other compound useful for selective growth of transformed cells is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell was transformed. For example, when the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. Other compounds for selective growth include ampicillin, tetracycline and neomycin.
The amount of an OspA polypeptide produced by a host cell can be assessed using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, chromatographic separation such as High Performance Liquid Chromatography (HPLC), immunodetection such as immunoprecipitation and/or immunoassay assays. activity such as the DNA binding gel displacement assays.
[00167] In some cases, an OspA polypeptide is not biologically active after isolation. Various methods of "refolding" or converting the polypeptide to its tertiary structure and generating disulfide bonds are used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH generally above 7 and in the presence of a particular concentration of a chaotropic agent. The selection of the chaotropic agent is very similar to the choices used for the solubilization of inclusion bodies, but generally the chaotropic agent is used at a lower concentration and is not necessarily the same as the chaotropic agent used for the solubilization. In some cases, the refolding/oxidation solution also contains a reducing agent or reducing agent plus its oxidized form in a specific proportion to generate a particular redox potential allowing disulfide shuffling to occur in the formation of the cysteine bridge(s) of the protein. Some of the commonly used redox pairs include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cuprous chloride, dithiothreitol (DTT)/dithiane DTT, and 2-2mercaptoethanol (bME)/dithio-b (ME). A co-solvent is generally used to increase refolding efficiency and the most common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.
[00168] If inclusion bodies are not formed to a significant degree after expression of an OspA polypeptide, then the polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate. The polypeptide is further isolated from the supernatants using methods such as those described herein or otherwise known in the art.
Purification of the OspA polypeptide from a solution can be carried out using a variety of techniques known in the art. Whether the polypeptide was synthesized to contain a tag such as hexa-histidine (OspA/hexaHis polypeptide) or another small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen, Carlsbad, CA ) either at its carboxyl or amino terminus, the polypeptide is usually purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag. For example, polyhistidine binds with high affinity and specificity to nickel, so a nickel affinity column (such as Qiagen® nickel columns) can be used for the purification of the OspA/polyHis polypeptide. See, for example, Ausubel et al., Eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York (1993).
[00170] Furthermore, the OspA polypeptide can be purified through the use of a monoclonal antibody that is able to specifically recognize and bind to the OspA polypeptide. Suitable procedures for purification therefore include, without limitation, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, high performance liquid chromatography (HPLC), electrophoresis (including native gel electrophoresis) followed by gel elution, and preparative isoelectric focusing ("Isoprime machine/technique", Hoefer Scientific, San Francisco, CA). In some cases, two or more purification techniques are combined to achieve increased purity.
OspA polypeptides are also prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art, such as those presented by Merrifield et al., J. Am. Chem. Soc., 85:2149 (1963), Houghten et al., Proc. Natl. Academic Sci. USA, 82:5132 (1985), and Stewart and Young, "Solid Phase Peptide Synthesis", Pierce Chemical Co., Rockford, IL (1984). Such polypeptides are synthesized with or without an amino-terminal methionine. Chemically synthesized OspA polypeptides, in some aspects, are oxidized using methods set forth in these references to form disulfide bridges. Chemically synthesized OspA polypeptides are expected to have a biological activity comparable to corresponding OspA polypeptides produced recombinantly or purified from natural sources, and thus are often used interchangeably with a recombinant OspA polypeptide. It is appreciated that a number of additional methods for producing nucleic acids and polypeptides are known in the art, and the methods can be used to produce OspA polypeptides. Chemical Derivatives of OspA Polypeptide Molecules
Chemically modified derivatives of OspA polypeptides are prepared by one of skill in the art, taking into account the disclosures set forth herein below. Derivatives of OspA polypeptides are modified in a manner that is different in either the type or location of molecules naturally bound to the polypeptide. Derivatives, in some respects, include molecules formed by the deletion of one or more naturally enrolled chemical groups. The polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173, or a polypeptide variant of OspA, in one aspect, is modified by the covalent linkage of a or more polymers. For example, the polymer selected is typically water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Included within the scope of suitable polymers is a blend of polymers. In certain aspects, for therapeutic use in the final product preparation, the polymer will be pharmaceutically acceptable.
[00173] Polymers are each, in various respects, of any molecular weight and are branched or unbranched. The polymers generally each have an average molecular weight of between about 2 kDa to about 100 kDa (the term "about" indicates that in preparations of a water-soluble polymer, some molecules will weigh more, others less, than than the indicated molecular weight). The average molecular weight of each polymer is, in various aspects, between about 5 kDa to about 50 kDa, between about 12 kDa to about 40 kDa, and between about 20 kDa to about 35 kDa.
Suitable water soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including those forms of PEG that have been used to transform proteins, including mono-(C1-C10)alkoxy-or aryloxy-polyethylene glycol); monomethoxy-polyethylene glycol; dextran (such as low molecular weight dextran of, for example, about 6 kDa); cellulose; or other carbohydrate-based polymers, poly(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol. Also encompassed by the present invention are bifunctional cross-linking molecules that are sometimes used to prepare covalently linked multimers of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171 , or 173, or a polypeptide variant of OspA.
[00175] In some aspects, chemical derivatization is performed under any suitable condition used to react a protein with an activated polymer molecule. Methods for preparing chemical derivatives of polypeptides generally comprise the steps of (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions where the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 169, 171, or 173, or a polypeptide variant of OspA becomes linked to one or more polymer molecules, and (b) obtain the reaction product(s). Optimum reaction conditions are determined based on known parameters and the desired result. For example, the greater the polymer molecules:protein ratio, the greater the percentage of fixed polymer molecules. In one embodiment, the OspA polypeptide derivative has a single polymeric molecule moiety at the amino terminus (see, for example, US Patent 5,234,784).
Pegylation of the polypeptide, in some respects, is specifically effected by any of the pegylation reactions known in the art, as described, for example, in the following references: Francis et al., Focus on Growth Factors, 3:4- 10 (1992); EP 0154316; EP 0401384 and US Patent 4,179,337. For example, pegylation is accomplished via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or a reactive water-soluble polymer analog) as described in this document. For acylation reactions, the polymer(s) selected should have a single reactive ester group. For reductive alkylation, the polymer(s) selected should have a single reactive aldehyde group. A reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is stable in water, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see US Patent 5,252,714).
[00177] In another embodiment, OspA polypeptides are chemically coupled to biotin, and the OspA/biotin polypeptide molecules that are conjugated are then able to bind to avidin, resulting in tetravalent avidin/biotin OspA polypeptide molecules . OspA polypeptides are also covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates with a valence of 10. The OspA Polypeptide Derivatives Described in this document, in certain respects, they have additional activities, enhanced or reduced biological activity, or other characteristics, such as an increase or decrease in half-life, compared to underived molecules. Immunogenic Compositions, Vaccines and Antibodies
[00178] Some aspects of the invention include immunogenic compositions and vaccines. The immunogenic chimeric OspA molecules of the present invention are used in combination as antigen(s) to induce an anti-OspA immune response in a subject (i.e., function as a vaccine). Examples of immunogenic OspA polypeptides (SEQ ID NOS: 2, 4, 6, 169, 171, and 173) are provided together to induce an immune response to any one or more of Borrelia serotypes 1-6, and more generally to many other species of Borrelia as discussed here. An immune response can also be heightened by the delivery of plasmid vectors encoding the OspA polypeptides of the invention (i.e., administration of "naked DNA"). In some aspects, OspA nucleic acid molecules (SEQ ID NOS: 1, 3, 5, 168, 170, and 172) are delivered by injection, via liposomes, or by other means of delivery described herein. Once immunized, the subject elicits an enhanced immune response against the OspA protein of Borrelia serotypes 1-6 and against other species of Borrelia.
As noted above, therefore, both OspA polypeptides and OspA nucleic acid molecules are included as antigens for use in immunogenic and/or vaccine compositions of the invention. In certain aspects, both nucleic acid and protein are delivered to the subject. In particular aspects, the immune response to a nucleic acid vaccine is proposed to be enhanced by the simultaneous administration of a cognate protein (see WO 99/30733). Nucleic acid and protein need not be administered in the same composition. Both should only be administered during the induction phase of the immune response, with the protein, in certain respects, being masked or maintained until after the nucleic acid has primed the immune system. In a particular aspect, vaccines are intended to deliver the antigen nucleic acid and protein to antigen presenting cells (see WO 97/28818). In various aspects, nucleic acid and protein are complexed, for example, through covalent conjugation. In other aspects, liposomal formulations are also included to improve the immunogenicity of antigens.
In some aspects, an immunogenic composition of the invention includes any one or more of the OspA molecules described herein, in combination with a pharmaceutical carrier, wherein the composition induces the production of an antibody that specifically binds to a protein protein The outer surface (OspA). In some aspects, the immunogenic composition also comprises an antimicrobial stabilizer or preservative. In particular aspects, the immunogenic composition induces the production of an antibody that specifically binds to Borrelia. In other aspects, the composition induces the production of an antibody that neutralizes Borrelia.
In certain aspects, the invention includes the use of adjuvants in immunogenic compositions comprising the chimeric OspA molecules (antigens) described herein. In certain aspects, immunogenicity is significantly improved if an antigen is co-administered with an adjuvant. In certain aspects, whether an adjuvant is used such as 0.001% to 50% phosphate buffered saline (PBS) solution. Adjuvants enhance the immunogenicity of an antigen but do not necessarily become immunogenic in themselves.
[00182] Adjuvants, in various respects, have a number of positive effects on vaccination. In some cases, adjuvants accelerate the generation of an immune response in robust individuals. Adjuvants, in other cases, increase the level of the immune response, prolong its duration and improve the immune memory. Adjuvants are often used to overcome the weakened immunity of specific groups of individuals (eg, elderly people or immunodeleted patients), or to improve the immunogenicity of a particular "at-risk group" (such as, but not limited to, very young and old). The immune-boosting effects of an adjuvant, in many cases, lead to a reduction in the amount of antigen needed in the final formulation to give a protective response (ie, a saving dose).
[00183] Generally speaking, adjuvants are classified, based on their dominant mechanism of action, into two main groups: the first group is receptor agonists or sensors of the innate immunity of the system, such as Toll-type receptor agonists (TLR), C-type lectin receptor agonists, gene I induced retinoic acid receptor (RLR) agonists (RIG-1) and nucleotide binding domain and leucine rich repeat (NLR)-containing receptor agonists. The second group is substances that act as delivery systems, also known as TLR-independent adjuvants. Examples of TLR adjuvant agonists are ASO4 (Glaxo Smith Kline), a TLR-4 agonist, used as an adjuvant in commercial Hepatitis B and papillioma virus vaccines; Vaxinate, a flagellin-TLR-5 fusion protein agonist, and numerous TLR-9 agonist adjuvants, such as those using double-stranded DNA (dsDNA), and CpG or ODN1a oligonucleotides. Other TLR agonists that fall into this category of adjuvants include glycolipids (TLR-1), lipoteichoic acid and lipoprotein lipopolysaccharide (TLR-1/TLR-2 and TLR-2/TLR-6), lipooligocacharides and monophosphoric lipid A (MPL ) (TLR-4), double-stranded RNA (TLR-3); peptide glycan (TLR-6), single-stranded RNA (TLR-7). Examples of two adjuvant type C lectin receptor agonists include β-glucans (Dectin-1) and mannans (Dectin-2), both derived from the cell wall of fungi. Agonist RLR receptor adjuvants include single-stranded viral RNA and double-stranded viral DNA, while agonistic NLR adjuvants include glycan peptide degradation products, microbial products, and non-infectious crystal particles. In all cases, agonists act by activating the innate immune system receptor directly to trigger an enhanced immune inflammatory response. The second group of adjuvants, TLR-independent adjuvants, act primarily as delivery systems and enhance antigen uptake and presentation by an antigen-presenting cell. In some cases, these adjuvants may also act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a sustained, slow release of the antigen to cells of the immune system. Adjuvants also attract immune system cells to an antigen depot and stimulate those cells to elicit immune responses. Examples of TLR-independent adjuvants include mineral salts such as aluminum hydroxide and aluminum phosphate (collectively referred to as alum) and calcium phosphate, oil-in-water emulsion (eg MF59, AS03 and ProVax), emulsion water in oil, (Montanide, TiterMax); biopolymers (Advax), derived from plants, in particular saponin fractions, a triterpenoid extract from the bark of the Quillaja saponaria Molina tree of South America (SFA-1, QS21, Quil A); immune stimulant complexes (ISCOM and ISCOM matrix) composed of fractions of saponin, sterol and, optionally, phospholipids (ISCOMATRIX and M-Matrix); liposomes, which are phospholipid spheres of various sizes and charge (Vaxfectin and Vaxisoma); virus-like particles and virosomes, which are liposomes containing viral surface antigens such as influenza hemagglutinin and neuraminidase, nanoparticles of various compositions, chitosan, peptides such as polyarginine and a peptide known as the KLK peptide.
[00184] The adjuvants listed here above are used alone or in combination. Combinations of TLR-dependent and TLK-independent adjuvants are often preferred as it is believed that the antigen and the TLR-dependent adjuvant are trafficked into the antigen-presenting cells of the TLR-independent adjuvant, which also stimulates absorption and stability, whereas the TLR-dependent adjuvant would directly enhance immunity through activation of TLR signaling.
[00185] Examples of combinations of TLR-dependent and TLR-independent adjuvants include AS01: a mixture of MPL (a TLR-4 agonist), liposomes and QS-21 (both TLR-independent adjuvants); AS04: MPL (a TLR-4 agonist) and aluminum hydroxide/phosphate; IC31: ODN1a (a TLR-9 agonist) and KLK peptide (a TLR-independent adjuvant) and Freund's complete adjuvant, a membrane extract of Mycobacterium tuberculosis (TLR-4 agonist) and an oil-in-water emulsion (a TLR-independent adjuvant).
[00186] Combinations consisting of multiple TLR-dependent adjuvants are also used to maximize the immune-boosting effect of adjuvant vaccine formulations. TLR agonists using different adapter proteins are often combined (eg a combination of an agonist for the TLR-3 receptor, or plasma membrane-bound TLR-4 using the TRIF (protein) adapter pathway adapter containing domain of interleukin 1/Toll receptor inducing INF-β) with an agonist of TLRs (TLR-7, TLR-8 and TLR-9), which are expressed in endosomal or lysosomal organelles and use the adapter pathway of the MyD88 protein (primary myeloid response differentiation protein).
[00187] These immunostimulating agents or adjuvants improve the host's immune response in vaccines as well. In some cases, substances such as lipopolysaccharides can act as intrinsic adjuvants since they are normally the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants, such as those listed herein above, are immunomodulators that are typically non-covalently linked to antigens and are formulated to potentiate the host's immune response.
[00188] A wide range of extrinsic adjuvants can elicit potent immune responses to antigens. These include saponins complexed with membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, mycobacteria killed in mineral oil, complete Freund's adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharides (LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune response (HIR) and cell-mediated immunity (CMI), immunogens are, in certain respects, emulsified in adjuvants.
[00189] Desirable characteristics of ideal adjuvants include any or all of: the absence of toxicity, ability to stimulate a long-lasting immune response; simplicity of fabrication and long-term storage stability, ability to elicit both CMI and HIR for antigens administered via multiple routes; synergy with other adjuvants; ability to selectively interact with populations of antigen presenting cells (APC), the ability to specifically induce specific immune responses from appropriate TH1 or TH2 cells; and ability to selectively increase appropriate antibody isotype levels (eg, IgA) against antigens.
[00190] US Patent 4,855,283, incorporated herein by reference, teaches therein that glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted at the sugar residue by an amino acid such as immunomodulators or adjuvants. US Patent 4,855,283 reported that N-glycolipid analogues showing structural similarities to naturally occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of inducing strong immune responses in both the herpes simplex virus vaccine and the pseudorabies virus vaccine . Some glycolipids have been synthesized from long-chain alkylamines and fatty acids that are directly linked to sugar through the anomeric carbon atom, to mimic the functions of naturally occurring lipid residues.
[00191] In some aspects, the immunogenic composition contains an amount of an adjuvant sufficient to potentiate the immune response to the immunogen. Suitable adjuvants include, but are not limited to, aluminum salts (aluminum phosphate or aluminum hydroxide), mixtures of squalene (SAF-1), muramyl peptide, saponin derivatives, mycobacterial cell wall preparations, monophosphoryl lipid A , mycolic acid derivatives, nonionic blocking copolymer surfactants, Quil A, cholera toxin B subunit, polyphosaazene and derivatives, and immunostimulant complexes (ISCOM) such as those described by Takahashi et al. (Nature 344:873-875, 1990). In some aspects, the adjuvant is a synthetic adjuvant. In a particular aspect, the synthetic adjuvant is glucopyranosyl lipid (GLA) adjuvant.
[00192] A further aspect of the present invention is a vaccine comprising the immunogenic composition of the invention and a pharmaceutically acceptable carrier. As discussed herein above, the vaccine, in certain aspects, includes one or more stabilizers and/or one or more preservatives.
In one aspect, there is provided a vaccine comprising at least one recombinant expression construct which comprises a promoter operably linked to a nucleic acid sequence encoding an antigen (chimeric OspA polypeptide described herein) and an adjuvant. In one embodiment, the recombinant expression construct (expression vector comprising the OspA polynucleotide) is present in a viral vector, which, in certain additional embodiments, is present in a virus that is selected from an adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus and a retrovirus.
Additional aspects of the present invention include antibodies against the chimeric OspA molecules described herein. In various aspects, the present invention includes chimeric OspA molecules to make anti-OspA antibodies and to provide immunity against Borrelia infection. In some aspects, these anti-OspA antibodies, e.g., murine, human or humanized monoclonal antibodies or single chain antibodies, are administered to a subject (e.g., passive immunization) to effect an immune response against the OspA protein of any one or more of Borrelia serotypes 1-6. As used herein, the term "antibodies" refers to a molecule that has specificity for one or more OspA polypeptides. Suitable antibodies are prepared using methods known in the art. In certain aspects, an OspA antibody is capable of binding to a particular portion of the polypeptide, thereby inhibiting the binding of the OspA polypeptide to the OspA polypeptide receptor(s). Antibodies and antibody fragments that bind to the chimeric OspA polypeptides of the present invention are within the scope of the present invention.
[00195] In some aspects, antibodies of the present invention include an antibody or fragment thereof that specifically binds to one or more OspA polypeptides produced by immunizing an animal with a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 169, 171, and 173. In other aspects, the invention includes an antibody or fragment thereof that specifically binds to a polypeptide encoded by a selected nucleic acid sequence from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 168, 170, and 172. In various aspects, the antibody or fragment thereof is human, humanized, polyclonal or monoclonal antibody. In further aspects, the antibody is a Fab or a Fab' antibody. In particular aspects, the antibody comprises a detectable marker. In some aspects, the antibody is a chemically modified derivative of the antibody.
Administration of chimeric OspA molecules according to the invention stimulates an immune or antibody response in humans or animals. In some aspects, the three chimeric OspA molecules (for example, OspA 1/2 251 lipidated, OspA 6/4 OspA lipidated, and OspA 5/3 lipidated; or OspA ^ original, OspA 6/4 original, and OspA 5/ 3original) are administered together to elicit antibody response against all six serotypes (1-6) discussed herein. This antibody response means that the methods of the invention are, in many respects, merely used to stimulate an immune response (as opposed to also being a protective response), because the resulting antibodies (unprotected) are nevertheless useful. From inducing antibodies, by means of techniques well known in the art, monoclonal antibodies are prepared, and these monoclonal antibodies are used in well known antibody binding assays, kits or diagnostic tests to determine the presence or absence of Borrelia burgdorferi sl or to determine whether an immune response to the spirochete was simply stimulated. Monoclonal antibodies, in certain aspects, are used in immunoadsorption chromatography to retrieve or isolate Borrelia antigens such as OspA.
The OspA antibodies of the present invention, in various aspects, are polyclonal, including monospecific, monoclonal, recombinant, chimeric, humanized polyclonal antibodies (MAbs), such as CDR-grafted, human, single-chain, and/or bispecific , as well as fragments, variants or derivatives thereof. Antibody fragments include those portions of the antibody that bind to an epitope on the OspA polypeptide. Examples of such fragments include Fab fragments and F(ab') fragments generated by enzymatic cleavage of full length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as expression from recombinant plasmids that contain nucleic acid sequences that encode the variable regions of antibodies.
Polyclonal antibodies directed to an OspA polypeptide are usually produced in a subject (including rabbits, mice or other animals or mammals) by multiple subcutaneous, intramuscular or intraperitoneal injections of OspA polypeptide and an adjuvant. It is useful, in certain aspects, to conjugate an OspA polypeptide of the present invention to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soy trypsin inhibitor. Additionally, adjuvants such as alum are used to boost the immune response. After immunization, blood samples are taken from the immunized subject and the serum is tested for anti-OspA polypeptide antibody titers.
Monoclonal antibodies directed to an OspA polypeptide are produced using any method that provides for the production of antibody molecules by continuous cell line in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et al., Nature, 256:495-497 (1975) and the human B-cell hybridoma method, Kozbor, J. Immunol., 133: 3001 (1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). Also provided by the present invention are hybridoma cell lines that produce monoclonal antibodies reactive with OspA polypeptides.
[00200] The monoclonal antibodies of the invention, in some cases, are modified for use as therapeutic agents. One embodiment is a "chimeric" antibody, in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical or homologous to a corresponding sequence in antibodies derived from other species or belonging to another class or subclass of antibodies. Fragments of such antibodies are also included, as long as they exhibit the desired biological activity. See, US Patent 4,816,567 and Morrison et al., Proc. Natl. Academic Sci. USA, 81:6851-6855 (1985).
[00201] In another embodiment, a monoclonal antibody of the invention is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art (see US Patent 5,585,089 and 5,693,762). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using methods described in the art (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting at least a portion of a rodent complementarity determining region (CDR) for the corresponding regions of a human antibody.
In an alternative embodiment, human antibodies are produced from phage display libraries ( Hoogenboom et al., J. Mol. Biol. 227:381 (1991) and Marks et al., J. Mol. Biol. 222 :581 (1991)). These processes mimic immune identification through the display of antibody repertoires on the surface of the filamentous bacteriophage, and subsequent phage selection for their binding to an antigen of choice. One such technique is described in Patent Application PCT/US98/17364 (Adams et al.), which describes the isolation of functionally agonistic and high-affinity antibodies to MPL and msk- receptors using this approach.
Chimeric, grafted and humanized CDR antibodies are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein or known in the art. In one embodiment, antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies are, in various aspects, produced by expression of recombinant DNA in host cells or by expression in hybridoma cells, as described herein. In some aspects, the monoclonal antibody or a fragment thereof is humanized. In a particular aspect, the monoclonal antibody is F237/BK2 as described herein.
[00204] In certain aspects, the invention includes methods for preventing or treating a Borrelia infection or Lyme disease, in a subject, the method comprising the step of administering an antibody or fragment thereof as described herein. to the subject, in an amount effective for preventing or treating Borrelia infection or Lyme disease. In particular aspects, the antibody or fragment thereof is a hyperimmune serum, a hyperimmune plasma, or a purified immunoglobulin fraction thereof. In other aspects, the antibody or fragment thereof is a purified immunoglobulin preparation or an immunoglobulin fragment preparation.
The anti-OspA antibodies of the present invention, in various aspects, are employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)) for the detection and quantification of OspA polypeptides. Antibodies will bind OspA polypeptides with an affinity that is appropriate for the assay method being used.
[00206] For diagnostic or clinical applications, in certain modalities, anti-OspA antibodies are labeled with a detectable fraction. The detectable fraction can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, in certain aspects, the detectable fraction is a radioisotope such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin, or an enzyme such as phosphatase alkaline, β-galactosidase, or horseradish peroxidase (Bayer et al., Meth. Enzym. 184:138-163 (1990)).
Competitive binding assays depend on the ability of a labeled standard (eg, an OspA polypeptide, or an immunologically reactive portion thereof) to compete with the test sample analyte (an OspA polypeptide) for binding with a limited amount of anti-OspA antibodies. The amount of an OspA polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies. To facilitate the determination of the amount of standard that remains bound, antibodies are generally insolubilized before or after competition, so that the standard and analyte that are bound to the antibodies are conveniently separated from the standard and analyte that remain unbound.
Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and/or quantified. In a sandwich assay, the test sample analyte is normally bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. See, for example, US Patent 4,376,110. The second antibody itself, in some cases, is labeled with a detectable fraction (direct sandwich assays) or is measured using an anti-immunoglobulin antibody that is labeled with a detectable fraction (indirect sandwich assays). For example, one type of sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable fraction is an enzyme.
Anti-OspA antibodies are also useful for in vivo imaging. An antibody labeled with a detectable fraction, in certain aspects, is administered to an animal in the bloodstream, and the presence and location of the labeled antibody in the host is assayed. The antibody, in various aspects, is labeled with any portion that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other means of detection known in the art. In some aspects of the invention, OspA antibodies are used as therapeutic agents. Chimeric OspA Compositions and Administration
To administer the chimeric OspA polypeptides described herein to subjects, the OspA polypeptides are formulated in a composition comprising one or more pharmaceutically acceptable carriers. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse allergic reactions, or other reactions when administered using routes well known in the art, as described below. "Pharmaceutically acceptable carriers" includes any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some aspects, the composition forms solvates with water or common organic solvents. Such solvates are also included.
The immunogenic composition or vaccine composition of the invention is, in various aspects, administered orally, topically, transdermally, parenterally, by spray inhalation, vaginally, rectally or by intracranial injection. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration via intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site is also contemplated. Generally, the compositions are essentially free of pyrogens as well as other impurities that could be harmful to the recipient.
[00212] The formulation of a pharmaceutical composition will vary according to the selected route of administration (eg solution, emulsion). An appropriate composition comprising the composition to be administered is prepared in a physiologically acceptable vehicle or carrier. In the case of solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles in some respects include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles, in certain aspects, include various additives, preservatives, or fluids, nutrients or electrolyte replenishers.
Pharmaceutical compositions useful for the compounds and methods of the present invention containing the OspA polypeptides as an active ingredient contain, in various aspects, the pharmaceutically acceptable carriers or additives, depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylic, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, Agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a surfactant pharmaceutically acceptable and the like. Additives which are chosen from, but not limited to, the above or combinations thereof as appropriate, depending on the dosage form of the present invention.
[00214] A variety of aqueous carriers, for example, water, buffered water, 0.4% saline, 0.3% glycine, or aqueous suspensions contain, in various aspects, the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersing or wetting agents in some cases are a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyl-ene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from hexitol fatty acids and anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions, in some aspects, contain one or more preservatives, for example, ethyl, or n-propyl, p-hydroxybenzoate.
[00215] In some aspects, OspA compositions are lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective with conventional immunoglobulins. Any suitable lyophilization and reconstitution techniques known in the art are employed. It is appreciated by those skilled in the art that lyophilization and reconstitution lead to varying degrees of loss of antibody activity and that usage levels are generally adjusted to compensate.
[00216] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.
[00217] In certain aspects, the concentration of OspA in such formulations varies widely, for example, from less than about 0.5%, usually to or at least about 1% to as much as 15 or 20% by weight and will be selected primarily on the basis of fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Thus, for example, and without limitation, a typical pharmaceutical composition for parenteral injection is prepared to contain 1 ml of sterile buffered water and 50 mg of blood clotting factor. A typical composition for intravenous infusion might be prepared to contain 250 ml of sterile Ringer's solution and 150 mg of blood clotting factor. Current methods for preparing parenterally administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980) . An effective dosage is generally within the range of 0.01 mg to 1000 mg per kg of body weight per administration.
[00218] In various aspects, the pharmaceutical compositions are in the form of a sterile injectable aqueous solution, oleaginous suspension, sterile dispersions or powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The suspension, in some aspects, is formulated in accordance with the known art using the suitable dispersants or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation, in certain aspects, is a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and the like), suitable mixtures thereof, vegetable oils, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil is employed, in various respects, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[00219] In all cases the form must be sterile and must be fluid to the extent that there is easy syringability. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Preservation of the action of microorganisms is brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sodium chloride or sugars. In certain aspects, prolonged absorption of the injectable compositions is brought about by the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.
Compositions useful for administration, in certain respects, are formulated with absorption or uptake enhancers to increase their effectiveness. Such enhancers include, for example, salicylate, glycocholate/linoleate, glycolate, aprotinin, bacitracin, SDS, caprate and the like. See, for example, Fix (J. Pharm. Sci., 85:1282-1285, 1996) and Oliyai et al. (Ann. Rev. Pharmacol. Toxicol., 32:521-544, 1993).
[00221] Furthermore, the hydrophilicity and hydrophobicity properties of the compositions used in the compounds and methods of the invention are well balanced, thus improving their usefulness for in vitro studies and especially in in vivo uses, while other compositions that do not present such a balance are substantially less useful. Specifically, the compositions of the invention have an appropriate degree of solubility in aqueous media, which allows for absorption and bioavailability in the body, while having a degree of solubility in lipids, which allows the compounds to cross the cell membrane from a site of putative action.
In particular aspects, the OspA polypeptides described herein are formulated in a vaccine composition comprising adjuvant. Any adjuvant known in the art is used in various aspects of vaccine composition, including oil-based adjuvants such as Complete Freund's Adjuvant and Incomplete Freund's Adjuvant, mycolate based adjuvants (eg trehalose dimycolate), bacterial lipopolysaccharide (LPS). ), peptideglycan (ie mureins, mucopeptides, or glycoproteins such as N-Opaque, muramyl dipeptides [MDP] or analogues of MDP), proteoglycans (eg, extracted from Klebsiella pneumoniae), streptococcal preparations (eg, OK432 ), Biostim™ (eg 01K2), the "ISCOMs" of EP 109 942, EP 180 564 and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oils (such as migliol), vegetable oils (such as such as peanut oil), liposomes, Pluronic® polyols, the Ribi adjuvant system (see, for example, GB-A-2 189 141), or interleukins, in particular those that stimulate cell-mediated immunity. An alternative adjuvant composed of extracts from Amycolata, a bacterial genus of the order Actinomycetales, has been described in US Patent 4,877,612. Furthermore, proprietary adjuvant mixtures are commercially available. The adjuvant used depends, in part, on the recipient subject. The amount of adjuvant to be administered depends on the type and size of the subject. Optimal dosages are easily determined by routine methods.
The vaccine composition optionally includes liquid diluents (i.e., sterile and non-toxic), semi-solids, or pharmaceutically acceptable vaccine-compatible solids that serve as pharmaceutical carriers, excipients, or media. Any diluent known in the art is used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, acacia gum, calcium phosphate, mineral oil , cocoa butter, and theobroma oil.
[00224] The vaccine composition is packaged in forms convenient for distribution. Compositions are enclosed in a capsule, tablet, sachet, troche, gelatin, paper, or other container. These forms of administration are preferred when compatible with the entry of the immunogenic composition into the recipient organism and, in particular, when the immunogenic composition is to be administered in the form of a unit dose. Dosage units are packaged, for example, in the form of tablets, capsules, suppositories, vials, or troches.
The invention includes methods for inducing an immune response in a subject, including OspA antibodies in a mammalian host which comprise administering an effective amount of the OspA compositions described herein. Likewise, the invention includes methods for preventing or treating a Borrelia infection or Lyme disease in a subject, the method comprising the step of administering an effective amount of the vaccine compositions described herein to the subject.
The vaccine composition is introduced into the subject to be immunized by any conventional method as described herein in detail above. In certain aspects, the composition is administered in a single dose or in a plurality of doses over a period of time (as described in more detail below). Dosage of a Chimeric OspA Composition/Methods for Inducing an Immune Response
[00227] The dosage of the immunogenic composition or useful vaccine composition to be administered will vary depending on various factors that modify the action of the drugs, for example, the age, condition, body weight, sex and diet of the subject, the severity of any infection, time of administration, mode of administration and other clinical factors.
In some aspects, the formulations or compositions of the present invention are administered by an initial dose followed by booster administration after a period of time has elapsed. In certain aspects, the formulations of the invention are administered as an initial bolus followed by a continuous infusion to maintain therapeutic circulating drug levels. In particular aspects, the immunogenic compositions or vaccine compositions of the invention are administered in a vaccination regimen after various periods of time. In some respects, vaccination is delivered on a rapid immunization schedule for travelers to regions that are prone to Borrelia infection. As another example, the composition or formulation of the present invention is administered as a single dose. Those of ordinary skill in the art can readily optimize effective doses and administration regimens as determined by good medical practice and the clinical condition of the individual subject. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
[00229] The pharmaceutical formulation is determined by one skilled in the art, depending on the route of administration and desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is incorporated herein by reference. Such formulations, in some cases, influence the physical state, stability, in vivo release rate and in vivo clearance rate of the administered composition. Depending on the route of administration, the appropriate dose is calculated, in particular respects, according to body weight, body surface area or organ size. In some aspects, appropriate doses are evaluated through the use of established assays to determine blood level dosages, in conjunction with appropriate dose-response data. In certain aspects, an individual's antibody titre is measured to determine optimal dosing and administration regimens. The final dosage regimen will be determined by the doctor or attending physician, taking into account various factors that modify the action of the pharmaceutical compositions, for example, specific activity of the composition, the subject's responsiveness, age, condition, body weight, the subject's sex and diet, the severity of any infection or malignant disease condition, the time of administration, and other clinical factors. As studies are conducted, additional information will emerge regarding appropriate dosage levels and duration of treatment for the prevention and/or treatment of relevant conditions.
In certain aspects, the OspA immunogenic or vaccine composition comprises any dose of OspA nucleic acid molecule(s) or polypeptide(s) sufficient to evoke an immune response in the subject. The effective amount of an OspA immunogenic composition or vaccine to be used therapeutically will depend, for example, on the context and therapeutic goals. One of skill in the art will appreciate that appropriate dosage levels for treatment or vaccination will thus vary depending, in part, on the molecule delivered, the indication for which the OspA molecule(s) are being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Thus, the clinician, in some cases, titrates the dosage and modifies the administration route to obtain the optimal therapeutic effect.
[00231] The typical dosage, in various respects, ranges from about 0.1 μg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage can range from 0.1 D g/kg to about 100 mg/kg; or 1 g/kg to about 100 mg/kg; or 5 g/kg to about 100 mg/kg. By way of example, a dose of an OspA polypeptide useful in the present invention is approximately 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg g/ml, 70 g/ml, 80 g/ml, 90 g/ml, 100 g/ml, 110 □g/ml, 120 g/ml, 130 □g/ml, 140 □g/ ml, 150 g/ml, 160 g/ml, 170 g/ml, 180 g/ml, 190 g/ml, 200 g/ml, 210 g/ml, 220 □g/ml, 230 g/ml, 240 g/ml, 250 □g/ml, 260 g/ml, 270 □g/ml, 280 g/ml, 290 g/ml, 300 □g/ml, 320 □ g/ml, 340 □g/ml, 360 g/ml, 380 g/ml, 400 g/ml, 420 g/ml, 440 g/ml, 460 □g/ml, 480 □g/ ml, 500 g/ml, 520 g/ml, 540 g/ml, 560 g/ml, 580 g/ml, 600 g/ml, 620 g/ml, 640 g/ml, In particular aspects, a typical dose comprises 0.1 to 5.0 ml per subject. In more specific aspects, a typical dose comprises 0.2 to 2.0 ml per subject. In certain aspects, the dose comprises 0.5 to 1.0 ml per subject.
The frequency of dosing will depend on the pharmacokinetic parameters of the OspA molecule in the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition, in various aspects, is therefore administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through implantation of a device or catheter. Further refinement of the appropriate dosage is routinely done by those skilled in the art and is within the scope of the tasks routinely performed by them. Appropriate dosages are often evaluated through the use of appropriate dose-response data that are routinely obtained. Kits
As a further aspect, the invention includes kits comprising one or more pharmaceutical formulations for administering OspA polypeptide(s) to a subject packaged in a manner that facilitates their use for administration to subjects.
[00234] In a specific embodiment, the invention includes kits for producing a single-dose administration unit. The kits, in various aspects, each contain a first container that holds a dry protein and a second container that contains an aqueous formulation. Also included within the scope of this invention are kits containing pre-filled single and multi-chamber syringes (eg, liquid syringes and lyosyringes).
[00235] In another embodiment, such a kit includes a pharmaceutical formulation described herein (for example, a composition comprising a therapeutic protein or peptide), packaged in a container such as a closed bottle or vessel, with a label affixed to the container or included on the packaging, which describes the use of the compound or composition in the practice of the method. In one embodiment, the pharmaceutical formulation is packaged in the container such that the amount of free space in the container (for example, the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of free space is negligible (that is, almost none).
[00236] In one aspect, the kit includes a first container that has a therapeutic protein or peptide composition and a second container that has a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in unit dosage form. The kit optionally further includes a device suitable for administering the pharmaceutical formulation according to the specific route of administration. In some aspects, the kit contains a label describing the use of pharmaceutical formulations.
[00237] Each publication, patent application, patents, and other references cited herein are incorporated by reference in their entirety, to the extent that they are not inconsistent with the present disclosure.
[00238] It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this requirement and scope of the claims attached. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. EXAMPLES
[00239] Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative and not limiting. Example 1: ANALYSIS OF THE OSPA SEQUENCE OF BORRELIA BURGDORFERI SENSU LATO EUROPEAN STRINGS (MOLECULAR EPIDEMIOLOGY) FOR THE DETERMINATION OF AN OSPA VACCINE FORMULATION
[00240] The aim of the study was to determine a suitable formulation for an OspA vaccine against Lyme disease for Europe. The study was based on the analysis of the OspA gene sequence (molecular epidemiology) of a collection of large and diverse strains of B. burgdorferi sensu lato, which adequately represent a wide geographic coverage of Europe, the various clinical syndromes associated with the disease, and each of the three pathogenic genospecies (B. afzelii, B. garinii and B. burgdorferi ss) associated with Lyme disease. Lyme disease is caused by Borrelia burgdorferi sensu lato, which comprises 13 genospecies in total, three of which (B. afzelii, B. garinii and B. burgdorferi s.s.) are recognized as being pathogenic in humans.
[00241] At baseline, a large-scale epidemiological study (see Table 3) was performed that evaluated strains of Borrelia burgdoferi sensu lato from patients with Lyme disease (and ticks) from 21 countries in Europe. A total of 553 European Borrelia isolates collected from 16 European countries were studied. Each species was determined by PCR using primer sets specific for the 16s rRNA genes of each species.
[00242] The isolates of each of the three species of Borrelia known to cause Lyme disease in humans in Europe were well represented: B. afzelii (n = 309, 55.9%), B. burgdorferi sensu stricto (n = 67, 12.1%), and B. garinii (n = 173, 31.3%). Of the 359 human isolates, 56.8% were from B. afzelii and B. afzelii was the predominant species determined from human isolates at most sites. Likewise, B. afzelii was isolated from 54.1% of tick isolates. B. burgdorferi s.s. was isolated from 11.7% of human strains and 12.9% of tick isolates. B. burgdorferi s.s. has been isolated from human isolates from Southeast Europe, namely Italy, Hungary, Slovenia and Austria. B. garinii strains were isolated from 30.4% of human isolates and accounted for 33% of tick isolates. B. garinii strains isolated from humans and ticks were obtained from most geographic regions across Europe. Data from this study correlated with data presented from other European studies and suggest that the collection of isolates studied represents an accurate picture of Lyme disease in Europe.
[00243] OspA sequencing was performed to determine an optimal vaccine formulation for Europe. Based on these data, a vaccine including OspA types 1 to 6 would cover 98.1% of strains and 96.7% of invasive disease cases. The results of the epidemiological study of European Borrelia isolates indicate that a vaccine based on OspA types 1, 2, 3, 4, 5 and 6 would provide theoretical coverage in Europe of 98% of Lyme disease and 96.7% of Lyme disease isolates. invasive neuroborreliosis.

Table 3. Results of Epidemiological Studies
[00244] 1 Predicted vaccine coverage based on numbers of isolates; totals are cumulative.
[00245] 2 Expected vaccine coverage of isolates from cases of neuroborreliosis; totals are cumulative.
Thus, a vaccine comprising three novel recombinant OspAs (1/2, 6/4, and 5/3), each representing two OspA serotypes, would retain structural elements necessary for protection against all six prevalent OspA serotypes (1-6) associated with Lyme borreliosis in Europe and against the unique OspA serotype associated with Lyme borreliosis in the US.
[00247] The inclusion of an OspA 5/3 construct, representing B. garinii OspA serotypes 5 and 3, (along with OspA serotypes 1/2 and 6/4), should protect against 98.1% of disease and 96.7% of invasive isolates. It would be expected that the vaccine without OspA 5/3 would protect against only about 88.9% of the disease, and only about 73.4% of invasive disease. Thus, a vaccine that comprises all six serotypes is more effective in preventing Lyme disease than a vaccine with only four serotypes. EXAMPLE 2 STRATEGY FOR THE CONSTRUCTION OF SYNTHETIC OSPA GENES THAT CODE LIPIDATED OSPA
[00248] The aim of this study was to prepare chimeric lipidated OspA constructs from several strains of Borrelia, in order to make a vaccine that protects the receptor from Lyme disease caused by any of these various strains of Borrelia. The general strategy is summarized in Fig. 1 and is described below.
[00249] For each new OspA gene, four sets of oligonucleotides of between 30-60 bases were synthesized. Each set of oligonucleotides consisted of between 8-12 complementary overlapping oligonucleotides. The oligonucleotides from each set were hybridized together, in separate experiments, to generate double-stranded DNA fragments with certain restriction enzyme recognition sites at both ends, ie, NH (Nde I - Hind III), HK fragments ( Hind III - Kpn I), KE (Kpn I - EcoR I) and EB (EcoR I - BamH I). Each of the four fragments was independently cloned into pUC18, cut with the corresponding restriction enzymes, and transformed into host E. coli DH5α, after which the sequence of the cloned fragment was verified.
[00250] The E. coli strain of DH5α [genotype: A1 end hsdR17 (rK-mK+) supE44 thi-1 recAl gyrA (Nalr) relAl Δ(lacZYA-argF)U169 deoR (Φ80dlacΔ(lacZ)M15] (Gibco BRL ) was used for all intermediate cloning steps. This strain is derived from the E. coli K12 strain, one of the most widely used hosts in genetic engineering. The strain is amp to allow selection of transformants with vectors containing the resistance gene to ampicillin (amp). E. coli HMS174 (DE3) was chosen as host for expression. E. coli HMS174 (DE3) host cells [genotype: F- recAl hsdR (rk12- mk12+) RifR (DE3)] were used for the final cloning steps.The strain is kan- to allow selection of transformants with vectors containing the kanamycin resistance gene (kan).
[00251] pUC18 (Gibco BRL, Basel, Switzerland) was used as cloning vector for all intermediate steps, because genetic manipulations and sequencing were easier than with this plasmid than with pET30a. The main features are namely, the lacZ gene fragment encoding the alpha peptide LacZ of base pairs 149 to 469 (lac promoter at 507 base pairs), the bla gene encoding the ampicillin resistance determinant at base pairs 1629 to 2486 (bla promoter at 2521 base pairs), the origin of replication at 867 base pairs and the multiple cloning sites from base pairs 185 to 451 (Fig. 12).
[00252] pET30a (Novagen) was used as an expression vector for the insertion of the final complete OspA gene. In pET the vector genes are cloned under the control of a T7 promoter and expression is induced by providing a source of T7 RNA polymerase in the host cell (no expression occurs until a source of T7 RNA polymerase is provided) . The main features are the gene encoding kanamycin resistance (kan) at base pairs 4048 to 4860, the lacI gene base pairs 826 to 1905, the F1 origin of replication at base pairs 4956 to 5411 and multiple sites cloning from base pairs 158 to 346 (Fig. 13).
[00253] The four fragments needed to make a full-length OspA gene were excised from a Miniprep DNA. DNA was isolated from each of the four clones using the same restriction enzymes used for the original cloning step. The DNA fragments were purified and ligated together with Nde I and BamH I cut pUC18 DNA and transformed into competent E. coli DH5α cells. The full-length OspA gene cloned into pUC18 was sequenced to confirm that no errors had been introduced at this stage.
The OspA gene was then subcloned into a pET-30a expression vector using the restriction enzymes Nde I and BamH I and transformed into the E. coli host HMS 174 (DE3). In the pET30a vector, the OspA gene is controlled by the bacteriophage T7 promoter.
[00255] Three synthetic OspA genes were designed to encode OspA molecules with the protective epitopes of OspAs serotype 1 and 2 (lipB sOspA 1/2251), OspAs serotype 6 and 4 (lipB sOspA 6/4) and serotype 5 and 3 of OspAs (lipB sOspA 5/3) from Borrelia. The primary amino acid sequences of these molecules (SEQ ID NOS: 2, 4 and 6, respectively) are shown in Figures 2-8 and described here with a full description of the main features incorporated in their design.
[00256] The oligonucleotides for the construct of lipB sOspA 1/2 were internally synthesized in an ABI 394 DNA/RNA synthesizer. Oligonucleotides for the lipB sOspA 5/3 and lipB sOspA 6/4 constructs were purchased from GenXpress (Wiener Neudorf, Austria) and were purified by HPLC. Table 4. Oligonucleotides for lipB sOspA 1/2* gene fragments

* A single amino acid change was introduced by PCR, lipB sOspA 1/2 was the construct name before the change was introduced and lipB sOspA 1/2251 was the name after the change was introduced. L Length of oligonucleotide in bases S Tape, C (coding) or complementary (C') Table 5. Oligonucleotides for gene fragments of lipB sOspA 5/3

L Length of oligonucleotide in bases S Tape, C (coding) or complementary (C') Table 6. Oligonucleotides for lipB sOspA 6/4 gene fragments

L Length of oligonucleotide in bases S Tape, C (coding) or complementary (C')
[00258] Preparation of competent E. coli cells. An isolated colony was used to inoculate 5 ml of modified LB broth (5.5 g of NaCl, 5 g of yeast extract, 10 g of soy peptone, which was not obtained from a genetically modified plant or animal source - per liter of water). The culture was incubated until it became cloudy, after which the culture was diluted to a volume of 25 ml with pre-warmed modified LB broth. The culture was further incubated until an OD600nm of 0.2 to 0.6 was reached (40 - 60 min), and was diluted to a volume of 125ml, transferred to a 500ml flask and incubated to an OD600nm of 0 .6 be reached. The culture was rapidly cooled by gentle shaking for 5 min in an ice bath and cells were pelleted directly (Beckman centrifuge, 4000 rpm for 10 min.), washed thoroughly with TfBI buffer (Teknova Hollister, CA) (30 mM acetate) of K-, 50 mM MnCl 2 of, 100 mM KCl, 10 mM CaCl 2 , 15% glycerol), resuspended in 5 ml TfBII (10 mM Na-MOPS, 75 mM CaCl 2 , 10 mM KCl, 15% glycerol) and kept on ice for 15 min. The cells were then pipetted into 100 μl aliquots and subjected to rapid freezing directly on dry ice.
[00259] Annealing oligonucleotide mixtures to form OspA gene fragments (de novo synthesis). Three synthetic OspA genes were designed to encode OspA molecules with the protective epitopes of OspAs serotype 1 and 2 (lipB sOspA 1/2), OspAs serotype 6 and 4 (lipB sOspA 6/4) and serotype 5 and 3 of OspAs (lipB sospA 5/3). For each new (lipidated) OspA gene, four sets of oligonucleotides of between 30 - 60 base pairs were synthesized (see Tables 4-6). Figures 16-18 show the optimized codon sequences for each of the constructs aligned with the predicted nucleotide sequences of the published sequences. Each set of oligonucleotides consisted of between 8-12 complementary overlapping oligonucleotides. The oligonucleotides from each set were hybridized together, in separate experiments, to generate double-stranded DNA fragments with certain restriction enzyme recognition sites at either end, ie NH (Nde I - Hind III), HK (Hind) fragments III - Kpn I), KE (Kpn I - EcoR I) and EB (EcoR I - BamH I).
The lyophilized oligonucleotides were reconstituted with distilled water, the OD260nm was measured and the concentration was adjusted to 10 μM. For each OspA fragment, 2 μl of each of the oligonucleotides were mixed with 1 μl of T4 polynucleotide kinase and T4 DNA ligase buffer (10x), and the mixture was incubated at room temperature for 30 minutes to allow phosphorylation of the oligos. (for the lipB sOspA 6/4 constructs this step was omitted as the oligos were phosphorylated). The mixture was heated at 95°C for 1 minute (denaturation step) and then the oligos were allowed to anneal, allowing the mixture to cool slowly to room temperature. The annealed mixture was used directly on the connections, or was stored at -20°C until additionally needed.
[00261] Cloning of OspA gene fragments. Each of the four fragments required to construct an individual synthetic OspA gene was independently cloned into pUC18 and transformed into the E. coli DH5α host (see Figure 1).
[00262] For each new OspA gene, four sets of oligonucleotides from bases between 30-60 were synthesized. Each set of oligonucleotides consisted of between 8-12 complementary overlapping oligonucleotides. The oligonucleotides from each set were hybridized together, in separate experiments, to generate double-stranded DNA fragments with certain restriction enzyme recognition sites at both ends, i.e., NH NH fragments (Nde I - Hind III), H - K (Hind III - Kpn I), KE (Kpn I - EcoR I) and EB (EcoR I - BamH I). Each of the four (4) fragments was cloned into pUC18 independently cut with the corresponding restriction enzymes and transformed into E. coli DH5α host, after which the sequence of the cloned fragment was verified.
Plasmid DNA (pUC18) was purified from an overnight E. coli culture (LB broth) with a QIAGEN plasmid purification system according to the manufacturer's protocol. The vector DNA was then digested with pairs of restriction enzymes; Nde I & Hind III, Hind III & Kpn I, Kpn I & EcoR I, EcoR I & BamH I, according to the manufacturers' protocols. The digested samples were applied to a 0.8% agarose gel and electrophoretically separated. Linearized vector DNA was excised and eluted using a commercial gel elution kit (QIAquick Gel Extraction Kit, Qiagen) according to the manufacturer's protocol and ligated, using T4 DNA ligase, to the annealed oligonucleotide mixture. Ligation products were transformed into competent E. coli DH5α cells and plasmid-containing transformants were selected on LB Agar containing ampicillin (100 µg/ml).
[00264] The presence of the insert of the expected size in the cloning vector, pUC18, was confirmed by purification of plasmid DNA, digestion of the DNA with the enzymes used for cloning and analysis of the DNA fragments by agarose gel electrophoresis , using the procedures described above. The cloned DNA fragment was sequenced using purified plasmid DNA as the template DNA and sequencing primers 5'-TCGGGGCTGGCTTAACTATG-3 (SEQ ID NO: 14) and 5'-GCTTCCGGCTCGTAT (SEQ ID NO: 15) ( which are in the pUC18 vector outside the multiple cloning sites, bp 130-150 and bp 530-515, respectively). Sequence reactions were performed on an automatic sequencer (ABI 310). The sequences were edited using SequenceEditor and the sequences were imported into VetorNTI for analysis. Clones with only the correct sequences were used as building blocks for the full-length OspA gene construct.
[00265] For the lipB sOspA 5/3 gene a different strategy was employed, since no suitable internal unique site can be found within the Kpn I - BamH I fragment and the amino acid sequence does not allow the internal use of a site EcoR I (see Figure 14). One Pvu II site exists within the Kpn I - BamH I fragment, however, there are two Pvu II sites in the pUC18 vector, which means that direct cloning of the fragments into pUC18 is not possible. Thus, the oligos for the constructs were designed to have an EcoR I site inserted outside and adjacent to the Pvu II site, to allow cloning of the Kpn I - EcoR I and EcoR I - BamH I fragments into pUC18. Subsequent digestion of the inserted fragments with Kpn I, EcoR I and BamH I generated fragments, which were subsequently digested with Pvu II. The Pvu II digested fragments (Kpn I-Pvu II and PvuII-BamH I) were then used in a triple ligation with pUC18 vector DNA cut with Kpn I and BamH I to generate the Kpn I - BamH I fragment.
[00266] Full-length OspA gene construct. In the next step, each of the four fragments necessary for the construction of an individual synthetic OspA gene was excised from the pUC18 vector and recloned, in a single step, in pUC18 to generate a full-length OspA gene vector (see Figure 1).
[00267] The four fragments needed to make full-length genes were excised from minipreps of DNA isolates using the same restriction enzymes used for the original cloning step. The digested samples were applied to an agarose gel by electrophoresis and separated. The DNA for each of the 4 respective insert fragments was excised and eluted using a commercial gel elution kit (QIAquick Gel Extraction Kit) according to the manufacturer's protocol and ligated using T4 DNA ligase for the Linearized vector DNA digested with Nde I and BamH I and purified using a QIAquick gel extraction kit. Ligated DNA was transformed into competent E. coli DH5α cells and clones containing the plasmid were selected on LB Agar containing ampicillin (100 µg/ml). Colonies were tested by PCR to detect the presence of inserts of the expected size (approximately 830 bp).
[00268] Single colonies were used as template DNA in PCR reactions comprising 10X buffer (15 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, Amplitaq DNA polymerase 1.25 U, 400 nM forward primer 5'-TCGGGGCTGGCTTAACTATG-3 (SEQ ID NO: 14) and 400 nM reverse primer 5'-GCTTCCGGCTCGTAT (SEQ ID NO: 15). PCR reaction conditions were as follows. 94°C for 5 min, 35 x (94°C for 30 s, 48°C for 30 s, 72°C for 1 min 30 s) followed by a 72°C bath for 5 min and held at 4° Ç. PCR products were used directly or stored at < 15°C until use. PCR products were analyzed by agarose gel electrophoresis for the presence of inserts of the correct size (about 980 bp). Correctly sized inserts were sequenced to confirm that no errors were introduced, ie, sequence reactions were established using plasmid DNA isolate (QIAGEN Plasmid Purification Kit) from overnight cultures (LB amp broth) and using sequencing primers flanking the cloning sites (5'-TCGGGGCTGGCTTAACTATG-3'(SEQ ID NO: 14) and 5'-GCTTCCGGCTCGTATGTTGT-3' (SEQ ID NO: 16), bp 130-150 and 530-510 , respectively). Sequence reactions were performed on an automatic sequencer (ABI 310). The sequences were edited using SequenceEditor and the sequences were imported into VetorNTI for analysis.
[00269] Subcloning of new OspA genes in the pET30a expression vector. Once the full-length OspA gene was verified in pUC18, the OspA genes were then subcloned into the pET-30a expression vector using the restriction enzymes Nde 1 and BamH I and transformed into the host E. coli HMS 174 (DE3).
Miniprep DNA from pUC18 clones with the correct sequence was digested with Nde I and BamH I. Similarly the vector DNA from pET30a was digested with Nde I and BamH I. The digested DNAs were run on an agarose gel and electrophoretically and separated. The approximately 830 bp insert and linearized vector DNA were excised and purified as described above. Insert DNA and vector were ligated using T4 DNA ligase and ligation products were transformed into competent E. coli HMS174 (DE3) cells (Novagen). Transformants were plated on LB plates containing kanamycin (30 µg/ml). Single colonies were screened by PCR using primers 5'-TTATGCTAGTTATTGCTCAGCG-3' (SEQ ID NO:17) and 5'-TTCCCCTCTAGAAATAATTTTGT-3' (SEQ ID NO: 18). PCR products were applied to an agarose gel and were separated by electrophoresis. Colonies that produced a product of the correct size (approximately 1 kb) were subsequently used to set up overnight cultures, from which Miniprep DNA was isolated using a QIAGEN Plasmid Purification Kit according to the QIAGEN protocol. manufacturer. The sequence was confirmed again (using primers 5'-TTATGCTAGTTATTGCTCAGCG-3' (SEQ ID NO: 17) and 5'-TTCCCCTCTAGAAATAATTTTGT-3' (SEQ ID NO:18), pp 65-86 and 395-373, respectively) and colonies were selected for the expression test.
[00271] Generation of lipB sOspA 1/2251 from lipB sOspA 1/2. A single amino acid was changed in the lipB sOspA 1/2 construct, that is, amino acid alanine at position 251 was changed to an asparagine residue, to increase immunogenicity. An amino acid change was introduced by PCR. First, PCR was defined with the outer forward primer and the inner reverse primer producing a product of about 730 bp with the introduced amino acid change (see Figure 15). Second, PCR was set up with the inner forward primer and the outer reverse primer to provide a 100 bp product containing the introduced amino acid change. The two overlapping PCR products in sequence were then used as template DNA in a final PCR reaction with the outer forward and outer reverse primers to produce the final full-length OspA product containing the introduced amino acid change.
[00272] The pET30a construct was used as the source of the template DNA. PCR reactions were created comprising 10X buffer [15 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2], 200 μm dNTPs, 1.25 U Amplitaq DNA polymerase, and 400 nM of each primer pair (5'-GGA ATT CCA TAT GCG TCT GTT GAT CGG CT (SEQ ID NO:19) & 5'-TTG GTG CCT GCG GAG TCG (SEQ ID NO:20) and primer pair 5 '- AAT ACG ACT CCG CAG GCA CC (SEQ ID NO:21) & 5'-CTG-GGA TCC GCT CAG CTT ATT TCA (SEQ ID NO:22)). PCR reactions were set up with the following conditions; 94°C for 5 min, 35 x (94°C for 30 sec, 48°C for 30 sec, 72°C for 1 min 30 sec) followed by a 72°C bath for 5 min and held at 4 °C. The reactions produced 2 separate overlapping products and the two products were used as template DNA in a third PCR reaction using the outer primer 5'- GGA ATT CCA TAT GCG TCT GTT GAT CGG CT (SEQ ID NO:19) and 5 '- CTG-GGA TCC GCT CAG CTT ATT TCA (SEQ ID NO: 22) which incorporates restriction sites for Nde I and BamH I. Reaction conditions were 94°C for 60 sec followed by 35 cycles of (30 sec 94 °C, 60 sec 49°C, 90 sec 72°C), followed by 72°C for 5 min. The amplified product was purified with a QiaQuick purification kit (Qiagen) according to the manufacturer's specifications and the product was digested with Nde I and BamH I and ligated to pET30a Nde I and BamH I vector DNA cut. Ligation products were transformed into competent E. coli DH5α cells. Transformants were plated on LB plates containing kanamycin (30 µg/ml). Isolated colonies were screened by PCR using primers 5'-TTATGCTAGTTATTGCTCAGCG-3' (SEQ ID NO:17) and 5'-TTCCCCTCTAGAAATAATTTTGT-3' (SEQ ID NO: 18). PCR products were applied to an agarose gel and were separated by electrophoresis. Colonies that produced a product of the correct size (approximately 1 kb) were then used to establish overnight cultures, from which miniprep DNA was isolated using a QIAGEN Plasmid Purification System according to the protocol of the manufacturer. The sequence was confirmed (using primers 5'-TTATGCTAGTTATTGCTCAGCG-3' (SEQ ID NO: 17) and 5-TTCCCTCTAGAAATAATTTTGT-3' (SEQ ID NO: 18)) and the resulting construct was transformed into E. coli HMS174 (DE3). ) Competent cells and the resulting positive transformants were named as lipB sOspA 1/2251.
[00273] Generation of constructs without leader sequence. Constructs were prepared with a lipB leader sequence, for which a lipid moiety is typically linked to the amino terminal cysteine residue. The experimental test of recombinant lipidated OspAs verified the presence of a lipid fraction. However, constructs that do not contain the lipB leader sequence were also prepared. Constructs that do not contain the lipB leader sequence were made by PCR amplification of each of the three lipB constructs (in pET30a), using selected primers to generate a final product of 769-771 bp lacking the nucleic acid sequence encoding the leader sequence and with the codon for the cysteine residue replaced by a codon for a methionine residue.
[00274] The PCR reactions comprised 10X buffer [15 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2], 200 μm dNTPs, 1.25 U Amplitaq DNA polymerase, 400 nM of forward primer 5'-CGTGCGTACCATATGGCACAGAAAGGTGCTGAGTCT-3' (SEQ ID NO:23) and 400 nM of reverse primer 5'-CTGGGATCCGCTCAGCTTATTTCA-3' (SEQ ID NO:22) and template DNA. PCR conditions were: 94°C for 5 min, 35 x (94°C for 30 s, 48°C for 30 s, 72°C for 1 min 30 s), followed by a 72°C bath for 5 min and held at 4°C. PCR reactions were used directly or stored at < - 15°C until use.
The PCR products were purified using a QiaQuick PCR purification kit (Qiagen), digested with Nde I and BamH I, and ligated to pET30a vector DNA digested with Nde I and BamH I. Ligation mixtures were used to transform E. coli HMS174(DE3) and colonies containing recombinant plasmids were selected for their resistance to kanamycin and the sequence was verified from the PCR products.
[00276] Evaluation of expression in E. coli HMS 174 (DE3). Selected colonies were tested for their ability to express the respective new OspA protein. In each case, individual colonies were used to inoculate LB broth containing kanamycin (30 μg/ml) and were incubated at 37°C for 1 to 5 hours until an OD value (600 nm) greater than 0.6 and less that 1 has been reached. At this point, a sample of the culture was kept (representing the uninduced sample) and the rest of the culture was induced by adding IPTG to a final concentration of 1 mM. The uninduced sample (1 ml) was centrifuged and the pellet was retained and stored at -20°C. The induced culture was allowed to grow for a further three hours, after which a 1 ml sample was taken, the OD (600 nm) was measured, the sample was centrifuged and the pellet retained and stored at -20°C.
[00277] Preparation of primary cells. Primary cells were prepared for each of the three lipidated constructs and for each of the three non-lipidated constructs. The primary cells comprised E. coli cells (HMS174 (DE3)) carrying a plasmid pET30a expressing the respective OspA. For the preparation of primary cells, a single colony from the respective stock was chosen from a plate containing kanamycin (30 μg/ml) and rifampicin (200 μg/ml) and was used to inoculate 500 μl of SMK medium (SOP 8114) and incubated during the night. One hundred microliters of this culture was then used to inoculate 100 ml of SMK medium (in duplicate) and the culture was incubated for 17 to 20 hours at 37°C with shaking. Sterile glycerol was then added to the culture at a final concentration of 15% and the material was pipetted in aliquots in amounts of 500 μl into 60 ampoules, thus producing 60 ampoules of primary cells that were directly stored at -80°C .
[00278] Three synthetic OspA genes were designed to encode OspA molecules with the protective epitopes of OspAs serotype 1 and 2 (lipB sOspA 1/2251), OspAs serotype 6 and 4 (lipB sOspA 6/4) and serotype 5 and 3 of OspAs (lipB sOspA 5/3). The primary amino acid sequences of these molecules and a description of the main features incorporated in their design are set out in the Examples that follow. EXAMPLE 3: DESCRIPTION OF 1/2251 LIPIDATED OSPA (LIPB SOSPA1/2251)
[00279] The aim of the study was to design a new OspA antigen, lipidated 1/2 251 OspA (lipB sOspA 1/2251), which includes serotypes 1 and 2. LipB sOspA 1/2251, comprises the proximal portion of the sequence of OspA from serotype 1 (strain B31, Gene Bank accession number X14407) fused to the distal portion of a sequence from serotype 2 (Strain Pko, Gene Bank accession number S48322). The start of the unique sequence for serotype 2 is the lysine (K) residue at position 216. The construct was originally designed to encode the amino acid alanine (A) at position 251. However, the construct was later changed by PCR to encode an asparagine (N) residue (the actual residue in the published Pko sequence) to enhance immunogenicity, hence the nomenclature lipB sOspA 1/2251.
[00280] Secondary characteristics of lipB sOspA 1/2251 are shown in the annotated amino acid sequence of lipB sOspA 1/2251 in Figure 2 and include: • putative arthritogenic epitope substitution (Gross et al, 1998.), hLFA-1 (YVLEGTLTA ) (SEQ ID NO:24), in the proximal portion of the molecule (amino acids 161 to 185), with an equivalent sequence (shown in italics and a flanking sequence) of an OspA deserotype 2 sequence (Strain Pko; Gene Bank accession; S48322): a sequence that is different from the hLFA-1 epitope; • an OspB leader sequence (amino acids 1 to 15 of Figure 2), and several substitutions to avoid the prior art. The asparagine (N) and aspartic acid (D) residues at positions 44 and 46 were replaced by an aspartic acid (D) and an asparagine (N), respectively, to produce the sequence KEKDKN (SEQ ID NO: 25). Alanine (A) and aspartic acid (D) residues at positions 78 and 79 were replaced by a threonine (T) and an asparagine (N), respectively, to produce the sequence of KTNKSK (SEQ ID NO: 26); • stabilizing mutations as described in international patent publication number WO 02/16421A2 (Luft & Dunn). For example, methionine (M) replaced arginine (R) at amino acid 136 (R139M); tyrosine (Y) replaced glutamic acid (E) at amino acid 157 (E160Y); and methionine (M), substituted for lysine (K) at amino acid 186 (K189M); and • additional stabilizing mutations. For example, threonine (T) replaced valine (V) at amino acid 173 (aa 176 of the disclosure). Removal of the putative arthritogenic epitope (position 161-185) by replacing a sequence from B. burgdorferi with a sequence from B. afzelii, breaking the hydrogen bond between amino acids 173 and 174 (aa 176 and 177 of the disclosure) . This led to a decrease in binding to protective monoclonal antibodies (105.5 and LA-2 (Jiang et al., J. Immunol. 144: 284-9, 1990; Golde et al., Infect. Immun. 65: 882- 9, 1997; and Ding et al., J. Mol. Biol. 302: 1153-64, 2000) Threonine (T) was introduced at position 173, instead of a valine (V), to re-establish hydrogen bonding and increase the reactivity to protective monoclonal antibodies 105.5 and LA2.
[00281] Furthermore, amino acids 16-25 (beginning of mature protein) are identical to the sequence of OspB (Gene Bank accession number X74810).
[00282] The amino acid and deduced nucleotide sequences of lipB sOspA 1/2251 are shown in Figure 3. The leader sequence (green) is cleaved during protein secretion. The mature OspA protein sequence begins with a cysteine residue (underlined), which forms the binding site for the protein's lipid anchor. EXAMPLE 4: DESCRIPTION OF 6/4 LIPIDATED OSPA (LIPB SOSPA 6/4)
[00283] The aim of the study was to design a new OspA antigen, lipidated sOspA 6/4 OspA (lipB sOspA 6/4), comprising serotypes 4 and 6. LipB sOspA 6/4 comprises the proximal portion of a sequence of OspA serotype 6 (strain K48, Gene Bank accession number I40098) fused to the distal portion of a serotype 4 sequence (Strain pTroB; Gene Bank accession I40089). The start of the unique sequence for serotype 4 is the asparagine (N) residue at position 217. Secondary features are shown in the annotated amino acid sequence of lipB sOspA 6/4 in Figure 4 and include: • stabilizing mutations described in the Application International Patent No. WO 02/16421A2 (Luft and Dunn): methionine (M) instead of an arginine (R) at amino acid 136, tyrosine (Y) instead of a glutamic acid (E) at amino acid 157 and methionine ( M) for a lysine (K) at amino acid 187; and • as lipB sOspA 1/2251, described above, an OspB leader sequence was used (amino acids 1-15 of Figure 4) and amino acids 16-25 are identical to the OspB sequence (Gene Bank accession number X74810).
[00284] Although the peptide sequence KEKNKD (SEQ ID NO: 27) was absent from the source OspA type 6 sequence (KEKDKD) (SEQ ID NO: 28), the aspartic acid residue (D) at position 46 was replaced with an asparagine (N) residue, in accordance with an equivalent change made in the construct lipB sOspA 1/2251 to produce the sequence KEKDKN (SEQ ID NO: 25).
[00285] Although the peptide sequence KADKSK (SEQ ID NO: 29) was absent from the source OspA type 6 sequence (KTDKSK) (SEQ ID NO: 30), the aspartic acid residue (D) at position 79 was replaced by an asparagine (N) residue, in accordance with an equivalent change made in the construct lipB sOspA 1/2251 to produce the KTNKSK sequence (SEQ ID NO: 26).
[00286] Amino acid 37 was changed from glutaminc acid (E), as present in the original sequence (Strain K48, Gene Bank accession number I40098), to a valine (V), since almost all type 6 sequences have a valine at this position.
[00287] The deduced amino acid and nucleotide sequences of lipB sOspA 6/4 are depicted in Figure 5. The leader sequence (green) is cleaved during protein secretion. The mature OspA protein sequence begins with a cysteine residue (underlined, see Figure 5), which forms the binding site for the protein's lipid anchor. EXAMPLE 5: DESCRIPTION OF 5/3 LIPIDATED OSPA (LIPB SOSPA 5/3)
[00288] The aim of the study was to design a new OspA antigen, lipidated sOspA 5/3 OspA (lipB sOspA 5/3), comprising serotypes 3 and 5. LipB sOspA 5/3 comprises the proximal portion of a sequence of OspA serotype 5 [No. Access Database emb|X85441|BGWABOSPA, OspA gene from B. garinii (substrain WABSou)] fused to the distal portion of a sequence of serotype 3 (strain PBr; Gene Bank accession number X80256, OspA gene from B. garinii) with modifications as shown in SEQ ID NOS: 5 and 6. The start of the unique sequence for serotype 3 is the aspartic acid residue (D) at position 216. Secondary features are shown in the amino acid sequence annotated from lipB sOspA 5/3 in Figure 6, and includes: • stabilizing mutations described in International Patent Application No. WO 02/16421A2 (Luft and Dunn): methionine (M) instead of an arginine (R) at the amino acid 136; tyrosine (Y) for a glutamic acid (E) at amino acid 157 and methionine (M) for a lysine (K) at amino acid 187; and • as lipB sOspA 1/2251 and lipB sOspA 6/4, described above, an OspB leader sequence was used (amino acids 1 to 15 in Figure 6), and amino acids 16-25 are identical to the OspB sequence (accession number from the X74810 Gene Bank.
[00289] Although the peptide sequence KEKNKD (SEQ ID NO: 27) was absent from the source OspA type 5 sequence (KEKDKD) (SEQ ID NO: 28), the aspartic acid residue (D) at position 46 was replaced by an asparagine (N) residue, in accordance with an equivalent change made in the construct lipB sOspA 1/2251 giving the sequence KEKDKN (SEQ ID NO: 25).
Although the peptide sequence KADKSK (SEQ ID NO:29) was absent from the source OspA type 5 sequence (KTDKSK) (SEQ ID NO:30), the aspartic acid residue (D) at position 79 was replaced by an asparagine (N) residue, in accordance with an equivalent change made in the construct lipB sOspA 1/2251 giving the sequence KTNKSK (SEQ ID NO:26).
[00291] The deduced amino acid and nucleotide sequences of lipB sOspA 5/3 are shown in Figure 7. The leader sequence (green) is cleaved during protein secretion. The mature OspA protein sequence begins with a cysteine codon (underlined, see Figure 7), which forms the binding site for the protein's lipid anchor. EXAMPLE 6: OPTIMIZATION OF CODON USE FOR HIGH LEVEL EXPRESSION IN E. COLT
[00292] Since the presence of codons that are rarely used in E. coli is known to present a potential impediment to the high level of expression of foreign genes, the low usage codons were replaced by codons that are used by highly expressed genes in E. coli. The nucleotide sequences of the new OspA genes were designed to utilize the most frequently found codons (preferred codons) among the highly expressed class II E. Coli genes (Guerdoux-Jamet et. al., DNA Research 4:257-65 ,1997). Data for usage codons between the novel OspA genes and for the highly expressed class II E. coli genes are summarized in Tables 7 and 8. Data for the less frequent amino acids for which tRNA molecules are less likely to be rate limiting are presented separately (Table 7) from the data for the most frequently occurring amino acids (Table 8). Table 7. Codon usage in new OspA genes (less common amino acids *)

* that is, Amino Acids which, individually, constitute < 2.5% of the total amino acids in number. Table 8. Codon for use in new OspA genes (most prevalent amino acids)


[00293] The high degree of agreement between the chosen use codon for the new OspA genes (common amino acids only) and between the E. coli class II genes is apparent (ie, the percentage plot is shown in Table 8 for class II genes against the individual new OspA genes, see Figure 8). For the three lipidated constructs, the original sequences had a GC content ranging from 32.8% to 33.8%, while the codon optimized sequences had a GC content ranging from 43.8% to 46.8%, which is similar to the 50% GC content of E. coli EXAMPLE 7: CONSTRUCTION OF SYNTHETIC UNLIPITED OSPA GENES
[00294] Constructs that do not contain the lipB leader sequence were also prepared. The two sets of constructs (lipidated and non-lipidated) are needed to assess their ease of production in the fermenter (biomass, stability, product yield, and so on), to assess the ease with which different types of antigen can be purified and compare their biological characteristics (security profile and protection potency).
[00295] Constructs (SEQ ID NOS: 7, 9, and 11) were generated by PCR amplification of each of the three lipB OspA constructs (SEQ ID NOS: 1, 3, and 5), using PCR primers with restriction sites incorporated. PCR products were purified, digested with Nde I and BamH I and ligated to digested pET30a vector DNA. Ligation mixes were used to transform E. coli DH5 α and the OspA sequences were verified. Miniprep DNA was prepared, isolated and used to transform HMS 174 (DE3) host cells. The sequences of the non-lipidated derivatives are identical to the lipidated versions, except that they lack the first 45 base pairs that encode the leader sequence and contain an Nde I site that contains a methionine codon that replaces the cysteine codon in the lipidated versions ( see Figure 9). EXAMPLE 8: EXPRESSION OF NEW OSPA RECOMBINANT ANTIGENS
To express/produce the new recombinant OspA genes for antigenic purposes, an E. coli expression system controlled by bacteriophage T7 RNA polymerase (Studier et al., J. Mol. Biol. 189:113-30, 1986 ) was used. In this expression system, the new OspA genes were cloned at the multiple cloning site, in one of the pET series of plasmids (eg, pET30a). Because foreign gene expression is under the control of a bacteriophage T7 promoter, which is not recognized by E. coli RNA polymerase, expression is dependent on a source of T7 RNA polymerase. This enzyme is provided when the recombinant plasmids are transferred to a suitable expression host, such as E. coli HMS174(DE3), which contains a chromosomal copy of the T7 RNA polymerase gene. Expression of the chromosomally integrated T7 RNA polymerase gene is under the control of a lacUV5 promoter that can be turned on (ie induced) by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) or lactose (see Figure 10). Consequently, foreign gene expression is also regulated by the addition of the inducer molecule.
[00297] Cells were induced in late log phase, and harvested 3-4 hours after induction. In induced cells, the chimeric OspA antigen was the most highly expressed protein, as determined by SDS-PAGE of cell lysates. Most OspA chimeras were found in the supernatant. Contaminant E. coli proteins were removed by anion exchange chromatography and the chimeric OspA proteins eluted in void volume were concentrated by ultrafiltration.
[00298] The expression of the new recombinant OspA proteins of each of the constructs was tested, and the samples of induced and non-induced cultures were run on an SDS polyacrylamide gel (Figure 11). For lipidated (SEQ ID NOS: 2, 4 and 6) and non-lipidated (SEQ ID NOS: 8, 10 and 12), an antigen band of approximately 31 kDa was observed in each case (see Figure 11). The proteins were characterized and the molecular weights determined correlated (+/- 0.5 daltons) with the theoretical molecular weights assuming that the terminal methionine is cleaved. Figure 11 shows that the expressed recombinant lipidated OspA proteins comprise at least 10% of the total protein yield, proving that the constructs are useful for the intended purpose. EXAMPLE 9: SINGLE RECOMBINANT OSPA ANTIGEN (R OSPA 1/2) PROTECTS AGAINST B. BURGDORFERI S.S. AND B.AFZELII INFECTION
[00299] The aim of this study was to determine whether a single recombinant antigen (rOspA 1/2; polypeptide comprising SEQ ID NO: 2 (lipB sOspA 1/2251)), intended to retain the protective properties of serotypes 1 and 2 of OspA, is able to induce antibody responses that protect mice against infection by either B.burgdorferi ss (OspA serotype 1) or B. afzelii (OspA serotype 2). Evidence is provided to demonstrate that the inclusion of additional rOspA antigens does not have an antagonistic effect on the protective immunity conferred by the rOspA 1/2 antigen.
[00300] Design and construction of rOspA 1/2. To eliminate the risk of introducing adventitious agents, complementary overlapping synthetic oligonucleotides were used to generate DNA fragments that were ligated together and cloned into the pET30a vector and the sequence verified. This approach also allowed usage codons to be optimized for the host E. coli HMS174 (DE3) used to express the OspA gene. The new gene is based on the proximal portion of a serotype-1 OspA sequence (amino acids 29 to 218, Strain B31, Gene Bank accession number X14407) fused to the distal portion of a serotype-2 sequence (amino acids 219 to 273, Strain PKo; Accession number S48322). The 25 amino acid fragment of B31 strain of B. burgdorferi (aa 164 to 188) was replaced with the sequence of Pko strain of B. afzelii (aa 164 to 188), because this region of OspA of B31 (aa 165 to 173) it is highly related to the region encompassing the hLFA-1 epitope (aa 332 to 340). The N-terminal sequence, including the leader sequence and the first 11 amino acids were obtained from OspB (Strain B31; Gene Bank Accession Number X74810) in order to optimize expression of the lipidated protein. Other specific amino acid changes were made to improve the immunogenicity and conformational stability of the rOspA 1/2 molecule and the sequence of rOspA 1/2 (lipB sOspA 1/2251) is shown in SEQ ID NO: 2.
[00301] Animal tests. The ability of a single recombinant OspA antigen (rOspA 1/2) to prevent infection with two Borrelia species expressing different OspA antigens was evaluated in C3H/HeJ mice immunized subcutaneously (days 0 and 28) with the OspA antigen purified (doses of 0.1 μg or 0.03 μg) formulated with 0.2% (w/v) aluminum hydroxide as an adjuvant. Mice were challenged 2 weeks after booster immunization, either by intradermal injection (needle challenge; 7 x 104 cells) or by natural route of infection (tick challenge). For further experiments, 8 nymphal ticks were applied per mouse and allowed to feed for up to 5 days. Nymphs were collected near Budweis (Czech Republic), an endemic area for Lyme disease. The majority of these ticks were infected with B. afzelii, as determined by PCR-unfed tick tests. The infectious status of the mice was determined four weeks later. In tick challenge experiments the presence of Borrelia was confirmed by culture (urinary bladder), and by detection of Borrelia DNA by real-time PCR (heart). Animal experiments were conducted in accordance with Austrian laws on animal testing and international guidelines (AAALAC and OLAW), and were reviewed by the Institutional Animal Care and Use Committee and approved by the Austrian regulatory authorities. Immunogenicity: Antibody response (IgG μg/ml) to rOspA 1/2 antigen was determined by ELISA using rOspA 1/2 as the coating antigen and an OspA specific monoclonal antibody (home-prepared) with an IgG content set as a pattern.
[00302] Diagnostic procedures. For the needle challenge experiments, the presence of antibodies against a conserved epitope on the surface-exposed lipoprotein VlsE protein (C6 ELISA; plates coated from Immunetics® C6 Lyme ELISA™) or Borrelia antigens other than the OspA immunogen (Western blotting) was used to diagnose the infection. Western blotting used a cell lysate prepared from ZS7 strain of B. burgdorferi s.s. this was already the challenge organism. Animals were considered infected if they were positive in both assays.
[00303] For the tick challenge experiments, the C6 ELISA and Western blotting were also done. However, Western blotting used lysates of B. burgdorferi s.s. ZS7, B. afzelii ACA1 and B. garinii KL11, because the identity of the infecting organism was unknown. Animals were considered to have seroconverted only if both assays were positive. In addition, Borrelia infection was evaluated by urinary bladder culture and by detection of B.burgdorferi s.l. in genomic DNA extracted from cardiac tissue using a real-time PCR assay targeting the 5' region of OspA and a 16S rRNA gene-based assay. Animals were scored as PCR positive only if a PCR product was detected with both assays. In general, to judge an animal as infected, mice had to be positive either by culture, PCR or serology.
[00304] Characterization of infective Borrelia. Wherever possible, the infecting organism was cultured and the OspA sequence and deduced amino acid sequence determined for residues 38-262 OspA (B. afzelii VS461, Gene Bank Accession Number Z29087). This information was compared to OspA reference sequences so that the type of OspA and the species of Borrelia can be inferred. For species expressing a single OspA serotype, the OspA sequence for the strain type for the species was chosen as a reference, eg B. afzelii VS461 and B. valaisiana VS116 (Gene Bank Accession Number Z29087 ; AF095940). As B. garinii has multiple types of OspA, the OspA sequences for OspA genotypes 3-7 were used (ie, strains PBr, PTrob, WABSou, TlsI and T25; Gene Bank Accession Numbers X80256, X80186, X85441, X85440 and X80254 respectively). For real-time PCR-based typing, OspA gene sequence alignments from 124 species of B. burgdorferi s.l. deposited in the Gene Bank were inspected for serotype-specific sequences and suitable primer-probe combinations were designed using Primer Express 3.0 (Applied Biosystems). All assays were run on an ABI Prism® 7900HT Sequence Detection Unit using universal cycling conditions.
[00305] Prevention of infection with B.burgdorferi s.s. (OspA serotype 1) by immunization with rOspA 1/2. All mice immunized with low doses of two different lots of rOspA 1/2 antigen developed specific IgG antibodies to the immunogen as determined by ELISA. Antibodies were not detected in control mice that were treated with vaccine formulation buffer containing aluminum hydroxide. To assess the ability to trigger this immune response to prevent infection with B. burgdorferi ss, a species that encodes an OspA serotype-1, mice were injected intradermally with 7 x 104 cells of the ZS7 strain of B. burgdorferi ss. All control mice treated with adjuvant-containing buffer solution showed serological evidence of infection as demonstrated by C6 ELISA and Western blotting. None of the mice immunized with the rOspA 1/2 antigen became infected and sera from these mice were negative in both assays. As little as 0.03μg of rOspA 1/2 antigen, when formulated with aluminum hydroxide as an adjuvant and administered in a two-dose immunization regimen, provided 100% protection (P < 0.0001, Fisher's exact test of two tails) against a needle challenge with the virulent ZS7 strain of B. burgdorferi ss.
[00306] Prevention of infection with B. afzelii (OspA serotype 2) by immunization with rOspA 1/2. To assess the ability of rOspA 1/2 antigen immunization to prevent infection with B. afzelii, a species that encodes an OspA serotype 2, mice were immunized, in two separate experiments, with the same lots of antigens and project of study as used in the needle challenge experiment described above. However, in this case, the immunized mice were challenged with wild ticks (nymphs) known to be mainly infected with B. afzelii. The ability of these wild ticks to transmit B. burgdorferi s.l. for mice was confirmed by challenge of unimmunized control animals.
[00307] The majority of control mice (total 11/14, 79%) became infected. All infected control animals were positive for Borrelia DNA from two independent real-time PCR analyzes (16S rRNA and OspA genes). In cases 10/11, it was possible to isolate Borrelia by urinary bladder culture. The remaining mouse was positive by serology and PCR. For 9 of 10 cultured isolates, OspA sequences were recovered and all were typed as B. afzelii (>99% OspA sequence identity). In addition, all infecting organisms were typed as B. afzelii by PCR analysis of DNA extracted from the heart using a real-time PCR assay specifically targeting the OspA genes of serotype 2. These data confirm that B. afzelii was the afzelii main species of Borrelia transmitted from infected wild ticks to their host mouse.
Few of the mice immunized with rOspA 1/2 (total 3/32.9%) became infected. Of these three mice, one was infected as determined by all three diagnostic criteria (serology, PCR and culture) and sequence analysis revealed that the infecting organism was B. garinii serotype-6 (>99% sequence identity of OspA). The two remaining animals considered infected were positive for only two of the three criteria. One mouse was positive by serology and PCR. However, the infecting organism could not be recovered in culture. However, this organism can be classified as B. garinii serotype-7 by PCR analysis of DNA extracted from the heart using specific PCR for the OspA gene of serotype-7. The third mouse was positive for PCR and culture, but serologically negative. The isolate cultured from this mouse was B. valaisiana as determined by sequencing (OspA sequence identity with the VS116 strain of B. valaisiana). It is important to note that none of the immunized mice (0/32) became infected with B. afzelii. As little as 0.03μg of the rOspA 1/2 antigen, when formulated with aluminum hydroxide as an adjuvant and given in a two-dose immunization regimen, provided full protection against wild tick-borne B. afzelii.
[00309] Conclusion. The only recombinant outer surface protein A (OspA) antigen designed to contain protective elements from two different OspA serotypes (1 and 2) was able to induce antibody responses that protect mice against infection with either B. burgdorferi sensu stricto (OspA serotype-1) or B. afzelii (OspA serotype-2). Protection against infection by the ZS7 strain of B. burgdorferi s.s. has been demonstrated in a needle test model. Protection against B. afzelii species was shown in a tick challenge model using wild ticks. In both models, as little as 0.03μg of antigen when administered in a two-dose immunization schedule with aluminum hydroxide as an adjuvant was sufficient to provide complete protection against the target species. As predicted, the protection afforded by this new antigen did not extend to other Borrelia species as demonstrated by the antigen's inability to provide protection against infection with B. garinii and B. valaisiana strains. This proof-of-principle study proves that knowledge of protective epitopes can be used for the rational design of effective genetically modified vaccines that require fewer OspA antigens and suggests that this approach can facilitate the development of an OspA vaccine for global use . EXAMPLE 10: EFFICIENCY OF MOUSE ANTI-OSPA ANTIBODIES TO BINDLY TO THE LIVE BORRELIA SURFACE OR INHIBIT ITS GROWTH CORRELATES WITH PROTECTION AGAINST NEEDLE CHALLENGE USING A B. BURGDORFERI S.S.
[00310] The purpose of this study was to establish protective correlations for mice immunized with the rOspA 1/2 antigen in a needle challenge model using a type 1 strain of OspA of Borrelia burgdorferi sensu stricto. The parameter analyzed was the potency of anti-OspA antibodies to bind to the surface of live Borreliae or to inhibit Borreliae growth.
Ninety-eight (98) mice were deliberately immunized with a 3 ng suboptimal dose of rOspA 1/2 antigen with 0.2% AI(OH)3 aa adjuvant, which was 10 times lower than that of lowest dose used in Example 9, in a prime-boost regimen such that, after challenge, infected and protected animals would be observed. Vaccination was performed subcutaneously using a dose volume of 100 µl on days 0, 14 and 28. On day 38, pre-challenge serum samples were taken from 96 mice, and the animals were challenged 10 days later with 19.4 x B.burgdorferi ss ZS7 grown culture ID50 and infection status was determined after four weeks. Seventy-one (71) of 96 mice (72%) were observed to be protected after immunization with this low dose antigen.
Four weeks after challenge blood was taken to identify infected mice by Western blotting analysis of their sera against a membrane fraction of B. burgdorferi ss strain ZS7. At the challenge doses used, only infected mice had a response of antibodies to ZS7-strain membrane antigens other than OspA (the vaccine-induced OspA response was not scored).
[00313] Quantification of OspA surface-binding antibody of live Borreliae. In this assay, the B31 strain of B. burgdorferi ss expressing OspA type 1 was incubated at a fixed dilution (1:100) with the pre-challenge mouse sera at room temperature in the presence of EDTA to prevent complement activation . After washing to remove unbound antibody, antibodies that were specifically bound to the cell surface were labeled by incubating the treated cells with an r-phycoerythrin-conjugated polyclonal anti-mouse Ig antibody. Subsequently, a DNA dye (LDS-751), which emits red fluorescence, thus enhancing detection, was used, and the bacteria were then analyzed by flow cytometry (FACSCalibur, Beckton-Dickinson). Fluorescence intensity, which correlates with the number of antibody molecules bound to the cell surface, was recorded for at least 2000 individual Borreliae, and the mean of the fluorescence intensities (MFI) was calculated. Normal mouse serum served as a negative control to assess the extent of non-specific binding of surface antibodies, whereas an OspA serotype 1 specific mAb served as a positive control to confirm the identity of the OspA type and to verify the level of OspA expression of cells in bacterial culture.
[00314] Bacterial growth inhibition assay. To measure the potency of pre-challenge sera to inhibit the growth of Borreliae, the B31 strain of B. burgdorferi s.s. expressing type 1 OspA was cultured at 33°C in the presence of serial dilutions of non-immune mouse serum or heat-inactivated pre-challenge (negative control) in the presence of complement (normal guinea pig serum). When bacteria in control cultures incubated with immune serum had not grown sufficiently, as determined microscopically, accurate cell counts were made by flow cytometric analysis. Cell cultures were mixed with a solution containing a defined number of fluorescently labeled beads and a DNA dye was added for fluorescently labeled Borrelia cells. Samples were processed through a FACSCalibur flow cytometer and up to 100 beads were counted and absolute cell concentrations were calculated (cells/ml) by comparing the number of events at the gate defining the beads and the gate defining the Borreliae . The dilution of serum that inhibited bacterial growth by 50% was calculated compared to the NMS control and reported as a GI-50 titer. The standard serum preparation was used to normalize titers between different assays. The distribution of parameters measured in serum was compared between infected and protected animals by the non-parametric Mann-Whitney U test (Graphpad Prism Vers. 5.0).
[00315] The results of this study (see Figure 19) clearly demonstrate that there is a highly significant correlation between the functional antibody content of the immune serum at the time of challenge and protection against infection with a high-dose needle challenge (19, 4 x ID50) from B. burgdorferi ss (ZS7). FACS-based fluorescence intensity measurements of live Borreliae expressing OspA type 1, which reflects the number of anti-OspA antibody molecules bound to the cell surface, performed after incubation of bacteria with pre-challenge sera with a fixed dilution, correlated better with protection (p<0.0001 Mann-Whitney U Test). However, growth inhibition titers also correlated highly significantly with protection (p = 0.0002 Mann-Whitney U Test, Figure 19). EXAMPLE 11: EFFICIENCY OF MOUSE ANTI-OSPA ANTIBODIES TO BIND TO LIVE BORRELIA SURFACE OR INHIBIT GROWTH CORRELATES WITH PROTECTION AGAINST TICK CHALLENGE USING A B. AFZELII CEPA TYPE 2
The aim of this study was to establish protective correlates of mice immunized with chimeric OspA 1/2 antigen in a tick challenge model, which uses the natural infection pathway using wild ticks collected from Budweis, Czech Republic to infect the mice. Since nymphal ticks from this endemic area are predominantly infected with B. afzelii, they are considered to provide a challenge with OspA type 2 strain of B. afzelii. As set out in Example 10, the parameters evaluated were the potency of anti-OspA for the antibodies to bind to the surface of live Borreliae or to inhibit the growth of Borreliae, both of which were shown to correlate well against Borrelia bugdorferi needle challenge. ss Thus, this study serves to extend the applicability of using these two parameters as correlates of protection against the natural infection of B. afzelii, the most prominent genospecies associated with human disease in Europe.
Forty mice were immunized with a suboptimal dose of 3 ng rOspA 1/2 antigen with 0.2% Al(OH)3 adjuvant, which was 10 times lower than the lowest dose used in the Example 9, on a prime-boost regimen. As in Example 10, this suboptimal dose was chosen in order to ensure that both protected and infected animals would be observed after challenge. Vaccination was performed subcutaneously using an injection volume of 100 µl on days 0, 14 and 28. On day 40, individual blood samples were taken from the mice to generate the pre-challenge sera. Because the limited number of ticks available did not allow all 40 mice to be challenged, 20 mice were selected based on anti-type 2 IgG concentrations and surface binding to cover a wide range of responses. Eight ticks were applied to each of the mice and were allowed to feed on the mice for 5 days. Four weeks after challenge, mice were sacrificed and the infectious status of control and immunized mice was determined by Western blotting of sera against membrane antigens from B. burgdorferi ss, B. afzelii and B. garinii, cultured organisms from Borrelia bladder, and real-time PCR detection of Borrelia from extracted bladder DNA.
[00318] Quantification of OspA surface-binding antibody of live Borreliae. In this assay, the Arcon strain of B. afzelii expressing OspA type 2 was incubated at a fixed dilution (1:100) with the pre-challenge mouse sera at room temperature in the presence of EDTA to prevent complement activation. After washing to remove unbound antibody, antibodies specifically bound to the cell surface were labeled by incubating the treated cells with an r-phycoerythrin-conjugated polyclonal anti-mouse Ig antibody. All subsequent steps in the assay were similar to those described in Example 10. Normal mouse serum served as a negative control for non-specific antibody binding. A high-titered mouse serum produced against the three-component rOspA vaccine formulation along with OspA serotype 2 specific mAbs served as positive controls to confirm the OspA serotype specificity and the expression level of OspA cells in the bacterial culture.
[00319] Bacterial growth inhibition assay. To measure the potency of pre-challenge sera to inhibit the growth of Borreliae, the Arcon strain of B. afzelii expressing OspA type 2 was grown at 33°C, in the presence of serial dilutions of non-immune mouse serum or of pre-challenge disabled by heat (negative control) without complement. When the bacteria in the control cultures, which were incubated with the non-immune sera, had grown sufficiently, as determined microscopically, accurate cell counts were made by flow cytometric analysis. The procedure used to count the bacteria was similar to that previously described for the growth inhibition assay in Example 10. The dilution of serum that inhibited bacterial growth by 50% was calculated compared to the NMS control and reported as GI titers -50. The standard serum preparation was used to normalize titers between different assays.
[00320] Statistical analysis. The distribution of measured serum parameters was compared in infected and protected animals by the non-parametric Mann-Whitney U test (Graphpad Prism Version 5.0).
[00321] Results. Of the 20 animals immunized three times with 0.003 pg of rOspA 1/2 and challenged with 8 wild type ticks, 7/20 (35%) were considered infected. Due to limited tick availability, it was not possible to determine the exact infection rate of the challenge, challenging a control group of unimmunized mice. However, this challenge was not necessary for the purposes of this study and typically an infection rate of 70-80% is obtained in wild Budweis tick challenge experiments.
[00322] Significant differences were detected between the protected and infected groups for the results of surface binding (p = 0.007) and growth inhibition (p = 0.03) assays (Figure 20).
[00323] Conclusion. In this study, it was demonstrated that there is a statistically significant correlation between functional antibody content in mouse serum at the time of challenge and protection against infection with a wild tick challenge applying 8 ticks per mouse. FACS-based fluorescence intensity measurements of live Borreliae expressing OspA type 2, which reflects the number of anti-OspA antibody molecules bound to the cell surface performed after incubation of the bacteria with the pre-challenge sera with a fixed dilution, correlated better with protection. Growth inhibition titers also correlated well with protection. In contrast to Borrelia burgdorferi s.s. strains, where complement is required to efficiently kill, the rOspA1/2 antigen induced antibodies that effectively inhibit Borrelia growth, even in the absence of complement.
[00324] The results of the studies presented in Examples 10 and 11, when taken together, establish the in vitro parameters of the mean fluorescence intensity (MFI) of surface-bound antibody to live Borreliae and the GI-50 titers of the serum from mouse immune as "protective correlates" in both examples where active mouse models of protection are currently available (eg, namely, a needle challenge model for type 1 strains of OspA of B. burgdorferi ss and a model of tick challenge for OspA type 2 strains of B. afzelii. Furthermore, in the absence of reliable asset protection models to assess protection against homologous B. garinii strains expressing OspA types 3-6, by inference, the aforementioned models can be used as "surrogate markers of protection" in vitro to assess the potential protection and cross-strain coverage of various vaccine formulations for strains that e. express all types of vaccine homologous OspA and even those that express heterologous OspA types. In fact, when functional studies using these immune response assays were performed on immune sera from mice immunized with the 3-component chimeric rOspA vaccine formulation, then comparable MFI and GI-50 titers were obtained for B. garinii (types) 3, 4, 5, 6 of OspA) (see examples 13), thus indicating through these surrogate protection markers that protective responses were also achieved against strains for which no active mouse model of protection currently exists. . Furthermore, when comparing the immune responses of mice immunized with either (a), individual chimeric rOspA antigens, (b), or to any of the possible 2-component chimeric rOspA antigen vaccine formulation combinations, or (c ), the 3-component rOspA antigen formulation, it was possible to show that the last three-component vaccine was needed to optimally cover strains expressing OspA types 1-6 (Example 14). Furthermore, through the use of these surrogate marker assays in vitro, it was possible to show the immune responses produced after immunization of mice with the 3-component chimeric rOspA vaccine formulation (rOspA 1/2, rOspA 6/4 and rOspA 5 /3) induce functional immune responses to all intra-type variants (or subtypes) of types 1, 2, 3, 5, and 6 tested to date (see Example 15), and even to the different, heterologous OspA types of the homologous OspA types 1-6 present in the vaccine (see Example 16). EXAMPLE 12: RECOMBINANT MULTIVALENT OSPUM FORMULATION COMPRISING 3 ANTIGENS (1/2, 6/4, AND 5/3) IS HIGHLY IMMUNOGENIC IN MICE
The multivalent OspA vaccine (rOspA 1/2, rOspA 5/3, and rOspA 6/4) was evaluated in a tick challenge model. Three recombinant OspA antigens containing the protective epitopes of OspA serotypes 1 and 2 (SEQ ID NO: 2), OspA serotypes 6 and 4 (SEQ ID NO: 4), and OspA serotypes 5 and 3 (SEQ ID NO: 6) were combined into a vaccine.
[00326] Groups of ten female C3H/HeJ mice (immunization age: 11 weeks) were immunized subcutaneously on days 0 and 28 with a fixed dose of 0.3 μg of multivalent vaccine (0.1 μg each, rOspA 1/2, rOspA 5/3, and rOspA 6/4). Tick challenge was done as described above with ticks from Budweis, Czech Republic. The ability of wild ticks to transmit B. burgdorferi s.l. to mice was confirmed by challenging the unimmunized control animals. The infection status of challenged mice was determined by Western blotting, real-time PCR, and by culture.
[00327] Interim blood samples were taken on day 41 by orbital puncture. Final blood samples (day 70/71) were collected by cardiac puncture. Individual sera were prepared from whole blood by centrifugation (10 minutes; 1000-2000xg, RT). Sera were stored at < - 20°C until use.
[00328] In this experiment the unfed ticks, taken from the same batch used to challenge the mice, were characterized to determine the rate of infection and to confirm the species of the infecting organisms. When 80 nymphal ticks were tested for the presence of B. burgdorferi s.l. by real-time PCR of 16S rRNA, 32.5% (26/80) were found to be infected. The OspA serotype could be determined by PCR-ELISA for 22 of the 26 infected nymphs, 86% (19/22) were typed as B. afzelii and 14% (3/22) as B. burgdorferi s.s..
[00329] All unimmunized control mice (100%, 10/10) became infected, whereas only one of the mice immunized with the multivalent rOspA vaccine became infected (10%, 1/10). There was 100% agreement between the different methods used to identify infected animals. The multivalent rOspA vaccine resulted in statistically highly significant protection (p = 0.00012; two-tailed Fisher's exact test) when compared to the control group.
These data demonstrate that immunization with a multivalent rOspA vaccine, which contains the rOspA 1/2 antigen, is able to prevent infection with B. afzelii, a species of Borrelia that expresses an OspA of serotype 2. In addition Furthermore, there is no evidence that the inclusion of additional rOspA antigens has an antagonistic effect on the protective immunity conferred by the rOspA 1/2 antigen.
This vaccine offered protection against tick-borne infection with B. afzelii that was equivalent to that seen with the OspA 1/2 antigen; 0.3μg of the vaccine (0.1 μg of each antigen) formulated with 0.2% Al(OH)3 and administered in a two-dose schedule provided 90% protection, as determined by Western blot, to the culture of Borrelia and detection of Borrelia DNA by PCR. EXAMPLE 13: A VACCINE COMPRISING THE THREE-COMPONENT VACCINE (OSPA 1/2, OSPA 6/4, AND OSPA 5/3) INDUCES HIGH LEVELS OF FUNCTIONAL ANTI-OSPA ANTIBODIES THAT BIND ON AND INHIBIT THE GROWTH OF BORRELIA STRIPS EXPRESS TYPES 1-6 OF OSPA
[00332] Since both surface binding (MFI) and growth inhibition (GI-50 titers) showed good correlations of protection, a needle challenge model (B. burgdorferi ss) (Example 10) and a mouse model of sapphire with tick (B. afzelii) (Example 11), the present study was performed to determine whether equivalent functional immune responses are induced by the 3-component chimeric rOspA antigen vaccine formulation against B. garinii OspA serotypes 3-6, for which no in vivo model of protection is available to investigate the efficacy of a vaccine.
[00333] Mouse Immunization. Groups of 10 female C3H/HeJ mice were immunized subcutaneously three times (day 0, day 14, day 28) with a 1:1:1 mixture of rOspA-1/2, rOspA-6/4 and rOspA-5 /3) in three different doses (1, 0.1, 0.03 μg protein per dose), combined with 0.2% Al(OH)3 as an adjuvant. Serum was generated from blood samples obtained on day 40.
[00334] Quantification of OspA antibody binding to the surface of live Borreliae. In this assay, in vitro grown cultures of six representative Borrelia strains expressing OspA types 1-6 (B31/OspA-1 from B. burgdorferi sensu stricto; Arcon/OspA-2 from B. afzelii; PBr/OspA-3 from B. garinii; DK6/OspA-4 from B. garinii; W/OspA-5 from B. garinii and KL11/0spA-6 from B. garinii) were incubated at a fixed dilution (1:100) with titration pellets of mouse sera at room temperature in the presence of EDTA to prevent complement activation. The subsequent washing, labeling, detection and analysis procedures are similar to those described in Example 10. Normal mouse serum served as a negative control for non-specific antibody binding.
[00335] Bacterial growth inhibition assay. To measure the potency of pre-challenge sera to inhibit the growth of Borreliae, six representative strains expressing types 1-6 of OspA (B31, Arcon, PBr, DK6, W, and KL11) were grown at 33°C in the presence of serial dilutions of non-immune mouse serum or heat-inactivated peak titration clusters (negative control). B31 was grown in the presence of complement (guinea pig serum), while the other five strains were tested in the absence of complement. Again, growth inhibition assays were performed as described in Example 10. The standard serum preparation was used to normalize titers between different assays.
[00336] Surface binding efficiency and growth inhibition of anti-OspA antibody responses. Intense fluorescence staining with MFI values ranging from 50 to 200 was observed for all six Borrelia strains when tested with the three clusters of sera from the different immunization dose groups (1.0, 0.1 and 0.03 μg protein per dose) of the 3-component vaccine at a 1:100 dilution (Figure 21). When serum clumps from the 3 dose groups were tested for their ability to inhibit bacterial growth, the 3-component vaccine was also found to have induced strong IG-50 titers for all six OspA-type strains, which they range from 1000 (type 4 strain, 0.03 μg dose) to 20,000 (type 6 strain).
[00337] Conclusion. Taken together, these results demonstrate that rOspA antigens are highly immunogenic and induce large amounts of functional antibodies that can bind to the surface of live Borreliae and inhibit Borreliae growth. Coverage among the six strains tested was completed, as titers of high growth inhibition and high fluorescence intensities were detected, comparable to the levels observed for OspA types 1 and 2. In summary, the results presented in this study indicate that antibody responses induced by the three-component rOspA vaccine (1/2 + 5/3 + 6/4), when formulated with Al(OH)3, prevents infections by strains expressing types 1-6 of OspA, a quaa, as epidemiological studies have shown theoretically covers over 99% of human disease-causing isolates in Europe and North America and is therefore highly effective in preventing Borreliosis of Lyme. EXAMPLE 14: A VACCINE COMPRISING THE THREE-COMPONENT VACCINE (OSPA 1/2, OSPA 6/4, AND OSPA 5/3) IS REQUIRED TO OPTIMUMLY COVER THE BORRELIA EXPRESSING TYPES 1-6 OF OSPA
[00338] The purpose of this study was to investigate and compare the immunogenicity and coverage of cross-strains of functional growth-inhibiting and/or surface-binding antibodies that inhibit growth induced by single and multicomponent rOspA vaccine formulations against Lyme borreliosis, again using the efficacy of anti-OspA antibodies to bind to the surface of live Borreliae and inhibit the in vitro growth of Borreliae as a protective correlate
[00339] Immunization of mice. Ten female mice (C3H) per group were immunized with 0.1 μg of a single-component vaccine comprising rOspA 1/2 antigen, rOspA 6/4 antigen, or rOspA 5/3 antigen, a two-component vaccine comprising 0.1 μg of antigens 1/2 + 5/3, antigens 1/2 + 6/4 or antigens 5/3 + 6/4; or a three-component vaccine comprising a 0.1 μg combination of all three antigens 1/2+ 5/3+ 6/4 adjuvanted with 0.2% Al(OH)3 in a primer-booster regimen . Vaccination was performed subcutaneously using a dose volume of 200 µl on days 0, 14 and 28. On day 42, individual blood samples were taken from each mouse to generate the sera.
[00340] Antibody surface binding and growth inhibition assays. A slightly modified version of the surface binding assay was used to determine the efficiency of anti-OspA IgG to bind to the surface of live Borreliae. Serial dilutions of a pool of sera with defined MFI titers were included in the analyzes to create a standard curve, from which the relative titers of the test sera were read after interpolation of a non-linear regression curve. The standard serum MFI titer for the individual strains expressing OspA types 1-6 was defined as the highest dilution at which the fluorescence intensity of Borreliae was determined to be at least 3 times the fluorescence intensity observed with normal mouse serum. All determinations were performed in duplicate.
The scatter plots shown in Figure 22 compare the MFI titers with six strains expressing homologous types of OspA observed for the immune sera of individual C3H mice after immunization with individual rOspA antigens or rOspA antigen combinations. The results showed that a formulation containing all three rOspA antigens (1/2, 5/3 and 6/4) was required to induce the high MFI titers against all six Borrelia strains expressing types 1-6 of OspA, and formulations composed of two rOspA antigens (ie, covering four strains) do not completely cover strains expressing the two types of OspA not present in the formulation.
To determine the potency of various vaccine combinations to induce growth-inhibiting antibodies, six representative strains of Borreliae (B31, Arcon, PBr, DK6, W, KL11) expressing OspA types 1-6, respectively , were cultured at 33°C in the presence of clumps of non-immune or heat-deactivated immune mouse serum. All sera were tested at a single dilution. The following dilutions were: B31, PBr and KL11 1:200, Arcon, DK6 and W 1:100 was grown in the absence of 20% complement, while another 5 strains were tested in the presence of complement. Baby rabbit complement was used for DK6, W and KL11, while guinea pig serum was used for B31 and Arcon. When bacteria in control cultures incubated with immune serum had not grown sufficiently, as determined microscopically, accurate cell counts were made as described above (see Example 10). The percentage of bacterial growth inhibition was calculated from the cell count observed with the test serum versus the normal mouse control serum. The overall growth inhibition observed for the different formulations tested was then presented (Figure 23) as the number of animals among the different groups of ten C3H mice that showed more than 50% growth inhibition. The results demonstrated that the 3-component formulation was the only formulation capable of inducing growth inhibitor antibodies with high titers against all six representative strains expressing OspA types 1-6 (Figure 23). In all cases, the 3-component vaccine formulation provided >50% growth inhibition in >90% of immunized animals. The 2-component vaccine formulations do not completely cover the two strains that express types of OspA not present in the vaccine. The formulation comprising rOspA 1/2 + 6/4 quarters does not cover the type 3 strain, the formulation comprising rOspA 1/2 + 5/3 does not cover types 4 or 6, and the formulation comprising rOspA 5/3 + 6/ 4 does not cover type 1. EXAMPLE 15: MULTIVALENT OSPA VACCINE FORMULATION COVERS BORRELIA WHICH EXPRESSES VARIANTS BETWEEN OSPA TYPES OR SUBTYPES 1-6
[00343] Although Borrelia OspA types 1-6 have been selected as the basis for the design and construction of the multivalent rOspA vaccine, Borreliae, which expresses type 1, 2, 3, 5 and OspA protein variants 6 was also isolated. These variants, while being classified as being within the same species, have slightly altered nucleotide gene sequences and amino acid protein sequences. Thus, type or subtype variants exist among OspA types 1, 2, 3, 5, and 6 (see Figure 24). No variants between types or subtypes have yet been observed for OspA type 4.
[00344] The aim of this study was to confirm that immune serum generated by immunization of mice with the multivalent 3-component rOspA vaccine contains functional antibodies that can bind to the surface of live Borreliae expressing these variants across types or subtypes.
[00345] For this study, a matched immune mouse serum was generated by immunizing 70 female C3H mice three times with 0.3 μg of the multivalent 3-component rOspA vaccine on days 0, 14 and 28. On day 42, the mice were bled and serum was obtained and pooled. The pooled immune serum was then used to test antibody binding to the surface of live Borreliae. Borrelia cultures were incubated with the pool of immune serum or normal control mouse serum at 1:100, in duplicate, and Borreliae fluorescence intensities measuring the binding of anti-OspA antibodies to the bacteria were monitored by analysis of FACS as described in this document above.
[00346] Elevated levels of surface binding antibodies (defined as fluorescence intensity above 10 times that observed for a mouse not immunized with control serum) at a 1:100 dilution of serum were detected for most of the strains expressing OspA 1-6 subtypes. In particular, high levels of antibody binding have been detected with Borreliae strains expressing OspA of subtypes 1a, 1b, 1c, 1d, 1F, 1H, 1J, 1K and 1L; 2a, 2b, 2e, 2g, 2k, 2l, and 2n; 3a, 3c, 3d, 3e and; 5a and 5c and 6a, 6e, 6f, 6g, and 6k (Figure 24). The weakest binding (defined as a fluorescence intensity of between 2-10 times that observed for an unimmunized mouse control serum) was observed with Borreliae strains expressing OspA subtypes 1g, 2j, 2m, 3b, 5d, and 61 (Figure 24), but this weaker binding was mainly due to the poor expression of the OspA protein under the growth conditions used.
[00347] Conclusion. The 3-component chimeric rOspA vaccine induces functional surface binding antibodies against all subtypes or between-type variants of OspA types 1, 2, 3, 5, and 6 in C3H mice EXAMPLE 16: THE MULTIVALENT OSPA VACCINE FORMULATION PROVIDES PROTECTION AGAINST OTHER TYPES OF DIE IN ADDITION TO THOSE THAT EXPRESS TYPES 1-6 OF OSPA
[00348] The purpose of this study was to determine whether the 3-component chimeric rOspA antigen vaccine formulation (which comprises all 3 chimeric antigens - 1/2, 6/4 and 3/5) can also provide protection against expressing Borrelia types of OspA from different types 1-6 of homologous OspA. 40 C3H mice were immunized three times with 0.3 μg of 3-component vaccine on days 0, 14 and 28. On day 42, mice were bled, and a pool of serum was made and used to assess binding efficiency of surface and growth inhibition against strains expressing heterologous OspA types.
The results of this study showed that the 3-component chimeric rOspA vaccine induces antibodies that bind to the surface of Borreliae and inhibit the growth of other types of Borreliae, including strains of B. spielmanii, B. valaisiania, B. lusitaniae and B. japonica (see Table 9). In the case of OspA type 7 expressing B. garinii, only weak surface binding and little or no growth inhibition was observed, however, this amount of weak binding and small growth inhibition may be due to low levels of OspA expression under the in vitro culture conditions used, rather than the lack of binding of the immune serum antibodies. Table 9. Surface binding and growth inhibition against other types of Borreliae
+: significant binding surface and/or growth inhibition -: no significant growth inhibition/binding (+-): low intensity binding surface EXAMPLE 17: MULTIVALENT OSPA VACCINE FORMULATIONS INDUCE ANTIBODIES TO A COMMON Epitope ON THE N-TERMINAL OF OSPA MOECULA WHICH MAY CONTRIBUTE TO THE PROTECTION AGAINST ANY OSPA EXPRESSING THE BORRELIA CEPA
During the course of investigating the protective efficacy of multivalent chimeric rOspA formulations, a monoclonal antibody (F237/BK2) was generated against a two-component rOspA vaccine comprising rOspA-1/2 and rOspA-6/4. F237/BK2 was shown by anti-OspA ELISA to bind to all OspA types investigated so far (OspA types 1-7), as well as to the 3 chimeric rOspA antigens (rOspA-1/2, rOspA-5 /3 and rOspA-6/4) This result indicates that F237/BK2 recognizes a common epitope found in all OspA molecules. Furthermore, preliminary epitope mapping studies indicate that this common epitope is located in the least N-terminal variable of half of the molecule (ie, at the N-terminal end of amino acid 130), where sequence homologies of OspA are most commonly observed.
Interestingly, F237/BK2 has also been shown to bind to the surface of Borreliae expressing homologous OspA types 1-6 and heterologous OspA types, including those expressed by B. spielmanii, B. valaisiania and B. japonica, albeit from B. japonica less efficiently than monoclonal antibodies directed against specific C-terminal type epitopes. Using methods similar to those described in the previous examples, F237/BK2 has also been found to inhibit the growth of representative strains expressing types 1, 2, 4, 5 and 6 of OspA.
[00352] When F237/BK2 was tested in an in vivo passive protection model in mice, F237/BK2 was observed to confer protection against wild tick challenge, corresponding to a B.afzelii type 2 challenge. Ticks were collected from Wundschuh (Styria, Austria), which are known to be predominantly infected with B. afzelii. Ten female C3H mice were injected intraperitoneally with 500 µg of affinity purified F237/BK2 mAb. Two hours later, 8 ticks were applied per animal to 10 passively immunized mice as well as to 10 mock immunized animals. Four days later, the fed ticks were removed. On day 90, mice were sacrificed and analyzed for infection by serological testing, PCR analysis and Borrelia culture as described in this document above. No animal was infected in the F237/BK2 treated group, whereas five animals (50%) were infected with B. afzelii in the control group. Thus, the F237/BK2 monoclonal antibody provided statistically significant (p = 0.0325) passive protection against a tick challenge when compared to sham immunized control mice. This is the first time that a monoclonal antibody that binds to a common epitope on the N-terminal half of the molecule has been reported to be involved in protection. Furthermore, if a vaccine can induce antibodies that recognize this common epitope, such an antibody would certainly contribute to the vaccine's cross-protective efficacy.
To test whether these antibodies were indeed induced by the 3-component chimeric rOspA vaccine formulation, a monoclonal antibody inhibition ELISA was performed employing peroxidase labeled F237/BK2. In these experiments, a GST-OspA type 3 protein was used as a coat antigen and either normal mouse serum or a pool of sera from C3H mice immunized three times with the 3-component chimeric rOspA vaccine was added to the wells, at a 1:100 dilution. Sixty minutes later, peroxidase-labeled F237/BK2 was added to a pre-optimized concentration to eventually give an optical density (OD) value of about 1 for the uninhibited normal mouse serum control, and the incubation was continued for another 60 minutes. Finally, ELISA plates were washed and developed with TMB substrate.
Using this monoclonal antibody from the ELISA inhibition assay, it can be demonstrated that the 3-component chimeric rOspA formulation actually induces antibodies that bind to an epitope identical to, or in close proximity to, the epitope recognized by mAb F237/BK2. OD values were significantly reduced (eg, typically 20-30%) by the anti-OspA immune serum compared to the uninhibited normal mouse control serum.
[00355] Conclusion. This study shows that the 3-component chimeric rOspA vaccine is capable of inducing both a specific type and a broad cross-protective immune response. EXAMPLE 18: ADDITIONAL SYNTHETIC OSP POLYPEPTIDES AND NUCLEIC ACID MOLECULES
The aim of the study was to design additional new OspA antigens comprising serotypes 1 and 2, 6 and 4, and 5 and 3, respectively. Three synthetic OspA genes (SEQ ID NOS: 168 (origin sOspA 1/2), 170 (origin sOspA 6/4), and 172 (origin sOspA 5/3)) were designed to encode OspA polypeptide molecules with protective epitopes from OspA serotypes 1 and 2 (origin sOspA 1/2), OspA serotypes 6 and 4 (origin sOspA 6/4) and OspA serotypes 5 and 3 (origin sOspA 5/3) from Borrelia. The primary amino acid sequences of these molecules (SEQ ID NOS: 169, 171, and 173, respectively) are shown in Table 1. These sequences comprise original chimeric constructs, that is, no mutations and no codon optimization. EXAMPLE 19: MULTIVALENT RECOMBINANT OSPA FORMULATION COMPRISING 3 ANTIGENS (1/2, 6/4, AND 5/3) IS IMMUNOGENIC IN MICE
The multivalent OspA vaccine comprising formulations of original constructs without codon optimization and without mutations (origin OspA 1/2, orig OspA 5/3, and orig OspA 6/4) is evaluated in a tick challenge model. Three recombinant OspA antigens containing the protective epitopes from OspA serotypes 1 and 2 (SEQ ID NO: 169), OspA serotypes 6 and 4 (SEQ ID NO:171), and between OspA serotypes 5 and 3 (SEQ ID NO: 173) are combined into a vaccine.
[00358] Groups of 10 female C3H/HeJ mice (immunization age: 11 weeks) were immunized subcutaneously on days 0 and 28 with a fixed dose of 0.3 μg of multivalent vaccine (0.1 μg each, orig OspA 1/2, Orig OspA 5/3, and Orig OspA 6/4). Tick challenge is done as described in this document above, with ticks from Budweis, Czech Republic. The ability of wild ticks to transmit B. burgdorferi s.l. to mice is confirmed by the challenge of unimmunized control animals. The infection status of challenged mice is determined by Western blotting, real-time PCR, and by culture.
[00359] Intercalated blood samples are taken on day 41 by orbital puncture. Final blood samples (day 70/71) are collected by cardiac puncture. Individual sera are prepared from whole blood by centrifugation (10 minutes; 1000-2000xg, RT). Sera are stored at < - 20°C until use.
[00360] In this experiment, unfed ticks, taken from the same batch used to challenge mice, are characterized to determine the rate of infection and to confirm the species of infecting organisms. EXAMPLE 20: THE VACCINE COMPRISING A THREE-COMPONENT VACCINE (ORIG OSPA 1/2, ORIG OSPA 6/4, AND ORIG OSPA 5/3) INDUCES HIGH LEVELS OF FUNCTIONAL ANTI-OSPA ANTIBODIES THAT BIND ON AND INHIBIT THE GROWTH OF EXPRESSING BORRELIA STRIPS TYPES 1-6 OF OSPA
The results presented in Example 13 indicate that the antibody responses induced by the three-component rOspA vaccine (lipB sOspA1/2 + lipB sOspA 5/3 + lipB sOspA 6/4) when formulated with Al(OH) 3, prevent infections by strains expressing OspA types 1-6 and therefore are effective in preventing Lyme borreliosis. Thus, the present study is carried out to determine whether functional equivalent immune responses are induced by the three-component OspA vaccine comprising chimeric (Orig) OspA antigens (Orig sOspA1/2 + Orig sOspA 5/3 + Orig sOspA 6/4 ).
[00362] Mouse immunization. Groups of 10 female C3H/HeJ mice were immunized subcutaneously three times (day 0, day 14, day 28) with a 1:1:1 mixture of Orig sOspA1/2 + Orig sOspA 5/3 + Orig sOspA 6/ 4) in three different doses (1, 0.1, 0.03 μg protein per dose), combined with 0.2% Al(OH)3 as adjuvant. Serum is generated from blood samples taken on day 40.
Quantification of OspA antibody binding to the surface of live Borreliae. In this assay, in vitro grown cultures of six representative Borrelia strains expressing OspA types 1-6 (B. burgdorferi sensu stricto B31/OspA-1; B. afzelii Arcon/OspA-2; B. garinii PBr/OspA -3; B. garinii DK6/OspA-4; B. garinii W/OspA-5; and B. garinii KL11/0spA-6) are incubated at a fixed dilution (1:100) with clusters of peak serum titers mouse, at room temperature, in the presence of EDTA to prevent complement activation. Subsequent washing, labeling, detection and analysis procedures are similar to those described in Examples 10 and 13. Normal mouse serum serves as a negative control for non-specific antibody binding.
[00364] Bacterial growth inhibition assay. To measure the potency of pre-challenge sera to inhibit the growth of Borreliae, six representative strains expressing OspA types 1-6 (B31, Arcon, PBr, DK6, W, and KL11) are grown at 33°C in presence of serial dilutions of clusters of heat-deactivated peak titration serum or non-immune mouse serum (negative control). B31 is grown in the presence of complement (guinea pig serum), while the other five strains are tested in the absence of complement. Growth inhibition assays are performed as described in Examples 10 and 13. The standard serum preparation is used to normalize titers between different assays.
[00365] Surface binding efficiency and growth inhibition of anti-OspA antibody responses. Fluorescence staining is measured in all six Borrelia strains when tested with the three clusters of sera from the different immunization dose groups (1.0, 0.1 and 0.03 μg protein per dose) of the vaccinia vaccine. 3 components at a 1:100 dilution. EXAMPLE 21: THE VACCINE COMPRISING THE THREE-COMPONENT VACCINE (OSPA 1/2, OSPA 6/4, AND OSPA 5/3) IS NECESSARY TO OPTIMUMLY COVER THE BORRELIA EXPRESSING OSPA 1-6 TYPES
[00366] The purpose of this study is to investigate and compare the immunogenicity and coverage of cross-strains of antibodies that inhibit the growth and/or functional surface binding induced by single and multicomponent formulations of the Orig sOspA Lyme Borreliosis vaccine using antibody efficiency anti-OspA to bind to the surface of live Borreliae and inhibit the in vitro growth of Borreliae as a protective correlate
[00367] Immunization of mice. Ten female mice (C3H) per group were immunized with 0.1 μg of a single-component vaccine comprising Orig sOspA1/2 antigen, Orig sOspA 5/3 antigen, or Orig sOspA 6/4 antigen, a two-component vaccine comprising 0.1 μg of the antigens of 1/2 + 5/3, antigens of 1/2 + 6/4, or antigens of 5/3 + 6/4, or a three-component vaccine comprising a combination of 0, 1 μg of all three antigens 1/2 + 5/3 + 6/4 with 0.2% AI(OH)3 adjuvant in a prime-boost regimen. Vaccination is performed subcutaneously, using a dose volume of 200 µl on days 0, 14 and 28. On day 42, individual blood samples are taken from each mouse to generate the sera.
[00368] Antibody growth inhibition and surface binding assays. A slightly modified version of the Surface Binding Assay described above is used to determine the efficiency of anti-OspA IgG to bind to the surface of live Borreliae. Serial dilutions of a pool of sera with defined MFI titers are included in the analyzes to create a standard curve, from which the relative titers of test sera are read after interpolation with a non-linear regression curve. The standard serum MFI titer for the individual strains expressing OspA types 1-6 is defined as the highest dilution at which the fluorescence intensity of Borreliae is determined to be at least 3-fold above the fluorescence intensity observed with normal mouse serum. All determinations are performed in duplicate.
To determine the potency of various vaccine combinations to induce growth-inhibiting antibodies, six representative strains of Borreliae (B31, Arcon, PBr, DK6, W, KL11), which express types 1-6 of OspA, respectively, are cultured at 33°C in the presence of clumps of non-immune or heat-deactivated immune mouse serum. All sera are tested at a single dilution. The following dilutions are used: B31, PBR and KL11 1:200, Arcon, DK6 and W 1:100. PBr is grown in the absence of 20% complement, while the other 5 strains are tested in the presence of complement. Baby rabbit complement is used for DK6, W and KL11, while guinea pig serum is used for B31 and Arcon. When bacteria in control cultures incubated with non-immune serum grow sufficiently, as determined microscopically, accurate cell counts are done as described above (see Example 10). The percentage of bacterial growth inhibition is calculated from the cell count observed with the test serum versus the normal mouse control serum. The overall growth inhibition observed for the different formulations tested is then presented as the number of animals among the different groups of ten C3H mice that showed more than 50% growth inhibition EXAMPLE 22: THE FORMULATION OF THE MULTIVALENT OSPA VACCINE COVERS THE BORRELIA WHICH EXPRESSES VARIANTS BETWEEN TYPES OR SUBTYPES OF TYPES 1-6 OF OSPA
The aim of this study was to confirm that the immune serum generated by immunization of mice with the 3-component multivalent OspA vaccine (orig sOspA 1/2, orig sOspA 6/4, and orig sOspA 5/3) contains functional antibodies that can bind Borreliae to the living surface expressing these variants across types or subtypes.
[00371] For this study, a cluster mouse immune serum is generated by immunizing 70 female C3H mice three times with 0.3 μg of the 3-component multivalent OspA orig vaccine on days 0, 14 and 28. On day 42, mice are bled and serum is obtained and pooled. The pooled immune serum is then used to test antibody binding to the surface of live Borreliae. Borrelia cultures are incubated with the 1:100 normal mouse immune serum or control serum pellet in duplicate, and the fluorescence intensities of Borreliae that measure the binding of anti-OspA antibodies to the bacteria are monitored by analysis of FACS as described in this document above.
The invention has been described in terms of particular embodiments found or proposed to comprise specific modes for practicing the invention. Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in relation to specific embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to fall within the scope of the claims which follow.

权利要求:
Claims (28)
[0001]
1. Polypeptide, characterized in that it is a polypeptide consisting of the amino acid sequence defined in SEQ ID NO: 173.
[0002]
2. Composition, characterized in that it comprises the polypeptide as defined in claim 1 and a pharmaceutically acceptable carrier.
[0003]
3. Composition according to claim 2, characterized in that it further comprises an additional polypeptide of an outer surface protein A protein (OspA) of Borrelia.
[0004]
4. Composition according to claim 3, characterized in that Borrelia is Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisitania, Borrelia Borrelia spielmanii, Borrelia miyamotoi or Borrelia lonestar.
[0005]
5. Composition according to claim 3 or 4, characterized in that the additional polypeptide comprises: (a) a polypeptide consisting of a first polypeptide fragment of a Borrelia outer surface protein A (OspA) serotype 4 protein garinii and a second polypeptide fragment of a Borrelia garinii OspA serotype 6 protein, (b) a polypeptide consisting of an N-terminal polypeptide fragment of the OspA serotype 6 protein and a C-terminal polypeptide fragment of the serotype 4 protein from OspA, (c) a polypeptide consisting of an N-terminal polypeptide fragment of the OspA serotype 4 protein and a C-terminal polypeptide fragment of the OspA serotype 6 protein; (d) a polypeptide consisting of the amino acid sequence defined in SEQ ID NO: 171; (e) a polypeptide consisting of a first polypeptide fragment of a Borrelia burgdorferi sensu stricto outer surface protein A (OspA) serotype 1 protein and a second polypeptide fragment of a Borrelia afzelii OspA serotype 2 protein, ( f) a polypeptide consisting of an N-terminal polypeptide fragment of the OspA serotype 1 protein and a C-terminal polypeptide fragment of the OspA serotype 2 protein, (g) a polypeptide consisting of an N-terminal polypeptide fragment of the protein OspA serotype 2 and a C-terminal polypeptide fragment of the OspA serotype 1 protein, and/or (h) a polypeptide consisting of the amino acid sequence defined in SEQ ID NO: 169.
[0006]
6. Composition according to any one of claims 3 to 5, characterized in that it comprises three polypeptides, in which the polypeptides have different sequences.
[0007]
7. Composition according to claim 6, characterized in that the polypeptides individually comprise the amino acid sequences defined in SEQ ID NOS: 169, 171 and 173.
[0008]
8. Nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide as defined in claim 1, wherein the nucleotide sequence consists of the nucleotide sequence defined in SEQ ID NO: 172 .
[0009]
9. Vector, characterized in that it comprises the nucleic acid molecule as defined in claim 8.
[0010]
10. Transgenic microorganism, characterized in that it comprises the vector as defined in claim 9.
[0011]
11. Process for producing a polypeptide, characterized in that it comprises culturing the host cell as defined in claim 10 under conditions suitable for expressing the polypeptide and, optionally, isolating the polypeptide from the culture.
[0012]
12. Composition, characterized in that it comprises the nucleic acid molecule as defined in claim 8 or the vector as defined in claim 9, and a pharmaceutically acceptable carrier.
[0013]
13. Composition according to claim 12, characterized in that it further comprises an additional nucleic acid molecule encoding a Borrelia outer surface protein A (OspA) protein, wherein the additional nucleic acid molecule comprises: ( a) a nucleic acid molecule consisting of the nucleotide sequence defined in SEQ ID NO: 170; (b) a nucleic acid molecule consisting of the nucleotide sequence defined in SEQ ID NO: 168.
[0014]
14. Composition according to claim 13, characterized in that Borrelia is Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisitania, Borrelia Borrelia spielmanii, Borrelia miyamotoi or Borrelia lonestar.
[0015]
15. Composition according to claim 13 or 14, characterized in that the additional nucleic acid molecule additionally comprises a nucleotide sequence of 5'-terminal outer surface protein B (OspB) fragment of Borrelia, wherein the OspB nucleotide sequence fragment comprises an OspB leader sequence.
[0016]
16. Composition, characterized in that it comprises two of the chimeric nucleic acid molecules in the composition as defined in any one of claims 12 to 15, wherein the nucleic acid molecules have different nucleotide sequences.
[0017]
17. Composition according to claim 16, characterized in that the nucleic acid molecules individually comprise the nucleotide sequences defined in SEQ ID NOS: 168, 170 and 172.
[0018]
18. Immunogenic composition, characterized in that it comprises the composition as defined in any one of claims 2 to 8 or 12 to 17 and a pharmaceutically acceptable carrier.
[0019]
19. Immunogenic composition according to claim 18, characterized in that the composition is selected from the group consisting of: (a) a composition in which Borrelia is Borrelia burgdorferi sensu lato; (b) a composition in which Borrelia is Borrelia afzelii, Borrelia garinii or Borrelia burgdorferi sensu stricto; and (c) a composition in which Borrelia is Borrelia japonica, Borrelia andersonii, Borrelia bissettii, Borrelia sinica, Borrelia turdi, Borrelia tanukii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii, Borrelia miyamotoi or Borrelia lonestar.
[0020]
20. Vaccine composition, characterized in that it comprises the immunogenic composition as defined in claim 18 or 19 and a pharmaceutically acceptable carrier.
[0021]
21. Combined vaccine, characterized in that it comprises the vaccine composition as defined in claim 20 in combination with a second vaccine composition.
[0022]
22. Combined vaccine according to claim 21, characterized in that the second vaccine composition protects against tick-borne disease.
[0023]
23. Combined vaccine according to claim 22, characterized in that the tick-borne disease is Rock Mountain spotted fever, babesiosis, recurrent fever, Colorado tick fever, human monocytic ehrlichiosis (HME), human granulocytic ehrlichiosis (HGE), Southern tick-associated rash disease (STARI), tularemia, Tick's palsy, Powassan's encephalitis, Q fever, Crimean-Congo hemorrhagic fever, Citauxzoonosis, scaronodular fever, or tick-borne encephalitis.
[0024]
24. Combined vaccine according to claim 21, characterized in that the second vaccine composition is a vaccine selected from the group consisting of: a tick-borne encephalitis vaccine, a Japanese encephalitis vaccine and a Spotted Fever vaccine of Rocky Mountain.
[0025]
25. Combined vaccine according to any one of claims 21 to 24, characterized in that the second vaccine composition has a seasonal immunization schedule compatible with immunization against infection by Borrelia or Lyme disease.
[0026]
26. Vaccine composition according to claim 20, characterized in that it is to prevent an infection or disease, the infection being by Borrelia and the disease being Lyme.
[0027]
27. Combined vaccine according to any one of claims 21 to 25, characterized in that it is for the prevention of an infection or disease, the infection being by Borrelia and the disease being Lyme.
[0028]
28. Use of a composition as defined in any one of claims 12 to 20, or a combined vaccine as defined in any one of claims 21 to 25, characterized in that it is for the preparation of a medicine.

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CA3095174A1|2018-04-03|2019-10-10|Sanofi|Antigenic ospa polypeptides|

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CN110483624B|2019-08-22|2021-02-09|中国疾病预防控制中心传染病预防控制所|Borrelia garinii OspA protein C-terminal peptide segment and application thereof|

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CN111196842A|2020-01-09|2020-05-26|济南大学|Expression and purification method of non-transmembrane structural domain of outer membrane transport channel protein|

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WO2021205022A1|2020-04-09|2021-10-14|Valneva Austria Gmbh|Improved methods of producing a lipidated protein|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-06| B25C| Requirement related to requested transfer of rights|Owner name: BAXTER INTERNATIONAL INC. (US) , BAXTER HEALTHCARE |

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-06-05| B25A| Requested transfer of rights approved|Owner name: BAXALTA INCORPORATED (US) ; BAXALTA GMBH (CH) |

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-11-17| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-03-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/05/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US33490110P| true| 2010-05-14|2010-05-14|

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US61/334,901|2010-05-14|

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PCT/US2011/036525|WO2011143617A1|2010-05-14|2011-05-13|Chimeric ospa genes, proteins, and methods of use thereof|

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