FUSION PROTEIN, IMMUNOGENIC COMPOSITION, VACCINE, AND, PROCESS TO PRODUCE PERIPLASMATIC EXPRESSION O

专利摘要:
fusion protein, immunogenic composition, vaccine, methods for treating or preventing disease, such as otitis media, pneumonia, an infection or illness associated with h. influenzae and exacerbations of acute chronic obstructive pulmonary disease (aecopd), and, processes for producing preriplasmatic expression of a fusion protein and for preparing a vaccine the present invention relates to compositions comprising haemophilus influenzae e protein and pilin a. more particularly, the present application relates to fusion proteins and immunogenic compositions comprising protein e and pila, vaccines comprising such immunogenic compositions and therapeutic uses thereof.
公开号:BR112013026175B1
申请号:R112013026175-7
申请日:2012-04-12
公开日:2020-05-12
发明作者:Normand Blais；Labbe Steve；Jan Poolman
申请人:Glaxosmithkline Biologicals S.A.；
IPC主号:

专利说明:

FUSION PROTEIN, IMMUNOGENIC COMPOSITION, VACCINE, AND, PROCESS TO PRODUCE PERIPLASMATIC EXPRESSION OF A FUSION PROTEIN [001] This application claims the priority of the patent application of
United States number 61/474779, filed on April 13, 2011, and United States patent application number 61/534012, filed on September 13, 2011.
FIELD OF THE INVENTION [002] The present invention relates to compositions comprising protein E and pilin A DE Haemophilus influenzae (H. influenzae). More particularly, the present application relates to fusion proteins and immunogenic compositions that comprise protein E and pilin A, vaccines that comprise such immunogenic compositions and therapeutic uses thereof.
BACKGROUND OF THE INVENTION [003] Protein E (PE) is an outer membrane lipoprotein with adhesive properties. It plays a role in the adhesion / invasion of non-typable Haemophilus influenzae (NTHi) in epithelial cells. (J. Immunology 183: 2593-2601 (2009); The Journal of Infectious Diseases 199: 522-531 (2009), Microbes and Infection 10: 87-96 (2008)). It is very conserved in both encapsulated Haemophilus influenzae and non-typable H. influenzae, and has a conserved epithelial binding domain. (The Journal of Infectious Diseases 201: 414-419 (2010)). Thirteen different point mutations have been described in different species of Haemophilus when compared to Haemophilus influenzae Rd as a reference strain. Its expression is observed both in the logarithmic growth and in the stationary phase of the bacteria. (WO2007 / 084053).
[004] Protein E is also involved in human complement resistance through binding to vitronectin. (Immunology 183:
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2593-2601 (2009)). PE, by the PKRYARSVRQ YKILNCANYH LTQVR binding domain (SEQ ID NO. 1, which corresponds to amino acids 84-108 of SEQ ID NO. 4), binds to vitronectin, which is an important inhibitor of the terminal complement pathway (J. Immunology 183 : 2593-2601 (2009)).
[005] Pilin A (PilA) is probably the main pilin subunit of H. influenzae type IV pili (Tfp), involved in contraction motility (Infection and Immunity, 73: 1635-1643 (2005)). NTHi PilA is a preserved adhesin and expressed in vivo. It was shown to be involved in NTHi adhesion, colonization and biofilm formation. (Molecular Microbiology 65: 1288-1299 (2007)).
[006] Haemophilus influenzae untypable is an important and common respiratory pathogen that causes otitis media in children and babies. NTHi is, after Streptococcus pneumoniae, the most common cause of acute otitis media in children (J. Immunology 183: 2593-2601 (2009), Pediatrics 113: 1451-1465 (2004)). It is an important cause of sinusitis in children and adults. (Current Infectious Disease Reports 11: 177-182 (2009)). It is associated with an increased risk of exacerbations in chronic obstructive pulmonary disease (COPD) in adults. (Journal of Chronic Obstructive Pulmonary Disease 3: 109-115 (2006)). In addition, H. influenzae causes community-acquired pneumonia in adults and can cause pneumonia in children in developing countries. (Current Infectious Disease Reports 11: 177182 (2009)).
[007] There is a need for NTHi vaccines.
SUMMARY OF THE INVENTION [008] As a first aspect, the present invention provides fusion proteins of formula (I).
(X) _m - (R1) n - A - (Y) o - B - (Z) _p (formula I) where:
X is a signal peptide or MHHHHHH (SEQ ID NO. 2);
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R ₁ is an amino acid;
n is 0, 1, 2, 3, 4, 5 or 6;
A is protein E of Haemophilus influenzae or an immunogenic fragment thereof, or PilA of Haemophilus influenzae or an immunogenic fragment thereof;
Y is selected from the group consisting of GG, SG, SS, GGG and (G) _h where h is 4, 5, 6, 7, 8, 9, or 10;
o is 0 or 1;
B is Haemophilus influenzae PilA or an immunogenic fragment thereof, or Haemophilus influenzae protein E or an immunogenic fragment thereof;
Z is GGHHHHHH (SEQ ID NO. 3); and p is 0 or 1.
[009] As a second aspect, the present invention provides immunogenic compositions comprising fusion proteins of formula (I). The composition may further comprise a pharmaceutically acceptable adjuvant. The composition can comprise an excipient.
[0010] In a third aspect, the present invention provides a method for the treatment or prevention of a condition or disease caused entirely or in part by Haemophilus influenzae. The method comprises administering to a subject who needs therapeutically efficient amount of the fusion protein of formula (I).
[0011] In a fourth aspect, the present invention provides a method for the treatment or prevention of otitis media. The method comprises administering to a subject who needs therapeutically efficient amount of the fusion protein of formula (I).
[0012] In a fifth aspect, the present invention provides a
Petition 870190129809, of 12/09/2019, p. 13/336 / 112 method for the treatment or prevention of exacerbations in chronic obstructive pulmonary disease. The method comprises administering to a subject who needs therapeutically efficient amount of the fusion protein of formula (I).
[0013] In a sixth aspect, the present invention provides a method for treating or preventing pneumonia. The method comprises administering to a subject who needs therapeutically efficient amount of the fusion protein of formula (I).
[0014] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a fusion protein of formula (I) for use in the treatment or prevention of a condition or disease caused entirely or in part by Haemophilus influenzae. The pharmaceutical compositions can additionally comprise a pharmaceutically acceptable adjuvant.
[0015] In an eighth aspect, the present invention provides nucleic acids that encode the proteins of the invention.
[0016] In a ninth aspect, the present invention provides a process for producing nucleic acids of the invention.
[0017] Additional aspects of the present invention are described in the detailed description of the modalities, examples and particular claims to follow.
BRIEF DESCRIPTION OF THE DRAWINGS [0018] Figure 1 shows SDS-PAGE of induced bacterial extracts for the LVL291, LVL268 and LVL269 fusion protein constructs. The insoluble fraction (I), soluble fraction (S) and fraction of culture medium (M) were loaded into LVL291, LVL268 and LVL269 before and after induction (ind).
[0019] Figure 2 shows SDS-PAGE and Western blot related to purification extracts for the LVL291 fusion protein constructs,
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LVL268 and LVL269. Flow fraction (Ft), wash fraction (W) and elution fraction (E) were loaded for purification of LVL291, LVL268 and LVL269. Anti-his tag was used to probe extracts.
[0020] Figure 3 shows SDS-PAGE of induced and purified bacterial extracts for the LVL291 and LVL315 fusion protein constructs. Culture medium fraction (M), soluble fraction (Sol), insoluble fraction (Ins), flow fraction (Ft), wash fraction # 1 (W1), wash fraction # 2 (W2) and elution fraction ( E) were loaded to LVL291 and LVL315.
[0021] Figure 4 shows SDS-PAGE of induced and purified bacterial extracts for the LVL312 fusion protein construct. Culture medium fraction (M), soluble fraction (Sol), insoluble fraction (Ins), flow fraction (Ft), wash fraction # 1 (W1), wash fraction # 2 (W2) and elution fraction ( E) were loaded into LVL312.
[0022] Figure 5 shows SDS-PAGE of induced bacterial extracts (IPTG 1mM and 10μΜ) for the LVL317 fusion protein construct. Extracts before (NI) and after induction (In), soluble fraction (S), insoluble fraction (I).
[0023] Figure 6 shows SDS-PAGE of induced bacterial extracts (IPTG 1mM and 10μΜ) for the LVL318 fusion protein construct. Extracts before (NI) and after induction (In), fraction of culture medium (M), soluble fraction (S), insoluble fraction (I).
[0024] Figure 7 shows the CD spectra of the PE, PilA and PE-PilA fusion proteins.
[0025] Figure 8 shows the combination of the CD spectrum of PE and PilA.
[0026] Figure 9 shows the PilA thermal denaturation curve.
[0027] Figure 10 shows the PE denaturation curve.
[0028] Figure 11 shows the thermal denaturation curve of the PE-PilA fusion protein.
Petition 870190129809, of 12/09/2019, p. 15/336 / 112 [0029] Figure 12 shows the typical SP Sepharose ™ fast flow chromatogram.
[0030] Figure 13 shows the typical Q Sepharose ™ fast flow chromatogram.
[0031] Figure 14 shows SDS-PAGE of samples in process, from the PE-PilA fusion protein purification process.
[0032] Figure 15 shows Western Blot of samples in process from the PE-PilA fusion protein purification process. Blot using rabbit polyclonal anti-PE.
[0033] Figure 16 shows Western Blot of samples in process, from the purification process of the PE-PilA fusion protein. Blot using rabbit polyclonal anti-E.coli (BLR).
[0034] Figure 17 shows the thermal transition of the PE-PilA fusion protein and the PE and PilA proteins. Curves: PilA (1), protein E (Prot E, PE) (2), volume of undiluted purified PE-PilA, 737pg / mL (3), and volume of purified PE-PilA diluted to final 60pg container concentration / ml (4).
[0035] Figure 18 shows the antibody responses against LVL291 PE-PilA fusion protein and against monovalent PE and PilA in the Balb / c mouse model.
[0036] Figure 19 shows the effect of vaccination with PE-PilA fusion protein on bacterial evidence of NTHi strain 86-028NP in the mouse nasopharynx.
[0037] Figure 20 shows the effect of vaccination with PE-PilA fusion protein on bacterial evidence of NTHi strain 3224A in the mouse nasopharynx.
[0038] Figure 21 shows the effect of PilA vaccination on bacterial evidence in the mouse nasopharynx.
[0039] Figure 22 shows the effect of PE vaccination on the evidence
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[0040] Figure 23 shows (a) LVL317 PE-PilA fusion protein that binds vitronectin and (b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.
[0041] Figure 24 shows the inhibition of binding to vitronectin by polyclonal antibodies against the PE-PilA fusion protein.
[0042] Figure 25 shows SDS-PAGE of the soluble fractions of bacterial extracts induced for the LVL291, LVL702, LVL736, LVL737, LVL738, LVL739, LVL739, LVL740, and vector pET26b (negative control) fusion constructs. (a) Experiment 1 (b) Experiment 2 (c) Experiment 3. The PE-PilA fusion protein is indicated by the arrow.
[0043] Figure 26 shows the average percentage of the fusion protein band in the soluble fraction of experiments 1, 2 and 3.
[0044] Figure 27 shows the response to the PE and PilA antibody for LVL317 and LVL735.
[0045] Figure 28 shows the effect of vaccination with LVL735 and LVL317 on bacterial evidence in a mouse model of nasopharyngeal colonization with non-typable Haemophilus influenzae.
DETAILED DESCRIPTION OF THE INVENTION [0046] Unless otherwise explained or defined herein, all technical and scientific terms used herein have the same meaning, in the manner commonly understood by those skilled in the art, to which this disclosure belongs. For example, definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Petition 870190129809, of 12/09/2019, p. 17/336 / 112 [0047] The singular terms "one", "one", "o" and "a" include plural referents, unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and", unless the context clearly indicates otherwise. It is further understood that all base sizes or sizes of amino acid, and all molecular weight or molecular weight values provided for nucleic acids or polypeptides are approximate, and are provided for description. In addition, the numerical limitations provided with respect to the concentrations or levels of a substance, such as an antigen, can be approximated. Thus, where a concentration is indicated to be (for example) approximately 200 pg, the concentration is intended to include values slightly higher or slightly lower than ("about" or "~") 200 pg.
[0048] Although methods and materials similar or equivalent to those described herein can be used in carrying out or testing this disclosure, suitable methods and materials are described below.
[0049] The term "comprises" means "includes". Thus, unless the context may require otherwise, the word "comprises" and variations such as "comprise" and "comprising" will be understood to imply the inclusion of a declared compound or composition (for example, nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not excluding any other compounds, composition, steps, or groups thereof. The abbreviation "e.g." it is derived from the Latin exempli gratia, and is used here to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term “for example”.
[0050] In order to facilitate the review of the various modalities of this disclosure, the following explanations of the terms are provided. Additional terms and explanations are provided in the context of this disclosure.
[0051] A "subject", as used here, is a mammal,
Petition 870190129809, of 12/09/2019, p. 18/336 / 112 including humans, non-human primates and non-primate mammals such as elements of the rodent genus (including, but not limited to, mice and rats), and elements of the order Lagomorpha (including, but not limited to, rabbits).
[0052] As used here, "Protein E", "Protein E", "Prot E" and "PE" mean H. influenzae protein E. Protein E may consist of or comprise the amino acid sequence of SEQ ID NO. 4 (MKKIILTLSL
GLLTACSAQI QKAEQNDVKL
APPTDVRSGY IRLVKNVNYY
IDSESIWVDN QEPQIVHFDA VVNLDKGLYV
YPEPKRYARS
VRQYKILNCA NYHLTQVRTD
FYDEFWGQGL RAAPKKQKKH
TLSLTPDTTL YNAAQIICAN YGEAFSVDKK), as well as strings with at least or exactly 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity, in relation to the full size, with SEQ ID NO. 4. Comparison of 53 Haemophilus influenzae protein E sequences (Table 1, SEQ ID NO. 5 - SEQ ID NO. 57) demonstrated approximately 77% to approximately 100% identity with protein E, as presented in SEQ ID AT THE. 4. For example, in the protein E amino acid sequence, amino acid # 20 can be isoleucine (I) or threonine (T); amino acid # 23 can be alanine (A) or valine (V); amino acid # 24 can be lysine (K) or glutamic acid (E); amino acid # 31 can be alanine (A) or threonine (T); amino acid # 32 can be proline (P) or alanine (A); amino acid # 34 can be threonine (T) or alanine (A); amino acid # 37 can be arginine (R) or glutamine (Q); amino acid # 47 can be valine (V) or alanine (A); amino acid # 57 may be tryptophan (W) or may be absent (-); amino acid # 70 can be alanine (A) or threonine (T); amino acid # 93 can be glutamine (Q) or absent (-); amino acid # 109 can be threonine (T) or isoleucine (I); amino acid # 119 can be glycine (G) or serine (S); amino acid # 153 can be glutamic acid (E) or lysine (K); amino acid # 156 can be serine (S) or leucine (L); amino acid # 160 can be lysine
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10/112 (K) or asparagine (N); amino acid # 161 can be lysine (K), isoleucine (I) or absent (-); amino acids # 162 - # 195 may be missing, or as shown in SEQ ID NO. 15 (with (-) indicating that amino acid # 166 is missing), or as presented in SEQ ID NO. 16; or any combination of these.
[0053] Protein E may consist of or comprise an amino acid sequence that differs from SEQ ID NO. 4 in any one or more amino acids selected from the group consisting of: amino acid # 20, amino acid # 23, amino acid # 24, amino acid # 31, amino acid # 32, amino acid # 34, amino acid # 37, amino acid # 47, amino acid # 57, amino acid # 70, amino acid # 93, amino acid # 109, amino acid # 119, amino acid # 153, amino acid # 156, amino acid # 160, amino acid # 161 and amino acids # 162- # 195, where amino acid # 20 is threonine (T); amino acid # 23 is valine (V); amino acid # 24 is lysine (K); amino acid # 31 is threonine (T); amino acid # 32 is alanine (A); amino acid # 34 is alanine (A); amino acid # 37 is glutamine (Q); amino acid # 47 is alanine (A); amino acid # 57 is absent (-); amino acid # 70 is threonine (T); amino acid # 93 is absent (-); amino acid # 109 is isoleucine (I); amino acid # 119 is serine (S); amino acid # 153 is lysine (K); amino acid # 156 is leucine (L); amino acid # 160 is asparagine (N); amino acid # 161 is lysine (K) or isoleucine (I); or amino acids # 162 - # 195 are as shown in SEQ ID NO. 15 (with (-) indicating that amino acid # 166 is missing), or as presented in SEQ ID NO. 16.
Table 1: Protein E amino acid sequences from 53 strains of
Haemophilus influenzae (SEQ ID NO. 5 - SEQ ID NO. 57). - indicates that the amino acid is missing.
Strain name protein E sequence 3224 ^a MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKTNA
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RdKW20 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDRGLYVYPEPKRYARSVRQYKILNCANYHLTQIRT DFYDEFWGQGLRAAPKKQKTTHKY 86-028NP MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKAHT R2846 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH R2866 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH 3655 MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH PittAA MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKQKAHTNA PittEE MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQID NO.12) PittHH MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH PittlI MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH R3021 MKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVQSGYVRLVKNVNYYIDSES IWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRI DFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKNKKI CT-LISLNFIQLLGCREYSIFLQLLLFYCWHF (SEQ ID NO.15) 22.4-21 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKKIKK ICTLISLNFIQLLGCREYSIFLQLLLFYCWHF (SEQ ID NO.16) 3219C MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO.17) 3185 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHT 3241A MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKAH 038144S1 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGFVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVR TDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFLVDKK (SEQ ID NO.20)
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810956 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKAHT 821246 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRT DFYDEFWGQGLRAAPKKQKTH 840645 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKQKAHT 902550Z19 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSES IWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVR TDFYDEFWGQGLRAAPKKKKHH A840177 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKQKAHT A860514 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPTDVRSGYVRLVKNANYYIDSE SIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQV RTDFYDEFWGQGLRAAPKKQKTTY A950014 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRI DFYDEFWGQGLRAAPKKQKTTKKTHKTKY 306543X4 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH A930105 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH 901905U MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH A920030 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKTNA 3221B MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKTHH 27W116791N MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSES IWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVR TDFYDEFWGQGLRAAPKKKKHH N218 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKKHH N163 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKAHT
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N162 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKAHT N107 MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRT DFYDEFWGQGLRAAPKKQKTTY N91 MKKIILTLSLGLLTACSAQTQKVEQNDVKLTAPADVRSGYVRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQV RTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQ ID NO.38) D211PG MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQII CANYGEAFSVDKK (SEQ ID NO.39) D211PD MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQII CANYGEAFSVDKK (SEQ ID NO.40) D201PG MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHT D201PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH D198PG MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH D198PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH D195PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQSLRATKKKKKKKY D189PG MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSES IWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVR TDFYDEFWGQGLRAAPKKKKHH D189PD MKKIILTLSLGLLTACSAQTQKVEQNDVKLTPPTDVRSGYVRLVKNVNYYIDSES IWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVR TDFYDEFWGQGLRAAPKKKKKHH D129CG MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKTHKA D124PG MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH D124PD MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH
Petition 870190129809, of 12/09/2019, p. 23/336 / 112
D58PG MKKIILTLSLGLLTACSAQTQKAEQNDVKLTPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKTH D33OD MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHT BS433 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDTVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKHH BS432 MKKIILTLSLGLLTACSAQTQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQIRT DFYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SE Q ID NO.54) 1714 MKKIILTLSLGLLTACSAQIQKAKQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKTNA 1128 MKKIILTLSLGLLTACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESI WVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRT DFYDEFWGQGLRAAPKKKKTH BS430 MKKIILTLSLGLLTACSAQIQKAEQNDMKLAPPTDVRSGYIRLVKNVNYYIDSESIVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTD FYDEFWGQGLRAAPKKQKKHTLSLTPDTTLYNAAQIICANYGKAFSVDKK (SEQID NO.57)
[0054] Protein E may be protein E of H. influenzae strain 3224A,
RdKW20, 86-028NP, R2846, R2866, 3655, PittAA, PittEE, PittHH, PittII,
R3021, 22.4-21, 3219C, 3185, 3241A, 038144S1, 810956, 821246, 840645, 902550Z19, A840177, A860514, A950014, 306543X4, A930105, 901905U, A920030, 3221B, N7, 1116, 91, N D211PG, D211PD, D201PG, D201PD, D198PG, D198PD, D195PD, D189PG, D189PD, D129CG, D124PG, D124PD, D58PG, D33OD, BS433, BS432, 1714, 1128 or BS430. Protein E can be protein E in the manner shown in any of SEQ ID NO. 5 - SEQ ID NO. 57.
[0055] Protein E can be a sequence with at least 95% identity, in relation to the full size, with any of SEQ ID NO. 4 - SEQ ID NO. 57. Protein E can be a sequence with at least 95% identity, in relation to the full length, with any of the sequences shown in table 1, SEQ ID NO. 5 - SEQ ID NO. 57.
[0056] Immunogenic protein E fragments comprise
Petition 870190129809, of 12/09/2019, p. 24/336 / 112 immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 4. Immunogenic fragments can elicit antibodies that can bind to SEQ ID NO. 4.
[0057] Immunogenic fragments of protein E may comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of any of SEQ ID NO. 4 - SEQ ID NO. 57. Immunogenic fragments can elicit antibodies that can bind to the full-length sequence, from which the fragment is derived.
[0058] Immunogenic fragments of protein E comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 5 - SEQ ID NO. 57. Immunogenic fragments can elicit antibodies that can bind to the full-length sequence, from which the fragment is derived.
[0059] As used here, “PilA” means H. influenzae pilin A. PilA can consist of or comprise the protein sequence of SEQ ID NO. 58 (MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ), as well as sequences with 80% to 100% identity with SEQ ID NO. 58. For example, PilA can be at least 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO. 58. The full-length comparison of 64 Haemophilus influenzae PilA sequences (Table 2, SEQ ID NO. 58 SEQ ID NO. 121) demonstrated approximately 80% to 100% identity with PilA, as presented in SEQ ID NO. 58. For example, in the amino acid sequence of PilA, amino acid # 6 can be glutamine (Q) or leucine (L); amino acid # 7 can be glutamine (Q) or threonine (T); amino acid # 37 can be glutamine (Q) or lysine (K); amino acid # 44 can be alanine (A) or serine (S); amino acid # 57 can be alanine (A) or serine
Petition 870190129809, of 12/09/2019, p. 25/336 / 112 (S); amino acid # 67 can be asparagine (N) or glycine (G); amino acid # 68 can be glutamic acid (E) or lysine (K); amino acid # 69 can be threonine (T) or proline (P); amino acid # 71 can be lysine (K), asparagine (N), serine (S) or threonine (T); amino acid # 73 can be threonine (T), serine (S) or methionine (M); amino acid # 76 can be lysine (K), serine (S) or asparagine (N); amino acid # 84 can be threonine (T) or lysine (K); amino acid # 86 can be alanine (A) or valine (V); amino acid # 91 can be lysine (K) or alanine (A); amino acid # 94 can be threonine (T), isoleucine (I) or lysine (K); amino acid # 96 can be serine (S) or glutamine (Q); amino acid # 97 can be asparagine (N) or serine (S); amino acid # 99 can be alanine (A) or glycine (G); amino acid # 103 can be alanine (A) or lysine (K); amino acid # 109 can be aspartic acid (D), alanine (A) or threonine (T); amino acid # 110 can be glycine (G), asparagine (N), or arginine (R); amino acid # 112 can be serine (S) or glutamic acid (E); amino acid # 114 can be threonine (T) or isoleucine (I); amino acid # 116 can be threonine (T) or glutamine (Q); amino acid # 118 can be glutamic acid (E), threonine (T), alanine (A), lysine (K) or serine (S); amino acid # 121 can be serine (S) or alanine (A); amino acid # 122 can be alanine (A) or threonine (T); amino acid # 123 can be lysine (K), threonine (T) or alanine (A); amino acid # 128 can be lysine (K) or threonine (T); amino acid # 135 can be aspartic acid (D) or glutamic acid (E); amino acid # 136 can be alanine (A) or threonine (T); amino acid # 145 can be glycine (G) or arginine (R); amino acid # 149 can be glutamine (Q) or lysine (K); or any combination of these.
[0060] Pil A may consist of or comprise an amino acid sequence that differs from SEQ ID NO. 58 in any one or more amino acids selected from the group consisting of amino acid # 6, amino acid # 7, amino acid # 37, amino acid # 44, amino acid # 57, amino acid # 67, amino acid # 68, amino acid # 69, amino acid # 71,
Petition 870190129809, of 12/09/2019, p. 26/336 / 112 amino acid # 73, amino acid # 76, amino acid # 84, amino acid # 86, amino acid # 91, amino acid # 94, amino acid # 96, amino acid # 97, amino acid # 99, amino acid # 103, amino acid # 109, amino acid # 110, amino acid # 112, amino acid # 114, amino acid # 116, amino acid # 118 amino acid, # 121, amino acid # 122, amino acid # 123, amino acid # 128, amino acid # 135, amino acid # 136, amino acid # 145 and amino acid # 149 , where amino acid # 6 is leucine (L); amino acid # 7 is threonine (T); amino acid # 37 is lysine (K); amino acid # 44 is serine (S); amino acid # 57 is serine (S); amino acid # 67 is glycine (G); amino acid # 68 is lysine (K); amino acid # 69 is proline (P); amino acid # 71 is lysine (K), serine (S) or threonine (T); amino acid # 73 is serine (S) or methionine (M); amino acid # 76 is serine (S) or asparagine (N); amino acid # 84 is lysine (K); amino acid # 86 is valine (V); amino acid # 91 is alanine (A); amino acid # 94 is isoleucine (I) or lysine (K); amino acid # 96 is glutamine (Q); amino acid # 97 is serine (S); amino acid # 99 is glycine (G); amino acid # 103 is alanine (A); amino acid # 109 is aspartic acid (D) or threonine (T); amino acid # 110 is glycine (G) or arginine (R); amino acid # 112 is serine (S); amino acid # 114 is threonine (T); amino acid # 116 is threonine (T); amino acid # 118 is glutamic acid (E), alanine (A), lysine (K) or serine (S); amino acid # 121 is serine (S); amino acid # 122 is threonine (T); amino acid # 123 is lysine (K) or alanine (A); amino acid # 128 is lysine (K); amino acid # 135 is glutamic acid (E); amino acid # 136 is threonine (T); amino acid # 145 is arginine (R); amino acid # 149 is lysine (K).
Table 2: Amino acid sequences of pilin A of 64 strains of Haemophilus influenzae (SEQ ID NO. 58 - SEQ ID NO. 121).
Strain name PilA sequence 86-028NP MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.58) NTHi3219C MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTKCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.59)
Petition 870190129809, of 12/09/2019, p. 27/336 / 112
NTHÍ3224A MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.60) NTHi12 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSSCSGGSNGIAADITTAKGYVASVITQSGGITVKGDGTLANME YILQAAGNAAAGVTWTTTGG NTHi44 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANM EYILQATGNAATGVTWTTTCKGD NTHi67 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKS DVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMS YTLTAEGDSAKGVTWTTTKGTTTTGG 1054MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANM EYILQATGNAATGVTWTTTKGTTQTQTGTDQTQTQTDQ 1729MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANM EYILQATGNAATGVTWTTTKGT5 1728MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANM EYILQATGNAATGVTWTTTKGTTQTTQTDQTQTTGT 1885MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSELLQASAPYKA DVELCVYSTNEITNCMGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAAAGVTQTTTTKK 1060MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANM EYILQAKGNATAGVTWTTASK RdKW20 MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANME YILQAKGNAAAGVTWTTTCKGTDASLFPANFCGSVTK (SEQ ID NO.69) 214NP MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANME YILQASGNAATGVTWTTTCKTD 1236MEE MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTSCTGGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANME YILQAKGNAAAGVTWTTTCKGD 1714MEE MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQATGNAATGVTQTTTTK2 1128MEE MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSD VELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEY ILQAKGNATAGVTTTTG R2846 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANM EYILQATGNAATGVTWTTTCKTTG R2866 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTQTTTKK 3655 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANM EYILQAKGNATAGVTWTTFKK PittAA MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQATGNAATGVTQTTTTKK PittGG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQAKGNATAGVTWTTTKK PittII MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTQTTTKK
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R3021 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTQTTTKKT 22.4-21 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKS DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTK (SEQ ID NO.81) 3185A MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.82) 3221B MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.83) 3241A MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.84) 038144S1 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAISELLQASAPYKSD VELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEY ILQAKGNATAGVTWTTTKKTTTKKT 821246 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTEASLFPANFCGSVTQ (SEQ ID NO.86) 840645 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.87) 902550Z19 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSDVELCVYSTGKPSTCSGGSNGIAADITTVKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.88) A840177 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.89) A920030 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANM EYILQATGNAATGVTQTTTKK A950014 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDRMSYTLTAEGDSAKGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.91) 901905U MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.92) A920029 MKLTTQTTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKSD VELCVYSTNETTNCTGGKNGIAADITTAKGYVASVITQSGGITVKGNGTLTNMEY ILQATGNAATGVTWTTTKK A930105 MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG 306543X4 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSSCSGGSNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.95) N218 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDTSLFPANFCGSVTQ (SEQ ID NO.96) N163 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.97) N162 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGNGTLANM EYILQATGNAATGVTQTTTTKK N120 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQAKGNATAGVTWTTTKK
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N107 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQAKGNATAGVTWTTTKK N92 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.101) N91 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQAKGNATAGVTWTTTKK.10 D219PG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.103) D211PG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.104) D211PD MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.105) D204CD MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILXATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.106) D198PG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.107) D198PD MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.108) D195PD MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG D195CD MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG D189PG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.111) D189PD MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTSCTGGKNGIAADITTAKGYVKSVTTSNGAITVAGNGTLDGMSYTLTAEGDSAKGVTWKTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.112) D124PG MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG D124PD MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG D124CG MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTTTG D58PG MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNETTNCTGGKNGIAADITTAKGYVASVKTQSGGITVKGDGTLANM EYILQATGNAATGVTQTTTKK BS433 MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.117) BS432 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANME YILQAKGNATAGVTWTTTKK BS430 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKA DVELCVYSTNEATKCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQASGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.119)
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1714 MKLTTLQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEYILQATGNAATGVTWTTTCKGTDASLFPANFCGSVTQ (SEQ ID NO.120) 1128 MKLTTQQTLKKGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSELLQASAPYKSD VELCVYSTGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANMEY ILQAKGNATAGVTTTTT
[0061] PilA can be PilA of H. influenzae strain NTHÍ3219C, NTHÍ3224A, NTHi12, NTHi44, NTHi67, 1054MEE, 1729MEE, 1728MEE, 1885MEE, 1060MEE, RdKW20, 214NP, 1236MEE, 1714MEE, 1128MEE, R282828 3655, PittAA, PittGG, PittII, R3021, 22.4-21, 3185A, 3221B, 3241A, 038144S1, 821246, 840645, 902550Z19, A840177, A920030, A950014, 901905U, A920029, A930105, N, 306543, N16, 3065 N107, N92, N91, D219PG, D211PG, D211PD, D204CD, D198PG, D198PD, D195PD, D195CD, D189PG, D189PD, D124PG, D124PD, D124CG, D58PG, BS433, BS432, amino acids of H. influenzae strain D204CD is presented in SEQ ID NO. 106, where X at position # 116 is both glutamine (Q) and leucine (L); ambiguously how the amino acid at position # 116 can be explained by technical resolution of the second nucleotide encoding amino acid # 116, explaining the PilA sequence for the D204CD strain. PilA can be PilA as presented in any of SEQ ID NO. 58 - SEQ ID NO. 121.
[0062] PilA can be a sequence with at least 95% identity, in relation to the full size, with any of SEQ ID NO. 58 - SEQ ID NO. 121 (as shown in table 2).
[0063] Immunogenic fragments of PilA comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58 - SEQ ID NO. 121. Immunogenic fragments can elicit antibodies that can bind to the full-length sequence, from which the fragment is derived.
[0064] For example, immunogenic fragments of PilA comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50
Petition 870190129809, of 12/09/2019, p. 31/336 / 112 contiguous amino acids of SEQ ID NO. 58. Immunogenic fragments can elicit antibodies that can bind to SEQ ID NO. 58.
[0065] The identity between the polypeptides can be calculated by several algorithms. For example, the Needle program, from the EMBOSS package (free software; EMBOSS: The European Molecular Biology Open Software Suite (2000). Trends in Genetics 16 (6): 276—277) and the Gap program from the GCG® package (Accelrys Inc .) can be used. This Gap program is an implementation of the Needleman-Wunsch algorithm described in: Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The BLOSUM62 classification matrix was used, and the opening and gap extension penalties were 8 and 2, respectively.
[0066] Observing the obtained alignment, the identical residues between two compared sequences can be observed. A percentage of identity can be computed (1) by calculating the number of identities divided by the size of the alignment, multiplied by 100 (for example, for the analysis of the Needle program), (2) calculating the number of identities divided by the size of the largest sequence , multiplied by 100, (3) calculating the number of identities divided by the size of the shortest sequence, multiplied by 100, or (4) calculating the number of identities divided by the number of aligned residues, multiplied by 100 (a residue is aligned if it is facing each other) (for example, for the analysis of the Gap program).
[0067] As used herein, "adjuvant" means a compound or substance that, when administered to a subject in conjunction with a vaccine, immunotherapeutic, or other composition containing antigen or immunogen, increases or improves the subject's immune response to the antigen or immunogen administered (compared to the immune response that can be obtained in the absence of adjuvant). This is to be distinguished from “adjuvant therapy”, defined by the National Cancer Institute of the United States Institutes of
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Health, in the context of cancer treatment as the additional treatment provided after primary treatment, to decrease the risk of cancer occurring. [0068] Conservative substitutions are well known and are generally established as the standard classification matrices in sequence alignment programs. These programs include PAM250 (Dayhoft MO et al., (1978), “A model of evolutionary changes in proteins”, In “Atlas of Protein sequence and structure” 5 (3) MO Dayhoft (ed.), 345-352), National Biomedical Research Foundation, Washington, and Blosum 62 (Steven Henikoft and Jorja G. Henikoft (1992), “Amino acid substitution matrices from protein blocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 1091510919. The invention additionally provides fusion proteins of formula (I) containing conservative amino acid substitutions. For example, the fusion proteins of formula (I) may contain a conservative substitution for any H. influenzae PE or PilA amino acid, as described in any of the sequences presented here (for example, any PE sequence shown in SEQ ID NO. 4 - SEQ ID NO. 57 and / or any PilA sequence shown in SEQ ID NO. 58 - SEQ ID NO. 121).
[0069] As used herein, "signal peptide" refers to a small polypeptide (less than 60 amino acids, for example, 3 to 60 amino acids) present in the precursor proteins (typically at the N-terminus), and which is typically absent from of mature protein. The signal peptide (sp) is typically rich in hydrophobic amino acids. The signal peptide directs the transport and / or secretion of the translated protein across the membrane. The signal peptides can also be called targeting signals, transit peptides, location signals, or signal sequences. For example, the signal sequence can be a cotranslational or post-translational signal peptide.
[0070] A heterologous signal peptide can be cleaved from a
Petition 870190129809, of 12/09/2019, p. 33/336 / 112 fusion protein construct by signal peptide peptidases, during or after protein transport or secretion. For example, the signal peptide peptidase is signal peptide I peptidase. A "heterologous" signal peptide is one that is not associated with proteins as they exist in nature.
[0071] As used herein, "treatment" means preventing the occurrence of symptoms of the condition or disease in a subject, preventing recurrence of symptoms of the condition or disease in a subject, delaying the recurrence of symptoms of the condition or disease in a subject, the decrease in the severity or frequency of symptoms of the condition or disease in a subject, slowing or eliminating the progression of the condition and the partial or total elimination of symptoms of the disease or condition in a subject.
[0072] As used herein, “optionally” means that the event (s) subsequently described may or may not occur, and includes both the event (s) that occur and the events that do not occur.
[0073] The pathogenesis of the disease caused by NTHi begins with nasopharyngeal colonization. The mechanisms for adhering to and staying in the nasopharynx microenvironment for a long time are considered 'virulence determinants' for NTHi.
[0074] The importance of NTHi that is able to adhere to the surfaces of the epithelial mucosa of a human host is reflected in the multiplicity of adhesins expressed by NTHi (Otitis media is one of the main causes of morbidity in 80% of all young children 3 years old (Expert Rev. Vaccines 5: 517-534 (2006)). Over 90% of children develop otitis media before the age of 7 (Current Opinion in Investigational Drugs 4: 953958 (2003)). In 2000 , there were 16 million visits to doctors' offices in relation to otitis media in the United States, and approximately 13 million antibacterial prescriptions were performed (Pediatrics 113: 14511465 (2004)). In European countries, reported rates of acute otitis media vary between 0.125 and 1.24 per child per year. (Expert Review of Vaccines
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8: 1479-1500 (2009)). Otitis media is an expensive infection and the most common reason children receive antibiotics. (Current Infectious Disease Reports 11: 177-182 (2009)). Bacteria are responsible for approximately 70% of cases of acute otitis media, with Streptococcus pneumoniae, not typeable Haemophilus influenzae, and Moraxella catarrhalis predominating as the etiological agents (Expert Review of Vaccines 5: 517-534 (2006)). A subset of children experience recurrent and chronic otitis media, and these otitis-prone children have prolonged middle ear effusions that are associated with hearing loss and delayed speech and language development. (Current Infectious Disease Reports 11: 177-182 (2009)).
[0075] After the introduction of the pneumococcal heptavalent vaccine in many countries, some studies have shown a significant increase in the proportion of acute otitis media caused by H. influenzae, with H. influenzae becoming the predominant pathogen. (Pediatric Infectious Disease Journal 23: 824-828; Pediatric Infectious Disease Journal 23: 829-833 (2004)).
[0076] Since otitis media is a multifactorial disease, the possibility of preventing otitis media using a vaccination strategy has been questioned. (Current Infectious Disease Reports 11: 177-182 (2009)). However, the results of a study suggest that it is possible for an antigen to induce at least partial protection against non-typable H. influenzae. (Lancet 367: 740-748 (2006)). One approach to developing vaccine antigens is to use antigenically conserved regions of genetically heterogeneous but abundantly expressed surface molecules. Another approach is to identify surface proteins that demonstrate conservation of sequence or functional epitope. A third consideration for a vaccine antigen may be to select an antigen that is expressed during infection and colonization in a human host. Murphy (Curr. Infect. Disease Reports 11: 177-182 (2009) states that, despite the existence of
Petition 870190129809, of 12/09/2019, p. 35/336 / 112 potential candidate non-typeable H. influenzae antigens, it cannot be predicted exactly whether the candidate antigen will be efficient. (Current Infectious Disease Reports 11: 177-182 (2009)). Some of the proteins described as potential vaccine antigens are: Haemophilus adhesin protein (Hap), high molecular weight proteins (HMW) 1 and 2, H. influnzae adhesion (Hia), D15 protein, HtrA heat shock protein, protein surface P2, lipoprotein D, peptides derived from fimbrin P5, outer membrane protein P4, outer membrane protein (OMP) 26, protein P6, protein E, pili type IV, lipo-oligosaccharide and phosphoryl choline. (Current Infectious Disease Reports 11: 177-182 (2009); Expert Review of Vaccines 5: 517-534 (2006)).
[0077] The chinchilla model is a robust and validated animal model of otitis media and its prevention (Expert Review of Vaccines 8: 1063-1082 (2009)). Although the chinchilla model can mimic the natural course of human infection, others suggest that the results in the chinchilla model may vary from laboratory to laboratory. (Current Opinion in Investigational Drugs 4: 953-958 (2003)).
[0078] Several other rodents have also been used for the induction of otitis media and are summarized in Vaccine 26: 1501-1524 (2008). The murine animal model is often studied in otitis media research.
[0079] The presence of bactericidal antibody is associated with the protection of otitis media due to non-typable H. influenzae. (Current Opinion in Infectious Disease 16: 129-134 (2003)). However, an immune response does not have to be bactericidal to be effective against NTHi. Antibodies that react only with NTHi surface adhesins can reduce or eliminate otitis media in chinchillas. (Current Opinion in Investigational Drugs 4: 953-958 (2003)).
[0080] Chronic obstructive pulmonary disease is a chronic inflammatory disease of the lungs and a major cause of morbidity and
Petition 870190129809, of 12/09/2019, p. 36/336 / 112 mortality worldwide. Approximately one in 20 deaths in 2005 in the USA had COPD as the underlying cause. (Drugs and Aging 26: 985999 (2009)). In 2020 COPD is projected to increase to the fifth leading cause of disability-adjusted life years, chronic diseases that cause disability, and the third most important cause of mortality (Lancet 349: 1498-1504 (1997)).
[0081] The COPD course is characterized by the progressive worsening of airflow limitation and a decline in lung function. COPD can be complicated by frequent and recurrent exacerbations (AE), which are associated with high health care costs and high morbidity. (Proceedings of the American Thoracic Society 4: 554-564 (2007)). One study suggests that approximately 50% of acute exacerbations of symptoms in COPD are caused by non-typable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Pseudomonas aeruginosa. (Drugs and Aging 26: 985-999 (2009)). H. influenzae is found in 20-30% of COPD exacerbations; Streptococcus pneumoniae in 10-15% of COPD exacerbations; and Moraxella catarrhalis in 10-15% of COPD exacerbations. (New England Journal of Medicine 359: 2355-2365 (2008)). Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were shown to be the main pathogens in acute bronchitis exacerbations in Hong Kong, South Korea and the Philippines, while Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. constitute a large proportion of pathogens in other Asian countries / regions, including Indonesia, Thailand, Malaysia and Taiwan (Respirology, (2011) 16, 532-539; doi: 10.1111 / j.1440.1843.2011.01943.x). In Bangladesh, 20% of COPD patients exhibited positive sputum culture for Pseudomonas, Klebsiella, Streptococcus pneumoniae and Haemophilus influenzae, while 65% of AECOPD patients exhibited positive cultures for Pseudomonas, Klebsiella, Acinetobacter, Enterobacter,
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Moraxella catarrhalis and combinations of these. (Mymensingh Medical Journal 19: 576-585 (2010)). However, it is suggested that the two most important measurements to prevent COPD exacerbation are active immunizations and chronic maintenance of pharmacotherapy. (Proceedings of the American Thoracic Society 4: 554-564 (2007)).
[0082] There is a need for efficient vaccines against NTHi. Using antigens that can act at different stages in the pathogenesis, one can improve the efficiency of a vaccine. The inventors have observed that PilA and PE can be beneficially present in the immunogenic compositions of the invention as fusion proteins.
[0083] The present invention relates to fusion proteins of formula (I).
(X) _m - (R1) n - A - (Y) o - B - (Z) _p (formula I) where:
X is a signal peptide or MHHHHHH (SEQ ID NO. 2);
m is 0 or 1;
R ₁ is an amino acid;
n is 0, 1, 2, 3, 4, 5 or 6;
A is protein E of Haemophilus influenzae or an immunogenic fragment thereof, or PilA of Haemophilus influenzae or an immunogenic fragment thereof;
Y is selected from the group consisting of GG, SG, SS and (G) _h where h is 4, 5, 6, 7, 8, 9, or 10;
o is 0 or 1;
B is Haemophilus influenzae PilA or an immunogenic fragment thereof, or Haemophilus influenzae protein E or an immunogenic fragment thereof;
Z is GGHHHHHH (SEQ ID NO: 3); and p is 0 or 1.
Petition 870190129809, of 12/09/2019, p. 38/336 / 112 [0084] In one embodiment, the fusion proteins of formula (I) are defined, where X is selected from the group consisting of the signal sequence of CcmH (cytochrome c membrane protein H), DsbA (isomerase periplasmic protein disulfide I), DsbB (membrane protein B disulfide), FlgI (flagellar peptideoglycan ring protein), FocC (chaperone F1c protein), MalE (maltose transporter subunit), NadA (quinolinate A subunit) synthase), NikA (ABC nickel transporter component A), NspA (Neisseria surface protein A), Omp26 (outer membrane protein 26), OmpA (outer membrane protein A), OspA (outer surface protein A), pelB (lyase B pectate), PhoA (bacterial alkaline phosphatase), PhtD (pneumococcal histidine triad protein D), PhtE (pneumococcal histidine triad protein E), SfmC (periplasmic pilin chaperone), Sip1 (immunogenic surface protein) , TolB (component B of the envelope complex c elular Tol-Pal), TorA (A subunit of the trimethylamine N-oxide reductase system), TorT (periplasmic protein T of the trimethylamine N-oxide reductase system) and Yral (probable periplasmic chaperone pilin); or any subgroup thereof. In one embodiment, X is a cotranslational signal peptide or post-translational signal peptide. In one embodiment, X is the signal sequence of FlgI (flgl sp). In another particular embodiment, X is the signal sequence of pelB (pelB sp). In another embodiment, X is a post-translational signal peptide. In another modality, X is selected from the group consisting of the signal sequence of FlgI, NadA and pelB.
[0085] In one embodiment, the fusion proteins of formula (I) are defined, where m is 1. In another embodiment, m is 0.
[0086] In a particular embodiment, R1 and n are defined wherein (R _{1) n} is 1 to 6 amino acids enriched in small amino acids, hydrophilic groups. Hydrophilic amino acids include glutamic acid (E), aspartic acid (D) and asparagine (N).
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30/112 [0087] In one embodiment, the fusion proteins of formula (I) are defined, where n is selected from the group consisting of 0, 1, 2 and 6. In a particular embodiment, R1 and n are defined, where (R1) n is selected from the group consisting of D, E, ATNDDD (SEQ ID NO. 178) and MD, or any subgroup thereof.
[0088] In a particular mode, n is selected from the group consisting of 1, 2 and 6. In a particular mode, n is 0.
[0089] In one embodiment, the fusion proteins of formula (I) are defined, where A is protein E of H. influenzae. In another embodiment, the fusion proteins of formula (I) are defined, where A is protein E encoded by an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subgroup of SEQ ID NO. 5 to SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are defined, where A is protein E, where protein E is approximately 75% to 100% identical to the amino acid protein E sequence shown in SEQ ID NO: 4. In another embodiment, A is protein E, where protein E is approximately 90% to 100% identical to the protein E sequence of amino acids shown in
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SEQ ID NO: 4. In another embodiment, A is protein E, where protein E is at least 95% identical to the protein E sequence of amino acids shown in SEQ ID NO: 4. In additional mode, A is protein E, in that protein E is at least 95% identical to protein E shown in any of SEQ ID NO. 4 - SEQ ID NO. 57. In a particular embodiment, A is protein E with the amino acid sequence shown in SEQ ID NO. 4.
[0090] In another embodiment, the fusion proteins of formula (I) are defined, in which A is an immunogenic fragment of protein E of H. influenzae. In another embodiment, A is an immunogenic fragment of protein E, in which protein E has an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subgroup of SEQ ID NO. 4 to SEQ ID NO. 57. In another embodiment, A is an immunogenic fragment of protein E, in which protein E is approximately 75% to 100% identical to the amino acid sequence shown in SEQ ID NO: 4. In another embodiment, A is an immunogenic fragment of protein And, where protein E is approximately 90% to 100% identical to SEQ ID NO. 4. In an additional modality, A is
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32/112 an immunogenic fragment of protein E, in which protein E is at least 95% identical to any of SEQ ID NO. 4 - SEQ ID NO. 57. More specifically, in one embodiment, A is an immunogenic fragment of protein E, where protein E is 93% to 100% identical to SEQ ID NO. 124. In a particular embodiment, A is an immunogenic fragment of protein E in which protein E is SEQ ID NO. 4.
[0091] In another embodiment, A is an immunogenic fragment of H. influenzae E protein selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, A is an immunogenic fragment of H. influenzae E protein selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). In an additional embodiment, A is an immunogenic fragment of H. influenzae protein E selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). More specifically, in one embodiment, A is SEQ ID NO. 124, amino acids 19160 of SEQ ID NO. 4. In an additional embodiment, A is SEQ ID NO.125, amino acids 20-160 of SEQ ID NO. 5. In another modality, A is a fragment
Petition 870190129809, of 12/09/2019, p. 42/336 / 112 immunogenic H. influenzae protein E selected from the group consisting of amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180).
Protein E - SEQ ID NO. 4
MKKIILTLSL GLLTACSAQI QKAEQNDVKL
APPTDVRSGY
VVNLDKGLYV
FYDEFWGQGL
IRLVKNVNYY
YPEPKRYARS
RAAPKKQKKH
IDSESIWVDN
VRQYKILNCA
TLSLTPDTTL
QEPQIVHFDA
NYHLTQVRTD
YNAAQIICAN
YGEAFSVDKK
Amino acids 17-160 of protein E of SEQ ID NO. 4 - SEQ ID NO. 122
SAQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTYNA
Amino acids 18-160 of protein E of SEQ ID NO. 4 - SEQ ID NO. 123
AQI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTYYNA
Amino acids 19-160 of protein E of SEQ ID NO. 4 - SEQ ID NO. 124
QI QKAEQNDVKL APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH
TLSLTPDTTL YNAAQIICAN YGEAFSVDKK
Amino acids 20-160 of protein E of SEQ ID NO. 4 - SEQ ID NO. 125
I QKAEQNDVKL APPTDVRSGY IRLVKNVNYY
IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK
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Amino acids 22-160 of protein E of SEQ ID NO. 4 - SEQ ID NO. 126
KAEQNDVKL APPTDVRSGY
IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK EQ IDA proteins 23-1 4 - SEQ ID NO. 179
AEQNDVKL APPTDVRSGY
IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK NO. 4 - SEQ ID NO. 180
EQNDVKL APPTDVRSGY IRLVKNVNYYIDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN (YGEAQIICAN). In another embodiment, the fusion proteins of formula (I) are defined, where A is H. influenzae PilA with an amino acid sequence selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101,
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SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO.105,
SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO.109,
SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO.113,
SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO.117,
SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subgroup of SEQ ID NO. 58 to SEQ ID NO. 121. In another embodiment, A is PilA where PilA is approximately 80% to 100% identical to SEQ ID NO. 58. In another embodiment, A is PilA where PilA is at least 95% identical to any of SEQ ID NO. 58 - SEQ ID NO. 121. In a particular embodiment, A is PilA of SEQ ID NO. 58.
[0093] In another embodiment, the fusion proteins of formula (I) are defined, in which A is an immunogenic fragment of H. influenzae PilA. In another embodiment, A is an immunogenic fragment of PilA in which PilA is approximately 80% to 100% identical to SEQ ID NO. 58. For example, A is an immunogenic fragment of PilA in which PilA has an amino acid sequence selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103,
SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO.107,
SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO.111,
SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO.115,
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SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any SEQ ID NO subgroup. 58 to SEQ ID NO. 121. In an additional embodiment, A is an immunogenic fragment of PilA in which PilA is at least 95% identical to any of SEQ ID NO. 58 - SEQ ID NO. 121. In a particular embodiment, A is an immunogenic fragment of H. influenzae strain 86-028NP PilA where PilA is SEQ ID NO. 58.
H. influenzae PilA strain 86-028NP - SEQ ID NO. 58
MKLTTQQTLK KGFTLIELMI VIAIIAILAT IAIPSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATog NO% [ANOTHER MODE] [% MODE] 127. More specifically, in one embodiment, A is SEQ ID NO. 127, a fragment consisting of amino acids 40-149 of SEQ ID NO. 58.
Amino acids 40-149 of PilA of H. influenzae strain 86-028NP - SEQ ID NO. 127.
T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNGIA ADITTAKGYV KSVTTSNGAI TVKGDGTLAN MEYILQATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ [0095] In another modality, it is a DNA fragment that consists of an immunogenic gene fragment. 58 - SEQ ID NO. 121. In an additional embodiment, A is an immunogenic fragment at least 95% identical to amino acids 40-149 of any of SEQ ID NO. 58 - SEQ ID NO. 121.
[0096] In one embodiment, the fusion proteins of formula (I) are defined, where Y is selected from the group consisting of GG, SG and SS. In another embodiment, the fusion proteins of formula (I) are defined, in
Petition 870190129809, of 12/09/2019, p. 46/336 / 112 that Y is GG or SG. In a particular embodiment, Y is GG.
[0097] In one embodiment, the fusion proteins of formula (I) are defined, where o is 1. In another embodiment, o is 0.
[0098] In one embodiment, the fusion proteins of formula (I) are defined, where B is H. influenzae PilA or an immunogenic fragment of H. influenzae PilA, when A is H. influenzae protein E or a immunogenic fragment of H. influenzae E protein. For example, B is PilA of H. influenzae strain 86-028NP. In another modality, B is PilA of H. influenzae with an amino acid sequence selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105,
SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO.109,
SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO.113,
SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO.117,
SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subgroup of SEQ ID NO. 58 to SEQ ID NO. 121. In another modality, B is PilA in which PilA is approximately 80% to 100% identical to SEQ ID NO. 58. In another modality, B is PilA in which PilA is at least 95% identical to any of SEQ ID NO. 58 - SEQ ID NO. 121. In a particular embodiment, B is PilA of SEQ ID NO. 58.
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38/112 [0099] In another modality, B is PilA in which PilA is at least 95% identical to any of SEQ ID NO. 58 - SEQ ID NO. 121 and A is PE where PE is at least 95% identical to any of SEQ ID NO. 4 - SEQ ID NO.
57.
[00100] In another embodiment, the fusion proteins of formula (I) are defined, where B is an immunogenic fragment of H. influenzae PilA when A is an immunogenic fragment of H. influenzae E protein. For example, B is an immunogenic fragment of the H. influenzae strain 86028NP PilA. In another embodiment, B is an immunogenic fragment of PilA in which PilA is approximately 80% to 100% identical to SEQ ID NO: 58. In another embodiment, B is an immunogenic fragment of PilA in which PilA has an amino acid selected from the group that consists of SEQ ID NO.
58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subgroup of SEQ ID NO. 58 to SEQ ID NO. 121. In another embodiment, B is an immunogenic fragment of PilA in which PilA is at least 95% identical to any of SEQ ID NO.
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- SEQ ID NO. 121. In a particular embodiment, B is an immunogenic fragment of H. influenzae PilA in which PilA has the amino acid sequence shown in SEQ ID NO. 58. In another embodiment, B is an immunogenic fragment of PilA that consists of amino acids 40-149 of any of SEQ ID NO. 58 - SEQ ID NO. 121. More specifically, in one embodiment, B is the PilA fragment as presented in SEQ ID NO. 127. In an additional embodiment, B is an immunogenic fragment at least 95% identical to amino acids 40-149 of any of SEQ ID NO. 58 - SEQ ID NO. 121.
[00101] In a particular embodiment, B is the PilA fragment as presented in SEQ ID NO. 127 and A is an immunogenic fragment of protein E selected from the group consisting of SEQ ID NO. 122, SEQ ID NO. 124, SEQ ID NO. 125 and SEQ ID NO. 126. More particularly, B is the PilA fragment as presented in SEQ ID NO. 127 and A is the protein fragment E as presented in SEQ ID NO. 124, amino acids 19-160 of protein E of SEQ ID NO. 4. In another embodiment, B is the PilA fragment as presented in SEQ ID NO. 127 and A is the protein fragment E as presented in SEQ ID NO. 125.
[00102] In another embodiment, B is an immunogenic fragment of PilA in which PilA is at least 95% identical to any of SEQ ID NO. 58 SEQ ID NO. 121 and A is an immunogenic fragment of PE in which PE is at least 95% identical to any of SEQ ID NO. 4 - SEQ ID NO. 57.
[00103] In another embodiment, the fusion proteins of formula (I) are defined, where B is protein E of H. influenzae when A is PilA of H. influenzae. For example, B is protein E with an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ
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ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subgroup of SEQ ID NO. 4 to SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are defined, where B is protein E, where protein E is approximately 75% to 100% identical to the amino acid protein E sequence shown in SEQ ID NO: 4. In another embodiment, B is protein E, where protein E is approximately 90% to 100% identical to the protein E sequence of amino acids shown in SEQ ID NO: 4. For example, B is protein E, where protein E it is at least 95% identical to protein E in the manner shown in SEQ ID NO. 4. In another embodiment, B is protein E in which protein E is at least 95% identical to any of SEQ ID NO. 4 - SEQ ID NO. 57. In a particular embodiment, B is protein E with the amino acid sequence shown in SEQ ID NO. 4.
[00104] In another embodiment, the fusion proteins of formula (I) are defined, where B is an immunogenic fragment of H. influenzae protein E when A is an immunogenic fragment of H. influenzae PilA. For example, B is an immunogenic fragment of protein E, in which protein E has an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21,
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SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subgroup of SEQ ID NO. 4 to SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are defined, in which B is an immunogenic fragment of protein E, in which protein E is approximately 75% to 100% identical to the protein E sequence of amino acids shown in SEQ ID NO. 4. In another embodiment, B is an immunogenic fragment of protein E in which protein E is approximately 90% to 100% identical to the protein E sequence of amino acids shown in SEQ ID NO: 4. In a particular embodiment, B is a fragment immunogenicity of protein E with the amino acid sequence shown in SEQ ID NO. 4. In an additional embodiment, B is an immunogenic fragment of protein E, where protein E is at least 95% identical to any of SEQ ID NO. 4 - SEQ ID NO. 57.
[00105] In another embodiment, B is a fragment of H. influenzae E protein selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22160 of SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, B is an immunogenic fragment of H. influenzae E protein selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19
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160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). More specifically, in one embodiment, B is the protein fragment E as presented in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4.
[00106] In a particular embodiment B is an immunogenic fragment of protein E in the manner presented in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4 when A is an immunogenic fragment of PilA as presented in SEQ ID NO. 127.
[00107] In one embodiment, the fusion proteins of formula (I) are defined, where p is 0. In another embodiment, the fusion proteins of formula (I) are defined, where p is 1.
[00108] In one embodiment, the fusion protein of formula (I) is selected from the group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO.146,
SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO.184,
SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO.192,
SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO.200,
SEQ ID NO. 202 and SEQ ID NO. 204; or any subgroup of these. In another embodiment, the fusion protein of formula (I) is approximately 95% identical to that of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO.140,
SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO.148,
SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO.186,
SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO.194,
SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO. 204.
[00109] The fusion proteins of formula (I) are used as
Petition 870190129809, of 12/09/2019, p. 52/336 / 112 immunogens in subjects such as mammals, particularly humans. In particular, the fusion proteins of the formula (I) are used to induce an immune response against H. influenzae in subjects, particularly humans. More specifically, the fusion proteins of formula (I) are used in the treatment or prevention of otitis media, and / or AECOPD, and / or pneumonia. [00110] The present invention relates to immunogenic compositions comprising H. influenzae E protein (or an immunogenic fragment thereof) and H. influenzae PilA (or an immunogenic fragment thereof), and immunogenic compositions comprising protein fusion proteins And H. influenzae (or an immunogenic fragment thereof) and PilA of H. influenzae (or an immunogenic fragment thereof). The present invention also relates to vaccines that comprise such immunogenic compositions and their therapeutic uses.
[00111] In one embodiment, the immunogenic compositions comprise H. influenzae protein E (or an immunogenic fragment thereof) and H. influenzae PilA (or an immunogenic fragment thereof). Protein E can be SEQ ID NO. 4 or a protein E sequence of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 4.
[00112] The immunogenic fragment of protein E can be SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126, or a sequence with at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126. The immunogenic protein E fragment can be SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO. 179 or SEQ ID NO. 180 or a sequence with at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID
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AT THE. 126, SEQ ID NO. 179 or SEQ ID NO. 180. Amino acid differences have been described in protein E of several species of Haemophilus when compared to protein E of Haemophilus influenzae Rd as a reference strain. Microbes & Infection (Corrected for “Identification of a novel Haemophilus influenzae protein important for adhesion to epithelia cells” [Microbes Infect. 10 (2008) 87-97], available on the internet on July 6, 2010, “Article in print”) , provides a sequence for protein E of H. influenzae strain 772. WO2002 / 28889 provides a sequence for protein E of H. influenzae strain 12085.
[00113] Protein E contains an epithelial cell binding region (PKRYARSVRQ YKILNCANYH LTQVR, SEQ ID NO. 128) that has been reported to be conserved among more than 100 encapsulated H. influenzae NTHi clinical isolates, and analyzed culture collection strains (Singh et al, J. Infect. Dis. 201 (3): 414-9 (2010)). Singh et al. reported that protein E was highly conserved in both H. influenzae NTHi and encapsulated (96.9% 100% identity without the signal peptide). In one embodiment, the protein fragment E comprises the binding region of SEQ ID NO. 128 (PKRYARSVRQ YKILNCANYH LTQVR).
[00114] PilA is a conserved adhesin expressed in vivo. The full-size comparison of 64 Haemophilus influenzae PilA sequences demonstrated approximately 80% to 100% identity.
[00115] In another embodiment, the immunogenic composition comprises a fusion protein in the manner defined by formula (I).
[00116] In one embodiment, the present immunogenic compositions can be administered with other H. influenzae antigens. For example, PE and PilA, or the fusion protein of formula (I) can be administered with H. influenzae protein D. Protein D can be in the manner described in WO91 / 18926. In another embodiment, the immunogenic composition can include the fusion protein of formula (I) and protein D of H. influenzae.
Petition 870190129809, of 12/09/2019, p. 54/336 / 112 [00117] In another embodiment, the immunogenic compositions of the invention can be administered with additional antigens from other bacterial species also known to cause otitis media, AECOPD or pneumonia.
[00118] The amount of the immunogenic composition that is required to achieve the desired therapeutic or biological effect will depend on numerous factors such as the intended use, the means of administration, the recipient and the type and severity of the condition being treated, and will be finally in the description of the attending physician or veterinarian In general, a typical dose for the treatment of a condition caused entirely or in part by H. influenzae in a human, for example, can be expected to be in the range of about 0.003 mg to about 0.090 mg. More specifically, a typical dose for the treatment of a condition caused entirely or partially by H. influenzae in a human can be in the range of about 0.01 mg to about 0.03 mg of fusion protein. The immunogenic composition may contain additional antigens; a typical dose for the treatment of a condition caused entirely or partially by H. influenzae in a human can be in the range of about 0.01 mg to about 0.03 mg for each additional antigen. This dose can be administered as a single dose. Several single doses can also be administered. For example, single separate doses can be administered as separate starting doses in the first year of life, or as separate booster doses delivered at regular intervals (for example, every 1, 5 or 10 years).
[00119] Formulations comprising the immunogenic compositions of the invention can be adapted for administration by an appropriate route, for example, by the intramuscular, sublingual, transcutaneous, intradermal or intranasal route. Such formulations can be prepared by any method known in the art.
[00120] The immunogenic compositions of the present invention can
Petition 870190129809, of 12/09/2019, p. 55/336 / 112 additionally comprise an adjuvant. When the term "adjuvant" is used in this specification, it refers to a substance that is administered together with the immunogenic composition to enhance the patient's immune response to the immunogenic component of the composition.
[00121] Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but they can also be a calcium, magnesium, iron or zinc salt, or they can be an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derived saccharides, or polyphosphazenes. In one embodiment, the fusion protein, PE or PilA can be adsorbed to aluminum phosphate. In another embodiment, the fusion protein, PE or PilA can be adsorbed to aluminum hydroxide. In a third embodiment, alum can be used as an adjunct.
[00122] Suitable adjuvant systems that promote a predominantly Th1 response include: non-toxic derivatives of lipid A, monophosphoryl lipid A (MPL) or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (for its preparation, see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably monophosphoryl lipid A 3-of-O-acylated, both together with an aluminum salt (e.g., aluminum phosphate or aluminum hydroxide) and an oil-in-water emulsion. In such combinations, the antigen and 3DMPL are contained in the same particulate structures, allowing a more efficient distribution of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further improve the immunogenicity of an alum adsorbed antigen (Thoelen et al. Vaccine (1998) 16: 708-14; EP 689454-B1).
[00123] AS01 is an adjuvant system that contains MPL (3-0desacil-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21) Antigenics, New York, NY, USA) and liposomes. AS01B is a system
Petition 870190129809, of 12/09/2019, p. 56/336 / 112 adjuvant containing MPL, QS21 and liposomes (50 g of MPL and 50 g of QS21). AS01E is an adjuvant system that contains MPL, QS21 and liposomes (25 g of MPL and 25 g of QS21). In one embodiment, the immunogenic composition or vaccine comprises AS01. In another embodiment, the immunogenic composition or vaccine comprises AS01B or AS01E. In a particular embodiment, the immunogenic composition or vaccine comprises AS01E.
[00124] AS03 is an adjuvant system that contains α-Tocopherol and squalene in an oil / water (o / w) emulsion. AS03A is an adjuvant system that contains α-Tocopherol and squalene in an o / a emulsion (11.86 mg tocopherol). AS03B is an adjuvant system containing α-Tocopherol and squalene in an o / w emulsion (5.93 mg tocopherol). AS03 _C is an adjuvant system that contains α-Tocopherol and squalene in an o / w emulsion (2.97 mg of tocopherol). In one embodiment, the immunogenic composition or vaccine comprises AS03.
[00125] AS04 is an adjuvant system containing MPL (50 qg MPL) adsorbed on an aluminum salt (500 qg Al ³⁺ ). In one embodiment, the immunogenic composition or vaccine comprises AS04.
[00126] A system involving the use of QS21 and 3D-MPL is disclosed in WO 94/00153. A composition in which QS21 is finished with cholesterol is disclosed in WO 96/33739. An additional adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. In one embodiment, the immunogenic composition further comprises a saponin, which may be QS21. The formulation can also comprise an oil-in-water and tocopherol emulsion (WO 95/17210). Unmethylated CpG containing oligonucleotides (WO 96/02555) and other immunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are also preferred inducers of a TH1 response, and are suitable for use in the present invention.
Petition 870190129809, of 12/09/2019, p. 57/336 / 112 [00127] Additional adjuvants are those selected from the group of metal salts, oil-in-water emulsions, Toll-type receptor agonists, (in particular Toll-type 2 receptor agonists, Toll-type 3 receptor agonists, agonists of the Toll type 4 receptor, Toll type 7 receptor agonist, Toll type 8 receptor agonist and Toll type 9 receptor agonist), saponins or combinations thereof.
[00128] The present invention provides a process for preparing an immunogenic composition comprising combining a fusion protein of formula (I) with an adjuvant.
[00129] The present invention further provides a vaccine containing an immunogenic composition of the invention and a pharmaceutically acceptable excipient.
[00130] Possible excipients include arginine, pluronic acid and / or polysorbate. In a preferred embodiment, polysorbate 80 (for example, TWEEN ^® 80) is used. In an additional embodiment, a final concentration of about 0.03% to about 0.06% is used. Specifically, a final concentration of about 0.03%, 0.04%, 0.05% or 0.06% of polysorbate 80 (w / v) can be used.
[00131] The present invention provides a process for preparing an immunogenic composition or vaccine comprising combining a fusion protein of formula (I) with a pharmaceutically acceptable excipient.
[00132] The present invention also provides nucleic acids that encode the proteins of the invention. The term "nucleic acid" refers to a polymeric form of nucleotides. The nucleotides can be ribonucleotides, deoxyribonucleotides, or form modified from both ribonucleotides and deoxyribonucleotides. The term includes single and double forms of DNA. Nucleic acids are preferably substantially free of other nucleic acids.
[00133] The present invention provides a process of producing acids
Petition 870190129809, of 12/09/2019, p. 58/336 / 112 nucleic compounds of the invention. Nucleic acids of the invention can be prepared by methods known to those skilled in the art. For example, the nucleic acids of the invention can be synthesized in part or in whole. Nucleic acids can be prepared by digesting longer amino acids or by joining shorter amino acids.
[00134] The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.
[00135] In the examples, the following terms have the determined meaning:
6xhis = six histidines;
xg = centrifugal force (number in gravities)
ATP = adenosine triphosphate;
BCA = bicinconinic acid;
BSA = bovine serum albumin;
° C = degrees Celsius;
CaCl2 = calcium chloride;
CV = column volume;
DNA = deoxyribonucleic acid;
DSC = differential calorimetry scanning;
DTT = dithiothreitol;
dNTP = deoxynucleoside triphosphate;
EDTA = ethylenediaminetetraacetic acid;
FT = complete flow;
HCl = hydrogen chloride;
His = his = histidine;
HEPES = 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid; IMAC = affinity chromoathography with immobilized metal; IPTG = isopropyl P-D-1-thiogalactopyranoside;
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KCl = potassium chloride;
K2HPO4 = dibasic potassium phosphate;
KH2PO4 = monobasic potassium phosphate;
LDS = dodecyl and lithium sulfate;
L = liter;
MES = 2- (A-morpholino) ethanesulfonic acid;
MgCl2 = magnesium chloride;
ml = milliliter;
RPM = revolutions per minute;
min = minute;
mM = millimolar;
pL = microliter;
NaCl = sodium chloride;
Na ₂ HPO ₄ = dibasic sodium phosphate;
NaH2PO4 = monobasic sodium phosphate;
ng = nanogram;
nm = nanometer;
O / N = all night;
PBS = phosphate buffered saline;
PCR = polymerase chain reaction;
SB = sample buffer;
sec = second; w / v = weight / volume.
EXAMPLES
Example 1: Fusion proteins [00136] Fusion proteins were produced with different signal peptides and amino acid linker sequences. These fusion proteins allowed the secretion of both protein E and PilA (or fragments thereof) without being restricted to a single bacterial strain. The protein
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[00137] The following table describes prepared fusion protein constructs.
Table 3: Fusion protein constructs containing PilA and protein E.
ConstrutoID S-have mine I ............................................. ...... C-Terminal LVL312 Ogl sp AND FragmentPilA(A.A .: 40-149 SEQ IDAT THE. 58, SEQID NO. 127) GG ProtE fragment(A.A .: 18 to 160 of SEQ ID NO. 4, SEQ IDNO.123) GGHH HHHH A.A. 1 19 21 130 133 275 276 283 LVL291 pelB sp ProtE fragment (A.A .: 19 to 160 of SEQ ID NO. 4, SEQ ID NO. 124) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) GGHHH HHH A.A. 1 22 23 164 167 276 277 284 LVL268 pelB sp D ProtE fragment(A.A .: 20 to 160 of SEQ ID NO. 4, SEQ ID NO. 125) GG FragmentPilA(A.A .: 40-149 SEQ IDAT THE. 58, SEQID NO. 127) GGHH HHHH A.A. 1 22 24 164 167 276 277 284 LVL269 nadA sp ATNDDD ProtE fragment(A.A .: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) GG FragmentPilA(A.A .: 40-149 SEQ IDAT THE. 58, SEQID NO. 127) GGHH HHHH A. A. 1 23 24-29 30 168 171 280 281 288 LVL270 MHHHHHH ProtE fragment(A.A .: 17 to 160 of SEQ ID NO. 4, SEQ ID NO. 122) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A.A. 17 8 151 154 263
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LVL315 pelB IVsp ID ProtE fragment(A.A .: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) GG FP(dΓ *I ragment • ilAA.A .: 40-149 and SEQ ID JO. 58, SEQ D NO. 127) GGHH HHHH 122 25 163 166 26 276 283 LVL317 pelB sp ProtE fragment(A.A .: 19 to 160 of SEQ ID NO. 4, SEQ ID NO. 124) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 164 167 276 LVL318 pelB sp MD ProtE fragment (A.A .: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) A. A. 1 22 25 163 166 275 LVL702 pelB sp ProtE fragment (A.A .: 20 to 160 of SEQ ID NO. 4, SEQ ID NO. 125) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) GGHHHH HH A. A. 1 22 23 163 166 275 283 LVL736 pelB sp ProtE fragment (A.A .: 17 to 160 of SEQ ID NO. 4, SEQ ID NO. 122) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) GGHHHH HH A.A. 1 22 23 166 169 278 286 LVL737 pelB sp ProtE fragment (A.A .: 18 to 160 of SEQ ID NO. 4, SEQ ID NO.123) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) GGHHHH HH A.A. 1 22 23 165 168 277 285 LVL738 pelB sp ProtE fragment (A.A .: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) GGHHHH HH A. A. 1 22 23 161 164 273 281 LVL739 pelB sp ragmen to ’rotEA.A .: 23 to 160 l and SEQ ID SO. 4, SEQ ID SO. 179) GG FPGdsI ilA ragmentoV.A .: 40-14 and SEQ II [Q. 58, SE (D NO. 127) GGHHHHHH) A.A. 1 22 23 160 163 272 280 LVL740 pelB sp ragmen to ’rotEA.A .: 24 to 160 l and SEQ ID SO. 4, SEQ ID SO. 180) GG FPGdsI ilA ragmentoV.A .: 40-14 and SEQ II [Q. 58, SEt. D NO. 127) GGHHHHHH) A. A. 1 22 23 159 162 271 279 LVL735 pelB sp ProtE fragment(A.A .: 20 to 160 of SEQ ID NO. 4, SEQ ID NO. 125) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127)
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A. A. 1 22 23 163 166 275 LVL778 pelB sp ProtE fragment(A.A .: 17 to 160 of SEQ ID NO. 4, SEQ ID NO. 122) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 166 169 278 LVL779 pelB sp ProtE fragment(A.A .: 18 to 160 of SEQ ID NO. 4, SEQ ID NO.123) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 165 168 277 LVL780 pelB sp ProtE fragment(A.A .: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 161 164 273 LVL781 pelB sp ProtE fragment(A.A .: 23 to 160 of SEQ ID NO. 4, SEQ ID NO. 179) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 160 163 272 LVL782 pelB sp ProtE fragment(A.A .: 24 to 160 of SEQ ID NO. 4, SEQ ID NO. 180) GG PilA fragment (A.A .: 40-149 of SEQ ID NO. 58, SEQ IDAT THE. 127) A. A. 1 22 23 159 162 271
sp = signal peptide; A.A. = amino acid
The DNA and amino acid sequences for each of the signal peptides and plasmids listed in table 3 are shown below.
SIGNAL SEQUENCES:
pelB signal peptide (DNA) - SEQ ID NO. 129: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggcc pelB signal peptide (Amino acid) - SEQ ID NO. 130: MKYLLPTAAA GLLLLAAQPA MA signal peptide Figi (DNA) - SEQ ID NO. 131: atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggct signal peptide Flgl (Amino Acid) - SEQ ID NO. 132: MIKFLSALIL LLVTTAAQA NadA signal peptide (DNA) - SEQ ID NO. 133: atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcg cactggca
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MKHFPSKVLT TAILATFCSG ALA
SEQUENCES OF THE FUSION PROTEIN CONSTRUCTION:
[00138] The simple underlined portion of the amino acid sequences is PilA from Haemophilus influenzae strain 86-028NP. The bold underlined position of the amino acid sequences was derived from Haemophilus influenza strain 772 protein E.
LVL312 (DNA) - SEQ ID NO. 135:
atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggctgagact aaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagc acaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatg taaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatatt ttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttc cagcaaatttttgcggaagtgtcacacaaggcggcgcgcagattcagaaggctgaacaaaatgatgtgaagct ggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatc gatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgttt atcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagt acgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgtt aagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgat aaaaaaggcggccaccaccaccaccaccactaa
LVL312 (protein): (flgI sp) (E) (PilA aa 40-149) (GG) (ProtE aa 18160) (GGHHHHHH) - SEQ ID NO. 136
MIKFLSALIL LLVTTAAQAE TKKAAVSELL
QASAPYKADV ELCVYSTNET TNCTGGKNGI AADITTAKGY VKSVTTSNGA ITVKGDGTLA NMEYILQATG NAATGVTWTT TCKGTDASLF PANFCGSVTQ GGAQIQKAEQ NDVKLAPPTD VRSGYIRLVK NVNYYIDSES IWVDNQEPQI VHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEF WGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF
Petition 870190129809, of 12/09/2019, p. 64/336 / 112
SVDKKGGHHH HHH
LVL291 (DNA) - SEQ ID NO. 137:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggata tatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaatt gtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttc gtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc atctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaaca aactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaa tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggt caagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggt aatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcg gaagtgtcacacaaggcggccaccaccaccaccaccactaa
LVL291 (Protein) (pelB sp) (ProtE aa 19-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 138
MKYLLPTAAA GLLLLAAQPA MAQIQKAEQN
DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF
CGSVTQGGHH HHHH
LVL268 (DNA) - SEQ ID NO. 139:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgatattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatat atacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattg tacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcg
Petition 870190129809, of 12/09/2019, p. 65/336/112 tcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacag ggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgct gctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggta tctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaa actgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaac aagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggta atgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcgg aagtgtcacacaaggcggccaccaccaccaccaccac
LVL268 (protein): (pelB sp) (D) (ProtE aa 20-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 140:
MKYLLPTAAA GLLLLAAQPA MADIQKAEQN
DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF
CGSVTQGGHH HHHH
LVL269 (DNA) - SEQ ID NO. 141:
atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcg cactggcagccacaaacgacgacgataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtac gaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaaga gccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgc acgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaat tttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacg ctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaa agcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaat gaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaat cagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaa
Petition 870190129809, of 12/09/2019, p. 66/336 / 112 gctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagca aatttttgcggaagtgtcacacaaggcggccaccaccaccaccacacacta
LVL269 (protein): (nadA sp) (ATNDDD) (ProtE aa 22-160) (GG) (PilA aa 40149) (GGHHHHHH) - SEQ ID NO.142
MKHFPSKVLT TAILATFCSG ALAATNDDDK
AEQNDVKLAP PTDVRSGYIR LVKNVNYYID SESIWVDNQE PQIVHFDAVV NLDKGLYVYP EPKRYARSVR QYKILNCANY HLTQVRTDFY DEFWGQGLRA APKKQKKHTL SLTPDTTLYN AAQIICANYG EAFSVDKKGG TKKAAVSELL QASAPYKADV ELCVYSTNET TNCTGGKNGI AADITTAKGY VKSVTTSNGA ITVKGDGTLA NMEYILQATG NAATGVTWTT TCKGTDASLF
PANFCGSVTQ GGHHHHHH
LVL270 (DNA) - SEQ ID NO. 143:
atgcaccaccaccaccaccacagcgcgcagattcagaaggctgaacaaaatgatgtga agctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtg aatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgta tgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactc aagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacat acgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttca gttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctg atgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagata taaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggca cattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaa aggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaataa
LVL270 (protein): (MHHHHHH) (ProtE aa 17-160) (GG) (PilA aa40-149) SEQ ID NO. 144:
MHHHHHHSAQ IQKAEQNDVK LAPPTDVRSG
YIRLVKNVNY YIDSESIWVD NQEPQIVHFD AVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRT DFYDEFWGQG
Petition 870190129809, of 12/09/2019, p. 67/336 / 112
LRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK
KGGTKKAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK
NGIAADITTA KGYVKSVTTS NGAITVKGDG TLANMEYILQ
ATGNAATGVT WTTTCKGTDA SLFPANFCGS VTQ
LVL315 (DNA) - SEQ ID NO. 145:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatata cgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtac attttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtca gtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgct cagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatct gaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaac tgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaa gcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaat gctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaa gtgtcacacaaggcggccaccaccaccaccaccactaa
LVL315 (protein): (pelB sp) (MD) (ProtE aa 22-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 146:
MKYLLPTAAA GLLLLAAQPA MAMDKAEQND
VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC
GSVTQGGHHH HHH
LVL317 (DNA) - SEQ ID NO. 147:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc
Petition 870190129809, of 12/09/2019, p. 68/336/112 gatggcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggata tatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaatt gtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttc gtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggt atctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaaca aactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaa caagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggt aatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcg gaagtgtcacacaataa
LVL317 (protein): (pelB sp) (ProtE aa 19-160) (GG) (PilA aa40-149) - SEQ ID NO. 148:
MKYLLPTAAA GLLLLAAQPA MAQIQKAEQN
DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF
CGSVTQ
LVL318 (DNA) - SEQ ID NO. 149:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccatggataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatata cgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtac attttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtca gtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgct cagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatct
Petition 870190129809, of 12/09/2019, p. 69/336/112 gaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaac tgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaa gcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaat gctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaa gtgtcacacaataa
LVL318 (protein): (pelB sp) (MD) (ProtE aa 22-160) (GG) (PilA aa40-149) SEQ ID NO. 150:
MKYLLPTAAA GLLLLAAQPA MAMDKAEQND
VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ
LVL702 (DNA) - SEQ ID NO. 181: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatata cgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtac attttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtca gtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgct cagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatct gaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaac tgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaa gcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaat gctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaa gtgtcacacaaggcggccaccaccaccaccaccac
LVL702 (protein): (pelB sp) (ProtE aa 20-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 182:
Petition 870190129809, of 12/09/2019, p. 70/336 / 112
MKYLLPTAAA GLLLLAAQPA MAIQKAEQND
VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC
GSVTQGGHHH HHH
LVL736 (DNA) - SEQ ID NO. 183:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaag cggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagcca caaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgtt ctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttgg ggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgcttta taatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagca gcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaa caacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagt gacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagcta caggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaattt ttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL736 (protein): (pelB sp) (ProtE aa 17-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 184:
MKYLLPTAAA GLLLLAAQPA MASAQIQKAE
QNDVKLAPPT DVRSGYIRLV KNVNYYIDSE SIWVDNQEPQ IVHFDAVVNL DKGLYVYPEP KRYARSVRQY KILNCANYHL TQVRTDFYDE FWGQGLRAAP KKQKKHTLSL TPDTTLYNAA QIICANYGEA FSVDKKGGTK KAAVSELLQA SAPYKADVEL CVYSTNETTN CTGGKNGIAA DITTAKGYVK SVTTSNGAIT
Petition 870190129809, of 12/09/2019, p. 71/336 / 112
VKGDGTLANM EYILQATGNA ATGVTWTTTC KGTDASLFPA NFCGSVTQGG HHHHHH
LVL737 (DNA) - SEQ ID NO. 185:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcg gatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccaca aattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttct gttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttat aatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagca gcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaa caacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagt gacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagcta caggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaattt ttgcggaagtgtcacacaaggcggccaccaccaccaccaccac
LVL737 (protein): (pelB sp) (ProtE aa 18-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 186:
MKYLLPTAAA GLLLLAAQPA MAAQIQKAEQ
NDVKLAPPTD VRSGYIRLVK NVNYYIDSES IWVDNQEPQI VHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEF WGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF SVDKKGGTKK AAVSELLQAS APYKADVELC VYSTNETTNC TGGKNGIAAD ITTAKGYVKS VTTSNGAITV KGDGTLANME YILQATGNAA TGVTWTTTCK GTDASLFPAN
FCGSVTQGGH HHHHH
LVL738 (DNA) - SEQ ID NO. 187:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgttggatatatatgatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatatat
Petition 870190129809, of 12/09/2019, p. 72/336/112 atgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtata agatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgc gggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcaga ttatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatt actgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtac gggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaac ggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgc aacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtc acacaaggcggccaccaccaccaccaccac
LVL738 (protein): (pelB sp) (ProtE aa 22-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 188:
MKYLLPTAAA GLLLLAAQPA MAKAEQNDVK
LAPPTDVRSG YIRLVKNVNY YIDSESIWVD NQEPQIVHFD AVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRT DFYDEFWGQG LRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK KGGTKKAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK NGIAADITTA KGYVKSVTTS NGAITVKGDG TLANMEYILQ ATGNAATGVT WTTTCKGTDA SLFPANFCGS
VTQGGHHHHHH
LVL739 (DNA) - SEQ ID NO. 189:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggta aagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatg cagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataaga tcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcggg cagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattat ttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattact gcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgg gtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacgg
Petition 870190129809, of 12/09/2019, p. 73/336 / 112 tgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaac aggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagacaactttgcgacacaggggacacacaggggacac
LVL739 (protein): (pelB sp) (ProtE aa 23-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 190:
MKYLLPTAAA GLLLLAAQPA MAAEQNDVKL
APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN YGEAFSVDKK GGTKKAAVSE LLQASAPYKA DVELCVYSTN ETTNCTGGKN GIAADITTAK GYVKSVTTSN GAITVKGDGT LANMEYILQA TGNAATGVTW TTTCKGTDAS LFPANFCGSV
TQGGHHHHHH
LVL740 (DNA) - SEQ ID NO. 191:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaag aatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagt ggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttg aattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagc acctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtgg aaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgca ataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggt gtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaag gcggccaccaccaccaccaccac
LVL740 (protein): (pelB sp) (ProtE aa 24-160) (GG) (PilA aa40149) (GGHHHHHH) - SEQ ID NO. 192:
MKYLLPTAAA GLLLLAAQPA MAEQNDVKLA
Petition 870190129809, of 12/09/2019, p. 74/336 / 112
PPTDVRSGYI RLVKNVNYYI DSESIWVDNQ EPQIVHFDAV VNLDKGLYVY PEPKRYARSV RQYKILNCAN YHLTQVRTDF YDEFWGQGLR AAPKKQKKHT LSLTPDTTLY NAAQIICANY GEAFSVDKKG GTKKAAVSEL LQASAPYKAD VELCVYSTNE TTNCTGGKNG IAADITTAKG YVKSVTTSNG AITVKGDGTL ANMEYILQAT GNAATGVTWT TTCKGTDASL FPANFCGSVT
QGGHHHHHH
LVL735 (DNA) - SEQ ID NO. 193:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatata cgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtac attttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtca gtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgct cagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatct gaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaac tgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaa gcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaat gctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaa gtgtcacacaa
LVL735 (protein): (pelB sp) (ProtE aa 20-160) (GG) (PilA aa40-149) - SEQ
ID NO. 194:
MKYLLPTAAA GLLLLAAQPA MAIQKAEQND
VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ
Petition 870190129809, of 12/09/2019, p. 75/336 / 112
LVL778 (DNA) - SEQ ID NO. 195: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccagcgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaag cggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagcca caaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgtt ctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttgg ggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgcttta taatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagca gcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaa caacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagt gacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagcta caggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaattt ttgcggaagtgtcacacaa
LVL778 (protein): (pelB sp) (ProtE aa 17-160) (GG) (PilA aa40-149) - SEQ ID NO. 196:
MKYLLPTAAA GLLLLAAQPA MASAQIQKAE QNDVKLAPPT DVRSGYIRLV KNVNYYIDSE SIWVDNQEPQ
IVHFDAVVNL DKGLYVYPEP KRYARSVRQY KILNCANYHL TQVRTDFYDE FWGQGLRAAP KKQKKHTLSL TPDTTLYNAA QIICANYGEA FSVDKKGGTK KAAVSELLQA SAPYKADVEL CVYSTNETTN CTGGKNGIAA DITTAKGYVK SVTTSNGAIT VKGDGTLANM EYILQATGNA ATGVTWTTTC KGTDASLFPA
NFCGSVTQ
LVL779 (DNA) - SEQ ID NO. 197:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgcccagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcg gatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccaca aattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttct gttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg
Petition 870190129809, of 12/09/2019, p. 76/336/112 gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttat aatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagca gcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaa caacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagt gacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagcta caggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaattt ttgcggaagtgtcacacaa
LVL779 (protein): (pelB sp) (ProtE aa 18-160) (GG) (PilA aa40-149) - SEQ
ID NO. 198:
MKYLLPTAAA GLLLLAAQPA MAAQIQKAEQ
NDVKLAPPTD VRSGYIRLVK NVNYYIDSES IWVDNQEPQI VHFDAVVNLD KGLYVYPEPK RYARSVRQYK ILNCANYHLT QVRTDFYDEF WGQGLRAAPK KQKKHTLSLT PDTTLYNAAQ IICANYGEAF SVDKKGGTKK AAVSELLQAS APYKADVELC VYSTNETTNC TGGKNGIAAD ITTAKGYVKS VTTSNGAITV KGDGTLANME YILQATGNAA TGVTWTTTCK GTDASLFPAN
FCGSVTQ
LVL780 (DNA) - SEQ ID NO. 199:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttg gtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttg atgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtata agatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgc gggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcaga ttatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatt actgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtac gggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaac ggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgc aacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtc
Petition 870190129809, of 12/09/2019, p. 77/336 / 112 acacaa
LVL780 (protein): (pelB sp) (ProtE aa 22-160) (GG) (PilA aa40-149) - SEQ
ID NO. 200:
MKYLLPTAAA GLLLLAAQPA MAKAEQNDVK
LAPPTDVRSG YIRLVKNVNY YIDSESIWVD NQEPQIVHFD AVVNLDKGLY VYPEPKRYAR SVRQYKILNC ANYHLTQVRT DFYDEFWGQG LRAAPKKQKK HTLSLTPDTT LYNAAQIICA NYGEAFSVDK KGGTKKAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK NGIAADITTA KGYVKSVTTS NGAITVKGDG
TLANMEYILQ ATGNAATGVT WTTTCKGTDA SLFPANFCGS VTQ
LVL781 (DNA) - SEQ ID NO. 201:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggta aagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatg cagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataaga tcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcggg cagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattat ttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattact gcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgg gtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacgg tgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaac aggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcaca hunting
LVL781 (protein): (pelB sp) (ProtE aa 23-160) (GG) (PilA aa40-149) - SEQ
ID NO. 202:
MKYLLPTAAA GLLLLAAQPA MAAEQNDVKL
APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN
Petition 870190129809, of 12/09/2019, p. 78/336 / 112
YGEAFSVDKK GGTKKAAVSE LLQASAPYKA DVELCVYSTN
ETTNCTGGKN GIAADITTAK GYVKSVTTSN GAITVKGDGT
LANMEYILQA TGNAATGVTW TTTCKGTDAS LFPANFCGSV TQ LVL782 (DNA) - SEQ ID NO. 203:
atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggc gatggccgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaag aatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagt ggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttg aattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagc acctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtgg aaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgca ataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggt gtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaa LVL782 (protein): (pelB sp) (protein aa 24-160) (GG) (PilA aa40-149) - SEQ ID NO. 204:
MKYLLPTAAA GLLLLAAQPA MAEQNDVKLA PPTDVRSGYI RLVKNVNYYI DSESIWVDNQ EPQIVHFDAV
VNLDKGLYVY PEPKRYARSV RQYKILNCAN YHLTQVRTDF YDEFWGQGLR AAPKKQKKHT LSLTPDTTLY NAAQIICANY GEAFSVDKKG GTKKAAVSEL LQASAPYKAD VELCVYSTNE TTNCTGGKNG IAADITTAKG YVKSVTTSNG AITVKGDGTL
ANMEYILQAT GNAATGVTWT TTCKGTDASL FPANFCGSVT Q [00139] The full length sequence for PE and PilA, from which the previous sequences were obtained, are presented in SEQ ID NO. 4 (PE) and SEQ ID NO. 58 (PilA), respectively.
Example 2: Construction and transformation of vector [00140] Oligonucleotides primers to amplify PE from
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70/112
H. influenzae strain 772 were designed based on the sequence of H. influenzae strain Hi Rd. The 5 'primer oligonucleotide sequence contains a nucleotide difference compared to the NTHi 772 sequence, introducing a different amino acid at position 24 when compared to the sequence genome of NTHi 772 currently reported. Amino acid # 24 in the fusion protein constructs is E (glutamic acid) instead of K (lysine), as seen in NTHi 772.
DNA sequence for PE of H. influenzae cepa Rd. - SEQ ID NO. 151 atgaaaaaaattattttaacattatcacttgggttacttaccgcttgttctgctcaaatccaaa aggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaat gtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgctgtggt gaatttagataggggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagattttgaatt gtgcaaattatcatttaactcaaatacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacct aaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcaaa ttatggtaaagcattttcagttgataaaaaataa
Protein sequence for PE of H. influenzae cepa Rd. - SEQ ID NO. 152
GLLTACSAQI QKAEQNDVKL
IDSESIWVDN QEPQIVHFDA
VRQYKILNCA NYHLTQIRTD
TLSLTPDTTL YNAAQIICAN
MKKIILTLSL
APPTDVRSGY IRLVKNVNYY VVNLDRGLYV YPEPKRYARS FYDEFWGQGL RAAPKKQKKH YGKAFSVDKK
DNA sequence for PE of H. influenzae strain 772 (as presented in: Microbes & Infection, corrected for “Identification of a novel Haemophilus influenzae protein important for adhesion to epithelium cells” [Microbes Infect. 10 (2008) 87-97] , available on July 6, 2010, “Printable article”)) - SEQ ID NO. 153 atgaaaaaaattattttaacattatcacttgggttacttactgcctgttctgctcaaatccaaa aggctaaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtagatgatgatgggggggggggggg
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71/112 tgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaa ttgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcac ctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcg aactatggtgaagcattttcagttgataaaaaa
Protein sequence for PE of H. influenzae strain 772 (as presented in: Microbes & Infection, corrected for “Identification of a novel Haemophilus influenzae protein important for adhesion to epithelium cells” [Microbes Infect. 10 (2008) 87-97] , available on July 6, 2010, “Article in print”)) - SEQ ID NO. 154
MKKIILTLSL GLLTACSAQI QKAKQNDVKL
APPTDVRSGY IRLVKNVNYY IDSESIWVDN QEPQIVHFDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYHLTQVRTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTL YNAAQIICAN
YGEAFSVDKK
Vector construction:
[00141] To generate LVL312, LVL291, LVL268, LVL269, LVL270, LVL702, LVL735, LVL778, LVL779, LVL780, LVL781 and LVL782, a preparation of the polymerase chain reaction (was performed) subsequently exemplified): 36.6 pL deionized water, 5 pL of 10X buffer # 1, 5 pL of 2mM dNTPs, 2 pL 25 mM MgCl ₂ , 0.4 pL of primer oligonucleotide # 1 (50 pM), 0.4 pL of primer # 2 oligonucleotide (50 pM), 0.5 pL of template (100 ng / pL) and 0.4 pL of KOD HiFi DNA polymerase 2.5 units / pL (NOVAGEN ^® ) was formulated. The polymerase chain reaction involved 25 cycles of 15 seconds of denaturation at 98 ° C, 2 seconds for annealing at 55 ° C and 20 seconds of oligonucleotide primer extension at 72 ° C. PCR products were purified using QIAQUICK® PCR purification kit (QIAGEN®). This product was used under conditions recommended by the supplier which were: the addition of buffer PB 5 volumes,
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72/112 supplied in the QIAQUICK® PCR purification kit, in 1 volume of the PCR preparation. The PCR preparation with PB buffer was subsequently vortexed. A QIAQUICK ^® column was placed in a 2 mL collection tube. To bind the DNA of the PCR preparation to the column, the mixed sample was applied to the QIAQUICK ® column and centrifuged for 30-60 seconds at 14,000 RPM. The flow was discarded and the QIAQUICK ^® column was placed back in the same tube. To wash the ligated DNA, 0.75 ml of PE buffer provided in the PCR purification kit QIAQUICK ^® PCR QIAQUICK ^® was added to the column, and the column was centrifuged for 30-60 seconds at 14,000 RPM. The flow was discarded and the QIAQUICK ^® column was collected again in the same tube. The QIAQUICK ^® column was centrifuged again in the 2 mL tube for 1 minute to remove the residual wash buffer. Each QIAQUICK ^® column was placed in a clean 1.5 mL microcentrifuge tube. To elute the DNA, 33 pL of water was added in the center of the QIAQUICK ® membrane and the column was centrifuged for 1 minute at 14,000 RPM. Restriction enzymes and related buffer were obtained from New England BioLabs. For example, approximately 5 pL of pET26b vector (100 ng / pL), 2 pL of NE 2 buffer (New England Biolabs, 1X NEBuffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9 at 25 ° C), 1 pL of Ndel (20,000 units / mL), 1 pL of Hindlll (20,000 units / mL) and 11 pL of deionized water were mixed and incubated for two hours at 37 ° C for digestion of DNA. From this point on, a second purification step was performed using the QIAQUICK ® PCR purification kit (QIAGEN®), with the procedure described previously.
[00142] The ligation was performed using Quick T4 DNA ligase and New England BioLabs Quick ligation reaction buffer. For example, around 10 ng of vector and 30 ng of insert in 10 pL of deionized water were mixed with 10 pL of Quick 2X binding reaction buffer (New
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England Biolabs, 132 mM Tris-HCl, 20 mM MgCl ₂ , ₂ mM dithiothreitol, 2 mM ATP, 15% polyethylene glycol, pH 7.6 at 25 ° C) and 1 gL of Quick T4 DNA ligase (New England Biolabs). The enzymatic reaction was incubated for 5 minutes at room temperature before transformation.
[00143] To generate LVL315, LVL317, LVL318, LVL736, LVL737, LVL738, LVL739 and LVL740, a PCR preparation of the following components was performed: 40 gL of deionized water, 5 gL of 10X buffer reaction, 1 gL of mixture mixture dNTPs, 1 gL of primer oligonucleotide # 1 (10 gM), 1 gL of primer oligonucleotide # 2 (10 gM), 1 gL of template (25 ng / gL) and 1 gL of PfuUltra High-Fidelity DNA polymerase 2.5 units / gL (QuikChange II Site-directed mutagenesis kit, Agilent Technologies, Stratagene Division) was formulated. The polymerase chain reaction involved a denaturation cycle at 95 ° C for 30 seconds, 18 cycles of 30 seconds of denaturation at 95 ° C, 1 minute for annealing at 55 ° C, and 5 minutes and 30 seconds of extension of primer oligonucleotide at 68 ° C. The PCR products were digested using 1 gL of Dpnl restriction enzyme at 37 ° C for one hour before transformation.
[00144] A detailed list of PCR primer oligonucleotide sequences used for amplifications is illustrated in Table 4.
[00145] To generate pRIT16711, the PE gene fragment encoding amino acids 22 to 160 of SEQ ID NO. 4, which excludes the sequence encoding its corresponding secretion signal, was amplified by PCR from the genomic DNA of the NTHi 772 strain. The oligonucleotide primers for amplification were designed based on the available sequence of the Hi Rd strain (at this time, the sequence 772 was not known). The 5 'primer oligonucleotide sequence contains a mutation compared to the NTHi 772 sequence (sequence as currently available), which introduces a different amino acid in the PE coding sequence at position 24, glutamic acid (E) instead of lysine (K) . After PCR amplification, the insert
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74/112 was cloned into the expression vector pET-26 (+) (NOVAGEN®) using the restriction sites BamHI and XhoI.
[00146] To generate pRIT16671, a DNA fragment that encodes a PilA gene fragment (amino acids 40 to 149 of SEQ ID NO. 58, SEQ ID NO. 127), which excludes its main peptide as well as a portion of the alpha helix predicted hydrophobic, was amplified from the genomic DNA of NTHi strain 86-028NP and cloned into the expression vector pET15. The vector pRIT16790 (which contains amino acids 40 to 149 of NTHi strain 86-028NP) was used as a template to generate the vector pRIT16671. The PilA gene fragment was amplified by PCR using the vector pRIT16790 and MDES PILA-3 and MDES PILA-4 primer oligonucleotides. The PilA fragment was cloned into the expression vector pET-26 using the NdeI / XhoI restriction sites. The DNA sequence encoding six histidine (his) amino acids was incorporated into the 5 'primer oligonucleotide to add six histidines (6xhis) at the N-terminal end of the PilA sequence (MDES PILA-3).
[00147] To generate LVL312 (signal peptide FlgI-E-Fragment PilAGG-PE fragment-GGHHHHHH), a polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / strain 86-028NP) using the vector pRIT16671 as a template and CAN534 and CAN537 primer oligonucleotides. The DNA sequence corresponding to the signal peptide Flgl (sp) and amino acid glutamic acid (E) was incorporated into the 5 'primer oligonucleotide (CAN534). To link the PilA sequence to the PE sequence, the DNA sequence corresponding to the two amino acid linkers glycine (GG) and the N-terminal amino acids of PE was incorporated into the 3 'primer oligonucleotide (CAN537). Another polymerase chain reaction was performed to amplify the PE gene (amino acids 18-160) using the vector pRIT16711 as a template and oligonucleotide primers CAN536 and CAN538. The DNA sequence corresponding to the C-terminal PilA amino acids and GG amino acids has been incorporated into the
Petition 870190129809, of 12/09/2019, p. 84/336 / 112 oligonucleotide 5 'primer to link the pilA sequence to PE (CAN536). The DNA sequence corresponding to the GG amino acid and 6xhis amino acid linkers was incorporated into the 3 'primer oligonucleotide (CAN538). Finally, to generate LVL312, a third polymerase chain reaction was performed to amplify the PilA and PE fusion genes with the signal peptide Flgl at the N-terminus, a glutamic acid (E) amino acid between Flgl and pilA, a GG linker between the PilA and PE sequences and a GG link between PE and the 6xhis amino acids at C-terminus. To achieve this amplification, the products of the two polymerase chain reactions described above were used as a template with the CAN534 and CAN538 primer oligonucleotides. The DNA sequence that corresponds to the NdeI restriction site has been incorporated into the 5 'primer oligonucleotide and the Hind111 restriction site has been incorporated into the 3' primer oligonucleotide. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN ^® ).
[00148] To generate LVL291 (signal peptide pelB-PE fragment-GGfragment PilA-GG-6xhis), a polymerase chain reaction was performed to amplify the PE gene (amino acids 19-160) using the vector pRIT16711 as a template and oligonucleotides CAN544 and CAN546 initiators. The DNA sequence that corresponds to the amino acids of the signal peptide pelB (sp) was incorporated into the 5 'primer oligonucleotide (CAN544). To link the PilA sequence to the PE sequence, the DNA sequence that corresponds to the GG amino acid linker and the PilA N-terminal amino acids was incorporated into the 3 'primer (CAN546). Another polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the vector pRIT16671 as a template, with the primers CAN545 and CAN535. The DNA sequence that corresponds to the C-terminal PE amino acids and GG amino acids was incorporated into the 5 'primer oligonucleotide (CAN545)
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76/112 to link the PilA sequence to the PE sequence. The DNA sequence that corresponds to the linker of amino acids GG and amino acids 6xhis was incorporated into the 3 'primer oligonucleotide (CAN535). Finally, to generate LVL291, a third polymerase chain reaction was performed to amplify the PE and PilA genes in fusion with the pelB signal peptide at the N termination, a GG link between the PE and PilA sequences and a GG link between PilA and 6xhis amino acids at the C-termination. To achieve this amplification, the products of two polymerase chain reactions described above were used as a template with the CAN544 and CAN535 primer oligonucleotides. The DNA sequence that corresponds to the NdeI restriction site has been incorporated into the 5 'primer oligonucleotide and the HindIII restriction site has been incorporated into the 3' primer oligonucleotide. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN ^® ).
[00149] To generate LVL268 (signal peptide pelB-D-PE fragment-GGfragment PilA-GG-6xhis), a polymerase chain reaction was performed to amplify the PE gene (amino acids 20-160) using the vector pRIT16711 as a template with the CAN547 and CAN546 primer oligonucleotides. The DNA sequence corresponding to the amino acids of the signal peptide pelB (sp) and the amino acid aspartic acid (D) was incorporated into the 5 'primer oligonucleotide (CAN547). To link the PilA sequence to the PE sequence, the DNA sequence that corresponds to the linker of the amino acids GG and the N-terminal amino acids PilA was incorporated into the 3 'primer (CAN546). Another polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using the vector pRIT16671 as a template with CAN545 and CAN535. The DNA sequence corresponding to the C-terminal PE amino acids and GG amino acids was incorporated into the 5 'primer oligonucleotide (CAN545) to link the PilA sequence to the PE sequence. The DNA sequence that corresponds to the
Petition 870190129809, of 12/09/2019, p. 86/336 / 112 amino acids GG and amino acids 6xhis were incorporated into the 3 'primer oligonucleotide (CAN535). Finally, to generate LVL268, a third polymerase chain reaction was performed to amplify the PE and PilA genes in fusion with the signal peptide pelB at the N-terminus, an D amino acid between signal peptide pelB and PE, a GG link between PE and pilA sequences and a GG link between PilA and 6xhis amino acids in C-term. To achieve this amplification, the products of the two polymerase chain reactions described above were used as a template with the CAN547 and CAN535 primer oligonucleotides. The DNA sequence that corresponds to the NdeI restriction site has been incorporated into the 5 'primer oligonucleotide and the Hind111 restriction site has been incorporated into the 3' primer oligonucleotide. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN ^® ).
[00150] To generate LVL269 (signal peptide NadA-ATNDDD-PE fragment-GG-fragment PilA-GG-6xhis), a polymerase chain reaction was performed to amplify the PE gene (amino acids 22-160 of SEQ ID NO. 4 ) using the pRIT16711 vector as a template with the CAN548 and CAN546 primer oligonucleotides. The DNA sequence that corresponds to the amino acids of the signal peptide pelB (sp) and amino acids ATNDDD was incorporated in the 5 'oligonucleotide primer (CAN548). To link the PilA sequence to the PE sequence, the DNA sequence corresponding to the GG amino acid linker and the N-terminal PilA amino acids was incorporated into the 3 'primer (CAN546). Another polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the vector pRIT16671 as a template with the CAN545 and CAN535 primer oligonucleotides. The DNA sequence that corresponds to the C-terminal PE amino acids and GG amino acids was incorporated into the 5 'primer oligonucleotide to link the PilA sequence to the PE sequence (CAN545). The DNA sequence that
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78/112 corresponds to the GG amino acid linker and 6xhis amino acids were incorporated into the 3 'primer oligonucleotide (CAN535). Finally, to generate LVL269, a third polymerase chain reaction was performed to amplify the PE and PilA gene in fusion with the NadA signal peptide at the N termination, ATNDDD amino acids between the pelB and PE signal peptide, a GG link between the sequences PE and PilA and a GG link between PilA and 6xhis amino acids at the C-termination. To achieve this amplification, the products of the two polymerase chain reactions described above were used as a template with the CAN548 and CAN535 primer oligonucleotides. The DNA sequence that corresponds to the NdeI restriction site has been incorporated into the 5 'primer oligonucleotide and the HindIII restriction site has been incorporated into the 3' primer oligonucleotide. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN ^® ).
[00151] To generate LVL270 (M-6xHis-PE fragment-GG-PilA fragment), a polymerase chain reaction was performed to amplify the PE gene (amino acids 17-160) using the vector pRIT16711 as a template with the primer oligonucleotides CAN540 and CAN542. The DNA sequence that corresponds to 6xhis amino acids was incorporated into the 5 'primer oligonucleotide (CAN540). To link the PilA sequence to the PE sequence, the DNA sequence corresponding to the GG amino acid linker and the N-terminal PilA amino acids was incorporated into the 3 'primer (CAN542). Another polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using vector pRIT16671 as a template with the CAN541 and CAN543 primer oligonucleotides. The DNA sequence that corresponds to the PE C-terminal amino acids and GG amino acids was incorporated into the 5 'primer oligonucleotide (CAN541) to link the PE PilA sequence. Finally, to generate LVL270, a third polymerase chain reaction was performed to amplify the fused 6-his-PE-GG-PilA gene. To reach
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79/112 this amplification, the products of the two polymerase chain reactions described above were used as a template with the primers oligonucleotides CAN540 and CAN543. The DNA sequence that corresponds to the NdeI restriction site has been incorporated into the 5 'primer oligonucleotide and the HindIII restriction site has been incorporated into the 3' primer oligonucleotide. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN®).
[00152] To generate LVL315 (signal peptide pelB-MD-PE fragment GG-Fragment PilA-GG-6xhis), targeted site mutagenesis was performed to change the N-terminal PE amino acid sequence from QIQ to MD using LVL291 as a template, with the CAN670 and CAN671 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00153] To generate LVL317 (signal peptide pelB-PE fragment-GG PilA fragment), site-directed mutagenesis was performed to incorporate a stop codon between the PilA gene and the DNA sequence corresponding to the amino acid residues GGHHHHHH (SEQ ID NO : 3) using LVL291 as a template, with the CAN678 and CAN679 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00154] To generate LVL318 (signal peptide pelB-MD-PE-GG-PilA), site-directed mutagenesis was performed to incorporate a stop codon between the PilA gene and the DNA sequence that corresponds to the amino acid residues GGHHHHHH (SEQ ID NO: 3) using LVL315 as a template, with CAN678 and CAN679 primer oligonucleotides and QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00155] To generate LVL702 (LVL291 AQ), a polymerase chain reaction was performed using the vector LVL291 as a template and the
Petition 870190129809, of 12/09/2019, p. 89/336 / 112 oligonucleotides primers CAN1517 and CAN1518. The elimination of three nucleotides corresponding to amino acid Q at position 23 in the LVL291 sequence was incorporated into the 5 'primer oligonucleotide. The only difference between LVL702 and LVL291 is the elimination of amino acid Q at position 23 in the LVL291 sequence. The Ndel and Hindlll restriction sites were incorporated into the 5 'and 3' primer oligonucleotides, respectively. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN®). [00156] To generate LVL735 (LVL317 AQ), a polymerase chain reaction was performed using the vector LVL317 as a template and the oligonucleotides primers CAN1517 and CAN1519. The elimination of three nucleotides corresponding to the Q amino acid at position 23 in the LVL317 sequence was incorporated into the 5 'primer oligonucleotide. The only difference between LVL735 and LVL317 is the elimination of amino acid Q at position 23 in the LVL317 sequence. The NdeI and HindIII restriction sites were incorporated into the 5 'and 3' primer oligonucleotides respectively. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN ^® ). [00157] To generate LVL736 (LVL291 + SA), site-directed mutagenesis was performed to add amino acids S and A between amino acids 22 and 23 in the LVL291 sequence. LVL291 was used as a template with the CAN1531 and CAN1532 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00158] To generate LVL737 (LVL291 + A), site-directed mutagenesis was performed to add amino acid A between amino acid 22 and 23 in the LVL291 sequence. LVL291 was used as templates with the CAN1529 and CAN1530 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00159] To generate LVL738 (LVL291 AQIQ), a site mutagenesis
Petition 870190129809, of 12/09/2019, p. Targeted 90/336 / 112 was performed to eliminate amino acids Q, I and Q at positions 23 to 25 in the LVL291 sequence. LVL291 was used as templates with the CAN1523 and CAN1524 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00160] To generate LVL739 (LVL291 AQIQK), site-directed mutagenesis was performed to eliminate amino acids Q, I, Q and K at positions 23 to 26 in the LVL291 sequence. LVL291 was used as templates with the CAN1525 and CAN1526 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00161] To generate LVL740 (LVL291 AQIQKA), site-directed mutagenesis was performed to eliminate amino acids Q, I, Q, K and A at positions 23 to 27 in the LVL291 sequence. LVL291 was used as templates with the CAN1527 and CAN1528 primer oligonucleotides and the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Stratagene Division).
[00162] To generate LVL778 (LVL736 A6xHis tag), LVL779 (LVL737 A6xHis tag), LVL780 (LVL738 A6xHis tag), LVL781 (LVL739 A6xHis tag) and LVL782 (the LVL740 chain was performed in vectors LVL736, LVL737, LVL738, LVL739 and LVL740 as a template, respectively, with the primers oligonucleotides CAN1669 and CAN543. The elimination of 6xHis tag corresponds to the amino acid sequence GGHHHHHH (SEQ ID NO. 3) in the C-terminal sequences. This deletion was incorporated into the 3 'primer oligonucleotide. The NdeI and HindIII restriction sites were incorporated into the 5 'and 3' primer oligonucleotides respectively. The generated PCR product was then inserted into the cloning vector pET-26b (+) (NOVAGEN®).
Table 4: PCR primer oligonucleotide sequences used for the
Petition 870190129809, of 12/09/2019, p. 91/336 / 112 amplifications of PE, PilA and PE-PilA.
Oligonucleotide primer ID DNA sequence5 ’- 3’ CAN534 CACACACATATGATTAAATTTCTCTCTGCATTAATTCTTCTACTG GTCACGACGGCGGCTCAGGCTGAGACTAAAAAAGCAGCGGTAT CTG (SEQ ID NO. 155) CAN535 TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTGTGTGACACTTCCGCAAAAATTTGC (SEQ ID NO. 156) CAN536 TTTGCGGAAGTGTCACACAAGGCGGCGCGCAGATTCAGAAGGC TGAACAAAATGATGT (SEQ ID NO. 157) CAN537 ACATCATTTTGTTCAGCCTTCTGAATCTGCGCGCCGCCTTGTGTG ACACTTCCGCAAA (SEQ ID NO. 158) CAN538 TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTTTTTATCAACTGAAAATG (SEQ ID NO. 159) CAN540 CACACACATATGCACCACCACCACCACCACAGCGCGCAGATTCAGAAGGCTGAACAAAATGATGT (SEQ ID NO. 160) CAN541 CATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGT ATC (SEQ ID NO. 161) CAN542 GATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAAT G (SEQ ID NO. 162) CAN543 TGTGTGAAGCTTTTATTGTGTGACACTTCCGCAAA (SEQ ID NO. 163) CAN544 CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCT GCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAGATTCAGAAGG CTGAACAAAATGATGT (SEQ ID NO. 164) CAN545 GCATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGG TATCTG (SEQ ID NO. 165) CAN546 CAGATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAA ATGC (SEQ ID NO. 166) CAN547 CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCT GCTGCTCCTCGCTGCCCAGCCGGCGATGGCCGATATTCAGAAGG CTGAACAAAATGATGT (SEQ ID NO. 167) CAN548 CACACACATATGAAACACTTTCCATCCAAAGTACTGACCACAGC CATCCTTGCCACTTTCTGTAGCGGCGCACTGGCAGCCACAAACG ACGACGATAAGGCTGAACAAAATGATG (SEQ ID NO. 168) CAN670 GCCGGCGATGGCCATGGATAAGGCTGAACAAAATG (SEQ ID NO. 169) CAN671 CATTTTGTTCAGCCTTATCCATGGCCATCGCCGGC (SEQ ID NO. 170) CAN678 GGAAGTGTCACACAATAAGGCGGCCACCACCACC (SEQ ID NO. 171) CAN679 GGTGGTGGTGGCCGCCTTATTGTGTGACACTTCC (SEQ ID NO. 172) CAN1517 GATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCT GCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATTCAGAAGGCTG AACAAAA (SEQ ID NO. 205) CAN1518 GGCCGCAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCC (SEQ ID NO. 206) CAN1519 GGCCGCAAGCTTTTATTGTGTGACACTTCC (SEQ ID NO. 207) CAN1523 GCTGCCCAGCCGGCGATGGCCAAGGCTGAACAAAATGATGTG (SEQ ID NO. 208) CAN1524 CACATCATTTTGTTCAGCCTTGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 209) CAN1525 GCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGTGAAGC (SEQ ID NO. 210) CAN1526 GCTTCACATCATTTTGTTCAGCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 211) CAN1527 GCTGCCCAGCCGGCGATGGCCGAACAAAATGATGTGAAGCTGG (SEQ ID NO. 212)
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CAN1528 CCAGCTTCACATCATTTTGTTCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 213) CAN1529 GCTGCCCAGCCGGCGATGGCCGCCCAGATTCAGAAGGCTGAAC (SEQ ID NO. 214) CAN1530 GTTCAGCCTTCTGAATCTGGGCGGCCATCGCCGGCTGGGCAGC (SEQ ID NO. 215) CAN1531 GCTGCCCAGCCGGCGATGGCCAGCGCCCAGATTCAGAAGGCTG AAC (SEQ ID NO. 216) CAN1532 GTTCAGCCTTCTGAATCTGGGCGCTGGCCATCGCCGGCTGGGCA GC (SEQ ID NO. 217) CAN1669 CACACACATATGAAATACCTGCTGCCGACC (SEQ ID NO. 218) MDesPILA-3 GAATTCCATATGCACCATCACCATCACCATACTAAAAAAGCAGC GGTATCTGAA (SEQ ID NO. 173) MDesPILA-4 GCGCCGCTCGAGTCATTGTGTGACACTTCCGC (SEQ ID NO. 174) MnoNTHi-44 GCCCAGCCGGCGATGGCCCAGATCCAGAAGGCTGAACAAAATG (SEQ ID NO. 175) MnoNTHi-45 CATTTTGTTCAGCCTTCTGGATCTGGGCCATCGCCGGCTGGGC (SEQ ID NO. 176)
Transformation [00163] Escherichia coli BLR (DE3) or E. coli HMS (DE3) cells were transformed with plasmid DNA according to standard methods with CaCl2 treated cells. (Hanahan D. «Plasmid transformation by Simanis.» In Glover, DM (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.). Briefly, the competent BLR (DE3) or HMS174 (DE3) cells were slowly thawed on ice. Approximately 4 pL of plasmid (10-100 ng) was mixed using 50-100 pL of competent cells. From this point, this formulation was incubated on ice for 30 minutes. To carry out the transformation reaction, the formulation was thermally pulsed at 42 ⁰ C for 45 seconds, and then incubated on ice for 2 minutes. Approximately 0.5 mL of SOC (Super Optimal broth with catabolite repression) was added to the transformed cells and the cell culture was incubated at 37 ° C for one hour before being plated on 50 Luria-Bertani agar (LB) ug / ml kanamycin. About 100 µl of transformed cell culture was plated and incubated overnight at 37 ° C.
[00164] BLR (DE3): BLR is a recA ~ derivative of BL21 (F- ompT hsdSB (rB- mB-) gal dcm (DE3). This strain of E. coli used for the expression of recombinant proteins improves the yields of monomer of
Petition 870190129809, of 12/09/2019, p. 93/336 / 112 plasmid and can assist in stabilizing target plasmids that contain repeated sequences, or whose products may cause the loss of the DE3 phage. (Studier, F.W. (1991) J. Mol. Biol. 219: 37-44). The detailed E.coli BLR (DE3) genotype was published by NOVAGEN®. (F- ompT hsdSB (rBmB-) gal dcm A (srl-recA) 306 :: Tn10 (TetR) (DE3).
[00165] HMS174 (DE3): HMS174 strains provide the recA mutation in a K-12 background. Like BLR, these strains can stabilize certain target genes whose products can cause the loss of the DE3 phage. The detailed E.coli HMS174 (DE3) genotype has been published by NOVAGEN®. (FrecAl hsdR (rK12-mK12 +) (DE3) (Rif R).
Production using BLR (DE3) and characterization of constructs marked with His are described in example 3 to example 6
Example 3: Protein expression using shake flask [00166] In general, a confluent agar plate inoculated with Escherichia coli BLR (DE3) transformed with recombinant plasmid was scraped, resuspended in culture medium and used to inoculate 800 ml of LB broth (Becton, Dickinson and Company) ± 1% (weight / volume, w / v) glucose (Laboratoire MAT, catalog number: GR-0101) and 50 pg / ml kanamycin (Sigma) to obtain OD _600nm between 0.1 and 0.2. The cultures were incubated at 37 ° C with shaking of 250 RPM to reach an OD _600nm of ~ 0.8.
[00167] One ml of each culture was then collected, centrifuged at 14,000 RPM for 5 minutes and the supernatants and precipitates were frozen at -20 ° C, separately.
[00168] At an OD _600nm ~ 0.8, the BLR (DE3) cultures were cooled (-20 ° C, 20 minutes or 4 ° C, 1 hour, preferably to 4 ° C for 1 hour) before inducing expression of recombinant protein by adding isopropyl PD-1-thiogalactopyranoside 1 mM (IPTG; EMD Chemicals Inc., catalog number: 5815) and overnight incubation at 16, 22 and 30 ° C, or 3
Petition 870190129809, of 12/09/2019, p. 94/336 / 112 hours at 37 ° C with 250 RPM agitation, preferably overnight at 22 ° C. After the induction period, the cultures were centrifuged at 14,000 RPM for 5 minutes or 6,000 RPM for 15 minutes, and the supernatant (sample of the medium fraction) and the precipitates (containing the soluble and insoluble fraction) were frozen at -20 °. C separately.
[00169] These conditions are used for the expression of periplasmic protein.
Example 4: Protein purification using shake flask, cell pastes, constructs marked with His tag [00170] Each bacterial precipitate obtained after induction was resuspended in 20 mM 4- (2-hydroxyethyl) -1piperazine ethanesulfonic acid (HEPES) buffer (pH 8.0) containing 500 mM NaCl, 10 mM imidazole and Roche COMPLETE® protease inhibitor cocktail (1 tablet / 50 mL of HEPES buffer containing 500 mM NaCl, Roche COMPLETE® ULTRA tablets, Roche Diagnostics Corporation).
[00171] Alternatively, 20 to 50 mM bicin buffer can be used instead of HEPES buffer containing NaCl. For example, 20 mM bicin buffer can be used. The bacteria were lysed using a constant 1.1 KW 2 X 30,000 PSI (pounds per square inch) system. The soluble (supernatant) and insoluble (precipitated) components were separated by centrifugation at 20,000g for 20 minutes at 4 ° C.
[00172] 6-His-tagged proteins were purified under natural conditions in immobilized metal affinity chromatography (IMAC) using the PROFINIA ™ protein purification protocol (BioRad Laboratories, Inc.). The soluble components were filled into a 5 mL His Trap column (Bio-Rad Laboratories, Inc.) pre-equilibrated with the same buffer used for bacterial resuspension; the soluble components were added at up to 5 mL / min (producing a “flow fraction”) After filling in the column, the column was washed with 10 volumes of
Petition 870190129809, of 12/09/2019, p. 95/336 / 112 column of the same buffer, at a rate of 10 mL / min (producing a “wash fraction # 1). A second wash using 20 mM bicin buffer or 20 mM HEPES buffer (pH 8.0) containing 500 mM NaCl and 20 mM imidazole was performed, producing a “wash fraction # 2). Elution was performed using 2 column volumes of 20mM HEPES buffer or 50mM bicin buffer (pH 8.0), containing 500 mM NaCl and 250 mM imidazole at a rate of 10 mL / min, producing an “elution fraction”.
[00173] To improve protein purity, IMAC positive elution fractions were pooled and filled in a pre-balanced size exclusion chromatography column (SEC) (HILOAD ^TM SUPERDEX ^TM 200 26/60 from GE Healthcare) phosphate buffered saline without calcium or magnesium (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO4, 1.47 mM KH2PO4, pH 7.4). The samples of the elution fractions were analyzed by electrophoresis in sodium dodecyl sulfate and polyacrylamide gel (SDS-PAGE). The samples were concentrated using Centricon 10,000 MW (Millipore).
[00174] The protein concentration was determined using a spectrometer.
Example 5: SDS-PAGE and Western Blot analysis of His-tagged constructs & SDS-PAGE analysis of LVL317 & LVL318 non-His constructs
Preparation of the soluble and insoluble fraction [00175] For example, 1 ml of culture after induction (see, for example, Example 3 above) was centrifuged at 14,000 RPM for 2 minutes. The precipitate was resolubilized using 40 gL of BUGBUSTER® protein extraction reagent (NOVAGEN ^® , EMD4 Biosciences, Merck), creating a cell suspension. The cell suspension was incubated on a rotation platform for 10 minutes at room temperature. The cell suspension was then centrifuged at 14,000 RPM for 2 minutes to separate the soluble fraction.
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The resulting precipitate (the insoluble fraction) was resolubilized using 70 pL of deionized water, 5 pL of 1M dithiothreitol (DTT) and 25 pL of NUPAGE® LDS (Lithium Dodecyl Sulphate) and 4X sample buffer (INVITROGEN ™). The soluble fraction (supernatant of the cell suspension of the resolubilized precipitate) was added in 30 pL of deionized water, 5 pL of 1M DTT and 25 pL of LDS 4X sample buffer.
Preparation of the _ fraction of the media [00176] For example, to prepare the fraction of the media, 100 pL of the supernatant from the culture of completely induced cells after centrifugation (see, for example, example 3 above) was concentrated by adding 500 pL of the RC I reagent (Bio-Rad Laboratories, Inc.); the sample was mixed and incubated for 1 minute at room temperature. Next, 500 µl of Reagent II (Bio-Rad Laboratories, Inc.) was added to the sample and mixed. This formulation was centrifuged at 14,000 RPM for 10 minutes. The precipitate was resolubilized using 28 pL of deionized water, 2 pL of 1M DTT and 10 pL of LDS SB 4X.
Preparation of the purification fraction [00177] For example, the purified proteins (for example, obtained in the manner described in example 4) were prepared by SDS-PAGE analysis by adding 70 µl of sample, 5 µl of 1M DTT and 25 µl of buffer of LDS 4X sample.
SDS-PAGE analysis and transfer to nitrocellulose membrane [00178] SDS-PAGE analysis and transfer to nitrocellulose membrane were performed according to the manufacturer's recommendations (Invitrogen) using NUPAGE® Bis-Tris 4-12% gels. Sample preparations, buffers and migration conditions were carried out under conditions recommended by the suppliers.
[00179] In one example, the gel was filled with a 20 μl sample of a master mix comprising 70 μl of a protein fraction
Petition 870190129809, of 12/09/2019, p. 97/336 / 112 purified, 5 µl 1M DTT and 25 µl LDS SB 4X.
[00180] After the samples are run on NUPAGE® Bis-Tris gels
4-12%, proteins were transferred to nitrocellulose membranes.
[00181] The nitrocellulose membranes were blocked for 30 minutes at 37 ^o C, 60 RPM, using a new 3% milk / 1X PBS solution. After blocking incubation, primary antibodies were added (6X His Tag® antibody, Abcam PLC, catalog number: ab9108) at a dilution of: 1: 1,000 in a new 3% milk / 1X PBS solution for 1 hour at 37 ° C ^o C, 60 RPM. After that, the membranes were washed three times, for 5 minutes each, at room temperature using 0.02% polysorbate 20 (eg TWEEN ^TM 20) / PBS 1X. Secondary antibodies (alkaline phosphatase (AP) anti-rabbit IgG (H + L), Jackson ImmunoResearch Laboratories, Inc.) were added in 1: 14,000 dilution using new 3% milk / 1X PBS solution. The membranes were incubated for 1 hour at 37 ^o C, 60 RPM. After that, the membranes were washed three times for 5 minutes at room temperature using 0.02% polysorbate 20 (eg TWEEN ^TM 20) / PBS 1X before exposing the membrane to 5-bromo-4chloro-3-indolyl phosphate / nitro blue tetrazolium (eg BCIP ^® / NBT by Sigma-Aldrich ^® , 1 tablet / 10 mL water).
[00182] See figure 1 for SDS-PAGE of induced bacterial extracts for the LVL291, LVL268 and LVL269 fusion protein constructs. The insoluble fraction (I), soluble fraction (S) and fraction of culture medium (M) were loaded into LVL291, LVL268 and LVL269 before and after induction (ind).
[00183] See figure 2 for SDS-PAGE and Western blot related to purification extracts for the LVL291, LVL268 and LVL269 fusion protein constructs. Flow fraction (Ft), wash fraction (W) and elution fraction (E) were loaded for purification of LVL291, LVL268 and LVL269. Anti-his tag was used to probe extracts.
Petition 870190129809, of 12/09/2019, p. 98/336 / 112 [00184] See figure 3 for SDS-PAGE of induced and purifying bacterial extracts for the LVL291 and LVL315 fusion protein constructs. Culture medium fraction (M), soluble fraction (Sol), insoluble fraction (Ins), flow fraction (Ft), wash fraction # 1 (W1), wash fraction # 2 (W2) and elution fraction ( E) were loaded to LVL291 and LVL315.
[00185] See figure 4 for SDS-PAGE of induced and purified bacterial extracts for LVL312 fusion protein construct. culture medium fraction (M), soluble fraction (Sol), insoluble fraction (Ins), flow fraction (Ft), wash fraction # 1 (W1), wash fraction # 2 (W2) and elution fraction ( E) were loaded into LVL312.
[00186] See figure 25 for SDS-PAGE of the soluble fractions of the bacterial extracts induced for the fusion protein vector constructs LVL291, LVL702, LVL736, LVL737, LVL738, LVL739, LVL740 and pET26b (negative control). (A) Experiment 1 (b) Experiment 2 (c) Experiment 3. The PE-PilA fusion protein indicated by the arrow.
[00187] See figure 26 for the average percentage of fusion protein band in the soluble fraction of experiments 1, 2 and 3.
[00188] The bacterial extracts LVL317 and LVL318 used in the SDS-PAGE analysis in figure 5 and figure 6, respectively, were prepared in general in the manner previously described.
[00189] Figure 5 describes SDS-PAGE of induced bacterial extracts (IPTG 1mM and 10μΜ) for the LVL317 fusion protein construct. The extracts from before (NI) and after induction (In), soluble fraction (S), insoluble fraction (I).
[00190] Figure 6shows SDS-PAGE of induced bacterial extracts (IPTG 1mM and 10μΜ) for the LVL318 fusion protein construct. The extracts from before (NI) and after induction (In), fraction of culture medium (M), soluble fraction (S), insoluble fraction (I).
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90/112 [00191] Proteins separated by SDS-PAGE were transferred to an Immobilon-P membrane. The protein bands stained with Coomassie blue were cut and placed in a sequencing reactor. The sequencing was performed according to the manufacturer's protocol, using an Applied Biosystems PROCISE® protein sequencer, model 494-cLC.
Table 5: Protein expression profiles in the shake flask and signal peptide cleavage for the fusion protein constructs.
Fusion protein construct ID Description termination N ~ ^> termination C Protein expression profiles Signal peptide cleavage LVL312 Figi sp - E - PilA fragment - GG - PE fragment -GGHHHHHH In: +++ So: + If: + Confirmed FVE291 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: + Confirmed FVE268 PelB sp - D - PE fragment - GG - PilA fragment -GGHHHHHH In: +++ So: ++ If: + Confirmed FVE269 NadA sp - ATNDDD - PE fragment - GG PilA fragment - GGHHHHHH In: +++ So: ++ If: + Confirmed FVE270 MHHHHHH - PE fragment - GG - PilA fragment In: + So: If: - Not tested LVL315 PelB sp - MD - PE fragment - GG - PilA fragment - GGHHHHHH In: +++ So: ++ If: + Confirmed LVL317 PelB - PE fragment - GG - PilA fragment In: +++ So: + Se: Nt Confirmed LVL318 PelB sp - MD - PE fragment - GG - PilA fragment In: +++ So: + If: -LVL702 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed FVE736 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed FVE737 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed FVE738 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed FVE739 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed FVE740 PelB sp - PE fragment - GG - PilA fragment GGHHHHHH In: +++ So: ++ If: Nt Confirmed
So = Soluble fraction. In = Insoluble fraction. Se = Protein secreted in the media fraction. Nt = Not tested. The following classification was based on visual inspection (coomassie) +: little expression; ++: medium expression; +++: a lot of expression; no expression
Example 6: Characterization of LVL291 fusion protein
PHYSICAL PROPERTIES OF LVL291: Bending PE and PilA in
EVL291 & melting point
Circular dichroism:
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Secondary Structure Analysis [00192] Circular dichroism (CD) is used to determine the secondary structure composition of a protein by measuring the difference in the absorption of polarized light on the left versus polarized light on the right, which is due to structural asymmetry. The shape and magnitude of the CD spectrum in the far-UV region (190-250nm) are different if a protein exhibits a leaf structure, alpha-helix or random coil. The relative abundance of each type of secondary structure in a given protein sample can be calculated by comparison with the reference spectra.
[00193] The far-UV spectra are measured using a 0.01cm optical path from 178 to 250nm, with a resolution of 1nm and bandwidth on a Jasco J-720 spectropolarimeter. The cell temperature is maintained at 23 ° C by a block of Peltier thermostated cells RTE-111. A nitrogen flow of 10 L / min is maintained during measurements.
Results:
[00194] The far-UV CD spectra obtained for PE (from construct pRIT16762), PilA (from construct pRIT 16790) and PE-PilA proteins are characteristic of folded proteins containing a mixture of alpha and beta structures, but PE is significantly more enriched in alpha helix than PilA and PE-PilA (Figure 7, CD spectra of fusion proteins PE, PilA and PE-PilA).
[00195] In order to evaluate the folding integrity of individual PE and PilA proteins, once linked together in a chimeric protein, and then to verify a possible interaction between both, the difference spectra were calculated.
[00196] When the PE and PilA far-UV spectra are combined, the resulting spectrum overlaps the PE-PilA chimera spectrum (Figure 8, Combination of PE and PilA CD spectrum). This result suggests that the PE-PilA chimera contains all secondary structures that are detected
Petition 870190129809, of 12/09/2019, p. 101/336 / 112 on the individual components. It is also suggested that the fusion of proteins has no major impact on the secondary structures of the individual components, and consequently that the folding of PE and PilA is not significantly different if the proteins are separated or in fusion.
Evaluation of the melting point:
[00197] In order to assess whether the fused expression has an impact on the thermodynamic properties of individual proteins, the melting points of PE, PilA and PE-PilA were evaluated by monitoring the split of the alpha helix with temperature by circular dichroism.
[00198] The presence of alpha helix is characterized by a minimum in the circular dichroism signal qm 222nm, thus, a significant increase in the CD signal by 222nm during the temperature increase is an indication of protein denaturation. The determination of the temperature at which the protein undergoes the loss of the secondary structure allows the determination of the melting point (Tm), which corresponds to the temperature at which half of the proteins shows loss of its structure.
[00199] The melting point can be determined by identifying the inflection point in the thermal denaturation curve, from a graph of temperature versus CD 222nm.
[00200] The melting point of PilA and PE, as determined by far-UV CD, are respectively 52 ° C and 68 ° C (Figure 9, PilA thermal denaturation curve; Figure 10, Thermal denaturation curve of PE).
[00201] The PE-PilA fusion protein exhibits two distinct Tm's at 48 ° C and 71 ° C (Figure 11, thermal denaturation curve of PEPilA fusion protein). These values indicate that the PE and PilA proteins are still independently folded when bound in a chimera, and that they are unfolded at a similar temperature if they are separated or fused. THE
Petition 870190129809, of 12/09/2019, p. 102/336 / 112 note that the unfolding of the PilA portion at 48 ° C does not cause precipitation or impact on the Tm of the PE portion at 71 ° C is a strong indication that the interaction between PE and PilA in the fusion is minimal, and that presents an observable main impact on each other. Protein melting points are sensitive in various external conditions, including buffer composition or the presence of interaction molecules; where no major variation is observed through the fusion of PE and PilA, which is a strong indication of the conservation of many structures and the properties of both PE and PilA when they are connected together.
Example 7: Fermentation process [00202] The fusion proteins of the invention can be prepared by methods known to those skilled in the art.
Example 8: Purification of PE, PilA, and LVL317 protein
Purification of PE protein from pR! T16762:
[00203] To generate the expression vector pRIT16762, the vector pRIT16711 was digested using the restriction enzymes BamHI and NcoI in order to eliminate amino acid residues 6 between the signal sequence (pelB) and PE. The vector obtained was called pRIT16712. In this vector, there are 3 amino acids between the signal sequence pelB and PE: MDP. In a second step, site-directed mutagenesis was performed to change the amino acid sequence from MDP to QIQ using pRIT16712 as a template, with the primers MnoNTHi-44 and MnoNTHi-45 (described in table 4) and the site-directed mutagenesis kit QuikChange II (Agilent Technologies, Stratagene Division).
[00204] The E. coli BLR (DE3) reference sample containing PE QIQ (from construct pRIT16762) was thawed from -80 ° C and was used to prepare 100 ml of pre-culture in LB broth, with incubation by all night at 37 ° C stirring at 215 RPM. After overnight incubation, eight vials containing 800 mL of LB APS were inoculated with 12.5 mL of pre
Petition 870190129809, of 12/09/2019, p. 103/336 / 112 culture and OD ₆₀₀ measured around 0.06. The cultures were incubated for 3 hours at 37 ° C with shaking. In an OD ₆₀₀ of around 0.9, 1mM IPTG was added to start the induction. During induction, cultures were incubated for 19 hours at 22 ° C with shaking. After induction, OD600 was around 2.2. Cell cultures were transferred to 1L centrifuge tubes placed in 1L bottles, and centrifuged at 4 ° C for 30 minutes at 6,000 xgeo supernatant was discarded. The 1mL aliquots of pre- and post-induction culture and the supernatant were kept for future analysis.
BLR lysis (DE3) induced with PE QIQ [00205] The centrifuge bags were removed from the centrifuge bottles, opened and the precipitate was removed from the bag into a beaker. The eight precipitates were pooled and resuspended in 100 mL of binding buffer (20mM Hepes, 10mM imidazole, 500mM NaCl, pH 8.01). The E.coli BLR (DE3) containing the PE QIQ construct was broken with Constant Systems Ltd.'s TS Series Bench Top cell disrupter (1 x 30 kPsi; 1 x 15 kPsi). The lysate was centrifuged 30 minutes, 6,000 RPM, 4 ° C. The supernatant was maintained and placed on an IMAC column.
IMAC purification of PE QIQ [00206] The IMAC column (BioRad, Bio-Scale Mini Profinity IMAC 5 ml cartridge) was equilibrated with 5CV of binding buffer (20mM HEPES, 10mM imidazole, 500mM NaCl, pH 8.01) at 5 mL / minutes. 100 ml of the lysed supernatant was placed in the IMAC at 2.5 ml / minute. The flow was collected in 50 mL fractions for future analysis. The column was washed with 3CV binding buffer to remove bound protein. The sample containing unbound proteins was collected in a 15 mL aliquot in a 50 mL tube. The column was washed with 2CV of wash buffer (20mM HEPES, 20mM imidazole, 500mM NaCl, pH 8.01) collected in 2 mL fractions in a 96-well plate. The bound protein was
Petition 870190129809, of 12/09/2019, p. 104/336 / 112 eluted with 6CV of 100% elution buffer (20mM HEPES, 250mM imidazole, 500mM NaCl, pH 8.01). The eluted protein was collected in 2 mL fractions in 96-well plates. Washing and elution were performed at 5 ml / minute.
Size exclusion chromatography (SECONDS) in the IMAC cluster of PE QIQ [00207] The SEC column (GE healthcare, HILOAD ™ 26/60 SUPERDEX ^TM 75 prep grade, 60 cm height, approximately 319 mL volume) was equilibrated with 3CV of SEC buffer (20mM HEPES, 150mM NaCl, pH 8.49). 11 mL of IMAC elution were placed on the column at a flow rate of 2.5 mL / minutes. The 2 mL fractions were collected from 0.3 CV to 0.9 CV. Two runs were performed and then the fractions were analyzed by SDS-PAGE. The fractions from the two runs containing Prot E protein were grouped (“SEC cluster”, total volume of approximately 48 mL). 500mM arginine was added to the SEC pool.
Dosage of the grouped PE QIQ samples generated in the previous SEC protocol [00208] The SEC grouping was dosed using the RCDC method (reducing agent and compatible detergent) from the Bio-Rad RC DC ^TM kit, following the manufacturer's protocol:
[00209] For each tested and standard sample, 25 pL were distributed in microcentrifuge tubes, in duplicate. 125 pL of I RC Bio-Rad reagent was added to each tube; each tube was vortexed and incubated for 1 minute at room temperature. 125 pL of Reagent II RC Bio-Rad is added to each tube; each tube is vortexed and then centrifuged at 14,000 x g for 5 minutes. Supernatants are discarded by inverting the tubes on clean, absorbent paper, allowing the liquid to be completely drained from the tubes. 25.4 pL of reagent A (already
Petition 870190129809, of 12/09/2019, p. 105/336 / 112 prepared by mixing 20gL of reagent S per 1 mL of reagent A) are added to each tube; each tube is vortexed and incubated at room temperature for 5 minutes, or until the precipitate is completely dissolved. Vortex before continuing to the next step. Add 200 gL of B DC reagent to each tube and vortex immediately. Incubate at room temperature for 15 minutes. Transfer all samples to a 96-well plate and read the absorbance at 750 nm to determine the protein concentration for each unknown protein sample.
[00210] The ProtE concentration was 1.069 mg / mL
Purification of His-tagged PilA protein:
[00211] PilA was purified after the following general procedure:
[00212] E. coli cells containing a construct encoding PilA or a fragment thereof are suspended in BUGBUSTER® and BENZONASE® nuclease (NOVAGEN®), for example, 10 ml BUGBUSTER® nuclease and 10 ul BENZONASE®. The cell lysate is mixed at room temperature on a rotation platform, for example, for 15 minutes. The cell lysate is centrifuged at 4 ° C, for example, at 16,000 g for 20 minutes. The protein-containing supernatant is added to a Ni NTA column containing Ni NTA HIS · BIND® resin and mixed at 4 ° C, for example, for 1 hour. The column can consist of 2 ml of Ni NTA HIS BIND® resin (NOVAGEN®) and 10 ml of 1X binding buffer (from the kit with Ni-NTA NOVAGEN® buffer). The column flow is then collected. The resin is washed twice with 1X wash buffer, for example, containing 300 mM NaCl, 50 mM NaH ₂ PO ₄ , 25 mM imidazole, pH 8.0). The wash is collected by gravity flow. The protein is eluted from the column with 1X elution buffer, for example, 300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0. The protein can be further purified by dialysis with the elution buffer and run again on a Ni NTA column, as
Petition 870190129809, of 12/09/2019, p. 106/336 / 112 described above.
PilA cleavage with thrombin.
[00213] PilA is then incubated with thrombin (diluted 1/50) at room temperature for 16 hours to remove the histidine mark.
Size exclusion chromatography (SEC) in PilA cleaved with thrombin.
[00214] The SEC column (GE healthcare, HILOAD ™ 26/60 SUPERDEX ^TM 75 prep grade, height 60 cm, volume approximately 319 mL) was equilibrated with 5CV SEC buffer (HEPES 20mM, NaCl 150mM, pH 8.52 ). Approximately 10 mL of cleaved PilA was placed on the column at a flow rate of 2.5 mL / minutes. The 2 mL fractions were collected from 0.3 CV to 0.9 CV. Two runs were performed and then the fractions were analyzed by SDS-PAGE. The two-run fractions containing cleaved PilA protein were grouped together (“SEC cluster”, approximate total volume of 52 mL).
PilA dosage, SEC grouping.
[00215] The SEC grouping was dosed with the RCDC method in the manner previously described. The concentration of cleaved PilA was 5.37 mg / mL.
PilA SEC cluster dialysis with PBS 1x pH 7.4 (dialysis factor = 1,600) and RCDC dosage [00216] The post-dialysis concentration determined by RCDC was 3.0 mg / mL.
Purification of LVL317
Osmotic shock [00217] Since the LVL317 fusion protein is expressed and processed in the bacterial periplasm, the protein was extracted by osmotic shock.
[00218] E. coli B2448 cell paste collected and frozen (-20 ° C)
Petition 870190129809, of 12/09/2019, p. 107/336 / 112 containing LVL317, from 4 L of fermentation culture, was pooled and resuspended in a hypertonic buffer consisting of 24 mM Tris-HCl, 16% (w / v) sucrose, 9.9% ( w / v) glucose, 10 mM EDTA, pH 8.0, up to a final volume of 4L. The suspension was mixed gently for 30 minutes at room temperature using a 3-blade propellant installed on a basic RW 16 agitator at medium speed. The suspension was centrifuged at 15,900 x g for 30 minutes at room temperature. The supernatant (SN1) was kept for gel analysis.
[00219] The resulting precipitate was resuspended in a hypotonic solution; 38 mM MgCl2 and mixed for 30 minutes at room temperature. The mixture was centrifuged at 15,900 x g for 30 minutes at room temperature, and the antigen was recovered in the supernatant (SN2).
[00220] A SN2 clarification was performed by filtration through a 0.45 / 0.2 pm polyethersulfone Sartorius Sartopore 2 MidiCap filter, at 600 mL / min flow rate.
[00221] SN2 was diluted 1: 3 with 20 mM NaH2PO4-Na2HPO4, pH 7.0. The pH was adjusted to 7.0 if necessary and another clarification with filtration through a 0.45 / 0.2 pm polyethersulfone filter Sartorius Sartopore 2 MidiCap, in 600 mL / min, was performed.
SP SEPHAROSE ™ rapid flow chromatography (SP FF) [00222] The diluted / filtered SN2 was loaded and captured in a strong cation exchange resin (SP SEPHAROSE ™ FF - GE Healthcare), in a 14 cm ID column (internal diameter ) x 20 cm in length (column volume 3,100 mlL) equilibrated with 2CV of NaH ₂ PO4 / Na ₂ HPO ₄ buffer 20 mM pH 7.0. After washing the column with 5CV of NaH ₂ PO4 / Na ₂ HPO ₄ buffer 20 mM pH 7.0, the antigen (contained in LVL317) was eluted by increasing the NaCl concentration by up to 100 mM in the same washing buffer.
[00223] See figure 12 for a typical SP SEPHAROSE ™ fast flow chromatogram.
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Q SEPHAROSE ™ rapid flow chromatography (Q FF) [00224] The antigen present in the eluted SP FF was diluted 1: 4 with a 20 mM Tris pH 8.5, pH adjusted to 8.5 if necessary, and passed through a resin of strong anion exchange (Q SEPHAROSE ™ FF - GE Healthcare) in a column of 14 cm ID x 11.8 cm in size (column volume 1,800 mL) equilibrated with 2CV of 20 mM Tris buffer pH 8.5. The antigen was recovered in the flow fraction.
[00225] See figure 13 for a typical Q SEPHAROSE ™ fast flow chromatogram.
Concentration, diafiltration, addition of polysorbate 80 and sterilization by filtration [00226] The Q FF flow containing the antigen was concentrated to up to 0.70.8mg / mL based on the UV chromatogram, and diafiltered with 5DV of KH ₂ PO4 / buffer K ₂ HPO ₄ 10 mM pH 6.5 using a 10 kDa cut Pellicon-2 ™ membrane (Millipore).
[00227] Using a 5% stock solution, polysorbate 80 (for example, TWEEN ^TM 80) was added in the ultrafiltration of the retentate and stirred for 30 minutes with a magnetic stirrer at 130 rpm at 4 ° C. The final concentration of polysorbate 80 was 0.04%. The ultrafiltration retentate was sterilized by filtration with a 0.45 / 0.2 pm cellulose acetate membrane (Sartobran 300, Sartorius). The purified volume was stored at -20 ° C or -80 ° C. The absolute protein concentration was measured by AAA (amino acid analysis) at 0.737mg / mL.
Example 9: Use of polysorbate 80 [00228] A titration experiment indicated that the addition of polysorbate 80, specifically TWEEN ^TM 80, in a final concentration of 0.04% (w / v) of the purified volume before filtration sterilization reduced the formation and aggregation of filamentous particle.
[00229] According to the DSC analysis, TWEEN ™ 80 reduced the degree
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100/112 structural change (30-45 ° C) observed after freeze / thaw cycles, after storage at -20 ° C and after storage for 4 days at 4 ° C, -20 ° C, -80 ° C and 37 ° C.
Example 10: SDS-PAGE and Western Blot analysis of LVL317
SDS-PAGE and Western analysis:
[00230] Bis-Tris 4-12% NUPAGE® Gel was loaded, as described below, with 10 pg of sample in LDS NUPAGE® sample buffer containing 50 mM DTT heated for 5 minutes at 95 ° C (20 pL samples were filled in samples with low concentration). Migration: 35 minutes at 200 Volts at room temperature (RT) in NUPAGE® MES running buffer. The gel was stained for 2 hours in Instant blue (Novexin cat .: ISB01L) and discolored all night in water.
[00231] Channel contents:
1: MW standard (10pL) 2: Start (total fraction) (10pg) 3: SN1 without filtration (10pg)
4: SN2 without filtration (10pg) 5: Not extracted (10pg) 6:
Load SP FF (10pg)
7: Flow SP FF (6.9pg) 8: Wash SPFF (20pL)
9: SP FF Elution (10pg)
10: SP FF Tape (10pg) 11: Q FF Load (8.9pg)
12: Q FF Elution (9.8pg)
13: FF Q tape (4.8pg) 14: TFF retentate before TWEEN ^tm 80 0.04% reinforced (10pg)
15: Purified unfiltered volume of 0.04% TWEEN ™ 80 reinforced (10pg)
16: Purified and sterilized volume by filtration of
TWEEN ™ 80 at 0.04% reinforced (10pg)
17: Purified and sterilized volume by filtration of
TWEEN ™ 80 at 0.04% reinforced (20 pg + E. Coli Rix cell lysate
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101/112 reinforced (1pg))
18: E. Coli Rix cell lysate (2pg) 19: E. Coli Rix cell lysate (1pg) 20: E. Coli Rix cell lysate (0.5pg) [00232] See figure 14 for an SDS-PAGE of in-process samples from the PE-PilA fusion protein purification process.
[00233] In relation to Western Blot, proteins were transferred to 4 ° C overnight at 30 Volts in NUPAGE® transfer buffer + 20% methanol, 0.1% SDS on nitrocellulose membrane. The membranes were blocked for 1 hour with 50mM Tris, 150mM NaCl pH 7.4 + 5% skimmed milk powder, incubated for 2 hours in primary rabbit antibody diluted in blocking buffer (anti-Prot-E 1/50 000 and anti E. coli (BLR) 1 / 1,000), washed 3 x 5 minutes in 50 mM Tris pH 7.4 + 0.05% Tween 20, incubated for 1 hour in secondary antibody (goat anti-rabbit conjugated with diluted alkaline phosphatase 1 / 5,000 in blocking buffer), washed 3 x 5 minutes in washing buffer and grown on BCIP / NBT substrate (1 tablet per 10 mL). All incubations were performed in 25 mL per membrane.
[00234] See figure 15 for a Western Blot of the samples in process, from the PE-PilA fusion protein purification process. Blotting was performed using polyclonal rabbit anti-PE.
[00235] Channel contents:
1: MW standard (10pL) 2: Start (total fraction) (10pg) 3: SN1 without filtration (10pg)
4: SN2 without filtration (10pg) 5: Not extracted (10pg) 6:
Load SP FF (10pg)
7: Flow SP FF (6.9pg) 8: Wash SPFF (20pL) 9:
SP FF Elution (10pg)
10: SP FF Tape (10pg) 11: Q Load FF (8.9pg) 12: Q Elution
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FF (9.8qg)
13: FF Q tape (4.8qg) 14: TFF retentate before TWEEN ^tm 0.04% reinforced (10qg)
15: Purified volume without filtration of TWEEN ^tm 80 to 0.04% reinforced (10qg)
16: Volume purified with TWEEN ^tm filtration sterilization 80 to 0.04% reinforced (10qg)
17: Volume purified with TWEEN ^tm filtration sterilization 80 to 0.04% reinforced (20qg + reinforced E. Coli Rix cell lysate (1qg))
18: E. Coli Rix cell lysate (2qg)
19: E. Coli Rix cell lysate (1qg)
20: E. Coli Rix cell lysate (0.5qg) [00236] See figure 16 for a Western Blot of in-process samples, from the PE-PilA fusion protein purification process. Blotting was performed using polyclonal rabbit anti-E.coli (BLR).
[00237] Channel contents:
1: MW standard (10qL) 2: Start (total fraction) (10qg) 3: SN1 without filtration (10qg)
4: SN2 without filtration (10qg) 5: Not extracted (10qg) 6: Load SP FF (10qg)
7: Flow SP FF (6.9qg) 8: Wash SPFF (20qL) 9:
SP FF Elution (10qg)
10: SP FF Tape (10qg) 11: Q FF Load (8.9qg) 12: Q FF Elution (9.8qg)
13: FF Q tape (4.8qg) 14: TFF retentate before TWEEN ^tm 80 to 0.04% reinforced (10qg)
15: Purified volume without filtration of TWEEN ^tm 80 to 0.04% reinforced (10qg)
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16: Volume purified with TWEEN ^tm filtration sterilization 80 to 0.04% reinforced (10gg)
17: Volume purified with sterilization by filtration of TWEEN ^Tm 80 at 0.04% reinforced (20pg + reinforced E. Coli Rix cell lysate (1μg))
18: E. Coli Rix cell lysate (2pg) 19: E. Coli Rix cell lysate (^ g) 20: E. Coli Rix cell lysate (0.5pg) [00238] Comments on the SDS-PAGE figures and Western Blot: The PE-PilA fusion protein migrates at 30kDa. Osmotic shock extraction extracted the expressed and processed fusion protein in the bacteria periplasm and reduced the contamination of the bacteria. A small loss of fusion protein occurred during hypertonic treatment (channel 3). A small proportion was not extracted by hypertonic treatment and remained associated with the cells (channel 5). There was a small loss in the SP FF flow (channel 7) and in the tape fraction of both columns (channels 10 and 13). Since the total volume of the strip fraction is less, the loss of fusion protein is not significant. Degraded bands are visible in the tape fractions, but not in the final product. There was no significant contamination of cellular proteins from the E. coli host in the purified volume (channel 16).
[00239] The analysis of LVL735 and LVL778 yielded profiles similar to LVL317.
Example 11: Melting point data for PE, PilA and LVL317 [00240] The thermal transition of the non-His-tagged PE-PilA fusion protein (LVL317) was compared with the thermal transition of both his-tagged PE proteins (in the same way). described in example 8) and PilA cleaved (in the manner described in example 8), purified in the manner previously described.
[00241] Previously, DSC, PE and PilA were dialyzed throughout the
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104/112 night in K ₂ HPO4 / KH ₂ PO ₄ 10mM pH 6.5 + 0.04% Tween 80 (volume ratio 1: 250 sample: buffer) so that they were in the same buffer as the fusion protein. After dialysis, the protein concentration was measured by BCA and adjusted to 300 pg / mL (PE) and 500 pg / mL (PilA).
[00242] The analysis was performed on VP ^TM -DSC from MicroCal, LLC (part of GE Healthcare). The final dialysis buffer was used as a reference and subtracted from the scans. The DSC scan rate was 90 ° C / hr. In order to assess the ability to measure the thermal transition in the final container (FC) after formulation, the fusion protein was diluted to the FC concentration (60 pg / mL). The final container data is not shown.
Results:
[00243] See figure 17 for the thermal transition of PE-PilA fusion protein and PE and PilA proteins. Curves: PilA (1), protein E (Prot E, PE) (2), PE-PilA PB undiluted 737pg / mL (3), and PE-PilA PB diluted in FC concentration of 60 pg / mL (4) .
[00244] 1 - PilA Tm: 53 ° C [00245] 2 - protein E Tm: 63 [00246] 3 - PE-PilA PB (Purified volume) undiluted 737 pg / mL
Tm1: 53.7 ° C and Tm2: 66.1 ° C [00247] 4 - PE-PilA PB diluted in FC concentration of 60 pg / mL
Tm1: 53.2 ° C and Tm2: 67.6 ° C [00248] Two transitions were detected in the purified fusion protein (LVL317) (curves 3 and 4).
[00249] The Tm ₁ (53.7 ° C) of the PE-PilA fusion protein is similar to the PilA transition (53 ° C).
[00250] The significant displacement of Tm2 in PE-PilA (66.1 ° C) was compared to the PE transition (63 ° C). The fusion of both domains appears to stabilize the PE fragment.
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105/112 [00251] The displacement of Tm ₂ in the diluted fusion protein compared to the undiluted is an artifact of concentration that increases with the decrease of the typical accentuated inclination of aggregation that depends on the concentration.
[00252] The analysis of the antigen folding of LVL735 and LVL778 was similar to that of LVL317.
Example 12: Response to anti-PilA immunogenicity in LVL291 PE-PilA fusion protein construct in Balb / c mice.
[00253] The immune response directed against the purified LVL291 PEPilA fusion protein (the LVL291 fusion protein without the heterologous signal peptide), formulated in AS03A, was evaluated in Balb / c mice. The animals (20 mice / group) were immunized intramuscularly on days 0, 14 and 28 with 10 pg of PE (from the vector pRIT16762), PilA (from the vector pRIT16790) or PE-PilA, each formulated in AS03A. The control group was vaccinated with AS03A only. The antibody response directed against each antigen was determined in the individual sera collected on day 42. No antibody response was obtained with the negative control. As shown in figure 18, the antibody response directed against PilA was greater in mice immunized with PE-PilA fusion, compared to the antibody response in mice immunized with monovalent PilA. Antibody responses directed against PE were similar in mice immunized with the fusion protein and in mice immunized with monovalent PE. GMT = geometric mean of the titration. The data were captured and analyzed with the SOFTMAX® Pro software (Molecular Devices) used in WINDOWS® (Microsoft); the four-parameter log-logistic function was used to calculate the standard curve. The four-parameter log-logistic function describes, with a high degree of accuracy, the reference serum curve that exhibits an accentuated sigmoidal shape when plotted in
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106/112 an optical density-versus-concentration (log) scale. Antibody concentrations were calculated at each dilution of mouse serum samples by interpolation from the standard curve. The antibody in the quality control sera and unknown serum samples is obtained by averaging the values of all dilutions that are in the functional range (10-80%) of the reference dilution curve.
[00254] The results are shown in figure 18, which graphically represents the antibody responses against the PE-PilA fusion protein LVL291, and against PE and monovalent PilA in the Balb / c mouse model.
Example 13: Murine nasopharyngeal colonization model. Immunization with PE-PilA. Challenge with NTHi strain 86-028NP and NTHi strain 3224A.
[00255] Female Balb / c mice (20 / group) were immunized intranasally on days 0 and 14 with 6 pg of a purified PE-PilA fusion protein (LVL291 for challenge with 86-028NP; LVL317 for challenge with strain 3224A ) formulated with LT (Escherichia coli thermolabile toxin), and on day 28 with 6 pg of a PE-PilA fusion protein purified in phosphate buffered saline (PBS). Control mice (20 / group) were vaccinated with LT only. The mice were subsequently challenged intranasally with 5 x 10 ⁶ CFU (colony forming units) of NTHi NTHi strain 86-028NP homologous and heterologous strain 3224. Homology and heterology are determined by reference to the NTHi strain, with which the mice were immunized. Colonies of bacteria were counted in the nasal cavities removed on days 1 and 2 after the challenge. D1 = day 1. D2 = day 2.
[00256] Vaccination with PE-PilA increased the separation of NTHi strain 86-028NP and strain 3224A in the nasopharynx on day 1 and day 2 after the challenge.
[00257] In relation to the experiment carried out with NTHi strain 86028NP: A fixed ANOVA of 2 variances was performed using the values of
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107/112 log10 of the counts as an answer, the fixed factors being the group (4 levels) and the day (2 levels). The assumption of heterogeneity of variance was rejected and a model with heterogeneous variances was fitted with the data. No significant interactions were detected between the 2 factors. The PEPilA fusion group (6 pg per mouse) significantly reduced the CFU compared to the control group (LT); the geometric mean ratio was 0.06, with a 95% confidence interval of 0.01, 0.25.
[00258] Regarding the experiment conducted with NTHi strain 3224A: A fixed ANOVA of 3 variances was performed using the log10 values as a response, the fixed factors being the group, the day and the experiment. The Shapiro-Wilk and Levene tests did not reject the assumptions of normality and homogeneity of variances. No significant interaction between any of the 2 factors or between the 3 factors was detected, and only the main factors were maintained in the analysis. PE-PilA / LT significantly reduced CFU compared to the control group; the geometric mean ratio being equal to 0.11 with a 95% confidence interval of 0.02, 0.61.
[00259] See figure 19 for the effect of vaccination with PE-PilA fusion protein on the evidence of the bacterium NTHi strain 86-028NP in the mouse nasopharynx.
[00260] See figure 20 for the effect of vaccination with PE-PilA fusion protein on the evidence of the bacterium NTHi strain 3224A in the mouse nasopharynx.
Example 14: Murine nasopharyngeal colonization model. Immunization with PilA. Challenge with NTHi strain 3219C.
[00261] Female OF1 mice (20 mice / group) were immunized intranasally on days 0 and 14 with 3 pg of PilA (from vector 16790) formulated with LT, and on day 28 with 3 pg of PilA in PBS. Control mice were vaccinated with LT only. The mice were subsequently challenged intranasally with 5 x 10 ⁶ CFU of
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NTHi strain 3219C. Bacterial colonies were counted in the nasal cavities removed 3 and 4 days after the challenge. D3 = day 3. D4 = day 4.
[00262] See figure 21 for the effect of PilA vaccination on the bacterial evidence in the mouse nasopharynx.
Example 15: Murine nasopharyngeal colonization model. Immunization with PE. Challenge with NTHi strain 3224A.
[00263] Female Balb / c mice (20 mice / group) were immunized intranasally on days 0 and 14 with 3pg of PE (from the vector pRIT16762) formulated with LT and on day 28 with 3 pg of PE in PBS. Control mice were vaccinated with LT only. The mice were subsequently challenged intranasally with 5 x 10 ⁶ CFU of NTHi strain 3224A. Bacterial colonies were counted in the nasal cavities removed 3 and 4 days after the challenge. 10 mice were examined on day 3 (D3). 10 mice were examined on day 4 (D4). Vaccination with PE significantly increased the evidence of NTHi in the nasopharynx on day 4 after the challenge (Figure 22), using the Dunn test for statistical analysis.
[00264] See figure 22 for the effect of PE vaccination on bacterial evidence in the nasopharynx of mice.
Example 16: Vitronectin binding. Vitronectin binding inhibition by LVL317 & LVL735 PE-PilA fusion protein.
[00265] The ability of PE in the purified LVL317 PE-PilA fusion protein construct to bind vitronectin was evaluated. The microtiter plates (POLYSORP ^™ , Nunc, Thermo Fisher Scientific) were coated with PE (from the pRIT16762 vector) or with purified LVL317 PE-PilA fusion protein (10 pg / ml). The plates were washed four times with 150mM NaCl-0.05% polysorbate 20 (e.g. TWEEN ^tm 20) and blocked for one to two hours with 1% PBS-BSA. After four washes, vitronectin (Vitronectin from human plasma, SIGMA
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ALDRICH®) was added (10 gg / mL), diluted twice (12 dilutions), and the plates were incubated for 1 hour at room temperature. The plates were then washed 4 times with 150mM NaCl-0.05% polysorbate 20 (for example TWEEN ^TM 20). After washes, bound vitronectin was detected using anti-human vitronectin in peroxidase sheet (US Biological), followed by the addition of the ortho-phenylene diamine / H ₂ O ₂ substrate. The color developed is directly proportional to the amount of antibody attached to the vitronectin.
[00266] See Figure 23 for (A) LVL317 PE-PilA fusion protein bound to vitronectin. PilA = NTHi PilA strain 86-028NP (described for pRIT16790); PE = protein E (described for pRIT16762) and (b) PE-PilA fusion protein from LVL317 and LVL735 bound to vitronectin.
Example 17: Vitronectin binding. Vitronectin binding inhibition by antibodies directed against the LVL291 PEPilA fusion protein.
[00267] The microtiter plates (POLYSORP ^TM , Nunc, Thermo Fisher Scientific) were coated with PE (from the vector pRIT16762), or with purified PE-PilA fusion protein (10 gg / mL). The plates were washed four times with 150mM NaCl-0.05% polysorbate 20 (eg TWEEN ^TM 20) and blocked for two hours with 1% PBS-BSA. After washing, vitronectin (human plasma vitronectin, SIGMAALDRICH®) was added at 50gg / mL and the purified anti-PE-PilA antibodies (produced and purified internally) were diluted twice in series and incubated for 1 hour at room temperature . The plates were then washed 4 times with 150 mM NaCl-0.05% polysorbate 20 (eg TWEEN ^TM 20). After four washes, the bound vitronectin was detected using an anti-citronectin peroxidase sheet (US Biological), followed by the addition of the ortho-phenylene diamine / H2O2 substrate. The color developed is directly proportional to the amount of antibody fixed in the
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110/112 vitronectin.
[00268] Inhibition of vitronectin that binds PE by polyclonal antibodies directed against PE-PilA has been observed.
[00269] See figure 24 for inhibition of vitronectin that binds polyclonal antibodies against PE-PilA fusion protein.
Example 18: Antigenicity of the LVL291 PE-PilA fusion protein. ELISA.
[00270] The purified LVL291 PE-PilA fusion protein was validated in an antigenicity test with monovalent proteins as a control. The fusion protein was tested in a sandwich ELISA developed with polyclonal antibodies (rabbit and guinea pig) generated against the PE gene fragment encoding amino acids 22 to 160 of SEQ ID NO: 4 (described for pRIT16711), or against NTHi PilA strain 86-028NP (from the vector pRIT16790).
[00271] PilA or PE was added at 100 ng / mL and diluted twice in series. After 30 minutes of incubation and after washing, the bound antigen was detected by a polyclonal rabbit serum obtained after immunization with PE or PilA. Bound antibodies were detected using an anti-rabbit Ig peroxidase (Jackson ImmunoResearch Laboratories, Inc.) followed by the addition of the ortho-phenylene-diamine / H2O2 substrate. The color developed is directly proportional to the amount of antigen present. Absorbance readings were measured using a spectrophotometer for microtiter plates. The antigenicity of the samples was determined by comparison with the reference antigen curve of full-size PE or full-size PilA, and is expressed in ug / mL. The reference represented 100% antigenicity.
[00272] As seen in Table 6, antigenicity was observed with the purified LVL291 PE-PilA fusion protein, compared to the PE and PilA monovalent antigens.
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Table 6: Relative antigenicity obtained with the PE-PilA fusion protein from
LVL291 in the
Relative antigenicity of PE (%) protein E as a reference 100 PE-PilA 130-148Relative antigenicity of PE (%) PilA as a reference 100 PE-PilA 120-152
Example 19: Immunogenicity of LVL735 PE-PilA fusion protein.
[00273] Female Balb / c mice (n = 34) were immunized intramuscularly on days 0, 14 and 28 with 50 pL of vaccine formulation containing 1, 0.2 or 0.04 pg of PE- PilA LVL317 or LVL735 formulated with AS01e or AlPO4 (aluminum phosphate). Antibody responses to PE and PilA were determined in individual sera collected on day 42 and the level of IgG against PE and PilA was measured and expressed in pg / mL.
[00274] See figure 27 for the PE and PilA antibody response to LVL317 and LVL735. GMC = geometric mean of the concentration. GMT = geometric mean of the titration. CI = confidence intervals.
Example 20: Protective efficacy of LVL735 and LVL317 fusion proteins in a mouse model of nasopharyngeal colonization of Haemophilus influenzae not typeable.
[00275] Female Balb / c mice were immunized intranasally on days 0 and 14 with 10 µl of vaccine formulation containing 5.8 µg of LVL735 or LVL317 mixed with 0.5 µg of labile E. coli toxin (LT). A booster dose of 5.8 pg of LVL735 or LVL317 without adjuvant was administered on day 28. Control mice were vaccinated with LT only on days 0 and 14, and PBS on day 28. Animals were challenged intranasally with 5 x 10 ⁶ cfu of NTHi 3224A strain on day 42. Bacterial colonies were counted in the nasal cavities removed 1 and 2 days after the challenge (n = 10 / time point).
[00276] The nasal cavities are homogenized in medium and one
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112/112 bacterial quantification is performed. The results are expressed in CFU / mL.
[00277] See figure 28 for the effect of vaccination with LVL735 and LVL317 on bacterial evidence in a mouse model of nasopharyngeal colonization with non-typable Haemophilus influenzae.

权利要求:
Claims (24)
[1]
1. Fusion protein of formula I:
(X) m - (R1) n - A - (Y) o - B - (Z) p (formula I) characterized by the fact that:
X is a signal peptide or MHHHHHH (SEQ ID NO. 2);
m is 0 or 1;
R1 is an amino acid;
n is 0, 1, 2, 3, 4, 5 or 6;
A is an immunogenic fragment of protein E selected from SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 179 or SEQ ID NO: 180 or a sequence having at least 95% sequence identity to any one of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO : 179 or SEQ ID NO: 180;
Y is selected from the group consisting of GG, SG, SS, GGG and (G) _h where h is 4, 5, 6, 7, 8, 9, or 10;
o is 0 or 1;
B is an immunogenic fragment of PilA at least 95% identical to amino acids 40-149 of any one of SEQ ID NO: 58 to SEQ ID NO: 121;
Z is GGHHHHHH (SEQ ID NO. 3); and p is 0 or 1.
[2]
2. Fusion protein, according to claim 1, characterized by the fact that X is the signal peptide of a protein selected from the group consisting of FlgI, NadA and pelB.
[3]
3. Fusion protein according to claim 1, characterized by the fact that m is 0.
[4]
Fusion protein according to any one of claims 1 to 3, characterized in that A is a fragment
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2/5 immunogenic H. influenzae protein E selected from the group consisting of amino acids 17-160 of SEQ ID NO: 4 (SEQ ID NO: 122), amino acids 18-160 of SEQ ID NO: 4 (SEQ ID NO: 123 ), amino acids 19160 of SEQ ID NO: 4 (SEQ ID NO: 124), amino acids 20-160 of SEQ ID NO: 4 (SEQ ID NO: 125), amino acids 22-160 of SEQ ID NO: 4 (SEQ ID NO : 126), amino acids 23-160 of SEQ ID NO: 4 (SEQ ID NO: 179) and amino acids 24-160 of SEQ ID NO: 4 (SEQ ID NO: 180).
[5]
Fusion protein according to any one of claims 1-4, characterized in that B is an immunogenic fragment of PilA consisting of amino acids 40-149 of any of SEQ ID NO: 58 through SEQ ID NO: 121 .
[6]
Fusion protein according to any one of claims 1-5, characterized by the fact that B is an immunogenic fragment of H. influenzae PilA as established in SEQ ID NO: 127.
[7]
Fusion protein according to any one of claims 1-6, characterized in that A is SEQ ID NO: 125.
[8]
8. Fusion protein according to any one of claims 1-6, characterized by the fact that A is SEQ ID NO: 124.
[9]
Fusion protein according to any one of claims 1-4, characterized in that B is a fragment of PilA as set out in SEQ ID NO: 127 and A is an immunogenic fragment of Protein E selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 125 and SEQ ID NO: 126.
[10]
10. Fusion protein according to claim 1, characterized in that it is selected from the group consisting of SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192,
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3/5
SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO. 204.
[11]
11. Fusion protein according to claim 1, characterized in that it is selected from the group consisting of SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200,
SEQ ID NO. 202 or SEQ ID NO. 204, in which the signal peptide was removed.
[12]
12. Fusion protein according to claim 1, characterized in that it is as in SEQ ID NO: 148.
[13]
13. Fusion protein according to claim 1, characterized in that it is as in SEQ ID NO. 177 (QIQKAEQN
DVKLAPPTDV HFDAVVNLDK VRTDFYDEFW ICANYGEAFS YSTNETTNCT GDGTLANMEY CGSVTQ).
[14]
14.
RSGYIRLVKN
GLYVYPEPKR
GQGLRAAPKK VDKKGGTKKA
GGKNGIAADI
ILQATGNAAT
VNYYIDSESI
YARSVRQYKI
QKKHTLSLTP
AVSELLQASA
TTAKGYVKSV
GVTWTTTCKG
WVDNQEPQIV
LNCANYHLTQ
DTTLYNAAQI
PYKADVELCV
TTSNGAITVK
TDASLFPANF
Fusion protein according to claim 1, characterized in that it is as in SEQ ID NO: 194.
[15]
15. Fusion protein according to claim 1, characterized in that it is as in SEQ ID NO. 219 (IQKAEQND
VKLAPPTDVR
FDAVVNLDKG RTDFYDEFWG CANYGEAFSV STNETTNCTG
SGYIRLVKNV
LYVYPEPKRY
QGLRAAPKKQ
DKKGGTKKAA
GKNGIAADIT
NYYIDSESIW
ARSVRQYKIL
KKHTLSLTPD
VSELLQASAP
TAKGYVKSVT
VDNQEPQ IVH
NCANYHLTQV
TTLYNAAQII
YKADVELCVY
TSNGAITVKG
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4/5
DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ).
[16]
16. Immunogenic composition, characterized by the fact that it comprises fusion proteins as defined in any one of claims 1 to 15.
[17]
17. Immunogenic composition according to claim 16, characterized by the fact that it further comprises H. influenzae protein D.
[18]
18. Immunogenic composition according to claim 16 or claim 17, characterized in that it further comprises an adjuvant, optionally wherein the adjuvant is AS01, for example, AS01B or AS01E.
[19]
19. Vaccine, characterized by the fact that it comprises the fusion protein as defined in any of claims 1-15, or immunogenic compositions as defined in any of claims 16-18.
[20]
20. Fusion protein according to claims 1-15, or immunogenic composition according to claims 16-18, or vaccine according to claim 19, characterized by the fact that it is for use in the treatment or prevention of otitis average.
[21]
21. Fusion protein, immunogenic composition, or vaccine according to claims 1-19, characterized by the fact that it is for use in the treatment or prevention of exacerbations of acute chronic obstructive pulmonary disease (AECOPD); and / or for use in treating or preventing pneumonia; and / or for use in the treatment or prevention of infection or illness associated with H. influenzae.
[22]
22. Fusion protein, immunogenic composition, or vaccine according to claims 1-19, characterized by the fact that it is for use in the treatment or prevention of exacerbations of obstructive pulmonary disease
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5/5 acute chronic (AECOPD).
[23]
23. Fusion protein according to claims 1-15, characterized by the fact that it is for use in inducing an immune response against H. influenzae in humans.
[24]
24. Process for producing periplasmic expression of a fusion protein as defined in any one of claims 1-15, characterized in that the process comprises inducing expression of proteins containing a signal peptide, wherein the signal peptide is FlgI or pelB.

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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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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2019-09-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-03-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-05-12| 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 12/04/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161474779P| true| 2011-04-13|2011-04-13|

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US61/474779|2011-04-13|

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US201161534012P| true| 2011-09-13|2011-09-13|

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US61/534012|2011-09-13|

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PCT/CA2012/050236|WO2012139225A1|2011-04-13|2012-04-12|Fusion proteins and combination vaccines comprising haemophilus influenzae protein e and pilin a|

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