IMMUNOGENIC COMPOSITION PRESENTING MULTIPLE ANTIGENS, AND METHODS AND USES OF THE SAME

专利摘要:
IMMUNOGENIC COMPOSITION PRESENTING MULTIPLE ANTIGENS, AND METHODS AND USES OF THE SAME. The present embodiments relate to an immunogenic system having multiple antigens comprising a polymer to which antigens are associated by complementary affinity molecules. For example, the polymer can be a polysaccharide, or antigenic polysaccharide, to which peptide or protein antigens from them or from different pathogens are indirectly linked. The present immunogenic compositions can elicit both humoral and cellular immune responses to one or multiple antigens at the same time
公开号:BR112013028887B1
申请号:R112013028887-6
申请日:2012-05-11
公开日:2020-10-06
发明作者:Richard Malley；Yingjie Lu；Fan Zhang
申请人:Children's Medical Center Corporation；
IPC主号:

专利说明:

GOVERNMENT SUPPORT
This invention was made with government support under Concessions number AI067737 01 and AI51526-01, granted by the National Institutes of Health. The Government of the United States of America has certain rights in the invention. CROSS REFERENCE TO RELATED ORDERS
This application claims benefit under 35 USC § 119 (e) of US Provisional Application 61 / 484,934 filed on May 11, 2011, Provisional Application US 61 / 608,168 filed on March 8, 2012, and Provisional Application US 61 / 609,974 filed on March 13, 2012, the contents of which are incorporated herein in full by reference. ARTICLE I. FIELD OF THE INVENTION
The present invention relates to molecular genetics, immunology and microbiology. The present application is generally directed to compositions and methods for the preparation of immunogenic compositions. More specifically, an embodiment of the present invention provides an immunogenic macro-complex comprising at least one protein or peptide antigen bound to a polymer, such as a polysaccharide, which can also be an antigen. In some embodiments, this complex can be used as an immunogenic composition, such as a vaccine, to confer a synergistic humoral and cellular immune response, and in some embodiments elicited antibody-mediated protection and synergistic cells against pathogens, for example, lethal infection and mucosal support of such pathogens. BACKGROUND OF THE INVENTION
Vaccines provide prevention and treatment of a variety of diseases, including infection by microorganisms, viral infection, and cancers. Current polysaccharide-based vaccines, however, are not always effective in more vulnerable populations. For example, infections by Streptococcus pneumonia (pneumococci) and Salmonella typhi are two of the main diseases for children in developing countries. For typhoid fever, licensed Vi polysaccharide vaccines are ineffective in children under two years of age. However, the success of polysaccharide-based vaccines and passive immunization for the prevention of colonization or disease have demonstrated the importance of capsular antibodies, particularly in the control of diseases caused by S. pneumoniae. In addition, studies in animals and humans demonstrated that antibodies elicited from pneumococcal vaccination can protect against pneumococcal nasopharyngeal colonization (PN), which precedes pneumococcal disease.
The limitation of current pneumococcal polysaccharide vaccines is that protection by anticapsular antibodies is limited by their serotype specificity. For example, although the 7-valued pneumococcal conjugate vaccine (PCV7) has significantly reduced the incidence of invasive pneumococcal disease due to vaccine-type (VT) strains, recent studies have shown that non-VT serotypes are gradually replacing pneumococcal VT populations , potentially limiting the vaccine's usefulness. This led to the assessment that pneumococcal colonization can be prevented by immunization with conserved antigens. In particular, several pneumococcal proteins have been evaluated as vaccine candidates in animal models of pneumococcal colonization. Mucosal immunization with some of these proteins has been shown to elicit systemic and mucosal antibodies to provide protection against pneumococcal disease and colonization. There remains a need for an immunogenic composition, which includes both polysaccharides and pneumococcal proteins, capable of creating robust cellular and humoral immune responses for all pneumococcal serotypes.
In addition, the innate immune response provides a rapid and generally effective defense against microbial pathogens. This response involves the cellular recognition of molecules associated with pathogens, triggering the production and release of inflammatory mediators, leukocyte recruitment and activation of antimicrobial effectors. Toll-like receptors (TLRs), of which at least eleven have been described for mammals, are capable of discriminating between a wide variety of molecules associated with pathogens and eliciting protective responses. For example, TLR4 recognizes many microbial products, including those from gram-negative bacteria, protein F from the respiratory syncytial virus and cholesterol-dependent cytolysins (CDC) from gram-positive bacteria. In addition, TLR2 recognizes a large number of synthetic and microbial compounds. Thus, the inclusion of such TLR agonists can enhance the immune response to vaccines. There remains a need to improve the effectiveness of vaccines by eliciting an innate immune response (mediated by TLR or others) against infections, such as colonization and pneumococcal disease. SUMMARY OF THE INVENTION
The present invention provides an immunogenic system featuring multiple antigens (MAPS), usable for the production of immunogenic compositions, such as those that are usable in vaccines. In particular, the present invention relates to compositions comprising an immunogenic complex comprising at least one type of polymer, for example, a polysaccharide, which can optionally be antigenic; at least one antigenic protein or peptide, and at least one pair of complementary affinity molecules comprising (i) a first affinity molecule that associates with the polymer, and (ii) a complementary affinity molecule that associates with protein or peptide; such that the molecules of first and complementary affinity serve as an indirect link between the polymer I and the antigenic protein or peptide. In this way, the polymer can fix at least 1, or at least 2, or a plurality of the same or different protein or peptide antigens. In some embodiments, the polymer is antigenic, for example, the polymer is a pneumococcal capsular polysaccharide. In some embodiments, protein or peptide antigens are recombinant protein or peptide antigens.
The immunogenic compositions as described herein can elicit both humoral and cellular responses to one or multiple antigens at the same time. Immunogenic compositions provide a long-term memory response, potentially protecting an individual from future infection. This makes it possible to obtain a single immunogenic composition that creates a high titer of functional anti-polysaccharide antibodies, and is similar or compares favorably with the level of antibodies induced by the conventional conjugate vaccine. In addition, there is no restriction on the specific support protein, and several antigen proteins can be used in the MAPS construct to generate a robust anti-polysaccharide antibody response. In addition, the antibody response and the strong Thl7 / Thl responses are specific for multiple protein antigens presented through the MAPS composition. This represents a great advantage, as a means to elicit two forms of immunity with a construct. In addition to a more conventional immune response to an antigenic polysaccharide conjugated to a protein support, the present invention provides a T cell response and, more specifically, Th17 and Th1 responses to systemically injected proteins. In addition, the present immunogenic composition can incorporate ligands on the dorsal structure of the polymer. This provides a potential to enhance the responses of specific B cells or T cells by modifying the protein / polymer ratio, complex size, or by incorporating a specific costimulatory factor, such as the TLR2 / 4 ligands, etc., in the composition.
In comparison with the typical conjugation technology, which involves the severe treatment of proteins, the present methods avoid the risk of denaturing another modification of the peptide antigen. This provides a substantial advantage in preserving the antigenicity of included proteins and increases the likelihood that the protein itself will serve as an antigen (rather than just as a support). Likewise, current methods prevent unnecessary modification / damage to the polysaccharide's dorsal structure, because there is no heavy chemical crosslinking: biotinylation can be precisely controlled to react with specific polysaccharide functional groups, and the level of biotinylation can be easily adjusted. This is advantageous to avoid the typical process of conjugation, which results in damage to the critical side chains or epitopes, which can cause reduced immunogenicity and protection.
The present affinity-based assembly offers easy and highly flexible preparation of the immunogenic composition. It is highly specific and stable, and it can stay in the cold for months and retain its potency.
The assembly process is simple enough to guarantee high reproducibility, there are only a few necessary steps, which reduces the risk of variation from batch to batch, of great industrial advantage. MAPS assembly is highly efficient (above 95%), even at low concentrations of protein and polysaccharide (such as 0.1 mg / ml), which is a great advantage, because inefficiencies in the manufacture of conjugates (typically efficiencies are in the range of <50%) represent a major obstacle and reason for high vaccine costs. For formulation: it is easy to adjust the composition and physical properties of the final product. The protein: polymer ratio in the complex is adjustable; with moderate biotinylation of polymer, protein: polymer can be 10: 1 (weight / weight) or more and, conversely, the ratio can be 1:10 or less, if this is the interest based on immunological targets. In addition, the size of the immunogenic MAPS composition can be adjusted by choosing the size of the polymer. The methods of making MAPS make it easy to combine proteins and polymers with little modification. The possible multivalence of the final product, through the loading of multiple protein antigens, from the same or different pathogens (for example, pneumococci and tuberculosis), in a single immunogenic construct, provides a composition that can be used to decrease the number of vaccines necessary to immunize an individual against more than one disease. In addition, the MAPS composition is highly stable; becoming dissociated only by boiling and maintaining immunogenicity even after several months at 4 ° C. The immunogenicity of the MAPS complex can be limited by the stability of the antigenic protein or peptide component, whose stability can be extended through inclusion in the MAPS complex. The specific antigens used here showed stability at room temperature and after at least one freeze-thaw cycle. This provides an important advantage over current vaccines that are compromised if the "cold chain" is not maintained carefully.
Thus, an aspect of the present invention relates to an immunogenic composition comprising a polymer, at least one protein or peptide antigen, and at least one pair of complementary affinity molecules, wherein the pair of complementary affinity molecules comprises a first affinity molecule that associates with the polymer and a complementary affinity molecule that associates with the protein or peptide antigen, so that when the first affinity molecule associates with the complementary affinity molecule, it indirectly binds the antigen to the polymer.
In some embodiments, the first affinity molecule is cross-linked to the polymer with a cross-linking reagent, for example, a cross-linking reagent selected from CDAP (l-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC (1- hydrochloride hydrochloride ethyl-3- [3-dimethylaminopropyl] carbodiimide), sodium cyanoborohydride; cyanogen bromide, or ammonium bicarbonate / iodoacetic acid. In some embodiments, the first affinity molecule is cross-linked to functional groups carboxyl, hydroxyl, amino, phenoxy, hemiacetal, polymer mercapto. In some embodiments, the first affinity molecule is covalently linked to the polymer.
In some embodiments, the first affinity molecule is biotin or a derivative thereof, or a molecule with a similar physical structure or property like biotin, for example, an amine-PEG3-biotin ((+) - biotinylation-3- 6, 9-trixaundecanediamine) or its derivative.
In some embodiments, the protein or peptide antigen of the immunogenic composition is a fusion protein comprising the antigenic protein or peptide fused to the complementary affinity binding molecule. The fusion can be a genetic construct, that is, a recombinant fusion peptide or protein. In some embodiments, an antigen can be covalently attached to a fusion protein with the complementary affinity molecule. In alternative embodiments, the antigen is non-covalently attached to the complementary affinity molecule.
In some embodiments, the complementary affinity molecule is a biotin-binding protein or a derivative or a functional portion thereof. In some embodiments, a complementary affinity molecule is a protein, of the avidin type or a derivative thereof or a functional portion thereof, for example, but not limited to, rhizavidine or a derivative thereof. In some embodiments, a complementary affinity molecule is avidin or streptavidin or a derivative or functional portion thereof.
In some embodiments, a secretion signal peptide is located at the N-terminus of the avidin-like protein. Any signal sequence known to those skilled in the art can be used, and, in some embodiments, the signal sequence is MKKIWLALAGLVLAFSASA (SEQ ID NO: 1) or a derivative or functional portion thereof. In some embodiments, the antigen can be fused to a complementary affinity molecule via a flexible linker peptide, wherein the flexible linker peptide attaches the antigen to the complementary affinity molecule.
In some embodiments, the polymer component of the immunogen comprises a polymer derived from a living organism, for example, a polysaccharide. In some embodiments, a polymer can be purified and isolated from a natural source, that is, it can be synthesized as with a natural composition / structure, or it can be a synthetic polymer (for example, with an artificial composition / structure) ). In some embodiments, a polymer is derived from an organism selected from the group consisting of: bacteria, archeobacteria, or eukaryotic cells, such as fungi, insects, plants, or chimeras thereof. In some embodiments, the polymer is a polysaccharide derived from a pathogenic bacterium. In specific forms, the polysaccharide is a pneumococcal capsular polysaccharide, a pneumococcal cell wall polysaccharide, or a Salmonella typhi Vi polysaccharide.
In some embodiments, a polymer of the immunogenic composition, as described herein, is a branched-chain polymer, for example, a branched polysaccharide, or, alternatively, it can be a straight-chain polymer, for example, a single-chain polymer , for example, polysaccharide. In some embodiments, the polymer is a polysaccharide, for example, dextran or a derivative thereof. In some embodiments, a polymer, for example, dextran polysaccharide can have an average molecular weight of 425kD-500kDa, inclusive, or in some embodiments, greater than 500kDa. In some embodiments, a polymer, for example, dextran polysaccharide can have an average molecular weight of 60kD-90kDa, inclusive, or in some embodiments, less than 70 kDa. The dextran polymer can be derived from a bacterium, such as Leuconostoc mesenteroides.
In some embodiments, an immunogenic composition, as described herein, comprises at least 2 antigens, or at least 3 antigens, or at least 5 antigens, or between 2-10 antigens, or between 10-15 antigens, or between 15 -20 antigens, or between 20-50 antigens, or between 50 100 antigens, or more than 100 antigens, inclusive. In some embodiments, where an immunogenic composition, as described herein comprises at least 2 antigens, the antigens can be the same antigen or at least 2 different antigens. In some embodiments, the antigens can be of the same or different pathogens, or they can be different epitopes or parts of the same antigenic protein, or they can be the same antigen that is specific for different serotypes or seasonal variations of the same pathogen (for example, influenza virus A, B, and C).
In some embodiments, an immunogenic composition, as described herein, comprises an antigen from a pathogenic organism or abnormal tissue. In some embodiments, the antigen is a tumor antigen. In some embodiments, an antigen can be at least an antigen selected from antigens of pathogens or parasites, such as the antigens of Streptococcus pneumoniae, Mycobacterium tuberculosis or M. tetanus, Bacillus anthracis, HIV, seasonal or epidemic flu antigens (such as H1N1 or H5N1), Bordetella pertussis, Staphylococcus aureus, Neisseria meningitides or N. gonorrhoeae, HPV, Chlamydia trachomatis, HSV or other herpes viruses, or Plasmodia sp. These antigens can include peptides, proteins, glycoproteins, or polysaccharides. In some embodiments, the antigen is a toxoid or portion of a toxin.
In some embodiments, an immunogenic composition, as described herein, comprises an antigenic polysaccharide, for example, such as Vi antigen (Salmonella typhi capsular polysaccharide), pneumococcal capsular polysaccharides, pneumococcal cell wall polysaccharides, Hib {Haemophilus capsular polysaccharide influenzaetype B), meningococcal capsular polysaccharide, Bacillus anthracis polysaccharide (the anthrax causing agent), and other bacterial capsular or cellular network polysaccharides, or any combinations thereof. The polysaccharide can have a protein component, for example, a glycoprotein, such as those from viruses.
In some embodiments, an immunogenic composition, as described herein, further comprises at least one costimulation factor associated with the polymer or polysaccharide, in which the costimulation factor can be associated directly or indirectly. For example, in some embodiments, a costimulation factor may be covalently attached to the polymer. For example, in some embodiments, a costimulation element can be covalently attached to the first affinity molecule, which is then cross-linked with the polymer. For example, in some embodiments, a costimulation element can be attached to a complementary affinity molecule, which associates with a first affinity molecule to bind the costimulation factor to the polymer.
In some embodiments, a costimulation factor is an adjuvant. In alternative embodiments, a co-stimulatory factor can be any known to the person skilled in the art, and includes any combination, for example, without limitation, Toll-type receptor agonists (TLR2, 3, 4, 5 7 agonists, 8, 9, etc.), NOD agonists, or inflammasome agonists.
Another aspect of the present invention relates to the use of the immunogenic composition, as described herein, to be administered to an individual to induce an immune response in the individual. In some embodiments, the immune response is an antibody / B cell response, a CD4 + T cell response (including Thl, Th2 and Thl7) and / or a CD8 + T cell response. In some embodiments, at least one adjuvant is administered in conjunction with the immunogenic composition.
Another aspect of the present invention relates to a method for inducing an immune response in an individual to at least one antigen, comprising administering to the individual the immunogenic composition, as described herein.
Another aspect of the present invention relates to a method of vaccinating an animal, for example, a bird, a mammal or a human, against at least one antigen, comprising administering a vaccine composition comprising the immunogenic composition, such as described here.
In all respects, as shown here, an animal or an individual can be a human. In some embodiments, the individual may be an agricultural or non-domestic animal, or a domestic animal. In some embodiments, a vaccine composition comprising the immunogenic composition as described herein can be administered subcutaneously, intranasally, buccally, sublingually, vaginally, rectally, intradermally, intraperitoneally, intramuscularly, or adhesive for transcutaneous immunization.
In all respects, as shown here, a Jimune response is an antibody / B cell response, a CD4 + T cell response (including Thl, Th2 and Thl7 responses) or a CD8 + T cell response against the antigen (s) ( s) protein / peptide. In some embodiments, an immune response is an antibody / B cell response against the polymer, for example, a pneumococcal polysaccharide. In some embodiments, at least one adjuvant is administered in conjunction with the immunogenic composition.
Another aspect of the present invention relates to the use of the immunogenic composition, as described herein, for use in a diagnosis for exposure to a pathogen or immunogenic agent.
Another aspect of the present invention relates to kits for the preparation of an immunogenic composition, as described herein. For example, such kits can comprise any one or more of the following materials: a container comprising a polymer, for example, a cross-linked polysaccharide with a plurality of first affinity molecules, and a container comprising a complementary affinity molecule that associates with the first affinity molecule, wherein the complementary affinity molecule associates with an antigen.
In another embodiment, the kit may comprise a container comprising a polymer, for example, a polysaccharide, a container comprising a plurality of first affinity molecules, and a container comprising a crosslinking molecule for the crosslinking of the first affinity molecules polymer. In some embodiments, the kit may comprise at least one costimulation factor, which can be added to the polymer. In some embodiments, the kit comprises a crosslinking reagent, for example, but not limited to, CDAP (l-caryl-4-dimethylaminopyridinium tetrafluoroborate), EDC (l-ethyl-3- [3-dimethylaminopropyl] hydrochloride] carbodiimide), sodium cyanoborohydride; cyanogen bromide; ammonium bicarbonate / iodoacetic acid to bind the cofactor to the polymer or polysaccharide. In some embodiments, the kit further comprises a means for fixing the complementary affinity molecule for the protein or peptide antigen, wherein the medium can be by a cross-linking reagent or by some intermediate fusion protein.
In some embodiments, the kit may comprise a container comprising an expression vector for the expression of a protein or peptide antigen fusion protein - affinity molecule, for example, an expression vector for expressing the protein antigen or peptide as a fusion protein with the complementary affinity molecule. In some embodiments, the vector may optionally comprise a sequence for a peptide linker, wherein the expression vector can express a complementary affinity antigen-molecule fusion protein comprising a linker peptide located between the antigen and the molecule of affinity.
In some embodiments, the kit may optionally comprise a container comprising a complementary affinity molecule that associates with the first affinity molecule, wherein the complementary affinity molecule associates with a peptide / protein antigen. In some embodiments, the kit may further comprise a means for fixing the complementary affinity molecule to the antigen, for example, using a cross-linking reagent, as described herein, or another intermediate protein, such as a divalent antibody or antibody fragment.
Also provided herein is a method of vaccinating an individual, for example, a mammal, for example, a human, with the immunogenic compositions, as described herein, the method comprising administering a vaccine composition, as described herein, to the individual. DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram of the system showing multiple antigens (MAPS). MAPS represents a new platform for a complex immunogenic composition, which is done by attaching a number of protein antigens to a polysaccharide or polysaccharide antigen through a stable interaction of an affinity pair, such as avidin-biotin pair. In an embodiment of the MAPS complex, protein antigens from one or more pathogens are recombinantly fused to an avidin-like protein and expressed in E. coli. The dorsal polysaccharide structure, which can be chosen from a variety of pathogens, is biotinylated and / or cross-linked with or without costimulation factors using 1- cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or l hydrochloride -ethyl-3- [3-dimethylaminopropyl] carbodiimide (EDC) as an activating reagent. A MAPS complex can be assembled easily by simply mixing and incubating the purified fusion antigens, one or multiple, in the desired ratio, with biotinylated polysaccharide. The assembled MAPS complex can be purified / separated according to size by gel filtration chromatography.
Figure 2 shows exemplary examples of polysaccharide biotinylation: the structures of the biotin derivative, amine-PEG3-biotin (also known as (+) biotinylation-3-6, 9-trixaundecanediamine), the structure of CDAP and the structure of EDC . The figure also shows a scheme for the polysaccharide biotinylation method using CDAP as the activation reagent, process (1) or using EDC as the activation reagent, process (2). Other procedures for biotinylation are included in the methods of the invention.
Figures 3A-3C show an embodiment of a recombinant rhizavidine and rhizavidine-antigen fusion protein. Figure 3A shows a schematic diagram of the construction of modified rhizavidine (top) and rhizavidine-antigen fusion protein (bottom). All constructs were cloned into the vector pET21b and transformed into E. coli BL21 (DE3) strain for expression. Figure 3B shows purified recombinant rhizavidine (rRhavi) SDS-PAGE. Figure 3C shows SDS-PAGE of purified rhavi-antigen fusion proteins. Lane 1, rhavi-Pdt; lane-2, rhavi-PsaA; lane 3, rhavi-spl733; lane 4, rhavi-spl534; lane 5, rhavi-sp0435; lane 6, rhavi-spl458; lane 7, rhavi-ESAT-6 / Cfpl0; lane 8, rhavi-TB9.8 / TB10.4; lane 9, rhavi-MPT64; lane 10, rhavi-MPT83.
Figures 4A-4C show the elution profile of an example of assembled MAPS. Figure 4 MAPS was mounted by incubating 0.5 pg of purified rRhavi with 1 pg of biotinylated dextran 90 (BD90, average MW 60-90 KD) at 4 ° C overnight and then applied to a column Superdex-200. Pico A and Pico B indicated the eluted fractions containing the MAPS complex, Pico C indicated the eluted fractions containing free rRhavi. Figure 4B shows SDS-PAGE of the peak fractions. All samples were boiled in SDS sample buffer with 10 mM DTT. Figure 4C shows the stability of the MAPS complex. Equal amounts of sample were treated and then applied to SDS - PAGE. The MAPS complex remained intact, even after treatment with SDS sample buffer containing a reducing reagent (lane 1) and can only be broken after boiling, attesting to the stability of the association. Lane 1, MAPS treated with SDS sample buffer containing 10 mM DTT, at room temperature, for 10 min; lane 2, MAPS treated with SDS sample buffer, without DTT, boiled for 1.0 minutes; lane 3, MAPS treated with SDS sample buffer containing 10 mM DTT, boiled for 10 min.
Figure 5 shows the assembly of the MAPS complex at a different temperature and at different concentrations of PS and protein antigen. MAPS complex can be effectively assembled over a wide range of concentrations of polysaccharides (PS) or protein antigen (as low as 0.1 mg / ml). Assembly can be done by incubating overnight at 4 ° C (Figure 5A) or at 25 ° C (Figure 5B), depending on the stability of the antigens. The assembly efficiency of the MAPS complex can be estimated by mixing the assembly using SDS -PAGE, with or without boiling the sample beforehand. Without boiling treatment, the protein antigens that were incorporated into the MAPS complex remained on PS and thus appear as bands of very high molecular weight on the gel (MAPS / PS), and only unbound proteins would bounce in a way lower in the gel and detected by the expected molecular weight of the antigen (monomer or dimer position). By comparing the protein antigen band before and after boiling, the percentage of antigens assembled on the MAPS complex can be estimated. In general, the assembly efficiency at 4 ° C is greater than 85%, and at 25 ° C, which is closer to 95% - 99%.
Figure 6 shows the elution profiles of MAPS assembled with different protein versus polysaccharide ratios. 0.5 pg of purified rRhavi was incubated overnight with either 1 mg, 0.5 pg or 0.1 pg of BD90, respectively; and then applied to gel filtration chromatography using a Superdex 200 column. The MAPS complex assembled in a higher protein versus polysaccharide ratio appeared to have a higher molecular weight than that assembled in a lower ratio. The fractions of the peak containing the MAPS complex for each sample (indicated by arrows) were collected. The protein-to-polysaccharide ratio in the purified MAPS complex was measured and correlated well with the input ratio.
Figure 7 shows the elution profiles of MAPS assembled with different polysaccharide sizes. 0.5 pg of fusion antigen was incubated with 0.25 pg biotinylated dextran with an average molecular weight of 425-500 KD (BD500), 150 KD (BD150) or 60-90 KD (BD90). The MAPS complex was separated using a Superpose 6 column, the chromatography profile showed that the complex assembled with a larger polysaccharide was larger in size.
Figure 8A-8D shows the assembly of MAPS with multiple antigens. Figure 8A shows the assembly of MAPS with two antigens in different ratios. Bivalent MAPS complexes were prepared by incubating biotinylated S. pneumoniae (SP) from capsular polysaccharide serotype 14 with two different pneumococcal fusion antigens rhavi-1652 and rhavi-0757 mixed at a molar ratio of 1: 4, 1: 2, 1 : 1, 2: 1, or 4: 1. SDS-PAGE showed that the amounts of each antigen incorporated into the MAPS complex were well correlated with the input proportions. Figures 8B-8D show a multivalent MAPS complex that was made with biotinylated polysaccharide (dextran, or serotype 3 pneumococcal capsular polysaccharide) connecting two (2V, Figure 8B), three (3V, Figure 8C) or five (5V, Figure 8D) different pneumococcal and / or tuberculosis antigens. SDS-PAGE showed antigens incorporated into the MAPS complex. All samples were boiled in SDS sample buffer with 10 mM DTT.
Figure 9 shows that immunization with MAPS complex induced a strong antibody response against polysaccharide antigens. Mice that were immunized with the MAPS complex from biotinylated dextran (9A), polysaccharide Vi (9B), or pneumococcal cell wall polysaccharide (CWPS) (9C) made from a significantly greater amount of anti-polysaccharide antibodies in comparison with groups of animals that received the adjuvant only (without Ag) or a mixture of non-copulated polysaccharides and proteins (Mixture). Figures 9D-9F show that the MAPS complex compares favorably with conventional conjugate vaccine in the generation of anti-PS Ab. The MAPS complexes were made from capsular polysaccharide SP serotype 1, 5, 14 (CPS), loaded with five protein antigens. Mice were immunized subcutaneously with MAPS or Prevenar 13 ® (13-valued pneumococcal conjugate vaccine [diphtheria CRM197 protein]; Wyeth / Pfizer) (PCV13) twice, two weeks apart, and the serum IgG antibody against CPS vaccine serotype was analyzed 2 weeks after the second ELISA immunization. The anti-CPS IgG titer in mice immunized with PCV13 was arbitrarily fixed at 1,200 units for comparison. For all serotypes tested, immunization with the MAPS complex generated either a similar level (serotype 5) or a much higher level of anti-CPS IgG antibodies (serotype 1 and serotype 14) than that generated by vaccination with PCV13. Serotype 1 (Figure 9D); serotype 5 (Figure 9E); serotype 14 (Figure 9F).
Figure 10 compares the anti-PS antibody induced by MAPS at different doses of immunization. MAPS complex was made from serotype 5 SP CPS loading with five protein antigens. The mice received the MAPS complex at 1 pg-16 pg of PS content per dose, for two immunizations, two weeks apart. Antibodies in serum against serotype 5 CPS were measured and compared between the different immunization groups two weeks after the second immunization. At all doses, immunization with MAPS includes robust IgG antibody against serotype 5 CPS. Giving 2 pg of PS per dose generated the highest antibody titer and increasing the dose to 16 pg of PS reduced the antibody titer about 4 times.
Figure 11 shows that anti-PS antibodies generated by immunization with the MAPS complex facilitate the death of target pathogens in vitro. Figure 11A demonstrates antibody-mediated death of the bacteria expressing Vi. The serum from animals immunized with MAPS complex (using Vi as the dorsal structure), but not from the other two groups, showed a potent extermination of the strain expressing Vi (more than 90% death) within 1 hour of incubation . The serum of mice immunized with alum (dashed line); mixture (black line), or MAPS (gray line). Figures 11B-11D demonstrate that the serum opsonophagocytic killing activity of mice immunized with MAPS compares favorably with the serum killing activity of mice immunized with the licensed PCV13 vaccine. The serum capacity of mice immunized with PCV13- or MAPS to mediate in vitro opsonophagocytic death of pneumococci by neutrophils was analyzed and compared. Human neutrophils were differentiated from cells of the HL-60 cell line. Opsonophagocytic death was performed by incubating the serum, at different dilutions, with serotype 1 (Figure 11B), serotype 5 (Figure 11C) or serotype 13 (Figure 11D), pneumococci and HL-60 cells differentiated at 37 ° C for 1 hour (in the presence of baby rabbit complement). An aliquot of the mixture was plated after incubation to count the surviving bacteria. The opsonophagocytic death unit was defined as the dilution of the serum at times, with 50% of bacterial death being observed. For all serotypes tested, serum from mice immunized with MAPS showed at least 4 times greater killing activity (OPA titer) than serum from mice immunized with PCV13. Figures 11B-11D: Serum from mice immunized with alum (dashed line); PCV13 (black line), or MAPS (gray line).
Figures 12A -12D demonstrate that immunization with a MAPS complex induces a robust antibody and cellular response against protein antigens. Bivalent MAPS complex was made from biotinylated dextran (BD500) and two pneumococcal antigens, rhavi-Pdt and rhavi- PsaA. Subcutaneous vaccines were given fortnightly, three times. Figure 12A shows the results of serum IgG antibodies measured against PsaA or Pdt 2 weeks after the last immunization. Mice immunized with the MAPS complex had significantly higher titers of anti-Pdt and anti-PsaA antibodies than mice that received the mixture. Antigen-specific T cell responses were assessed by in vitro stimulation of whole blood of the immunized animals. The production of IL-17A (Figure 12B) and IFN-y (Figure 12C) in vitro was measured in blood samples incubated for six days with either purified PsaA, Pdt, or pneumococcal whole cell antigen (WCA). In comparison to the mice immunized with the mixture, the animals that received the MAPS complex showed a significantly stronger response to IL-17A and IFN-y. Figure 12D shows a correlation of IL-17A and IFN-y production by WCA stimulation. For all panels, bars represent means with standard deviation and statistical analysis was performed using the Mann-Whitney test, or using Spearman R for correlation.
Figure 13 shows the evaluation of the immunogenicity of the MAPS complex in different sizes. MAPS complexes were made from two pneumococcal fusion antigens, rhavi-PsaA and rhavi-PdT, and using dextran of different length as a dorsal structure (BD500, Mw of 425-500kDa; BD90, Mw of 60-90 kDa) . Antibody responses to dextran, and two protein antigens PdT and PsaA, as well as antigen-specific T cell responses were measured and compared after three immunizations. As shown, mice that were immunized with the larger complex (MAPS BD500) generated a similar level of anti-PsaA and anti-Pdt antibodies (Figure 13B), but the significantly higher anti-dextran antibody titer (Figure 13A), as well as the T-cell response associated with IL-17A (Figure 13C) than the animals that received the minor complex (MAPS BD90).
Figure 14 shows that the addition of costimulatory factors (TLR ligands) to the MAPS complex facilitates the responses of T cells associated with IL-17A and IFN-y. Complex MAPS were made from biotinylated dextran and a pneumococcal protein antigen, rhavi- 0435, with or without TLR ligand / agonist: rhavi-Pdt, TLR4 ligand; Pam3CSK4, additional TLR2 agonist. The incorporation of rhavi-TFD is through the affinity interaction between rhavi and biotin, while Pam3CSK4 is covalently fixed to the dorsal dextran structure. Immunization was administered subcutaneously three times, and T cell responses against the 0435 protein were measured and compared. She showed that the addition of the TLR2 agonist or a combination of TLR4 and TLR2 ligands significantly increased the T cell responses associated with IL-17A and IFN-y to the protein antigen.
Figure 15 shows an example of a multivalent combination vaccine against pneumococcus / tuberculosis (TB) mycobacteria. The multivalent SP / TB combination MAPS vaccine was prepared using serotype SP 3 and carrying an SP protein (pneumolysin toxoid, PdT) and six TB proteins (in four fusion constructs) (Figure 15A). Immunization of mice with SP / TB MAPS induced a higher IgG antibody titer for type 3 CPS (Figure 15B, left panel), as well as for Pdt (Figure 15B, right panel), and led to 100% protection of mice with fatal pulmonary infection by serotype 3 pneumococcus (Figure 15C). Figures 15D-15J show the B cell and T cell immunity antigens induced by SP / TB MAPS vaccination. Figure 15D shows the antibody responses to different TB protein antigens. Figures 15E-15F show strong responses of T cells associated with IL-17A (Figure 15E) and IFN-y (Figure 15F) in a whole blood sample from MAPS-immunized mice after in vitro stimulation with purified TB protein antigens. Figures 15G and 15H show T-cell responses associated with IL-17A (Figure 15G) and IFN-y (Figure 15H) from splenocytes from animals immunized with MAPS for the mixture of purified TB protein antigens or for complete TB cell extract. Figures 151 and 15J provide more data on TB-specific memory / effector T cells induced by MAPS immunization. The results showed that depletion of CD4 + T cells, but not CD8 + T cells, had a significant impact on the production of specific TB antigen cytokines, indicating that immunization with the MAPS vaccine had primarily initiated an immune response from a CD4 + T cell (helper T cells).
Figure 16 demonstrates that a multivalent immunogenic composition based on prototype MAPS prevents invasive infection and nasopharyngeal colonization of pneumococci. Multivalent SP MAPS was made using SP cell wall polysaccharide (CWPS) as the dorsal structure and loaded with five pneumococcal protein antigens (Figure 16a). Mice were immunized with this MAPS SP three times, two weeks apart, and antibodies and specific T cell responses against pneumococcus were analyzed two weeks after the last immunization. Figure 16B shows the serum IgG antibody against CWPS (left panel) or against pneumococcal full cell antigen (WCA) (right panel). Mice immunized with MAPS SP obtained significantly higher antibody titer for either CWPS or WCA than mice in the control group that received adjuvant only (without Ag) or PS / uncoated protein mixture (Mixture). Figures 16C and 16D show SP-specific T cell responses induced by vaccination with MAPS SP. The peripheral blood of mice from different immunization groups was stimulated with either purified pneumococcal proteins (mixture of antigens) or WCA. Cells from mice vaccinated with MAPS, but not from control groups, responded to SP antigens greatly and gave robust production of IL-17A (Figure 16C) and IFN-Y (Figure 16D). Figures 16E and 16F show that MAPS complex vaccination protects mice from invasive infection, as well as nasopharyngeal colonization of pneumococcus. Mice from different immunization groups were challenged either with SP serotype 3 strain WU2 in a lung aspiration model (Figure 16E), or with pneumococcal serotype 6 strain 603 in a nasal colonization model (Figure 16F). Protection against sepsis or colonization was only observed in mice immunized with MAPS. DETAILED DESCRIPTION OF THE INVENTION
It should be understood that this invention is not limited to the methodology, protocols and reagents, particulars, etc., described herein and, as such, may vary. The terminology used here is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
As used herein and in the claims, the singular forms include the reference in the plural and vice versa, unless the context clearly indicates otherwise. The term "or" is inclusive unless modified, for example, by "or". Except in the examples of operation, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all cases by the term "about". It should also be understood that all sizes of bases or sizes of amino acids and all values of molecular weights or molecular masses, provided for nucleic acids or polypeptides are approximate, and are provided for description.
All patents and other publications identified are hereby expressly incorporated by reference, for the purpose of describing and revealing, for example, the methodologies described in these publications that can be used in connection with the present invention. These publications are given for publication only before the filing date of this application. Nothing in this regard should be construed as an admission that inventors are not entitled to anticipate this description by virtue of the previous invention, or any other reason. All statements regarding date or representation regarding the contents of these documents are based on information available to applicants, and do not constitute any admission as to the accuracy of the dates or the content of the documents.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those typically understood by one skilled in the art to which this invention belongs. Although any known methods, devices and materials can be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.
The present invention relates to immunogenic compositions and compositions comprising an immunogenic complex, comprising at least one antigen, or multiple antigens, attached to a polymer scaffold for use in eliciting an immune response to each of the antigens attached to the polymer , and optionally to the polymer itself, when administered to an individual. This system featuring multiple antigens (MAPS) stimulates a humoral and cellular immune response: it can generate anti-polysaccharide antibodies and B / Thl / Thl7 cell responses to multiple protein antigens using a single MAPS immunogenic construct. A combination of B and T cell immunity in the body may represent an optimal vaccine strategy against many diseases, including the invasive infection associated with pneumococcal disease and nasopharyngeal transport. In some embodiments, the immunogenic composition is a vaccine or is included in a vaccine.
That way; one aspect of the present invention relates to an immunogenic composition (system having multiple antigens, or MAPS) comprising at least one polymer, for example, a polysaccharide, at least one protein or peptide antigen, and at least one pair of molecules complementary affinity molecule comprising (i) a first affinity molecule associated with the polymer, and (ii) a complementary affinity molecule associated with the antigen, which serves to indirectly fix the antigen to the polymer (for example, the first affinity molecule associates with the complementary affinity molecule to bind the antigen to the polymer). Thus, as the polymer can be used as a scaffold to fix at least 1, or at least 2, or more than one (for example, a plurality) of the same or different antigens. The immunogenic compositions, as described herein, can be used to elicit both humoral and cellular immunity to multiple antigens at the same time.
Consequently, the embodiments here provide an immunogenic composition and methods usable to create an immune response in an individual, which can be used alone or together or in mixture with, essentially, any existing vaccine approaches. Definitions:
For convenience, certain terms used throughout the application (including the specification, examples, and attached claims) are gathered here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those typically understood by one skilled in the art to which this invention belongs.
The term "immunogenic composition" used herein is defined as a composition capable of eliciting an immune response, such as an antibody or cellular immune response, when administered to an individual. The immunogenic compositions of the present invention may or may not be immunoprotective or therapeutic. When the immunogenic compositions of the present invention prevent, ameliorate, alleviate or eliminate the individual's disease, then the immunogenic composition can optionally be referred to as a vaccine. As used herein, however, the term immunogenic composition is not intended to be limited to vaccines.
As used here, the term "antigen" refers to any substance that initiates an immune response directed against the substance. In some embodiments, an antigen is a peptide or polypeptide, and in other embodiments, it can be any chemical or portion, for example, a carbohydrate, which elicits an immune response directed against the substance.
The term "associates" as used herein refers to the bonding of two or more molecules by non-covalent or covalent bonds. In some embodiments, where the bonding of two or more molecules occurs by covalent attachment, the two or more molecules can be fused together, or crosslinked together. In some embodiments, where the bonding of two or more molecules occurs by a non-covalent bond, the two or more molecules can form a complex.
The term "complex" as used herein refers to a set of two or more molecules, spatially connected by different means than a covalent interaction, for example, they can be connected through electrostatic interactions, hydrogen bonded or by hydrophobic interactions (ie van der Waals forces).
The term "crosslinked" as used herein refers to a covalent attachment formed between a polymer chain and a second molecule. The term "cross-linking reagent" refers to an entity or an agent that is an intermediate molecule to catalyze the covalent attachment of a polymer to an entity, for example, the first affinity molecule or costimulatory factor.
As used here, the term "fused" means that at least one protein or peptide is physically associated with a second protein or peptide. In some embodiments, fusion is typically a covalent fixation, however, other types of bonds are included in the term "fused" and include, for example, bonding through an electrostatic interaction, or a hydrophobic interaction and the like. Covalent attachment can encompass bonding as a fusion protein or chemically copulated bond, for example, by means of a linked disulfide formed between two cysteine residues.
As used herein, the term "fusion polypeptide" or "fusion protein" means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include the translation products of a chimeric gene construct, which joins DNA sequences encoding one or more antigens, or fragments or mutants thereof, with the DNA sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins that are joined by a peptide bond or across several peptides. In some embodiments, the second protein to which the antigens are fused to is a complementary affinity molecule that is capable of interacting with a first affinity molecule of the complementary affinity pair.
The terms "polypeptide" and "protein" can be used interchangeably to refer to a polymer of amino acid residues linked by peptide bonds, and for the purposes of the claimed invention, have a typical minimum length of at least 25 amino acids. The term "polypeptide" and "protein" can encompass a multimeric protein, for example, a protein containing more than one domain or subunit. The term "peptide" as used herein refers to a sequence of amino acids linked to the peptide bond containing less than 25 amino acids, for example, between about 4 amino acids and 25 amino acids in length. Proteins and peptides can be composed of amino acids arranged linearly by peptide bonds, whether produced biologically, recombinantly, or synthetically and whether compounds of naturally occurring or non-naturally occurring amino acids are included within this definition. Both full-length proteins and their fragments greater than 25 amino acids are encompassed by the definition of protein. The terms also include polypeptides that have co-translational modifications (e.g., signal peptide divage) and post-translational modifications of the polypeptide, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation , proteolytic divination (e.g., metalloprotease divination), and the like. In addition, as used herein, a "polypeptide" refers to a protein that includes modifications, such as deletions, additions and substitutions (generally of a conservative nature as would be known to the person skilled in the art), for the native sequence, provided that the protein maintains the desired activity. These changes may be deliberate, such as through site-directed mutagenesis, or they may be accidental, such as through mutations in the hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.
The term "signal sequence" means a nucleic acid sequence which, when operably linked to a nucleic acid molecule, facilitates the secretion of the product (e.g., protein or peptide) encoded by the nucleic acid molecule. In some embodiments, the signal sequence is preferably located 5 'to the nucleic acid molecule.
As used herein, the term "N-glycosylated" or "N-glycosylation" refers to the covalent attachment of a portion of sugar to asparagine residues in a polypeptide. Portions of sugar may include, but are not limited to, glucose, mannose and N-acetylglucosamine. Glycan modifications are also included, for example, sialization.
A "cell containing antigens" or "APC" is a cell that expresses the major histocompatibility complex (MHC) molecules and may have foreign antigen complexed with MHC on its surface. Examples of cells showing antigen are dendritic cells, macrophages, B cells, fibroblasts (skin), thymus epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells and vascular endothelial cells.
The term "functional portion" or "functional fragment", as used in the context of a "functional portion of an antigen" refers to a portion of the antigen or antigen polypeptide that mediates the same effect as the portion of the complete antigen, for example, it elicits an immune response in an individual, or mediates an association with another molecule, for example, comprises at least one epitope.
A "portion" of a target antigen, as the term is used here, will be at least three amino acids in length, and can be, for example, at least 6, at least 8, at least 10, at least 14, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 25 amino acids or more, inclusive.
The terms "cytotoxic T lymphocyte" or "CTL" refer to lymphocytes that induce death by apoptosis or other mechanisms in target-tagged cells. CTLs form antigen-specific conjugates with target cells through interaction of TCRs with processed antigen (Ag) on the surface of the target cells, resulting in apoptosis of the target-tagged cell. Apoptotic bodies are eliminated by macrophages. The term "CTL response" is used to refer to the primary immune response mediated by CTL cells.
The term "cell-mediated immunity" or "IMC" as used herein refers to an immune response that does not involve antibodies or complement but involves the activation of, for example, macrophages, natural killer cells (NK), cytotoxic T lymphocytes specific antigens (T cells), helper T cells, neutrophils and the release of various cytokines in response to a target antigen. In other words, CMI refers to immune cells (such as T cells and other lymphocytes) that bind to the surface of other cells that exhibit a target antigen (such as cells presenting antigens (APC)) and trigger a response. The answer may involve other lymphocytes and / or any of the other white blood cells (leukocytes) and the release of cytokines. Cellular immunity protects the body by: (i) activation of antigen-specific cytotoxic T lymphocytes (CTLs) that are capable of destroying body cells by displaying foreign antigen epitopes on their surface, such as cells infected with viruses and cells with intracellular bacteria, (2) activation of macrophages and NK cells, allowing them to destroy intracellular pathogens, and (3) stimulating cells to secrete a variety of cytokines or chemokines, which influence the function of other cells, such as T cells, macrophages or neutrophils involved in adaptive immune responses and innate immune responses.
The term "immune cell" as used herein refers to any cell that can release a cytokine, chemokine or antibody in response to a direct or indirect antigenic stimulus. Included in the term "immune cells" here are lymphocytes, including natural killer cells (NK), T cells (CD4 + and / or CD8 +), 6 cells, macrophages, leukocytes, dendritic cells; mast cells; monocytes and any other cell that is capable of producing a cytokine or chemokine molecule in response to direct or indirect antigen stimulation. Typically, an immune cell is a lymphocyte, for example, a T-cell lymphocyte.
The term "cytokine", as used herein, refers to a molecule released from an immune cell in response to stimulation with an antigen. Examples of these cytokines include, but are not limited to: GM-CSF; IL-lα; IL-lβ; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F or other members of the IL-17, IL-22, IL-23, IFN-ot family; IFN-β; IFN-y; MIP-lα; MIP-lβ; TGFβ; TNFa or TNFβ. The term "cytokine" does not include antibodies
The term "individual" as used herein refers to any animal in which it is useful in eliciting an immune response. The individual may be a wild, domestic, commercial or companion animal, such as a bird or mammal. The individual may be a human. Although in an embodiment of the invention it is contemplated that immunogenic compositions, as described herein, may also be suitable for therapeutic or prophylactic treatment in humans, but it is also applicable to warm-blooded vertebrates, for example, mammals, such as primates nonhumans (particularly upper primates), sheep, dogs, rodents (eg mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals such as chickens, ducks or turkeys. In another setting, the individual is a wild animal, for example, a bird, such as for the diagnosis of avian influenza. In some embodiments, the individual is an experimental animal or animal or an animal substitute as a model of disease. The individual may be an individual in need of veterinary treatment, where eliciting an immune response to an antigen is useful to prevent a disease and / or to control the spread of a disease, for example, SIV, STL1, SFV, or in the case of livestock, foot-and-mouth disease, or in the case of birds, Marek's disease or avian influenza, and other such diseases.
As used here, the term "pathogen" refers to an organism or molecule that causes a disease or disorder in an individual. For example, pathogens include, but are not limited to, viruses, fungi, bacteria, parasites and other infectious organisms or molecules thereof, as well as by taxonomically related macroscopic organisms within the categories of algae, fungi, yeasts, protozoa, or the like.
A "cancer cell" refers to a precancerous cell, either cancerous or transformed, either in vivo, ex vivo, or in tissue culture, which has spontaneous or induced phenotypic changes that do not necessarily involve the absorption of new material genetic. Although the transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or the absorption of exogenous nucleic acid, it can also appear spontaneously or after exposure to a carcinogen, thereby mutating an endogenous gene. Transformation / cancer is associated with, for example, morphological changes, immortalization of cells, control of aberrant growth, formation of foci, anchorage independence, malignancy, loss of contact inhibition and limitation of growth density, growth factor or independence of serum, specific tumor markers, invasiveness or metastasis, and tumor growth in appropriate animal hosts, such as nude mice. See, for example, Freshney, CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994). !
The term "wild type" refers to the sequence of naturally occurring, normal polynucleotides encoding a protein, or a portion thereof, or protein sequence, or a portion thereof, respectively, as typically exists in vivo.
The term "mutant" refers to an organism or cell with any change in its genetic material, in particular, a change (i.e., deletion, substitution, addition or alteration) in relation to a wild-type polynucleotide sequence or any change relative to a sequence of wild type proteins. The term "variant" can be used interchangeably with "mutant". While it is generally assumed that a change in genetic material results in a change in protein function, the terms "mutant" and "variant" refer to a change in the sequence of a wild-type protein, regardless of whether the change changes the protein function (for example, increases, decreases, gives a new function), or if the change has no effect on the function of the protein (for example, mutation or variation is silent).
The term "pharmaceutically acceptable" refers to compounds and compositions that can be administered to mammals without undue toxicity. The term "pharmaceutically acceptable carriers" excludes tissue culture medium. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and others, and organic acid salts such as acetates, propionates, malonates, benzoates, and others. Pharmaceutically acceptable vehicles are well known in the art.
It will be appreciated that proteins or polypeptides often contain amino acids other than the 20 amino acids typically referred to as the 20 naturally occurring amino acids and that many amino acids, including terminal amino acids, can be modified in a given polypeptide by natural processes, such as glycosylation and other post-translational modifications, or by chemical modification techniques that are well known in the art. Known modifications that may be present in polypeptides of the present invention include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of flavin, covalent fixation of a heme portion, covalent fixation of a polynucleotide or polynucleotide derivative , covalent fixation of a lipid or lipid derivative, covalent phosphotidylinositol fixation, crosslinking, cyclization, disulfide bonding, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formulation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer - mediated RNA addition of amino acids to proteins, such as arginylation, and ubiquitination.
As used here, the terms "homologues" or "homologues" are used interchangeably, and, when used to describe a polynucleotide or polypeptide, indicate that two polynucleotides or polypeptides, or designated sequences thereof, when optimally aligned and compared, for example, using BLAST programs, version 2.2.14 with default parameters for alignment are identical, with appropriate insertions or deletions of nucleotides or insertions or deletions of amino acids or, typically at least 70% of the nucleotides of the high homology nucleotides. For a polypeptide, there must be at least 30% amino acid identity with the polypeptide, or at least 50% greater homology. The term "homologous" or "homologous" as used herein also refers to homology with respect to structure. Determination of gene or polypeptide homologs can be easily determined by one skilled in the art. When in the context with a defined percentage, the percentage of defined homology means, at least, the percentage of similarity of amino acids. For example, 85% homology refers to at least 85% amino acid similarity.
As used herein, the term "heterologous" with reference to sequences of nucleic acids, proteins or polypeptides means that these molecules are not naturally occurring in this cell. For example, the nucleic acid sequence encoding a fusion antigen polypeptide described herein that is inserted into a cell, for example, in the context of a protein expression vector, is a heterologous nucleic acid sequence.
For sequence comparison, typically a sequence acts as a reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are assigned, if necessary, and the program parameters of the sequence algorithm are designed. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the projected program parameters. When necessary or desired, optimal alignment of sequences for comparison can be conducted by any variety of approaches, as they are well known in the art.
The term "variant" as used herein may refer to a polypeptide or nucleic acid that differs from the naturally occurring polypeptide or nucleic acid by one or more deletions, additions, substitutions or modifications of the side chain of amino acids or nucleic acids, while still retaining one or more naturally occurring biological functions or activities of the molecule. Amino acid substitutions include changes in which an amino acid is replaced by one or a different amino acid residue that is naturally occurring or unconventional. Such substitutions can be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced by another naturally occurring amino acid of a similar character in terms of polarity, side chain functionality or size. Substitutions encompassed by the variants, as described herein, can also be "non-conservative", in which an amino acid residue that is present in a peptide is replaced with an amino acid having different properties (for example, replacing a charged or hydrophobic amino acid with alanine) or alternatively, where a naturally occurring amino acid is replaced by an unconventional amino acid. Also included within the term "variant" when used with reference to a polynucleotide or polypeptide, are variations in the primary, secondary or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (for example, in comparison with a polynucleotide or polypeptide wild type).
The term "substantially similar" when used in reference to a variant of an antigen or a functional derivative of an antigen in comparison to the original antigen means that a sequence of the particular individual varies from the antigen polypeptide sequence by one or more substitutions, deletions or additions, but retains at least 50%, or more, for example, at least 60%, 70%, 80%, 90% or more, including, the function of the antigen to elicit an immune response in an individual. To determine polynucleotide sequences, all of the individual's polynucleotide sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference polynucleotide sequence, despite differences in the codon sequence. A nucleotide sequence that is "substantially similar" to a given nucleic acid sequence of the antigen if: (a) the nucleotide sequence hybridizes to the coding regions of the native antigen sequence, or (b) the nucleotide sequence is capable of hybridization to the native antigen nucleotide sequence under moderately stringent conditions and has a biological activity similar to that of the native antigen protein, or (c) the nucleotide sequences that are degenerated as a result of the genetic code relative to the nucleotide sequences defined in (a) or (b). Substantially similar proteins will typically be greater than about 80% similar to the corresponding sequence of the native protein.
Variants can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Variants may also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that typically do not occur in the peptide sequence that underlies the variant, for example, but is not limited to the insertion of ornithine that it does not typically occur in human proteins. "Conservative amino acid substitutions" result from replacing one amino acid with another that has similar structural and / or chemical properties. Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, one of the following six groups each contains amino acids that are conservative substitutions for each other: (1) Alanine (A), Serine (S), Threonine (T), (2) Aspartic acid (D), glutamic acid (E ), (3) Asparagine (N), Glutamine (Q), (4) Arginine (R), Lysine (K), (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See, for example, Creighton, PROTEINS (W.H. Freeman & Co., 1984).
The choice of conservative amino acids can be selected based on the location of the amino acid to be replaced in the peptide, for example, whether the amino acid is on the outside of the peptide and exposed to solvents, or on the inside and not exposed to solvents. Selection of such conservative amino acid substitutions is within the skill of one skilled in the art. Therefore, conservative amino acid substitutions and properties can be selected for amino acids on the outside of a protein or peptide (that is, amino acids exposed to a solvent). These substitutions include, but are not limited to, the replacement of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.
Alternatively, it is also possible to select the appropriate conservative amino acid substitutions for amino acids within a protein or peptide (i.e., the amino acids are not exposed to a solvent). For example, you can use the following conservative substitutions: where Y is replaced with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, LF polypeptides including non-amino acid substitutions Conservative approaches are also included within the term "variants". As used herein, the term "non-conservative" substitution refers to the replacement of an amino acid residue with a different amino acid residue that has different chemical properties. Non-limiting examples of non-conservative substitutions include aspartic acid (D), being replaced by glycine (G); asparagine (N), being replaced by lysine (K) and alanine (A) being replaced by arginine (R).
The term "derivative" as used herein refers to proteins or peptides that have been chemically modified, for example, by ubiquitination, labeling, pegylation (derivatization with polyethylene glycol) or the addition of other molecules. A molecule is also a "derivative" of another molecule when it contains additional chemical moieties that are not typically part of the molecule. Such portions can improve the solubility of the molecule, absorption, biological half-life, etc. The portions can alternatively decrease the toxicity of the molecule, or eliminate or mitigate an undesirable side effect of the molecule, etc. The portions capable of mediating such effects are described in REMINGTON'S PHARMACEUTICAL SCIENCES (21st ed., Tory, ed., Lippincott Williams & Wilkins, Baltimore, MD, 2006).
The term "functional" when used in conjunction with "derivative" or "variant" refers to a protein molecule that has a biological activity that is substantially similar to a biological activity of the molecule or entity that is a derivative or variant. "Substantially similar" in this context means that the biological activity, for example, the antigenicity of a polypeptide, is at least 50% as active as a reference, for example, a corresponding wild-type polypeptide, for example, at least 60 % as active, 70% as active, 80% as active, 90% as active, 95% as active, as 100% active, or even higher (that is, the variant or derivative has greater activity than the wild type) , for example, 110% active, 120% active, or more, inclusive.
The term "recombinant" when used to describe a nucleic acid molecule, means a polynucleotide of genomic origin, cDNA, viral, semi-synthetic, and / or synthetic, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used in connection with a recombinant peptide, polypeptide, protein, or fusion protein, means a polypeptide produced by expression of a recombinant polynucleotide. The term recombinant as used in connection with a host cell means a host cell into which a recombinant polynucleotide has been introduced. Recombinant is also used here to refer, with reference to the material (for example, a cell, nucleic acid, protein, or vector) that the material has been modified by the introduction of a heterologous material (for example, a cell, a nucleic acid, a protein, or a vector).
The term "vectors" refers to a nucleic acid molecule capable of transporting or mediating the expression of a heterologous nucleic acid, to which it has been linked to a host cell, a plasmid is a species of the genus encompassed by the term "vector" ". The term "vector" typically refers to a nucleic acid sequence containing an origin of replication and other entities necessary for replication and / or maintenance of a host cell. Vectors capable of directing the expression of genes and / or nucleic acid sequence to which they are operationally linked are referred to herein as "expression vectors". In general, utility expression vectors are often in the form of "plasmids", which refer to circular double-stranded DNA molecules that, in their vector form, are not linked to the chromosome and typically comprise entities for stable or transient expression or encoded DNA. Other expression vectors that can be used in the methods, as described herein, include, but are not limited to plasmids, episomes, artificial bacterial chromosomes, artificial yeast chromosomes, bacteriophages or viral vectors, and such vectors can integrate into the host genome or replicate autonomously in the particular cell. A vector can be a DNA or RNA vector. Other forms of expression vectors known to those skilled in the art that serve equivalent functions can also be used, for example, self-replicating extrachromosomal vectors or vectors that integrate into a host genome. The preferred vectors are those capable of replication and / or autonomous expression of nucleic acids to which they are attached.
The term "reduced" or "reduce" or "decrease", as used herein, generally means a reduction of a statistically significant amount in relation to a reference. For the avoidance of doubt, "reduced" means a statistically significant decrease of at least 10% compared to a reference level, for example, a decrease of at least 20%, at least 30%, at least 40%, at least t 50%, or at least 60%, or at least 70%, or at least 80%, at least 90% or more, up to and including a 100% decrease (ie, missing level, compared to a reference sample ), or any decrease between 10-100%, compared to a reference level, as the term is defined here.
The term "low", as used herein, generally means lower by a statistically significant amount, for the avoidance of doubt, "low" means a statistically significant value of at least 10% lower than a reference level, for example, a value at least 20% lower than a reference level, at least 30% lower than a reference level, at least 40% lower than a reference level, at least 50% lower than that a reference level at least 60% lower than a reference level, at least 70% lower than a reference level, at least 80% lower than a reference level, at least 90% lower than a reference level, up to and including 100% lower than a reference level (ie, missing level, compared to a reference sample).
The terms "increased" or "increase", as used herein, generally mean an increase by a statistically significant amount, such as a statistically significant increase of at least 10%, compared to a reference level, including an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, including, including, for example, at least 2 times, at least 3 times, at least four times, at least 5 times, increase of at least 10 times or more, compared to a reference level, as the term is defined herein.
The term "elevated" as used herein generally means a higher by a statistically significant amount in relation to a reference, such as a statistically significant value at least 10% greater than a reference level, for example, by least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, at least 100% greater, inclusive, such as at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 10 times greater or more, compared to a level of reference.
As used here, the term "comprising" means that other elements may also be present, in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.
The term "consisting of" refers to compositions, methods, and components thereof, as described herein, which are exclusive of any element not recited in this description of the embodiment.
As used herein, the term "consisting essentially of" refers to the elements necessary for a given embodiment. The term allows for the presence of elements that do not materially affect the basic (s) and (new) or functional (s) characteristic (s) of the embodiment of the invention.
The present invention provides a flexible and versatile composition, which can be designed and manufactured to elicit a particular, broad-spectrum target, or a variety of antigenic targets. Table 1 presents a simple example guide foreseeing the flexibility of MAPS forms: Table 1. Versatility of the MAPS platform
Polymers
A MAP component is made up of a "back structure", typically a polymer. The polymer can be antigenic or non-antigenic. It can be made from a wide variety of substances, as described here, with the proviso that the polymer serves as a means of presenting the associated antigen (s) to the immune system in an immunogenic manner. In some embodiments, the polymer is a synthetic polymer. In some embodiments, the polymer is a naturally occurring polymer, for example, a polysaccharide derivative or purified from bacterial cells. In some embodiments, the polysaccharide is derived or purified from eukaryotic cells, for example, cells from plants, fungi, or insects. In still other embodiments, the polymer is derived from mammalian cells, such as virus-infected cells or cancer cells. In general, these polymers are well known in the art and are encompassed for use in the methods and compositions, as described herein.
In some embodiments, a polymer is a polysaccharide selected from any of the following, dextran, Salmonella typhi polysaccharide Vi, pneumococcal capsular polysaccharide, pneumococcal cell wall polysaccharide (CWPS), meningococcal polysaccharide, Haemophilus polysaccharide, influenzae or haemophilus influenzae any other polysaccharide of viral, prokaryotic or eukaryotic origin.
In some embodiments, the polysaccharide consists of, or comprises, a portion of antigenic sugar. For example, in some embodiments, a polysaccharide for use in immunogenic methods and compositions, as described herein, is a Salmonella typhi Vi polysaccharide. Capsular polysaccharide Vi was developed against bacterial enteric infections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis. 436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993). Vi is a polymer of α 1 -> 4-galacturonic acid, with an N-acetyl at the C-2 position and 0- variable acetylation at C-3. The virulence of S. typhi correlates with the expression of this molecule. Sharma et al., PNAS 101 17492 (2004). The polysaccharide Vi vaccine for S. typhi has several advantages: Side effects are rare and mild, a single dose produces consistent and effective immunogenicity. Polysaccharide Vi can be safely standardized using physicochemical methods verified for other polysaccharide vaccines, Vi is stable at room temperature and can be administered simultaneously with other vaccines, without affecting immunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).
Thus, polysaccharide Vi from S. typhi can be cross-linked to a molecule of first affinity, as described herein, for the attachment of at least one antigen to the polysaccharide. In some embodiments, the antigen can be from the same or another organism, such that the resulting immunogenic composition confers at least some level of immunity against one pathogen, or two different pathogens: if the antigen provides protection against the pneumococcus, an immunogenic composition, where the polymer scaffold is a polysaccharide Vi can create an immunogenic response against both S. typhi and pneumococci. Other examples include combining sugars from encapsulated bacteria (such as meningococci, S. aureus, pneumococci, Hib, etc.) and tuberculosis antigens, to provide an immunogenic composition that creates an immune response against two different pathogens.
Other portions of polysaccharides (PS) that can be used in the present invention, as an alternative to dextran, bacterial cell wall polysaccharides (CWPS), etc., include cancer carbohydrate antigens.
In addition, in relation to pneumococcal polysaccharides, the polysaccharide can be derived from any of the more than 93 pneumococcal serotypes that have been identified so far, for example, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Additional serotypes can be identified and included in the present immunogenic composition, as described herein. More than one pneumococcal polysaccharide can be included as the polymer backbone of the present immunogenic compositions or in a vaccine comprising the present MAPS compositions.
The polysaccharide can also be derived from the invention, the immunogenic composition comprising N. meningitidis capsular polysaccharides of at least one, two, three or four of serogroups A, C, W, W135, or Y.
Another embodiment comprises Type 5, Type 8, or any of the polysaccharides or oligosaccharides of Staphylococcus aureus.
In some embodiments, the polymer is a chimeric polymer comprising more than one type of polymer. For example, a polymer of the immunogenic composition, as described herein, may comprise a portion of polymer A, and the remaining portion of polymer B. There is no limit to the number of different types of polymers that can be used in a single entity. dorsal structure of MAPS. In some embodiments, where the polymer is a branched polymer, the polymer chain can be a polymer, and the branches can be at least 1 or at least 2 or at least 3 or more different polymers .
In some embodiments, the polymer is a branched polymer. In some embodiments, the polymer is a single chain polymer.
In some embodiments, the polymer is a polysaccharide comprising at least 10 carbohydrate repeat units, or at least 20, or at least 50, or at least 75, or at least 100, or at least 150, or at least 200 , or at least 250, or at least 300, or at least 350, or at least 4 00, or at least 450, or at least 500, or more than 500 repetition units, inclusive.
In one aspect of the invention, the polysaccharide (PS) can have a molecular mass of <500kDa or> 500kDa. In another aspect of the invention, PS has a molecular mass of <70 kDa.
In some embodiments, a polymer is a large molecular weight polymer, for example, a polymer can have an average molecular weight of between about 425-500kDa, including, for example, at least 300kDa, or at least 350kDa , or at least 400kDa, or at least 425kDa, or at least 450kDa, or at least 500kDa or more than 500kDa, inclusive, but typically less than 500kDa.
In some embodiments, a polymer can be a small molecular weight polymer, for example, a polymer can have an average molecular weight of between about 60kDa to about 90kDa, for example, at least 50kDa, or at least less than 60kDa, or at least 70 kDa, or at least 80 kDa, or at least 90 kDa, or at least 100 kDa, or greater than 100 kDa, inclusive, but generally less than about 120kDa.
In some embodiments, the polymer is collected and purified from a natural source, and in other embodiments, the polymer is synthetic. Methods for producing synthetic polymers, including synthetic polysaccharides, are known to those skilled in the art and are encompassed in the compositions and methods as described herein.
Only a few of the polysaccharide polymers, which can serve as a dorsal structure of one or more types of antigens or antigens are exemplified in Table 2: Table 2. Example of a MAPS dorsal structure of polysaccharide polymer and associated example antigens

Additional polymers that can be used in the immunogenic MAPS compositions described herein include the 5 polymers based on polyethylene glycol, poly (ortho esters), Polyacryl, PLGA, polyethylenimine (PEI), polyamidoamine (PAMAM), dendrimer, ester polymer polymers. β-amino, polyphosfoester (PPE), liposomes, polymerosomes, nucleic acids, phosphorothioated oligonucleotides, chitosan, silk, polymeric micelles, protein polymers, virus particles, virus-like particles (VLPs-particles) or other micro-particles. See, for example, El-Sayed et al. , Smart Polymer Carriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5 Exp. Op. Biol. Therapy, 23 (2005). Biocompatible polymers, developed for distribution of nucleic acid can be adapted for use as a backbone here. See, for example, BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., Eds., John Wiley & Sons, Inc. Hoboken, NJ, 2011).
For example, VLPs resemble viruses, but are not infectious because they do not contain any viral genetic material. Expression, including recombinant expression of viral structural proteins, such as envelope components or capsids, can result in self-assembly of VLPs. VLPs were produced from components of a wide variety of virus families, including Parvoviridae (eg, adeno-associated virus), Retroviridae (eg, HIV), and Flaviviridae (eg, hepatitis B or C virus). VLPs can be produced in a variety of cell culture systems including mammalian cell lines, insect cell lines, yeast and plant cells. Recombinant VLPs are particularly advantageous because the viral component can be fused to recombinant antigens, as described herein. Antigens
The immunogenic compositions, as described herein, can comprise any antigen that elicits an immune response in an individual. In some embodiments, at least one or more antigens are associated with the polymer of the composition. In some embodiments, at least 2, or at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or at least 50, or at least 100, or more than 100 antigens can be associated with the polymer, as described herein.In some embodiments, where the immunogenic composition comprises more than one antigen, the antigens can be the same antigen, or they can be a variety of different antigens associated with the polymer . In some embodiments, where the immunogenic composition comprises more than one antigen, the antigens may be antigens from the same pathogen, or from different pathogens, or, alternatively, they may be different antigens from the same pathogen, or similar antigens from different pathogen serotypes.
An antigen for use in immunogenic compositions and methods described herein can be any antigen, including, but not limited to, pathogenic peptides, toxins, toxoids, subunits thereof, or combinations thereof (eg, cholera toxin, tetanus toxoid).
In some embodiments, an antigen, which can be fused to the complementary affinity molecule, can be any antigen associated with an infectious disease, or cancer or immune disease. In some embodiments, an antigen can be an antigen expressed by any of a variety of infectious agents, including viruses, bacteria, fungi or parasites.
In some embodiments, an antigen is derived (for example, obtained) from a pathogenic organism. In some embodiments, the antigen is a cancer or tumor antigen, for example, an antigen derived from a tumor or cancer cell.
In some embodiments, an antigen derived from a pathogenic organism is an antigen associated with an infectious disease, it can be derived from any of a variety of infectious agents, including viruses, bacteria, fungi or parasites.
In some embodiments, a target antigen is any antigen associated with a condition, for example, an infectious disease or pathogen, or cancer or an immune disease, such as an autoimmune disease. In some embodiments, an antigen can be expressed by any one of a variety of infectious agents, including viruses, bacteria, fungi or parasites. A target antigen for use in the methods and compositions, as disclosed herein, may also include, for example, pathogenic peptides, toxins, toxoids, subunits thereof, or combinations thereof (for example, cholera toxin, tetanus toxoid).
Non-limiting examples of infectious viruses include: Retroviridae; Picornaviridae (for example, polio virus, hepatitis A virus; enterovirus, coxsackie virus, rhinovirus, human echovirus); Caleiviridae (such as strains that cause gastroenteritis); Togaviridae (for example, equine encephalitis virus, rubella virus); Flaviridae (for example, dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (for example, coronaviruses); Rhabdoviridae (for example, vesicular stomatitis virus, rabies virus); Filoviridae (for example, Ebola virus); Paramyxoviridae (for example, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza virus); Bungaviridae (for example, Hantaan virus, bunga virus, phlebovirus and Nairo virus); Arena viridae (hemorrhagic fever virus); Reoviridae (for example, reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papilloma virus, polyoma virus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zoster virus, cytomegalovirus (CMV), Marek's disease virus, herpes virus); Poxviridae (smallpox virus, vaccine virus, smallpox virus); and Iridoviridae (cow the African swine fever virus), and unclassified viruses (for example, the etiological agents of spongiform encephalopathies, the agent of hepatitis delta (probably a defective hepatitis B virus satellite), non-hepatitis agents A, non-B (class 1 = transmitted internally; class 2 = transmitted parenterally (ie, Hepatitis C); Norwalk and related viruses, and astrovirus). The compositions and methods described herein are contemplated for use in the treatment of infections with these viral agents.
Examples of fungal infections that can be treated by including antigens in the present embodiments include aspergillosis, candidiasis (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis. Thus, examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulation, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. The components of these organisms can be included as antigens in the MAPS described here.
In one aspect of the invention, an antigen is derived from an infectious microbe such as Bordatella pertussis, Brucella, Enterococci sp. , Neisseria meningitidis, Neisseria gonorrheae, Moraxella, Haemophilustable or non-typable, Pseudomonas, Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, E. coli, Helicobacter pylori, Clostridia, Bacteroides, Chlamydioneae, Burgio, Trema, Vibrio cholera pneumophilia, Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. graxonae, M. leprae), Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes (Streptococcus Group A), Streptococcus agalactia Group B), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, Campylobacter sp. pathogenic, Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella sp. , Treponema pallidium, Treponema pertenue, and Actinomyces israelii. The compositions and methods described herein are contemplated for use in the treatment or prevention of infections against these bacterial agents.
Additional parasitic pathogens from which antigens can be derived include, for example: Entamoeba histolytica, Plasmodium falciparum, Leishmania sp., Toxoplasma gondii, Rickettsia, and Helminths.
In another aspect of the invention, an antigen is a truncated pneumococcal PsaA protein, serine / threonine pneumococcal toxoid kinase pneumolysin (StkP), serine / threonine repeat unit pneumococcal kinase protein (StkPR), pneumococcal PcsB protein, staphylococcal hemolysin, mtb Mycobacterium tuberculosis ESAT-6 protein, antigen from the nucleus of the M. tuberculosis cell wall, Chlamydia polypeptides or fragments thereof CT144, CT242 or CT812, Chlamydia DNA gyrase subunit B, Chlamydia sulfite / bisphosphate synthesis, FtsY Chlamydia cell division protein, Chlamydia methionyl-tRNA synthase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthase I subunit (atpl) or Chlamydia metal-dependent hydrolase.
One embodiment of the present invention provides an immunogenic composition targeting the Myocobacterium tuberculosis (TB) pathogen, a bacterial intracellular parasite. An example of a TB antigen is TbH9 (also known as Mtb 3 9A). Other TB antigens include, but are not limited to, DPV (also known as Mtb8.4), 381, Mtb41, Mtb40, Mtb32A, Mtb64, Mtb83, Mtb9.9A, Mtb9.8, Mtbl6, Mtb72f, Mtb59f, Mtb88f, Mtb71f, Mtb46f and Mtb31f, where "f" indicates that it is a fusion of two or more proteins.
As mentioned above, an antigen can be derived from a species of Chlamydia for use in the immunogenic compositions of the present invention. Chlamydiaceae (consisting of Chlamydiae and Chlamydophila) are mandatory intracellular gram-negative bacteria. Chlamydia trachoma infections are among the most prevalent sexually transmitted bacterial infections and perhaps 89 million new cases of genital Chlamydia infections occur each year. The present invention Chlamydia includes, for example, C. trachomatis, Chlamydophila pneumoniae, C. muridarum, C. suis, Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila caviae, Chlamydophila felis, Chlamydophila pecorum, and C. pneumoniae. Chlamydia has established that T cells play a critical role in both clearing initial infection and protecting re-infection from susceptible hosts. Thus, immunogenic compositions, as described herein, can be used to provide the value determined by eliciting cellular immune responses against chlamydial infection.
More specifically, the Chlamydialy useful antigens in the present invention include DNA gyrase subunit B, sulfite / bisphosphate phosphatase synthesis, cell division protein FtsY, methionyl-tRNA synthase, DNA helicase (uvrD); ATP synthase subunit I (atpl) or a metal-dependent hydrolase (U.S. Pub. Patent application No. 20090028891). Additional Chlamyidia trachomatis antigens include polypeptide CT144, a peptide with amino acid residues 67-86 of CT144, a peptide with amino acid residues 77- 96 of CT144, protein CT242, a peptide having amino acids 109-117 of CT242, a peptide having amino acids 112 -120 of CT242 polypeptide, protein CT812 (from the pmpD gene), a peptide with amino acid residues 103-111 of the CT812 protein; and several other antigenic peptides from C. trachomatis: NVTQDLTSSTAKLECTQDLI (SEQ ID NO: 2), AKLECTQDLIAQGKLIVTNP (SEQ ID NO: 3), SNLKRMQKI (SEQ ID NO: 4), AALYSTEDL (SEQ ID NO: 5) SEQ ID NO: 6), QSVNELVYV (SEQ ID NO: 7), LEFASCSSL (SEQ ID NO: 8), SQAEGQYRL (SEQ ID NO: 9), GQSVNELVY (SEQ ID NO: 10), and QAVLLLDQI (SEQ ID NO: 11). See WO 2009/020553. In addition, Chlamydia pneumonia antigens, including homologues of the previous polypeptides (see US Patent No. 6,919,187), can be used as antigens in the immunogenic compositions and methods as described herein.
Fungal antigens can be derived from Candida species and other yeasts, or other fungi (Aspergillus, other environmental fungi). In relation to other parasites, malaria, as well as worms and amoebae can provide the antigenic antigen for use in immunogenic compositions and methods as described herein.
In some embodiments, where the antigen is intended to generate an anti-influenza immunogen, hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins are generally the antigens of choice. Both the nucleoprotein (NP) polypeptide and matrix (M) are internal viral proteins and are therefore not generally considered in the design of vaccines for antibody-based immunity. Influenza vaccines are used routinely in humans, and include vaccines derived from inactivated complete influenza viruses, live attenuated influenza viruses, or materials purified and inactivated from viral strains. For example, a traditional influenza vaccine can be manufactured using three potentially threatening strains of the flu virus. These strains are typically grown on fertilized chicken eggs, which requires extensive processing, including egg inoculation and incubation, egg harvesting, virus purification and inactivation, processing and assembly of the virus or viral components for vaccine formulation. final, and aseptic placement in appropriate containers. Typically, this egg-based production cycle takes more than 70 weeks. In the event of a flu epidemic, the availability of a potent and safe vaccine is a major concern. In addition, there are risks associated with impurities in eggs, such as antibiotics and contaminants, that negatively impact the sterility of the vaccine. In addition, egg-derived flu vaccines are contraindicated for people with severe allergies to egg proteins and people with a history of Guillain-Barré syndrome. The present invention provides an alternative to egg-based influenza vaccines, not only avoiding egg-related sequelae, but providing a platform for the use of multiple influenza antigens on a highly controlled platform.
In some embodiments, an antigen for use in immunogenic compositions as disclosed herein can also include those used in biological warfare, such as ricin, which can elicit an IMC response.
In addition, the present invention also provides immunogenic compositions comprising antigens that create an immune response against cancer. In these conjugates, an antigen is an antigen expressed by a cancer or tumor, or derived from a tumor. In some embodiments, these antigens are referred to herein as a "cancer antigen" and are typically a protein expressed predominantly on cancer cells, such that the conjugate elicits both a potent and a potent cellular immunity to this protein. A large number of cancer-associated antigens have been identified, some of which are now being used to make experimental vaccines for the treatment of cancer and are therefore suitable for use in the present embodiments. Antigens associated with more than one type of cancer include carcinoembryonic antigen (CEA); cancer / testicular antigens, such as NY-ESO-1; mucin-1 (MUC1), such as Sialyl Tn (STN); gangliosides, such as GM3 and GD2; p53 protein and HER2 / neu protein (also known as ERBB2). Unique antigens for a specific type of cancer include a mutant form of the epidermal growth factor receptor, called EGFRvIII, melanocyte / melanoma differentiating antigens, such as tyrosinase, MARTI, gplOO, the strain related to the cancer / testis group with the do (MAGE) and tyrosinase-related antigens; prostate specific antigen; leukemia-associated antigens (LAAs), such as the BCR -ABL fusion protein, Wilms tumor protein and proteinase-3, and idiotype (Id) antibodies. See, for example, Mitchell, 3 Curr. Opin. Investig. Drugs 150 (2002); Dao & Scheinberg, 21 Best Practice. Res. Clin. Haematol. 391 (2008).
Another approach in generating an immune response against cancer employs antigens from microorganisms that cause or contribute to the development of cancer. These vaccines have been used against cancers, including hepatocellular carcinoma (hepatitis B virus, hepatitis C virus, Opisthorchis viverrin), lymphoma and nasopharyngeal carcinoma (Epstei-Barr virus), colorectal cancer, stomach cancer (Helicobacter pylori), cancer of the bladder (Schisosoma hematobium), T cell leukemia (human T cell lymphotropic virus), cervical cancer (human papillomavirus), and others. To date, clinical trials have been conducted for vaccines targeting bladder cancer, brain tumors, breast cancer, cervical cancer, kidney cancer, melanoma, multiple myeloma, leukemia, lung cancer, pancreatic cancer, prostate cancer and solid tumors. SeePardoll et al., ABELOFF'S CLIN. ONCOL. (4th ed., Churchill Livingstone, Philadelphia 2008); Sioud, 360 Methods Mol. Bio. 277 (2007); Pazdur et al., 30 J. Infusion Nursing 30 (3): 173 (2007); Parmiani et al., 178 J. Immunol. 1975 (2007); Lollini et al., 24 Trends Immunol. 62 (2003); Schlom et al., 13 Clin. Cancer Res. 3776 (2007); Banchereau et al., 392 Nature 245 (1998); Finn, 358 New Engl. J. Med. 2704 (2008); Curigliano et al., 7 Exp. Rev. Anticancer Ther. 1225 (2007). Marek's disease virus, a herpes virus that causes tumors in poultry, has been controlled by vaccine. Thus, the present embodiments include anti-cancer immunogenic compositions, both prevented and prophylactic and vaccines for cancer treatment / therapy. Diseases and proliferative cancers contemplated include AIDS-related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocytic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft tissue sarcoma, anal cancer, angiosarcoma, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brain and CNS tumors breast, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T cell lymphoma, dermatofibrosarcoma- protuberance , small round-cell tumor - desmoplá sicas, ductal carcinoma, cancers of the endocrine system, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, including, for example, eye melanoma and retinoblastoma, fallopian tube cancer , Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal carcinoid tumor, genitourinary tract cancers, germ cell tumors, trophoblastic gestational disease, glioma, gynecological cancer, hematological malignancies, hair cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, Hodgkin's disease, cervical cancer related to the human papilloma virus, hydatidiform mole, hypopharyngeal cancer, islet cell cancer, Kaposi's sarcoma, kidney cancer, cancer larynx, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, lung cancer, lymphedema, lymphoma, lin non-Hodgkin's disease, male breast cancer, malignant rhabdoid kidney tumor, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungi, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen's rupture syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), oral cavity cancer, oropharyngeal cancer, osteosarcoma , ostomy ovarian cancer, pancreatic cancer, paranasal sinus cancer, parathyroid cancer, parotid gland cancer, penis cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, renal cell carcinoma, retinoblastoma , rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, Schwannoma, Sezary syndrome, skin cancer, lung cancer small cell (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous cell carcinoma (skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional cell cancer (bladder), transitional cell cancer (renal pelvis / urethra), trophoblastic cancer, urethral cancer, urinary system cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulva cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.
In some embodiments, an antigen for use in immunogenic compositions, as described herein, can include antigens from autoimmune diseases, for example, they can be "autoantigens". Autoimmune diseases contemplated for diagnosis according to the tests described here include, but are not limited to alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, Addison's disease, aplastic anemia, multiple sclerosis, autoimmune adrenal disease, anemia autoimmune hemolytic, autoimmune hepatitis, autoimmune oophoritis and orchitis, Behçet's disease, bullous pemphigoid, cardiomyopathy, dermatitis with celiac psilosis, chronic fatigue syndrome, chronic inflammatory demyelinating syndrome (CFIDS), chronic inflammatory polyneuropathy, chronic inflammatory polyneuropathy Churg-Strauss, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, herpetiform dermatitis, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis , idiopathic purple thrombocytopenia (ITP), IgA nephropathy, insulin-dependent diabetes (Type I), li chenlate, lupus, Ménière's disease, mixed connective tissue disease, myasthenia gravis, myocarditis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, rheumatic polymyalgia, polymyositis and dermatomyositis, primary aggmaglobulinaemia, primary psoriasis Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, rigid man's syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, Wegener syndrome, vasculitis and vitiligo. In general, it is important to assess the actual or potential response capacity of IMC in individuals having, or suspected of having or being susceptible to, an autoimmune disease.
In some embodiments, an antigen for use in immunogenic compositions, as described herein, can be an antigen that is associated with an inflammatory disease or condition. Examples of inflammatory disease conditions in which antigens may be useful include, but are not limited to, acne, angina, arthritis, aspiration pneumonia, empyema, gastroenteritis, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pleuritis, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, and chronic inflammatory demyelinating polyneuropathy, among others.
In some embodiments, an antigen may be an intact (i.e., whole or complete) antigen, or a functional portion of an antigen that comprises more than one epitope. In some embodiments, an antigen is a peptide functional portion of an antigen. By "intact", in this context, it is understood that the antigen is the full-length antigen, as that antigen polypeptide occurs in nature. This is in direct contrast to the distribution of only a small portion or peptide of the antigen. The distribution of an intact antigen to a cell allows or facilitates the elicitation of an immune response to a wide range of epitopes of the intact antigen, rather than just a single or a few selected peptide epitopes. Thus, the immunogenic methods and compositions described herein encompass intact antigens associated with the polymer for a more sensitive and greater specificity of the immune response, compared to the use of a single epitope peptide-based antigen.
Alternatively, in some embodiments, an intact antigen can be divided into several parts, depending on the size of the initial antigen. Typically, when an entire antigen is a multimer polypeptide, the complete protein can be divided into subunits and / or domains where each subunit or domain of the antigen can be associated with the polymer according to the methods as described herein. Alternatively, in some embodiments, an intact antigen can be divided into functional fragments, or parts, of the entire antigen, for example, at least 2, or at least 3, or at least four, or at least 5, or at least at least 6, or at least seven, or at least eight, or at least nine, or at least 10, or at least 11, or at least 12, or at least 13, or at least 15, or at least 20, or at least minus 25, or more than 25 parts (e.g., parts or fragments), inclusive, and wherein each individual functional fragment of the antigen can be associated with the polymer according to the methods as described herein.
The fragmentation or division of a full-length antigen polypeptide can be an equal division of the full-length antigen polypeptide, or alternatively, in some embodiments, the fragmentation is asymmetric or uneven. As a non-limiting example, where an antigen is divided into two overlapping fragments, an antigen can be divided into fragments of approximately the same (equal) size, or alternatively, a fragment can be about 45% of the complete antigen and the other fragment can be about 65%. Like other non-limiting examples, a complete antigen can be divided into a combination of fragments of different sizes, for example, where an antigen is divided into two fragments, fragments can be divided by about 40% and about 70%, or about 45% and about 65%, or about 35% and about 75%, or about 25% and about 85%, inclusive, of the complete antigen. Any combination of overlapping fragments of a full-length total antigen is encompassed for use in generating a panel of overlapping polypeptides of an antigen. As an illustrative example only, when an antigen is divided into 5 portions, the portions can be divided equally (that is, each overlapping fragment has about 21% to 25% of the entire full-length antigen) or unevenly (for example , an antigen can be divided into the following five overlapping fragments: fragment 1 of about 25%, fragment 2 of about 5%, fragment 3 of about 35%, fragment 4 of about 10% and fragment 5 of about 25% of the size of the full-length antigen, provided that each fragment overlaps with at least one other fragment).
Typically, a panel of antigen portions can substantially cover the entire length of the complete (or intact) antigen polypeptide. Therefore, in some embodiments, an immunogenic composition comprises a polymer with many different fragments, and / or overlapping the same intact antigen. Overlapping protein fragments of an antigen can be produced much faster and more economically, and with greater stability compared to using peptide antigens alone. In addition, in some embodiments, antigens that are polypeptides larger than single peptides are preferred as conformation is important for the recognition of epitopes, and polypeptides from antigens or larger fragments will provide a benefit over the peptide fragments.
One skilled in the art can divide an entire antigen into proteins overlapping an antigen to create a panel of antigen polypeptides. By way of an illustrative example only, TB-specific TB1 antigen (CFP also known as culture-10 or CFP-10 filtrate) can be divided into, for example, at least seventeen portions to generate a panel of seventeen different polypeptides, each comprising a different but overlapping TBP TB1 antigen-specific TB1 fragment. Culture filtrate protein (CFP-10) (GenBank AAC83445) is a protein fragment of 100 kDa 100 amino acid residues from M. tuberculosis. It is also known as L45 antigen homologous protein (LHP).
A target antigen for use in the methods and compositions described herein can be expressed by recombinant means, and can optionally include an affinity tag or epitope to facilitate purification, which are methods well known in the art. The chemical synthesis of an oligopeptide, either free or conjugated to support proteins, can be used to obtain the antigen of the invention. Oligopeptides are considered a type of polypeptide. An antigen can be expressed as a fusion with a complementary affinity molecule, for example, but not limited to rhizavidine or a fragment or functional derivative thereof. Alternatively, it is also possible to prepare the target antigen and then conjugate it to a complementary affinity molecule, for example, but not limited to rhizavidine or a fragment or functional derivative thereof.
Polypeptides can also be synthesized as branched structures, such as those shown in US Patent No. 5,229,490 and No. 5,390,111. Antigenic polypeptides include, for example, synthetic or recombinant T cell B cell epitopes, universal T cell epitopes, and mixed T cell epitopes from one organism or disease and B cell epitopes from others.
An antigen can be obtained by recombinant means or by chemical polypeptide synthesis, as well as antigen obtained from natural sources or extracts, can be purified by means of physical and chemical characteristics of the antigen, such as by fractionation or chromatography. These techniques are well known in the art.
In some embodiments, an antigen can be solubilized in water, a solvent such as methanol, or a buffer. Suitable buffers include, but are not limited to, free Ca2 + / Mg2 + phosphate buffered saline (PBS), normal saline (150 mM NaCl in water), and Tris buffer. Antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of antigen soluble only in acidic pH, acetate-PBS at acidic pH can be used as a diluent, after solubilization in dilute acetic acid. Glycerol can be a non-aqueous solvent suitable for use in the compositions, methods and kits described herein.
Typically, when designing a protein vaccine against a pathogen, an extracellular protein or one exposed to an environment in a virus is often the ideal candidate as the antigen component in the vaccine. The antibodies generated against the extracellular protein that make it the first line of defense against the pathogen during infection. The antibodies bind to the protein on the pathogen to facilitate the opsonization of the antibody and to mark the pathogen for ingestion and destruction by a phagocyte such as a macrophage. Antibody opsonization can also kill the pathogen by antibody-dependent cell cytotoxicity. The antibody triggers the release of lysis products from cells, such as monocytes, neutrophils, eosinophils and natural killer cells.
In one embodiment of the invention described herein, the antigens for use in the compositions, as described herein, include all wild type proteins, as amino acid residues have the sequences found in the virus that occur naturally and that have not been altered by conditions of selective growth or molecular biology methods.
In one embodiment, the immunogenic compositions, as described herein, can comprise antigens that are glycosylated proteins. In other words, an antigen of interest can each be a glycosylated protein. In an embodiment of the immunogenic compositions as described herein, antigens, or antigen fusion polypeptides are 0-linked glycosylates. In another embodiment, the immunogenic compositions as described herein, antigens, or antigen fusion polypeptides are N-linked glycosylates. In yet another embodiment of the immunogenic compositions, as described herein, antigens or antigen fusion are glycosylated both N-linked and O-linked. In other embodiments, other types of glycosylation are possible, for example, C-mannosylation. Protein glycosylation occurs predominantly in eukaryotic cells. N-glycosylation is important for the duplication of some eukaryotic proteins, providing a mechanism for modifying co-translation and post-translation that modulates the structure and function of the membrane and secreted proteins. Glycosylation is the enzymatic process that binds saccharides to produce glycans, and fix them to proteins and lipids. In N-glycosylation, glycans are attached to the amide nitrogen of the asparagine side chain during protein translation. The three main saccharides that form glycans are glucose molecules, mannose, and N-acetylglucosamine. The consensus for N-glycosylation is Asn-Xaa-Ser / Thr, where Xaa can be any of the known amino acids. The 0-linked glycosylation occurs at a later stage, during protein processing, probably in the Golgi apparatus. In 0-linked glycosylation, N-acetyl-galatosamine, O-fucose, 0-glucose, and / or N-acetylglucosamine is added to serine or threonine residues. One skilled in the art can use bioinformatics software, such as NetNGlyc 1.0 and NetOGlyc prediction software from the Technical University of Denmark to find the N- and O-glycosylation sites in a polypeptide of the present invention. The NetNglyc server predicts protein N-glycosylation sites, using artificial neural networks, which examine the context of the Asn-Xaa-Ser / Thr sequence. The Forecast software NetNGlyc 1.0 and NetOGlyc 3.1 can be accessed on the ExPASy website. In one embodiment, N-glycosylation occurs in the target antigen polypeptide of the fusion polypeptide described herein. Affinity molecule pairs:
As described herein, in some embodiments, an antigen is connected to a polymer via complementary affinity pairs. This connection of the antigen to the polymer is mediated by the polymer being connected to a first affinity molecule, which associates a second affinity molecule (for example, complementary), which is attached to the antigen. An example complementary affinity pair is the biotin / biotin binding protein.
Illustrative examples of the complementary affinity affinity pairs include, but are not limited to, biotin binding proteins or avidin-like proteins that bind biotin. For example, where the first binding affinity molecule is biotin (which associates with the polymer), the complementary affinity molecule can be a biotin binding protein or an avidin-like protein or a derivative thereof, for example, but not limited to, avidin, rhizavidine or streptavidin or variants, derivatives or functional portions thereof.
In some embodiments, the first binding affinity molecule is biotin, a biotin derivative, or a biotin mimetic, for example, but not limited to, amine-PEG3-biotin (((+) biotinylation-3-6 , 9-trixaundecanediamine) or a functional derivative or fragment thereof A specific biotin mimetic has a specific peptide motif containing the sequence of DXaAXbPXc (SEQ ID NO: 12), or CDXaAXbPXcCG (SEQ ID NO: 13), where Xa is R or L, Xb is S or T, and Xc is Y or W. These motifs can link avidin and neutravidine, but streptavidin, see, for example, Gaj et al., 56 Prot. Express. Purif. 54 (2006).
The binding of the first affinity molecule to the polymer, and the complementary affinity molecule to the antigen can be a non-covalent bond, or a chemical mechanism, for example, covalent bond, affinity bond, intercalation, coordinate bond and complexation . Covalent bonding provides a very stable bond, being particularly well suited for the present embodiments. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporating external bridge bonding molecules.
For example, in some embodiments, an antigen may be linked non-covalently to one of the pairs of a complementary display pair. In alternative embodiments, an antigen can be covalently linked or fused to one of the pairs of a complementary display pair. Methods for generating fusion proteins are well known in the art, and are discussed here.
In other embodiments, a first binding affinity molecule is attached to the polymer by means of a non-covalent bond, or by a covalent bond. In some embodiments, a cross-linking reagent is used to covalently bind the first binding affinity molecule to the polymer, as described herein.
In some embodiments, the first binding affinity molecule associates with the complementary affinity molecule by non-covalent binding association, as is known in the art, including, but not limited to, electrostatic interaction, bound hydrogen, hydrophobic interaction ( (van der Waals forces), hydrophilic interactions, and other non-covalent interactions. Other interactions of a higher order, with intermediate portions, are also contemplated.
In some embodiments, the complementary affinity molecule is an avidin-related polypeptide. In specific embodiments, the complementary affinity molecule is rhizavidine, such as recombinant rhizavidine. In particular, recombinant rhizavidine is a modified rhizavidine that can be expressed in E. coli, in high yield. The typical yield is> 30 P9 per liter of E. coli culture. Rhizavidina has a lower sequence homology for egg avidin (22.4% sequence identity and 35.0% similarity) compared to other avidin-like proteins. The use of modified rhizavidine reduces the risk of MAPS inducing an egg-related allergic reaction in an individual. In addition, modified recombinant rhizavidine has no apparent cross-reactivity to egg avidin (and vice
More specifically, some embodiments comprise a modified rhizavidine designed for recombinant expression in E. coli. The coding sequence for the rhizavidine gene was optimized using E. coli expression codons, to avoid any difficulties during expression in E. coli due to the rare codons present in the original gene. To simplify the construct, after a bioinformatics and structure-based analysis, the first 44 full-length rhizavidine residues were removed, as these were considered unnecessary for the structure and function of the nucleus. Correct duplication of the recombinant protein was improved by adding an E. coli secretion signal sequence to the N-terminal of the shortened rhizavidine (45- 179), in order to facilitate the translocation of the recombinant protein in the periplasmic space of the cells of E. coli, where the functionally important disulfide bond in rhizavidine can form correctly. The modified recombinant rhizavidine forms a dimer, in comparison with the known avidin-like proteins that form tetramers, further improving the expression of the recombinant rhizavidine antigen fusion, as a protein soluble in E. coli.
In addition, as discussed in greater detail here elsewhere, to improve the expression and solubility of fusion antigens in E. coli, a flexible linker region was added between rhizavidine and the antigen protein. In addition, based on structural analysis and "biotinformatics", different antigen constructs were cloned and expressed: either a full-length antigen, or the important functional domain, or and chimera proteins were made up of two different antigens.
Additional affinity pairs that may be usable in the methods and compositions described herein include antibody-antigen, metal / metal ion / ion binding protein, lipid / lipid binding protein, saccharide / saccharide binding protein, amino acid binding protein / peptide / amino acid or peptide, enzyme substrate or enzyme inhibitor, binding agonist / receptor, or biotin mimetic. When using alternative affinity pairs, alternative means of fixing the respective polymer and antigen can also be employed, such as enzymatic reactions in vitro, instead of genetic fusion. More specifically, antigen-antibody affinity pair provides a very strong and specific interaction. The antigen can be any epitope including protein, peptide, nucleic acid, lipid, poly / oligosaccharide, ion, etc. The antibody can be of any type of immunoglobulin, or the Ag binding portion of an immunoglobulin, such as a Fab fragment. With respect to metal / metal ion / ion-binding protein, examples include Ni NTA versus histidine-tagged protein, or Zn versus Zn binding protein. With respect to the lipid / lipid binding protein, examples include cholesterol versus cholesterol binding protein. With respect to the saccharide / saccharide binding protein, examples include maltose versus maltose binding protein, mannose / glucose / oligosaccharide versus lectin. Enzyme substrates / inhibitors include substrates from a wide range of substances, including proteins, peptides, amino acids, lipids, sugars, or ions. The inhibitor can be the real substrate analog, which generally can bind enzymes more firmly and even irreversibly. For example, trypsin versus soybean trypsin inhibitor. The inhibitor can be a natural or synthetic molecule. With respect to another ligand / agonist - receptor, ligand can be of a wide range of substances, including proteins, peptides, amino acids, lipids, sugars, ions, agonist can be the analogue of the real ligand. Examples include the LPS interaction Cross-linking reagents:
Many divalent or polyvalent binding agents are usable in copulating protein molecules to other molecules. For example, representative copulating agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, disocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of copulating agents known in the art, but instead is exemplary of the most common copulating agents. SeeKillen & Lindstrom, 133 J. Immunol. 1335 (1984); Jansen et al., 62 Imm. Rev. 185 (1982); Vitetta et al.
In some embodiments, the crosslinking reagents described in the literature are encompassed for use in the methods, immunogenic compositions and kits, as described herein. See, for example, Ramakrishnan, et al. , 44 Cancer Res. 201 (1984) (describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester)); Umemoto et al., U.S. Patent No. 5,030,719 (describes the use of a halogenated acetyl hydrazide derivative copulated to an antibody via an oligopeptide linker). Specific linkers include.- (a) EDO (l-ethyl-3- (3-dimethylamino-propyl) hydrochloride) carbodiimide; (b) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pyridyl-dithio) - toluene (Pierce Chem. Co., Cat. (21558G); (c) SPDP (succinimidyl-6- [3- (2-pyridyldithio) propionamide hexanoate] (Pierce Chem. Co., Cat # 21651G); (d ) Sulfo- LC-SPDP (sulfosuccinimidyl-6- [3- (2-pyridyldithio) -propianamide] hexanoate (Pierce Chem. Co. Cat. # 2165-G); and (f) sulfo-NHS (N-hydroxysulfo- succinimide: Pierce Chem. Co., Cat. # 24510) conjugated to EDC.
The bonds or binding agents described above contain components that have different attributes, thus leading to conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. In addition, the SMPT linker contains a stereochemically hindered disulfide bond, and can form conjugates with increased stability. Disulfide bonds are, in general, less stable than other bonds because the disulfide bond can be cleaved in vitro, which results in less available conjugate. Sulfo-NHS, in particular, can enhance the stability of carbodiimide copulations. Carbodiimide copulations (such as EDC), as used in conjunction with sulfo-NHS, form esters that are more resistant to hydrolysis than the carbodiimide copulation reaction alone.
Exemplary cross-linking molecules for use in immunogenic methods and compositions as described herein include, but are not limited to, those listed in Tables 3 and 4. Table 3. Exemplary homobifunctional crosslinkers *
Table4. Heterobifunctional crosslinkers
Co-stimulatory factor
In some embodiments, the immunogenic composition as described herein comprises at least one costimulatory molecule. In some embodiments, the costimulatory factor is cross-linked to the polymer. In some embodiments, the costimulatory factor is associated with the polymer by a complementary affinity pair similar to how an antigen is associated with the polymer. In some embodiments, where the complementary affinity pair that binds the costimulatory factor to the polymer is the same, or a different complementary affinity pair that binds the antigen to the polymer.
In some embodiments, at least one, or at least 2, or at least 3, or at least 5, or at least 10, or at least 15, or at least 20, or at least 50, or at least 100, or more than about 100 inclusive, co-stimulatory factors may be associated with the polymer, as described herein.In some embodiments, the co-stimulatory factors may be the same co-stimulatory factor, or they may be a variety of different costimulatory factors associated with the polymer.
In some embodiments, the co-stimulatory factor is a ligand / agonist of Toll-type receptors, for example, but not limited to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, etc. In some embodiments, a co-stimulatory factor is a NOD ligand / agonist, or an inflammasome activator / agonist. Without intending to be limited by theory, inflammasome is a multiprotein oligomer consisting of caspase 1, PYCARD, NALP and, sometimes, caspase 5 or caspase 11 and promotes the maturation of inflammatory cytokines interleukin 1-β and interleukin 18.
In some embodiments, a co-stimulatory factor is a cytokine. In some embodiments, a cytokine is selected from the group consisting of: GM-CSF; IL-lü; IL-lü; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-23; IFN-Q; IFN-Ü; IFN-EJ; IFN-Y; MiP-lQ; MIP-1D; TGF-D; TNFEü and TNFD. In some embodiments, the co-stimulatory factor is an adjuvant, which can be associated with the polymer, as just discussed, or can be added to the MAPS composition prior to or concurrent with administration to an individual. Adjuvants are further described here elsewhere. Production of antigens and antigens fused with the complementary affinity molecule
Recombinant proteins can be conveniently expressed and purified by one skilled in the art, or using commercially available kits, for example, ProBond ™ Purification System (Invitrogen Corp, Carlsbad, CA). In some embodiments, recombinant antigens can be synthesized and purified by protein purification methods, using bacterial expression systems, yeast expression system, baculovirus / insect cell expression system, cell expression systems mammals, or systems of transgenic plants or animals, as known to those skilled in the art.
The fusion polypeptides described herein can be synthesized and purified by protein and molecular methods that are well known to one skilled in the art. Molecular biology methods and recombinant heterologous protein expression systems are used. For example, the recombinant protein can be expressed in bacteria, mammals, insects, yeast, or plant cells, or in transgenic plants or animal hosts.
In one embodiment, an isolated polynucleotide encoding a fusion polypeptide or a non-fusion polypeptide described herein is provided herein. Conventional polymerase chain reaction (PCR) cloning techniques can be used to construct a chimeric or fusion coding sequence encoding a fusion polypeptide, as described herein. A coding sequence can be cloned into a cloning vector for general purposes, such as vectors pUC19, pBR322, pBLUESCRIPT® (Stratagene, Inc.) or pCR TOPO® (Invitrogen). The resulting recombinant vector carrying the nucleic acid encoding a polypeptide, as described herein, can then be used for other molecular biology manipulations, such as site-directed mutagenesis to create a variant fusion polypeptide, as described herein, or can be subcloned into protein expression vectors or viral vectors for protein synthesis in a variety of protein expression systems using host cells selected from the group consisting of mammalian cell lines, insect cell lines, yeasts, bacteria and plants.
Each PCR primer must have at least 15 nucleotides that overlap with their corresponding templates in the region to be amplified. The polymerase used in PCR amplification must have high fidelity, such as PfuULTRA® polymerase (Stratagene) to reduce sequence errors during the PCR amplification process. For ease of ligation of several PCR fragments separated together, for example, in the construction of a fusion polypeptide, and subsequently inserting into a cloning vector, the PCR primers must also have distinct and unique restriction digest sites at their flank ends that do not recombine to template DNA during PCR amplification. The choice of restriction digest sites for each pair of specific primers should be such that the DNA sequence encoding the fusion polypeptide is in the frame and will encode the fusion polypeptide from the beginning to the end without stop codons. At the same time, the chosen restriction digest sites should not be found within the DNA sequence encoding the fusion polypeptide. The coding DNA sequence for the desired polypeptide can be ligated into a pBR322 cloning vector or one of its derivatives for amplification, fidelity verification and authenticity of the chimeric coding sequence, substitutions / or site-directed mutagenesis specific for specific amino acids and substitutions in the polypeptide.
Alternatively, the DNA sequence encoding the polypeptide can be cloned by PCR into a vector using, for example, the TOPO® cloning method comprising TA vectors assisted by topoisomerase, such as pCR®-TOPO, pCR®-Blunt II- TOPO, pENTR / D-TOPO®, and pENTR / SD / D-TOPO®. (Invitrogen, Inc., Carlsbad, CA). Both pENTR / D-TOPO®, and pENTR / SD / D-TOPO® are directional TOPO input vectors that allow cloning of the DNA sequence in the 5 '-> 3' orientation into a GATEWAY® expression vector. Directional cloning in the 5 '-> 3' orientation facilitates unidirectional insertion of the DNA sequence into a protein expression vector such that the promoter is upstream of 1 ATG 5 'start codon of the DNA sequence encoding the fusion polypeptide , allowing the expression of protein directed by the promoter. The recombinant vector carrying the DNA sequence encoding the fusion polypeptide can be transfected into and propagated in general E. coli cloning such as XLIBlue, SURE® (STRATAGENE®) and TOP-10 (Invitrogen) cells.
A person skilled in the art would be able to clone and link the coding region of the antigen of interest with the coding region of the complementary affinity molecule to construct a chimeric coding sequence for a fusion polypeptide comprising the antigen or a fragment thereof and the complementary affinity molecule of a derivative thereof, using specially designed oligonucleotide probes and polymerase chain reaction (PCR) methodologies that are well known in the art. One skilled in the art will also be able to clone and link the chimeric coding sequence to a fusion protein in a selected vector, for example, bacterial expression vector, insect expression vector or baculovirus expression vector. The antigen coding sequences and the target antigen polypeptide or fragment thereof must be linked in frame and the chimeric coding sequence must be linked downstream of the promoter, and between the promoter and the transcription terminator. Subsequent to this, the recombinant vector is transfected into E. coli of regular cloning, such as XL1 Blue. Recombinant E. coli harboring the transfer vector DNA is then selected through antibiotic resistance to remove any E. coli harboring the non-recombinant plasmid DNA. E. selected colitransformants are cultured and the recombinant vector DNA can be further purified by transfection in S. frugiperda cells.
In some embodiments, the antigens, as described herein, may comprise a signal peptide for translocation into the bacterium's periplasmic space. The signal peptide is also called an N-terminal leader peptide, which may or may not be cleaved after translocation across the membrane. An example of a signal peptide is MKKIWLALAGLVLAFSASA (SEQ ID NO: 1), as described herein. Another signal sequence is MAPFEPLASGILLLLWLIAPSRA (SEQ ID NO: 14). Other examples of signal peptides can be found in SPdb, a database of signal peptides, which is found on the worldwide network website "proline.bic.nus.edu.sg/spdb/".
In some embodiments, where the antigen is fused to a complementary affinity protein, the signal sequence may be located at the N-terminus of the complementary affinity protein. For example, if an antigen is fused to an avidin-like protein, the signal sequence may be located at the N-terminus of the complementary affinity protein. In some embodiments, the signal sequence is cleaved from the complementary affinity protein before the complementary affinity protein associates with the first affinity molecule.
In some embodiments, a complementary affinity antigen and / or protein, as described herein, lacks a signal sequence.
The polypeptides described herein can be expressed in a variety of expression host cells, for example, bacteria, yeast, mammalian cells, insect cells, plant cells, algal cells, such as Chlamadomonas, or in expression-free systems. cells. In some embodiments, the nucleic acid can be subcloned from the cloning vector into a recombinant expression vector that is appropriate for the expression of the fusion polypeptide in bacteria, mammalian cells, insects, yeast, or a plant or system cell-free expression, such as a rabbit reticulocyte expression system. Some vectors are designed to transfer nucleic acid encoding for expression in mammalian cells, insect cells in a single recombination reaction. For example, some of the GATEWAY ® (Invitrogen) target vectors are designed for the construction of baculoviruses, adenoviruses, adeno-associated viruses (AAV), retroviruses, and lentiviruses that, through infection of their respective host cells, allow heterologous expression of fusion polypeptides in the appropriate host cells. The transfer of a gene in a destination vector is carried out in just two steps, according to the manufacturer's instructions. There are GATEWAY ® expression vectors for the expression of proteins in insect cells, mammalian cells and yeast. After transformation and selection in E. coli, the expression vector is ready to be used for expression in the appropriate host.
Examples of other expression vectors and host cells are pcDNA3.1 based on a strong CMV promoter (Invitrogen) and pCIneo vectors (Promega) for expression in mammalian cell lines, such as CHO, COS, HEK-293, Jurkat, and MCF -7; incompetent adenoviral vector in replication vectors pADENO-X ™, pAd5F35, pLP-ADENO ™ -X-CMV (CLONTECH®), pAd / CMV / V5-DEST, vector pAd-DEST (Invitrogen) for adenovirus-mediated gene transfer and expression in mammalian cells; retroviral vectors pLNCX2, pLXSN, and pLAPSN for use with the RETRO-X ™ system from Clontech for retrovirus-mediated gene transfer and expression in mammalian cells; pLenti4 / V5-DEST ™, pLenti6 / V5-DEST ™, and pLentiβ.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors, such as vector pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC (Stratagene) for gene transfer mediated by adeno-associated viruses, and expression in mammalian cells; baculovirus BACpakβ (Clontech) and pFASTBAC ™ HT (Invitrogen) for expression in insect cell lines 9 (Sf9), Sfll, Tn-368 and BTI-TN-5B4-1 from S. frugiperda; pMT / BiP / V5-His (Invitrogen) for expression in Drosophila schneider- S2 cells, Pichia expression vectors pPICZÜ, pPICZ, pFLDÜ and pFLD (Invitrogen) for expression in P. pastoris and pMETÜ and pMET vectors for expression in P methanolica; vectors pYES2 / GS and pYDl (Invitrogen) for expression in yeast S. cerevisiae.
Recent advances in large scale heterologous expression proteins in Chlamydomonas reinhardtii are described. Griesbeck., 34 Mol. Biotechnol. 213 (2006); Fuhrmann, 94 Methods Mol Med. 191 (2006). The foreign heterologous coding sequences are inserted into the genome of the nucleus, chloroplast and mitochondria, by homologous recombination. The chloroplast expression vector p64 carrying the selectable chloroplast marker the most versatile aminoglycoside adenyltransferase (aadA), which confers resistance to spectinomycin and streptomycin, can be used to express a foreign protein in the chloroplast. The pistol method of biological genes can be used to introduce the vector into algae. After entering into chloroplasts, the foreign DNA is released from the gene gun particles and is integrated into the chloroplast genome through homologous recombination.
Also included in the invention is the complementary affinity molecule fused with an antigen. In some embodiments, the fusion construct may also optionally comprise purification tags, and / or secretion signal peptides. These fusion proteins can be produced by any standard method. For example, for the production of a stable cell line expressing a complementary affinity molecule fusion protein - antigen, the nucleic acids - PCR amplified antigens can be cloned into the restriction site of a derivative of a mammalian expression vector. For example, KA, which is a derivative of pcDNA3 (Invitrogen) contains a fragment of DNA encoding an influenza virus (HA) hemagglutinin tag. Alternatively, derivatives of vectors encoding other tags, such as the c-myc or polyhistidine tags, can be used. The antigen-complementary affinity molecule expression construct can be co-transfected with a marker plasmid, into an appropriate mammalian cell line (for example, COS, HEK293T, or NIH-3T3 cells) using, for example, LIPOFECTAMINE ™ (Gibco -BRL, Gaithersburg, MD) according to the manufacturer's instructions, or any other appropriate transfection technique known in the art. Suitable transfection markers include, for example, β-galatosidase expression plasmids or green fluorescent protein (GFP) or any plasmid that does not contain the same detectable marker as the antigen-complementary affinity molecule fusion protein. Cells expressing the fusion protein can be classified and further cultured, or the antigen - complementary affinity molecule fusion protein can be purified. In some embodiments, antigen - complementary affinity molecule fusion protein is amplified with a signal peptide. In alternative embodiments, a cDNA encoding the antigen - complementary affinity molecule fusion protein can be amplified without the signal peptide and subcloned into a vector (pSecTagHis) having a strong secretion signal peptide. In another example, the antigen - complementary affinity molecule fusion protein may have an alkaline phosphatase (AP) tag, or a histadine (His) tag for purification. Any method known to those skilled in the art for the purification of antigen protein and / or antigen - complementary affinity molecule fusion protein is encompassed for use in the methods of the invention.
In some embodiments, any of the polypeptides described herein is produced by expression from a recombinant baculovirus vector. In another embodiment, any of the polypeptides described herein is expressed by an insect cell. In yet another embodiment, any of the polypeptides described herein is isolated from an insect cell. There are several benefits of baculovirus protein expression in insect cells, including high levels of expression, ease of scaling, protein production with post-translational modifications, and simplified cell growth. Insect cells do not require CO2 for growth and can be easily adapted for high-density suspension culture for large-scale expression. Many of the post-translational modification pathways present in mammalian systems are also used in insect cells, allowing the production of recombinant protein that is antigenically, immunogenically, and functionally similar to native mammalian protein.
Baculoviruses are DNA viruses in the Baculoviridae family. These viruses are known to have a narrow host range that is mainly limited to Lepidopteran insect species (butterflies and moths). The Autographa californica baculovirus nuclear polyhedrosis virus (AcNPV), which has become the prototype baculovirus, replicates efficiently in susceptible cultured insect cells. AcNPV has a closed circular double-stranded DNA genome of about 130,000 base pairs and is well characterized with respect to host range, molecular biology and genetics. The baculovirus expression vector system (BEVS) is a safe and fast method for the abundant production of recombinant proteins in insect and insect cells. Baculovirus expression systems are powerful and versatile systems for high-level recombinant protein expression in insect cells. Expression levels up to 500 mg / 1 have been reported using the baculovirus expression system, making it an ideal system for high-level expression. Recombinant baculoviruses that express foreign genes are constructed by homologous recombination between baculovirus DNA and chimeric plasmids containing the gene sequence of interest. Recombinant viruses can be detected due to their distinct plaque morphology and plaque purified until homogeneous.
The recombinant fusion proteins described herein can be produced in insect cells, including, but not limited to, cells derived from the species Lepidopteran S. frugiperda. Other insect cells that can be infected with baculovirus, such as Bombyx mori, Galleria mellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as an appropriate substrate for the production of recombinant proteins described here. Baculovirus expression of recombinant proteins is well known in the art. See U.S. Patent No. 4,745,051; No. 4,879,236; No. 5,179,007; No. 5,516,657; No. 5,571,709; No. 5,759,809. It will be understood by those skilled in the art that the expression system is not limited to a baculovirus expression system. What is important is that the expression system directs the N-glycosylation of expressed recombinant proteins. The recombinant proteins described herein can also be expressed in other expression systems, such as entomopox virus (insect poxviruses), cytoplasmic polyhedrosis virus (CPV), and transformation of insect cells with the recombinant gene or constitutive expression of genes. A good number of baculovirus transfer vectors and the corresponding appropriately modified host cells are commercially available, for example, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 by BD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 by NOVAGEN®, and pPolh-FLAG and pPolh-MAT by SIGMA ALDRICH®.
The region between the promoter and the transcription terminator may have multiple restriction enzyme digestion sites to facilitate cloning of the foreign coding sequence, in this example, the DNA sequence coding for an antigen polypeptide, and a complementary affinity molecule . Additional sequences can be included, for example, signal peptides and / or tag encoding sequences (tags), such as His tag, MAT- tag, FLAG tag, recognition sequence for enterokinase, bee melitin secretion signal, beta -galatosidase, glutathione S-transferase (GST) tag upstream of MCS to facilitate secretion, identification, correct insertion, positive selection of recombinant virus, and / or purification of the recombinant protein.
In some embodiments, the fusion protein may comprise an N-terminal signal sequence, as described herein. In some embodiments, the signal sequence is attached to the N-terminal of the complementary affinity molecule as described herein.
In some embodiments, a fusion polypeptide, as described herein, has a spacer peptide, for example, a 14-residue spacer (GSPGISGGGGGILE) (SEQ ID NO: 15) separating the antigen from the complementary affinity molecule. The coding sequence for such a short spacer can be constructed by annealing a complementary pair of primers. One skilled in the art can design and synthesize oligonucleotides, which will code for the selected spacer. Spacer peptides should generally have non-polar amino acid residues, such as glycine and proline.
Standard techniques known to those skilled in the art can be used to introduce mutations (to create amino acid substitutions in a polypeptide antigen sequence of the fusion polypeptide described here, for example, in the antigen in the nucleotide sequence encoding the fusion polypeptide described herein, including , for example, site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusion polypeptide has less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions, including with respect to the fusion polypeptides described herein.
Some silent or wrong-sense mutations can also be made in the DNA coding sequence that do not change the encoded amino acid sequence, or the ability to promote transmembrane distribution. These types of mutations are usable to optimize the use of codons, or to improve the expression and production of the recombinant protein.
Specific site-directed mutagenesis of a coding sequence for the fusion polypeptide in a vector can be used to create specific amino acid mutations and substitutions. Site-directed mutagenesis can be performed using, for example, the QUICKCHANGE® site-directed mutagenesis kit from Stratagene according to the manufacturer's instructions.
In one embodiment, expression vectors comprising the DNA sequence encoding the polypeptides described herein for the expression and purification of the recombinant polypeptide produced from a protein expression system using host cells selected from, are described herein. for example, bacteria, mammalian cells, insects, yeast, or plants. The expression vector must have the necessary regulatory elements 51 upstream and 31 downstream, such as promoter sequences, ribosome recognition and TATA box and 3'UTR AAUAAA transcription termination sequence for the transcription and translation of the efficient gene in your cell respective host. The expression vector is preferably a vector with the transcription promoter selected from the group consisting of CMV promoter (cytomegalovirus), RSV promoter (Rous sarcoma virus), □ -actin promoter, VS40 promoter (virus 40 of simian) and muscle creatine kinase promoter, and the transcription terminator selected from a group consisting of SV40 0 poly (A) and BGH terminator, more preferably, an expression vector having the cytomegalovirus premature promoter / enhancer sequence and the adenovirus leader / tripartite intron sequence and containing the origin of replication and poly (A) sequence of SV4 0. The expression vector may have additional coding regions, such as encoding them, for example, 6X-histidine, V5, thioredoxin , glutathione -S- transferase, c-Myc, VSV-G, HSV, FLAG, maltose-binding peptide, metal-binding peptide, HA and "secretion" signals (bee melitin, O-factor, PHO, Bip ), which can be incorporated into the polypeptide the expressed fusion. In addition, there may be enzyme digestion sites incorporated after these coding regions to facilitate their enzymatic removal, if not required. These complementary nucleic acids are useful for the detection of expression of the fusion polypeptide, for the purification of the protein by affinity chromatography, increased solubility of the recombinant protein in the host cytoplasm, and / or for the secretion of the expressed fusion polypeptide in medium. yeast cell culture or spheroplasts. The expression of the fusion polypeptide can be constitutive in host cells, or it can be induced, for example, with copper sulfate, sugars, such as galactose, methanol, methylamine, thiamine, tetracycline, infection with baculovirus, and IPTG (isopropyl-beta- D-thiogalatopyranoside), a stable synthetic analog of latose.
In another embodiment, the expression vector comprising a polynucleotide described herein is a viral vector, such as an adenovirus, adeno-associated virus (AAV), retrovirus, and lentiviral vectors, among others. Recombinant viruses provide a versatile system for studies of gene expression and therapeutic applications.
In some embodiments, the fusion polypeptides described herein are expressed from viral infections of mammalian cells. Viral vectors can be, for example, adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus. A simplified system for generating recombinant adenoviruses is presented by He et al., 95 PNAS 2509 (1998). The gene of interest is first cloned into a shuttle vector, for example, pAdTrack-CMV. The resulting plasmid is linearized by digestion with restriction endonuclease Pmel, and subsequently co-transformed into E. coli. BJ5183 cells with a dorsal adenoviral structure plasmid, for example. pADEASY-1 from Stratagene's ADEASY ™ adenoviral vector system. recombinant adenovirus vectors are selected for kanamycin resistance, and recombination confirmed by endonuclease restriction analyzes. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, for example, HEK 293 cells (E1 transformed human embryonic kidney cells) or 911 (E1 transformed human embryonic retinal cells). Fallaux, et al. 7 Human Gene Ther. 215 (1996). Recombinant adenoviruses are generated within HEK 293 cells.
Recombinant lentiviruses have the advantage of distribution and expression of fusion polypeptides in dividing and non-dividing mammal cells. HIV-1-based lentiviruses can effectively transduce a broader host range than retroviral systems based on Moloney leukemia virus (MoMLV). Preparation of the recombinant lentivirus can be achieved using, for example, the vectors pLenti4 / V5-DEST ™, pLenti6 / V5-DEST ™ or pLenti together with VIRAPOWER ™ lentiviral expression systems from Invitrogen, Inc.
Recombinant adeno-associated virus (rAAV) vectors are applicable to a wide variety of host cells, including several human and non-human cell lines or different tissues. rAAVs are capable of transducing a wide range of cell types and the transduction is not dependent on the cell division of the active host. High titers,> 108 viral particles / ml, are easily obtained in the supernatant and 1011-1012 viral particles / ml, with a higher concentration. The transgene is integrated into the host genome so that expression is long-term and stable.
Large-scale preparation of AAV vectors is done by co-transfecting three plasmids from a packaging cell line: AAV vector carrying the encoded nucleic acid, AAV RC vector containing AAV rep and cap genes, and adenovirus helper plasmid pDF6, in plates 50 x 150 mm of 293 confluent cells. The cells are collected three days after transfection, and the viruses are released through three freeze-thaw cycles or by sonication.
AAV vectors can be purified using two different methods, depending on the serotype of the vector. The AAV2 vector is purified by the single-stage gravity flow column purification method, based on its affinity for heparin. Auricchio et. al. , 12 Human Gene Ther. 71 (2001); Summerford & Samulski, 72 J. Virol. 1438 (1998); Summerford & Samulski, 5 Nat. Med. 587 (1999). The AAV2 / 1 and AAV2 / 5 vectors are currently purified by three sequential CsCl gradients.
Without wishing to be bound by theory, when proteins are expressed by a cell, including a bacterial cell, the proteins are targeted to a particular part of the cell or secreted from the cell. Thus, protein targeting or protein classification is the mechanism by which a cell transports proteins to the appropriate positions in or outside the cell. The target classification can be in the inner space of an organelle, any one of the various inner membranes, outer membrane of the cells, or the outer one via secretion. This distribution process is carried out based on information contained in the protein itself. Correct classification is fundamental for the cell; mistakes can lead to illness.
With some exceptions, bacteria do not have membrane-bound organelles as found in eukaryotes, but they can assemble proteins on various types of inclusions, such as gas vesicles and storage granules. Also, depending on the species of bacteria, bacteria can have a single plasma membrane (Gram-positive bacteria), or both an inner membrane (plasma) and an outer cell wall membrane, with an aqueous space between the two called periplasm (Gram bacteria) negative). Proteins can be secreted into the environment, depending on whether an external membrane exists or not. The basic mechanism in the plasma membrane is similar to the eukaryotic. In addition, bacteria can target-mark proteins on or across the outer membrane. Protein secretion systems across the outer bacterial membrane can be quite complex and play key roles in pathogenesis. These systems can be described as type I secretion, type II secretion, etc.
In most Gram-positive bacteria, some proteins are targeted for export through the plasma membrane and subsequent covalent attachment to the bacterial cell wall. A specialized enzyme, sortase, cleaves the target protein at a characteristic recognition site close to the extermination protein, such as an LPXTG motif (SEQ ID NO: 16) (where X can be any amino acid), then transfers the protein over the wall of the cell. A system analogous to sortase / LPXTG, having the motif PEP-CTERM (SEQ ID NO: 17), called exosortase / PEP-CTERM, is proposed to exist in a wide range of Gram-negative bacteria.
Proteins with appropriate N-terminal target-tagging signals are synthesized in the cytoplasm and then directed to a specific protein transport pathway. During, or immediately after its translocation across the cytoplasmic membrane, the protein is processed and duplicated in its active form. Then, the translocated protein is either retained on the periplasmic side of the cell or released into the environment. Since the signal peptides that target the membrane proteins are key determinants of the specificity of the transport path, these signal peptides are classified according to the transport path to which they direct the proteins. Classification of the signal peptide is based on the type of signal peptidase (SPase) that is responsible for the removal of the signal peptide. Most exported proteins are exported from the cytoplasm via the general "secretory pathway (Sec)". Most well-known virulence factors (eg, Staphylococcus aureus exotoxins, Bacillus anthracis protective antigen, Listeria monocytogenes O listeriolisin) that are secreted by gram-positive pathogens have a typical N-terminal signal peptide that would take them to the via See. The proteins that are secreted in this way are translocated across the cytoplasmic membrane in an unduplicated state. Subsequent processing and duplication of these proteins takes place in the cell wall environment on the trans side of the membrane. In addition to the See system, some gram-positive bacteria also contain the Tat system which is capable of translocating the duplicated proteins across the membrane. Pathogenic bacteria can contain certain special export systems that are specifically involved in the transport of only a few proteins. For example, several clusters of genes have been identified in microbacteria that encode proteins that are secreted into the environment via specific pathways (ESAT 6) and are important for mycobacterial pathogenesis. Specific ATP binding cassette (ABC) carriers direct the export and processing of small antibacterial peptides called bacteriocins. Genes for endolysins that are responsible for initiating bacterial lysis are often located close to genes that code for holine-like proteins, suggesting that these holines are responsible for exporting endolysin to the cell wall. Wooldridge, BACT. SECRETED PROTS: SECRETORY MECHS. & ROLE IN PATHOGEN. (Caister Academic Press, 2009)
In some embodiments, the signal sequence usable in the present invention is an OmpA signal sequence, however, any signal sequence commonly known to those skilled in the art, which allows the transport and secretion of antimicrobial agents outside the infected bacteriophage cell is encompassed for use in the present invention.
Signal sequences that direct the secretion of proteins from bacterial cells are well known in the art, for example, as described in international patent application WO 2005/071088. For example, one can use some of the non-limiting examples of signal peptides represented in Table 5, which can be attached to the amino or carboxyl terminal of the antimicrobial peptide (Amp) or antimicrobial polypeptide to be expressed by the bacteriophage engineered by the antimicrobial agent, for example, bacteriophage engineered by AMP. Fixation can be through fusion or chimera composition with the selected antigen or antigen-complementary fusion molecule of complementary affinity, resulting in the secretion of bacteria infected with the bacteriophage engineered by the antimicrobial agent, for example, AMP-engineered bacteriophage. Table 5: Exemplary signal peptides to direct the secretion of a protein or peptide antigen or fusion protein antigen-molecule complementary to a bacterial cell


The polypeptides described herein, for example, antigen or complementary affinity antigen-molecule fusion protein can be expressed and purified by a variety of methods known to one skilled in the art, for example, the fusion polypeptides described herein can be purified from any suitable expression system. Fusion polypeptides can be purified to substantial purity by conventional techniques, including selective precipitation with substances such as ammonium sulfate; column chromatography, immunopurification methods, and others; that are well known in the art. See, for example, Scopes, PROTEIN PURIFICATION: PRINCIPLES & PRACTICE (1982); US Patent No. 4,673,641.
A number of procedures can be employed when recombinant proteins are purified. For example, proteins having established molecular adhesion properties can be reversibly fused to the protein of choice. With the appropriate ligand, the protein can be selectively adsorbed to a purification column and then released from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, the protein of choice can be purified using affinity or immunoaffinity columns.
After the protein is expressed in host cells, the host cells can be lysed to release the expressed protein for purification. Methods of lysing the various host cells are carried out in "Sample Preparation-Tools for Protein Research" EMD Bioscience and in Current Protocols in Protein Sciences (CPPS). An example of a purification method is affinity chromatography, such as metal-ion affinity chromatography using nickel, cobalt, or zinc affinity resins for histidine-tagged fusion polypeptides. Methods of purifying histidine-tagged recombinant proteins are described by Clontech using their cobalt resin TALON® and by NOVAGEN® in their pET system manual, 10th edition. Another preferred purification strategy is immunoaffinity chromatography, for example, resin conjugated to anti-myc antibody can be used to affinity purify myc-tagged fusion polypeptides. When suitable protease recognition sequences are present, the fusion polypeptides can be cleaved from the histidine or myc tag, releasing the fusion polypeptide from the affinity resin while the histidine and myc tags are left attached to the affinity resin .
Standard separation techniques for the purification of recombinant and naturally occurring proteins are well known in the art, for example, solubility fractionation, gel filtration by size exclusion, and various column chromatographies.
Solubility fractionation: Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture medium) from the protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mix. The proteins then precipitate based on their solubility. The more hydrophobic a protein is, the more likely it is to precipitate in low concentrations of ammonium sulfate. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resulting ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant at a known concentration, to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or through diafiltration. Other methods that rely on protein solubility, such as precipitation with cold ethanol, are well known to those skilled in the art and can be used to fractionate complex protein mixtures.
Filtration by size exclusion: The molecular weight of the protein of choice can be used to isolate it from larger and smaller proteins using ultrafiltration through membranes of different pore sizes (eg AMICON® or MILLIPORE® membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a cut molecular weight less than the molecular weight of the protein of interest. The product retained from ultrafiltration is then ultrafiltered against a membrane with a molecular cut greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed, as described below.
Column chromatography: The protein of choice can also be separated from other proteins based on its size, liquid surface charge, hydrophobicity and affinity for ligands. In addition, antibodies produced against recombinant or naturally occurring proteins can be conjugated to spine arrays and immunopurified proteins. All of these methods are well known in the art. It will be evident to an expert that the chromatographic techniques can be performed on any scale and using equipment from different manufacturers (for example, Pharmacia Biotech). For example, an antigen polypeptide can be purified using a PA63 heptomer affinity column. Singh et al., 269, J. Biol. Chem. 29039 (1994).
In some embodiments, a combination of purification steps comprising, for example: (a) ion exchange chromatography, (b) hydroxyapatite chromatography, (c) hydrophobic interaction chromatography, and (d) size exclusion chromatography can be used to purify the fusion polypeptides described herein.
Cellular free expression systems are also contemplated. Cell-free expression systems offer several advantages over traditional cell-based expression methods, including easy modification of reaction conditions to favor duplication of proteins, decreased sensitivity to product toxicity and adaptation of strategies for high yield, such as rapid screening of expression or production of large amounts of protein with reduced reaction volumes and process time. The cell-free expression system can use plasmid or linear DNA. In addition, improvements in translation efficiency have resulted in yields in excess of one milligram of protein per milliliter of the reaction mixture. Commercially available cell-free expression systems include TNT-copulated reticulocyte lysate systems (Promega), which use in vitro systems based on rabbit reticulocytes. Formulations of an immune composition and methods of use
Specific embodiments of the present invention provide for the use of immunogenic compositions, as described herein, to induce an immune response in an animal. More specifically, the compositions elicit humoral and cellular immunity, and in many cases mucosal immunity. Embodiments of the present invention provide at least partial protection from partial improvement or after infection by, in particular, pneumococci. Pneumococci cause a number of diseases, such as meningitis, pneumonia, bacteremia and otitis media. Nearly a million children die from pneumococcal disease worldwide each year. S. pneumonia has been studied extensively, and at least some of the sequenced genomes. See, for example, US Patent No. 7,141,418. Although antibodies against capsular polysaccharides, which define known serotypes, confer specific serotype protection, other mechanisms of immunity protection have been described. VerMalley et al. , 88 J. Mol. Med. 135 (2010). These other protective mechanisms include, but are not limited to, antibodies to non-capsular antigens and T cell responses to pneumococcal constituents. The application of the protein-polysaccharide conjugate vaccine, PCV7, significantly reduced diseases. Black et al., 24 (S2) Vaccine 79 (2006); Hansen et al., 25 Pediatr. Infect. Dis. J. 779 (2006)). However, recent studies have shown that the absence of other serotypes in PCV7 has resulted in the appearance of replacement pneumococcal serotypes. Pichichero & Casey, 26 (S10) Pediatr. Infect. Dis. J. S12 (2007).
Some pneumococcal antigens common to all species serotypes have been shown to have immunoprotective potential despite encapsulation, for example, PspA, PspC, PsaA surface proteins and cytotoxin pneumolysin or pneumolysoid mutants (Basset et al., 75 Infect. Immun. 5460 (2007); Briles et al., 18 Vaccine 1707 (2000)); the use of genomic and mutational libraries has identified several common proteins from dozens of additional species (Hava & Camilli, 45 Mol. Microbiol. 1389 (2002); Wizemann et al., 60 Infect. Immun. 1593 (2001)). Immunity was induced by individual antigens in animal models (Alexander et al., 62 Infect. Immun. 5683 (1994);
Balachandran et al. , 70 Infect. Immun. 2526 (2002); Chung et al. , 170 J. Immunol. 1958 (2003); Glover et al. 76 Infect. Immun. 2767 (2008); Wu et al. , 175 J. Infect. Dis. 839 (1997)), but no vaccine based on a common antigen has been approved for human use to date.
In one embodiment, a method of vaccinating a mammal is provided herein, comprising administering the immunogenic composition comprising at least one, or multiple antigens attached to at least one type of polymer scaffold, for example, a polysaccharide polymer or carbohydrates for use in eliciting an immune response to one or more antigens attached to the polymer when administered to an individual. In some embodiments, the immune response is a humoral and / or cellular immune response.
Therefore, an aspect of the present invention relates to methods for eliciting an immune response in an individual, comprising administering to the individual an immunogenic composition comprising at least one type of polymer, for example, a polysaccharide, at least one antigen, and at least one pair of complementary affinity molecules comprising (i) a first affinity molecule that associates with the polymer, for example, a polysaccharide, and (iij a complementary affinity molecule that associates with the antigen, for attaching the antigen to the polymer, for example, a polysaccharide, (for example, the first affinity molecule associates with the complementary affinity molecule to bind the antigen to the polymer, for example, polysaccharide).
Therefore, an aspect of the present invention relates to methods for eliciting humoral and / or cellular immunity to multiple antigens, at the same time, for example, when the immunogenic composition administered to the individual comprises a polymer comprising at least 1, or at least 2, or one more, for example, a plurality of the same or different antigens.
One aspect of the present invention relates to a method of immunizing or vaccinating an individual, for example, a bird or a mammal, for example, a human against a pathogen, comprises administering an immune composition, as described herein, comprising at least least one antigen derived from one or more pathogens. In some embodiments, an individual can be immunized against at least 1, or at least 2, or at least 2, or at least 3, or at least 5, or at least 10, or at least 15, or at least about of 20, or at least 50, or at least about 100, or more than 100 different pathogens at the same time, in which the polymer of the immunogenic composition as the corresponding different antigens fixed.
In some embodiments, an individual can be administered with several different immunogenic compositions, as described herein, for example, an individual can be administered with a composition comprising a polymer with an antigen, or a plurality of antigens, for example, antigens A, B, C, and D, etc., and also administered with a composition comprising a polymer comprising a different antigen or a different set of antigens, for example, W, X, Y, and Z antigens, etc. Alternatively, an individual can be administered with a composition comprising a polymer A with an antigen, or a plurality of antigens, for example, antigens A, B, C, and D, etc., and also administered with a composition comprising a polymer B comprising the same , for example, antigens A, B, C, and D etc., or a different set of antigens. It is envisaged that the present invention provides methods for immunizing an individual with as many antigens as desired, for example, with a variety of different immunogenic complexes, as described herein, to allow immunization with a maximum of 100 or more antigens.
In one embodiment, the immunogenic compositions as described herein comprise a pharmaceutically acceptable carrier. In another embodiment, the immunogenic composition described herein is formulated for administration to a bird, mammal, or human, as or in a vaccine. Appropriate formulations can be found in, for example, Remington's Pharmaceutical Sciences (2006), or Introduction to Pharmaceutical Dosage Forms (4th ed., Lea & Febiger, Philadelphia, 1985).
In one embodiment, the immunogenic compositions as described herein comprise pharmaceutically acceptable vehicles that are inherently non-toxic and non-therapeutic. Examples of such vehicles include ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable fatty acids saturated, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium phosphate, sodium chloride, zinc salts, colloidal silica, magnesium tri-silicate, polyvinylpyrrolidone, cellulose-based substances, and polyethylene glycol. For all administrations, conventional forms of deposit are appropriately used. Such forms include, for example, microcapsules, nanocapsules, liposomes, plasters, forms for inhalation, nasal sprays, sublingual tablets, and sustained release preparations. For examples of controlled release compositions, see U.S. Patent No. 3,773,919, No. 3,887,699, EP 58,481A, EP 158,277A, CA Patent No. 1176565; Sidman et al., 22 Biopolymers 547 (1983); Langer et al. , 12 Chem. Tech. 98 (1982). Proteins will generally be formulated at a concentration of about 0.1 mg / ml to 100 mg / ml per application per patient.
In one embodiment, other ingredients can be added to the vaccine formulations, including antioxidants, for example, ascorbic acid; low molecular weight polypeptides (less than about ten residues), for example, polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins, chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.
In some embodiments, the present MAPS immunogenic compositions are administered with at least one adjuvant. Adjuvants are a heterogeneous group of substances that enhance the immune response against an antigen that is administered simultaneously. In some cases, adjuvants improve the immune response so that less vaccine is needed. Adjuvants serve to bring the antigen - the substance that stimulates the specific protective immune response - into contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (size or duration). Adjuvants can also decrease the toxicity of certain antigens, and provide solubility for some components of the vaccine. Almost all of the adjuvants used today to improve the immune response against antigens are particles or were particles in conjunction with the antigen. In the book VACCINE DESIGN-SUBUNIT & ADJUVANT APPROACH (Powell & Newman, Eds., Plenum Press, 1995), many known adjuvants are described both in terms of their immunological activity and in relation to their chemical characteristics. The type of adjuvants that do not form particles are a group of substances that act as immune signal substances and that, under normal conditions, consist of substances that are formed by the immune system as a consequence of immune activation after administration of particulate adjuvant systems.
Adjuvants for immunogenic compositions and vaccines are well known in the art. Examples include, but are not limited to, monoglycerides and fatty acids (for example, a mixture of mono-olein, oleic acid, and soybean oil); mineral salts, for example, aluminum and aluminum hydroxide or calcium phosphate gels; oil emulsions and surfactant-based formulations, for example, MF59 (oil-in-water emulsion stabilized in microfluidized detergent), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + SQ 21), MPL - SE, Montanide ISA - 51 and ISA - 720 (stabilized water-in-oil emulsion); particulate adjuvants, for example, virosomes (unilamellar liposome vehicles incorporating influenza hemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactic co-glycolide (PLG); microbial derivatives (natural and synthetic), for example, monophosphoryl lipid A (MPL), Detox (dorsal structure of the cell wall MPL + M. phlei), AGP [RC-529] (synthetic acylated monosaccharides), Detox PC, DC_Chol (immuno lipoidal stimulators capable of self-organizing into liposomes), OM-174 (derived from lipid A), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), or other modified DNA, LT and CT structures (bacterial toxins genetically modified to give non-toxic adjuvant effects); endogenous human immunomodulators, for example, hGM - CSF UO - hIL 12 (cytokines that can be administered either as a protein or encoded plasmid), Immudaptin (C3d tandem matrix), MoGM CSF, TiterMax -G, CRL -1005, GERBU, TERamide , PSC97B, Adjumer, PG 026, GSK I, GcMAF, B - alethine, MPC - 026, Adjuvax, CpG ODN, Betafectin, Alúme and MF59 and inert vehicles, such as gold particles. Additional adjuvants are known in the art, see, for example, US Patent No. 6,890,540; US Patent Publication No. 2005; 0244420; PCT / SE97 / 01003.
In some embodiments an adjuvant is a particulate and may have a feature of being slowly biodegradable. Care must be taken to ensure that the adjuvant does not form toxic metabolites. Preferably, in some embodiments, such adjuvants that can be used matrices are mainly substances originating from a body. These include polymers of lactic acid, poly-amino acids (proteins), carbohydrates, lipids and biocompatible polymers with low toxicity.
Combinations of these groups of substances originating from a body or combinations of substances originating from a body of biocompatible polymers can also be used. Lipids are the preferred substances, as they exhibit structures that make them biodegradable, as well as the fact that they are an essential element in all biological membranes.
In one embodiment, the immunogenic compositions as described herein for administration must be sterile for administration to an individual. Sterility is easily achieved by filtration through sterile filtration membranes (for example, 0.2 micron membranes), or by gamma irradiation.
In some embodiments, the immunogenic compositions described herein further comprise pharmaceutical excipients, including, but not limited to biocompatible oils, physiological saline solutions, preservatives, carbohydrates, proteins, amino acids, osmotic pressure control agents, carrier gases, to controlling agents pH, organic solvents, hydrophobic agents, enzyme inhibitors, water-absorbing polymers, surfactants, absorption promoters and anti-oxidants. Representative examples of carbohydrates include water-soluble sugars such as hydropropyl cellulose, carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid, chitosan, alginate, glucose, xylose, galactose, fructose, maltose, sucrose, dextran, chondroitin sulfate, etc. Examples representative proteins include albumin, gelatin, etc. Representative examples of amino acids include glycine, alanine, glutamic acid, arginine, lysine, and their salts. Such pharmaceutical excipients are well known in the art.
In some embodiments, the immunogenic composition of MAPS is administered in combination with other therapeutic ingredients, including, for example, □ - interferon, cytokines, chemotherapeutic agents, or anti-inflammatory or anti-viral agents. In some embodiments, the immunogenic composition, as described herein, can be administered with one or more coherent and / or adjuvant molecules, as shown herein.
In some embodiments, the immunogenic composition is administered in a pure or substantially pure form, but it can be administered as a pharmaceutical composition, formulation or preparation. Such a formulation comprises the MAPS described herein together with acceptable vehicles from one or more pharmaceutically vehicles and, optionally, other therapeutic ingredients. Other therapeutic ingredients include compounds that enhance antigen presentation, for example, interferon gamma, cytokines, chemotherapeutic agents, or anti-inflammatory agents. The formulations can conveniently be presented in unit dosage form and can be prepared by methods well known in the pharmaceutical art. For example, Plotkin and Mortimer, in VACCINES (2nd ed., WB Saunders Co., 1994) describe the vaccination of animals or humans to induce a specific immune response to certain pathogens, as well as methods for the preparation of antigens, determining a appropriate dose of antigen, and testing for induction of an immune response.
Formulations suitable for intravenous, intramuscular, intranasal, buccal, sublingual, vaginal, rectal, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions that are preferably isotonic with the recipient's blood. Such formulations can be conveniently prepared by dissolving the solid active ingredient in water containing physiologically compatible substances such as sodium chloride (eg, 0.1 M-2.0 M), glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and making the solution sterile. These can be present in single or multiple dose containers, for example, sealed ampoules or vials.
Liposomal suspensions can also be used as pharmaceutically acceptable vehicles. These can be prepared according to methods known to those skilled in the art, for example, as described in US Patent 4,522,811.
Formulations for intranasal administration are described in US Patents 5,427,782; No. 5,843,451; No. 6,398,774.
The formulations of the immunogenic compositions can incorporate a stabilizer. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids that can be used, alone or as mixtures. Two or more stabilizers can be used in aqueous solutions at the appropriate concentration and / or pH value. The specific osmotic pressure of such an aqueous solution is generally in the range of 0.1-3.0 osmoses, preferably in the range of 0.80-1.2. The pH of the aqueous solution is adjusted to be within the pH range 5.0-9.0, preferably within the pH range 6, 8.
When oral preparations are desired, immunogenic compositions can be combined with typical vehicles, such as latose, sucrose, starch, magnesium stearate, crystalline cellulose, methylcellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic, among others.
In some embodiments, the immunogenic compositions, as described herein, can be administered intravenously, intranasally, intramuscularly, subcutaneously, intraperitoneally, sublingually, vaginally, rectally or orally. In some embodiments, the route of administration is oral, intranasal, subcutaneous, or intramuscular. In some embodiments, the route of administration is intranasal administration.
Vaccination can be carried out by conventional methods. For example, immunogenic compositions can be used in an appropriate diluent, such as saline or water or complete or incomplete adjuvants. The immunogenic composition can be administered by any suitable route of administration to induce an immune response. The immunogenic composition can be administered once or at periodic intervals until an immune response is elicited. Immune responses can be detected by a variety of methods known to those skilled in the art, including, but not limited to, antibody production, cytotoxicity assay, proliferation assays and cytokine release assays. For example, blood samples can be extracted from the immunized mammal, and analyzed for the presence of antibodies against the antigens of the immunogenic composition by ELISA (see de Boer et. Al., 115 Arch Virol. 147 (1990) and the title of these antibodies can be determined by methods known in the art.
The exact dose to be used in the formulation will also depend on the route of administration and should be decided according to the judgment of the doctor and the circumstances of each patient. For example, a range of 25 dg-900 Dg of total protein can be administered monthly for three months.
Finally, the attending physician will decide the amount of immunogenic composition or vaccine composition to be administered to particular individuals. As with all immunogenic compositions or vaccines, the immunogenically effective amounts of the immunogens must be determined empirically. Factors to be considered include immunogenicity, whether or not immunogen will be complexed with or covalently attached to an adjuvant or supporting protein or other carrier, routes of administration and the number of immunizing doses to be administered. Such factors are known in the art of vaccines and it is well within the ability of immunologists to make these determinations without undue experimentation. Kits
The present invention also provides kits for the production of an immunogenic composition, as described herein, which is useful for an investigator to adapt an immunogenic composition with his preferred antigens, for example, for research purposes to evaluate the effect of an antigen, or a combination of antigens in the immune response. Such kits can be prepared from readily available materials and reagents. For example, such kits may comprise any one or more of the following materials: a container comprising a polymer, for example, a polysaccharide, cross-linked with a plurality of first affinity molecules, and a container comprising a complementary affinity molecule that is associated with the first affinity molecule, where the complementary affinity molecule associates with an antigen.
In another embodiment, the kit may comprise a container comprising a polymer, for example, a polysaccharide, a container comprising a plurality of first affinity molecules, and a container comprising a crosslinking reagent for crosslinking the first affinity molecules to the polymer.
In some embodiments, the kit further comprises a means for attaching the complementary affinity molecule to the antigen, where the means can be made by a cross-linking reagent or any intermediate fusion protein. In some embodiments, the kit may comprise at least one costimulation factor, which can be added to the polymer. In some embodiments, the kit comprises a cross-linking reagent, for example, but not limited to, CDAP (l-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC (l-ethyl-3- [3-dimethylaminopropyl] hydrochloride] carbodiimide), sodium cyanoborohydride; cyanogen bromide; ammonium bicarbonate / iodoacetic acid to bind the cofactor to the polymer.
A variety of kits and components can be prepared for use in the methods described here, depending on the intended use of the kit, the particular target antigen and the user's needs.
In one embodiment, an immunogenic composition or vaccine composition, as described herein, when administered to mice, can elicit an immune response that prevents a disease symptom in at least 20% of animals challenged with 5 LD5o of the immunogenic composition comprising antigens for which the symptom of the disease is avoided. The methods of vaccination and challenge of an immunized animal are known to one skilled in the art. For example, a 10 üg aliquot of an immunogenic composition or vaccine composition, as described herein, can be prepared in 100 Dl of PBS and / or with the addition of incomplete Freund's adjuvant and injected intramuscularly, by vaccinated mouse. . Alternatively, parenteral, intraperitoneal and plantar pad injections can be used. Volumes of injections in the pads are reduced to 50 Dl. Mice can be immunized with an immunogenic composition or vaccine composition, as described herein, on three separate occasions at intervals of several days, for example, 14 days between them.
The effectiveness of vaccination can be tested by challenge with the pathogen. Seven days after the last dose of an immunogenic composition, the immunized mice are challenged intranasally with a pathogenic organism from which the antigen was derived. Ether anesthetized mice (10 g to 12 g) can be infected intranasally with 50 pl of allantoic fluid diluted in PBS containing 5 LD5o of the pathogenic organism. Protection can be measured by monitoring animal survival and body weight, which is assessed over an observation period of 21 days. Severely affected mice are euthanized. An LD50 of A / Mallard / Pennsylvania / 10218/84 is equal to 100-1000 of the tissue culture infectious dose 50 test (TCID50).
In other embodiments, the immunized mice can be challenged with a variety of different pathogenic organisms, for example, different pathogenic organisms from which each of the polymer-attached antigens are derived. For example, an immunogenic composition comprises five different antigens attached to the polymer, for example, polysaccharide, where each antigen is derived from five different pathogenic organisms, the immunized mice can be challenged with each of the five different pathogenic organisms, or sequentially (in any order) or simultaneously. One skilled in the art would be able to determine the LD50 for each pathogenic organism used to challenge immunized mice by methods known in the art. See, for example, LaBarre & Lowy, 96 J. Virol. Meths. 107 (2001); Golub, 59J. Immunol. 7 (1948).
Although the above invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications can be made without departing from the spirit or scope of the attached claims. The following is intended to be illustrative of the present invention, however, the practice of the invention is not limited or restricted in any way by the examples. EXAMPLES
The examples presented here refer to methods for generating an immunogenic complex, as described herein, and methods and compositions thereof. In particular, the examples refer to methods for producing a multiple antigen presentation complex (MAP), as shown here, and methods of use to generate an immune response in an individual. Example 1. Construction of recombinant rhizavidine and Rhizavidine-antigen fusion proteins
Recombinant rhizavidine (rRhavi) used in these studies is a modified N-terminal version that contains only residues 45 to 179 of the wild-type protein. To optimize the level of rRhavi expression in E. coli, the gene sequence encoding the Rhizavidina polypeptides (45-179) was re-designed using E. coli preferred expression codons, then synthesized and cloned into the pET21b vector. To facilitate correct duplication and obtain a high yield of soluble recombinant protein, a DNA sequence encoding an E. coli periplasmic coli location signal sequence (19 amino acids, MKKIWLALAGLVLAFSASA, SEQ ID NO: 1) was introduced at the 5 'end of the synthetic rRhavi gene. This signal sequence is expected to be automatically deleted from the recombinant protein, after its target-tagging for the E. coli periplasm during the expression process.
To construct a Rhizavidina-antigen fusion protein, a DNA sequence encoding a flexible linker region consisting of seven amino acids (GGGGSSS, SEQ ID NO: 27) was directly inserted into the 3 'end of the synthetic rRhavi gene, to help stabilize the fusion protein. The genes encoding candidate antigens (full length or desired fragments) were amplified from the genomic DNA of pathogens of interest by routine PCR procedures and inserted into the rRhavi expression vector just beyond the linker region.
For protein expression, plasmids containing target constructs were transformed into the E. coli BL21 (DE3) strain using the normal heat shock procedure. A single colony was chosen recently from the plate (or a glycerol filler was used later) and inoculated in 30 ml of Luria-Bertani (LB) medium containing ampicillin (Amp +) for a night culture at 37 ° C. On day 2, 5 ml of culture was inoculated in 1 liter of LB / Amp + medium and cultured at 37 ° C until ODeoo = 1 was reached. After cooling the medium to 16 ° C, 0.2 mM final IPTG concentration was added to the cultures for overnight induction.
The proteins were purified from the periplasmic fraction using a modified osmotic shock protocol. Briefly, bacterial cells from the 6-liter culture were collected and resuspended in 120 ml of buffer containing 30 mM Tris (pH 8.0), 20% sucrose and 1 mM EDTA. After stirring at room temperature for 20 min, the cells were re-granulated by centrifugation at 10,000 rpm for 10 min. The supernatant was collected as fraction 1, and the cells were resuspended in 80 ml of ice-cold solution containing 5 mM MgClz, protease inhibitor and DNase. After stirring at 4 ° C for 20 min, the mixture was centrifuged at 13,000 rpm for 20 min and the supernatant was collected as fraction 2. After the addition of a final concentration of 150 mM NaCl, 10 mM MgC12 and 10 mM imidazole, the supernatant combining fraction 1 and fraction 2 was applied to a Ni-NTA column. The proteins eluted from the NTA column were further purified by gel filtration using a Superdex 200 column by cycling in an AKTA purifier. Peak fractions containing target protein were pooled and concentrated. Protein concentration was measured using Bio-Rad's BCA protein assay kit. The purified proteins were aliquoted, frozen instantly in liquid nitrogen and kept at 80 ° C for future use. Example 2. Polysaccharide biotinylation
Biotinylation of polysaccharides was performed using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as an activation reagent. Briefly, the polysaccharides were dissolved in 10 mg / ml LPS-free water (or other concentration as indicated). At t = 0, a volume of CDAP (made recently at 100 mg / ml in acetonitrile) was slowly added to the polysaccharide solution at a rate of 1 - 2 mg CDAP / mg of polysaccharide, when vortexing. Thirty seconds later, a 0.2 M volume of triethylamine (TEA) was added (equal to or twice the volume of CDAP, depending on the different types of polysaccharides) to raise the pH. In 2.5 min, a volume of biotin derivative (Pierce's EZ-Link Amine-PEG3-biotin, solubilized in 20 mg / ml in LPS-free water) was added to a final ratio of 1-1.5 pg of biotin / mg polysaccharide for overnight copulation at 4 ° C (or 1-3 hours copulation at 25 ° C). On day 2, 50 mM of final concentration of glycine or serine was added to end the reaction, and then the mixture was desalted by passing through a column or dialyzed against a large volume of PBS to remove free biotin derivatives. The biotin content in the biotinylated polysaccharide was measured using Pierce's biotin quantification kit and the polysaccharide concentration was determined by the anthrone test. Example 3. Assembly and purification of MAPS
To assemble a MAPS complex, a volume of biotinylated polysaccharide was mixed with the candidate - rRHAvi-antigen fusion proteins in a desired ratio and then incubated at 4 ° C or 25 ° C overnight. After incubation, the mixture was centrifuged at 13,200 rpm for 3 minutes to remove insoluble aggregates. The supernatant was applied to gel filtration chromatography, using superose-6 or sperdex-200 column, with PBS, Tris buffer, or saline as the cycling solution. Peak fractions containing high molecular weight complexes were collected and concentrated. The protein contents and the ratio of different antigens in the MAPS complex were tested by SDS-PAGE with Coomassie blue staining, and the MAPS protein / polysaccharide ratio was determined using the BCA protein assay kit and the anthrone assay. Example 4. Immunization; analysis of antibodies and cytokines; challenge in mice
All immunogenic compositions and vaccines were prepared one day before immunization. Pneumococcal whole cell vaccine, MAPS an equimolar mixture containing all specific antigens, rhizavidine, polysaccharide and biotin were diluted using saline with the indicated concentration and then mixed with Al (OH) 3 in a 15 ml conical tube for adsorption night at 4 ÜC.
C57BL / 6J mice (Jackson Laboratories, Bar Harbor, Maine) were used in all immunization experiments. The age at the time of the first immunization was between 4-6 weeks. Gently restricted, the non-anesthetized mice received three subcutaneous injections of 200 pl of adjuvant, with or without the indicated amount of antigen in the lower back, at 2-week intervals. The blood was collected 2 weeks after the second and / or third immunization, and analyzed for antibodies and for the production of cytokines in vitro, after stimulation with whole cell pneumococcal antigen (WCA), TB extract, or particular protein antigen.
Challenge was carried out two weeks after the last immunization, or bleeding. In the NP colonization model, mice were challenged intranasally with 2 x 107 colony forming units (CFU) of serotype 6B strain 0603 in 20 pl of PBS. To determine the presence and degree of NP colonization, an upper respiratory culture was made 10 days later by instilling sterile retrograde saline through the transected trachea, collecting the first 6 drops (about 0.1 ml) from the nostrils, and placing in plates of pure samples or diluted in blood agar plates containing 2.5 mg / ml gentamicin.
In the aspiration-sepsis challenge model, the mice were anesthetized gently with isoflorane, in the supine position, and received an intranasal inoculation of 100 pl containing 10sCFU of pneumococcus serotype 3 strain WU-2. The mice were monitored twice a day and euthanized by CO2 inhalation and terminal bleeding when showing signs of disease, after which a blood culture was obtained.
Assays for murine antibodies to WCA or different protein antigens were performed on 96-well Immulon 2 HB plates (Thermo Scientific, Waltham, MA) coated with WCA (100 pg protein / ml PBS) or with protein antigens (1 Dg protein / ml PBS). The plates were blocked with 1% BSA in PBS. Antibody diluted in PBS-T was added and incubated at room temperature for 2 hours. The plates were washed with PBS-T, and antibody conjugated to secondary HRP for mouse immunoglobulin G (Sigma) and incubated at room temperature for one hour. The plates were washed and developed with SureBlue TMB Microwell Peroxidase Substrate substrate (KPL, Gaithersburg, MD).
For cytokine stimulation, the stimulants were diluted in stimulation medium (DMEM medium (BioWhittaker, Walkersville, MD) containing 10% defined low endotoxin FBS (Hyclone, Logan, UT), 50 pM 2-mercaptoethanol (Sigma ) and ciprofloxacin (10 pg / ml, Cellgro, Manassas, VA)), at a concentration of 1 Dg / ml-10 üg / ml for all protein antigens, or for pneumococcal WCA. 25 ml of heparinized blood was added to 225 pl of DMEM medium with / without stimulants and cultured at 37 ° C for 6 days. Supernatants were collected after centrifugation and stored at - 80 ° C until analyzed by ELISA for IL-17A or IFN-y concentration (R & D Systems, Minneapolis, MN).
For splenocyte stimulation, mouse splenocytes were isolated, resuspended in stimulation medium again, and then seeded in 48-well plates (3 x 106 cells / well, in 300 pl of volume). After incubation at 37 ° C for 2 h, stimuli were added to the indicated concentration, for stimulation at 37 ° C for 3 days. Supernatants were collected after centrifugation and stored at - 80 ° C until analyzed by ELISA for IL-17A or IFN-y concentration.
The concentrations of antibodies and IL-17A and colonization densities of NP were compared by the Mann-Whitney U test using PRISM (version 4.0a for Macintosh, GraphPad Software, Inc). Differences in survival were analyzed using the Kaplan-Meier test, also using PRISM.

权利要求:
Claims (23)
[0001]
1. Immunogenic composition comprising an immunologically effective amount of at least one antigenic polysaccharide, an immunologically effective amount of at least one peptide or polypeptide antigen, and at least one pair of complementary affinity molecules characterized by the fact that the pair comprises a first molecule of affinity and a second affinity molecule complementary to the first affinity molecule, where: the first affinity molecule is associated with at least one antigenic polysaccharide, and the second complementary affinity molecule is associated with at least one peptide or polypeptide antigen, and wherein the first affinity molecule non-covalently associates with the complementary affinity molecule to bind the peptide or polypeptide antigen and the antigenic polysaccharide.
[0002]
2. Immunogenic composition according to claim 1, characterized by the fact that the first affinity molecule is cross-linked or covalently linked to the antigenic polysaccharide.
[0003]
3. Immunogenic composition according to claim 1 or 2, characterized by the fact that the first affinity molecule is cross-linked with the antigenic polysaccharide using a cross-linking reagent selected from any of the group consisting of: CDAP (l- tetrafluoroborate) cyano-4-dimethylaminopyridinium); EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride); sodium cyanoborohydride; cyanogen bromide; and ammonium bicarbonate / iodoacetic acid.
[0004]
4. Immunogenic composition according to any one of claims 1 to 3, characterized by the fact that the pair of affinity molecules can be selected from the group consisting of: biotin / biotin-binding protein, antibody / antigen, enzyme / substrate, receptor / ligand, metal / metal binding protein, carbohydrate / carbohydrate binding protein, lipids / lipid binding protein, polyhistidine tag / polyhistidine tag binding substance.
[0005]
Immunogenic composition according to any one of claims 1 to 4, characterized by the fact that the first affinity molecule is biotin or a derivative or mimetic molecule thereof, and / or in which the second complementary affinity molecule is selected of the group consisting of: a biotin-binding protein, an avidin-like protein, such as rhizavidine, avidin, streptavidin or a homologous, derivative or functional portion thereof.
[0006]
Immunogenic composition according to any one of claims 1 to 5, characterized in that the peptide or polypeptide antigen is a fusion protein comprising the peptide or polypeptide antigen fused to the second complementary affinity molecule or a binding molecule with protein affinity.
[0007]
Immunogenic composition according to any one of claims 1 to 6, characterized in that the peptide or polypeptide antigen is non-covalently or covalently linked to the second complementary affinity molecule.
[0008]
8. Immunogenic composition according to any one of claims 1 to 5, characterized in that the antigenic polysaccharide is a branched-chain polysaccharide or a linear-chain polysaccharide.
[0009]
Immunogenic composition according to any one of claims 1 to 8, characterized in that it comprises a flexible linker peptide linked to the peptide or polypeptide antigen, wherein the flexible linker peptide fixes the peptide or polypeptide antigen to the second affinity molecule complementary.
[0010]
10. Immunogenic composition according to any one of claims 1 to 9, characterized in that the peptide or polypeptide antigen originates from a pathogenic organism or a tumor.
[0011]
11. Immunogenic composition according to any one of claims 1 to 10, characterized in that it comprises between 1 and 10 different peptide or polypeptide antigens or between 10 and 20 different peptide or polypeptide antigens, or between 20 and 50 peptide or polypeptide antigens different, or between 50 and 100 different peptide or polypeptide antigens, or more than 100 different peptide or polypeptide antigens, in which different peptide or polypeptide antigens are selected from the group consisting of different peptides or polypeptides from the same or different organisms pathogenic or tumors, different variants of the same peptide or polypeptide, or different domains or portions of the same polypeptide antigen or antigen.
[0012]
12. Immunogenic composition according to claim 10, characterized by the fact that the peptide or polypeptide antigen of a pathogenic organism is selected from a group consisting of: pneumococcal antigens, tuberculosis antigens, anthrax antigens, HIV, seasonal or epidemic influenza antigens, influenza antigens, Pertussis antigens, Staphylococcus aureus antigens, meningococcal antigens, Haemophilus antigens, HPV antigens, E. Coli antigens, Salmonella antigens, Enterobacter antigens, Pseudomonas antigens , Klebsiella antigens, Citrobacter antigens, Clostridia antigens, Shigella antigens, Campylobacter antigens, Vibrio cholera antigens, toxoids, toxins or portions of toxins and their combinations.
[0013]
13. Immunogenic composition according to any one of claims 1 to 12, characterized by the fact that the antigenic polysaccharide is selected from the group consisting of: Vi polysaccharide, pneumococcal capsular polysaccharides, cell wall pneumococcal polysaccharide, Haemophilus polysaccharide influenzae type b, meningococcal polysaccharide, Gram-positive bacterial lipopolysaccharide and other capsular or cell wall bacterial polysaccharides.
[0014]
14. Immunogenic composition according to claim 1, characterized by the fact that (i) still comprises at least one costimulation factor associated with the antigenic polysaccharide, the peptide or polypeptide antigen, and / or (ii) further comprises at least one adjuvant.
[0015]
15. Immunogenic composition according to claim 14, characterized by the fact that the costimulation factor is selected from the group consisting of: Toll-type receptor ligand or agonists, NOD ligand or agonists, and activators / inflammation agonists.
[0016]
16. Use of an immunogenic composition as defined in any one of claims 1 to 15, characterized in that it is for the preparation of a medicament to induce the immune response in an individual to at least one antigenic polysaccharide in the immunogenic composition or to induce an immune response in the individual at least one peptide or polypeptide antigen present in the immunogenic composition.
[0017]
17. Use of an immunogenic composition as defined in any one of claims 1 to 15, characterized in that it is for the preparation of a medicament to induce the immune response in an individual to at least one antigenic polysaccharide in the immunogenic composition and to induce an immune response in the individual at least one peptide or polypeptide antigen present in the immunogenic composition.
[0018]
18. Use according to claims 16 or 17, characterized by the fact that the immune response is an antibody-mediated response or B cells.
[0019]
19. Use according to claims 16 or 17, characterized by the fact that the immune response is a response mediated by CD4 + T cells, including Thl, Th2, or Thl7, or a response mediated by CD8 + T cells or a mediated response by CD4 + / CD8 + T cells.
[0020]
20. Use according to claims 16 or 17, characterized by the fact that the immune response is an antibody-mediated response or a B-cell-mediated response and a T-cell-mediated response
[0021]
21. Kit characterized by the fact that it comprises: i. a container comprising an antigenic polysaccharide cross-linked or covalently linked to a plurality of first affinity molecules; and ii. (a) a container comprising a peptide or polypeptide antigen fused to a second complementary affinity molecule, which binds to one of the first affinity molecules; or (b) a container comprising at least one expression vector for expressing a peptide or polypeptide antigen fused to a second complementary affinity molecule, which binds to one of the first affinity molecules.
[0022]
22. Kit, according to claim 21, characterized by the fact that it also comprises a container that comprises at least one costimulation factor and, optionally, at least one cross-linking reagent selected from the group consisting of: CDAP ( 1-cyano-4-dimethylaminopyridinium tetrafluoroborate); EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride); sodium cyanoborohydride; cyanogen bromide; and ammonium bicarbonate / iodoacetic acid to bind the cofactor to the antigenic polysaccharide.
[0023]
23. Kit according to claim 21, characterized in that the expression vector can comprise a binding peptide sequence, wherein the expression vector can express a complementary affinity antigen-molecule fusion protein, comprising a peptide linker between the peptide or polypeptide antigen and the second complementary affinity molecule.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US20210332090A1|2021-10-28|Multiple antigen presenting immunogenic composition, and methods and uses thereof

---

CA3061709A1|2018-10-04|A multiple antigen presenting system |-based staphylococcus aureus vaccine, immunogenic composition, and uses thereof

---

US20200121777A1|2020-04-23|Combined vaccine against mycobacterium tuberculosis

同族专利:

---

公开号 | 公开日

---

JP6366501B2|2018-08-01|

---

KR102234933B1|2021-04-05|

---

KR20190104631A|2019-09-10|

---

MX2013013111A|2015-03-04|

---

CA2835630A1|2012-11-15|

---

US10611805B2|2020-04-07|

---

AU2012253327B2|2017-05-25|

---

CN103732222A|2014-04-16|

---

MX2013013112A|2014-06-23|

---

CN103732222B|2018-12-28|

---

US20190119335A1|2019-04-25|

---

RU2013154489A|2015-06-20|

---

EP2709603A1|2014-03-26|

---

US10017548B2|2018-07-10|

---

AU2012253359A1|2013-11-28|

---

WO2012155053A1|2012-11-15|

---

KR102084509B1|2020-03-05|

---

JP2019104758A|2019-06-27|

---

JP2014514363A|2014-06-19|

---

EP3912619A1|2021-11-24|

---

EP2706991A4|2015-05-06|

---

US20210332090A1|2021-10-28|

---

KR20140053903A|2014-05-08|

---

US10766932B2|2020-09-08|

---

US20140154286A1|2014-06-05|

---

CA2835628A1|2012-11-15|

---

JP6502420B2|2019-04-17|

---

RU2632651C2|2017-10-06|

---

KR20210037018A|2021-04-05|

---

ZA201308465B|2019-03-27|

---

CN108714212A|2018-10-30|

---

US20200407404A1|2020-12-31|

---

AU2012253327A1|2013-11-28|

---

EP2709603A4|2015-08-26|

---

JP2014517835A|2014-07-24|

---

US20160090404A1|2016-03-31|

---

JP6152378B2|2017-06-21|

---

EP3881836A1|2021-09-22|

---

JP2020079300A|2020-05-28|

---

KR20140146993A|2014-12-29|

---

JP2017193557A|2017-10-26|

---

ZA201308461B|2020-05-27|

---

JP2018197237A|2018-12-13|

---

EP3912619A4|2021-11-24|

---

EP2709603B1|2021-03-24|

---

WO2012155007A1|2012-11-15|

---

EP2706991A1|2014-03-19|

---

CN103648489A|2014-03-19|

---

ES2864764T3|2021-10-14|

---

AU2012253359B2|2017-06-08|

---

US20140154287A1|2014-06-05|

---

BR112013028892A2|2017-01-31|

---

RU2013154706A|2015-06-20|

---

BR112013028892B1|2021-06-15|

---

RU2619176C2|2017-05-12|

---

EP2706991B1|2021-03-24|

---

JP6666389B2|2020-03-13|

---

CN108840912A|2018-11-20|

---

US9499593B2|2016-11-22|

---

ES2864765T3|2021-10-14|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US244420A|1881-07-19|Stove pipe threadeb and beader |

---

US3887699A|1969-03-24|1975-06-03|Seymour Yolles|Biodegradable polymeric article for dispensing drugs|

---

US3773919A|1969-10-23|1973-11-20|Du Pont|Polylactide-drug mixtures|

---

PH19942A|1980-11-18|1986-08-14|Sintex Inc|Microencapsulation of water soluble polypeptides|

---

IE52535B1|1981-02-16|1987-12-09|Ici Plc|Continuous release pharmaceutical compositions|

---

US4522811A|1982-07-08|1985-06-11|Syntex Inc.|Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides|

---

US4673641A|1982-12-16|1987-06-16|Molecular Genetics Research And Development Limited Partnership|Co-aggregate purification of proteins|

---

US4745051A|1983-05-27|1988-05-17|The Texas A&M University System|Method for producing a recombinant baculovirus expression vector|

---

DE3413608A1|1984-04-11|1985-10-24|Hoechst Ag, 6230 Frankfurt|IMPLANTABLE PREPARATIONS OF REGULATORY PEPTIDES WITH CONTROLLED RELEASE AND METHOD FOR THE PRODUCTION THEREOF|

---

US4879236A|1984-05-16|1989-11-07|The Texas A&M University System|Method for producing a recombinant baculovirus expression vector|

---

DE3788058T2|1986-08-28|1994-04-21|Teijin Ltd|CELL TOXIC ANTIBODY COMPLEX AND METHOD FOR THE PRODUCTION THEREOF.|

---

NZ224422A|1987-05-05|1990-11-27|Molecular Eng Ass|Composition adapted for intranasal immunisation against viral infection comprising a glycoprotein complexed with a lipid|

---

US5229490A|1987-05-06|1993-07-20|The Rockefeller University|Multiple antigen peptide system|

---

US5179007A|1989-07-07|1993-01-12|The Texas A & M University System|Method and vector for the purification of foreign proteins|

---

FR2664905B1|1990-07-18|1994-08-12|Agronomique Inst Nat Rech|MODIFIED BACULOVIRUS, PROCESS FOR OBTAINING SAME, AND EXPRESSION VECTORS OBTAINED FROM SAID BACULOVIRUS.|

---

US5516657A|1992-05-11|1996-05-14|Cambridge Biotech Corporation|Baculovirus vectors for expression of secretory and membrane-bound proteins|

---

AU712981B2|1995-03-22|1999-11-18|Henry M. Jackson Foundation For The Advancement Of Military Medicine|Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents|

---

US5390111A|1993-11-12|1995-02-14|General Electric Company|Method and system for processing cone beam data for reconstructing free of boundary-induced artifacts a three dimensional computerized tomography image|

---

WO1995017515A1|1993-12-23|1995-06-29|University Technologies International Inc.|Methods of expressing proteins in insect cells and methods of killing insects|

---

IL112372A|1994-02-07|2001-08-26|Res Dev Foundation|Non-viral vector for the delivery of genetic information to cells|

---

US8246945B2|2000-04-06|2012-08-21|University Of Arkansas|Methods and reagents for decreasing clinical reaction to allergy|

---

SE506793C2|1996-09-19|1998-02-09|Atlas Copco Rocktech Ab|Coupling piece for use in simultaneous ground and / or rock drilling with an upper rotary impact device and a lower impact device|

---

DE69737125T3|1996-10-31|2015-02-26|Human Genome Sciences, Inc.|Streptococcus pneumoniae antigens and vaccines|

---

ES2230687T3|1997-04-24|2005-05-01|Henry M. Jackson Foundation For The Advancement Of Military Medicine|COPULATION OF PROTEINS NOMODIFIED WITH POLYACARIDS DERIVED FROM HALOACILO OR DIHALOACILO FOR THE PREPARATION OF VACCINES THAT INCLUDE PROTEINS-POLISACARIDS.|

---

AU9310298A|1997-09-09|1999-03-29|Trustees Of Columbia University In The City Of New York, The|T-independent conjugate-vaccines|

---

SE9900496D0|1999-02-12|1999-02-12|Pharmatrix Ab|Vaccine formulation|

---

US6398774B1|1999-09-29|2002-06-04|Heska Corporation|Intranasal delivery system|

---

US6919187B2|2000-04-21|2005-07-19|Corixa Corporation|Compounds and methods for treatment and diagnosis of chlamydial infection|

---

US20020051794A1|2000-08-09|2002-05-02|Alk-Abello A/S|Novel parenteral vaccine formulations and uses thereof|

---

MXPA04011248A|2002-05-14|2005-02-17|Chiron Srl|Mucosal combination vaccines for bacterial meningitis.|

---

EP1708741B1|2003-12-24|2016-03-30|Aduro Biotech|Recombinant nucleic acid molecules encoding fusion proteins comprising antigens and bacterial secretory signal polypeptides, expression cassettes, and bacteria, and methods of use thereof|

---

CA2519511A1|2003-03-17|2004-09-30|Wyeth Holdings Corporation|Mutant cholera holotoxin as an adjuvant and an antigen carrier protein|

---

US20070014807A1|2003-09-03|2007-01-18|Maida Anthony E Iii|Multiplex vaccine|

---

US20050214318A1|2003-10-23|2005-09-29|Nmk Research, Llc|Immunogenic composition and method of developing a vaccine based on fusion protein|

---

WO2005103073A2|2004-04-27|2005-11-03|Intercell Ag|Td antigens|

---

GB0410220D0|2004-05-07|2004-06-09|Kirkham Lea Ann|Mutant pneumolysin proteins|

---

AU2005274628A1|2004-08-18|2006-02-23|Governors Of The University Of Alberta|Protein production method utilizing YebF|

---

US20090148894A1|2005-08-15|2009-06-11|Broedel Sheldon E|Methods for optimizing the secretion of protein in prokaryotes|

---

FI20041396A0|2004-10-29|2004-10-29|Vesa Pekka Hytoenen|New microbial avidin|

---

US20090028891A1|2004-11-11|2009-01-29|Queensland University Of Technology|Chlamydia Antigens and Uses Thereof|

---

GB0428394D0|2004-12-24|2005-02-02|Chiron Srl|Saccharide conjugate vaccines|

---

EP2308505A3|2005-09-01|2011-11-30|Novartis Vaccines and Diagnostics GmbH|Multiple vaccines including serogroup C meningococcus|

---

JP2009518427A|2005-12-08|2009-05-07|ユニバーシティーオブルーイビルリサーチファンデーション，インコーポレーテッド|In vivo cell surface manipulation|

---

WO2007081583A2|2006-01-04|2007-07-19|Children's Medical Center Corporation|Anti-pneumococcal preparations and methods of use|

---

US7943133B2|2006-02-02|2011-05-17|Boston Biocom Llc|Mesothelin antibody protein fusions and methods of use|

---

US20080032340A1|2006-06-09|2008-02-07|University Of Arizona|Peptide motifs for binding avidin or neutravidin|

---

WO2007149982A2|2006-06-22|2007-12-27|The Brigham And Women's Hospital, Inc.|Diagnosis of autoimmune disease|

---

CA2690973A1|2006-06-23|2007-12-27|Paul M. Simon|Targeted immune conjugates|

---

US7452985B2|2006-08-17|2008-11-18|Visgeneer, Inc.|Human Ron-related gene variant associated with cancers|

---

EP2104512A2|2006-12-21|2009-09-30|Emergent Product Development UK Limited|Streptococcus proteins, and their use in vaccination|

---

CN100579152C|2007-01-29|2010-01-06|华为技术有限公司|Method, system and device for playing group multimedia ring|

---

EP2108047B1|2007-01-31|2012-10-24|Pfenex Inc.|Bacterial leader sequences for increased expression|

---

CA2690710C|2007-06-26|2016-10-18|Glaxosmithkline Biologicals S.A.|Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates|

---

AU2008284352A1|2007-08-03|2009-02-12|President And Fellows Of Harvard College|Chlamydia antigens|

---

EP2176291A1|2007-08-10|2010-04-21|Wacker Chemie AG|Expression of full length igg and secretion into the culture medium of prokaryotic cells|

---

WO2009029831A1|2007-08-31|2009-03-05|University Of Chicago|Methods and compositions related to immunizing against staphylococcal lung diseases and conditions|

---

WO2010071986A1|2008-12-24|2010-07-01|Sanofi Pasteur Limited|Modified steptococcus pneumonia pneumolysin polypeptides|

---

EP2208787A1|2009-01-19|2010-07-21|Université de Liège|A recombinant alpha-hemolysin polypeptide of Staphylococcus aureus, having a deletion in the stem domain and heterologous sequences inserted|

---

US8828953B2|2009-04-20|2014-09-09|NaZura BioHealth, Inc.|Chemosensory receptor ligand-based therapies|

---

CN102548572A|2009-06-29|2012-07-04|健诺西生物科学公司|Vaccines and compositions against streptococcus pneumoniae|

---

EP2563396A2|2010-04-30|2013-03-06|Augmenta Biologicals LLC|Delivery proteins|

---

BR112013028887B1|2011-05-11|2020-10-06|Children's Medical Center Corporation|IMMUNOGENIC COMPOSITION PRESENTING MULTIPLE ANTIGENS, AND METHODS AND USES OF THE SAME|

---

AU2014214844B2|2013-02-07|2017-12-14|Children's Medical Center Corporation|Protein antigens that provide protection against pneumococcal colonization and/or disease|JP3576627B2|1995-01-25|2004-10-13|キヤノン株式会社|Method for manufacturing color filter and liquid crystal display device|

---

EP1790660B1|2000-02-28|2012-06-20|Novartis Vaccines and Diagnostics S.r.l.|Heterologous expression of neisserial proteins|

---

US9125863B2|2008-05-22|2015-09-08|Children's Medical Center Corporation|Synergistic immunogenic fusion protein-polysaccharide conjugate|

---

BR112013028887B1|2011-05-11|2020-10-06|Children's Medical Center Corporation|IMMUNOGENIC COMPOSITION PRESENTING MULTIPLE ANTIGENS, AND METHODS AND USES OF THE SAME|

---

WO2012164480A1|2011-05-30|2012-12-06|National Institute Of Immunology|A vaccine composition capable of inducing memory antibody response from single point immunization|

---

US9265819B2|2011-09-21|2016-02-23|St. Jude Children's Research Hospital|Live, attenuated Streptococcus pneumoniae strain and vaccine for protection against pneumococcal disease|

---

WO2014059144A1|2012-10-10|2014-04-17|Arizona Board Of Regents Acting For And On Behalf Of Arizona State University|Systems and devices for molecule sensing and method of manufacturing thereof|

---

AU2014214844B2|2013-02-07|2017-12-14|Children's Medical Center Corporation|Protein antigens that provide protection against pneumococcal colonization and/or disease|

---

WO2015065985A1|2013-10-31|2015-05-07|Arizona Board Of Regents On Behalf Of Arizona State University|Chemical reagents for attaching affinity molecules on surfaces|

---

US10080790B2|2014-09-19|2018-09-25|The Regents Of The University Of Michigan|Staphylococcus aureus materials and methods|

---

CN104548101B|2015-01-31|2017-02-22|广东海大畜牧兽医研究院有限公司|Immunopotentiator for inducing mammary mucous membranes of mammals to produce IgAand preparation method and applications thereof|

---

CN105646681B|2016-01-21|2021-07-16|浙江海隆生物科技有限公司|Preparation method and application of staphylococcus aureus alpha-hemolysin subunit vaccine for dairy cows|

---

US20190192691A1|2016-04-11|2019-06-27|Obsidian Therapeutics, Inc.|Regulated biocircuit systems|

---

JP2019524639A|2016-05-04|2019-09-05|ザ チルドレンズ メディカル センター コーポレーション|Methods for purifying avidin-like proteins and their fusion proteins|

---

US10751398B2|2016-10-07|2020-08-25|Loyola University Chicago|Inflammasome activators and methods of use to treat tumors|

---

US11180535B1|2016-12-07|2021-11-23|David Gordon Bermudes|Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria|

---

US20180161433A1|2017-03-22|2018-06-14|Timothy Andrew Erickson|Size tunable microbial mimetics for immunotherapy of thyroid carcinomas and solid tumors|

---

AU2018243910A1|2017-03-28|2019-11-14|The Children's Medical Center Corporation|A multiple antigen presenting system -based staphylococcus aureus vaccine, immunogenic composition, and uses thereof|

---

KR20200018787A|2017-05-22|2020-02-20|더 칠드런스 메디칼 센터 코포레이션|Combination vaccine against mycobacterium tuberculosis|

---

CN111132691A|2017-06-10|2020-05-08|创赏有限公司|Multivalent conjugate vaccines with divalent or multivalent conjugate polysaccharides providing improved immunogenicity and avidity|

---

US10729763B2|2017-06-10|2020-08-04|Inventprise, Llc|Mixtures of polysaccharide-protein pegylated compounds|

---

RU2020102449A|2017-06-23|2021-07-26|Носокоумиал Вэксин Корпорейшн|IMMUNOGENIC COMPOSITIONS|

---

EP3694490A4|2017-10-13|2021-08-18|The Research Foundation for The State University of New York|Comprehensive vaccine design for commensal disease progression|

---

CN108339115B|2018-05-11|2020-07-10|武汉博沃生物科技有限公司|Pneumococcal combined vaccine using recombinant carrier protein and preparation method thereof|

---

WO2020056127A1|2018-09-12|2020-03-19|The Children's Medical Center Corporation|Pneumococcal fusion protein vaccines|

---

TW202019470A|2018-09-12|2020-06-01|美商艾芬尼維克斯公司|Multivalent pneumococcal vaccines|

---

WO2020086742A1|2018-10-24|2020-04-30|Obsidian Therapeutics, Inc.|Er tunable protein regulation|

---

CN109400704B|2018-11-14|2020-07-21|珠海泰诺麦博生物技术有限公司|Antibody against staphylococcus aureus α -hemolysin and application thereof|

---

WO2020185632A1|2019-03-08|2020-09-17|Obsidian Therapeutics, Inc.|Human carbonic anhydrase 2 compositions and methods for tunable regulation|

---

WO2020223240A1|2019-04-29|2020-11-05|The Broad Institute, Inc.|Affinity-based multiplexing for live-cell monitoring of complex cell populations|

---

WO2020252404A1|2019-06-12|2020-12-17|Obsidian Therapeutics, Inc.|Ca2 compositions and methods for tunable regulation|

---

AU2020290522A1|2019-06-12|2022-01-20|Obsidian Therapeutics, Inc.|CA2 compositions and methods for tunable regulation|

---

WO2021046451A1|2019-09-06|2021-03-11|Obsidian Therapeutics, Inc.|Compositions and methods for dhfr tunable protein regulation|

---

WO2021092206A1|2019-11-05|2021-05-14|Beth Israel Deaconess Medical Center, Inc.|Mycobacterial compositions and biomarkers for use in treatment and monitoring of therapeutic responsiveness|

---

WO2021133142A1|2019-12-27|2021-07-01|주식회사 아모라이프사이언스|Cell culture substrate and manufacturing method thereof|

---

WO2021133143A1|2019-12-27|2021-07-01|주식회사 아모라이프사이언스|Cell culture substrate and manufacturing method therefor|

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

---

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

---

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 |

---

2020-05-12| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2020-10-06| 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 11/05/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US201161484934P| true| 2011-05-11|2011-05-11|

---

US61/484,934|2011-05-11|

---

US201261608168P| true| 2012-03-08|2012-03-08|

---

US61/608,168|2012-03-08|

---

US201261609974P| true| 2012-03-13|2012-03-13|

---

US61/609,974|2012-03-13|

---

PCT/US2012/037412|WO2012155007A1|2011-05-11|2012-05-11|Multiple antigen presenting immunogenic composition, and methods and uses thereof|

---