Research and development of carbohydrate-based anti-salmonella vaccines

Abstract

Provided herein are vaccine compositions comprising a Salmonella antigen conjugated to a capsid wherein the capsid comprises a wild-type or native sequence. Also provided herein are vaccine compositions comprising a Salmonella antigen conjugated to a capsid wherein the capsid comprises at least one mutation, such as a non-natural mutation. Such compositions are useful in the prevention or treatment of Salmonella infection (salmonellosis), in the treatment and prevention of gastroenteritis, typhoid and / or paratyphoid; and the method can effectively aim at various salmonella strains.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 392,274, filed on July 26, 2022, the entire contents of which are incorporated herein by reference in their entirety. Background Art

[0003] The emergence of multidrug-resistant microbial species indicates an urgent need for new prevention and treatment strategies. Vaccines are a powerful tool to reduce infection rates. It is well understood that carbohydrates on the surface of microbial cells are attractive targets for vaccines. However, there are multiple challenges associated with the development of carbohydrate-based vaccines. The first obstacle is to obtain microbially associated glycans, because carbohydrates from microbial species generally have unique structures compared to mammalian counterparts and can present unique reactivity in glycosylation reactions. It is necessary to establish the synthesis of conjugated forms of these compounds. In addition, carbohydrates alone generally have low immunogenicity, which does not induce high-level and long-lasting antibody responses. Therefore, methods for strongly enhancing the immune response to carbohydrate antigens are needed.

[0004] Carrier systems are essential for delivering carbohydrate antigens to the immune system and inducing strong anti-carbohydrate antigen antibody responses. However, a potential disadvantage of protein carriers is that glycoconjugates can induce high anti-carrier antibody responses. As an example, GD3-KLH produced an anti-GD3 IgG titer of 300, while KLH produced a titer of 1,800,000. High anti-carrier antibodies can significantly curb the production of anti-glycan antibodies. This phenomenon has been reported for carbohydrate-based vaccines against microbial diseases. It has been shown that an ideal carrier should induce high levels of anti-glycan antibodies without strong anti-self antibodies.

[0005] Therefore, there is a great need in the art to identify potential therapeutic strategies and compositions for activating immune responses in the treatment and prevention of diseases and infections. Salmonella infection (Salmonellosis) is a major public health problem worldwide. Developing an effective vaccine is a very attractive strategy to combat salmonellosis, especially with the emergence of multi-drug resistant Salmonella strains. Salmonella bacteria have characteristic glycan structures on the cell surface that define specific serotypes and can be used as antigenic targets at the same time. Strategies and vaccines for targeting carbohydrates from the surface of Salmonella are provided herein that can effectively combat a variety of pathogenic strains of Salmonella. Summary of the invention

[0006] Provided herein is a vaccine composition comprising an antigen conjugated to a capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type phage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence as shown in SEQ IDNO: 1. In some embodiments, the vaccine composition comprises an antigen conjugated to a capsid having at least one mutation from a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having at least one mutation from a wild-type phage Qβ capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation.

[0007] In certain embodiments of the vaccine composition, the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty mutations.

[0008] In certain embodiments of the vaccine composition, the at least one non-natural mutation is a disulfide bond mutation.

[0009] In some such embodiments of the present invention disclosed herein, the antigen conjugated to the capsid is a Salmonella antigen. Such Salmonella antigens can be derived from surface polysaccharides of Salmonella. For example, but not limited to, the antigen can be a polysaccharide, such as a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, a non-sugar or a dodecasaccharide. In some preferred embodiments, the Salmonella antigen comprises a trisaccharide, a hexasaccharide or a nonasugar of the Salmonella O-polysaccharide backbone. Most preferably, the Salmonella antigen comprises a trisaccharide of Man-Rha-Gal. The vaccine composition disclosed herein can be used to prevent or treat Salmonella infection (salmonellosis), gastroenteritis, typhoid and / or paratyphoid in a subject in need. The vaccine can be directed against a variety of Salmonella strains, such as Salmonella Enteritidis, Salmonella Paratyphi A, Salmonella Typhimurium and Salmonella Newport (e.g., providing protection or treatment against them). DETAILED DESCRIPTION

[0010] Salmonella infection (Salmonellosis) is a major public health problem worldwide. There are multiple pathogenic strains of Salmonella bacteria. Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A, B and C (S. Paratyphi A, B and C) serotypes cause enteric fever, while non-typhoidal Salmonella (NTS) serotypes generally cause gastroenteritis, which may develop into invasive disease. In Southeast Asia, typhoid and paratyphoid fever caused by S. Paratyphi are endemic. Although typhoid and paratyphoid fever are rare, there are >1.2 million cases of NTS in the United States each year, resulting in 10,000-20,000 hospitalizations and hundreds of deaths, with Salmonella Typhimurium (S. Typhimurium) and Salmonella Enteritidis (S. Enteritidis) being the most common.

[0011] The development of effective vaccines is an attractive strategy to combat salmonellosis, especially with the emergence of multidrug-resistant Salmonella strains. Salmonella bacteria have characteristic glycan structures on the cell surface that define specific serotypes and can be used as antigenic targets at the same time. Salmonella typhi expresses Vi capsular polysaccharides that cover the bacterial surface and protect the bacteria from the host immune system. Salmonella paratyphi and most NTS do not express such capsules, and therefore the surface polysaccharides in these serotypes are O polysaccharides of lipopolysaccharides (COPS). Carbohydrate-based vaccine constructs have been shown to be effective in protecting the host, and an example of a carbohydrate-based vaccine construct is a ViCPS vaccine targeting the Vi polysaccharide on Salmonella typhi. Although ViCPS can provide 50% to 80% protective efficacy in the first year, the protective effect weakens after two years. At present, no vaccine has been approved for any other Salmonella strains. Therefore, there is an urgent need to develop new anti-Salmonella vaccines.

[0012] As mentioned above, enteric fever caused by S. Paratyphi A and S. Typhi, as well as invasive non-typhoidal Salmonella (iNTS) disease caused primarily by S. Enteritidis and S. Typhi, are responsible for a high disease burden worldwide. Multidrug resistance is prevalent and, with the exception of S. Typhi, no licensed vaccines are available to prevent salmonellosis. Vaccines targeting polysaccharides on the surface of pathogenic bacteria have been shown to be an effective preventive strategy, as exemplified by the S. Typhi capsular polysaccharide vaccine. Salmonella serotypes Paratyphi A, Typhimurium, and Enteritidis are coated with a dense O-polysaccharide layer containing a repeating α-D-mannose (Man)-1,4-α-L-rhamnose (Rha)-1,3-α-D-galactose (Gal)-1,2-motif. As disclosed herein, synthetic chemistry and vaccinology were used to develop carbohydrate-based vaccines against the major invasive species Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Paratyphi A. The studies disclosed herein explored whether the Man-Rha-Gal motif constitutes a broadly protective antigen by constructing a glycoconjugate vaccine based on this antigen and characterizing its immunogenicity and efficacy against Salmonella infection.

[0013] The synthesis of Salmonella-related carbohydrate antigens is provided herein. Traditionally, the research on anti-Salmonella vaccines based on carbohydrates relies on natural polysaccharides isolated from pathogenic strains. The polysaccharides obtained are heterogeneous mixtures with variable lengths and structures. In addition, there are problems with batch-to-batch differences and potential contaminants. Independent of separation, a synthetic approach has been developed to produce Salmonella-related polysaccharides with well-defined structures, including tetrasaccharide repeating units of O-antigen polysaccharides from two pathogenic serotypes of Salmonella enteritidis and Salmonella paratyphi A. Since the optimal immunogenic size of polysaccharide antigens is unknown, the synthesis of trisaccharides and tetrasaccharides to twelve-sugar polysaccharide antigens from Salmonella enteritidis, Salmonella typhimurium and Salmonella paratyphi A is provided herein. In addition, since the O polysaccharides of these Salmonella strains have a common backbone, polysaccharides corresponding to the backbone structure are synthesized to help develop potential broad-spectrum vaccines.

[0014] Also provided herein is the development of Qβ-glycan conjugates as strain-specific anti-Salmonella vaccines. One of the main challenges in developing carbohydrate-based vaccines is the poor immunogenicity of antigens. Previously, it has been demonstrated that virus-like particles exemplified by bacteriophage Qβ are capable of displaying carbohydrate antigens in a highly organized manner. (See WO 2019 / 045791, which is incorporated herein by reference in its entirety.) Patterned antigen display leads to a strong IgG antibody response to tumor-associated carbohydrate antigens. Based on these results, it is hypothesized that Qβ can significantly enhance the humoral response to Salmonella-associated glycans.

[0015] Disclosed herein are strain-specific antigenic glycans conjugated to bacteriophage Qβ as potential vaccines. The bioconjugation reaction was optimized to obtain high yields of glycoconjugates without resorting to large excesses of valuable carbohydrate substrates. For example, the Enteritidis glycan Qβ-conjugate induced very high titers of IgG antibodies against the glycan (IgG titers exceeded 80,000,000 ELISA units in rabbits). The induced antibodies recognized the native O-polysaccharide, both as an isolated antigen and when present on bacterial cells. In addition, transfer of antisera from immunized rabbits provided 100% protection to mice against challenge with a lethal dose of Salmonella Enteritidis. Based on these encouraging results, the optimal structure of the glycan-based antigen was established. In addition, the novel Qβ vector was designed to activate Salmonella-specific helper T cells and produce higher levels of anti-glycan IgG antibodies. An effective strain-specific anti-Salmonella vaccine will be developed against Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphi A.

[0016] The present invention further provides the establishment of Qβ-glycan conjugates as broad-spectrum anti-Salmonella murine vaccines. Since S. Enteritidis, S. Typhimurium and S. Paratyphi A have a common polysaccharide backbone structure and their dideoxyhexose branches are different, the common backbone glycan is the target of the vaccine. Antibodies induced by a single construct consisting of Qβ conjugated to a backbone glycan may provide broad-spectrum protection against multiple Salmonella strains, which will provide an exciting tool for salmonellosis prevention.

[0017] To this end, a Man-Rha-Gal oligosaccharide was synthesized and conjugated to a bacteriophage Qβ vector to enhance anti-polysaccharide immunity. New Zealand white rabbits (n=7) were then immunized intramuscularly three times with these conjugates. Vaccination resulted in a robust serum IgG response that recognized native O-polysaccharides from Salmonella Paratyphi A, Salmonella Typhimurium, and Salmonella Enteritidis by both ELISA and flow cytometry (geometric mean titers of 6.3×10 4 , 2.1×10 4 and 3×10 6 EU / mL; P<0.05 compared with baseline). Passive protection studies in naive CD-1 mice (n=18 / group) showed that immune sera were effective against a lethal dose of S. enteritidis R11 (1.3×10 6 CFU) (a bloodstream isolate from Mali) provided excellent protection (vaccine efficacy = 94.4%, P < 0.0001 compared with baseline). Taken together, these findings highlight that a single repeat unit of the common Man-Rha-Gal motif is sufficient to induce a protective antibody response.

[0018] Aspects of the invention provided herein include vaccine compositions comprising a Salmonella antigen conjugated to a capsid. In some embodiments, the capsid comprises at least one non-natural mutation. In some embodiments, the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty mutations. In some such embodiments, at least one non-natural mutation is a disulfide bond mutation.

[0019] In some embodiments, the Salmonella antigen is derived from the surface polysaccharide of Salmonella. The Salmonella antigen can be a polysaccharide. For example, but not limited to, the Salmonella antigen comprises a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, a nonasugar or a dodecasaccharide. In some preferred embodiments, the Salmonella antigen comprises a trisaccharide, a hexasaccharide or a nonasugar of the Salmonella O-polysaccharide backbone. Most preferably, the Salmonella antigen comprises at least one trisaccharide of Man-Rha-Gal. For example, the Salmonella antigen may comprise a repeating sequence of a Man-Rha-Gal trisaccharide (such as two, three or more Man-Rha-Gal trisaccharides).

[0020] In some embodiments of the present invention, the capsid is a phage capsid. The phage may be selected from the group consisting of: (a) phage Qβ; (b) phage R17; (c) phage fr; (d) phage GA; (e) phage SP; (f) phage MS2; (g) phage M11; (h) phage MX1; (i) phage NL95; (j) phage f2; (k) phage PP7; (l) phage AP205; and (m) phage P22. Preferably, the phage is phage Qβ. In some such embodiments, the mutation includes at least one mutation selected from the following: N10K, A38K, A40C, A40S, T75K, D102C, D102S or A117K or a combination thereof. In some preferred embodiments, the mutation includes A38K. In other embodiments, the capsid comprises at least two mutations selected from A40C / D102C, A40S / D102S or A43C / Q98C. In other embodiments, the capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C.

[0021] Embodiments of the present invention include methods for preventing or treating Salmonella infection (Salmonellosis) in a subject, comprising administering to the subject a vaccine composition disclosed herein. Also provided are methods for preventing or treating gastroenteritis, typhoid fever and / or paratyphoid fever, comprising administering to the subject a vaccine composition provided herein. In some such embodiments, the gastroenteritis is chronic or acute.

[0022] The envisaged vaccine composition may be systemically administered. Such systemic administration may be selected from the group consisting of oral, intravenous, intradermal, intraperitoneal, subcutaneous and intramuscular administration.

[0023] In some embodiments, the vaccines contemplated herein are directed against a plurality of Salmonella strains selected from the group consisting of: Salmonella Enteritidis, Salmonella Paratyphi A, Salmonella Typhimurium, and Salmonella Newport. In some embodiments, the Salmonella antigens are derived from the O-polysaccharide backbone present in one or more of Salmonella Enteritidis, Salmonella Paratyphi A, Salmonella Typhimurium, or Salmonella Newport. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The cell surface core O-polysaccharides (COPS) of Salmonella Paratyphi, Salmonella Typhimurium, and Salmonella Enteritidis are depicted and show a common backbone structure with repeating trisaccharides of α-D-mannose (Man)-1,4-α-L-rhamnose (Rha)-1,3-α-D-galactose (Gal)-1,2-.

[0025] FIG2 shows the synthetic scheme of the structures of (A) the Salmonella Enteritidis tetrasaccharide, (B) the Salmonella Paratyphi A tetrasaccharide, and (C) the pentasaccharide with a glucoside at the reducing terminal Gal and the tetrasaccharide without a poratose unit.

[0026] Figure 3 A synthetic scheme for the tetrasaccharide of S. typhimurium is shown.

[0027] Figure 4 The synthetic schemes of common trisaccharides, hexasaccharides, and nonasaccharides of the O-polysaccharide backbone are shown.

[0028] Figure 5 A protocol is presented for the synthesis of strain-specific Salmonella glycans by conversion of common trisaccharides, hexasaccharides, and nonasaccharides of the O-polysaccharide backbone.

[0029] Figure 6 Depicted is the conjugation of Salmonella-associated glycans (SAGs) to phage vectors.

[0030] Figure 7 High and long-lasting anti-β-glycan 1 antibody responses induced by Qβ-glycan 1 in mice are depicted. Figure 7A shows the anti-glycan 1 IgG titers induced by Qβ-glycan 1 (1 μg and 4 μg glycan) and the control group containing Qβ on day 35 after the first immunization. Each symbol represents one mouse. Figure 7 B shows the mean IgG titers induced by Qβ-glycan 1 (4 μg) over 477 days. The persistence trend was similar in the 1 μg group.

[0031] Figure 8 ELISA data for Qβ-glycan 1-induced IgG antibodies against related glycans 2, 17, and 18 are shown.

[0032] Fig. 9 Depicted is an ELISA analysis of various sera against COPS, showing that antiserum from rabbits immunized with Qβ-glycan 1 exhibits strong binding to COPS (all sera were diluted 25,000-fold). X-axis: pre-immune rabbit serum (pre-immune serum from rabbit); post-immune rabbit serum (day 56 serum from rabbits immunized with Qβ-glycan 1); mouse (day 56 serum from two selected mice immunized with Qβ-glycan 1); mAb (mAb 6347 against Salmonella paratyphi A polysaccharide; mAb 6391 and 6393 against the common core of COP). Each mAb was used at a 1:20 dilution.

[0033] Fig.10 Strong binding of Qβ-glycan1-induced antibodies to S. enteritidis and enhanced uptake of bacteria by macrophages were shown. Fig.10 A depicts flow cytometry analysis showing that IgG antibodies in post-immune sera (1:10,000 dilution) from rabbits immunized with Qβ-glycan 1 exhibited significant recognition of S. Enteritidis clinical strain R11. Fig.10 B shows that post-immunization sera from rabbits immunized with Qβ-glycan 1 enhanced the uptake of S. enteritidis by macrophage J774 cells. Post-immunization sera from Qβ-immunized rabbits or pre-immunized rabbits did not have much effect on macrophage uptake.

[0034] Fig.11 Depicted are Kaplan-Meier survival curves of mice challenged with a lethal dose of S. Enteritidis R11. Post-immune sera from Qβ-glycan1-immunized rabbits (at 1:100 or 1:500 dilutions) provided complete protection to mice from challenge (n=12 per group). In contrast, all mice in the group receiving PBS (n=7) and the group receiving pre-immune sera at a 1:100 dilution (n=12) died of bacterial infection by day 8. Eleven of the 12 mice receiving pre-immune sera at a 1:500 dilution also died by day 8.

[0035] Fig.12 We demonstrated that anti-carrier antibody titers were reduced and anti-glycan IgG responses were significantly improved when rationally designed Qβ mutants were used. Fig.12 A depicts the relative solvent exposure of the amino acid residues of Qβ (1QBE) calculated based on the crystal structure of Qβ. The N-terminal residues K2, K13, and K16 are sites for glycan conjugation. Residues A38, T75, Q99, and P119 are highly exposed to the solvent and serve as potential mutation sites. Fig.12 B shows that mutant Qβ / A38K / A40C / D102C conjugated to the representative carbohydrate antigen Tn induced a stronger anti-Tn IgG response than wild-type (WT) Qβ-Tn. Fig.12 C shows that Qβ / A38K / A40C / D102C conjugated to Tn induced much less antibodies against WT Qβ or Qβ / A38K / A40C / D102C than WT Qβ-Tn (at 409,600-fold serum dilution). p values ​​were determined by two-tailed t-test.

[0036] Fig.13 The encapsidation scheme is depicted. Fig.13 A shows the RNA-guided encapsidation technology for encapsulating proteins in Qβ. Plasmid 1 contains the sequence of the anti-Rev aptamer (aR), the coat protein (CP), and the hairpin (hp) structure for binding to the coat protein. Plasmid 2 contains the sequence of the Rev tag and the target protein (TP). The plasmid is transformed into E. coli and transcribes and expresses the Rev-tagged TP cargo protein and bifunctional mRNA. The anti-Rev aptamer in the mRNA binds to the Rev tag, and the Qβ genome encapsulating the hp binds to the inside of the CP monomer, thereby encapsulating the target protein into the interior of Qβ, where the coat protein RNA sequence is used as a linker. Fig.13 B shows that Qβ@(RFP) was successfully produced by RNA encapsidation technology, resulting in a more than 100-fold increase in the RFP fluorescence of the capsid.

[0037] Fig.14 Depicted are anti-COPS IgG ELISA titers against COPS from S. Paratyphi A, S. Enteritidis, S. Typhimurium, and S. Newport in sera of rabbits immunized with conjugates of WT Qβ with backbone glycans 25, 26, 27, respectively. Significantly higher levels of IgG antibodies were found against COPS from S. Paratyphi A, S. Enteritidis, and S. Typhimurium, whereas lower levels were found against the unrelated S. Newport.

[0038] A. definition

[0039] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0040] The term "administering" refers to providing an agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0041] As used herein, "bacteriophage" refers to a virus that infects and replicates in bacteria. In certain embodiments, the phage is selected from, but not limited to, the group consisting of: phage Qβ; (b) phage R17; (c) phage fr; (d) phage GA; (e) phage SP; (f) phage MS2; (g) phage M11; (h) phage MX1; (i) phage NL95; (j) phage f2; (k) phage PP7; (l) phage AP205; and (m) phage P22. As used herein, "phage Qβ" (also referred to as "phage Qb," "Qβ," and "Qb") is one of many small RNA phages that infect Escherichia coli.

[0042] As used herein, "carbohydrate antigen" refers to an antigenic species that elicits a strong antibody response. In certain embodiments, the carbohydrate antigen is selected from, but is not limited to, mucin ("MUC1"), ganglioside GD2 (Ahmed M. et al. FEBS Letters 588:288-297 (2014)), fucosyl GM1, GD2 (including acetylated GD2), GD3 (including acetylated GD3), GM2, Globo-H, Lewis A, Lewis Y, mucin (e.g., MUC1, MUC4), polysialic acid, sialyl-Lewis A, carbohydrates from Salmonella bacteria (or any of the bacteria, viruses or pathogenic diseases provided herein), Tf, Tn or sTn (Tn, Tf and STn are usually found on glycoproteins such as MUC1).

[0043] The term "inhibit" or "inhibits" refers to reducing, suppressing, attenuating, reducing, preventing, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% or stabilizing the development or progression of a disease, disorder or condition, the activity of a biological pathway or biological activity (such as the growth of a solid malignant tumor) compared to an untreated control subject, cell, biological pathway or biological activity or compared to a target subject before treatment (such as the growth of a solid malignant tumor). The term "reducing" refers to inhibiting, suppressing, attenuating, reducing, preventing or stabilizing the symptoms of a cancer disease, disorder or condition. It should be understood that, although not excluded, treating a disease, condition or disorder does not require the complete elimination of the disease, condition, disorder or symptoms associated therewith.

[0044] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0045] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, that is involved in carrying or transporting the subject compound from one organ or part of an organ of the body to another organ or part of an organ of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0046] "Pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds.

[0047] The terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the likelihood of developing a disease, disorder or condition in a subject who does not have the disease, disorder or condition but is at risk or susceptible to the disease, disorder or condition.

[0048] "Subjects" may include human subjects for medical purposes, such as for the treatment of existing diseases, disorders, conditions or for preventive treatment to prevent the onset of diseases, disorders or conditions, or animal subjects for medical, veterinary or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates, such as humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques, etc.; bovines, such as cattle, bulls, etc.; sheep, such as sheep, etc.; goats, such as goats, etc.; pigs, such as pigs, fat pigs, etc.; equines, such as horses, donkeys, zebras, etc.; felines, including wild cats and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, etc.; and rodents, including mice, rats, guinea pigs, etc. The animal may be a transgenic animal. In some embodiments, the subject is a human, including but not limited to fetuses, newborns, infants, adolescents, and adult subjects. Further, "subjects" may include patients suffering from or suspected of having a disease, disorder, or condition. Therefore, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (eg, rats or mice used in experiments, etc.).

[0049] The term "subject in need thereof" means a subject identified as being in need of therapy or treatment.

[0050] The terms "systemic administration" and "administered systemically" and "peripheral administration" or "administered peripherally" mean administration of a compound, drug, or other material other than directly to the central nervous system so that it enters the patient's system and thereby undergoes metabolism and other similar processes, e.g., subcutaneous administration.

[0051] The term "therapeutic agent" or "pharmaceutical agent" refers to an agent capable of producing a desired biological effect on a host.

[0052] The term "therapeutic effect" refers to a local or systemic effect caused by a pharmacologically active substance in an animal, particularly a mammal, and more particularly a human.

[0053] As used herein, the terms "therapeutically effective amount" and "effective amount" mean an amount of a compound, material, or composition comprising a compound of the invention effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0054] The term "treating" a disease in a subject or "treating" a subject having a disease refers to administering a drug treatment to the subject, such as administering a vaccine composition as described herein, such that at least one symptom of the disease is alleviated, prevented from worsening, or delayed from worsening.

[0055] B. Capsid

[0056] Provided herein is a vaccine composition comprising an antigen conjugated to a capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type phage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence as shown in SEQ IDNO: 1. In some embodiments, the vaccine composition comprises an antigen conjugated to a capsid having at least one mutation from a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having at least one mutation from a wild-type phage Qβ capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation. In some embodiments, the vaccine composition provided herein comprises an antigen (e.g., a carbohydrate antigen, a polypeptide, a peptide, a protein, and a small molecule) conjugated to a capsid (e.g., a bacteriophage Qb), wherein the capsid comprises at least one mutation (e.g., at least one point mutation or at least one non-natural disulfide bond). In some embodiments, the capsid is a fragment or a portion of a capsid amino acid sequence of sufficient length, which, when conjugated to an antigen, can induce an enhanced and strong immune response. In certain embodiments, the capsid polypeptide further comprises amino acids that do not correspond to a naturally occurring capsid amino acid sequence (e.g., comprising at least one point mutation, or at least one non-natural disulfide bond mutation, or comprising a fusion protein of a capsid amino acid sequence and an amino acid sequence corresponding to a non-capsid protein or polypeptide).

[0057] In some embodiments, the capsid is derived from a bacteriophage. In certain embodiments, the bacteriophage is selected from the group consisting of: bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; (l) bacteriophage AP205; and (m) bacteriophage P22.

[0058] In some embodiments, the capsid has the sequence shown in Table A of the coat protein.

[0059] Table A List of capsids and related GenBank numbers

[0060]

[0061]

[0062] In some embodiments, the bacteriophage Qβ capsid comprises the sequence shown in SEQ ID NO:1.

[0063] (M)AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQ PSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELA ALLASPLLIDAIDQLNPAY(SEQ IDNO:1)

[0064] In some embodiments, the phage Qβ capsid comprises at least one mutation shown in Table B.

[0065] Table B. Qβ mutants reported to assemble into capsids.

[0066]

[0067]

[0068] Note: Yield ++: >80 mg / L, +: >20-80 mg / L, -: <20 mg / L, na: not applicable. 7a corresponds to Fiedler, J et al. Biomacromolecules, 2012, 13(8), 2339-2348; 42a corresponds to Prasuhn, D et al. Journal of the American Chemical Society (JACS), 2008, 130(4), 1328-1334; 42b corresponds to Udit, A et al. ChemBioChem, 2009, 10(3), 503-510. 43 corresponds to Hovlid, ML et al. The Scripps Research Institute, La Jolla, 2014.

[0069] In certain embodiments, the capsid comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty mutations. In some embodiments, the mutation is a non-natural disulfide bond mutation. In some embodiments, the capsid comprises but is not limited to the following mutations (Table C).

[0070] Table C: List of bacteriophage Qβ capsids

[0071]

[0072]

[0073]

[0074] In certain embodiments, the phage Qb mutant may have the following physical properties.

[0075] Table D: Physical properties of Qβ mutants.

[0076]

[0077] In some embodiments, the phage Qb capsid comprises a polypeptide comprising an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1-15. In some embodiments of the methods, compositions and kits provided herein, the Qb mutant has at least one mutation shown in Table C. In some embodiments, the phage Qβ capsid comprises a wild-type or native sequence. In some embodiments, the phage Qβ capsid comprises a sequence consisting essentially of the sequence shown in SEQ ID NO: 1. In some embodiments, the Qb mutant has an amino acid sequence consisting essentially of the mutations shown in SEQ ID NO: 2-15. In some embodiments, the capsid comprises a polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in the GenBank accession number shown in Table A.

[0078] In some embodiments of the methods, compositions, and kits provided herein, the vaccine composition comprises a capsid having an amino acid sequence consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 of SEQ ID Nos: 1-15, or a biologically active variant thereof, or a combination thereof, or with an amino acid sequence of the capsid (e.g., SEQ ID NO:1-15, or any GenBank number shown in Table A) is composed of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical contiguous amino acids. In some embodiments, the bacteriophage Qβ capsid comprises a wild-type or native sequence. In some embodiments, the bacteriophage Qβ capsid comprises a sequence consisting essentially of the sequence shown in SEQ ID NO:1.

[0079] As well known to those skilled in the art, polypeptides with basic sequence similarity can cause identical or very similar immune responses in host animals. Therefore, in some embodiments, derivatives, equivalents, variants, fragments or mutants of Qb capsid or its fragments are also applicable to methods, compositions and test kits provided herein.

[0080] In some embodiments, the altered polypeptide may have an altered amino acid sequence, such as by conservative substitution, but still induces an enhanced immune response and is considered to be a functional equivalent. As used herein, the term "conservative substitution" means that an amino acid residue is replaced by another biologically similar residue. It is well known in the art that amino acids within the same conservative group can generally replace each other without substantially affecting the function of the protein. According to certain embodiments, the derivative, equivalent, variant or mutant of Qb is at least 85% homologous to the sequence shown in SEQ ID NO:1-15 or its biologically active variant or a combination thereof. In some embodiments, the homology is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In some embodiments, the phage Qβ capsid comprises a wild-type or native sequence. In some embodiments, the phage Qβ capsid comprises a sequence consisting essentially of the sequence shown in SEQ ID NO:1.

[0081] C. Vaccine composition and pharmaceutical composition / preparation thereof

[0082] Provided herein is a vaccine composition comprising an antigen conjugated to a capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild-type phage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having a wild-type or native sequence as shown in SEQ IDNO: 1. In some embodiments, the vaccine composition comprises an antigen conjugated to a capsid having at least one mutation from a wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a phage Qβ capsid having at least one mutation from a wild-type phage Qβ capsid. In some embodiments, the at least one mutation comprises a non-natural mutation. In some embodiments, the non-natural mutation comprises a non-natural amino acid mutation. In some embodiments, the vaccine composition provided herein comprises an antigen (e.g., a carbohydrate antigen, a polypeptide, a peptide, a protein, and a small molecule) conjugated to a capsid (e.g., a phage Qb), wherein the capsid comprises at least one mutation (e.g., at least one point mutation, at least one non-natural amino acid mutation, or at least one non-natural disulfide bond mutation). The capsid is described in Section B above. In some embodiments, the antigen is a fragment or a portion of a carbohydrate antigen of sufficient length, which, when conjugated to the capsid, can induce an enhanced and strong immune response. The capsid can be conjugated to multiple multiple antigens that are the same or different antigens. In certain embodiments, the antigen is a protein, and the peptide is selected from, but not limited to, TNFα, IL1α, IL1β, tau protein, PCSK9, or amyloid β. In some embodiments, the antigen is a small molecule selected from, but not limited to, nicotine, cocaine, or advanced glycosylation products.

[0083] In some embodiments, the present invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more vaccine compositions (e.g., one or more Qb wild-type or Qβ mutants, antigen conjugates as described above) formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. On the other hand, the composition can be administered as is or mixed with a pharmaceutically acceptable carrier, and can also be administered in combination with other therapies. Therefore, combination therapy includes sequential, simultaneous and separate or co-administration of the composition, wherein the therapeutic effect of the first administration is not completely eliminated when the subsequent compound is administered. In some embodiments, at least one vaccine composition (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) can be provided to a subject alone or in combination with at least one therapeutic drug, chemotherapeutic agent, scavenger compound, antibiotic, antiviral agent, antifungal agent, anti-inflammatory agent, vasoconstrictor and anticoagulant, antigen that can be used for vaccine application or corresponding prodrug.

[0084] As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as those for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection in the form of a sterile solution suspension or sustained release formulation; (3) topical administration, for example, application to the skin in the form of a cream, ointment or controlled release patch or spray; (4) intravaginal or intrauterine, for example as a pessary, cream or foam; (5) sublingual; (6) ocular; (7) transdermal; or (8) nasal.

[0085] As described above, certain embodiments of the one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) may contain a basic functional group, such as an amino or alkylamino group, and thus be able to form a pharmaceutically acceptable salt with a pharmaceutically acceptable acid. These salts can be prepared in situ during the administration of the vehicle or the dosage form manufacturing process, or by reacting a purified compound of the invention in free base form with a suitable organic or inorganic acid and isolating the salt thus formed during the subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, and dodecylsulfonate, among others (see, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19).

[0086] Pharmaceutically acceptable salts of the subject compounds include, for example, conventional nontoxic salts or quaternary ammonium salts of compounds derived from nontoxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochlorides, hydrobromides, sulfates, sulfamates, phosphates, nitrates, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothiosulfonic acid, and the like.

[0087] In other cases, the one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can also be prepared in situ during administration of a vehicle or during the manufacture of a dosage form, or by reacting a purified compound in the form of a free acid with a suitable base (e.g., a pharmaceutically acceptable hydroxide, carbonate, or bicarbonate of a metal cation), with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali metal or alkaline earth metal salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts, among others. Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, e.g., Berge et al., supra).

[0088] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0089] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0090] The one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) can be formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

[0091] In certain embodiments, the formulation of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) may include other carriers to allow for greater stability, to allow for greater stability, different in vivo release characteristics, targeting to specific sites, or any other desired characteristics that will allow for more efficient delivery of the one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) to a subject or target in a subject, such as, but not limited to, liposomes, microspheres, nanospheres, nanoparticles, bubbles, micelle formers (e.g., bile acids), and polymer carriers (e.g., polyesters and polyanhydrides). In certain embodiments, the formulations mentioned above render the compounds of the invention orally bioavailable.

[0092] Liquid dosage formulations of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0093] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0094] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, mixtures thereof, and the like.

[0095] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules; or as a solution or suspension in an aqueous or non-aqueous solution; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; or as a pastille (using an inert base such as gelatin and glycerin or sucrose and acacia); and / or as a mouthwash, etc., each containing a predetermined amount of the active ingredient. One or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) can also be administered as pills, elixirs, or pastes.

[0096] In solid dosage forms (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) wetting agents such as glycerol; 4) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol, glyceryl monostearate and nonionic surfactants; (8) adsorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the composition may also contain a buffer. Solid compositions of a similar type may also be used as fillers in soft and hard shell gelatin capsules using excipients such as lactose (lactose or milk sugar), and high molecular weight polyethylene glycols and the like.

[0097] Tablets can be manufactured by optionally compressing or molding with one or more adjuvants. Compressed tablets can be prepared using a binding agent (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0098] Tablets and other solid dosage forms, such as sugar coatings, capsules, pills and granules, can optionally be coated or prepared with coatings and shells (e.g., enteric coatings and other coatings well known in the field of pharmaceutical preparations). The dosage form can also be formulated to allow the active ingredient to be released slowly or controlled therein, for example, using different proportions of hydroxypropyl methylcellulose to provide the desired release curve, other polymer matrices, liposomes and / or microspheres. The composition can also be formulated for rapid release, for example, freeze drying. The dosage form can be sterilized by filtering, for example, through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or other certain sterile injectable media immediately before use. These compositions can also optionally contain an emulsifier and can belong to a composition that releases one or more active ingredients only or preferentially in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. If appropriate, the active ingredient can also be in microencapsulated form with one or more of the above-mentioned excipients.

[0099] Formulations for rectal or vaginal administration may be presented as suppositories which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylates and which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0100] Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0101] Dosage forms for topical or transdermal administration of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0102] Ointments, pastes, salves and gels may contain, in addition to the active compounds of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0103] Powders and sprays may also contain excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powders, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0104] Transdermal patches have the additional advantage of providing controlled delivery in vivo. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0105] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this invention.

[0106] Pharmaceutical compositions suitable for parenteral administration may comprise sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or combinations of sterile powders which may be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes or suspending agents which render the formulation isotonic with the blood of the intended recipient, or thickening agents.

[0107] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters, such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0108] In certain embodiments, according to the methods and compositions provided herein, the above-mentioned pharmaceutical compositions can be combined with other pharmacologically active compounds known in the art ("second active agents"). The second active agent can be a macromolecule (e.g., a protein) or a small molecule (e.g., a synthetic inorganic, organometallic or organic molecule). In one embodiment, the second active agent independently or synergistically helps treat cancer.

[0109] In another embodiment, the compositions of the present invention may contain other biologically active substances, including therapeutic drugs or prodrugs, for example, other chemotherapeutic agents, scavenger compounds, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, vasoconstrictors and anticoagulants, antigens useful for cancer vaccine applications or corresponding prodrugs.

[0110] Exemplary scavenger compounds include, but are not limited to, thiol-containing compounds such as glutathione, thiourea, and cysteine; alcohols such as mannitol, substituted phenols; quinones, substituted phenols, aryl amines, and nitro compounds.

[0111] Various forms of chemotherapeutic agents and / or other biologically active agents may be used. These include, but are not limited to, biologically active uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like.

[0112] D. Treatment

[0113] Provided herein are vaccine compositions for treating and / or preventing diseases and conditions for which an enhanced immune response may be beneficial. Such diseases include, but are not limited to, pathogenic infections (e.g., bacterial, viral, or fungal infections) and cancer. In some embodiments, the vaccine compositions described herein can be used as immunotherapy.

[0114] In some embodiments, vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) can be used to treat diseases including, but not limited to, persistent infectious diseases, sexually transmitted diseases, gastrointestinal diseases, lung diseases, cardiovascular diseases, stress and fatigue-related disorders, fungal diseases, pathogenic diseases, viral infections, or bacterial infections.

[0115] Viral infectious diseases include human papillomavirus (HPV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2), herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, influenza virus, hepatitis A and B, FIV, lentivirus, pestivirus, West Nile virus, measles, smallpox, vaccinia, Ebola, coronavirus, retrovirus, herpes virus, potato S virus, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, Moloney virus, ALV, cytomegalovirus (CMV), Epstein Barr virus (EBV), Zika or Rous sarcoma virus (RSV).

[0116] In addition, it also includes bacterial, fungal and other pathogenic diseases, such as Aspergillus, Brugia, Candida, Chikungunya, Chlamydia, Coccidia, Cryptococcus, Dengue, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma. coplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Anopheles-vectored Plasmodium vivax, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma, and Vibrio cholerae. Exemplary species include Neisseriagonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa, Trypanosoma equiperdum, Clostridium tetani, Clostridium botulinum; or fungi, such as Paracoccidioides brasiliensis; or other pathogens, such as Plasmodium falciparum.

[0117] Also included are National Institute of Allergy and Infectious Diseases (NIAID) priority pathogens. These include Category A pathogens such as Variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever), and related viruses); Category B pathogens such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), alphaviruses (Venezuelan encephalomyelitis), and related viruses. encephalomyelitis, eastern and western equine encephalomyelitis, ricin from Ricinus communis (castor bean), epsilon toxin from Clostridium perfringens; Staphylococcus enterotoxin B, Salmonella species, Shigella dysenteriae, Escherichia coli strain O157:H7, Vibrio cholerae, Cryptosporidium parvum; Category C pathogens such as Nipah virus, hantavirus, Aedes yellow fever, and multidrug-resistant tuberculosis; helminths such as schistosomes and tapeworms; and protozoa such as Leishmania (e.g., Leishmania mexicana) in sand flies, Plasmodium, and Chagas disease in Echinochloa crusgalli.

[0118] Bacterial pathogens include, but are not limited to, pathogenic Gram-positive cocci, including but not limited to: Pneumococcus; Staphylococcus; and Streptococcus. Pathogenic Gram-negative cocci include: Meningococcus; and Neisseria gonorrhoeae. Pathogenic enteric gram-negative bacilli include: Enterobacteriaceae; Pseudomonas, acinetobacteria, and eikenella; melioidosis; Salmonella; shigellosis; Haemophilus; chancroid; brucellosis; tularemia; Yersinia (pasteurella); Streptobacillus moniliformis and spiral; Listeria monocytogenes; erysipelothrix rhusiopathiae; diphtheria; cholera; anthrax; and Donovan's disease (granuloma inguinale). Pathogenic anaerobes include tetanus; Clostridium botulinum; other Clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochetes include syphilis; treponematoses: yaws, pinta, and endemic syphilis; and leptospirosis. Other infections caused by highly pathogenic bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis, and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidioidomycosis, coccidioidomycosis, coccidioidomycosis, mycetoma, and chromomycosis; and dermatophytosis. Rickettsial infections include rickettsiae and rickettsial diseases. Examples of mycoplasma and chlamydial infections include: Mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections. Pathogenic protozoa and helminths and infecting eukaryotic organisms include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.

[0119] In one embodiment, the method of treatment comprises administering an effective amount of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) to a subject (e.g., a subject in need thereof). Subjects in need thereof may include, for example, subjects who have been diagnosed with a tumor (including a precancerous tumor, a cancer) or subjects who have received treatment, including subjects who are refractory to previous treatments.

[0120] In "therapeutically effective amount", the term "effective amount" of a therapeutic agent refers to the amount of the agent necessary to induce the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on factors such as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, etc. More specifically, the term "effective amount" refers to an amount sufficient to produce a desired effect (e.g., to reduce or improve the severity, duration, progression or onset of a disease, disorder or condition, or one or more symptoms thereof; to prevent the progression of a disease, disorder or condition, to cause the disease, disorder or condition to regress; to prevent the recurrence, development, onset or progression of symptoms associated with a disease, disorder or condition, or to enhance or improve the preventive or therapeutic effect of another therapy).

[0121] The compositions described herein can be delivered by any suitable route of administration, including oral, nasal, transmucosal, ocular, rectal, intravaginal, parenteral (including intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection), intracisternal, topical, such as by powder, ointment or drops (including eye drops), including buccal and sublingual, transdermal, by inhalation spray, or other modes of delivery known in the art.

[0122] As used herein, the terms "systemic administration" and "administered systemically" and "peripheral administration" and "administered peripherally" mean administering a vaccine composition described herein so that it enters the patient's system and thereby undergoes metabolism and other similar processes.

[0123] As used herein, the terms "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0124] In certain embodiments, the pharmaceutical composition is typically delivered (e.g., by oral or parenteral administration). In certain other embodiments, the pharmaceutical composition is delivered locally by direct injection into a tumor or directly into the blood supply (e.g., arterial or venous blood supply) of the tumor. In some embodiments, the pharmaceutical composition is delivered by systemic administration and topical administration. For example, a subject suffering from a tumor can be treated by direct injection of a composition containing the composition described herein into a tumor or the blood supply of the tumor, and in combination with oral administration of the pharmaceutical composition of the present invention. If local and systemic administration are used simultaneously, local administration can occur before, simultaneously with, and / or after systemic administration.

[0125] In some embodiments, the subject pharmaceutical composition of the present invention will be incorporated into one or more substances to be delivered, and its amount is enough to deliver the therapeutic agent or other materials incorporated into the patient's effective amount of treatment, as a part of preventive or therapeutic treatment. The desired concentration of the active compound in the granule will depend on the absorption rate, inactivation rate and excretion rate of the drug and the delivery rate of the compound. It should be noted that the dosage value can also change with the severity of the condition to be alleviated. It should be further understood that for any specific subject, the specific administration regimen should be adjusted in time according to the professional judgment of the personnel of the use of the composition or the supervision composition according to personal needs. Usually, technology known to those skilled in the art is used to determine administration.

[0126] The dosage can be based on the amount of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) per kg of patient body weight. For example, a range of amounts of the composition or compound encapsulated therein is contemplated, including about 0.001 mg, 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, or 250 mg or more of such compositions per kg of patient body weight. Other amounts will be known to those skilled in the art and are readily determined.

[0127] In certain embodiments, the dosage of the one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) will generally be in the range of about 0.001 mg / kg body weight to about 250 mg / kg body weight, specifically in the range of about 50 mg / kg to about 200 mg / kg, and more specifically in the range of about 100 mg / kg to about 200 mg / kg. In one embodiment, the dosage is in the range of about 150 mg / kg to about 250 mg / kg. In another embodiment, the dosage is about 200 mg / kg.

[0128] In some embodiments, the molar concentration of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) in the pharmaceutical composition will be less than or equal to about 2.5M, 2.4M, 2.3M, 2.2M, 2.1M, 2M, 1.9M, 1.8M, 1.7M, 1.6M, 1.5M, 1.4M, 1.3M, 1.2M, 1.1M, 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M or 0.2M. In some embodiments, the concentration of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) will be less than or equal to about 0.10 mg / ml, 0.09 mg / ml, 0.08 mg / ml, 0.07 mg / ml, 0.06 mg / ml, 0.05 mg / ml, 0.04 mg / ml, 0.03 mg / ml or 0.02 mg / ml.

[0129] Actual dosage levels of the active ingredients in the compositions of the invention may be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0130] The selected dosage level will depend upon a variety of factors, including the activity of the specific therapeutic agent, or its ester, salt or amide, in the formulation employed; the route of administration; the time of administration; the rate of excretion or metabolism of the specific therapeutic agent employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the specific compound employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0131] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can prescribe and / or administer the dosage of the compound of the present invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0132] In general, an appropriate daily dose of a compound of the invention will be that amount of the compound which is the lowest dose that produces an effective therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0133] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form.

[0134] The precise time and amount of administration of any particular compound that produces the most effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the particular compound, the patient's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose and type of drug), route of administration, etc. The guidelines presented herein can be used to optimize treatment, e.g., determine the optimal time and / or amount of administration, which will require only routine experimentation consisting of monitoring the subject and adjusting the dose and / or time.

[0135] When the subject is treated, the health of the patient can be monitored by measuring one or more relevant indices at predetermined times during a 24-hour period. All aspects of treatment, including supplements, amounts, number of applications, and formulations, can be optimized based on the results of such monitoring. By measuring the same parameters, the patient can be reassessed periodically to determine the degree of improvement, with the first such reassessment typically occurring at the end of four weeks after the start of treatment, and subsequent reassessments being performed every four to eight weeks during treatment, and once every three months thereafter. Therapy can last for several months or even several years, with a typical treatment time for humans being a minimum of one month. Based on these reassessments, the amount of, for example, the medicament administered and the number of applications can be adjusted.

[0136] Treatment may be initiated with smaller doses than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is reached.

[0137] Toxicity and therapeutic efficacy of the subject compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 and ED 50 Compounds that exhibit large therapeutic indices are preferred. In some embodiments, LD 50 (lethal dose), and for one or more vaccine compositions described herein (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above), LD 50 The decrease may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. Similarly, ED may be measured. 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms), and for one or more vaccine compositions described herein (e.g., one or more Qβ wild-type, Qβ mutant antigen conjugates as described above), the ED 50The increase may be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. Similarly, the IC 50 (i.e., the concentration that achieves half-maximal cytotoxic or cytostatic effect on cancer cells), and for one or more vaccine compositions described herein (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above), the IC 50 Can increase for example at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more.Although can use the compound that shows toxic side effect, should pay attention to design the delivery system that will compound target desired part, in order to reduce side effect.

[0138] In some embodiments, the presently disclosed methods produce at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% inhibition of cancer cell growth in an assay.

[0139] In any of the above methods, administration of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) can reduce the solid malignancy in the subject by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% compared to the solid malignancy before administration of the vaccine composition.

[0140] In some embodiments, a therapeutically effective amount of one or more vaccine compositions (eg, one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) is administered prophylactically to prevent the formation of solid malignancies in a subject.

[0141] In some embodiments, the subject is a human. In other embodiments, the subject is non-human, such as a mammal.

[0142] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of any supplement or alternatively any component thereof is preferably within a range that includes little or no toxicity to the ED50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For the agents of the invention, the therapeutically effective dose may be estimated initially from cell culture assays. The dose may be formulated in animal models to achieve an IC value as determined in cell culture. 50 The circulating plasma concentration range of the drug can be determined by the method of high performance liquid chromatography.

[0143] Example

[0144] The following examples have been included to provide guidance to those of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. Based on the present disclosure and the general level of those skilled in the art, it will be appreciated by those skilled in the art that the following examples are intended to be exemplary only, and that various changes, modifications, and variations may be made without departing from the scope of the presently disclosed subject matter. The following examples are provided in an illustrative manner and not in a limiting manner.

[0145] Example 1: Qβ Salmonella glycan conjugate as a potential anti-Salmonella vaccine

[0146] Salmonella is a gram-negative bacterium that can cause serious infection (salmonellosis) through ingestion of contaminated food or water. The genus Salmonella encompasses >2500 serovars, differentiated by serotype based on the structure of the O-polysaccharide on the cell surface and by clinical syndromes. Typhoid and paratyphoid serovars (S. Typhi and S. Paratyphi) cause invasive enteric fever, whereas nontyphoidal Salmonella typically cause gastroenteritis, and their invasive disease may occur due to immune immaturity, immunosuppression, or aging. S. Typhi causes typhoid fever, which causes more than 200,000 deaths each year, primarily in developing countries, especially Southeast Asia, Latin America, and Africa. In Asia, paratyphoid fever caused by S. Paratyphi is also prevalent. In the United States, nontyphoidal Salmonella (NTS) are more common and are frequently associated with multistate outbreaks resulting in >1.2 million infections, tens of thousands of hospitalizations, and hundreds of deaths each year. Of the different NTS serovars associated with clinical illness in the United States, S. Enteritidis and S. Typhimurium are the most common. According to 2010 data from the Centers for Disease Control and Prevention (CDC) Foodborne Disease Active Surveillance Network, salmonellosis accounts for 54% of hospitalizations and 43% of deaths from food poisoning. Worldwide, there are an estimated 94 million cases and 155,000 deaths each year. While the incidence of foodborne illnesses, such as Shiga toxin-producing E. coli O157 infection, has decreased by more than 50% over the past two decades, no progress has been made in the incidence of Salmonella spp., with the incidence of salmonellosis increasing by 10% over the same period.

[0147] Currently, invasive salmonellosis is treated with antimicrobial agents. However, multidrug-resistant strains of Salmonella have emerged, with the CDC classifying drug-resistant Salmonella with a severe threat level, which requires "rapid and sustained action to ensure the problem does not grow." Despite the frequency and severity of salmonellosis and public health efforts to control disease incidence, there are currently no vaccines against strains of Salmonella other than S. Typhi. Of particular note for S. Typhi is the ViCPS vaccine, which uses purified Vi capsular polysaccharide directly from S. Typhi as an immunogen. This vaccine provides protection with 50% to 80% efficacy in the first year, but loses its protection after two years. There is an urgent need to develop vaccines against Salmonella, especially against strains other than S. Typhi, such as S. Enteritidis, S. Typhimurium, and S. Paratyphi A.

[0148] Vaccines targeting polysaccharides on the cell surface of pathogenic bacteria have been shown to be an effective strategy for preventing a variety of bacterial pathogens, as exemplified by the ViCPS vaccine against Salmonella Typhi. For other Salmonella strains, although they lack Vi, there are characteristic surface polysaccharides. The cell surface core O-polysaccharide (COPS) of Salmonella Paratyphi, Salmonella Typhimurium, and Salmonella Enteritidis share a common backbone structure with repeating trisaccharides of α-D-mannose (Man)-1,4-α-L-rhamnose (Rha)-1,3-α-D-galactose (Gal)-1,2-. A dideoxyhexose is linked to the 3-OH of Man, and the structure of the dideoxy sugar distinguishes different serovars. Salmonella Paratyphi A contains boractose, Salmonella Typhimurium contains abecidose, and the dideoxy sugar is tyvese on Salmonella Enteritidis. (See Figure 1 . ) In some polysaccharides, the 6-OH of the galactose backbone can be linked to an α-glucosyl unit. In addition, some of the 2-OH and 3-OH groups of rhamnose are acetylated, leading to further heterogeneity of natural polysaccharides.

[0149] To date, all carbohydrate-based anti-Salmonella vaccines have directly utilized purified polysaccharides, or as digestion products of polysaccharides derived from cultures of corresponding serovars. These processes require strict control to remove endotoxins and other harmful contaminants. In addition, polysaccharides are mixtures with uneven lengths and structures, which may have batch-to-batch variability, making it challenging to better understand the key structures of protective epitopes. This article considers the possibility of developing vaccines against Salmonella using synthetic oligosaccharides with well-defined structures. These glycan antigens can be delivered using a carrier system based on powerful virus-like particles (VLP) phage Qβ to produce a strong and lasting humoral immune response.

[0150] One of the main obstacles to the development of polysaccharide-based anti-Salmonella vaccines is that carbohydrates are weakly immunogenic T-cell-independent antigens. When administered alone, they can only produce low titers of IgM antibodies, which usually have low affinity and only last for a short time. For an effective and long-lasting vaccine, it is best to induce IgG antibodies. To achieve this, polysaccharides can be linked to immunogenic protein carriers that contain the necessary helper T (Th) cell epitopes to induce Th cell activation and antibody isotype conversion from IgM to IgG. The loss of ViCPS immune protection after two years may be due to the fact that the administered ViCPS does not have any Th epitopes.

[0151] Another key parameter in carbohydrate-based vaccine design is how antigens are presented to B cells. This is because B cell activation and differentiation require two signals: an antigen-specific first signal delivered by binding and cross-linking of cell surface B cell receptors (BCRs) and a second co-stimulatory cytokine signal from homologous Th cells. Hapten arrays spaced 5-10 nm apart have been shown to cross-link BCRs well, and antigens displayed in a highly organized manner may lead to earlier B cell expansion for effective IgM and IgG responses. Antigen organization can also greatly affect B cell tolerance, with B cells being unresponsive to poorly organized antigens and responding rapidly to the same antigens presented in an orderly manner. Therefore, a carrier that can display antigens in an organized manner would be very advantageous.

[0152] VLP can be a powerful platform for antigen delivery. This is because compared with amorphous protein carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), VLP is composed of self-assembling protein subunits, thereby giving them highly ordered structures. The repeated B cell epitopes presented on the VLP surface can effectively crosslink BCR. In addition, VLP can contain a large amount of Th epitopes, which can coordinate BCR crosslinking and activate the stimulation of Th cells. Other advantages of VLP include being easy to carry out genetic manipulation with engineering for better immunoenhancement particles, their high stability to various chemical modification conditions, the speed and scale of production, and the ability of immunoactivators incorporated into capsids.

[0153] Recently, VLPs have been used as carriers of tumor-associated carbohydrate antigens (TACAs). Similar to Salmonella-associated glycans (SAGs), TACAs are T cell-independent antigens. Among the various types of VLPs studied, bacteriophage Qβ is the most potent in terms of carrier function. By displaying TACAs in an organized manner on Qβ, an enhancement of antibody responses was observed even against very weak TACAs (i.e., monomeric Tn antigens, with anti-Tn IgG titers exceeding 250,000). In contrast, Tn-KLH conjugates did not induce anti-Tn IgG antibodies. Importantly, immunization with Qβ-Tn significantly protected mice from the development of aggressive tumors, while Qβ alone did not provide any survival benefit, indicating that the nonspecific immune response caused by Qβ itself was insufficient.

[0154] For another tumor-associated antigen, mucin-1 (MUC1), a head-to-head comparison was performed between Qβ-MUC1 and a MUC1 conjugate with KLH, the gold standard used in multiple clinical trials of carbohydrate-based anticancer vaccines. In agreement with the results of the Tn study, Qβ was superior, as Qβ-MUC1 was able to induce three times more anti-MUC1 IgG antibodies and was more effective in reducing tumor burden in a mouse tumor model than the corresponding KLH-MUC1 construct.

[0155] Through cancer vaccine studies, several valuable lessons have been learned in applying Qβ to glycan-based vaccine design. 1) High-density antigen display is essential for high IgG antibody responses. A Qβ construct with approximately 140 copies of MUC1 was able to induce 4 times more anti-MUC1 IgG antibodies compared to a corresponding construct with 30 copies of MUC1, even though both groups of mice received the same total amount of MUC1. The same density-dependent phenomenon was observed in Tn vaccine studies; 2) Th cell activation is important, as ablation of Th cells before immunization almost completely abolished the anti-glycan IgG response; 3) Covalent conjugation of the antigen to the Qβ carrier is critical, as a mixture of Qβ and MUC1 produced less than 3% of the anti-MUC1 antibodies compared to those induced by covalently conjugated Qβ-MUC1; 4) The structure of the linker used to conjugate the antigen to Qβ is important. Qβ-Tn constructs formed using a copper-catalyzed azide-alkyne cycloaddition click reaction were much less effective in inducing tumor-binding antibodies than those formed with flexible alkylamide linkers. This was attributed to potential antigenic competition from the rigid triazole ring and steric exclusion / epitope inhibition effects due to the anti-triazole antibodies formed.

[0156] Despite its success in anticancer vaccine development, Qβ has not been systematically investigated as a carrier of bacteria-associated glycans. We hypothesized that organized display of SAGs on Qβ could result in strong and long-lasting anti-SAG IgG responses as a potential anti-Salmonella vaccine.

[0157] Innovation:

[0158] 1) The research disclosed in this article is based on the combination of immunology and modern synthetic and bioconjugation chemistry.

[0159] 2) The VLP Qβ disclosed herein represents an exciting new vector for antimicrobial vaccine development. The Qβ-SAG conjugate has the potential to generate high titers of anti-SAG IgG antibodies and immune memory, two key factors in preventing Salmonella infection.

[0160] 3) This article discloses novel Qβ vectors that are associated with reduced antibody production against the vector itself while retaining the ability to activate bacteria-specific Th cells to induce even higher levels of anti-SAG IgG antibodies than wild-type Qβ. These vectors can become a platform technology for vaccine development for diseases other than salmonellosis.

[0161] 4) The availability of synthetic SAG oligosaccharides removes the reliance on pathogenic bacterial cultures to isolate polysaccharides for use in vaccine constructs, thus improving batch-to-batch consistency and reducing the potential for highly active bacterial contaminants in the vaccine. The well-characterized synthetic glycan structures allow for the study of the minimum length and structure of protective SAG epitopes.

[0162] 5) Targeting the common polysaccharide backbone of S. Enteritidis, S. Typhimurium, and S. Paratyphi A is a new direction for vaccine design, enabled by synthetic backbone glycans, as it is practically infeasible to access these structures by enzymatic or chemical digestion. Backbone-based vaccines could be broad-spectrum, providing protection against multiple medically important Salmonella serotypes.

[0163] Synthesis of oligosaccharide antigens associated with Salmonella spp.

[0164] Traditionally, the development of carbohydrate-based vaccines against Salmonella has relied on polysaccharides isolated from pathogenic strains. The glycans obtained by this method exist as a heterogeneous mixture of different lengths and structures. In addition, there are problems with batch-to-batch variability and potential contaminants. Synthetic routes are disclosed to produce Salmonella-related glycans with well-defined structures, independent of isolation from natural sources.

[0165] Strain-specific Salmonella tetrasaccharide synthesis:

[0166] Tetrasaccharides of Salmonella Enteritidis and Salmonella Paratyphi A.

[0167] The first synthetic target was the S. enteritidis tetrasaccharide 1, corresponding to a repeating unit with an aminopropyl linker at the reducing end. Four monosaccharide building blocks 3-6 were designed, in which benzyl (Bn) and acetonide groups were mainly used as protecting groups, because these electron-donating protecting groups can potentially enhance the reactivity of the building blocks in glycosylation. The key rare tyvesicose donor 3 was efficiently prepared from readily available thiomannosides, which were selectively protected and then subjected to Barton-McCombie deoxygenation to afford 3 (Scheme 1a; Figure 2A Building blocks 4–6 were prepared in a straightforward manner. As deoxysugars are generally more anomeric than their fully oxidized counterparts, selective activation of tadalafil 3 over bifunctional mannoside 4 was achieved by N-iodosuccinimide (NIS, 1.2 equiv.) and TMSOTf (Scheme 1b; Figure 2A ), providing 85% yield of α-tyveline glycosylated disaccharide 9 and a small amount of β-anomer (about 5%), which was separated by column chromatography. The new α-glycosidic bond in compound 9 was confirmed by the coupling constant values ​​of 13C-1 and H-1 [δ 99.3 (JC1 / H1=167.8 Hz, C-1tyveline), 85.8 (JC1 / H1=167.8 Hz, C-1Man)]. Glycosylation of acceptor 6 with rhamnoside donor 5 produced the only α-anomer, disaccharide 10 anomer (Scheme 1c; Figure 2A ). Removal of the non-reducing terminal OAc from 10 followed by glycosylation with disaccharide donor 9 gave protected tetrasaccharide 12, which was subsequently deprotected to yield tetrasaccharide 1 (75 mg). The structure of 1, including the stereochemistry of all glycosidic bonds, was confirmed by NMR analysis. Alternatively, the 2-OAc in 12 was selectively deprotected (compound 13) for future extension to longer glycan chains. (See Figure 2A . )

[0168] To synthesize the Salmonella paratyphi tetrasaccharide 2, the puratose building block 14 was prepared from the glucoside 15 by the same selective protection and Barton-McCombe deoxygenation as used to synthesize the tyvesuline donor 3. The protected Salmonella paratyphi tetrasaccharide 16 was then synthesized in good overall yields using puratose 14 and building blocks 4-6 following a similar chemoselective glycosylation approach as used to prepare 12. (See Figure 2B. ) Subsequent deprotection of 16 produced the S. paratyphi tetrasaccharide 2a (72 mg) with two OAc in its rhamnoside unit for future studies on the role of OAc in inducing antibody responses. In addition, to better understand the role of glycan structure in antibody recognition, pentasaccharide 17 with a glucoside on the reducing Gal and tetrasaccharide 18 without a pyruvose unit were also synthesized (90 mg each). (See Figure 2C . )

[0169] In order to develop a vaccine against S. Typhimurium, another major strain of the genus Salmonella, S. Typhimurium tetrasaccharides 19 and 20 were synthesized. (See Figure 3 . ) The preparation of 19 and 20 will begin with the abecidose donor 21 (Scheme 2), which in turn will be prepared by selective protection of the galactoside 22 followed by Barton-McCombe deoxygenation. Chemoselective glycosylation of the mannoside 4 by abecidose 21 will produce the disaccharide 23. Similar to the synthesis of the S. Enteritidis and S. Paratyphi A tetrasaccharides, it is expected that the predominantly α-anomer will be formed, which will be confirmed by NMR. Coupling of the disaccharide 23 with the disaccharide 11 will produce the tetrasaccharide 24, which will be deprotected to produce the S. Typhimurium tetrasaccharide 20. Skipping the acetylation step in the deprotection of 24 will give the tetrasaccharide 19.

[0170] Synthesis of strain-specific octasaccharides and Dodecasaccharides and common main chain oligosaccharides.

[0171] Since naturally occurring Salmonella O-polysaccharides are primarily composed of tetrasaccharide repeats, oligosaccharides longer than one tetrasaccharide unit may result in a more effective anti-Salmonella antibody response. Therefore, octasaccharides and dodecasaccharides of strain-specific Salmonella glycans were synthesized, containing two and three repeat units, respectively. Targeting the polysaccharide backbone may produce a broad-spectrum vaccine against a variety of Salmonella strains. To conduct this study, a synthetic route was developed to prepare the backbone oligosaccharides.

[0172] Common trisaccharides, hexasaccharides, and nonasaccharides of the O-polysaccharide backbone have been synthesized.

[0173] The common backbone of Salmonella species is composed of repeating Man-Rha-Gal trisaccharides. Backbone oligosaccharides 25-27 were synthesized using one, two, and three repeating trisaccharide units, respectively (Scheme 3; Figure 4). The monosaccharide building blocks 28-31 established a protecting group pattern with high α-selectivity in glycosylation. Pre-activation-based glycosylation of mannoside donor 28 54-55 (1.2 equiv.) with bifunctional rhamnoside acceptor 29 (1 equiv.) produced disaccharide 32 in 82% yield. This was followed by glycosylation of galactoside 31 protected by di-tert-butylsilylene 56 to produce trisaccharide 34 in 78% yield (Scheme 3a). Similarly, trisaccharide 33 was synthesized in 74% yield using galactoside 30 as the final acceptor. The Bz group of 33 was removed and the resulting trisaccharide acceptor was glycosylated by donor 34 to produce hexasaccharide 35 (80% yield) as the only anomer isolated (Scheme 3b). The α configuration of the newly formed glycosidic bond was confirmed by its 1JCH value of 173.5 Hz, which was likely assisted by the di-tert-butylsilylene moiety. Repeated removal of Bz and glycosylation by trisaccharide donor 34 produced fully protected nonasaccharide 36 (81% yield), which was successfully deprotected to yield Salmonella backbone nonasaccharide 27. Trisaccharide 25 and hexasaccharide 26 were similarly synthesized (10 mg each) and fully characterized by NMR and MS.

[0174] With the success of the synthesis of common backbone 25-27, the most direct way to obtain strain-specific Salmonella glycans is to transform these backbone glycans. Figure 5 ), selective removal of the PMB group from nonasaccharide 36, followed by glycosylation with >3 equivalents of tyvesucan donor 3, will produce dodecasaccharide 37, which will be deprotected to produce S. Enteritidis dodecasaccharide 38a. Similarly, S. Enteritidis octasaccharide 39a, S. Paratyphi A octasaccharide and dodecasaccharide, and S. Typhimurium dodecasaccharide and octasaccharide 38b,c and 39b,c will be synthesized. This gives a different route that enables rapid access to the SAG array. The synthesis of all target glycans outlined in goal 1 will be scaled up to 1 g each to support vaccine studies. Since glycosylation is performed in overall high yields without the need for large excesses of building blocks, no significant difficulties are expected in achieving this goal. Triple glycosylation of 36×3 may not result in high alpha selectivity for all three sites. If this is the case, tetrasaccharide modules with deoxysugar units such as 40 will be synthesized and then used to assemble larger oligosaccharides.

[0175] Development of strain-specific anti-Salmonella vaccines.

[0176] SAG itself is only weakly immunogenic and cannot induce effective antibody responses when administered alone. Therefore, it is crucial that SAG is covalently conjugated to an immunogenic carrier. Bacteriophage Qβ has been shown to be a powerful carrier for carbohydrate-based anti-cancer vaccines. It was tested whether phage Qβ with SAG can induce a strong protective antibody response as a strain-specific anti-Salmonella vaccine.

[0177] Conjugation of SAG to bacteriophage Qβ vectors can be performed efficiently.

[0178] To synthesize the necessary strain-specific glycans, bioconjugation with wild-type (WT) Qβ was performed. The S. enteritidis tetrasaccharide 1 was functionalized with di-N-hydroxysuccinimide adipate (NHS) ester 41 (Scheme 5; Figure 6 ) and then incubated with Qβ (5 equivalents of glycan per amine moiety on the outer surface of Qβ; a total of 20 equivalents of glycan per Qβ monomer). The resulting Qβ-glycan 1 conjugate was purified. The excess unconjugated glycan was recovered in near quantitative yield in its free carboxylic acid form, which could be converted back into NHS esters and reused for bioconjugation. LC-MS characterization of the Qβ-glycan conjugate showed an average of 334 glycan copies per Qβ particle. 20 mg of Qβ-glycan 1 conjugate was synthesized. Following a similar synthetic scheme, a Qβ conjugate with Salmonella paratyphi (paratose) tetrasaccharide conjugate 2 was synthesized (average of 290 glycan 2 copies per Qβ particle, 10 mg of conjugate was prepared). Glycans 1 and 2 were also conjugated to BSA to aid in immunological analysis.

[0179] Immunization with Qβ-glycan 1 induces potent anti-glycan IgG responses in both mice and rabbits

[0180] Humoral responses were evaluated against Qβ-glycan 1 and Qβ-glycan 2. These conjugates were injected subcutaneously into groups of 5 mice at doses of 1 μg and 4 μg of glycan, respectively (day 0), followed by two booster injections on days 14 and 28. Control groups (5 mice per group) received the same protein levels of unconjugated Qβ.

[0181] Enzyme-linked immunosorbent assay (ELISA) analysis using BSA-glycan 1 as the coating antigen showed that Qβ-glycan 1 induced high titers of IgG antibodies. On day 35, the mean IgG titers of the groups receiving 1 μg and 4 μg of glycan reached 487,000 and 980,000 ELISA units, respectively ( Figure 7 A). In contrast, the control group immunized with Qβ generated only anti-glycan IgG titers of less than 5,000 ELISA units, indicating that the inclusion of glycan 1 in the vaccine is essential for a high anti-glycan antibody response.

[0182] For an effective vaccine, it is important that the immune response induced is durable. Monitoring the antibody response induced by Qβ-glycan1 over time (e.g. Figure 7(4 μg group data shown in B). 56 days after the first immunization, the anti-glycan 1 IgG antibody level reached its maximum value (6,000,000 ELISA units), which was maintained for 120 days. The antibody titer gradually decreased, and at day 477, half of the IgG peak level was still present. These results indicate that Qβ-glycan 1 induces a persistent anti-glycan 1 IgG response. No obvious side effects were observed in the immunized animals throughout the period, emphasizing the safety of the vaccine.

[0183] To determine the selectivity and specificity of antibodies induced by Qβ-glycan 1, the tetrasaccharide backbone 18 as well as the S. paratyphi tetrasaccharide 2 and pentasaccharide 17 were conjugated to BSA using linker 41 for ELISA. Analysis using these BSA-glycan conjugates demonstrated that the strongest binding of immunized sera to BSA-glycan 1 was observed ( Figure 8 ), indicating the high selectivity for glycan 1 and the key role of the non-reducing end tyvesican in glycan 1 for antibody recognition.

[0184] In addition to mice, rabbits are another commonly used animal model for immune assessment. Two rabbits were immunized subcutaneously with Qβ-glycan 1 (4 μg of glycan, injected once every two weeks). Serum was collected on days 0, 35, 49, and 56. ELISA analysis for BSA-glycan 1 showed that a strong anti-glycan 1 IgG response was induced, with IgG titers reaching 83,000,000 and 150,000,000 ELISA units by day 56 (Table 1), which was more than 6,000 times the titer of the Qβ-immunized control or pre-immunized rabbits. These results show that Qβ-glycan 1 is effective in multiple species.

[0185] Table 1. ELISA titers of anti-Glycan 1 antibodies induced by Qβ-Glycan 1 in rabbits. Potent anti-Glycan 1 IgG antibodies were induced compared to control rabbits receiving Qβ alone.

[0186]

[0187] For a successful vaccine, it is critical that the antibodies generated by the vaccine construct are able to recognize the native polysaccharide. To test this, the homologous serotype native core O-polysaccharide (COPS) from Salmonella Enteritidis was isolated and immobilized in ELISA wells. Antisera from rabbits and mice were evaluated for their ability to bind to COPS. Three anti-Salmonella monoclonal antibodies (mAb 6347 against S. Paratyphi A, mAbs 6391 and 6393 against the core polysaccharide) were also added to the same ELISA plates for comparison. Fig. 9As shown, mAbs 6391 and 6393 recognize COPS well, whereas mAb 6347 does not. Sera from immunized rabbits bind strongly to native COPS. Higher COPS binding was observed for rabbit sera compared to mouse sera. Similar species differences in anti-glycan antibody responses have been reported by the Cummings group.

[0188] Qβ-glycan1-induced antibodies strongly bind to S. enteritidis and enhance bacterial uptake by macrophages.

[0189] S. Enteritidis strain R11 isolated from the blood of infected young children in Mali was used to assess whether postimmune sera could bind to cognate antigens on intact bacterial cells. Postimmune sera from rabbits immunized with Qβ and Qβ-glycan 1 were analyzed for binding to R11 by flow cytometry. Whereas binding by preimmune sera or sera from rabbits immunized with the Qβ vector alone was negligible, antisera from rabbits immunized with Qβ-glycan 1 effectively recognized R11 ( Fig.10 A), which indicates the potential of Qβ-glycan1 as a vaccine against S. enteritidis.

[0190] Opsonophagocytic uptake of antibody-bound bacteria by macrophages represents an important mechanism for antibody-mediated clearance of Salmonella. To determine whether post-immune sera possess functional anti-Salmonella activity, uptake of S. enteritidis R11 by J774 mouse macrophages was assessed following incubation of the bacteria with pre-immune sera, antisera from Qβ, or sera from rabbits immunized with Qβ-glycan 1. While pre-immune and Qβ-immunized rabbit sera did not induce significant bacterial uptake relative to medium alone, antisera from rabbits immunized with Qβ-glycan 1 enhanced macrophage opsonization of the bacteria by 400% ( Fig.10 B).

[0191] Passive transfer of serum from immunized rabbits protected mice from lethal challenge with Salmonella Enteritidis.

[0192] The protective efficacy of antiserum against Salmonella infection was evaluated in vivo. Mice were passively administered PBS (n = 7), mixed pre-immune sera of different dilutions (n ​​= 12 / group) or vaccine-induced antiserum (n = 12 / group), and then challenged intraperitoneally with a lethal dose (LD100) of R11 (1×106CFU). Although all mice in the PBS group died of bacterial infection by day 7, and all mice except one group of the two groups of mice receiving pre-immune sera died of infection by day 8, it was exciting that 100% of the mice receiving post-immune sera (1:100 or 1:500 dilution, a total of 24) survived the bacterial challenge ( Fig.11 ).

[0193] Establishing the optimal length and structure of Salmonella Enteritidis glycans for antibody responses.

[0194] Initial immunological evaluations focused on the S. Enteritidis tetrasaccharide antigen 1. Since native S. Enteritidis COPS consists of a polymer of multiple repeats of tetrasaccharide units, it was hypothesized that longer glycans could better mimic the conformational properties of native COPS, resulting in a stronger antibody response. Since the level of anti-Salmonella glycan antibodies produced in mice was lower than that in rabbits, it will be tested whether including octasaccharides and dodecasaccharides as antigens in the vaccine can generate better antibodies in mice.

[0195] The octasaccharide 39a and dodecasaccharide 38a will be conjugated to Qβ via a bifunctional linker 41 in a similar manner to tetrasaccharide 1. Acetylation will be studied by conjugating acetylated tetrasaccharide 1a to Qβ. The resulting construct will be extensively purified to remove free glycans. The glycan loading level will be quantified by LC-MS assay. If the loading level is low due to the larger size of the glycans, the bioconjugation reaction can be repeated to increase the loading to approximately 300, as in Qβ-glycan 1. Excess unconjugated glycans can be recovered and recycled.

[0196] Mice will be immunized with the new conjugates, and IgG antibody levels in the post-immunization sera will be determined by ELISA against the immunizing antigen as well as against native COPS from S. enteritidis. Comparison of the antibody response with that induced by Qβ-glycan1 will help determine the effects of antigen length and acetylation on COPS recognition.

[0197] Vaccine dose is an important parameter to optimize. In the Qβ-Tn study, it was observed that the higher dose used in vaccination (20 μg) resulted in stronger binding of antisera to Tn-expressing tumor cells than the lower doses (1 μg and 4 μg). In preliminary studies, it was observed that mice immunized with 4 μg of Qβ glycan 1 tended to have higher mean IgG antibody levels compared to the 1 μg group ( Figure 7 A). Higher doses (8 μg and 40 μg antigen per injection) will be evaluated to determine the dose that produces the strongest glycan-specific antibody titers. For the 40 μg dose, 10 mg of Qβ-glycan conjugate will be sufficient to fully immunize 20 mice.

[0198] Adjuvants can significantly affect the immune response. In preliminary studies, Freund's adjuvant was used. However, since complete Freund's adjuvant cannot be used in humans due to its toxicity, three adjuvants that have been used as part of human vaccines will be tested: unmethylated cytosine-guanine dinucleotide, alum, and monophosphoryl lipid A (MPLA), which will be co-administered with the Qβ-glycan conjugate. These adjuvants interact with Nod-like receptors (NLRs) and Toll-like receptors (TLRs), which are key activators of the innate immune system. Previously, studies have shown that Freund's adjuvant and MPLA induce similar levels of IgG antibodies. The adjuvant that produces the highest titer of antibodies against COPS from Salmonella enteritidis will be selected.

[0199] Following immunization, mice will be monitored for up to 2 years to assess the durability of the IgG response. Figure 7 Based on preliminary data in B, anti-glycan IgG levels may remain high for the first 6 months and then gradually decline. If this is the case, a booster injection could be given again at 1 year. This is similar to the elderly population in humans, who are more susceptible to Salmonella infection. The potential persistence and boostability of the IgG response would be attractive features of the vaccines disclosed herein.

[0200] Qβ-glycan immunization is well tolerated. To date, the only side effect observed as a result of immunization is the formation of ulcers at the injection site due to the toxicity of complete Freund's adjuvant, despite the induction of high IgG titers. In cancer vaccine studies, switching the adjuvant to MPLA completely eliminated this side effect. Any possible side effects are monitored for up to 2 years after immunization.

[0201] For constructs that generate high antibody responses, the specificity of the induced antibodies will be analyzed by screening the antisera on a microbial glycan microarray provided by the National Glycomics Center. This microarray contains more than 300 representative carbohydrate structures from 36 species of 15 genera, including many Salmonella strains. Based on the initial screening ( Figure 8 ), it is expected that the antibodies generated will be highly selective for S. Enteritidis OPS glycans.

[0202] The ability of the antisera to bind to bacterial cells was tested by incubating the immunized sera with Salmonella enteritidis followed by flow cytometric analysis. For sera that reacted strongly with bacteria, the binding capacity of the antisera to bacterial cells was determined by macrophage opsonophagocytosis assay ( Fig.10 B) and complement-mediated cytotoxicity assays (SBA) to determine the ability of the serum to clear cells. The constructs that produced IgG antibodies that showed the strongest binding and cytotoxic effects on S. enteritidis were identified. This provided key information for the preferred epitope structure.

[0203] Deepening our understanding of immune responses through Qβ-glycan conjugates.

[0204] To better understand the immune response and immune memory induced by vaccination, memory B cells and long-lasting antibody-secreting plasma B cells will be analyzed. Cells will be isolated from the lymph nodes and spleens of mice immunized with Qβ or Qβ-glycan 1 alone. These cells will be subjected to flow cytometric analysis after incubation with fluorescently labeled BSA-glycan 1 conjugates. The memory B cell population will be quantified as SAG+IgG+B220+CD38+ cells. If the frequency of these cells is too low for accurate quantification, memory B cells can be differentiated into plasma cells by stimulation with lipopolysaccharide (LPS) for six days and transferred to BSA-glycan 1-coated enzyme-linked immunospot assay (ELISPOT) plates to quantify the number of B cells secreting glycan-specific IgG. Long-lasting glycan-specific plasma B cells will also be evaluated by ELISPOT using cells from the bone marrow. Qβ-glycan 1 immunization is expected to produce greater numbers of glycan-specific memory B cells and long-lasting plasma B cells compared to controls.

[0205] Th cell activation is crucial for the production and long-term memory of high-affinity IgG antibodies. In order to analyze the Th activation caused by Qβ-polysaccharide 1, lymphocytes from spleen or lymph nodes will be isolated from immunized mice and original mice, and the lymphocytes will be subsequently labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Bone marrow dendritic cells (BMDC) will be produced by culturing bone marrow cells from the tibia and femur of original mice with DC culture medium containing granulocyte / macrophage colony stimulating factor and interleukin-4. After stimulating with LPS, mature BMDC will be incubated with Qβ-polysaccharide 1, and then added to the splenocytes labeled with CFSE. In the presence of BMDC processed by Qβ-polysaccharide 1, the proliferation of CD4+T cells will cause the intracellular CFSE concentration to decrease. Flow cytometric analysis of the CFSE intensity of CD4+ cells will allow the CD4+T cells of vaccine specificity to be quantified. As expected, a greater percentage of CD4+ T cells in immunized mice were Qβ-glycan 1 specific compared to naive mice, suggesting that Qβ-glycan 1 vaccination induced CD4+ T cell activation.

[0206] Qβ mutant vectors were generated that were associated with reduced anti-vector responses while increasing anti-glycan antibody levels.

[0207] A potential disadvantage of using immunogenic protein carriers is that anti-carrier antibody levels may increase dramatically under a prime-boost immunization regimen. For example, the KLH-GD3 construct (1 prime injection and 2 booster injections) resulted in an anti-GD3 IgG titer of 400, with the anti-KLH IgG titer being 200 times (819,200) the anti-GD3 IgG titer. For Qβ-Tn, the difference was smaller at 12X: the average IgG titer for Qβ was 3,100,000 relative to the average anti-Tn IgG titer of 263,000.36. The endogenous epitopes of the carrier may compete with the desired epitopes for a limited number of Th cells for cytokine signaling, which is essential for B cell activation and IgG production. In addition, antibodies that bind to non-essential epitopes may spatially block adjacent targeted epitopes from being recognized by B cells, or lead to immune complex removal. Therefore, in order to help the immune system focus on the glycan epitopes in the conjugate, it is desirable to remove non-essential carrier epitopes. Qβ mutant strategies that reduce anti-Qβ antibody responses and further enhance anti-glycan antibody production were investigated.

[0208] Removal of the Qβ B cell epitope reduced anti-Qβ titers and increased antibody levels against carbohydrate antigens.

[0209] It was hypothesized that B cell epitopes might reside in flexible and accessible loops on the outside of Qβ, as they are required to bind to the large B cell receptor on the surface of B cells. Based on the near-atomic resolution crystal structure of Qβ, the triple mutant A38K / A40C / D102C was designed. A38 was chosen because it is on the outside and exposed to the solvent ( Fig.12 A). The A40C and D102C mutations were introduced because these two residues are spatially close in the crystal structure and can form a disulfide bond bridging the two subunits to enhance capsid stability. The A38K / A40C / D102C mutant was expressed in E. coli and self-assembled to form particles with high yield (about 20 mg / L) and similar size (28 nm diameter) to wild-type (WT) Qβ.

[0210] IgG induced by the carbohydrate antigen Tn (400 Tn per capsid) conjugated to Qβ / A38K / A40C / D102C was evaluated. Two notable findings were: (1) the triple mutant induced significantly higher anti-Tn IgG antibody levels ( Fig.12 B); and (2) anti-vector IgG titers against WT Qβ and Qβ / A38K / A40C / D102C were much lower than those induced by WT Qβ-Tn ( Fig.12 C). This indicates that some intrinsic B cell epitopes of Qβ were successfully removed without generating new dominant B cell epitopes.

[0211] According to the rationale for generating the Qβ / A38K / A40C / D102C triple mutant, the above optimized glycan structure will be conjugated to the Qβ / A38K / A40C / D102C triple mutant, and mice and rabbits will be immunized with the new construct with optimized doses and adjuvants. IgG levels against COPS as well as IgG levels against the vector will be determined by ELISA. Similar to the results with Tn ( Fig.12 B), Conjugates with the triple mutant vector will generate better IgG antibody responses compared to the corresponding WT-Qβ conjugate.

[0212] In addition to the triple mutants, other exposed residues will also be mutated, including T75, Q99, A117, and P119 to amino acids such as lysine, alanine, and serine to further disrupt the intrinsic B cell epitopes of Qβ. Based on past experience, about 70% of Qβ mutants can self-assemble and form particles with high yields because the selected mutation sites are not present at the interface between the coat protein subunits. These new mutants (mQβ, m represents mutant) will be conjugated to S. enteritidis glycans and immunized mice and rabbits. The mQβ constructs that produce the highest anti-glycan IgG antibody response, the strongest COPS, and bacterial binding will be identified as leads for further studies.

[0213] Establish a heterologous prime-boost strategy.

[0214] Qβ may have multiple major B cell epitopes. Therefore, effective Qβ mutations were incorporated into one coat protein to test whether they could further reduce anti-Qβ IgG titers. However, in the case of too many mutations, the coat protein may not be able to assemble into particles. A heterologous prime-boost strategy using multiple mQβ containing different major B cell epitopes can be developed. One mQβ-glycan conjugate will be used to prime the immune system, and the second mQβ-glycan conjugate will be used for booster injections. In this strategy, since the B cell epitopes of various mQβ are different, the glycan antigen is the only common component of all injections. Therefore, anti-glycan immunity should benefit the most from booster injections. This heterologous prime-boost strategy should result in stronger anti-glycan antibody production and lower unwanted responses to the carrier.

[0215] Generating novel Qβ vectors encapsulating exogenous Salmonella-associated Th cell epitopes to further enhance anti-Salmonella Antibody levels and protective efficacy against Bacillus spp.

[0216] The ability of the Qβ-glycan conjugate to induce high levels of anti-glycan IgG antibodies suggests that activation of Th cells leads to B cell maturation and antibody isotype switching to IgG. However, since carbohydrates do not generally contain Th cell epitopes, the activated Th cells are most likely directed against the Qβ carrier. In addition, it has been shown that mice immunized with relevant Salmonella Th epitopes had significantly reduced bacterial loads in mice after challenge with Salmonella. Therefore, in order to enhance anti-Salmonella vaccine efficacy, it is desirable to include Salmonella-specific Th cell epitopes.

[0217] In order to activate Salmonella-specific Th cells, it is necessary to introduce Th cell epitope sequences from Salmonella into the Qβ-glycan conjugate. Although this can be achieved by linking the Th epitope peptide to Qβ through linker 41 in the same way as the glycan, introducing the Th epitope to the outer surface of Qβ has several possible disadvantages: 1) it will reduce the number of sites available for glycan conjugation. Since the high density of glycans on the surface of Qβ is very important for high anti-glycan antibody levels, reducing the number of glycans on the capsid may have an adverse effect on anti-glycan antibody levels; 2) antibodies against exogenous Th epitopes may be induced, which may compete with the production of anti-glycan antibodies.

[0218] To avoid the potential complications of anchoring Th epitopes on the outside of Qβ, we considered an RNA-guided encapsidation strategy to encapsulate Salmonella Th epitopes inside Qβ. Due to steric hindrance, these Th epitopes inside will not be able to interact with B cells, thereby minimizing the induction of anti-Th antibodies. In addition, the Qβ capsid can be disassembled once inside the cell, thereby releasing the cargo. This is supported by the fact that after uptake of Qβ, the RNA encapsulated within Qβ activates the Toll-like receptor-9 pathway in antigen-presenting cells.

[0219] The RNA-guided encapsidation strategy is based on the understanding of the Qβ self-assembly process. After expression in E. coli, the monomeric Qβ coat protein (CP) can bind to the single-stranded RNA genome with high affinity between the RNA hairpin (hp) structure and the positively charged residues of the coat protein. The RNA sequence acts as a template and guides the monomers to assemble into nanoparticles with a diameter of 28 nm.

[0220] By first introducing the two binding domains into a plasmid (plasmid 1, Fig.13A) is used to carry out RNA-guided encapsidation by targeting the mRNA sequence of the Qβ coat protein on the Qβ coat protein. An RNA aptamer (aR) capable of binding to an arginine-rich peptide (Rev) is inserted just upstream of the ribosome binding site of the coat protein sequence. The sequence of the Qβ encapsulation hp is directly downstream of the stop codon. At the same time, the target protein (TP) sequence will be tagged with a Rev peptide at the N-terminus and inserted into plasmid 2. Transformation with the two plasmids and expression in Escherichia coli produces Qβ, which carries the Rev-tagged target protein that is encapsidated by binding to the anti-Rev aptamer. This type of Qβ is named Qβ@(protein)n, where n indicates the average number of proteins within the particle. This system was successfully used to encapsulate red fluorescent protein (RFP) inside the interior of Qβ ( Fig.13 B).

[0221] To apply the method to Salmonella vaccine development, a plasmid was constructed with a sequence corresponding to an N-terminal Rev-tagged peptide containing the Th epitope AAQYVAAHPGEVCPA from the Salmonella alkyl hydroperoxide reductase subunit C (AhpC154-168). Co-expression of this plasmid with a plasmid containing the mRNA of the best Qβ mutant identified in objective 2.3 and flanked by anti-Rev aptamer sequences and hp sequences in E. coli will encapsidate the Rev-tagged Th epitope within mQβ, thereby generating mQβ@(Th)n.

[0222] To evaluate the performance of mQβ@(Th)n, it will be functionalized externally with optimized S. enteritidis glycans. The resulting mQβ@(Th)n-glycan conjugates will be used to immunize mice using optimized doses and adjuvants. Anti-COPS IgG titers and bacterial binding will be determined. Additionally, activation of AhpC154-168-specific Th cells will be determined using the CFSE assay outlined herein.

[0223] The protective efficacy of vaccine constructs with the new mQβ@(Th)n-glycan conjugates will next be evaluated. Mice will be fully immunized with the mQβ@(Th)n-glycan construct. A control group of WT Qβ-glycan and mQβ-glycan will be administered under the same conditions to account for the effects of mQβ and mQβ@(Th)n. Four weeks after the last boost, all mice will be injected with LD100 (1×106 CFU) of Salmonella Enteritidis R11. The number of mice surviving the Salmonella challenge will be monitored over time to construct a Fig.11 Kaplan-Meier survival curves are shown.

[0224] In another experiment, two groups of mice immunized with mQβ@(Th)n-glycan and mQβ-glycan, respectively, will be kept for one year and then challenged with S. enteritidis. The recall responses of the mQβ@(Th)n-glycan immunized groups will be evaluated to determine the relative protection against bacterial challenge.

[0225] In order to enhance Th activation, a new polypeptide can be designed to contain multiple repeats of Th epitope AhpC154-168. In addition, other Th epitopes such as EutC243-257, STM1540262-276 and FliC429-443 can also be introduced into polypeptide. As part of a vaccine, the ability to introduce various types of Salmonella Th epitopes is attractive for future conversion to humans because the human leukocyte antigen (HLA) structure is diverse, with different Th epitope structural preferences. Multiple Th epitopes covering major human HLA subtypes can be incorporated into Qβ for future conversion.

[0226] Since it is difficult to generate high levels of anti-S. Enteritidis glycan IgG responses in mice compared to rabbits, if the antibody levels induced in mice are not sufficient to be protective, the mQβ@(Th)n-glycan conjugates in rabbits will be evaluated. Sera will be collected from immunized rabbits. Serial dilutions of post-immunization sera from immunized rabbits will be administered to mice, and they will then be infected with a lethal dose of S. Enteritidis R11. The maximum dilution of serum that provides 100% protection will be determined, which will indicate the strength of the protective response. The passive transfer model is a useful alternative to active immunization strategies.

[0227] Glycan-based vaccines against S. Typhimurium and S. Paratyphi A were generated. Based on the experience gained in the S. Enteritidis vaccine studies, glycan-based vaccines against S. Typhimurium and S. Paratyphi A were developed using the optimal mQβ@(Th)n platform. Synthetic glycan antigens 2, 2a, 19, 20, 38b,c and 39b,c will be conjugated to mQβ@(Th)n using the same linker 41 as used for the S. Enteritidis glycans.

[0228] Mice will be immunized with these constructs by three injections at two-week intervals. Control mice will receive mQβ@(Th)n mixed with polysaccharides that are not covalently linked. Blood will be collected before immunization and on days 7, 21, 35, 56, and 77 after the initial vaccination. COPS will be purified from Salmonella typhimurium and Salmonella paratyphi A for determination of anti-polysaccharide antibody titers by ELISA. Anti-COPS antibody titers and subtypes (IgG versus IgM, IgG subclasses) will be determined and compared at different time points to determine the kinetics of the humoral response and the necessity of booster injections.

[0229] mQβ@(Th)n-glycan conjugates will generate higher anti-COPS IgG titers compared to controls receiving mQβ mixed with glycans. As epitope length preference and acetylation preference may differ from those of the S. Enteritidis vaccine, the optimal length of the glycan epitope will be determined by comparing binding to COPS from S. Typhimurium and S. Paratyphi A, binding to bacteria, and promotion of opsonization and complement-mediated cytotoxicity by IgG antibodies induced by vaccines containing tetrasaccharides 2 or 19 versus the corresponding octasaccharides and dodecasaccharides 38b,c and 39b,c. The role of acetylation will be determined by comparing 2 versus 2a and 19 versus 20.

[0230] Based on the results of the immunogenicity evaluation and in vitro analysis of COPS and bacterial binding / opsonization, the two best constructs against S. typhimurium will be selected. Groups of mice will be immunized with the constructs containing the adjuvants identified herein (3 doses every two weeks). The control group will receive a mixture of mQβ@(Th)n and polysaccharides. The immunized mice will then be inoculated with S. typhimurium D65 (clinical strain isolated from a patient in Mali, 1×10 5 The survival rate will be compared with a control group that received PBS injection only and a control group immunized with a mixture of mQβ@(Th)n and polysaccharide with the same adjuvant. The construct that provides the best protection will be identified.

[0231] There are currently no good animal models to assess vaccine-mediated protection against S. Paratyphi A. Therefore, in vitro bacterial binding, complement-mediated cytotoxicity, and opsonization promoted by postimmune sera will be used as surrogates for in vivo protection.

[0232] Disclosed herein is a novel mQβ(Th)n vector with reduced anti-vector response, which, after conjugation with optimized Salmonella glycans, can activate Salmonella-specific Th cells and induce potent strain-specific protective immunity against lethal infection with Salmonella typhimurium and Salmonella enteritidis.

[0233] Development of broad-spectrum anti-Salmonella vaccines

[0234] In addition to developing vaccines targeting specific strains, it would be attractive if a single vaccine construct could potentially protect against multiple common Salmonella strains. Since COPS from S. Paratyphi A (serogroup A), S. Typhimurium (serogroup B), and S. Enteritidis (serogroup D) share a common trisaccharide backbone (Man-Rha-Gal), a single vaccine could potentially be effective against all three serogroups (serogroups A, B, D) if a strong functional antibody response could be induced against this structure.

[0235] Evaluation of Qβ conjugates with Salmonella backbone oligosaccharides.

[0236] The common backbone trisaccharides, hexasaccharides, and nonasaccharides 25-27 were conjugated to wild-type Qβ using the bifunctional linker 41. Based on LC-MS analysis, the average loading levels of 25-27 were 246, 215, and 208, respectively. Mice (5 per group) and rabbits (2 per group) were immunized with these conjugates with three biweekly injections (4 μg of glycan per injection). One week after the last injection, antisera were collected and analyzed for IgG antibody titers against the immunizing antigen. Consistent with the strain-specific glycan conjugates with Qβ, both mice and rabbits produced a strong IgG response (average IgG titer of 1,500,000 for mice and 20,000,000 for rabbits).

[0237] To analyze whether the antibodies generated can recognize multiple Salmonella strains, COPS from S. Enteritidis, S. Paratyphi A, S. Typhimurium, and S. Newport (serogroups C2-3) were purified and used as ELISA antigens. While S. Enteritidis, S. Paratyphi A, and S. Typhimurium have the same COPS backbone, S. Newport has a different COPS structure. The binding of the antisera to these four types of COPS was tested by ELISA ( Fig.14 Significantly higher levels of IgG antibodies were observed in antisera recognizing COPS from S. Enteritidis, S. Paratyphi A, and S. Typhimurium, whereas much lower levels (approximately 500-fold lower) were observed in antisera recognizing COPS from S. Newport. These results emphasize the broad reactivity and selectivity of antibodies raised against the backbone structure.

[0238] Based on these results, it was hypothesized that conjugation of backbone oligosaccharides to the novel mQβ(Th)n vectors identified above would further enhance the strength of the anti-Salmonella response.Trisaccharides, hexasaccharides, and nonasaccharides 25-27 would be conjugated to the mQβ(Th)n vectors.

[0239] Both mice and rabbits will be immunized with the new construct and adjuvant (3 injections biweekly). A control group will receive mQβ(Th)n mixed with no covalently linked glycans and adjuvant.

[0240] Post-immunization sera were collected from immunized animals and the ability of the induced IgG antibodies to bind to COPS was compared with that from the corresponding conjugates with WT Qβ and the control group of mQβ(Th)n mixed with glycans without covalent bonds. In preliminary studies, it was observed that the conjugate of the trisaccharide 25 could already generate a strong IgG response targeting COPS, and the hexasaccharide antigen 26 induced the highest IgG antibody titers against COPS binding ( Fig.14 ). For new vectors, antigen length preference will be established in a similar manner. Antigens that produce the strongest response will be further evaluated in challenge studies.

[0241] Clinical strains of S. Enteritidis, S. Typhimurium, and S. Paratyphi A will be cultured in addition to S. Newport as a control. Binding of the sera to these bacteria will be measured by flow cytometry. COPS binding results indicate that the antiserum will strongly recognize S. Enteritidis, S. Typhimurium, and S. Paratyphi A strains, while binding to S. Newport will be poor. Binding will be determined by opsonin assay in the presence of macrophages ( Fig.10 B) Functional analysis of the antisera on bacteria by complement-mediated cytotoxicity assays. Strong binding of the antisera to all three strains (S. Enteritidis, S. Typhimurium and S. Paratyphi A) will significantly enhance their in vitro uptake and killing, while the effect on the S. Newport strain should not be significantly different from the control serum.

[0242] With the establishment of antiserum capacity against S. Enteritidis, S. Typhimurium and S. Paratyphi A strains, in vivo protection studies will be performed. One group of mice was immunized with mQβ(Th)n-glycan construct and adjuvant, while the control group received a mixture of mQβ and glycan. The immunized mice were divided into two groups and challenged with a lethal dose of S. Typhimurium D65 or S. Enteritidis R11.

[0243] Statistical analysis and ensuring robust and unbiased results.

[0244] Logarithmic transformation of antibody titers and cell mean fluorescence intensity will allow examination of the multiplicative effect of new vaccine constructs due to antibody binding. At a given dose level, the ANOVA model with step-down Dunnett test is used for paired treatments to control the familywise Type I error rates of multiple tests while maximizing statistical power. The log-rank test will be used to compare survival rates between groups. If necessary, more advanced analysis will be conducted in consultation with the MSU Statistical Training and Consulting Center.

[0245] To assess immune responses, each sample will be tested on 3 different occasions (biological replicates) to ensure reproducible results. The processing protocols will be kept confidential from the researchers performing the measurements.

[0246] Sex as a biological variable.

[0247] The IgG responses induced by the Qβ constructs from male and female mice were compared. Anti-glycan IgG levels were comparable in both sexes. Several other constructs will also be tested similarly. If no significant differences occur, both sexes will be used in the cohort.

[0248] There are currently no vaccines against the major Salmonella strains (i.e., S. Enteritidis, S. Typhimurium, and S. Paratyphi A). The development of an effective carbohydrate-based anti-Salmonella vaccine by combining synthetic carbohydrate chemistry and Qβ engineering will fill this gap. Qβ conjugates of Salmonella-associated glycans will be evaluated for their ability to induce potent and durable anti-Salmonella immunity and protect immunized subjects from lethal challenge with Salmonella; thereby laying the foundation for understanding the various factors that influence the anti-Salmonella glycan immune response and providing an exciting new direction for the prevention of salmonellosis.

[0249] Common abbreviations: BCR (B cell receptor); BSA (bovine serum albumin); CFSE (carboxyfluorescein diacetate succinimidyl ester); COPS (core O-polysaccharide); CP (coat protein), ELISA (enzyme-linked immunosorbent assay); ELISPOT (enzyme-linked immunospot); hp (hairpin); KLH (keyhole limpet hemocyanin); LD100 (lethal dose); MPLA (monophosphoryl lipid A); SAG (Salmonella-associated glycan); TACA (tumor-associated carbohydrate antigen); Th (helper T cell); TLR (Toll-like receptor); VLP (virus-like particle); WT (wild type)

[0250] Incorporated by Reference

[0251] All publications, patent applications, patents and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter belongs. All publications, patent applications, patents and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent and other reference was specifically and individually indicated to be incorporated by reference. It should be understood that although many patent applications, patents and other references are cited herein, such citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0252] Equivalent form

[0253] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will understand that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A vaccine composition comprising a Salmonella antigen conjugated to a capsid, wherein the capsid comprises at least one non-natural mutation.

2. The vaccine composition of claim 1, wherein the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty mutations.

3. The vaccine composition of claim 1, wherein the at least one non-natural mutation is a disulfide bond mutation.

4. The vaccine composition of claim 1, wherein the Salmonella antigen is derived from a surface glycan of Salmonella.

5. The vaccine composition of claim 4, wherein the Salmonella antigen is a polysaccharide.

6. The vaccine composition of claim 5, wherein the Salmonella antigen comprises a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, a nonasaccharide or a dodecasaccharide.

7. The vaccine composition of claim 4, wherein the Salmonella antigen comprises a trisaccharide, a hexasaccharide or a nonasaccharide of the Salmonella O-polysaccharide backbone.

8. The vaccine composition of claim 7, wherein the Salmonella antigen comprises the trisaccharide of Man-Rha-Gal.

9. The vaccine composition of claim 1, wherein the capsid is a bacteriophage capsid.

10. The vaccine composition of claim 9, wherein the phage is selected from the group consisting of: (a) phage Qβ; (b) phage R17; (c) phage fr; (d) phage GA; (e) phage SP; (f) phage MS2; (g) phage M11; (h) phage MX1; (i) phage NL95; (j) phage f2; (k) phage PP7; (l) phage AP205; and (m) phage P22. The vaccine composition according to claim 10 , wherein the bacteriophage is bacteriophage Qβ.

12. The vaccine composition of claim 2, wherein the mutation comprises at least one mutation selected from the group consisting of N10K, A38K, A40C, A40S, T75K, D102C, D102S or A117K or a combination thereof.

13. The vaccine composition of claim 12, wherein the mutation comprises A38K.

14. The vaccine composition of claim 2, wherein the capsid comprises at least two mutations selected from A40C / D102C, A40S / D102S or A43C / Q98C.

15. The vaccine composition of claim 2, wherein the capsid comprises at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C.

16. A method of preventing or treating Salmonella infection (Salmonellosis) in a subject, the method comprising administering to the subject the vaccine composition of claim 1.

17. A method for preventing or treating gastroenteritis, typhoid fever and / or paratyphoid fever, the method comprising administering the vaccine composition according to claim 1 to a subject.

18. The method of claim 17, wherein the gastroenteritis is chronic or acute.

19. The method according to any one of claims 16 to 18, wherein the vaccine composition is administered systemically.

20. The method of claim 19, wherein the systemic administration is selected from the group consisting of oral, intravenous, intradermal, intraperitoneal, subcutaneous, and intramuscular administration.

21. The method of claim 16, wherein the vaccine is directed against a plurality of Salmonella strains selected from the group consisting of: Salmonella Enteritidis, Salmonella Paratyphi A, Salmonella Typhimurium, and Salmonella Newport.

22. The method of claim 7, wherein the Salmonella antigen is derived from an O-polysaccharide backbone present in one or more of Salmonella Enteritidis, Salmonella Paratyphi A, Salmonella Typhimurium, or Salmonella Newport.

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