Development of a Carbohydrate-Based Salmonella Vaccine

Abstract

A vaccine composition comprising a Salmonella antigen conjugated to a capsid, wherein the capsid comprises a wild-type or native sequence, is provided herein. Also provided herein is a vaccine composition 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 for the treatment and prevention of Salmonella infection (salmonellosis), gastroenteritis, typhoid fever, and / or paratyphoid fever; and may be effective against multiple strains of Salmonella.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 392,274, filed Jul. 26, 2022; the entire contents of the foregoing application are incorporated herein by reference.

Background Art

[0002] Background of the Invention The emergence of multi - drug resistant microbial species suggests that new prevention and treatment strategies are urgently needed. Vaccines are a powerful tool for reducing the infection rate. It is well recognized that carbohydrates on the surface of microbial cells can be attractive targets for vaccines. However, there are numerous difficulties associated with carbohydrate - based vaccine development. The first barrier is access to microbe - associated glycans, as carbohydrates from microbial species often have unique structures compared to mammalian counterparts and may exhibit unique reactivity in glycosylation reactions. It is necessary to establish the synthesis of these compounds in a conjugatable form. Furthermore, carbohydrates alone are typically poorly immunogenic and do not elicit high - level, long - lasting antibody responses. Therefore, a method for potently boosting the immune response to carbohydrate antigens is needed.

[0003] Carrier systems are essential for delivering carbohydrate antigens to the immune system and inducing a strong anti - carbohydrate antigen antibody response. A potential drawback of protein carriers is that glycoconjugates can induce a high anti - carrier antibody response. As an example, GD3 - KLH generated an anti - GD3 IgG titer of 300, while that of KLH was 1,800,000. High anti - carrier antibodies can significantly suppress the production of anti - glycan antibodies. This phenomenon has been reported for carbohydrate - based anti - microbial disease vaccines. It has been suggested that an ideal carrier is one that induces high levels of anti - glycan antibodies without a strong anti - self antibody.

[0004] Accordingly, there is a great need in the art to identify potential therapeutic strategies and compositions that activate the immune response in the treatment and prevention of diseases and infections. Salmonella infection (salmonellosis) is a major public health problem worldwide. The development of an effective vaccine is a very attractive strategy for combating salmonellosis, especially with the increasing number of multidrug-resistant Salmonella strains. Salmonella bacteria possess characteristic glycan structures on their cell surface, which define specific serotypes and can function as antigenic targets at the same time. Strategies and vaccines for targeting Salmonella surface-derived carbohydrates that can be effective against multiple pathogenic strains of Salmonella are provided herein. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] Vaccine compositions comprising an antigen conjugated to a capsid are provided herein. 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 bacteriophage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having the wild-type or native sequence set forth in SEQ ID NO: 1. In some embodiments, the vaccine composition comprises an antigen conjugated to a capsid having at least one mutation from the wild-type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having at least one mutation from the wild-type bacteriophage 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.

[0006] In certain embodiments of the vaccine composition, the capsid comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations.

[0007] In certain embodiments of the vaccine composition, at least one non-natural mutation is a disulfide bond mutation.

[0008] In some such embodiments of the invention disclosed herein, the antigen conjugated to the capsid is a Salmonella antigen. Such a Salmonella antigen may be derived from the surface glycan of Salmonella. For example, without limitation, the antigen may be a polysaccharide (such as a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, or dodecasaccharide). In some preferred embodiments, the Salmonella antigen comprises a trisaccharide, hexasaccharide, or nonasaccharide of the Salmonella O-polysaccharide backbone. Most preferably, the Salmonella antigen comprises the trisaccharide Man-Rha-Gal. The vaccine composition disclosed herein can be used in the prevention or treatment of Salmonella infection (salmonellosis) gastroenteritis, typhoid fever, and / or paratyphoid fever in a subject in need thereof. The aforementioned vaccine can be directed to (e.g., provide protection or treatment against) multiple strains of Salmonella (such as S. Enteritidis, S. Paratyphi A, S. Typhimurum, and S. Newport).

[0009] Detailed Description of the Invention 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 nontyphoidal Salmonella (NTS) serotypes generally cause gastroenteritis but can progress to invasive disease. In Southeast Asia, enteric and paratyphoid fevers caused by S. Paratyphi A are endemic. In the United States, enteric and paratyphoid fevers are rare, but NTS cases exceed 1.2 million per 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 causes.

[0010] The development of an effective vaccine is a very attractive strategy to combat salmonellosis, especially with the increasing prevalence of multidrug-resistant Salmonella strains. Salmonella bacteria possess characteristic glycan structures on their cell surfaces, which define specific serotypes and can function as antigenic targets at the same time. S. Typhi expresses Vi capsular polysaccharide that covers the bacterial surface and protects the bacteria from the host immune system. S. Paratyphi A and most NTS do not express such capsules, and thus the surface polysaccharides in these serotypes are the O polysaccharides of lipopolysaccharide (COPS). Carbohydrate-based vaccine constructs have been shown to be effective in host protection, and an example is the ViCPS vaccine that targets the Vi polysaccharide on S. Typhi. Although ViCPS provides protection with an efficacy of 50% - 80% in the first year, the protection declines after 2 years. Currently, there is no approved vaccine against any other Salmonella strain. Therefore, the development of new anti-Salmonella vaccines is urgently needed.

[0011] As described above, enteric fevers caused by Salmonella Paratyphi A and S. Typhi, as well as invasive non-typhoidal Salmonella (iNTS) diseases caused mainly by S. Enteritidis and S. Typhimurium, are major causes of the global disease burden. The spread of multidrug resistance is increasing, and there are no licensed vaccines to prevent salmonellosis other than for S. Typhi. Vaccines targeting polysaccharides on the surface of pathogenic bacteria have proven to be an effective preventive strategy, as exemplified by the S. Typhi capsular polysaccharide vaccine. The Salmonella serotypes Paratyphi A, Typhimurium, and Enteritidis are covered with a dense layer of O-polysaccharide containing the 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 S. Typhimurium, S. Enteritidis, and S. Paratyphi A. The studies disclosed herein explore whether the Man-Rha-Gal motif constitutes a broad-spectrum protective antigen by constructing glycoconjugate vaccines based on this antigen and characterizing its immunogenicity and efficacy against Salmonella infection.

[0012] The synthesis of Salmonella - related carbohydrate antigens is provided herein. Traditionally, research on carbohydrate - based Salmonella vaccines has relied on native polysaccharides isolated from pathogenic strains. The resulting glycans are heterogeneous mixtures with variable lengths and structures. Furthermore, there are concerns regarding batch - to - batch variability and potential contaminants. Instead of relying on isolation, a synthetic pathway was developed to produce structurally well - defined Salmonella - related glycans, including the tetrasaccharide repeating unit of the O - antigen polysaccharide from two pathogenic serotypes, S. Enteritidis and S. Paratyphi A. Since the optimal immunogenic size of the glycan antigen is not known, the synthesis of glycan antigens from trisaccharides and tetrasaccharides to dodecasaccharides from S. Enteritidis, S. Typhimurum, and S. Paratyphi A is provided herein. Furthermore, since the O - polysaccharides of these strains of Salmonella share a common backbone, glycans corresponding to that backbone structure were synthesized to potentially aid in the development of a broad - spectrum vaccine.

[0013] The development of Qβ - glycan conjugates as strain - specific anti - Salmonella vaccines is also provided herein. One of the major challenges in carbohydrate - based vaccine development is the low immunogenicity of the antigen. To date, it has been demonstrated that virus - like particles exemplified by bacteriophage Qβ can present carbohydrate antigens in a highly organized manner. (See WO2019 / 045791, which is incorporated herein by reference in its entirety.) Patterned antigen presentation led to a strong IgG antibody response against tumor - associated carbohydrate antigens. Based on these results, the hypothesis that Qβ can significantly enhance the humoral response against Salmonella - related glycans was established.

[0014] 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 relying on a large excess of precious carbohydrate substrates. For example, the Enteritidis glycan Qβ - conjugate induced IgG antibodies with very high titers against the glycan (IgG titers exceeding 80,000,000 ELISA units in rabbits). The induced antibodies recognized the native O - polysaccharide whether as an isolated antigen or when present on bacterial cells. Furthermore, when antiserum from immunized rabbits was introduced, mice were 100% protected against a lethal challenge of S. Enteritidis. Based on these promising results, the optimal structure of the glycan - based antigen was established. Additionally, a novel Qβ carrier was designed to enable the activation of Salmonella - specific helper T cells and the production of higher levels of anti - glycan IgG antibodies. A strain - specific anti - Salmonella vaccine effective against S. Enteritis, S. Typhimurium, and S. Paratyphi A will be developed.

[0015] Further provided herein is the establishment of Qβ - glycan conjugates as broad - spectrum anti - Salmonella vaccines. Since S. Enteritidis, S. Typhimurium, and S. Paratyphi A share a common polysaccharide backbone structure and differ in their dideoxyhexose branches, the common backbone glycan was targeted for the vaccine. Antibodies induced by a single construct made with Qβ conjugated to the backbone glycan may provide broad - spectrum protection against multiple Salmonella strains, offering an exciting tool for Salmonella prevention.

[0016] For that purpose, Man-Rha-Gal oligosaccharide was synthesized and this glycan was conjugated to bacteriophage Qβ carrier to enhance anti-polysaccharide immunity. Then, New Zealand white rabbits (n = 7) were immunized intramuscularly three times with these conjugates. Vaccination resulted in a robust serum IgG response recognizing native O-polysaccharides from S. Paratyphi A, S. Typhimurium, and S. Enteritidis in both ELISA (geometric mean titers = 6.3×10 4 , 2.1×10 4 , and 3×10 6 EU / mL; P < 0.05 versus baseline) and flow cytometry formats. In a passive protection assay in naive CD-1 mice (n = 18 / group), immune sera were shown to provide excellent protection against systemic challenge with a lethal dose (1.3×10 6 CFU) of S. Enteritidis R11, a bloodstream isolate from a mare (vaccine efficacy = 94.4%, P < 0.0001 versus baseline). Overall, these findings emphasize that a single repeat unit of the common Man-Rha-Gal motif is sufficient to induce a protective antibody response.

[0017] Aspects of the invention provided herein include vaccine compositions comprising a Salmonella antigen conjugated to a capsid. In some embodiments, the capsid described above comprises at least one non-natural mutation. In some embodiments, the capsid comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations. In some such embodiments, at least one non-natural mutation is a disulfide bond mutation.

[0018] In some embodiments, the Salmonella antigen is derived from the surface glycan of Salmonella. The Salmonella antigen can be a polysaccharide. For example, without limitation, the Salmonella antigen can include a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, a nonasaccharide, or a dodecaccharide. In some preferred embodiments, the Salmonella antigen includes a trisaccharide, a hexasaccharide, or a nonasaccharide of the Salmonella O-polysaccharide backbone. Most preferably, the Salmonella antigen includes at least one Man-Rha-Gal trisaccharide. For example, the Salmonella antigen can include repeats of the Man-Rha-Gal trisaccharide (such as 2, 3, or more Man-Rha-Gal trisaccharides).

[0019] In some embodiments of the present invention, the capsid is a bacteriophage capsid. The bacteriophage can be selected from the group consisting of (a) 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. Preferably, the bacteriophage is bacteriophage Qβ. In some such embodiments, the mutation includes at least one mutation selected from N10K, A38K, A40C, A40S, T75K, D102C, D102S, or A117K or combinations thereof. In some preferred embodiments, the mutation includes A38K. In other embodiments, the aforementioned capsid includes at least two mutations selected from A40C / D102C, A40S / D102S, or A43C / Q98C. In further embodiments, the aforementioned capsid includes at least three mutations selected from A40C / D102C / K13R or A38K / A40C / D102C.

[0020] Embodiments of the present invention include a method for preventing or treating Salmonella infection (salmonellosis) in a subject, the method comprising administering to the subject the vaccine composition disclosed herein. Also provided is a method for preventing or treating gastroenteritis, typhoid fever, and / or paratyphoid fever, the method comprising administering to the subject the vaccine composition disclosed herein. In some such embodiments, the gastroenteritis is chronic or acute.

[0021] The contemplated vaccine composition can be administered systemically. Such systemic administration can be selected from the group consisting of oral administration, intravenous administration, intradermal administration, intraperitoneal administration, subcutaneous administration, and intramuscular administration.

[0022] In some embodiments, the vaccines contemplated herein are directed against multiple strains of Salmonella selected from S. Enteritidis, S. Paratyphi A, S. Typhimurum, and S. Newport. In some embodiments, the Salmonella antigen is derived from the O-polysaccharide backbone present in one or more of S. Enteritidis, S. Paratyphi A, S. Typhimurum, or S. Newport.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0037] A. Definitions The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0038] The term "administering" means providing a pharmaceutical or composition to a subject and includes, but is not limited to, administration by a medical professional and self - administration.

[0039] As used herein, "bacteriophage" refers to a virus that infects bacteria and replicates within the bacteria. In certain embodiments, the bacteriophage is selected from the group consisting of, but not limited to: (a) 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. As used herein, "bacteriophage Qβ" (also referred to as "bacteriophage Qb", "Qβ", and "Qb") is one of many small - molecule RNA bacteriophages that infect Escherichia coli.

[0040] As used herein, "carbohydrate antigen" refers to a class of antigens that elicit a strong antibody response. In certain embodiments, the carbohydrate antigen is selected from, but 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 (or any of the bacterial, viral, or pathogenic diseases provided herein), Tf, Tn or sTn (Tn, Tf, and STn are commonly found in glycoproteins such as MUC1).

[0041] The terms "inhibit" or "inhibits" mean to reduce, suppress, attenuate, abate, arrest, or stabilize a disease, disorder, or condition, a biological pathway activity, or a biological activity, e.g., the growth of a solid malignant tumor, by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or even 100% as compared to an untreated control subject, cell, biological pathway, or biological activity, or as compared to a target in a subject prior to the subject being treated, e.g., the growth of a solid malignant tumor. The term "reduce" means to inhibit, suppress, attenuate, abate, arrest, or stabilize the symptoms of a cancer disease, disorder, or condition. It will be appreciated that treatment of a disease, disorder, or condition, although not excluded, does not require that the disease, disorder, condition, or associated symptoms be completely eliminated.

[0042] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications within the scope of sound medical judgment and in commensurate with a reasonable benefit / risk ratio.

[0043] 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, involved in the conveyance or transport of a subject compound from one organ or body part to another. 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 function 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (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 glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0044] "Pharmaceutically acceptable salts" refer to relatively non-toxic inorganic and organic acid addition salts of a compound.

[0045] Terms such as "prevent", "preventing", "prevention", "preventive treatment" and the like refer to reducing the probability of the onset of a disease, disorder or condition in a subject who does not have the disease, disorder or condition but is at risk of or predisposed to developing the disease, disorder or condition.

[0046] "Subject" may include human subjects in medical purposes, for example, in the treatment of existing diseases, disorders, conditions, or in preventive treatment to prevent the onset of diseases, disorders or conditions, or may include animal subjects for medical, veterinary or development purposes. Suitable animal subjects include, but are not limited to, primates such as humans, monkeys, apes, langurs, chimpanzees, orangutans, macaques, etc.; bovine such as cattle, bulls, etc.; ovine such as sheep, etc.; caprine such as goats, etc.; porcine such as pigs, boars, etc.; equine such as horses, donkeys, zebras, etc.; felines including wild cats and domestic cats; canines including dogs; lagomorphs including rabbits, hares, etc.; and mammals including rodents such as mice, rats, guinea pigs, etc. The animal can be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile and adult subjects. Further, "subject" may include a patient suffering from or suspected of suffering from a disease, disorder or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (e.g., rats and mice used in experiments).

[0047] The term "subject in need thereof" means a subject identified as in need of treatment or therapy.

[0048] The terms "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" mean administering a compound, drug or other material other than directly into the central nervous system, for example, subcutaneously, such that it enters the patient's system and thus is subjected to metabolism and other similar processes.

[0049] The term "therapeutic agent" or "pharmaceutical" refers to an agent that can have a desired biological effect on a host.

[0050] The term "therapeutic effect" refers to a local or systemic effect in an animal, particularly a mammal, more particularly a human, that is caused by a pharmacologically active substance.

[0051] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a compound, material, or composition comprising a compound of the present invention that, at a reasonable benefit / risk ratio applicable to any medical treatment, is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal.

[0052] The term "treatment" of a disease in a subject or "treatment" of a subject having a disease refers to subjecting the subject to a medical treatment, e.g., administration of a vaccine composition described herein, so as to reduce at least one symptom of the disease, prevent its worsening, or delay its worsening.

[0053] B. Capsid Vaccine compositions comprising an antigen conjugated to a capsid are provided herein. 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 bacteriophage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having the wild-type or native sequence set forth in SEQ ID NO: 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 bacteriophage Qβ capsid having at least one mutation from a wild-type bacteriophage 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 compositions provided herein comprise an antigen (e.g., a carbohydrate antigen, a polypeptide, a peptide, a protein, and a small molecule) conjugated to a capsid (e.g., 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 portion of a capsid amino acid sequence of sufficient length that can induce an enhanced strong immune response when conjugated to an antigen. In certain embodiments, the capsid polypeptide also 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 a fusion protein comprising a capsid amino acid sequence and an amino acid sequence corresponding to a non-capsid protein or polypeptide).

[0054] In some embodiments, the capsid is derived from a bacteriophage. In certain embodiments, the bacteriophage is selected from the group consisting of: (a) 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.

[0055] In some embodiments, the capsid has the sequences described in Coat Protein Table A. [Table A-1] [Table A-2]

[0056] In some embodiments, the bacteriophage Qβ capsid contains the sequence set forth in SEQ ID NO: 1. [Chemical Structure]

[0057] In some embodiments, the bacteriophage Qβ capsid contains at least one mutation described in Table B. [Table B-1] [Table B-2] [Table B-3] 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., JACS 2008, 130(4), 1328 - 1334; 42b corresponds to Udit, A et al., ChemBioChem 2009, 10(3), 503 - 510. 43 corresponds to Hovlid, M. L et al., The Scripps Research Institute, La Jolla, 2014.

[0058] In certain embodiments, the capsid comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mutations. In some embodiments, the mutations are non - native disulfide bond mutations. In some embodiments, the capsid comprises, but is not limited to, the following mutations (Table C).

Table C - 1

Table C - 2

[0059] In certain embodiments, the bacteriophage Qb variant may have the following physical characteristics.

Table D

[0060] In some embodiments, the bacteriophage Qb capsid comprises a polypeptide having 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 sequences set forth in SEQ ID NOs: 1-15. In some embodiments of the methods, compositions and kits provided herein, the Qb variant has at least one mutation as set forth in Table C. 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 set forth in SEQ ID NO: 1. In some embodiments, the Qb variant has an amino acid sequence consisting essentially of the mutations set forth in SEQ ID NOs: 2-15. In some embodiments, the capsid comprises a polypeptide having 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 sequences set forth in the GenBank numbers set forth in Table A.

[0061] In some embodiments of the methods, compositions, and kits provided herein, the vaccine composition comprises 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 a capsid amino acid sequence (e.g., any of SEQ ID NOs: 1-15 or the GenBank numbers listed in Table A) and having 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 set forth in SEQ ID NO: 1.

[0062] As is well known to those skilled in the art, polypeptides having substantial sequence similarity can elicit the same or very similar immune responses in host animals. Thus, in some embodiments, derivatives, equivalents, variants, fragments or mutants of the Qb capsid or fragments thereof may also be suitable for the methods, compositions, and kits provided herein.

[0063] In some embodiments, the altered polypeptide can have an amino acid sequence altered, for example, by conservative substitutions, yet still elicit an enhanced immune response and be considered a functional equivalent. As used herein, the term "conservative substitution" means the replacement of an amino acid residue with another biologically similar residue. It is well known in the art that amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of the protein. According to certain embodiments, a derivative, equivalent, variant or mutant of Qb is at least 85% homologous to the sequences set forth in SEQ ID NOs: 1-15, or a biologically active variant thereof, 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 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 set forth in SEQ ID NO: 1. C. Vaccine Compositions and Their Pharmaceutical Compositions / Formulations

[0064] A vaccine composition comprising an antigen conjugated to a capsid is provided herein. 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 bacteriophage Qβ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having a wild-type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Qβ capsid having the wild-type or native sequence set forth in SEQ ID NO: 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 bacteriophage Qβ capsid having at least one mutation from a wild-type bacteriophage 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, polypeptide, peptide, protein, and small molecule) conjugated to a capsid (e.g., bacteriophage Qb), wherein the capsid comprises at least one mutation (e.g., at least one point mutation, or at least one non-natural amino acid mutation, or at least one non-natural disulfide bond mutation). The capsid is described in the foregoing Section B. In some embodiments, the antigen is a fragment or portion of a carbohydrate antigen of sufficient length to elicit an enhanced strong immune response when conjugated to the capsid. The capsid can be conjugated to a plurality of 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, TNF alpha, IL1α, IL1β, tau protein, PCSK9, or amyloid beta. In some embodiments, the antigen is a small molecule selected from, but not limited to, nicotine, cocaine, or advanced glycation products.

[0065] In some embodiments, the invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of one or more vaccine compositions (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above), formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In another aspect, the composition can be administered by itself or mixed with a pharmaceutically acceptable carrier and can also be administered in combination with other therapies. Thus, combination therapy includes sequential, simultaneous, individual or co-administration of the composition, and the therapeutic effect of the first administration is not completely lost if subsequent compounds are administered. In some embodiments, at least one vaccine composition (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above) can be provided to a subject alone or in combination with at least one therapeutic agent, chemotherapeutic agent, scavenger compound, antibiotic, antiviral agent, antifungal agent, anti-inflammatory agent, vasoconstrictor and anticoagulant, antigen useful for vaccine use, or the corresponding prodrug.

[0066] As described in detail below, the pharmaceutical compositions of the invention are adapted for administration by: (1) oral, e.g., as a solution (aqueous or non-aqueous liquid or suspension), tablet, e.g., for buccal, sublingual and systemic absorption, bolus, powder, granule, paste for application to the tongue; (2) parenteral, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, e.g., as a sterile solution or suspension, or as a sustained release formulation; (3) topical, e.g., as a cream, ointment or controlled release patch or spray applied to the skin; (4) intravaginally or rectally, e.g., as a pessary, cream or foam; (5) sublingually; (6) to the eye; (7) transdermally; or (8) nasally, and can be particularly formulated for administration in solid or liquid form.

[0067] As described above, certain embodiments of one or more vaccine compositions (e.g., one or more Qβ wild types or Qβ mutants, antigen conjugates as described above) may contain basic functional groups such as amino or alkylamino, and thus may form pharmaceutically acceptable salts with pharmaceutically acceptable acids. These salts can be prepared in situ in the dosing vehicle or dosage form manufacturing process, or by reacting the purified compound of the invention in its free base form separately with a suitable organic or inorganic acid and isolating the salt thus formed during subsequent purification. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate, etc. (see, for example, Berge et al., (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

[0068] Pharmaceutically acceptable salts of the subject compound include, for example, conventional non-toxic salts of the compound from non-toxic organic or inorganic acids or quaternary ammonium salts. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid; 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, isothionic acid, etc.

[0069] In other cases, one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants as described above, antigen conjugates) may contain one or more acidic functional groups and thus may form pharmaceutically acceptable salts with pharmaceutically acceptable bases. Similarly, these salts can be prepared in situ in the dosage vehicle or dosage form manufacturing process or by reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, etc. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine and piperazine, etc. (see, for example, Berge et al., supra).

[0070] Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, and coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0071] 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 chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0072] One or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variants, antigen conjugates as described above) can be in a formulation 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 the active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the host to be treated and the particular mode of administration. The amount of the active ingredient that can be combined with the carrier materials to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect.

[0073] In certain embodiments, the formulation of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variants, antigen conjugates as described above) allows for greater stability, different release characteristics in vivo, targeting to a particular site, or any other desired feature that allows for more effective delivery of the one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variants, antigen conjugates as described above) to a subject or a target in a subject, and can include other carriers such as, but not limited to, liposomes, microspheres, nanospheres, nanoparticles, bubbles, micelle formers such as bile acids, and polymeric carriers such as polyesters and polyanhydrides. In certain embodiments, the above formulations make the compounds of the present invention orally bioavailable.

[0074] Liquid dosage forms of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ mutants, antigen conjugates as described above) may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0075] In addition to the inert diluent, the oral composition may also include adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents, coloring agents, perfumes and preservatives.

[0076] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0077] Formulations suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using flavor bases, usually sucrose and gum arabic or tragacanth), powders, granules, or as solutions or suspensions of aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as troches (using inert bases such as gelatin and glycerin, or sucrose and gum arabic), and / or as gargles, etc., each containing a predetermined amount of the active ingredient. One or more vaccine compositions (e.g., one or more of the Qβ wild-type or Qβ mutants, antigen conjugates as described above) can also be administered as a bolus, a lickable preparation or a paste.

[0078] 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 with 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, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retardants, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (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 buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard shell gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0079] Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants or dispersing agents. Molded tablets can be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0080] Tablets and other solid dosage forms, such as dragees, capsules, pills and granules, may be scored, if desired, or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide for sustained or controlled release of the active ingredient therein using, for example, various proportions of hydroxypropyl methylcellulose, other polymeric matrices, liposomes and / or microspheres to provide the desired release profile. The compositions may also be formulated, for rapid release, for example, by lyophilization. They may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also contain, if desired, opacifying agents and may be compositions which release only the active ingredient(s) or which preferentially release the active ingredient(s) in a particular part of the gastrointestinal tract in a delayed manner, if desired. Examples of embedding compositions which may be used include polymeric substances and waxes. The active ingredient may also, where appropriate, be in microencapsulated form with one or more of the above excipients.

[0081] Formulations for rectal or vaginal administration may be presented as suppositories, prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax or salicylates, which are solid at room temperature but liquid at body temperature and melt in the rectal or vaginal cavity to release the active compound.

[0082] Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, for example, carriers known to be appropriate in the art.

[0083] 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, powders for external use, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compounds can be mixed, under sterile conditions, with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants as may be required.

[0084] Ointments, pastes, creams and gels can contain, in addition to the active compounds of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0085] Powders for external use and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can further contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0086] Transdermal patches have the additional advantage of providing controlled delivery to the body. 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 either by providing a rate-controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0087] Ophthalmic preparations, eye ointments, powders for external use, solutions, etc. are also contemplated as being within the scope of the present invention.

[0088] A pharmaceutical composition suitable for parenteral administration may contain a sterile isotonic aqueous or non-aqueous liquid, a dispersing agent, a suspending agent or an emulsion, or a sterile powder or powder that can be reconstituted into a sterile injection solution or dispersing agent immediately before use. This sterile powder or powder may contain sugar, alcohol, antioxidant, buffer, bacteriostatic agent, solute that is isotonic with the blood of the recipient for which the preparation is intended, or a suspending agent or thickening agent.

[0089] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of coating materials such as lecithin, in the case of dispersing agents, by maintaining the required particle size, and by the use of surfactants.

[0090] In certain embodiments, the pharmaceutical composition can be combined with other pharmacologically active compounds (“second active agents”) known in the art according to the methods and compositions provided herein. The second active agent can be a large molecule (such as a protein) or a small molecule (such as a synthetic inorganic, organometallic or organic molecule). In one embodiment, the second active agent independently or synergistically aids in the treatment of cancer.

[0091] In another embodiment, the compositions of the present invention can include a therapeutic agent or prodrug, such as other chemotherapeutic agents, scavenger compounds, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, vasoconstrictors and anticoagulants, and other biologically active substances including antigens useful for cancer vaccine applications or corresponding prodrugs.

[0092] 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, arylamines and nitro compounds.

[0093] Various forms of chemotherapeutic agents and / or other biologically active agents can be used. These include, but are not limited to, forms such as biologically active uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like.

[0094] D. Treatment methods Vaccine compositions are provided herein for the treatment and / or prevention of diseases and conditions in which an enhanced immune response can 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.

[0095] In some embodiments, vaccine compositions (e.g., one or more Qβ wild-type or Qβ variants, antigen conjugates as described above) are useful in the treatment of 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.

[0096] 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), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes viruses such as HSV-1 and HSV-2, influenza virus, hepatitis A and B, FIV, lentivirus, pestivirus, West Nile virus, measles virus, variola virus, vaccinia virus, Ebola virus, 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 virus or Rous sarcoma virus (RSV).

[0097] In addition, there are bacterial diseases, fungal diseases and other pathogenic diseases, such as Aspergillus, Brugia, Candida, Chikungunya, Chlamydia, Coccidia, Cryptococcus, Dengue, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, P. vivax in Anopheles mosquitoes, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibrio cholerae.Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus species, 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, 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.

[0098] Also included are the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogens. These include Category A pathogens such as smallpox, Bacillus anthracis, Yersinia pestis, Clostridium botulinum toxin, Francisella tularensis, filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever) and related viruses); Category B pathogens such as Coxiella burnetii (Q fever), Brucella species, Burkholderia mallei, alphaviruses (Venezuelan equine encephalitis, Eastern and Western equine encephalitis), ricin toxin from Ricinus communis, epsilon toxin of 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, jungle yellow fever, and multidrug-resistant tuberculosis; helminths such as Schistosoma and Taenia; and protozoa such as sandfly-transmitted Leishmania (e.g., L. mexicana), Plasmodium, and snail-transmitted Chagas disease.

[0099] Examples of bacterial pathogens include, but are not limited to, bacterial pathogenic Gram-positive cocci, such as, but not limited to, pneumococci, staphylococci, and streptococci. Pathogenic Gram-negative cocci include meningococci and gonococci. Pathogenic enteric Gram-negative bacilli include Enterobacteriaceae; Pseudomonas, Acinetobacter, and Eikenella; melioidosis; Salmonella; Shigella; Haemophilus; chancroid; brucellosis; tularemia; Yersinia (Pasteurella); Streptobacillus moniliformis and Spirillum; Listeria monocytogenes; Erysipelothrix rhusiopathiae; diphtheria, cholera, anthrax; and Donovanosis (inguinal granuloma). Pathogenic anaerobic bacteria include tetanus; botulinum; other Clostridium species; Mycobacterium tuberculosis, Mycobacterium leprae, and other Mycobacteria. Pathogenic spirochetal diseases include syphilis; treponematosis; chancroid, pinta, and endemic syphilis; and leptospirosis. Other infectious diseases caused by higher pathogenic bacteria and pathogenic fungi include actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis, and coccidioides mycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsis, mycetoma, and phaeohyphomycosis; and dermatophytosis. Rickettsial infections include Rickettsia and rickettsiosis. Examples of Mycoplasma and Chlamydia infections include Mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal Chlamydia infections. Pathogenic protozoa and helminths and infections eukaryotes caused thereby include amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; Pneumocystis carinii; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.

[0100] In one embodiment, the treatment method includes administering to a subject (e.g., a subject in need thereof) an effective amount of one or more vaccine compositions (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above). Subjects in need thereof can include, for example, subjects diagnosed with pre-cancerous tumors, tumors including cancer, or treated subjects who were refractory to previous treatments.

[0101] The term "effective amount" in the "therapeutically effective amount" of a therapeutic agent refers to the amount of the agent necessary to induce a desired biological response. As recognized by those skilled in the art, the effective amount of an agent can 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" means, for example, 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 and cause regression of a disease, disorder or condition; to prevent the recurrence, occurrence, onset or progression of symptoms associated with a disease, disorder or condition; or to be a sufficient amount to enhance or improve the prophylactic or therapeutic effect(s) of another treatment.

[0102] The compositions described herein can be delivered orally, nasally, transmucosally, ophthalmically, rectally, intravaginally, parenterally, e.g., intramuscularly, subcutaneously, intramedullary injection, as well as intracerebroventricularly, directly into the ventricle, intravenously, intra-articularly, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal or intraocular injection, intrabursal, topically, as powders, dusts, ointments or drops (including eye drops), e.g., buccally and sublingually, transdermally, by inhalation spray, or by any other suitable route of administration including other delivery modes known in the art.

[0103] As used herein, the terms "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" mean administering the vaccine composition described herein such that it enters the patient's system and is subject to metabolism and other similar processes.

[0104] As used herein, the terms "parenteral administration" and "parenterally administered" mean a mode of administration usually by injection other than enteral and topical administration, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal and intrasternal injection and infusion.

[0105] In certain embodiments, the pharmaceutical composition is systemically delivered (e.g., by oral or parenteral administration). In certain other embodiments, the pharmaceutical composition is locally delivered by direct injection into the tumor or into the blood supply of the tumor (e.g., arterial or venous blood supply). In some embodiments, the pharmaceutical composition is delivered by both systemic and local administration. For example, a subject having a tumor can be treated by directly injecting a composition containing the composition described herein into the tumor or the blood supply of the tumor, in combination with oral administration of the pharmaceutical composition of the invention. When both local and systemic administration are used, the local administration can be performed before, together with and / or after the systemic administration.

[0106] In some embodiments, the subject pharmaceutical composition of the invention incorporates, in an amount sufficient to deliver to a patient a therapeutically effective amount of a therapeutic agent or other material as part of a prophylactic or therapeutic treatment, the single or multiple substances to be delivered. The desired concentration of the active compound in the particles depends on the absorption, inactivation, and excretion rates of the drug and the delivery rate of the compound. It should be noted that the dosage values can also vary with the severity of the condition to be alleviated. For any particular subject, it should be further understood that the specific dosage regimen should be adjusted over time according to the individual requirements and the professional judgment of the administrator or supervisor of the composition. Typically, administration is determined using techniques known to those of ordinary skill in the art.

[0107] The dosage can be based on the amount of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variant, antigen conjugate as described above) per kg of the patient's body weight. For example, the amount of the composition or compound encapsulated therein can be contemplated to include from about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20, 25, 50, 75, 100, 150, 200 or 250 mg or more per kg of the patient's body weight. Other amounts are known to those of ordinary skill in the art and can be readily determined.

[0108] In certain embodiments, the dosage of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variant, antigen conjugate as described above) is generally in the range of about 0.001 mg to about 250 mg per kg of body weight, specifically in the range of about 50 mg to about 200 mg per kg, more specifically in the range of about 100 mg to about 200 mg per kg. In one embodiment, the dosage is in the range of about 150 mg to about 250 mg per kg. In another embodiment, the dosage is about 200 mg per kg.

[0109] In some embodiments, the molar concentration of one or more vaccine compositions (e.g., one or more Qβ wild-type or Qβ variant as described above, antigen conjugate) in the pharmaceutical composition is 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 variant as described above, antigen conjugate) is 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.

[0110] The actual dosage level of the active ingredient in the composition of the present invention can be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for the patient, the composition, and the mode of administration, without undue toxicity to a particular patient.

[0111] The selected dosage level depends on a variety of factors including the activity of the particular therapeutic agent, or its esters, salts or amides, used in the formulation, the route of administration, the time of administration, the rate of excretion or metabolism of the particular therapeutic agent used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0112] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can prescribe and / or administer the dosage of the compound of the present invention used 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.

[0113] Generally, the appropriate daily dose of the compounds of the present invention is that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective doses generally depend on the above factors.

[0114] If desired, the effective daily dose of the active compound can be administered as two, three, four, five, six or more fractional doses, which are administered separately at appropriate intervals throughout the day, in unit dosage forms as necessary.

[0115] The exact timing and amount of administration of any particular compound that provides the most effective treatment in a given patient depends on the activity, pharmacokinetics, and bioavailability of the particular compound, the patient's physiological state (including age, sex, type and stage of disease, general physical condition, responsiveness to the dose administered and type of drug therapy), route of administration, and the like. The guidance provided herein can be used to optimize treatment, for example, to determine the optimal time and / or amount of administration, which does not require more than routine experimentation consisting of monitoring the subject and adjusting the dose and / or timing.

[0116] While the subject is being treated, the patient's health can be monitored by measuring one or more relevant indicators at predetermined times throughout the 24 hours. All aspects of the treatment, including supplements, amounts, time of administration, and formulation, can be optimized based on the results of such monitoring. The patient can be periodically re-evaluated to determine the degree of improvement by measuring the same parameters, and the first such re-evaluation is typically performed at the end of 4 weeks from the start of treatment, and subsequent re-evaluations are performed every 4 - 8 weeks during treatment and then every 3 months thereafter. Treatment can continue for several months or even years, and a minimum of 1 month is typical for treatment in humans. For example, adjustments to the amount(s) of drug administered and the time of administration can be made based on these re-evaluations.

[0117] Treatment can be initiated at a dosage less than the optimal dosage of the compound. Thereafter, the dosage can be increased gradually until an optimal therapeutic effect is achieved.

[0118] The toxicity and therapeutic efficacy of the subject compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, LD 50 and ED 50 Compositions showing a large therapeutic index are preferred. In some embodiments, LD 50 (lethal dosage) can be measured. For example, in one or more vaccine compositions described herein (e.g., one or more Qβ wild-type or Qβ variant, antigen conjugate as described above), it can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more lower compared to the control. Similarly, ED 50 (i.e., the concentration that achieves half of the maximum inhibition of symptoms) can be measured. For example, in one or more vaccine compositions described herein (e.g., one or more Qβ wild-type or Qβ variant, antigen conjugate as described above), it can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more greater compared to the control. Also similarly, IC 50(i.e., the concentration that achieves half of the maximum cytotoxic or cytostatic effect against cancer cells) can be measured. For example, in one or more vaccine compositions described herein (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above), it can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more greater compared to the control. Compounds that exhibit toxic side effects can be used, but care should be taken to design a delivery system to direct the compound to the desired site in order to reduce side effects.

[0119] In some embodiments, the methods of the present disclosure result in 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.

[0120] In any of the above methods, administration of one or more vaccine compositions (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above) can result in at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% reduction of solid malignant tumors in a subject compared to the solid malignant tumors before administration of the vaccine composition.

[0121] In some embodiments, a therapeutically effective amount of one or more vaccine compositions (e.g., one or more of the Qβ wild-type or Qβ variants, antigen conjugates as described above) is administered prophylactically to prevent the formation of solid malignant tumors in a subject.

[0122] In some embodiments, the subject is human. In other embodiments, the subject is non-human, such as a mammal.

[0123] Data obtained from cell culture assays and animal studies can be used in the formulation of a range of dosages for use in humans. The dosage of any supplement or any component therein is preferably within a range of circulating concentrations that have little or no toxicity and an ED 50 within. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. In the agents of the present invention, a therapeutically effective dosage can first be estimated from cell culture assays. The dosage can be formulated in an animal model to achieve a range of circulating plasma concentrations including the IC 50 determined in cell culture. Such information can be used to more accurately determine useful dosages in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Example

[0124] Exemplification The following examples are included to provide guidance to those skilled in the art for carrying out representative embodiments of the subject matter of the present disclosure. Considering the present disclosure and the general level of skill in the art, those skilled in the art can recognize that the following examples are for illustrative purposes only and that numerous changes, modifications, and variations can be used without departing from the scope of the subject matter of the present disclosure. The following examples are provided as illustrative, not limiting.

[0125] Example 1: Qβ Salmonella Glycan Conjugate as a Potential Anti-Salmonella Vaccine Salmonella is a Gram-negative bacterium that can cause severe infectious diseases (salmonellosis) upon ingestion of contaminated food or water. Salmonella includes over 2500 serotypes that are distinguished by serotype based on the structure of O polysaccharide on the cell surface and clinical syndromes. Typhoid and paratyphoid serotypes (S. Typhi and S. Paratyphi) cause invasive enteric fever, while non-typhoidal Salmonella generally causes gastroenteritis, but invasive diseases can occur as a result of immunological immaturity, suppression, or aging. S. Typhi causes typhoid fever and is responsible for over 200,000 deaths annually, mainly in developing countries, especially in Southeast Asia, Central and South America, and Africa. In Asia, paratyphoid caused by S. Paratyphi A is also epidemic. In the United States, non-typhoidal Salmonella (NTS) is more common and is frequently associated with large epidemics in multiple states, causing over 1.2 million infections, tens of thousands of hospitalizations, and hundreds of deaths annually. Among the various NTS serotypes associated with clinical diseases in the United States, S. Enteritidis and S. Typhimurium are the most common. According to 2010 data from the Foodborne Diseases Active Surveillance Network of the Centers for Disease Control and Prevention (CDC) in the United States, salmonellosis accounted for 54% of hospitalizations and 43% of deaths due to reported food poisoning. Worldwide, an estimated 94 million cases and 155,000 deaths occur annually. The incidence of foodborne diseases such as Shiga toxin-producing Escherichia coli O157 infection has decreased by over 50% in the past 20 years, but there has been no progress in Salmonella, and salmonellosis has increased by 10% during the same period.

[0126] Currently, invasive salmonellosis is treated with antibiotics. However, multidrug-resistant strains of Salmonella have emerged (9-10), and the CDC has classified drug-resistant Salmonella as a serious threat level that requires "rapid and continuous response to ensure that the problem does not spread." Despite the frequency and severity of salmonellosis and the public health efforts to control the occurrence of this disease, currently, there is no vaccine available for Salmonella strains other than S. Typhi. In the case of S. Typhi, the ViCPS vaccine, which directly uses Vi capsular polysaccharide purified from S. Typhi as an immunogen, is particularly notable 13. This vaccine provides protection with an effectiveness between 50% and 80% in the first year, but its protective effect is lost after two years. The development of a vaccine against Salmonella, particularly against strains other than S. Typhi such as S. Enteritidis, S. Typhimurium, and S. Paratyphi A, is urgently needed.

[0127] Vaccines that target polysaccharides on the cell surface of pathogenic bacteria have proven to be an effective strategy for protection against multiple bacterial pathogens, as exemplified by the ViCPS vaccine against S. Typhi. In the case of other Salmonella strains, they lack Vi but have characteristic surface polysaccharides. The cell surface core O-polysaccharides (COPS) of S. Paratyphi, S. Typhimurium, and S. Enteritidis share a common backbone structure with a repeating trisaccharide of α-D-mannose (Man)-1,4-α-L-rhamnose (Rha)-1,3-α-D-galactose (Gal)-1,2-. Dideoxyhexose is linked to the 3-OH of Man, and different serotypes are distinguished by the structure of the dideoxy sugar. S. Paratyphi A contains paratose, S. Typhimurium has abequose, while the dideoxy sugar is tyvelose in S. Enteritidis. (See Figure 1.) In some polysaccharides, an α-glucosyl unit may be attached to the 6-OH of the backbone galactose. Furthermore, some of the 2-OH groups and 3-OH groups of rhamnose are acetylated, resulting in further heterogeneity of the native glycan.

[0128] To date, all carbohydrate-based Salmonella vaccines have utilized purified polysaccharides, either directly or as digestion products of polysaccharides from cultures of the corresponding serotypes. These processes need to be tightly controlled to remove endotoxins and other harmful contaminants. Furthermore, polysaccharides are mixtures with heterogeneous lengths and structures and can have batch-to-batch variability, making it difficult to more deeply understand the important structures of protective epitopes. The possibility of developing vaccines against Salmonella using synthetic oligosaccharides with well-defined structures is contemplated herein. These glycan antigens can be delivered using a potent virus-like particle (VLP) bacteriophage Qβ-based carrier system to generate a strong and long-lasting humoral immune response.

[0129] One of the major barriers in developing glycan-based Salmonella vaccines is that carbohydrates are weakly immunogenic T cell-independent antigens. When administered alone, they can generate only low-titer IgM antibodies, typically with low affinity and short-lived persistence. For an effective and persistent vaccine, it is highly desirable that IgG antibodies can be induced. To achieve this, the glycan can be attached to an immunogenic protein carrier that contains helper T (Th) cell epitopes necessary to induce Th cell activation and antibody isotype switching from IgM to IgG. The loss of immunoprotection from ViCPS after two years is likely due to the fact that ViCPS, which does not contain any Th epitopes, is administered.

[0130] Another important parameter in carbohydrate-based vaccine design is how the antigen is presented to B cells. This is because two signals are required for B cell activation and differentiation: the first antigen-specific signal delivered through the binding and cross-linking of the B cell receptor (BCR) on the cell surface, and the second co-stimulatory cytokine signal from cognate Th cells. Arrays of haptens spaced 5 - 10 nm apart have been shown to cross-link the BCR sufficiently, and antigens presented in a highly organized manner may lead to earlier B cell proliferation for strong IgM and IgG responses. Also, antigen organization can have a major impact on B cell tolerance, with B cells not responding to poorly organized antigens while responding rapidly to the same antigens presented in an ordered manner. Thus, carriers that enable organized antigen presentation would be highly advantageous.

[0131] VLPs can be a powerful platform for antigen delivery. This is because, in contrast to amorphous protein carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and tetanus toxoid (TT), VLPs are composed of self-organizing protein subunits, giving a highly ordered structure. Repeated B cell epitopes presented on the VLP surface can efficiently cross-link the BCR. Furthermore, VLPs can contain multiple Th epitopes, allowing the cross-linking of the BCR and stimulation by activated Th cells to act synergistically. Other advantages of VLPs include the ease of genetic manipulation for particle engineering for enhanced immunogenicity, high stability against various chemical modification conditions, the speed and scale of production, and the ability to incorporate immunostimulatory agents within the capsid.

[0132] Recently, VLPs have been utilized as carriers for tumor-associated carbohydrate antigens (TACAs). Similar to Salmonella-associated glycans (SAGs), TACAs are T cell-independent antigens. Among the multiple types of VLPs investigated, bacteriophage Qβ is the most potent in terms of carrier function. By presenting TACAs in an organized manner on Qβ, enhancement of the antibody response was observed even against very weak TACAs, namely monomeric Tn antigen, and the anti-Tn IgG titer exceeded 250,000. In comparison, the Tn-KLH conjugate did not induce anti-Tn IgG antibodies. Importantly, immunization with Qβ-Tn significantly protected mice from the development of invasive tumors, while Qβ alone provided no survival advantage, suggesting that the non-specific immune response attributable to Qβ alone was insufficient.

[0133] Regarding another tumor-associated antigen, mucin-1 (MUC1), a direct comparison was made between Qβ-MUC1 and a MUC1 conjugate with KLH, the gold standard utilized in multiple clinical trials of carbohydrate-based cancer vaccines. Consistent with the results of the Tn study, Qβ-MUC1 was superior as it was able to induce more than three times as much anti-MUC1 IgG antibody than the corresponding KLH-MUC1 construct and was significantly effective in reducing tumor burden in a mouse tumor model.

[0134] Through cancer vaccine research, several valuable lessons have been learned in applying Qβ to glycan-based vaccine design. 1) High-density antigen presentation is important for a high IgG antibody response. A Qβ construct carrying approximately 140 copies of MUC1 was able to induce four times more anti-MUC1 IgG antibodies compared to the corresponding construct with 30 copies of MUC1, even when both mouse groups received the same amount of total MUC1. The same density-dependent phenomenon was observed in Tn vaccine research; 2) Activation of Th cells is important because depletion of Th cells before immunization almost completely abolished the anti-glycan IgG response; 3) Covalent conjugation of the antigen to Qβ is important because a mixture of Qβ and MUC1 produced less than 3% anti-MUC1 antibodies compared to the anti-MUC1 antibodies induced by covalently conjugated Qβ-MUC1; 4) The structure of the linker used for conjugation of the antigen to Qβ is important. The Qβ-Tn construct formed using a copper-catalyzed azide-alkyne cycloaddition click reaction was less effective in inducing tumor-binding antibodies compared to that formed using a flexible alkylamide linker. This was thought to be due to potential antigen competition with the rigid triazole ring and the effect of steric exclusion / epitope suppression by the anti-triazole antibodies formed.

[0135] Despite the success of anti-cancer vaccine development, Qβ has not been systematically studied as a carrier for bacterial-related glycans. We hypothesized that by tissue-specifically presenting SAG on Qβ, a strong and long-lasting anti-SAG IgG response could be obtained as a potential anti-Salmonella vaccine.

[0136] Innovations: 1) The study disclosed herein is constructed by integrating immunology with modern synthetic chemistry and bioconjugation chemistry. 2) The VLP Qβ disclosed herein represents an exciting new carrier for antimicrobial vaccine development. The Qβ-SAG conjugate has the potential to elicit high titers of anti-SAG IgG antibodies and immunological memory, two elements essential for Salmonella infection prevention. 3) Disclosed herein are novel Qβ carriers that retain the ability to activate bacterial-specific Th cells to induce even higher levels of anti-SAG IgG antibodies compared to wild-type Qβ, while being associated with reduced antibody production against the carrier itself. These carriers could be a platform technology applicable to vaccine development for diseases other than Salmonellosis. 4) The availability of synthetic SAG oligosaccharides eliminates the reliance on pathogenic bacterial cultures for polysaccharide isolation for vaccine constructs, thus improving batch-to-batch consistency and reducing the likelihood of 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. 5) Targeting the common polysaccharide backbone of S. Enteritidis, S. Typhimurium, and S. Paratyphi A represents a new direction in vaccine design made possible by synthetic backbone glycans, as obtaining these structures by enzymatic or chemical digestion is not practically feasible. The backbone-based vaccine has the potential to be broad-spectrum and confer protection against multiple medically important Salmonella serotypes.

[0137] Synthesis of Salmonella-related oligosaccharide antigens. Conventionally, research on carbohydrate-based anti-Salmonella vaccines has relied on polysaccharides isolated from pathogenic strains. The glycans obtained by this approach exist as heterogeneous mixtures with variable lengths and structures. Furthermore, there are concerns regarding batch-to-batch variability and potential contaminants. Instead of relying on isolation from natural sources, a synthetic route for producing Salmonella-related glycans with well-defined structures is disclosed.

[0138] Synthesis of strain-specific Salmonella tetrasaccharide: Tetrasaccharides from S. Enteritidis and S. Paratyphi A. The first synthetic target was the S. Enteriditis tetrasaccharide 1 corresponding to one repeating unit with an aminopropyl linker at the reducing end. Four monosaccharide components 3 - 6 were designed, and here, benzyl (Bn) groups and acetonide groups were mainly used as protecting groups. This is because these electron - donating protecting groups may enhance the reactivity of the components in glycosylation. The important and rare tyvelose donor 3 was efficiently prepared from readily available thiomannoside. Here, thiomannoside was selectively protected and then converted to 3 via Barton - McCombie deoxygenation (Scheme 1a; Figure 2A). Components 4 - 6 were prepared in a straightforward manner. Since deoxysugars are typically more anomeric - reactive than their fully oxygenated counterparts, selective activation of tyvelose 3 over the bifunctional mannoside 4 was achieved by N - iodosuccinimide (NIS, 1.2 equiv) and TMSOTf (Scheme 1b, Figure 2A), and then separated by column chromatography to give α - tyvelosylated disaccharide 9 in 85% yield along with a minor amount of the β - anomer (ca. 5%). The new α - glycosidic bond in compound 9 was confirmed by 13C - 1 and H - 1 coupling constant values [δ 99.3 (JC1 / H1 = 167.8 Hz, C - 1 tyvelose), 85.8 (JC1 / H1 = 167.8 Hz, C - 1 Man)]. Glycosylation of acceptor 6 with rhamnoside donor 5 yielded disaccharide 10 as the exclusive alpha anomer (Scheme 1c; Figure 2A). Removal of the non - reducing end 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 for future chain elongation to longer glycans (compound 13). (See Figure 2A.)

[0139] To synthesize the S.Paratyphi A tetrasaccharide 2, the paratose component 14 was prepared from the glucoside 15 by the same selective protection and Barton-McCombie deoxygenation applied to the synthesis of the tibulose donor 3. The protected S.Paratyphi A tetrasaccharide 16 was then synthesized in good overall yield using the paratoside 14 and components 4 - 6 according to a chemo-selective glycosylation approach similar to that for the preparation of 12. (See Figure 2B.) Subsequent deprotection of 16 yielded the S.Paratyphi A tetrasaccharide 2a (72 mg), which bears two OAc on its rhamnoside unit for future investigation of the role of OAc in antibody response induction. Furthermore, to gain a deeper understanding of the role of glycan structure in antibody recognition, the pentasaccharide 17 bearing a glucoside on the reducing end Gal and the tetrasaccharide 18 without a paratose unit were also synthesized (90 mg each). (See Figure 2C.)

[0140] S.Typhimurium is another major strain of Salmonella. To develop a vaccine against S.Typhimurium, the S.Typhimurium tetrasaccharides 19 and 20 were synthesized. (See Figure 3.) The preparation of 19 and 20 would start from the abequose donor 21 (Scheme 2) prepared by the selective protection of galactoside 22 followed by Barton-McCombie deoxygenation. Chemo-selective glycosylation of mannoside 4 with abequose 21 would afford the disaccharide 23. Similar to the synthesis of the S.Enteriditis and S.Paratyphi A tetrasaccharides, the major anomer expected to be formed is alpha, which would be confirmed by NMR. Coupling of the disaccharide 23 with the disaccharide 11 would yield the tetrasaccharide 24, which upon deprotection would give the S.Typhimurium tetrasaccharide 20. Omission of the acetylation step in the deprotection of 24 would afford the tetrasaccharide 19.

[0141] Synthesis of strain-specific octasaccharides and dodecasaccharides and common backbone oligosaccharides from S.Enteritidis, S.Paratyphi A, and S.Typhimurium Since naturally occurring Salmonella O-polysaccharides mainly consist of tetrasaccharide repeats, oligosaccharides longer than one tetrasaccharide unit may be able to elicit a stronger anti-Salmonella antibody response. Therefore, octasaccharides and dodecasaccharides of strain-specific Salmonella glycans containing two or three repeat units each were synthesized. By targeting the polysaccharide backbone, it may be possible to develop a broad-spectrum vaccine against multiple strains of Salmonella. To enable this research, a synthetic route for preparing the backbone oligosaccharides was developed.

[0142] The common trisaccharide, hexasaccharide, and nonasaccharide of the O-polysaccharide backbone were synthesized. The common backbone of Salmonella consists of a repeating trisaccharide of Man-Rha-Gal. Backbone oligosaccharides 25 - 27 were synthesized using one, two, and three repeating trisaccharide units, respectively (Scheme 3; Figure 4). Monosaccharide components 28 - 31 were established using a protecting group pattern for high alpha selectivity in glycosylation. The pre-activated base glycosylation 54 - 55 of the mannoside donor 28 (1.2 equiv) with the bifunctional rhamnoside acceptor 29 (1 equiv) gave the disaccharide 32 in 82% yield. Subsequent glycosylation of the galactoside 31 protected with di-t-butylsilylene 56 gave the trisaccharide 34 in 78% yield (Scheme 3a). Similarly, using galactoside 30 as the final acceptor, the trisaccharide 33 was synthesized in 74% yield. The Bz group of 33 was removed, and the resulting trisaccharide acceptor was glycosylated with donor 34 to produce the hexasaccharide 35 (80% yield) as the only isolated anomer (Scheme 3b). The alpha configuration of the newly formed glycosidic bond was confirmed by its 1JCH value of 173.5 Hz, which was presumably assisted by the di-t-butylsilylene moiety. Repeating the Bz removal and glycosylation with the trisaccharide donor 34 led to the fully protected nonasaccharide 36 (81% yield), which was successfully deprotected to produce the Salmonella backbone nonasaccharide 27. The trisaccharide and hexasaccharides 25 and 26 were synthesized similarly (10 mg each) and fully characterized by NMR and MS.

[0143] Since the synthesis of the common backbones 25 - 27 was successful, the simplest way to access strain - specific Salmonella glycans would be to convert these backbone glycans. As illustrated in Scheme 4 (Figure 5), the selective removal of the PMB group from the nonasaccharide 36, followed by glycosylation with more than 3 equivalents of the tibulose donor 3, would generate the dodecaccharide 37, which upon deprotection would yield the dodecaccharide 38a of S. Enteritidis. Similarly, the octasaccharide 39a of S. Enteritidis, the octasaccharides and dodecaccharides of S. Paratyphi A, and the dodecaccharides and octasaccharides 38b,c and 39b,c of S. Typhimurium would be synthesized. This would provide a branched pathway and enable rapid access to a series of SAGs. The synthesis of all the target glycans outlined in Objective 1 would be scaled up to 1 g each to support vaccine research. Since glycosylation proceeds with generally good yields without requiring a large excess of components, no major difficulties are expected in achieving this goal. The triple glycosylation of 36 by 3 may not provide high alpha - selectivity at all three sites. In this case, tetrasaccharide modules such as 40 bearing deoxysugar units would be synthesized and then used to assemble larger oligosaccharides.

[0144] Development of strain - specific anti - Salmonella vaccines. SAG itself is weakly immunogenic and cannot induce a strong antibody response when administered alone. Therefore, it is important that SAG be 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 bacteriophage Qβ bearing SAG could induce a strong protective antibody response as a strain - specific anti - Salmonella vaccine.

[0145] The conjugation of SAG with the bacteriophage Qβ carrier can be efficiently carried out. After the necessary strain-specific glycans were synthesized, bioconjugation to wild-type (WT) Qβ was performed. The tetrasaccharide 1 of S. Enteritidis was functionalized with adipic acid di-N-hydroxysuccinimide (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 its free carboxylic acid form in nearly quantitative yield and converted back to the NHS ester for reuse in bioconjugation. Characterization of the Qβ-glycan conjugate by LC-MS showed the presence of an average of 334 copies of the glycan per Qβ particle. 20 mg of the Qβ-glycan 1 conjugate was synthesized. After a similar synthetic scheme, a Qβ conjugate with the S. Paratyphi A (paratose) tetrasaccharide conjugate 2 was synthesized (an average of 290 copies of glycan 2 per Qβ particle, 10 mg of the conjugate was prepared). Also, to assist in immunological analysis, glycans 1 and 2 were conjugated to BSA.

[0146] Immunization with Qβ-glycan 1 induced a strong anti-glycan IgG response in both mice and rabbits. The humoral responses to Qβ-glycan 1 and Qβ-glycan 2 were evaluated. These conjugates were subcutaneously injected into groups of 5 mice (day 0) at doses of 1 μg and 4 μg of glycan, respectively, followed by two booster injections on days 14 and 28. Control groups (5 mice each) received unconjugated Qβ at the same protein level.

[0147] Enzyme-linked immunosorbent assay (ELISA) analysis using BSA-glycan 1 as the coating antigen showed that high-titer IgG antibodies were induced by Qβ-glycan 1. The mean IgG titers for the groups receiving 1 μg and 4 μg of glycan reached 487,000 and 980,000 ELISA units, respectively, on day 35 (Figure 7A). In comparison, the anti-glycan IgG titers obtained in the control group immunized with Qβ alone were less than 5,000 ELISA units, suggesting that including glycan 1 in the vaccine is important for a high anti-glycan antibody response.

[0148] For an effective vaccine, it is important that the induced immune response persists. The antibody response induced by Qβ-glycan 1 was monitored over time (data for the 4 μg group shown in Figure 7B). The level of anti-glycan 1 IgG antibodies reached its maximum value (6,000,000 ELISA units) 56 days after the first immunization and was maintained for 120 days. The antibody titer gradually decreased, and half of the peak IgG level remained on day 477. These results suggested that a persistent anti-glycan 1 IgG response was induced by Qβ-glycan 1. No obvious harmful effects were observed in the immunized animals over the entire period, highlighting the safety of the vaccine.

[0149] To establish the selectivity and specificity of the antibodies induced by Qβ-glycan 1, the tetrasaccharide backbone 18, as well as the tetrasaccharide 2 and pentasaccharide 17 of S. Paratyphi A, were conjugated to BSA using linker 41 for ELISA. Analysis using these BSA-glycan conjugates demonstrated that the post-immune sera bound most strongly to BSA-glycan 1 (Figure 8), indicating high selectivity for glycan 1 and suggesting an important role of the non-reducing terminal tibulose in glycan 1 for antibody recognition.

[0150] In addition to mice, rabbits are another common animal for immune evaluation. Two rabbits were immunized subcutaneously with Qβ-glycan 1 (4 μg glycan, injections every two weeks). Serum was collected on days 0, 35, 49, and 56. ELISA analysis against BSA-glycan 1 induced a robust anti-glycan 1 IgG response, with IgG titers reaching 83,000,000 and 150,000,000 ELISA units by day 56 (Table 1), indicating that these were 6,000-fold higher than those from Qβ-immunized control rabbits or rabbits before immunization. These results show that Qβ-glycan 1 is effective in multiple species.

Table 1

[0151] For a successful vaccine, it is important that the antibodies generated by the vaccine construct can recognize the native polysaccharide. To test this, the native core O-polysaccharide (COPS) of the homologous serotype was isolated from S. Enteritidis and immobilized on ELISA wells. Antisera from rabbits and mice were evaluated for their ability to bind to COPS. Also, three anti-Salmonella monoclonal antibodies (mAb6347 against S. Paratyphi A, mAb6391 and 6393 against the core polysaccharide) were added to the same ELISA plate for comparison. As shown in Figure 9, mAb6391 and 6393 recognized COPS well, while mAb6347 did not. Serum from immunized rabbits bound strongly to native COPS. Higher COPS binding was observed for rabbit serum compared to mouse serum. A similar species difference in the anti-glycan antibody response has been reported by the Cummings group.

[0152] Antibodies induced by Qβ-glycan 1 bound strongly to S. Enteritidis and enhanced the macrophage uptake of the bacteria. Whether post-immune sera could bind to homologous antigens on intact bacterial cells was evaluated using S. Enteritidis strain R11 isolated from the blood of infected infant monkeys. Binding of post-immune sera from rabbits immunized with Qβ and Qβ-glycan 1 to R11 was analyzed by flow cytometry. While only very weak binding was seen in either pre-immune sera or sera from rabbits immunized with Qβ carrier alone, anti-sera from Qβ-glycan 1-immunized rabbits strongly recognized R11 (Figure 10A), suggesting the potential of Qβ-glycan 1 as an anti-S. Enteritidis vaccine.

[0153] Opsonophagocytic uptake of antibody-bound bacteria by macrophages represents an important mechanism for antibody-mediated clearance of Salmonella. To determine whether post-immune sera had functional anti-Salmonella activity, uptake of S. Enteritidis R11 by J774 mouse macrophages was evaluated after incubation of the bacteria with pre-immune sera, anti-sera from Qβ-immunized rabbits, or sera from Qβ-glycan 1-immunized rabbits. While pre-immune and Qβ-immunized rabbit sera did not cause significant bacterial uptake compared to medium alone, anti-sera from Qβ-glycan 1-immunized rabbits enhanced bacterial macrophage opsonization 400% (Figure 10B).

[0154] Passive transfer of sera from immunized rabbits protected mice from lethal challenge with S. Enteritidis. The protective efficacy of antiserum against Salmonella infection was evaluated in vivo. Mice were passively administered PBS (n = 7), pooled pre-immune antiserum at different dilutions (n = 12 / group), or vaccine-induced antiserum (n = 12 / group), and then challenged intraperitoneally with a lethal dose (LD100) dose (1 × 106 CFU) of R11. All mice in the PBS group died of bacterial infection by day 7, and all but one of the mice in the two groups that received pre-immune serum died of infection by day 8. Interestingly, 100% of the mice (1:100 or 1:500 dilution, a total of 24 mice) that received post-immune serum survived the bacterial challenge (Figure 11).

[0155] Establishment of the optimal length and structure of S. Enteritidis glycan for antibody response. In a preliminary immunological evaluation, S. Enteritidis tetrasaccharide antigen 1 was focused on. Since native S. Enteritidis COPS consists of a polymer of multiple repeats of tetrasaccharide units, a hypothesis was put forward that longer glycans could better mimic the conformational properties of native COPS and result in a stronger antibody response. Since the anti-Salmonella glycan antibody levels generated in mice were lower than those in rabbits, it will be tested whether excellent antibodies can be obtained in mice by including octasaccharide and dodecaccharide as antigens in the vaccine.

[0156] Octasaccharide and dodecaccharide 39a and 38a will be conjugated to Qβ via a bifunctional linker 41 in a manner similar to tetrasaccharide 1. Acetylation will be investigated by conjugating acetylated tetrasaccharide 1a to Qβ. The resulting constructs will be purified completely to remove free glycans. The glycan loading level will be quantified by LC-MS assay. If the loading level is low due to the longer size of the glycan, the bioconjugation reaction can be repeated to increase the loading to approximately 300 as in the case of Qβ-glycan 1. Excess unconjugated glycan can be recovered and recycled.

[0157] Mice will be immunized with the novel conjugate, and the IgG antibody levels in the post-immunization sera against the immunizing antigen and native COPS from S. Enteritidis will be determined by ELISA. Comparison of the antibody responses against those induced by Qβ-glycan 1 will help establish the influence of antigen length and acetylation on the recognition of COPS.

[0158] The vaccine dose is an important parameter to be optimized. In the Qβ-Tn study, it was observed that using a higher dose (20 μg) in vaccination resulted in antisera that bound more strongly to Tn-expressing tumor cells than lower doses (1 μg and 4 μg). In preliminary studies, mice immunized with Qβ-glycan 1 at a glycan level of 4 μg tended to have higher mean IgG antibody levels compared to the 1 μg group (Figure 7A). Higher dosages (8 and 40 μg of antigen per injection) will be evaluated to establish the dose that gives the strongest glycan-specific antibody titer. For a dose of 40 μg, 10 mg of the Qβ-glycan conjugate will be sufficient for complete immunization of 20 mice.

[0159] Adjuvants can significantly affect the immune response. In preliminary studies, Freund's adjuvant was utilized. However, since complete Freund's adjuvant cannot be used in humans due to its toxicity, the following three adjuvants that are already 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 important activators of the innate immune system. Previously, it has been shown that Freund's adjuvant and MPLA induce similar levels of IgG antibodies. The adjuvant that gives the highest titer of antibodies against COPS from S. Enteritidis will be selected.

[0160] Mice will be monitored up to 2 years after immunization for the longevity of the IgG response. According to the preliminary data in Figure 7B, anti-glycan IgG levels may remain high for the first 6 months and then gradually decline. In this case, another booster injection can be given at 1 year. This mimics the cohort of elderly humans who are susceptible to Salmonella infection. The potential persistence and boostability of the IgG response would be attractive features of the vaccines disclosed herein.

[0161] Qβ-glycan immunization is well tolerated. To date, despite the induction of high IgG titers, the only side effect observed due to immunization was ulceration at the injection site due to the toxicity of complete Freund's adjuvant. By switching the adjuvant to MPLA, this side effect in cancer vaccine studies was completely eliminated. Any possible adverse effects will be monitored up to 2 years after immunization.

[0162] For constructs showing a high antibody response, the specificity of the induced antibodies will be analyzed by screening the antiserum on a microbial glycan microarray provided by the National Glycomics Center. This microarray contains over 300 representative carbohydrate structures from 36 species across 15 genera, including many Salmonella strains. Based on preliminary screening (Figure 8), it is expected that the antibodies generated will be highly selective for the OPS glycan of S. Enteritidis.

[0163] The binding ability of the antiserum to bacterial cells is tested by incubation of the post-immune serum with S. Enteritidis and subsequent flow cytometry analysis. For sera that react strongly with the bacteria, the ability of the serum to eliminate the cells is determined by the macrophage opsonization assay (Figure 10B) and the complement-mediated cytotoxicity assay (SBA). Constructs that generate IgG antibodies showing the strongest binding and cytotoxic effects against S. Enteritidis are identified. This provides important information regarding the preferred epitope structure.

[0164] A deeper understanding of the immune response by the Qβ-glycan conjugate. To better understand the immune response and immune memory induced by vaccination, memory B cells and long-lived antibody-secreting plasma cells will be analyzed. Cells will be isolated from the lymph nodes and spleens of mice immunized with Qβ alone or Qβ-glycan 1. These cells will be subjected to flow cytometry analysis upon incubation with a fluorescently labeled BSA-glycan 1 conjugate. The memory B cell population will be quantified as cells that are SAG+IgG+B220+CD38+. If the frequency of these cells is too low to be accurately quantified, memory B cells will be differentiated into plasma cells by stimulation with lipopolysaccharide (LPS) for 6 days and transferred to an enzyme-linked immunospot assay (ELISPOT) plate coated with BSA-glycan 1 to quantify the number of glycan-specific IgG-secreting B cells. Also, long-lived glycan-specific plasma cells will be evaluated by ELISPOT using bone marrow-derived cells. Qβ-glycan 1 immunization is predicted to result in a greater number of glycan-specific memory B cells and long-lived plasma cells compared to the control.

[0165] Th cell activation is important for the production of high-affinity IgG antibodies and long-term memory. To analyze Th activation by Qβ-glycan 1, lymphocytes derived from the spleen or lymph nodes will be isolated from immunized and naive mice and then labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Bone marrow dendritic cells (BMDC) will be generated by culturing bone marrow cells from the tibia and femur of naive mice using DC medium containing granulocyte / macrophage colony-stimulating factor and interleukin-4. After stimulation with LPS, mature BMDC will be incubated with Qβ-glycan 1 and then added to the CFSE-labeled spleen cells. The proliferation of CD4+ T cells in the presence of Qβ-glycan 1-treated BMDC will reduce the intracellular CFSE concentration. Flow cytometric analysis of the CFSE intensity of CD4+ cells will enable quantification of vaccine-specific CD4+ T cells. Compared to naive mice, immunized mice have a significantly higher percentage of CD4+ T cells that are expected to be specific for Qβ-glycan 1, suggesting CD4+ T cell activation induced by Qβ-glycan 1 vaccination.

[0166] Generation of Qβ mutant carriers with enhanced anti-glycan antibody levels while associated with a reduced anti-carrier response. A potential drawback in the use of immunogenic protein carriers is that anti-carrier antibody levels can increase dramatically under prime-boost vaccination protocols. For example, with the KLH-GD3 construct (one prime injection and two boost injections), the anti-GD3 IgG titer reached 400, and the anti-KLH IgG titer was 200-fold higher (819,200). In the case of Qβ-Tn, the difference was smaller, at 12-fold: an average IgG titer of 3,100,000 against Qβ versus an average anti-Tn IgG titer of 263,000. 36. Endogenous epitopes from the carrier can compete for a limited number of Th cells for the desired epitope and for cytokine signals important for B cell activation and IgG production. Furthermore, antibodies that bind to non-essential epitopes can sterically block adjacent targeted epitopes from being recognized by B cells or can result in immune complex removal. Therefore, it is desirable to remove non-essential carrier epitopes to assist the immune system in focusing on the glycan epitopes in the conjugate. A Qβ variant strategy was investigated to reduce the anti-Qβ antibody response and further enhance anti-glycan antibody production.

[0167] Removal of Qβ B cell epitopes decreased the anti-Qβ titer and increased antibody levels against the carbohydrate antigen. Since B cell epitopes need to bind to the bulky B cell receptors on the B cell surface, it was hypothesized that they are likely to be present within flexible and accessible loops on the outside of Qβ. Based on the near-atomic resolution crystal structure of Qβ, the triple mutant A38K / A40C / D102C was designed. A38 was selected because it is located on the outside and is solvent-exposed (Figure 12A). The A40C mutation and the D102C mutation were introduced because these two residues are spatially close in the crystal structure and can form a disulfide bond that crosslinks the two subunits and enhances capsid stability. The A38K / A40C / D102C mutant was expressed in E. coli and self-assembled to form particles in good yield (about 20 mg / L) and of a size similar to wild-type (WT) Qβ (28 nm in diameter).

[0168] IgG induced by carbohydrate antigen Tn (400 Tn per capsid) conjugated to Qβ / A38K / A40C / D102C was evaluated. There were two notable findings: (1) the triple mutant induced significantly higher levels of anti-Tn IgG antibodies (Figure 12B); and (2) the anti-carrier IgG titers against both WT Qβ and Qβ / A38K / A40C / D102C were much lower than those induced by WT Qβ-Tn (Figure 12C). This suggests that some of the native B cell epitopes of Qβ were successfully removed without creating new dominant B cell epitopes.

[0169] According to the rationale in the generation of the Qβ / A38K / A40C / D102C triple mutant, the optimized glycan structure from the above source will be conjugated to the Qβ / A38K / A40C / D102C triple mutant, and mice and rabbits will be immunized with the new construct at optimized doses and adjuvants. IgG levels against COPS and the carrier will be determined by ELISA. Similar to the results of Tn (Figure 12B), the conjugate with the triple mutant carrier will yield an excellent IgG antibody response compared to the corresponding WT-Qβ conjugate.

[0170] In addition to the triple mutant, to further disrupt the native B cell epitopes of Qβ, other exposed residues including T75, Q99, A117, and P119 will be mutated to amino acids such as lysine, alanine, and serine. Based on past experience, since the selected mutation sites do not exist at the interface between coat protein subunits, approximately 70% of the Qβ mutants can self-organize and form particles in good yields. These new mutants (mQβ, m indicates mutant) will be conjugated to S. Enteritidis glycan and used to immunize mice and rabbits. The mQβ construct that yields the highest anti-glycan IgG antibody response, the strongest COPS, and bacterial binding will be identified as a lead for further studies.

[0171] Establishment of a heterologous prime-boost strategy. Qβ can have multiple major B cell epitopes. Therefore, it will be tested whether combining effective Qβ mutations in one coat protein can further reduce the anti-Qβ IgG titer. However, if there are too many mutations, the coat protein may not organize into particles. A heterologous prime-boost strategy using multiple mQβs containing different major B cell epitopes can be developed. One mQβ-glycan conjugate will be used to prime the immune system and a second mQβ-glycan conjugate will be used for booster injection. In this strategy, since the B cell epitopes from various mQβs are different, the glycan antigen is the only component common to all injections. Therefore, anti-glycan immunity should benefit most from the booster injection. This heterologous prime-boost strategy should result in stronger anti-glycan antibody production and a decrease in the unwanted response to the carrier.

[0172] Creation of a novel Qβ carrier encapsulating exogenous Salmonella - related Th cell epitopes that can further enhance anti - Salmonella antibody levels and protective efficacy. The ability of Qβ - glycan conjugates to induce high levels of anti - glycan IgG antibodies suggests the activation of Th cells leading to B cell maturation and antibody isotype switch to IgG. However, since carbohydrates typically do not contain epitopes for Th cells, the activated Th cells are most likely against the Qβ carrier. Furthermore, immunization with appropriate Salmonella Th epitopes has been shown to directly cause a significant decrease in bacterial load in mice after Salmonella challenge. Therefore, it would be desirable to include epitopes for Salmonella - specific Th cells to enhance anti - Salmonella vaccine efficacy.

[0173] To activate Salmonella - specific Th cells, Salmonella - derived Th cell epitope sequences need to be introduced into the Qβ - glycan conjugate. This can be achieved by linking the Th epitope peptide to Qβ in the same manner as the glycan via linker 41. However, introducing Th epitopes on the outer surface of Qβ may give rise to several drawbacks: 1) The number of sites available for glycan conjugation will be reduced. Since the high - density glycans on the Qβ surface are important for high levels of anti - glycan antibodies, reducing the number of glycans on the capsid may adversely affect anti - glycan antibody levels; 2) Antibodies against exogenous Th epitopes that may compete with the generation of anti - glycan antibodies can be induced.

[0174] To avoid the possible complications that can occur with the immobilization of Th epitopes outside Qβ, an RNA - directed encapsidation strategy for packaging Salmonella Th epitopes inside Qβ is contemplated herein. Due to steric hindrance, these internal Th epitopes cannot interact with B cells and, thus, the induction of anti - Th antibodies will be minimized. Furthermore, the Qβ capsid can dissemble and release its cargo once it is taken up inside the cell. This is supported by the fact that RNA encapsulated inside Qβ activates the intracellular Toll - like receptor - 9 pathway in antigen - presenting cells after uptake of Qβ.

[0175] The RNA - directed encapsidation strategy is based on the understanding of the Qβ self - assembly process. When expressed inside Escherichia 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 serves as a template and guides the assembly of monomers into 28 - nm - diameter nanoparticles.

[0176] RNA-directed encapsidation is carried out by first introducing two binding domains into the mRNA sequence of the Qβ coat protein on a plasmid (plasmid 1, Figure 13A). An RNA aptamer (aR) that can bind to the arginine-rich peptide (Rev) is inserted immediately upstream of the ribosome binding site for the coat protein sequence. The sequence of the Qβ packaging hp is placed immediately downstream of the stop codon. In parallel, the N-terminus of the target protein (TP) sequence will be tagged with the Rev peptide and inserted into plasmid 2. Transformation with both plasmids and expression in E. coli will generate Qβ with Rev-tagged target protein encapsulated by binding to the anti-Rev aptamer. Such Qβ is named Qβ@(protein)n, where n represents the average number of proteins inside the particle. This system was successfully utilized to encapsulate the red fluorescent protein (RFP) inside Qβ (Figure 13B).

[0177] To apply the method for developing Salmonella vaccines, a plasmid carrying a sequence corresponding to an N-terminal Rev-tagged peptide containing the Th epitope AAQYVAAHPGEVCPA (AhpC154-168) derived from Salmonella alkyl hydroperoxide reductase subunit C is constructed. When this plasmid is co-expressed in E. coli with a plasmid containing the mRNA of the best mutant Qβ identified in Objective 2.3, flanked by the anti-Rev aptamer sequence and the hp sequence, the Rev-tagged Th epitope should be encapsulated inside mQβ, and mQβ@(Th)n should be obtained.

[0178] To evaluate the performance of mQβ@(Th)n, the outside will be functionalized with optimized S. Enteritidis glycan. The resulting mQβ@(Th)n-glycan conjugate will be used to immunize mice with optimized doses and adjuvants. Anti-COPS IgG titers and bacterial binding will be determined. Furthermore, the activation of AhpC154-168-specific Th cells will be determined using the CFSE assay outlined herein.

[0179] Next, the protective efficacy of the vaccine construct having the novel mQβ@(Th)n-glycan conjugate will be evaluated. Mice will be sufficiently immunized with the mQβ@(Th)n-glycan construct. To interpret the effects of mQβ and mQβ@(Th)n, WTQβ-glycan and mQβ-glycan will be administered to the comparison groups under the same conditions. Four weeks after the last booster, all mice will be injected with LD100 (1×106CFU) of S. Enteritidis R11. The number of mice that survived the Salmonella challenge will be monitored over time to construct a Kaplan-Meier survival curve as shown in FIG. 11.

[0180] In another experiment, two groups of mice immunized with mQβ@(Th)n-glycan and mQβ-glycan, respectively, will be maintained for one year and then challenged with S. Enteritidis. The recall response from the mQβ@(Th)n-glycan immunized group will be evaluated to determine the relative protection against bacterial challenge.

[0181] To enhance Th activation, a novel polypeptide can be designed to include multiple repeats of the Th epitope AhpC154-168. Additionally, other Th epitopes such as EutC243-257, STM1540262-276, and FliC429-443 can also be introduced into the polypeptide. Since the human leukocyte antigen (HLA) structure is diverse and has various Th epitope structure priorities, the ability to introduce multiple types of Salmonella Th epitopes as part of the vaccine is attractive for future human applications. Multiple Th epitopes covering the major human HLA subtypes can be incorporated into Qβ for future applications.

[0182] Since it is difficult to generate a high anti-S. Enteritidis glycan IgG response in mice compared to rabbits, if the level of antibodies induced in mice is not sufficient for protection, the mQβ@(Th)n-glycan conjugate in rabbits will be evaluated. Serum will be recovered from immunized rabbits. Mice will be administered serial dilutions of post-immunization serum from immunized rabbits and then 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.

[0183] Generation of glycan-based vaccines against S. Typhimurium and S. Paratyphi A. Using the experience obtained in S. Enteritidis vaccine research, the development of glycan-based vaccines against S. Typhimurium and S. Paratyphi A will be carried out using the best 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 the S. Enteritidis glycan.

[0184] Mice will be immunized with these constructs using three injections every two weeks. The control group of mice will receive mQβ@(Th)n mixed with a non-covalently bound glycan. Blood will be collected before immunization and on days 7, 21, 35, 56, and 77 after the first inoculation. COPS will be purified from S. Typhimurium and S. Paratyphi A to determine the anti-glycan antibody titers by ELISA. The anti-COPS antibody titers and subtypes (IgG vs IgM, IgG subclasses) will be determined and compared at various time points to establish the kinetics of the humoral response and the need for booster injections.

[0185] The mQβ@(Th)n-glycan conjugate will likely result in superior anti-COPS IgG titers compared to the control group that received mQβ mixed with glycans. Since the priorities of epitope length and acetylation may differ from those of the S. Enteritidis vaccine, the optimal length of the glycan epitope will be established by comparing the binding to COPS derived from S. Typhimurium and S. Paratyphi A, the binding to bacteria, and the promotion of opsonization and complement-mediated cytotoxicity by IgG antibodies induced by vaccines containing tetrasaccharide 2 or 19 with those containing the corresponding octasaccharide and dodecasaccharides 38b,c and 39b,c. The role of acetylation will be determined by comparing 2 with 2a and 19 with 20.

[0186] Based on the results from the evaluation of immunogenicity and in vitro analysis of COPS and bacterial binding / opsonization, two of the best constructs against S. Typhimurium will be selected. Mouse groups will be immunized with the constructs together with the adjuvants identified herein (3 doses every 2 weeks). The control group will receive a mixture of mQβ@(Th)n and glycans. Then, the immunized mice will be challenged with LD100 of S. Typhimurium D65 (a clinical strain isolated from a human patient, 1×10 5 CFU). The survival rate will be compared with that of a control group that received only PBS injection and those immunized with a mixture of mQβ@(Th)n and glycans with the same adjuvant. The construct that results in the best protection will be identified.

[0187] In the case of S. Paratyphi A, currently, there is no good animal model for evaluating vaccine-mediated protection. Therefore, in vitro bacterial binding, complement-mediated cytotoxicity, and opsonization promoted by post-immunization sera will be used as an alternative means of in vivo protective effect.

[0188] A novel mQβ(Th)n carrier with reduced anti-carrier response is disclosed herein, which can activate Salmonella-specific Th cells upon conjugation with optimized Salmonella glycan and induce strong strain-specific protective immunity against lethal infection by S. Typhimurum and S. Enteritidis.

[0189] Development of a broad-spectrum anti-Salmonella vaccine In addition to the development of vaccines targeting specific strains, it would be attractive if a single vaccine construct could protect against multiple common Salmonella strains. Since the COPS from S. Paratyphi A (serogroup A), S. Typhimurum (serogroup B), and S. Enteritidis (serogroup D) share a common trisaccharide backbone (Man-Rha-Gal), if a strong functional antibody response can be induced against this structure, a single vaccine might be effective against all three serogroups (serogroups A, B, D).

[0190] Evaluation of Qβ conjugates with Salmonella skeletal oligosaccharides. The common backbone trisaccharide, hexasaccharide, and nonasaccharide 25 - 27 were conjugated to wild-type Qβ using a bifunctional linker 41. The average loading levels were 246, 215, and 208 for 25 - 27, respectively, based on LC-MS analysis. Mice (5 per group) and rabbits (2 per group) were immunized with these conjugates using three bi-weekly injections (4 μg of glycan per injection). One week after the last injection, antisera were collected and the IgG antibody titers against the immunizing antigens were analyzed. Consistent with the strain-specific glycan conjugates with Qβ, both mice and rabbits generated strong IgG responses (the average IgG titer in mice was 1,500,000, while for rabbits it was 20,000,000).

[0191] To analyze whether the generated antibodies could potentially recognize multiple strains of Salmonella, 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 share the same COPS backbone, S. Newport has a different COPS structure. Binding of the antiserum to these four types of COPS was tested by ELISA (Figure 14). Significantly higher levels of IgG antibodies were observed in the antiserum recognizing COPS from S. Enteritidis, S. Paratyphi A, and S. Typhimurium, while it was much (approximately 500-fold) lower for S. Newport. These results highlight the broad reactivity and selectivity of the antibodies produced against the backbone structure.

[0192] Based on these results, a hypothesis was established that conjugation of the backbone oligosaccharides with the novel mQβ(Th)n carrier identified above would further enhance the strength of the anti-Salmonella response. Trisaccharide, hexasaccharide, and nonasaccharide 25-27 would be conjugated with the mQβ(Th)n carrier.

[0193] Both mice and rabbits would be immunized with the novel constructs and adjuvant (three injections every two weeks). The control group would receive mQβ(Th)n mixed with a non-covalently bound glycan and adjuvant.

[0194] The immune sera will be recovered from the immunized animals and the ability of the induced IgG antibodies to bind to COPS will be compared to that from a control group of mQβ(Th)n mixed with the corresponding conjugate with WTQβ and a glycan without a covalent bond. In preliminary studies, the conjugate of the trisaccharide 25 was already able to generate a strong IgG response targeting COPS, while the hexasaccharide antigen 26 was found to induce the highest titer of IgG antibodies against COPS binding (Figure 14). Using the new carrier, the priority of the antigen length will be established in a similar manner. The antigen that generates the strongest response will be further evaluated in the challenge study.

[0195] Clinical strains of S.Enteritidis, S.Typhimurum, and S.Paratyphi A will be cultured as controls in addition to S.Newport. The binding of these bacteria by the immune sera will be measured by flow cytometry. The results of COPS binding suggest that the antiserum strongly recognizes the S.Enteritidis strain, S.Typhimurum strain, and S.Paratyphi A strain, while binding very little to S.Newport. The functional assay of the antiserum against the bacteria will be analyzed by an opsonin assay (Figure 10B) and a complement-mediated cytotoxicity assay in the presence of macrophages. The strong binding of the antiserum to all three strains (S.Enteritidis, S.Typhimurum, and S.Paratyphi A) will significantly enhance their uptake and killing in vitro, while the effect on the S.Newport strain should not be significantly different compared to the control serum.

[0196] Since the ability of the antiserum against S. Enteritidis strain, S. Typhimurium strain, and S. Paratyphi A strain has been established, in vivo protection studies will be conducted. A group of mice will be immunized with the mQβ(Th)n-glycan construct and an adjuvant, and the control group will receive a mixture of mQβ and glycan. The immunized mice will be divided into two groups and challenged with a lethal dose of S. Typhimurium D65 or S. Enteritidis R11, respectively.

[0197] Statistical analysis and ensuring robust and unbiased results. It will be possible to test the multiplicative effect of the novel vaccine construct by logarithmic transformation of the antibody titer and the mean fluorescence intensity of the cells due to antibody binding. At a given dose level, the familywise error rate of the first kind for multiple testing will be controlled while maximizing the power by a step-down Dunnett's test for pairwise treatment after the ANOVA model. The log-rank test will be used to compare the survival between groups. If necessary, more advanced analysis will be conducted in consultation with the Center for Statistical Training and Consulting at MSU.

[0198] To evaluate the immune response, each sample will be tested on three independent occasions (biological replicates) to ensure reproducible results. The treatment regimen will be blinded to the researchers performing the measurements.

[0199] Gender as a biological variable. The IgG responses induced by the Qβ constructs from male and female mice were compared. Comparable levels of anti-glycan IgG were obtained from both genders. Several other constructs will be tested similarly. If no significant difference is observed, both genders will be used in the cohort.

[0200] Currently, there are no vaccines available 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 manipulation will fill this gap. Qβ conjugates with Salmonella-related glycans will be evaluated for their ability to induce strong and persistent anti-Salmonella immunity and protect immunized subjects from lethal Salmonella challenge; an understanding of the various factors that influence the anti-Salmonella glycan immune response will thereby lay the foundation for providing exciting new directions in Salmonella prevention.

[0201] Common abbreviations used: 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-related glycan); TACA (tumor-associated carbohydrate antigen); Th (helper T cell); TLR (Toll-like receptor); VLP (virus-like particle); WT (wild type)

[0202] Incorporation by reference All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference were specifically and individually indicated to be incorporated by reference. Although numerous patent applications, patents, and other references are mentioned herein, it should be understood that such references do not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0203] Equivalents For the sake of clarity of understanding, the foregoing subject matter has been described in some detail by way of illustration and example, but it will be understood by those skilled in the art that certain changes and modifications may be made within the scope of the appended claims.

Claims

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

2. The vaccine composition according to claim 1, wherein the capsid contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations.

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

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

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

6. The vaccine composition according to claim 5, wherein the Salmonella antigen contains a trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, or dodecasaccharide.

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

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

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

10. The vaccine composition according to claim 9, wherein the bacteriophage is selected from the group consisting of (a): 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.

11. The vaccine composition according to claim 10, wherein the bacteriophage is bacteriophage Qβ.

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

13. The vaccine composition according to claim 12, wherein the mutation contains A38K.

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

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

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

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

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

21. The method according to claim 16, wherein the vaccine is directed against multiple strains of Salmonella selected from S. Enteritidis, S. Paratyphi A, S. Typhimurium, and S. Newport.

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

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