WO2024025936A2 - Développement de vaccins anti-salmonella à base de glucides - Google Patents

Développement de vaccins anti-salmonella à base de glucides Download PDF

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WO2024025936A2
WO2024025936A2 PCT/US2023/028672 US2023028672W WO2024025936A2 WO 2024025936 A2 WO2024025936 A2 WO 2024025936A2 US 2023028672 W US2023028672 W US 2023028672W WO 2024025936 A2 WO2024025936 A2 WO 2024025936A2
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Prior art keywords
bacteriophage
salmonella
glycan
vaccine composition
capsid
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PCT/US2023/028672
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English (en)
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WO2024025936A3 (fr
Inventor
Xuefei Huang
Xingling PAN
Chang-xin HUO
Scott Michael BALIBAN
Sharon Mei TENNANT
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Board Of Trustees Of Michigan State University
The University Of Maryland, Baltimore
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Publication of WO2024025936A2 publication Critical patent/WO2024025936A2/fr
Publication of WO2024025936A3 publication Critical patent/WO2024025936A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the first obstacle lies in the access to microbial associated glycans as carbohydrates from microbial species often have distinctive structures compared to the mammalian counterparts, and can present unique reactivities in glycosylation reactions.
  • the synthesis of these compounds in conjugatable forms needs to be established.
  • carbohydrates alone typically have low immunogenecities, which do not elicit high level and long-lasting antibody responses. Therefore, methods are needed to powerfully boost the immune responses to carbohydrate antigens.
  • a carrier system is essential to deliver carbohydrate antigens to the immune system and elicit strong anti-carbohydrate antigens antibody responses.
  • a potential drawback of protein carrier is that high anti-carrier antibody responses can be induced by the glyco-conjugate.
  • GD3-KLH generated an-antiGD3 IgG titer of 300, while that for KLH were 1,800,000.
  • the high anti-carrier antibodies can significantly suppress the generation of anti-glycan antibodies. This phenomenon has been reported for carbohydrate based anti-microbial disease vaccines. It has been suggested that an ideal carrier should induce high levels of anti-glycan antibodies without strong anti-self antibodies. Accordingly, there is a great need in the art to identify potential therapeutic strategies and compositions that activate immune responses in the treatment and prevention of disease and infection. Salmonella infections (salmonellosis) are a major public health problem throughout the world. The development of an effective vaccine is a highly attractive strategy to combat salmonellosis, especially with the rise of multidrug resistant Salmonella strains.
  • Salmonella bacteria bear characteristic glycan structures on the cell surface, which define the specific serovars and at the same time can serve as antigenic targets.
  • strategies and vaccines for targeting the carbohydrates from Salmonella surface which can be effective against multiple pathogenic strains of Salmonella.
  • Summary of the Invention Provided herein are vaccine compositions comprising an antigen conjugated to a capsid.
  • the vaccine composition comprises an antigen conjugated to a wild type capsid.
  • the vaccine composition comprises an antigen conjugated to a wild type bacteriophage Q ⁇ capsid.
  • the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or native sequence.
  • the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or natural 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 Q ⁇ capsid having at least one mutation from the wild type bacteriophage Q ⁇ 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.
  • the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mutations.
  • the at least one non-natural mutation is a disulfide bond mutation.
  • the antigen conjugated to the capsid is a Salmonella antigen.
  • Salmonella antigens may derived from a surface glycan of Salmonella.
  • the antigen may be a polysaccharide, such as a tri-, tetra-, penta-, hexa-, hepta-, octa-, non, or dodeca-saccharide.
  • the Salmonella antigen comprises tri-, hexa-, or nona- saccharides of Salmonella O-polysaccharide backbone.
  • the Salmonella antigen comprises the trisaccharide of Man-Rha-Gal.
  • the vaccine compositions disclosed herein may be used in preventing or treating a Salmonella infection (salmonellosis) gastroenteritis, typhoid fever, and/or paratyphoid fever in a subject in need thereof.
  • Said vaccines may birected to (e.g., provide protection against or treatment for) multiple strains of Salmonella, such as S. Enteritidis, S. Paratyphi A, S. Typhimurum, and S. Newport.
  • Salmonella infections are a major public health problem throughout the world. There are multiple pathogenic strains of Salmonella bacteria. Salmonella Typhi (S.
  • Typhi and Salmonella Paratyphi A, B and C serovars cause enteric fever, while nontyphoidal Salmonella (NTS) serovars generally cause gastroenteritis that can progress to invasive diseases.
  • NTS nontyphoidal Salmonella
  • typhoid and paratyphoid due to S. Paratyphi A are endemic.
  • typhoid and paratyphoid are rare, there are > 1.2 million NTS cases annually, resulting in 10,000-20,000 hospitalizations and hundreds of deaths, for which Salmonella Typhimurium (S. Typhimurium) and Salmonella Enteritidis (S. Enteritidis) are most common.
  • Salmonella bacteria bear characteristic glycan structures on the cell surface, which define the specific serovars and at the same time can serve as antigenic targets.
  • S. Typhi expresses a Vi capsule polysaccharide that covers the bacterial surface and protects the bacterium from the host immune system.
  • S. Paratyphi A and most NTS do not express such a capsule, and hence the surface polysaccharide in these serovars is the O polysaccharide of lipopolysaccharide (COPS).
  • COPS lipopolysaccharide
  • Carbohydrate based vaccine constructs have been shown to be effective in protecting the host, an example of which is the ViCPS vaccine targeting the Vi polysaccharide on S. Typhi.
  • ViCPS offers protection with between 50% to 80% efficacy in the first year, protection wanes after two years.
  • enteric fever caused by Salmonella Paratyphi A and S. Typhi, as well as invasive nontyphoidal Salmonella (iNTS) disease, primarily caused by S. Enteritidis and S. Typhimurium, are responsible for a high burden of disease globally.
  • Multidrug resistance is increasing in prevalence, 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 prevention strategy, as exemplified by S. Typhi capsular polysaccharide vaccines.
  • Salmonella serovars Paratyphi A, Typhimurium, and Enteritidis are coated with a dense layer of O-polysaccharide that contains a repeating ⁇ -D-mannose (Man)-1,4- ⁇ -L-rhamnose (Rha)-1,3- ⁇ -D-galactose (Gal)-1,2- motif.
  • Enteritidis glycan Q ⁇ -conjugate induced very high titers of IgG antibodies (IgG titers over 80,000,000 ELISA units in rabbits) against the glycan.
  • the induced antibodies recognized native O-polysaccharide both as an isolated antigen as well as when present on bacterial cells.
  • transfer of anti-sera from immunized rabbits provided 100% protection to mice against the challenge of a lethal dose of S. Enteritidis.
  • novel Q ⁇ carriers were designed to enable the activation of Salmonella-specific helper T cells and the generation of higher levels of anti- glycan IgG antibodies.
  • a Man-Rha-Gal oligosaccharide was synthesized and this glycan was conjugated to a bacteriophage Q ⁇ carrier to enhance anti-polysaccharide immunity.
  • S. Enteritidis R11 1.3 x 10 6 CFU
  • vaccine efficacy 94.4%, P ⁇ 0.0001 vs. baseline.
  • a single repeating unit of the common Man-Rha-Gal motif is sufficient to induce protective antibody responses.
  • Aspects of the invention provided herein include a vaccine composition comprising a Salmonella antigen conjugated to a capsid.
  • said capsid comprises at least one non-natural mutation.
  • the capsid comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mutations.
  • at least one non-natural mutation is a disulfide bond mutation.
  • the Salmonella antigen is derived from a surface glycan of Salmonella.
  • the Salmonella antigen may be a polysaccharide.
  • the Salmonella antigen comprises tri-, tetra-, penta-, hexa-, hepta-, octa-, non, or dodeca-saccharides.
  • the Salmonella antigen comprises tri-, hexa-, or nona-saccharides of Salmonella O-polysaccharide backbone. Most preferably the Salmonella antigen comprises at least one trisaccharide of Man-Rha-Gal.
  • the Salmonella antigen may comprise repeats of the Man-Rha-Gal trisaccharide, such as two, three, or more Man-Rha-Gal trisaccharides.
  • the capsid is a bacteriophage capsid.
  • Said bacteriophage may be selected from the group consisting of (a): bacteriophage Q ⁇ ; (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.
  • the bacteriophage is bacteriophage Q ⁇ .
  • the mutation comprises at least one mutation selected from N10K, A38K, A40C, A40S, T75K, D102C, D102S, or A117K, or combination thereof. In some preferred embodiments the mutation comprises A38K. In other embodiments, said capsid comprises at least two mutations selected from A40C/D102C, A40S/D102S, or A43C/Q98C. In further embodiments, said capsid comprises at least three mutations selected from A40C/D102C/K13R or A38K/A40C/D102C.
  • Embodiments of the invention include methods of preventing or treating a Salmonella infection (salmonellosis) in a subject, the method comprising administering to the subject a vaccine composition disclosed herein. Also provided are methods of preventing or treating gastroenteritis, typhoid fever, and/or paratyphoid fever, the method comprising administering to the subject a vaccine composition provided herein. In some such embodiments, the gastroenteritis is chronic or acute.
  • the contemplated vaccines compositions may be administered systematically. Such systematic administration may be selected from the group consisting of oral, intravenous, intradermal, intraperitoneal, subcutaneous, and intramuscular administration. In some embodiments, the vaccines contemplated herein are directed against multiple strains of Salmonella, selected from S.
  • the Salmonella antigen is derived from a O- polysaccharide backbone present in one or more of S. Enteritidis, S. Paratyphi A, S. Typhimurum, or S. Newport.
  • Figure 1 Depicts the cell surface core O-polysaccharides (COPS) of S. Paratyphi, S. Typhimurium, and S.
  • COPS cell surface core O-polysaccharides
  • FIG. 1 illustrates the common backbone structure with repeating trisaccharides of ⁇ -D-mannose (Man)-1,4- ⁇ -L-rhamnose (Rha)-1,3- ⁇ -D-galactose (Gal)-1,2.
  • Figure 2 illustrates the synthesis schemes for (A) S. Enteriditis tetrasaccharide, (B) S. Paratyphi A tetrasaccharide, and (C) the structures of penta-saccharide bearing a glucoside on the reducing end Gal, and tetra-saccharide without the paratose unit.
  • Figure 3 illustrates the synthesis scheme for S. Typhimurium tetrasaccharides.
  • Figure 4 illustrates the synthesis scheme for common tri-, hexa- and nona- saccharides of O-polysaccharide backbone.
  • Figure 5 illustrates the synthesis scheme for strain-specific Salmonella glycans via conversion of the common tri-, hexa- and nona-saccharides of O-polysaccharide backbones.
  • Figure 6 depicts the conjugation of Salmonella associated glycans (SAGs) to bacteriophage carrier.
  • Figure 7 depicts high and long-lasting anti-glycan 1 antibody responses induced by Q ⁇ -glycan 1 in mice.
  • Figure 8 shows ELISA data for IgG antibodies induced by Q ⁇ -glycan 1 against related glycans 2, 17, and 18.
  • Figure 9 depicts ELISA analysis of various sera against COPS, showing that anti- sera from rabbits immunized with Q ⁇ -glycan 1 exhibited strong binding to COPS (all sera were diluted 25,000 times.
  • X-axis Pre-rabbit (pre-immunized sera from rabbits); Rabbit (day 56 sera from rabbits immunized with Q ⁇ -glycan 1); Mouse (day 56 sera from selected two mice immunized with Q ⁇ -glycan 1); mAb (mAb 6347 was against S. Paratyphi A glycan; mAb 6391 and 6393 were against the common core of COPS. Each mAb was used at 1:20 dilution.
  • Figure 10 shows strong binding of S.
  • FIG 10A depicts flow cytometry analysis showing that IgG antibodies in post-immune sera (1:10,000 dilution) from rabbits immunized with Q ⁇ -glycan 1 exhibited significant recognition of S. Enteritidis clinical strain R11.
  • Figure 10B shows that post-immune sera from rabbits immunized with Q ⁇ -glycan 1 enhanced uptake of S. Enteritidis bacteria by macrophage J774 cells. Post- immune sera from Q ⁇ immunized or pre-immunized rabbits did not have much impact on macrophage uptake.
  • Figure 12 illustrates reduced anti-carrier antibody titers and significantly improved anti-glycan IgG responses when using rationally designed Q ⁇ mutants.
  • Figure 12A depicts the calculated relative solvent exposure of amino acid residues of Q ⁇ (1QBE) based on its crystal structure.
  • N-terminal residues K2, K13, and K16 are the sites for glycan conjugation.
  • Residues A38, T75, Q99, and P119 are highly solvent exposed and serve as potential sites for mutation.
  • Figure 12B shows mutant Q ⁇ /A38K/A40C/D102C conjugated to a representative carbohydrate antigen Tn elicited stronger anti-Tn IgG responses than wildtype (WT) Q ⁇ -Tn.
  • Figure 12C shows Q ⁇ /A38K/A40C/D102C conjugated to Tn elicited much less antibody against against WT Q ⁇ or Q ⁇ /A38K/A40C/D102C than WT Q ⁇ -Tn (at 409,600 fold serum dilution). The p values were determined through a two-tailed t test.
  • Figure 13 depicts encapsidation scheme.
  • Figure 13A illustrates the RNA-directed encapsidation technique used to package protein inside Q ⁇ .
  • Plasmid 1 contains the sequence for anti-Rev aptamer (aR), coat protein (CP), and hairpin (hp) for binding coat protein.
  • Plasmid 2 contains sequence fo Rev tag and the target protein (TP).
  • Transformation and transcription of the plasmids into E. coli result in the expression of Rev-tagged TP cargo, and bifunctional mRNA.
  • the anti-Rev aptamer in the mRNA binds to the Rev tag and Q ⁇ genome packaging hp binds to the interior of the CP monomers, thus encapsidating the target protein to Q ⁇ interior with the coat protein RNA sequence acting as the linker.
  • Figure 13B shows Q ⁇ @(RFP) was successfully produced through the RNA encapsidation technique leading to more than 100 fold increase in RFP fluorescence of the capsid.
  • Figure 14 depicts anti-COPS IgG ELISA titers in sera from rabbits immunized with the conjugates of WT Q ⁇ with backbone glycans 25, 26, 27 respectively, against COPS from S. Paratyphi A, S. Enteritidis, S. Typhimurum, and S. Newport. Significantly higher levels of IgG antibodies were found against COPS from S. Paratyphi A, S. Enteritidis, and S. Typhimurum, while those against the unrelated S. Newport were low.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • bacteriophage refers to viruses that infect and replicate within bacterium.
  • the bacteriophage is selected from, but not limited to, the group consisting of bacteriophage Q ⁇ ; (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.
  • bacteriophage Q ⁇ is one of many small RNA bacteriophages infecting Escherichia coli.
  • carbohydrate antigen refers to classes of antigens that elicit elicit strong antibody responses.
  • carbohydrate antigens are selected from, but not limited to, Mucins (“MUC1”), Ganglioside GD2 (Ahmed M. et al.
  • inhibitor means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease, disorder, or condition, the activity of a biological pathway, or a biological activity, such as the growth of a solid malignancy, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to an untreated control subject, cell, biological pathway, or biological activity or compared to the target, such as a growth of a solid malignancy, in a subject before the subject is treated.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as 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 hydrox
  • “Pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • a “subject” can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like.
  • primates e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like
  • an animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a “subject” can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition.
  • Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).
  • the term “subject in need thereof” means a subject identified as in need of a therapy or treatment.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • therapeutic agent or “pharmaceutical agent” refers to an agent capable of having a desired biological effect on a host.
  • therapeutic effect refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • terapéuticaally-effective amount and “effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • treating a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a vaccine composition as described herein, such that at least one symptom of the disease is decreased, prevented from worsening, or delayed from worsening.
  • a pharmaceutical treatment e.g., the administration of a vaccine composition as described herein, such that at least one symptom of the disease is decreased, prevented from worsening, or delayed from worsening.
  • 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 Q ⁇ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or native sequence. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or natural 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.
  • the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having at least one mutation from the wild type bacteriophage Q ⁇ capsid.
  • the at least one mutation comprises a non-natural mutation.
  • the non-natural mutation comprises a non-natural amino acid mutation.
  • the vaccine compositions provided herein comprises an antigen (e.g., carbohydrate antigen, polypeptides, peptides, proteins and small molecules) conjugated to a capsid (e.g., bacteriophage Qb), wherein said capsid comprises at least one mutation (e.g., at least one point mutation, or at least one non-natural disulfide bond).
  • the capsid are fragments or a portion of the capsid amino acid sequence of sufficient length, that when conjugated to the antigen, can elicit an enhanced and strong immune response.
  • the capsid polypeptide also includes amino acids that do not correspond to the 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).
  • the capsid is derived from bacteriophage.
  • the bacteriophage is selected from the group consisting of bacteriophage Q ⁇ ; (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.
  • the capsid has a sequence set forth in coat protein Table A. Table A Listing of capsid and associated GenBank numbers
  • the bacteriophage Q ⁇ capsid comprises a sequence set forth in SEQ ID NO: 1.
  • the bacteriophage Q ⁇ capsid comprises at least one mutation set forth in Table B. Table B. Q ⁇ mutants reported that assemble to form the capsid.
  • the capsid comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty mutations.
  • the mutation is a non- natural disulfide bond mutation.
  • the capsid comprises, but not limited to, the following mutations (Table C). Table C: Listing of bacteriophage Q ⁇ capsid In certain embodiments, bacteriophage Qb mutants may possess the following physical characteristics. Table D: Physical characteristics of Q ⁇ mutants.
  • the bacteriophage Qb capsid comprises a polypeptide comprising amino acid sequences at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NOs: 1-15.
  • the Qb mutant has at least one mutation set forth in Table C.
  • the bacteriophage Q ⁇ capsid comprises a wild type or native sequence.
  • the bacteriophage Q ⁇ capsid comprises a sequence consisting essentially of the sequence set forth in SEQ ID NO: 1.
  • the Qb mutant has an amino acid sequence that consists essentially of the mutations set forth in SEQ ID NOs: 2-15.
  • the capsid comprises a polypeptide comprising amino acid sequences at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in the GenBank numbers set forth in Table A.
  • the vaccine composition comprises a capsid having an amino acid sequence that consists 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 biologically active variant thereof, or combinations thereof, or consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence for a capsid (e.g., SEQ ID NOs: 1-15, or any of the GenBank numbers set forth in Table A).
  • a capsid e.g., SEQ ID NOs: 1-15, or any of the GenBank numbers set forth in Table A.
  • the bacteriophage Q ⁇ capsid comprises a wild type or native sequence. In some embodiments, the bacteriophage Q ⁇ capsid comprises a sequence consisting essentially of the sequence set forth in SEQ ID NO: 1. As is well-known to those skilled in the art, polypeptides having substantial sequence similarities can cause identical or very similar immune reaction in a host animal. Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of the Qb capsid, or fragment thereof, can also suitable for the methods, compositions and kits provided herein. In some embodiments, the altered polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still elicits an enhanced immune response, and are considered functional equivalents.
  • the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein.
  • the derivative, equivalents, variants, or mutants of the Qb are at least 85% homologous to a sequence set forth in SEQ ID NOs: 1- 15, or biologically active variant thereof, or combinations thereof.
  • 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%.
  • the bacteriophage Q ⁇ capsid comprises a wild type or native sequence. In some embodiments, the bacteriophage Q ⁇ capsid comprises a sequence consisting essentially of the sequence set forth in SEQ ID NO: 1.
  • C. Vaccine compositions and pharmaceutical compositions/formulations of same Provided herein are vaccine compositions comprising an antigen conjugated to a capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild type capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a wild type bacteriophage Q ⁇ capsid. In some embodiments, the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or native sequence.
  • the vaccine composition comprises an antigen conjugated to a bacteriophage Q ⁇ capsid having a wild type or natural 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 Q ⁇ capsid having at least one mutation from the wild type bacteriophage Q ⁇ 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.
  • the vaccine compositions provided herein comprises an antigen (e.g., carbohydrate antigen, polypeptides, peptides, proteins, and small molecules) conjugated to a capsid (e.g., bacteriophage Qb), wherein said capsid comprises at least one mutation (e.g., at least one point mutation, at least one non-natural amino acid mutation, or at least one non-natural disulfide bond mutation).
  • a capsid e.g., bacteriophage Qb
  • the antigens are fragments or a portion of the carbohydrate antigen of sufficient length, that when conjugated to the capsid, can elicit an enhanced and strong immune response.
  • the capsid may be conjugated to a plurality of multiple antigens that are the same or different antigen.
  • the antigen is a protein and peptide is selected from, but not limited to, TNFalpha, IL1 ⁇ , IL1 ⁇ , tau protein, PCSK9, or amyloid ⁇ .
  • the antigen is a small molecule selected from, but not limited to, nicotine, cocaine, or advanced glycation product.
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more vaccine compositions (e.g., one or more Qb wild type, or Q ⁇ mutant, antigen conjugate as described above), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the compositions can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other therapies. Conjunctive therapy thus includes sequential, simultaneous and separate, or co-administration of the composition, wherein the therapeutic effects of the first administered has not entirely disappeared when the subsequent compound is administered.
  • At least one vaccine compositions may be provided to the subject alone or in combination with at least one therapeutic drug, chemotherapeutic agents, scavenger compounds, antibiotics, anti-virals, anti-fungals, anti- inflammatories, vasoconstrictors and anticoagulants, antigens useful for vaccine applications or corresponding pro-drugs.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets
  • certain embodiments of the one or more vaccine compositions may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids.
  • a basic functional group such as amino or alkylamino
  • These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).
  • the pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • the one or more vaccine compositions may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • the one or more vaccine compositions may be formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • a formulation of one or more vaccine compositions can comprise other carriers to allow more stability, to allow more stability, different releasing properties in vivo, targeting to a specific site, or any other desired characteristic that will allow more effective delivery of the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above) to a subject or a target in a subject, such as, without limitation, liposomes, microspheres, nanospheres, nanoparticles, bubbles, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides.
  • other carriers to allow more stability, to allow more stability, different releasing properties in vivo, targeting to a specific site, or any other desired characteristic that will allow more effective delivery of the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above) to a subject or a target in a subject, such as,
  • an aforementioned formulation renders orally bioavailable a compound of the present invention.
  • Liquid dosage formulations of one or more vaccine compositions include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an active ingredient.
  • an inert base such as gelatin and glycerin, or sucrose and acacia
  • One or more vaccine compositions may also be administered as a bolus, electuary or paste.
  • solid dosage forms e.g., capsules, tablets, pills, dragees, powders, granules and the like
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. Compositions may also be formulated for rapid release, e.g., freeze-dried.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of one or more vaccine compositions include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • compositions suitable for parenteral administration can comprise sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • the above-described pharmaceutical compositions can be combined with other pharmacologically active compounds (“second active agents”) known in the art according to the methods and compositions provided herein.
  • Second active agents can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules).
  • second active agents independently or synergistically help to treat cancer.
  • the composition of the invention may comprise other biologically active substances, including therapeutic drugs or pro-drugs, for example, other chemotherapeutic agents, scavenger compounds, antibiotics, anti-virals, anti-fungals, anti- inflammatories, vasoconstrictors and anticoagulants, antigens useful for cancer vaccine applications or corresponding pro-drugs.
  • Exemplary scavenger compounds include, but are not limited to thiol-containing compounds such as glutathione, thiourea, and cysteine; alcohols such as mannitol, substituted phenols; quinones, substituted phenols, aryl amines and nitro compounds.
  • chemotherapeutic agents and/or other biologically active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like, which are biologically active.
  • Therapeutic Methods Provided herein are vaccine compositions for the treatment of and/or prevention of diseases and conditions for which an enhanced immune response may be beneficial.
  • Such diseases include, but not limited to, pathogenic infections (e.g., bacterial, viral, or fungal infections) and cancer.
  • the vaccine compositions described herein may be use as an immunotherapy.
  • the vaccine compositions e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above
  • the vaccine compositions are useful in the treatment of diseases including, but not limited to, persistent infectious disease, sexually transmitted diseases, gastro-intestinal diseases, pulmonary diseases, cardiovascular diseases, stress- and fatigue- related disorders, fungal diseases, pathogenic diseases, viral infections, or bacterial infections.
  • Viral infectious diseases including human papilloma virus (HPV), hepatitis A Virus (HAV), hepatitis B Virus (HBV), hepatitis C Virus (HCV), retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2), herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2, influenza virus, Hepatitis A and B, FIV, lentiviruses, pestiviruses, West Nile Virus, measles, smallpox, cowpox, ebola, coronavirus, retrovirus, herpesvirus, potato S virus, simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Moloney virus, ALV, Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Zika, or Rous Sarcoma Virus (RSV).
  • HPV human papillo
  • bacterial, fungal and other pathogenic diseases are included, 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 mosquito vectors, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibriocholerae.
  • Aspergillus Brugia, Candida, Chikungunya, Chlamydia, Coccidia, Cryptococcus, Dengue, Dirofilaria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium,
  • Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.
  • NIAID National Institute of Allergy and Infectious Diseases
  • Category A agents such as variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola hemorrhagic fever, Marburg hemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever) and related viruses);
  • Category B agents such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), alphaviruses (Venezuelan encephalomyelitis, eastern & western equine encephalomyelitis), ricin toxin from Ricinus communis (castor beans), epsilon
  • Bacterial pathogens include, but are not limited to, such as bacterial pathogenic gram-positive cocci, which include but are not limited to: pneumococci; staphylococci; and streptococci.
  • Pathogenic gram-negative cocci include: meningococci; and gonococci.
  • Pathogenic enteric gram-negative bacilli include: enterobacteriaceae; pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigellosis; hemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella); streptobacillus moniliformis and spirilum; listeria monocytogenes; erysipelothrix rhusiopathiae; diphtheria; cholera; anthrax; and donovanosis (granuloma inguinale).
  • Pathogenic anaerobic bacteria include; tetanus; botulism; other clostridia; tuberculosis; leprosy; and other mycobacteria.
  • Pathogenic spirochetal diseases include: syphilis; treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.
  • infections caused by higher pathogen bacteria and pathogenic fungi include: actinomycosis; nocardiosis; cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma and chromomycosis; and dermatophytosis.
  • Rickettsial infections include rickettsial and rickettsioses.
  • mycoplasma and chlamydial infections include: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis; and perinatal chlamydial infections.
  • Pathogenic protozoans and helminths and infections eukaryotes thereby include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; pneumocystis carinii; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm) infections.
  • a method of treatment comprises 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 Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above).
  • a subject in need thereof may include, for example, a subject who has been diagnosed with a tumor, including a pre-cancerous tumor, a cancer, or a subject who has been treated, including subjects that have been refractory to the previous treatment.
  • the term “effective amount,” as in “a therapeutically effective amount,” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response.
  • the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. More particularly, the term “effective amount” refers to an amount sufficient to produce the desired effect, e.g., to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
  • compositions described herein may be delivered by any suitable route of administration, including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, intracisternally, topically, as by powders, ointments or drops (including eyedrops), including buccally and sublingually, transdermally, through an inhalation spray, or other modes of delivery known in the art.
  • suitable route of administration including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous,
  • systemic administration means the administration of the vaccine compositions described herein such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the pharmaceutical compositions are delivered generally (e.g., via oral or parenteral administration).
  • the pharmaceutical compositions are delivered locally through direct injection into a tumor or direct injection into the tumor’s blood supply (e.g., arterial or venous blood supply).
  • the pharmaceutical compositions are delivered by both a general and a local administration.
  • a subject with a tumor may be treated through direct injection of a composition containing a composition described herein into the tumor or the tumor’s blood supply in combination with oral administration of a pharmaceutical composition of the present invention. If both local and general administration is used, local administration can occur before, concurrently with and/or after general administration.
  • the subject pharmaceutical compositions of the present invention will incorporate the substance or substances to be delivered in an amount sufficient to deliver to a patient a therapeutically effective amount of an incorporated therapeutic agent or other material as part of a prophylactic or therapeutic treatment.
  • the desired concentration of the active compound in the particle will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the compound. It is to be noted that dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Typically, dosing will be determined using techniques known to one skilled in the art.
  • Dosage may be based on the amount of the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above) per kg body weight of the patient.
  • the one or more vaccine compositions e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above
  • a range of amounts of compositions or compound encapsulated therein are contemplated, including about 0.001, 0.01, 0.1, 0.5, 1, 10, 15, 20, 25, 50, 75, 100, 150, 200 or 250 mg or more of such compositions per kg body weight of the patient.
  • Other amounts will be known to those of skill in the art and readily determined.
  • the dosage of the one or more vaccine compositions will generally be in the range of about 0.001 mg to about 250 mg per kg body weight, specifically in the range of about 50 mg to about 200 mg per kg, and 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.
  • the molar concentration of the one or more vaccine compositions in a pharmaceutical composition will be less than or equal to about 2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M, 1.5 M, 1.4 M, 1.3 M, 1.2 M, 1.1 M, 1 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M or 0.2 M.
  • the concentration of the one or more vaccine compositions will be less than or equal to about 0.10 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07 mg/ml, 0.06 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml or 0.02 mg/ml.
  • Actual dosage levels of the active ingredients in the compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular therapeutic agent in the formulation employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular therapeutic agent being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the precise time of administration and amount of any particular compound that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.
  • the guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing. While the subject is being treated, the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period.
  • All aspects of the treatment may be optimized according to the results of such monitoring.
  • the patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters, the first such reevaluation typically occurring at the end of four weeks from the onset of therapy, and subsequent reevaluations occurring every four to eight weeks during therapy and then every three months thereafter.
  • Therapy may continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. Adjustments, for example, to the amount(s) of agent administered and to the time of administration may be made based on these reevaluations.
  • Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound.
  • Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred.
  • the LD 50 (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above) described herein relative to the control.
  • the one or more vaccine compositions e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above
  • the ED 50 i.e., the concentration which achieves a half-maximal inhibition of symptoms
  • the concentration which achieves a half-maximal inhibition of symptoms can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, Q ⁇ mutant, antigen conjugate as described above) described herein relative to control.
  • the one or more vaccine compositions e.g., one or more Q ⁇ wild type, Q ⁇ mutant, antigen conjugate as described above
  • the IC50 i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells
  • the IC50 can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the one or more vaccine compositions (e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above)described herein relative to control.
  • the one or more vaccine compositions e.g., one or more Q ⁇ wild type, or Q ⁇ mutant, antigen conjugate as described above
  • the presently disclosed methods produce at least about a 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.
  • the administering of the one or more vaccine compositions can result in at least about a 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a solid malignancy in a subject, compared to the solid malignancy before administration of the vaccine compositions.
  • the therapeutically effective amount of one or more vaccine compositions is administered prophylactically to prevent a solid malignancy from forming in the subject.
  • the subject is human.
  • the subject is non-human, such as a mammal.
  • the data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans.
  • the dosage of any supplement, or alternatively of any components therein, lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information may be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • Example 1 Q ⁇ salmonella glycan conjugate as a potential anti-salmonella vaccine
  • Salmonella is a Gram-negative bacterium, which can cause serious infections (salmonellosis) through ingestion of contaminated food or water.
  • Salmonella encompasses > 2500 serovars distinguished by serotype based on the structure of the O polysaccharide on cell surface and by clinical syndrome.
  • Typhoid and paratyphoid serovars S. Typhi and S. Paratyphi
  • nontyphoidal Salmonella generally cause gastroenteritis for which invasive disease can occur as a consequence of immunological immaturity, suppression, or senescence.
  • S. Typhi and S. Paratyphi cause invasive enteric fever
  • nontyphoidal Salmonella generally cause gastroenteritis for which invasive disease can occur as a consequence of immunological immaturity, suppression, or senescence.
  • Typhi causes typhoid fever that kills over 200,000 people every year mainly in developing countries especially in Southeast Asia, Latin America and Africa.2 In Asia, paratyphoid due to S. Paratyphi A is also prevalent.
  • NTS nontyphoidal Salmonella
  • US nontyphoidal Salmonella
  • S. Enteritidis and S. Typhimurium are most common.
  • Vaccines targeting polysaccharides on cell surface of pathogenic bacteria have proven to be an effective strategy for protection from multiple bacterial pathogens, as exemplified by the ViCPS vaccine against S. Typhi.
  • ViCPS vaccine against S. Typhi For other Salmonella strains, while they lack Vi, there are characteristic surface polysaccharides.
  • the cell surface core O- polysaccharides (COPS) of S. Paratyphi, S. Typhimurium, and S. Enteritidis share a common backbone structure with repeating trisaccharides of ⁇ -D-mannose (Man)-1,4- ⁇ -L- rhamnose (Rha)-1,3- ⁇ -D-galactose (Gal)-1,2-.
  • COPS O- polysaccharides
  • a dideoxy hexose is linked to the 3-OH of the Man and the structure of the dideoxy sugar differentiates the various serovars.
  • S. Paratyphi A contains paratose, S. Typhimurium has abequose, while the dideoxy sugar is tyvelose on S. Enteritidis. (See Figure 1.)
  • the 6-OH of the backbone galactose can have an ⁇ -glucosyl unit attached.
  • some of the 2- and 3-OH groups of the rhamnose are acetylated, resulting in further heterogeneities of natural glycans.
  • glycan based anti-Salmonella vaccines are weakly immunogenic T cell independent antigens. When administered alone, they can only generate low titers of IgM antibodies, which typically have low affinity and only last for a short time. For an effective and durable vaccine, it is highly desirable that IgG antibodies can be elicited.
  • glycans can be attached to immunogenic protein carriers, which contain the necessary helper T (Th) cell epitopes to induce Th cell activation and antibody isotype switching from IgM to IgG.
  • Th helper T
  • B cell activation and differentiation requires two signals: an antigen-specific first signal delivered via binding and cross-linking of cell surface B cell receptors (BCRs) and a second co-stimulatory cytokine signal from cognate Th cells.
  • BCRs cell surface B cell receptors
  • Arrays of haptens spaced by 5 to 10 nm have been shown to cross-link BCRs well and antigens displayed in a highly organized manner can lead to earlier B cell amplification for potent IgM and IgG responses.
  • Antigen organization can also greatly influence B cell tolerance, with B cells unresponsive to poorly organized antigens while responding promptly to the same antigen presented in an orderly manner.
  • VLPs can be powerful platforms 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-assembled protein subunits giving them highly ordered structures. Repetitive B cell epitopes presented on the VLP surface can efficiently cross-link BCRs. Furthermore, VLPs can contain a large number of Th epitopes, which can synergize BCR cross-linking with stimulation by activated Th cells.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • TT tetanus toxoid
  • VLPs have been utilized as carriers of tumor associated carbohydrate antigens (TACAs). Similar to Salmonella associated glycans (SAGs), TACAs are T cell independent antigens. Out of the multiple types of VLPs investigated, bacteriophage Q ⁇ is the most powerful in terms of carrier function.
  • TACAs By displaying TACAs in an organized manner on Q ⁇ , enhancement of antibody responses was observed even against a very weak TACA, i.e., monomeric Tn antigen, with anti-Tn IgG titers exceeding 250,000. In comparison, Tn-KLH conjugates elicited no anti-Tn IgG antibodies. Importantly, immunization with Q ⁇ -Tn significantly protected mice from the development of an aggressive tumor, while Q ⁇ alone did not provide any survival benefits suggesting the non- specific immune response due to Q ⁇ itself was not sufficient.
  • MUC1 mucin-1
  • head-to-head comparison has been performed between Q ⁇ -MUC1 and MUC1 conjugate with KLH, a gold standard utilized in multiple clinical trials of carbohydrate based anti-cancer vaccines.
  • Q ⁇ was superior as Q ⁇ -MUC1 was able to induce three times more anti-MUC1 IgG antibodies, and was significantly more effective in reducing tumor load in mouse tumor models than the corresponding KLH-MUC1 construct.
  • Q ⁇ was superior as Q ⁇ -MUC1 was able to induce three times more anti-MUC1 IgG antibodies, and was significantly more effective in reducing tumor load in mouse tumor models than the corresponding KLH-MUC1 construct.
  • Q ⁇ was superior as Q ⁇ -MUC1 was able to induce three times more anti-MUC1 IgG antibodies, and was significantly more effective in reducing tumor load in mouse tumor models than the corresponding KLH-MUC1 construct.
  • High density antigen display is critical for high IgG antibody responses.
  • Q ⁇ - Tn constructs formed using the copper catalyzed azide-alkyne cycloaddition click reaction were much less effective in inducing tumor binding antibodies vs those formed with a flexible alkyl amide linker. This was attributed to potential antigen competition from the rigid triazole ring and steric exclusion/epitopic suppression effects due to anti-triazole antibodies formed.
  • Q ⁇ has not been systematically studied as a carrier for bacterium associated glycans. It was hypothesized that organized display of SAGs on Q ⁇ can lead to strong and long-lasting anti-SAG IgG responses as potential anti-Salmonella vaccines.
  • VLP Q ⁇ disclosed herein represent an exciting new carrier for anti-microbial vaccine development.
  • Q ⁇ -SAG conjugates can potentially generate high titers of anti-SAG IgG antibodies and immunological memory, two crucial factors in preventing Salmonella infections.
  • Novel Q ⁇ carriers are disclosed herein, which are associated with reduced antibody generation against the carrier itself while retaining the ability to activate bacterium specific Th cells for induction of even higher levels of anti-SAG IgG antibodies vs wild type Q ⁇ . These carriers can be a platform technology applicable to vaccine development for diseases beyond salmonellosis.
  • Enteriditis tetrasaccharide 1 corresponding to one repeat unit with an amino propyl linker at the reducing end.
  • Four monosaccharide building blocks 3-6 were designed, where benzyl (Bn) and acetonide groups were primarily used as protective groups as these electron-donating protective groups can potentially enhance building block reactivities in glycosylations.
  • the key rare tyvelose donor 3 was efficiently prepared from the readily available thiomannoside which was selectively protected and then underwent Barton-McCombie deoxygenation leading to 3 (Scheme 1a; Figure 2A). Building blocks 4 – 6 were prepared in a straightforward fashion.
  • Paratyphi A tetrasaccharide 16 in good overall yields following a similar chemoselective glycosylation approach for the preparation of 12. (See Figure 2B.) Subsequent deprotection of 16 generated S. Paratyphi A tetrasaccharide 2a (72 mg), which bears two OAcs in its rhamnoside unit for future investigation of the role of OAc in eliciting antibody responses. In addition, to better understand the roles of glycan structures in antibody recognition, penta-saccharide 17 bearing a glucoside on the reducing end Gal, and tetra-saccharide 18 without the paratose unit were also synthesized (90 mg each). (See Figure 2C.) S.
  • Typhimurium is another major strain of Salmonella.
  • S. Typhimurium tetrasaccharides 19 and 20 are synthesized. (See Figure 3.) Preparation of 19 and 20 will start from abequose donor 21 (Scheme 2), which in turn will be prepared by selective protection of galactoside 22 followed by Barton- McCombie deoxygenation. Chemoselective glycosylation of mannoside 4 by abequose 21 will lead to disaccharide 23. Analogous to synthesis of S. Enteriditis and S. Paratyphi A tetrasaccharides, the expected major anomer formed will be alpha, which will be confirmed by NMR.
  • the common backbone of Salmonella consists of repeating trisaccharide of Man- Rha-Gal.
  • the backbone oligosaccharides 25-27 were synthesized with one, two and three repeating trisaccharide units respectively (Scheme 3; Figure 4).
  • Monosaccharide building blocks 28 - 31 were established with protective group patterns for high alpha-selectivities in glycosylation.
  • Pre-activation based glycosylation54-55 of mannoside donor 28 (1.2 eq.) with the bifunctional rhamnoside acceptor 29 (1 eq.) produced disaccharide 32 in 82% yield.
  • Bacteriophage Q ⁇ has been shown to be a powerful carrier for carbohydrate based anti-cancer vaccines. It was tested whether bacteriophage Q ⁇ with SAGs can elicit powerful protective antibody responses as strain-specific anti-Salmonella vaccines. Conjugation of SAG with bacteriophage Q ⁇ carrier can be carried out efficiently. With the requisite strain-specific glycans synthesized, bio-conjugation to wild type (WT) Q ⁇ was performed. The S.
  • Enteritidis tetrasaccharide 1 was functionalized with adipic acid di-N-hydroxysuccinimide (NHS) ester 41 (Scheme 5; Figure 6), and then incubated with Q ⁇ (5 eq of glycan per amine moiety on Q ⁇ external surface; total 20 eq of glycan per Q ⁇ monomer). The resulting Q ⁇ -glycan 1 conjugate was purified. The excess of unconjugated glycan was recovered in near quantitative yield in its free carboxylic acid form, which could be converted back to the NHS ester and re-used for bioconjugation.
  • NHS adipic acid di-N-hydroxysuccinimide
  • conjugates were injected subcutaneously into groups of 5 mice (day 0) at doses of 1 and 4 ⁇ g of glycans respectively followed by two booster injections on days 14 and 28.
  • Control groups (5 mice each) received unconjugated Q ⁇ at the same protein level.
  • Enzyme linked immunosorbent assay (ELISA) analysis using BSA-glycan 1 as the coating antigen showed that high titers of IgG antibodies were induced by Q ⁇ -glycan 1.
  • the average IgG titers for the groups receiving 1 ⁇ g and 4 ⁇ g of glycan reached 487,000 and 980,000 ELISA units respectively on day 35 ( Figure 7A).
  • ELISA analysis against BSA-glycan 1 showed that robust anti-glycan 1 IgG responses were induced, with IgG titers reaching 83,000,000 and 150,000,000 ELISA units by day 56 (Table 1), which were more than 6,000-fold higher than those from Q ⁇ immunized control or pre-immunized rabbits. These results indicate that Q ⁇ -glycan 1 is effective in multiple species. Table 1. ELISA titers of anti-glycan 1 antibodies induced by Q ⁇ -glycan 1 in rabbits. Robust anti-glycan 1 IgG antibodies were elicited compared to control rabbits receiving Q ⁇ only. For successful vaccines, it is critical that antibodies generated by the vaccine construct are capable of recognizing the native polysaccharides.
  • COPS homologous serotype native core O-polysaccharide
  • Enteritidis R11 by J774 mouse macrophages was assessed after incubation of the bacteria with pre- immune sera, anti-sera from Q ⁇ or sera from Q ⁇ -glycan 1 immunized rabbits. While the pre-immune and Q ⁇ immunized rabbit sera did not cause significant bacterial uptake relative to media alone, antisera from Q ⁇ -glycan 1 immunized rabbits enhanced macrophage opsonisation of bacteria by 400% ( Figure 10B). Passive transfer of sera from immunized rabbits protected mice from lethal challenge by S. Enteritidis. The protective efficacy of anti-sera against Salmonella infection was evaluated in vivo.
  • LD100 lethal dose
  • mice in the PBS group succumbed to bacterial infection by day 7 and all but one out of the two groups of mice receiving pre-immune sera died from infection by day 8, excitingly, 100% of the mice receiving post-immune sera (1:100 or 1:500 dilution, 24 total) survived the bacterial challenge (Figure 11).
  • LD100 lethal dose
  • Enteritidis tetrasaccharide antigen 1 As the native S. Enteritidis COPS consist of a polymer of multiple repeats of the tetrasaccharide unit, it was hypothesized that longer glycans may mimic the conformational properties of native COPS better resulting in stronger antibody responses. Whether inclusion of octa- and dodeca-saccharides as antigens in the vaccine can result in superior antibodies in mice will be tested as the anti-Salmonella glycan antibody levels generated in mice were lower than those in rabbits. The octa- and dodeca-saccharides 39a and 38a will be conjugated with Q ⁇ through the bifunctional linker 41 in a similar manner as tetrasaccharide 1.
  • Acetylation will be investigated by conjugating the acetylated tetrasaccharide 1a with Q ⁇ .
  • the resulting constructs will be thoroughly purified to remove free glycans.
  • the glycan loading level will be quantified by LC-MS assay. If the loading levels are lower due to the larger sizes of the glycans, the bioconjugation reaction can be repeated to increase the loading to around 300 as in Q ⁇ -glycan 1.
  • the excess unconjugated glycans can be recovered and recycled. Mice will be immunized with the new conjugates and the IgG antibody levels in the post-immune sera will be determined by ELISA against the immunizing antigen, as well as against the native COPS from S. Enteritidis.
  • Adjuvants can significantly influence the immune responses. In preliminary studies, Freund’s adjuvant was utilized. However, as Complete Freund’s Adjuvant cannot be used in humans due to its toxicity, three adjuvants that are already used as part of human vaccines will be tested: unmethylated Cytosine-Guanine dinucleotides, Alum, and monophosphoryl lipid A (MPLA), which will be co-administered with Q ⁇ -glycan conjugates. These adjuvants interact with Nod-like receptors (NLR) and Toll-like receptors (TLRs), which are key activators of the innate immune system.
  • NLR Nod-like receptors
  • TLRs Toll-like receptors
  • memory B cells and long-lasting antibody secreting plasma B cells will be analyzed.
  • Cells will be isolated from lymph nodes and spleens of mice immunized with Q ⁇ only or Q ⁇ -glycan 1. These cells will be subjected to flow cytometry analysis upon incubation with fluorescently labeled BSA-glycan 1 conjugate.
  • Memory B cells populations will be quantified as cells that are SAG+IgG+B220+CD38+.
  • the memory B cells can be differentiated into plasma cells by stimulation with lipopolysaccharide (LPS) for six days, and transferred to BSA- glycan 1 coated enzyme-linked immunospot assay (ELISPOT) plates to quantify the number of glycan specific IgG secreting B cells.
  • LPS lipopolysaccharide
  • ELISPOT enzyme-linked immunospot assay
  • Long-lasting glycan specific plasma B cells will also be evaluated by ELISPOT with cells from bone marrow.
  • Q ⁇ -glycan 1 immunization is expected to lead to a larger number of glycan specific memory B and long- lasting plasma B cells compared to controls. Th cell activation is crucial for the generation of high affinity IgG antibodies and long term memory.
  • lymphocytes from spleen or lymph nodes will be isolated from immunized as well as na ⁇ ve mice, which will be followed by labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • Bone marrow dendritic cells will be generated by culturing bone marrow cells from the tibias and femurs of na ⁇ ve mice with DC-media containing granulocyte/macrophage colony-stimulating factor and interleukin-4. After stimulation with LPS, the mature BMDCs will be incubated with Q ⁇ -glycan 1 and then added to CFSE labeled spleen cells.
  • CD4+ T cells in the presence of Q ⁇ -glycan 1 treated BMDCs will cause reduction of intracellular CFSE concentrations.
  • Flow cytometry analysis of CFSE intensities of CD4+ cells will allow quantification of CD4+ T cells specific to the vaccine.
  • a much greater percentage of CD4+ T cells are expected to be Q ⁇ -glycan 1-specific in immunized mice compared to naive mice, which suggests CD4+ T cell activation induced by Q ⁇ -glycan 1 vaccination.
  • a potential drawback in using immunogenic protein carriers is that anti-carrier antibody levels may drastically increase under prime-boost vaccination protocols.
  • a KLH-GD3 construct (1 prime and 2 boost injections) led to an anti-GD3 IgG titer of 400 with a 200X higher anti-KLH IgG titer (819,200).
  • the difference was smaller at 12X: a mean IgG titer of 3,100,000 against Q ⁇ vs. a mean anti-Tn IgG titer of 263,000.36
  • the endogenous epitopes from the carrier may compete with the desired epitope for the limited number of Th cells for cytokine signals, critical for B cell activation and IgG production.
  • antibodies binding to the non-essential epitopes may sterically block the adjacent targeted epitope from being recognized by B cells or lead to immune complex removal.
  • Q ⁇ mutant strategy to reduce anti-Q ⁇ antibody responses and further enhance anti-glycan antibody production was investigated. Removing Q ⁇ B-cell epitopes lowered anti-Q ⁇ titers and increased antibody levels against a carbohydrate antigen. It was hypothesized that B cell epitopes likely reside in flexible and accessible loops on Q ⁇ exterior as they need to bind to bulky B cell receptors on the surface of B cells.
  • A38K/A40C/D102C was designed.
  • A38 was selected since it is on the exterior and solvent exposed ( Figure 12A).
  • the A40C and D102C mutations were introduced as these two residues are close in space in crystal structure, and can form a disulfide bond bridging two subunits to enhance capsid stability.
  • the A38K/A40C/D102C mutant was expressed in E. coli and self-assembled to form particles in good yields ( ⁇ 20 mg/L) and similar sizes as wild type (WT) Q ⁇ (28 nm diameter).
  • IgGs induced by a carbohydrate antigen Tn conjugated with Q ⁇ /A38K/A40C/D102C 400 Tn per capsid were evaluated. There were two notable findings: (1) the triple mutant elicited significantly higher anti-Tn IgG antibody levels (Fig. 12B); and (2) anti-carrier IgG titers against both WT Q ⁇ and Q ⁇ /A38K/A40C/D102C were much lower than those induced by WT Q ⁇ -Tn (Fig.12C). This suggests some inherent B- cell epitopes of Q ⁇ was successfully removed without creating new dominant B-cell epitopes.
  • the optimized glycan structure from above will be conjugated with Q ⁇ /A38K/A40C/D102C triple mutant, and mice and rabbits will be immunized with the new constructs with the optimized dose and adjuvant.
  • the IgG levels against COPS as well as those against the carrier will be determined by ELISA.
  • the conjugate with the triple mutant carrier will produce superior IgG antibody responses compared to the corresponding WT-Q ⁇ conjugate.
  • Q ⁇ may have multiple major B cell epitopes. Therefore, effective Q ⁇ mutations will be combined into one coat protein to test whether it can further reduce anti-Q ⁇ IgG titers. However, with too many mutations, the coat protein may not assemble into particles.
  • a heterologous prime-boost strategy using multiple mQ ⁇ s containing different major B cell epitopes may be developed. One mQ ⁇ -glycan conjugate will be used to prime the immune system and a second mQ ⁇ -glycan conjugate for booster injections. In this strategy, as B-cell epitopes from various mQ ⁇ are different, glycan antigen is the only component common to all injections.
  • anti-glycan immunity should benefit the most from booster injections.
  • This heterologous prime-boost strategy should result in stronger anti-glycan antibody production and lower unwanted responses against the carrier.
  • the ability to induce high levels of anti-glycan IgG antibodies by Q ⁇ -glycan conjugates suggests activation of Th cells leading to B-cell maturation and antibody isotype switch to IgG.
  • carbohydrates typically do not contain epitopes for Th cells, activated Th cells are most likely against the Q ⁇ carrier.
  • Th epitopes for Salmonella specific Th cells.
  • Th cell epitope sequences from Salmonella need to be introduced into Q ⁇ -glycan conjugates. While this can be accomplished by linking Th epitopic peptides to Q ⁇ in the same manner as the glycans through linker 41, introduction of Th epitopes to the external surface of Q ⁇ has several possible disadvantages: 1) it would reduce the number of sites available for glycan conjugation.
  • RNA directed encapsidation strategy is contemplated herein to package the Salmonella Th epitopes in the Q ⁇ interior. Due to steric hindrance, these Th epitopes in the interior will not be able to interact with B cells, thus minimizing induction of anti-Th antibodies.
  • RNA encapsulated inside Q ⁇ activates intracellular Toll like receptor-9 pathway in antigen presenting cells after ingestion of Q ⁇ .
  • the RNA directed encapsidation strategy is based on the understanding of the Q ⁇ self-assembly process.
  • the monomeric Q ⁇ coat protein (CP) can bind with single-stranded RNA genome with high affinity between a RNA hairpin (hp) structure and positively charged residues of the coat protein.
  • the RNA sequence serves as a template, and guides the assembly of the monomers into a 28 nm diameter nanoparticle.
  • RNA-directed encapsidation is carried out by first introducing two binding domains to the mRNA sequence for Q ⁇ coat protein on a plasmid (plasmid 1, Figure 13A).
  • An RNA aptamer (aR) capable of binding an arginine-rich peptide (Rev) is inserted just upstream of the ribosome binding site for the coat protein sequence.
  • the sequence of the Q ⁇ packaging hp is positioned immediately downstream of the stop codon.
  • the target protein (TP) sequence will be N-terminally tagged with the Rev peptide and inserted into plasmid 2. Transformation with both plasmids and expression in E.
  • Q ⁇ with the Rev- tagged target protein encapsidated by binding with the anti-Rev aptamer.
  • Q ⁇ is designated Q ⁇ @(protein)n with n indicating the average number of proteins inside a particle.
  • This system was successfully utilized to encapsulate red fluorescence protein (RFP) in the interior of Q ⁇ ( Figure 13B).
  • RFP red fluorescence protein
  • a plasmid bearing the sequence corresponding to an N-terminus Rev tagged peptide containing Th epitope AAQYVAAHPGEVCPA from Salmonella alkyl hydroperoxide reductase subunit C (AhpC154-168) is constructed.
  • the activation of AhpC154-168 specific Th cells will be determined using the CFSE assay outlined herein.
  • the protective efficacy of the vaccine construct with the new mQ ⁇ @(Th)n-glycan conjugate will be evaluated next. Mice will be fully immunized with the mQ ⁇ @(Th)n- glycan construct.
  • the comparison groups will be administered with WT Q ⁇ -glycan and mQ ⁇ -glycan under identical conditions to decipher the impacts of mQ ⁇ and mQ ⁇ @(Th)n.
  • All mice will be injected with LD100 (1 x 106 CFU) of S. Enteritidis R11.
  • mice surviving Salmonella challenge over time will be monitored to construct Kaplan-Meier survival curves as in Figure 11.
  • two groups of mice immunized with mQ ⁇ @(Th)n-glycan and mQ ⁇ -glycan respectively will be kept for one year, and then challenged with S. Enteritidis.
  • the recall responses from the mQ ⁇ @(Th)n-glycan immunized group will be assessed to determine relative protection to bacterial challenge.
  • a new polypeptide may be designed to contain multiple repeats of Th epitopes AhpC154-168.
  • Th epitopes such as EutC243–257, STM1540262–276 and FliC429–443 can be introduced into the polypeptide as well.
  • HLA human leukocyte antigen
  • Th epitopes covering the major human HLA subtypes can be incorporated into Q ⁇ for future translation. As it is difficult to generate high anti-S.
  • Enteritidis glycan IgG responses in mice compared to rabbits if the levels of antibody elicited in mice are not sufficient to be protective, mQ ⁇ @(Th)n-glycan conjugates in rabbits will be evaluated.
  • Sera will be collected from immunized rabbits. Mice will be administered with serial dilutions of post- immune sera from immunized rabbits and then infected with lethal doses of S. Enteritidis R11. The maximum dilution of sera giving 100% protection will be determined, which will indicate the strength of the protective responses.
  • the passive transfer model is a useful alternative to the active immunization strategy. Creating glycan based vaccines against S. Typhimurium and S. Paratyphi A.
  • COPS will be purified from S. Typhimurium and S. Paratyphi A.
  • 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 humoral responses and the necessity of booster injections.
  • the mQ ⁇ @(Th)n-glycan conjugates will lead to superior anti-COPS IgG titers compared to control groups receiving mQ ⁇ mixed with glycans.
  • the epitope length preference and acetylation preference may be different from those of the S. Enteritidis vaccine
  • the optimum length of the glycan epitope will be established by comparing binding to COPS from S. Typhimurium and S. Paratyphi A, binding to bacteria, and promotion of opsonization and complement mediated cytotoxicity by IgG antibodies induced by vaccines containing tetrasaccharide 2 or 19 vs the corresponding octa- and dodeca-saccharides 38b,c and 39b,c.
  • the role of acetylation will be determined by comparing 2 vs 2a and 19 vs 20.
  • mice will be immunized with the constructs with the adjuvant identified herein (3 biweekly doses).
  • the control groups will receive admixture of mQ ⁇ @(Th)n and the glycan.
  • the immunized mice will then be challenged with LD100 of S. Typhimurium D65 (a clinical strain isolated from a Malian patient, 1 x 10 5 CFU).
  • the survival rate will be compared with control group receiving only PBS injection as well as those immunized with admixture of mQ ⁇ @(Th)n and the glycan with the same adjuvant.
  • mice and rabbits were immunized with these conjugates with three biweekly injections (4 ⁇ g of glycan per injection).
  • anti-sera were collected and IgG antibody titers were analyzed against the immunizing antigen. Consistent with the strain specific glycan conjugates with Q ⁇ , both mice and rabbits produced strong IgG responses (average IgG titers in mice are 1,500,000, while those for rabbits are 20,000,000).
  • mice will be immunized with the mQ ⁇ (Th)n-glycan construct and the adjuvant with the control group receiving admixture of mQ ⁇ and glycan.
  • the immunized mice will be divided into two groups and be challenged with lethal doses of S. Typhimurium D65 or S. Enteritidis R11 respectively. Statistical analysis and ensuring robust and unbiased results.
  • BCR B cell receptor
  • BSA bovine serum albumin
  • CFSE carboxyfluorescein diacetate succinimidyl ester
  • COPS core O-polysaccharides
  • CP coat protein
  • ELISA enzyme linked immunosorbent assay
  • ELISPOT enzyme- linked immunospot
  • hp hairpin
  • KLH keyhole limpet hemocyanin
  • LD100 lethal dose
  • MPLA monophosphoryl lipid A
  • SAG Salmonella associated glycan
  • TACA tumorumor associated carbohydrate antigen
  • Th helper T cells
  • TLR Toll like receptor
  • VLP virus like particle
  • WT wildtype Incorporation by Reference All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une composition de vaccin comprenant un antigène de Salmonella conjugué à une capside, la capside comprenant une séquence native ou de type sauvage. L'invention concerne également une composition de vaccin comprenant un antigène de Salmonella conjugué à une capside, ladite capside comprenant au moins une mutation, telle qu'une mutation non naturelle. De telles compositions sont utiles dans le traitement et la prévention pour prévenir ou traiter une infection à Salmonella (salmonellose), la gastroentérite, la fièvre typhoïde et/ou la fièvre paratyphoïde; et peuvent être efficaces contre de multiples souches de Salmonella.
PCT/US2023/028672 2022-07-26 2023-07-26 Développement de vaccins anti-salmonella à base de glucides WO2024025936A2 (fr)

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US202263392274P 2022-07-26 2022-07-26
US63/392,274 2022-07-26

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WO2024025936A3 WO2024025936A3 (fr) 2024-03-28

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102596219B (zh) * 2009-09-03 2014-04-30 Cj第一制糖株式会社 噬菌体和包含所述噬菌体的抗菌组合物
US9011871B2 (en) * 2011-11-07 2015-04-21 University Of Maryland, Baltimore Broad spectrum vaccine against typhoidal and non-typhoidal Salmonella disease
CN111278457A (zh) * 2017-08-28 2020-06-12 密西根州立大学校董会 使用噬菌体及其突变体治疗癌症和感染的组合物和方法

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