A multivalent shigella vaccine and methods of making and using thereof

Abstract

The present invention, in embodiments, relates to a multivalent vaccine comprising a combination of two or more different monovalent Shigella oligosaccharide protein conjugates and an adjuvant. One of the vaccine components can be a Shigella sonnei oligosaccharide- protein conjugate and the other three can be Shigella flexneri oligosaccharide-protein conjugates, specifically, S. flexneri 2a, S. flexneri 3a and S. flexneri 6, respectively. All conjugates feature sOS components acting as surrogates of the heterogeneous Shigella surface polysaccharide antigens characteristic of each one of the selected Shigella serotypes. The multivalent vaccine of the present invention is made for the prevention and / or treatment of disease caused by Shigella, in particular S. sonnei and S. flexneri.

Description

A MULTIVALENT SHIGELLA VACCINE AND METHODS OF MAKING AND USING THEREOFFIELD OF THE INVENTION

[0001] The present invention relates to a multivalent Shigella vaccine composition comprising a combination of two or more, including, e.g., four, monovalent synthetic oligosaccharide(sOS)-based Shigella conjugates. In particular, the proposed multivalent Shigella vaccine composition includes sOS components acting as surrogates of the heterogeneous Shigella surface polysaccharide (PS) antigens characteristic of serotypes S.flexneri 2a (SF2a), S.flexneri 3a (SF3a), S.flexneri 6 (SF6), and S. sonnei (Sson), respectively.BACKGROUND OF THE INVENTION

[0002] Shigellosis is a major diarrheal disease. It is responsible for a large burden of disease worldwide and is a leading cause of diarrheal deaths due to bacterial infection. Shigella affects especially children under 5 years of age in low- and middle-income countries (LMICs). Besides direct mortality and morbidity, it is a direct cause of stunting in this population. Shigellosis is a general problem also causing disease among travelers, military and non-governmental organization staff deployed in endemic areas. In high-income countries, it occurs in the form of outbreaks with some communities, such as young children in daycare and people in locations with poor sanitation, being particularly affected. Of additional concern in terms of public health threat is the intercontinental spread of antimicrobial resistant strains, including multidrug resistant strains.

[0003] The disease is caused by Gram-negative bacteria of the genus Shigella. Bacteria are transmitted via the fecal-oral route, through direct person-to-person contact or indirectly upon contact with contaminated food or water. The Shigella bacillus comprises four species, or groups A-D, which are subdivided in more than 50 serotypes and subtypes. S.flexneri (group B) consists of 15 or more serotypes and subtypes, whereas S. sonnei (group D) features only 1 serotype. In combination, these two species (S.flexneri (SF) and S. sonnei (Sson)) account for approximately 90% of all cases of endemic shigellosis worldwide. While the latter dominates in transitional and high-income countries, SF remains prevalent in LMICs, which makes it the most important species globally. The spread of resistance among Shigella isolates increasingly compromisesantibiotic treatment and emphasizes the need for alternatives among which disease prevention through vaccination is an attractive option. Altogether, epidemiological data call for a broad serotype coverage vaccine conferring primarily protective immunity to the pediatric population in LMICs. This is a challenging issue considering the diversity of circulating strains responsible for disease.

[0004] Shigella serotypes are differentiated by their surface polysaccharide (PS) and, in particular, by the O-antigen (O-Ag) component, or O-specific polysaccharide (O-SP) part, of their lipopolysaccharide (LPS). The O-Ag consists of oligomers and polymers of oligosaccharide (OS) repeating units. Protective immunity against shigellosis is largely serotype-specific. Despite a diversity of vaccine candidates being designed to prevent infection by Shigella and evaluated for safety and immunogenicity in clinical trials, many of which are currently ongoing, there is yet no licensed Shigella vaccine with broad serotype coverage for infants. A major challenge resides in the need for a vaccine that is well tolerated, immunogenic and efficacious against moderate-to-severe diarrhea caused by circulating Shigella strains representative of a large diversity of serotypes among infants and children under 5 years of age in LMICs.SUMMARY OF THE INVENTION

[0005] One embodiment of this invention pertains to a multivalent Shigella vaccine composition comprising two or more different monovalent sOSs (sOS) conjugated to a carrier, wherein the two or more different monovalent sOSs comprise (a) a sOS that comprises one or more O-Ag repeating units (RUs) for SF2a, (b) a sOS that comprises one or more O-Ag RUs fromRUs from SF3a, (c) a sOS that comprises one or more O-Ag RUs fromRUs from SF6, (d) a sOSs that comprises one or more O-Ag RUs from Sson, or (e) a combination of the sOSs of (a), (b), (c), and / or (d).

[0006] In the framework of the invention, the multivalent composition is thus a combination of monovalent OSs and not a chimeric OS.

[0007] In one embodiment, the carrier comprises tetanus toxoid (TT), tetanus toxin C fragment (TTc), recombinant TT, cholera toxin b subunit (CTB), diphtheria toxoid (DT), non-toxic diphtheria toxin mutant (e.g., CRM197 or CRM9), P. aeruginosa exotoxin A (EP A), recombinant Exoprotein A (rEPA), C. jejuni Acriflavine resistance protein A (CjAcrA), E. coli Acriflavine resistance protein A (EcAcrA), heat labile enterotoxigenic Escherichia coli (ETEC)subunit B (LTB), QP, outer membrane vesicles (OMVs), glycoengineered proteins, virus-like particles (VLPs), nanocarriers or nanoparticles, recombinant TT with certain mutations, Generalized Modules for Membrane Antigens (GMMA), P. aeruginosa PcrV (PcrV), Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), the recombinant fragment of tetanus toxin heavy chain (rTTHc), a cross-reactive Shigella Ipa protein, a mutated IpaB protein, or T-helper peptides (with natural or not peptide sequence), all in a mono- or multivalent fashion.

[0008] In one embodiment, the carrier comprises TT or CRM197.

[0009] In one embodiment, the two or more different monovalent sOSsOSs are each independently conjugated to a carrier via one or more points of attachment (e.g., covalent attachment). In another embodiment, the two or more different monovalent sOSsOSs are each independently conjugated to a carrier via a single point of attachment (e.g., covalent attachment).

[0010] In another embodiment, the two or more different monovalent sOSsOSs are each independently conjugated to a carrier via chemoselective chemistry. In one embodiment, the two or more different monovalent sOSsOSs are each independently conjugated to a carrier via Michael reaction. In another embodiment, the two or more different monovalent sOSsOSs are each independently conjugated to a carrier via thiol chemistry.

[0011] In one embodiment, the two or more different monovalent sOSs are each independently conjugated to a carrier via a linker.

[0012] In one embodiment, the one or more O-Ag RUs from SF2a comprise the following:

[0013] In one embodiment, at least one of Ri, R2, or R3 is acetyl (Ac).

[0014] In one embodiment, Ri or R2 is Ac.

[0015] In one embodiment, at least one of Ri, R2, and R3 is H.

[0016] In one embodiment, Ri, R2, and R3 are H.

[0017] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF2a. In another embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF2a.

[0018] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF2a is conjugated to a carrier with a linker.

[0019] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following

[0020] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following structure:

[0021] In one embodiment, the carrier is TT.

[0022] In one embodiment, the one or more O-Ag RUs from SF3a comprise the following:

[0023] In one embodiment, at least one of R4 or R5 is Ac.

[0024] In one embodiment, R4 or R5 is Ac.

[0025] In one embodiment, at least one of R4 or R5 is H.

[0026] In one embodiment, R4 is H.

[0027] In one embodiment, R5 is Ac.

[0028] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF3a. In another embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF3a.

[0029] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF3a is conjugated to a carrier with a linker.

[0030] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

[0031] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

[0032] In one embodiment, the carrier is TT.

[0033] In one embodiment, the one or more O-Ag RUs from SF6 comprise the following:X may represent H+or a pharmaceutically acceptable cation (i.e. forming a pharmaceutically acceptable salt). When X is H+, -CCh’, X+corresponds to -COOH.

[0034] In one embodiment, at least one of Re or R? is Ac.

[0035] In one embodiment, Re or R? is Ac.

[0036] In one embodiment, at least one of Re or R? is H.

[0037] In one embodiment, Re and R? is H.

[0038] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF6. In another embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF6.

[0039] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF6 is conjugated to a carrier with a linker.

[0040] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

[0041] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

[0042] In one embodiment, the carrier is TT.

[0043] In one embodiment, the one or more O-Ag RUs from Sson comprise the following:

[0044] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from Sson. In another embodiment, the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 4 O-Ag RUs from Sson.

[0045] In one embodiment, the sOS that comprises one or more O-Ag RUs from Sson is conjugated to a carrier with a linker.

[0046] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the following structure:

[0047] In one embodiment, the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the followingstructure:

[0048]

[0049] In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 5.

[0050] In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 5.

[0051] In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and thecarrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 5.

[0052] In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 5.

[0053] Another embodiment of this invention pertains to a quadrivalent Shigella vaccine composition comprising four different monovalent sOSs conjugated to a carrier, wherein the fourdifferent monovalent sOSs comprise (a) a sOS that comprises one or more O-Ag RUs from SF2a, (b) a sOS that comprises one or more O-Ag RUs from SF3a, (c) a sOS that comprises one or more O-Ag RUs from SF6, and (d) a sOS that comprises one or more O-Ag RUs from Sson.

[0054] All the embodiments related to the composition apply here as well, alone or in combination.

[0055] In one embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via one or more points of attachment (e.g., covalent attachment). In another embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via a single point of attachment (e.g., covalent attachment).

[0056] In another embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via chemoselective chemistry. In one embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via Michael reaction. In another embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via thiol chemistry.

[0057] In one embodiment, the four different monovalent sOSs are each independently conjugated to a carrier via a linker.

[0058] All the embodiments mentioned above apply here as well, alone or in combination.

[0059] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF2a comprises 2 to 10 O-Ag RUs from SF2a. In another embodiment, the sOS that comprises one or more O-Ag RUs from SF2a comprises 3 O-Ag RUs from SF2a.

[0060] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF2a is conjugated to a carrier with a linker.

[0061] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF2a conjugated to a carrier with a linker has the following structure:

[0062] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF2a conjugated to a carrier with a linker has the following structure:

[0063] In one embodiment, the carrier is TT.

[0064] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF3a comprises 2 to 10 O-Ag RUs from SF3a. In another embodiment, the sOS that comprises one or more O-Ag RUs from SF3a comprises 3 O-Ag RUs from SF3a.

[0065] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF3a is conjugated to a carrier with a linker.

[0066] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF3a conjugated to a carrier with a linker has the following structure:

[0067] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF3a conjugated to a carrier with a linker has the following structure:

[0068] In one embodiment, the carrier is TT.

[0069] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF6 comprises 2 to 10 O-Ag RUs from SF6. In another embodiment, the sOS that comprises one or more O-Ag RUs from SF6 comprises 3 O-Ag RUs from SF6.

[0070] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF6 is conjugated to a carrier with a linker.

[0071] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF6 conjugated to a carrier with a linker has the following structure:

[0072] In one embodiment, the sOS that comprises one or more O-Ag RUs from SF6 conjugated to a carrier with a linker has the following structure:

[0073] In one embodiment, the carrier is TT.

[0074] In one embodiment, the sOS that comprises one or more O-Ag RUs from Sson comprises 2 to 10 O-Ag RUs from Sson. In another embodiment, the sOS that comprises one or more O-Ag RUs from Sson comprises 4 O-Ag RUs from Sson.

[0075] In one embodiment, the sOS that comprises one or more O-Ag RUs from Sson is conjugated to a carrier with a linker.

[0076] In one embodiment, the sOS that comprises one or more O-Ag RUs from Sson conjugated to a carrier with a linker has the following structure:

[0077] In one embodiment, the sOS that comprises one or more O-Ag RUs from Sson conjugated to a carrier with a linker has the following structure:

[0078] In one embodiment, the carrier is TT.

[0079] Another embodiment of this invention pertains to a vaccine formulation comprising (a) a multivalent Shigella vaccine composition comprising two or more different monovalent synthetic oligosaccharides conjugated to a carrier; and (b) an adjuvant, wherein the two or more different monovalent synthetic oligosaccharides comprise (i) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF2a, (ii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF3a, (iii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF6, (iv) a synthetic oligosaccharide that comprises one or more O-Ag RUs for Sson, or (v) a combination of the synthetic oligosaccharides of (i), (ii), (iii), and / or (iv).

[0080] All the embodiments mentioned above apply here as well, alone or incombination.

[0081] In one embodiment, the two or more monovalent sOSs are each independently conjugated to a carrier via one or more points of attachment (e.g., covalent attachment). In another embodiment, the two or more monovalent sOSs are each independently conjugated to a carrier via a single point of attachment (e.g., covalent attachment).

[0082] In another embodiment, the two or more different monovalent sOSs are each independently conjugated to a carrier via chemoselective chemistry. In one embodiment, the two or more different monovalent sOSs are each independently conjugated to a carrier via Michael reaction. In another embodiment, the two or more different monovalent sOSs are each independently conjugated to a carrier via thiol chemistry.

[0083] In one embodiment, the two or more different monovalent sOSs are each independently conjugated to a carrier via a linker.

[0084] In one embodiment, the adjuvant is selected from at least one of MATRIX M®, cytosine phospho-guanine (CPGs), QS-21 (derived from the soap bark tree (Quillaja saporiaria))^ amorphous aluminum hydroxyphosphate sulfate (AAHS), potassium aluminum sulfate, aluminum phosphate, aluminum hydroxide, double mutant heat-labile toxin (dmLT), or combinations thereof.

[0085] In one embodiment, the vaccine is free of an adjuvant.

[0086] In one embodiment, the vaccine formulation further comprises a preservative. In some embodiments the preservative is at least one of 2 -phenoxy ethanol (2 -PE), thimerosal, m-cresol, phenol, benzyl alcohol, or combinations thereof.

[0087] In one embodiment, the vaccine formulation further comprises one or more excipients. In some embodiments, the one or more excipients include at least one of a surfactant, a buffer, a salt, a tonicity modifier, a preservative, or combinations thereof. In one embodiment, the surfactant is a non-ionic surfactant. In some embodiments, the non-ionic surfactant includes polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-80 (PS80), poloxamer, or a combination thereof. In one embodiment, the one or more excipients comprise PS-80, Tris (tromethamine), sodium chloride, 2-phenoxyethanol (2 -PE), or combinations thereof.

[0088] All the embodiments mentioned above apply here as well, alone or incombination.

[0089] Another embodiment of this invention pertains to a method of making a multivalent Shigella vaccine composition that includes obtaining two or more different monovalent synthetic oligosaccharides; conjugating each of the two or more different monovalent synthetic oligosaccharides to a carrier to prepare two or more different monovalent synthetic oligosaccharide carrier conjugates, wherein the two or more different monovalent synthetic oligosaccharides comprise (i) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF2a, (ii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF3a, (iii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF6, and / or (iv) a synthetic oligosaccharide that comprises one or more O-Ag RUs for Sson; and combining the two or more different monovalent synthetic oligosaccharide carrier conjugates.

[0090] In one embodiment, the method further comprises mixing the two or more different monovalent synthetic oligosaccharide carrier conjugates with an adjuvant.

[0091] Yet another embodiment of this invention pertains to a method of administering a multivalent Shigella vaccine composition to a subject that includes obtaining a multivalent Shigella vaccine composition comprising two or more different monovalent synthetic oligosaccharides conjugated to a carrier; and administering the multivalent Shigella vaccine composition to a subject, wherein the two or more different monovalent synthetic oligosaccharides comprise (i) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF2a, (ii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF3a, (iii) a synthetic oligosaccharide that comprises one or more O-Ag RUs for SF6, (iv) a synthetic oligosaccharide that comprises one or more O-Ag RUs for Sson, or (v) a combination of the synthetic oligosaccharides of (i), (ii), (iii), and / or (iv).

[0092] Yet another embodiment of this invention pertains to a multivalent composition comprising two or more different monovalent sOSs conjugated to a carrier for its use as a vaccine against Shigella, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv). Yet another embodiment of this invention pertains to a multivalent composition comprising two or more different monovalent sOSs conjugated to a carrier for its use for the prevention and / or treatment of disease caused by Shigella, in particular Sson and SF, wherein thetwo or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).

[0093] All the embodiments mentioned above apply here as well, alone or in combination.

[0094] According to one embodiment, the multivalent Shigella vaccine composition further comprises an adjuvant.BRIEF DESCRIPTION OF THE FIGURES

[0095] For a more complete understanding of the invention and the advantages thereof, reference is made to the following descriptions, taken in conjunction with the accompanying figures, in which:

[0096] Figures 1A and IB are graphs showing IgG titers against SF2a LPS and Sson LPS of mice that received injections of a monovalent SF2a-TT vaccine conjugate and a monovalent Sson-TT vaccine conjugate separately or combined in a 1:1 ratio to achieve 2.0 pg of OS each per dose, according to one embodiment of the invention.

[0097] Figures 2A and 2B are graphs showing IgG titers of mice that received three injections of a monovalent SF2a-TT vaccine conjugate and a monovalent SF3a-TT vaccine conjugate separately or combined in a 1:1 ratio to achieve 2.0 pg of OS each per dose, with and without alum as an adjuvant, according to one embodiment of the invention.

[0098] Figure 3 is a graph showing IgG titers against SF2a LPS and SF3a LPS of mice that received injections of a monovalent SF2a-TT vaccine conjugate and a monovalent SF3a-TT vaccine conjugate separately or combined in a 1:1 ratio to achieve 1.0 pg of OS each per dose with alum as an adjuvant, according to one embodiment of the invention.

[0099] Figure 4A is an illustration of a quadrivalent Shigella vaccine composition having SF2a, SF3a, SF6 and Sson monovalent glycoconjugates as components of the quadrivalent Shigella vaccine formulation, with TT as the carrier and where “n” refers to the exact number of RUs per sOS, and “m” refers to the average number of sOS per carrier (OS: TT loading), according to one embodiment of the invention.[000100] Figure 4B is an illustration of a quadrivalent Shigella vaccine composition having SF2a, SF3a, SF6 and Sson monovalent glycoconjugates as components of the quadrivalent Shigella vaccine formulation, with TT as the carrier, wherein “n=3” (i.e., exact number of RUs per sOS) and OS: TT is approximately “m” (average number of sOS per carrier), according to one embodiment of the invention.[000101] Figures 5A-5D are graphs showing IgG titers against SF2a LPS, SF3a LPS, SF6 LPS and Sson LPS, respectively, of mice that received three injections of a trivalent or quadrivalent vaccine composition comprising SF2a-TT, SF3a-TT, SF6-TT and / or Sson-TT glycoconjugates combined in a 1: 1: 1:0, 1: 1:0: 1 or 1: 1: 1: 1 ratio to achieve 2.0 pg of OS each per dose, adjuvanted or not with aluminum hydroxide (H) or aluminum phosphate (P), according to one embodiment of the invention.[000102] Figures 6A-6D are graphs showing IgG titers against SF2a LPS, SF3a LPS, SF6 LPS and Sson LPS, respectively, of mice that received three injections of vaccine compositions comprising SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT glycoconjugates each by itself, or in a quadrivalent formulation and adjuvanted with aluminum phosphate (P); the four monovalent conjugates were combined in a 1: 1: 1: 1 wt:wt ratio in the quadrivalent vaccine composition, to achieve 2.0 pg of OS each per dose, to show influence of the carrier (TT) content on the IgG titers, according to one embodiment of the invention.[000103] Figure 7 is a graph showing anti-SF2a LPS IgG titers of mice that received three injections of vaccine compositions comprising SF2a-TT glycoconjugates at different pg amounts of OS each per dose, adjuvanted with alum or not, to show the influence of the relative OS content on the IgG titers, according to one embodiment of the invention.[000104] Figure 8 is a graph showing anti-SF3a LPS IgG titers of mice that received three injections of vaccine compositions comprising SF3a-TT glycoconjugates at different pg amounts of OS each per dose, adjuvanted with alum or not, to show the influence of the relative OS content on the IgG titers, according to one embodiment of the invention.[000105] Figures 9A-9D are graphs showing anti-SF2a, anti-SF3a, anti-SF6, and anti-Son LPS IgG titers, respectively, of mice that received three injections of vaccine compositions comprising SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT glycoconjugates each by itself, or in a quadrivalent formulation, and adjuvanted with aluminum phosphate (P); the four monovalent conjugates were combined in a 1: 1: 1: 1, 1:1:1:0.25, 1:1:1:0.1, or 1:1:0.5:0.1 wt:wt ratio (in theorder of SF2a: SF3a: SF6: Sson) to achieve 2.0 pg of OS per dose for SF2a-TT and SF3a-TT, to show the influence of the relative OS content on the IgG titers, according to one embodiment of the invention.[000106] Figures 10A-10D are graphs showing anti-SF2a, anti-SF3a, anti-SF6, and antiSon LPS IgG titers, respectively, of mice that received injections of quadrivalent vaccine compositions comprising SF2a-TT, SF3a-TT, SF6-TT and Sson-TT glycoconjugates combined in a 1: 1: 1: 1 ratio to achieve 2.0 pg of OS each per dose, adjuvanted or not with aluminum hydroxide (A1H) or aluminum phosphate (A1P), according to one embodiment of the invention.[000107] Figure 11 is a table illustrating a study design to test rabbit immunization with a quadrivalent Shigella vaccine composition according to one embodiment of the invention.[000108] Figures 12A-12C are tables of results from a study of rabbit immunization with a quadrivalent Shigella vaccine composition according to one embodiment of the invention.[000109] Figure 13 is a table illustrating a study design to test rabbit immunization with a quadrivalent Shigella vaccine composition according to one embodiment of the invention.[000110] Figures 14A-14C are tables of results from a study of rabbit immunization with a quadrivalent Shigella vaccine composition according to one embodiment of the invention.[000111] Figure 15 is a flow chart of an exemplary method of preparing a monovalent Shigella OS-carrier conjugate according to one embodiment of the invention.DETAILED DESCRIPTION OF THE INVENTION[000112] Among those benefits and improvements that have been disclosed, other objects and advantages of this disclosure will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given regarding the various embodiments of the disclosure are intended to be illustrative, and not restrictive.[000113] Throughout the specifications and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases "in one embodiment," “in an embodiment,” and "in some embodiments" as used herein do not necessarily refer to the same embodiment s), though they may. Furthermore, the phrases "inanother embodiment" and "in some other embodiments" as used herein do not necessarily refer to a different embodiment, although they may. All embodiments of the disclosure are intended to be combinable without departing from the scope or spirit of the disclosure.[000114] As used herein, the term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a”, “an”, and “the” include plural references. The meaning of “in” includes “in” and “on”.[000115] As used herein, terms such as “comprising”, “including” and “having” do not limit the scope of a specific claim to the materials or steps recited by the claim.[000116] As used herein, terms such as “consisting of’ limit the scope of a specific claim to the materials and steps recited by the claim.[000117] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit / risk ratio.[000118] In another aspect, the present invention is directed to pharmaceutically acceptable salts of the compounds described above. As used herein, “pharmaceutically acceptable salts” includes salts of compounds of the present invention derived from the combination of such compounds with non-toxic acid or non-toxic base.[000119] In addition to pharmaceutically acceptable salts, other salts are included in the invention. They may serve as intermediates in the purification of the compounds, in the preparation of other salts, or in the identification and characterization of the compounds or intermediates.[000120] As used herein, the term “oligosaccharide” more particularly refers to a saccharide containing from 2 to 20 O-Ag.[000121] As used herein, a range of values in the form “x-y” or “x to y”, or “x through y”, include integers x, y, and the integers there between. For example, the phrases “1-6”, or “1 to 6” or “1 through 6” are intended to include the integers 1, 2, 3, 4, 5, and 6. Preferred embodiments include each individual integer in the range, as well as any subcombination of integers. For example, preferred integers for “1-6” can include 1, 2, 3, 4, 5, 6, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 2-6, etc.[000122] Abbreviation: S.flexneri (SF); S.flexneri 2a (SF2a); S.flexneri 3a (SF3a); S. flexneri 6 (SF6); S. sonnei (Sson); repeating units (RUs); oligosaccharide (OS); synthetic OS (sOS); tetanus toxoid (TT); tetanus toxin C fragment (TTc); cholera toxin b subunit (CTB); diphtheria toxoid (DT); non-toxic diphtheria toxin mutant (e.g., CRM197 or CRM9), Pseudomonas aeruginosa exotoxin A (EP A); recombinant Exoprotein A (rEPA); Campylobacter jejuni Acriflavine resistance protein A (Cj AcrA); Escherichia coli Acriflavine resistance protein A (EcAcrA); heat labile enterotoxigenic Escherichia coli (ETEC) subunit B (LTB); Generalized Modules for Membrane Antigens (GMMA); Pseudomonas aeruginosa PcrV (PcrV);Haemophilus influenzae protein D (PD); the outer membrane protein complex of serogroup B meningococcus (OMPC); recombinant fragment of tetanus toxin heavy chain (rTTHc); low- and middle-income countries (LMICs); O-antigen (O-Ag); O-specific polysaccharide (O-SP); lipopolysaccharide (LPS); acetyl (Ac); aluminum hydroxide (A1H or H or A1OH, or ALOH); aluminum phosphate (P or A1P or A1PO, or ALPO); MATRIX M® (MM); double mutant heat- labile toxin (dmLT); quadrivalent Shigella vaccine (QSV), KI: killing index, wt: weight.[000123] As used herein, the term “free of adjuvant” means the formulation includes no adjuvant or 0% of adjuvant.[000124] As used herein, the term “O-antigen” means the polysaccharide component of the LPS.[000125] As used herein, the term “O-antigen repeating unit” means the biological carbohydrate repeating unit of the O-Ag.[000126] As used herein, the term “basic O-antigen repeating unit” means the biological carbohydrate repeating unit of the O-Ag, with the repeating unit lacking any non-stoichiometric carbohydrate substitutions (i.e., (9-acetylation).[000127] As used herein, the term “carrier” means any molecule to which an OS or PS of the invention can be bound (e.g., covalently bound) to form the glycoconjugate of the invention. A carrier can be a natural, modified-natural, synthetic, semi-synthetic or recombinant material containing one or more functional groups, for example primary and / or secondary amino groups, alkynyl groups, azido groups, thiol groups, phenol groups (e.g. of tyrosine), or carboxyl groups. The carrier can be water soluble or insoluble. Carriers that fulfill these criteria are well-known to those of ordinary skill in the art. Suitable carriers according to the present invention include, but are not limited to, proteins, peptides, lipopeptides, zwitterionic PSs, lipid aggregates (such as oildroplets or liposomes), inactivated virus particles, nanocarriers or nanoparticles (including, e.g., self-assembling and non-self-assembling nanoparticles, including, e.g., ferritin nanoparticles, liposomes, lipid nanoparticles, gold nanoparticles, etc.), virus-like particles (VLPs), for example bacteriophage QP, and Generalized Modules for Membrane Antigens (GMMA). The carrier may be a protein to which the OS or PS is coupled or attached or conjugated, typically for the purpose of enhancing or facilitating detection of the antigen by the immune system. OSs and PSs are T-independent antigens that are poorly immunogenic and do not lead to long-term protective immune responses. Conjugation of OSs or PSs to a protein carrier changes the context in which immune effector cells respond to OSs or PSs. The term carrier protein is intended to cover both small peptides and large polypeptides (e.g., >10 kDa).[000128] As used herein, the term “linker” or “spacer” does not contain any carbohydrate residue; thus, it is neither a carbohydrate residue nor an OS or a PS compound. The OS or PS is preferably conjugated to a carrier using a linking molecule (i.e., a “linker” or “spacer”). A linker, spacer or crosslinking agent, as used in the present invention, is generally a small molecule, linear or not, having a molecular weight of approximately <500 daltons and is non-pyrogenic and non-toxic in the final product form, in particular, in the framework of an in vitro use, or when the final product is an immunogenic composition for use in vaccination.[000129] As used herein, the term “conjugate” means an OS or PS linked (e.g., covalently linked) to a carrier. In one embodiment, the OS or PS is bound to the carrier via the reducing end of said OS or PS. Such a conjugation is thus site-selective and corresponds to a conjugate wherein the carrier is attached to the OS or PS via at least one single anchoring point. The OS or PS can be bound (e.g., covalently bound) to the carrier with or without a linking molecule, a linker or a spacer.[000130] As used herein, the term “adjuvant” refers to substances that enhance the immune response to the presence of an antigen, in the context of vaccine formulations that are well known to a person skilled in the art. Generally recognized examples of adjuvants include, e.g.:mineral-containing compositions, including calcium salts and aluminum salts (or mixtures thereof). Calcium salts include calcium phosphate. Aluminum salts include hydroxides, phosphates, sulfates, etc., with the salts taking any suitable form (e.g., gel, crystalline, amorphous, etc.). According to one embodiment, adsorption to these salts ispreferred. The mineral containing compositions may also be formulated as a particle of metal salt. The adjuvants known as aluminum hydroxide and aluminum phosphate may be also used. The invention can use any of the "hydroxide" or "phosphate" adjuvants that are generally used as adjuvants. The adjuvants known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline. The adjuvants known as "aluminum phosphate" are typically aluminum hydroxyphosphates, often also containing a small amount of sulfate (i.e., aluminum hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Mixtures of both an aluminum hydroxide and an aluminum phosphate can be employed in the formulation according to the present invention;saponins, which are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins from the bark of the Quillaia saponaria. Molina tree have been widely studied as adjuvants. Saponins can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria oficianalis (soap root). Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as Immune stimulating complexes (ISCOMs). Saponin compositions have been purified using High-performance liquid chromatography (HPLC) and RP-HPLC (Reversed phase HPLC). Specific purified fractions using these techniques have been identified, including QS 7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Saponin formulations may also comprise a sterol, such as cholesterol. Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs). ISCOMs generally include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA, QH-A & QH-C; microparticles (i.e., a particle of 100 nm to 150 pm in diameter, 200 nm to 30 pm in diameter, or 500 nm to 10 pm in diameter) formed from materials that are biodegradable and non-toxic. Such non-toxic and biodegradable materials include, but are not restrictedto, poly(a-hydroxy acid), polyhydroxybutyric acid, polyorthoester, polyanhydride, polycaprolactone;CD Id ligands, such as an a-glycosylceramide, phytosphingosine-containing a- glycosylceramides, OCH, KRN7000 [(2S,3S,4R)-l-O-(a-D-galactopyranosyl)-2-(N- hexacosanoylamino)-l,3,4-octadecanetriol], CRONY- 101, 3"-sulfo-galactosyl-ceramide; immunostimulatory oligonucleotides, such CPG motif containing ones (a dinucleotide sequence containing an unmethylated cytosine residue linked by a phosphate bond to a guanosine residue), or Cpl motif containing ones (a dinucleotide sequence containing cytosine linked to inosine), or a double-stranded RNA, or an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.Immunostimulatory oligonucleotides can include nucleotide modifications / analogs such as phosphorothioate modifications and can be double-stranded or (except for RNA) single-stranded;compounds containing lipids linked to a phosphate-containing acyclic backbone, such as the Toll-like receptor 4 (TLR4) antagonist E5564; andoil emulsions (e.g., Freund's adjuvant).[000131] According to one embodiment, adjuvants may be chosen from aluminum salts (e.g., aluminum hydroxide, aluminum phosphate), oil-in-water emulsion formulations with or without specific stimulating agents (such as, e.g., TLR agonists), muramyl peptides, saponin adjuvants, cytokines, or detoxified mutants of bacterial toxins (such as, e.g., the cholera toxin, the pertussis toxin, or the E. coli heat labile toxin (e.g., dmLT)).[000132] As used herein, the term “toxoid” means a bacterial toxin (usually an exotoxin), whose toxicity has been inactivated or suppressed either by chemical (formalin) or heat treatment, while other properties, typically immunogenicity, are maintained. A “mutated toxoid” (or “mutated toxin”) as used herein is a recombinant bacterial toxin, which has been modified to be less toxic or even non-toxic by modifying the wild-type amino acid sequence. Such a mutation could be a substitution of one or more amino acids. Such a mutated toxoid presents on its surface a functionality that can react with the functional group of the interconnecting molecule to provide a modified toxoid. Said functionality is known to the person skilled in the art and includes, but is not restricted to, the primary amino functionality of a lysine residue that can react with activated esters, an isocyanate group or an aldehyde in the presence ofa reducing agent, the carboxylate functionality of a glutamate or aspartate residue that can be activated by carbodiimides, or to the thiol functionality of a cysteine residue.[000133] As used herein, the term “synthetic oligosaccharide” means a carbohydrate chain comprising at least 2 monosaccharide residues that are assembled either by chemical synthesis or by chemoenzymatic synthesis. As used herein, the term “synthetic glycan” refers to carbohydrates that are made by chemical or chemoenzymatic synthesis, as opposed to carbohydrates that are isolated from natural sources (e.g. bacterial cell components, including capsular PSs and lipopolysaccharides, etc.). As used herein, the terms “synthetic oligosaccharide” and “synthetic glycan” can be used interchangeably.[000134] As used herein, the term “subject” means a mammalian subject, such as a mouse, a rat, a rabbit, a non-human primate, a guinea pig, a ferret, a horse, a llama, a goat, or a sheep, a pig, preferably a human.[000135] As used herein, the term “monovalent” refers to a unique type of OS mimicking a natural O-Ag. This does not necessary mean that said monovalent entity would protect against a unique type of serotype.Shigellosis[000136] As discussed above, Shigellosis is a major diarrheal disease. It is responsible for a large burden of disease worldwide and is a leading cause of diarrheal deaths due to bacterial infection. Shigella affects especially children under 5 years of age in Low- or Middle-Income Countries (LMICs). Besides direct mortality and morbidity, it is a direct cause of stunting in this population. Shigellosis is a general problem also causing disease among travelers, military and non-governmental organization staff deployed in endemic areas. In high-income countries, it occurs in the form of outbreaks with some communities, such as young children in daycare and people in locations with poor sanitation, being particularly affected. Of additional concern in terms of public health threat is the intercontinental spread of antimicrobial resistant strains, including multidrug resistant strains, that is facilitated by travelers.[000137] The disease is caused by Gram-negative bacteria of the genus Shigella. Bacteria are transmitted via the fecal-oral route, through direct person-to-person contact or indirectly upon contact with contaminated food or water. The Shigella bacillus comprises four species, or groups A-D, which are subdivided in more than 50 serotypes and subtypes. SF (group B) consists of 15 and more serotypes and subtypes whereas Sson (group D) features only 1 serotype.In combination, they account for approximately 90% of all cases of endemic shigellosis worldwide. While the latter dominates in transitional and high-income countries, SF remains prevalent in LMICs, which makes it the most important species globally. The spread of resistance among Shigella isolates increasingly compromises antibiotic treatment and emphasizes the need for alternatives among which disease prevention through vaccination is an attractive option. Altogether, epidemiological data call for a broad serotype coverage vaccine conferring primarily protective immunity to the pediatric population in LMICs. This is a challenging issue considering the diversity of circulating strains responsible for disease.Shigella Vaccine Strategies[000138] Shigella serotypes are differentiated by their surface PS and in particular by the O-Ag component, or O-specific polysaccharide (O-SP) part, of their LPS. The O-Ag consists of oligomers and polymers of OS RU(s). Protective immunity against shigellosis is largely serotype-specific. A large diversity of Shigella vaccine candidates have been proposed over the years. They are classified into three main categories: killed and live attenuated vaccines or subunit vaccines. Early read out of efficacy in small trials was demonstrated among military recruits and children for representatives of both live attenuated vaccines and subunit vaccines. However, there is yet no broadly licensed Shigella vaccine. One significant challenge resides in the need for a vaccine that is safe, immunogenic and efficacious against moderate-to-severe diarrhea caused by several Shigella serotypes among infants and children under 5 years of age in LMICs. A lattice-type Sson PS-protein conjugate whereby the chemically detoxified Sson LPS is covalently linked in a random fashion to the genetically detoxified exoprotein A of P. aeruginosa (rEPA) was found to be well tolerated, immunogenic, and efficacious in young Israeli adults and in children three years-of-age and older. However, the efficacy was markedly reduced in the 2-3 years of age cohort and the conjugate was not efficacious in the most vulnerable population of children 1-2 years of age. An association between serum O-Ag IgG and protective efficacy was found, suggesting this endpoint to be potentially a correlate of protection against shigellosis. Therefore, improvement relying on alternative vaccine design approaches is needed to advance a Shigella vaccine.[000139] Several other Shigella conjugate vaccine candidates encompassing a PS component of biological origin have been developed. They differ in terms of compositionand production process. The main categories include (i) conjugates made of the chemically detoxified LPS covalently attached to a protein carrier or non-covalently attached to a protein carrier by means of the avidin-streptavidin complex (MAPS), (ii) O-Ag-rEPA bioconjugates engineered in recombinant A. coli, (iii) partially deacylated LPS extracted from Shigella culture combined to outer membrane proteins in the form of Generalized Modules for Membrane Antigens or GMMA, or (iv) low reactogenic LPS associated to Shigella Ipa proteins as in InvaplexAR-detox or to outer membrane proteins from meningococcus as in a proteosome-LPS Shigella vaccine. Currently, all of the LPS-based vaccine candidates are developed to be administered parenterally with the exception of the latter that was delivered by intranasal spray. While original developments considered monovalent vaccine candidates, in recent years all strategies have addressed the need for vaccines covering multiple serotypes.sOSs as Vaccine Components[000140] Several vaccine candidates using synthetic glycans as surrogates of highly heterogeneous bacterial PSs isolated from natural sources in conjugate vaccine development have been explored for decades. The most used strategy involves the covalent coupling of synthetic glycans to a protein carrier. Other strategies were proposed, albeit less explored, whereby the synthetic glycan haptens are combined to T-helper peptides in a non-covalent manner, for example, in the form of liposomes or glyconanoparticles. Despite its licensing two decades ago, Quimi-Hib® appears to be the only licensed bacterial vaccine based on synthetic glycans, to date, that was found to be well tolerated and immunogenic and has demonstrated efficacy against disease in the target population. Although featuring synthetic haptens, it is well-acknowledged that Quimi-Hib® is made of synthetic polyribitol phosphate fragments comprising an average of eight RUs, albeit differing in terms of chain length. In contrast, the synthetic glycan-protein conjugates described in the instant application comprise synthetic glycans of a well-defined number of repeating units and an even more well-defined number of residues.[000141] In addition, bacterial vaccines often need to address diseases caused by multiple serotypes of a targeted pathogen, as exemplified with PREVNAR 20® (a pneumococcal 20- valent conjugate vaccine), meningococcal tetravalent conjugate vaccine MENVEO® (Groups A, C, Y and W135), and meningococcal multivalent conjugate vaccine PENBRAY A®(Groups A, B, C, W, and Y). Yet, there are few reports on multivalent synthetic carbohydrate-based vaccines except for those related to pneumococcal vaccines. The development of multivalent vaccine formulations presents many challenges, especially related to chemistry, manufacturing, and controls. For the clinical development of a synthetic glycan-based Shigella vaccine, an additional challenge relates to the poor understanding of the molecular basis, at the antigen level, that governs the induction of an immune response correlated to protection.The Invention[000142] Aiming at broadening serotype coverage to answer the need in the field, this invention relates to vaccine compositions comprising synthetic Shigella O-Ag conjugates. In particular, the proposed vaccine compositions comprise two or more different monovalent O- Ag OS conjugates targeting Sson, SF6, SF3a, and / or SF2a. The RUs of the corresponding Shigella O-Ags are described further below.[000143] Accordingly, this invention, in embodiments, relates to a multivalent Shigella vaccine composition, including a quadrivalent Shigella vaccine composition, comprising two or more different monovalent sOSs conjugated to a carrier, wherein the two or more different monovalent sOSs comprise (a) a sOS that comprises one or more O-Ag RUs from SF2a, (b) a sOS that comprises one or more O-Ag RUs from SF3a, (c) a sOS that comprises one or more O-Ag RUs from SF6, (d) a sOS that comprises one or more O-Ag RUs from Sson, or (e) a combination of the sOSs of (a), (b), (c), and / or (d).[000144] Another embodiment of this invention pertains to a quadrivalent Shigella vaccine composition comprising four different monovalent sOSs conjugated to a carrier, wherein the four different monovalent sOSs comprise (a) a sOS that comprises one or more O-Ag RUs from SF2a, (b) a sOS that comprises one or more O-Ag RUs from SF3a, (c) a sOS that comprises one or more O-Ag RUs from SF6, and (d) a sOS that comprises one or more O-Ag RUs from Sson.[000145] Another embodiment of this invention pertains to a vaccine formulation comprising (a) a multivalent Shigella vaccine composition comprising two or more different monovalent sOSs conjugated to a carrier; and (b) an adjuvant, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprisesone or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).[000146] Another embodiment of this invention pertains to a method of making a multivalent Shigella vaccine composition that includes obtaining two or more different monovalent sOSs; conjugating each of the two or more different monovalent sOSs to a carrier to prepare two or more different monovalent sOS carrier conjugates, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, and / or (iv) a sOS that comprises one or more O-Ag RU for Sson; and combining the two or more different monovalent sOS carrier conjugates.[000147] Yet another embodiment of this invention pertains to a method of administering a multivalent Shigella vaccine composition to a subject that includes obtaining a multivalent Shigella vaccine composition comprising two or more different monovalent sOSs conjugated to a carrier; and administering the multivalent Shigella vaccine composition to a subject, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).[000148] Yet another embodiment of this invention pertains to a multivalent composition comprising two or more different monovalent sOSs conjugated to a carrier for its use as a vaccine against Shigella, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).[000149] Yet another embodiment of this invention pertains to a multivalent composition comprising two or more different monovalent sOSs conjugated to a carrier for its use for the prevention and / or treatment of disease caused by Shigella, in particular Sson and SF, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOSthat comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv). All the embodiments related to the composition apply here as well, alone or in combination.S. sonnei (Sson)[000150] The naturally-occurring Sson LPS O-Ag, which is shown in the figure below, has a disaccharide repeating unit consisting of two rare amino sugars: 2-acetamido-2-deoxy-L- altruronic acid (l.-Alt NAcA) and 2-acetamido-4-amino-2,4-dideoxy-D-fucose (D- FucpNAc4N):4)-a-L-AltoNAcA-A°Structure of the RU of the O-Ag in the naturally occurring LPS of Sson (A°B°).[000151] The presence of the l.-Alt NAcA (A°) and D-FucpNAc4N (B°) residues in the same repeating unit renders this structure zwitterionic.[000152] Embodiments of the synthetic Sson OS carrier conjugate in the present disclosure comprises a carrier with a spacer (e.g., an aminopropyl spacer) or linker linked to the D- FucpNAc4N residue, as shown in the example figures below (n = number of Sson disaccharide Rus, m = the average number of sOS per carrier, R = a linker, spacer or crosslinking reagent):[000153] The minimum number of Sson disaccharide RUs (n) necessary to achieve sufficient immune responses in animal models is generally 1 or 2. In some embodiments, the number of disaccharide RUs present in the Sson sOS hapten can be from 1 to 500, in particular from 2 to 50; in some embodiments, the number of disaccharide RUs present in the Sson sOS hapten can be from 2 to 10; in some embodiments, the number of disaccharide RUs present in the Sson sOS hapten can be from 2 to 8; in some embodiments, the number of disaccharide RUs present in the Sson sOS hapten can be from 2 to 5; in one embodiment, the number of disaccharide RUs present in the Sson sOS hapten is 4.S. flexneri serotype 6 (SF6):[000154] The O-Ag repeating unit (RU) of SF6, which is shown in the figure below, is a tetrasaccharide consisting of two L-rhamnose residues, one D-galacturonic acid residue, and one A-acetyl-D-galactosamine residue, rendering this an anionic PS:2)-a-L-Rha / ?3Ac / 4Ac-(l — >2-a-L-Rha / ?-(l — >4)-0-D-Gal / ? A-(l ^3)-P-D-Gal / ? NAc-(l — >Structure of the RU of the O-Ag in the naturally occurring LPS of SF6 (ACABC1D1).[000155] An embodiment of the synthetic SF6 OS is shown in the example figure below:X may represent H+or a pharmaceutically acceptable cation (i.e. forming a pharmaceutically acceptable salt). When X is H+, -CO?’, X+corresponds to -COOH.[000156] In one embodiment, at least one of Re or R7 is Ac.[000157] In one embodiment, Re or R7 is Ac.[000158] In one embodiment, at least one of Re or R7 is H.[000159] In one embodiment, Re and R7 is H.[000160] More detailed embodiments of a synthetic SF6 OS carrier conjugate are shown in the example figures below (n = 3, number of SF6 tetrasaccharide RUs; m = average number of sOS (OS) per carrier), in which a carrier and a linker, spacer or crosslinking agent (e.g., a thioether-type spacer) is linked to the D-Gal NAc residue:[000161] The minimum number (n) of SF6 tetrasaccharide RUs necessary to achieve sufficient immune responses in animal models is generally 1 or 2. In some embodiments, the number of tetrasaccharide RUs present in the SF6 sOS hapten can be from 2 to 500, in particular from 2 to 50; in some embodiments, the number of tetrasaccharide RUs present in the SF6 sOS hapten can be from 2 to 10; in some embodiments, the number of tetrasaccharide RUs present in the SF6 sOS hapten can be from 2 to 5; in one embodiment, the number of tetrasaccharide RUs present in the SF6 sOS hapten is 3.[000162] In naturally occurring SF6 LPS, positions 3A and 4A are (9-acetylated at about -60% and -30%, respectively. While the presence of (9-acetylation is frequently an important quality attribute in glycoconjugate vaccines to effect desired immune responses, it was determined that non-O-acetylated SF6 sOSs can elicit sufficient immune responses in animal models. The degree and location of (9-acetylation present in the SF6 sOS can be variable and different combinations are possible, wherein, in some embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to about 100%, respectively; in some embodiments, positions 3 A and 4A are independently (9-acetylated at about 0% to 30%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to 60%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to 80%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 30% to 60%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 60% to 100%, respectively; in some embodiments, both positions 3 A and 4A are not (9-acetylated. In some embodiments, both positions 3A and 4A are (9-acetylated stoichiometrically. In some embodiments, position3A is not (9-acetylated, whereas position 4A is (9-acetylated stoichiometrically. In some embodiments, position 4A is not (9-acetylated, whereas position 3A is (9-acetylated stoichiometrically.S. flexneri serotype 3a (SF3a):[000163] The O-Ag repeating unit (RU) of SF3a, which is shown in the figure below, is a pentasaccharide consisting of three L-rhamnose residues, one D-glucose residue, and one N- acetyl-D-glucosamine residue:2)-[a-D-Glcp-(1 -^3)]-<z-L-Rhap-(1 -^2)-a-L-Rhap-(1 -^3)-<z-L-Rhap2Ac-(1 -^3)-f-DGIcpNAc6Ac-(1 E A B AcC ACDStructure of the RU of the O-Ag in the naturally occurring SF3a LPS ([EJABACCACD)[000164] An embodiment of the synthetic SF3a OS is shown in the example figure below:[000165] In one embodiment, at least one of R4 or R5 is Ac.[000166] In one embodiment, R4 or R5 is Ac.[000167] In one embodiment, at least one of R4 or R5 is H.[000168] In one embodiment, R4 is H.[000169] In one embodiment, R5 is Ac.[000170] More detailed embodiments of a synthetic SF3a OS carrier conjugate are shown in the example figures below (n = 3; number of SF3a pentasaccharide RUs; m = average number of sOS per carrier), in which a carrier and a linker, spacer or crosslinking agent ( a thioether-type spacer) is linked to the D-GlcpNAc residue:[000171] The minimum number of SF3a pentasaccharide RUs (n) necessary to achieve sufficient immune responses in animal models is generally 1 or 2. In some embodiments, thenumber of pentasaccharide RUs present in the SF3a sOS hapten can be from 2 to 500, in particular from 2 to 50; in some embodiments, the number of pentasaccharide RUs present in the SF3a sOS hapten can be from 2 to 10; in some embodiments, the number of pentasaccharide RUs present in the SF3a sOS hapten can be from 2 to 4; in one embodiment, the number of pentasaccharide RUs present in the SF3a sOS hapten is 3.[000172] In naturally occurring SF3a LPS, position 2Cis (9-acetylated at about 100% (stoichiometric) and position 6D is (9-acetylated at about 40%, respectively. The degree and location of (9-acetylation present in the SF3a sOS can be variable and different combinations are possible, wherein, in some embodiments, positions 2c and 6D are independently O- acetylated at about 0% to about 100%, respectively; in some embodiments, positions 2c and 6D are independently (9-acetylated at about 0% to 30%, respectively; in some embodiments, positions 2c and 6D are independently (9-acetylated at about 0% to 60%, respectively; in some embodiments, positions 2c and 6D are independently (9-acetylated at about 0% to 80%, respectively; in some embodiments, positions 2c and 6D are independently (9-acetylated at about 30% to 60%, respectively; in some embodiments, positions 2c and 6D are independently (9-acetylated at about 60% to 100%, respectively; in some embodiments, both positions 2c and 6D are not (9-acetylated. In some embodiments, both positions 2c and 6D are (9-acetylated stoichiometrically. In some embodiments, position 2c is not (9-acetylated, whereas position 6ois (9-acetylated stoichiometrically. In some embodiments, position 6D is not (9-acetylated, whereas position 2c is (9-acetylated stoichiometrically.S. flexneri serotype 2a (SF2a):[000173] The O-Ag repeating unit (RU) of SF2a, which is shown in the figure below, is a pentasaccharide consisting of three L-rhamnose residues, one D-glucose residue, and one N- acetyl-D-glucosamine residue:2)-a-L-Rhap3Ac / 4Ac-(1 -^2)-<z-L-Rhap-(1 -^3)-[<z-D-Glcp-(1 -^4)]-<z-L-Rhap-(1 -^3)-f-DGIcpNAc6Ac-(1 - ACA B E C ACDStructure of the RU of the O-Ag in the naturally occurring SF2a LPS (ACAB[E]CACD)[000174] The monosaccharide composition of the SF2a RU is the same as that of SF3a, however, the linkages between the monosaccharides are different.[000175] An embodiment of the synthetic SF2a OS is shown in the example figure below:[000176] In one embodiment, at least one of Ri, R2, or R3 is Ac.[000177] In one embodiment, Ri or R2 is Ac.[000178] In one embodiment, at least one of Ri, R2, and R3 is H.[000179] In one embodiment, Ri, R2, and R3 are H.[000180] A more detailed embodiment of a synthetic SF2a OS carrier conjugate is shown in the example figures below (n = 3; number of SF2a O-Ag RUs; m = average number of sOS per carrier), in which a carrier and a linker or spacer (e.g., a thioether-type spacer) is linked to the D-GlcpNAc residue:[000181] The minimum number of SF2a pentasaccharide RUs necessary to achieve sufficient immune responses in animal models is generally 1 or 2. In some embodiments, the number of pentasaccharide RUs present in the SF2a sOS hapten can be from 2 to 500; in some embodiments, the number of pentasaccharide RUs present in the SF2a sOS hapten can be from 2 to 10; in some embodiments, the number of pentasaccharide RUs present in the SF2a sOS hapten can be from 2 to 4; in one embodiment, the number of pentasaccharide RUs present in the SF2a sOS hapten is 3.[000182] In naturally occurring SF2a LPS, positions 3A and 4A are (9-acetylated at about 65% and 25%, respectively, and position 6D is (9-acetylated at about 60%. The degree and location of (9-acetylation present in the SF2a sOS can be variable and different combinations are possible, wherein, in some embodiments, positions 3A and 4A are independently O-acetylated at about 0% to about 100%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to 30%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to 60%, respectively; insome embodiments, positions 3A and 4A are independently (9-acetylated at about 0% to 80%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 30% to 60%, respectively; in some embodiments, positions 3A and 4A are independently (9-acetylated at about 60% to 100%, respectively; in some embodiments, both positions 3A and 4A are not (9-acetylated. In some embodiments, both positions 3A and 4A are (9-acetylated stoichiometrically. In some embodiments, position 3A is not (9-acetylated, whereas position 4A is (9-acetylated stoichiometrically. In some embodiments, position 3A is not (9-acetylated, whereas position 4A is (9-acetylated stoichiometrically.Vaccine Conjugates[000183] As a directing concept to the proposed invention that diverges from all multivalent Shigella vaccines reported to date, is the fact that the proposed multivalent or quadrivalent Shigella vaccine of the present disclosure includes two, three or all four monovalent conjugates, which, once combined, provide the proposed multivalent or quadrivalent Shigella vaccine of the present disclosure and are made of sOSs designed to act as functional mimics of the homologous natural type-specific Shigella O-Ag. However, many variables in the design, development, and production of glycoconjugate vaccines influence their immunogenicity and presumably their efficacy. In this regard, for each monovalent component, possible impacting variables, include, for example, the exact OS hapten for each valence, with further key parameters including, at least, the chain length, whether corresponding to an exact number of basic O-Ag RUs or extended with short fragments thereof; the nature of the reducing end residue; the nature of the end chain residue; the presence / absence of non-carbohydrate substitutions, especially ( -acetyl groups, whose influence on immunogenicity is known but does not obey any established rule; and / or the presence / absence of the natural charges. Additional possible impacting variables, include, for example, the nature of the carrier, whether a protein (e.g., cross-reactive, chemically detoxified / recombinant) or otherwise; and the selected linker (if used), in terms of, for example, its size, conjugation chemistry (e.g., amino acids serving for covalent attachment in the case of a protein carrier), chemical composition and / or the site of linker-attachment on the glycan.Carrier Selection[000184] According to embodiments described herein, the carrier for the vaccine conjugate comprises TT. In this regard, TT was determined to be a suitable carrier for SF2a-TT. For example, a monovalent synthetic carbohydrate-based monovalent SF2a vaccine candidate, in which the carrier was TT was developed by Institut Pasteur, which has successfully undergone clinical trials in Kenya (see, NCT04602975). TT is obtained upon chemical detoxification of tetanus toxin, a 150 kDa protein isolated from culture of Clostridium tetani.[000185] According to some embodiments, the carrier, e.g., TT, is covalently linked to a certain number (m) of OS chains of n repeat units (RUs) of O-Ag. According to one embodiment, the number (m) of pentasaccharide RUs of O-Ag is 3 (i.e., a 15mer OS), thus leading to 15mer OS-TT conjugates, e.g., SF2a-TT15 or SF3a-TT15. According to one embodiment, the number (n) of tetrasaccharide RUs of O-Ag is 3 (i.e., a 12mer OS), resulting in 12mer-protein conjugates, e.g., SF6-TT12. According to one embodiment, the number (n) of disaccharide RUs of O-Ag is 4 (i.e., an 8mer OS), resulting in an 8mer-protein conjugate, e.g., Sson-TT8.[000186] Other potential carriers and / or carrier proteins that could be used in the Shigella vaccine composition include, for example, tetanus toxin C fragment (TTc), non-toxic diphtheria toxin mutant (including, e.g., CRM197 or CRM9), rEPA, recombinant TT or recombinant TT with certain mutations (including, e.g., a recombinant full-length 8MTT corresponding to a non-toxic tetanus toxin with eight mutations that was engineered in E. coli), rTTHc (52 kDa), or a cross-reactive Shigella Ipa protein. In addition, a mutated IpaB protein incorporating non-natural azido-amino acids was recently demonstrated to be a potent cross-reactive carrier for Sson and SF2a PSs. Thus, according to one embodiment, the carrier comprises TT, TTc, CTB, DT, non-toxic diphtheria toxin mutant (including, e.g., CRM9 or CRM 197), EP A, rEPA, CjAcrA, EcAcrA, heat labile enterotoxigenic Escherichia coli (ETEC) subunit B (LTB), QP (Qubevirus durum), outer membrane vesicles (OMVs), glycoengineered proteins, virus-like particles (VLPs), nanocarriers or nanoparticles (such as, e.g., self-assembling and non-self-assembling nanoparticles, including, e.g., ferritin nanoparticles, liposomes, lipid nanoparticles, gold nanoparticles, etc.), recombinant TT and / or recombinant TT with certain mutations, Generalized Modules for Membrane Antigens (GMMA), PcrV, Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), rTTHc (52 kDa), a cross-reactive ShigellaIpa protein, or a mutated IpaB protein (including, e.g., a mutated IpaB incorporating nonnatural azido-amino acids).[000187] According to some embodiments, other potential carriers include, e.g., Neisseria meningitidis outer membrane protein, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, human serum albumin (including, e.g., recombinant), universal CD4+ cell epitopes, in particular artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N19 or tetanus toxoid, pneumococcal surface protein PspA, pneumolysin, iron-uptake proteins, toxin A or B from Clostridium difficile, a GBS protein, e.g., GBS80, GBS67 and GBS59 from Streptococcus agalactiae and fusion proteins, for example, GBS59(6xD3), synthetic peptides bearing immunodominant T-helper cell epitopes (with natural or not peptide sequence), lipopeptides, for example, Pam(3)CAG, zwitterionic PSs, the peptide PADRE, among others.[000188] According to some embodiments, the carrier proteins and / or polypeptides (such as, e.g., TT and CRM) may be present in a mono- or multivalent fashion.[000189] According to some embodiments, the carrier proteins and / or polypeptides (such as, e.g., TT and CRM) may be expressed in native organisms or recombinantly, and may contain both natural and non-natural amino acids (e.g., mutations, chemical modifications, etc.).[000190] According to some embodiments, each of the sOSs is independently conjugated to the same type of carrier (e.g., TT) or different types of carriers.[000191] According to some embodiments, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to 0.1 pg (TT) to 200 pg (TT) per total amount of each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 0.5 pg (TT) to about 50 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to from about 1 pg (TT) to about 100 pg (TT) per each OS.According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to from about1 pg (TT) to about 150 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to from about 1 pg (TT) to about 175 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to from about 1 pg (TT) to about 10 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 10 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 20 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 80 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 125 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 150 pg (TT) per each OS. According to one embodiment, the carrier (e.g., TT) included with the multivalent Shigella vaccine composition is provided in the conjugates in an amount corresponding to about 175 pg (TT) per each OS.Conjugation Chemistry[000192] In traditional glycoconjugate vaccines, the carbohydrate and protein components are usually modified to install chemoselective functional groups to allow chemical conjugation. Bacterial PSs are typically produced via fermentation, followed by harvesting and purification and chemical activation prior to conjugation to carrier protein. As a result, the purified PSs are a heterogeneous mixture of different sized molecules, which can undergo size reduction via enzymatic degradation, chemical hydrolysis, mechanical means, or otherwise, if desired. Depending on the size, structure, and physicochemical properties of the carbohydrate, and the nature of the carrier, different chemistries can be employed to produce glycoconjugates. Examples of common chemistries used in carbohydrate activation known in the art include, but are not limited to, modification of hydroxy groups with cyanylatingagents (e.g., CNBr and CDAP) or carbonyldiimidazole to form active esters and / or periodate oxidation of vicinal diols or trans-diols in the sugar ring or at the glycerol moiety of sialic acid residues to generate aldehydes ready for conjugation to the amino groups of carrier proteins. Alternatively, amino groups can be introduced with ammonium salts or with dihydrazide spacers to the carbohydrate, which can react directly with carboxylic acids of the protein through carbodiimide chemistry or coupled to a variety of bifunctional linkers to incorporate squaric ester, maleimide, thiol, azide, hydrazide, hydrazine, or alkyne moieties for further conjugation. In cases where chemoselective chemistry (e.g., Michael reaction, click chemistry) is used in the preparation of glycoconjugates, the carrier protein is modified accordingly to introduce the appropriate orthogonal chemical groups to react with the linker introduced onto the carbohydrate. In many cases, the activation of the carbohydrates and / or modification of the carrier protein is random, multiple sites of attachment are possible, resulting in a heterogeneous mixture of cross-linked, lattice-type conjugates.[000193] By contrast, the conjugates included in the multivalent vaccine compositions in the instant disclosure comprise chemically well-defined and homogeneous OSs acting as surrogates of the O-Ag, and wherein a linker (e.g., an aminoethyl and / or aminopropyl linker) is installed at the reducing end of the sOSs, which can be further modified to include suitable chemical groups for conjugation to the carrier protein via a single point of attachment, yielding “sun-type” conjugates. Different linkers with various lengths can be used, including but are not limited to, adipic acid dihydrazide, aminoalkyl, hydrazine-PEG-hydrazine, etc., where the PEG moieties in the linker have been implicated to prolong circulation and could enhance immunogenicity in vaccine formulations.[000194] The linker is in particular a linker aglycone (i.e., linked to the reducing end of the OS, for example the aminopropyl group).[000195] The linker (in this case, it may also be referred to as a spacer) is in particular a bioorthogonal bifunctional species, which allows for reaction via the formation of an amide bond on the one hand and a thiol maleimide reaction on the other.[000196] For example, in one embodiment, two or more monovalent sOSs (including, e.g., four monovalent sOSs) are each independently conjugated to a carrier via one or more points of attachment (e.g., covalent attachment). In another embodiment, two or more monovalentsOSs (including, e.g., four monovalent sOSs) are each independently conjugated to a carrier via a single point of attachment (e.g., covalent attachment).Degree of Conjugation (molar ratio between OS hapten and carrier protein)[000197] In selecting the conjugation chemistry, it can be appreciated by a skilled artisan that using a single carrier protein and the same conjugation chemistry to conjugate different Shigella haptens would greatly simplify the manufacturing processes. However, it is also known that, in multivalent vaccine compositions, the accumulation of carrier protein and linker from multiple glycoconjugates may inadvertently divert immune recognition towards epitopes on the carrier protein and linker, thereby suppressing the desired immune responses towards the target carbohydrate haptens. Therefore, the molar ratio of carbohydrate haptens to carrier protein (degree of conjugation) must be fine-tuned to achieve desired immunological responses, while balancing conjugation reaction yields, cost of production, and achieving a reproducible and robust manufacturing process. Through extensive experimentation and systematic optimization of the conjugation reaction condition parameters (e.g., reaction buffer, pH, duration, concentration of reaction components, and input molar ratio of OS to a carrier (e.g., TT)), the Shigella OS-carrier conjugates disclosed herein possess a range of degree of conjugation that achieves desired immunological responses, while balancing the other factors discussed above.[000198] For example, according to one embodiment, for the monovalent Sson-carrier conjugate, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier (e.g., TT) can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 10 to 20. In someembodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier can be from 1 to 5.[000199] In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 5 to 30. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 10 to 30. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 12 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RU for Sson and carrier can be from 10 to 20. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 10 to 18. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 10 to 15. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 12 to 18. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier can be from 12 to 15. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 16-17. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 18-19. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 20-21. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 10. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 12. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 13. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 14. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 15. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 16. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 17. In some embodiments, thedegree of conjugation between synthetic Sson OS and carrier is about 18. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 19. In some embodiments, the degree of conjugation between synthetic Sson OS and carrier is about 20.[000200] According to another embodiment, for the monovalent SF6-carrier conjugate, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier (e.g., TT) can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier can be from 1 to 5.[000201] In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier can be from 5 to 30. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier can be from 10 to 20. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier can be from 15 to 25. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 16-17. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 18-19. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 20-21. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 22-23. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 10. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 15. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 16. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 17. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 18. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 19. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 20. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 21. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 22. In some embodiments, the degree of conjugation between synthetic SF6 OS and carrier is about 23.[000202] According to some embodiments, for the monovalent SF3a-carrier conjugate, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier (e.g., TT) can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 30. In some embodiments, the degree ofconjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier can be from 1 to 5.[000203] In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier can be from 5 to 30. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier can be from 10 to 30. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier can be from 15 to 30. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier can be from 15 to 25. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier can be from 10 to 20. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 16-17. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 18-19. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 20-22. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 26-28. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 18-28. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 18. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 19. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 20. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 21. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 22. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 23. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 24. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 25. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 26. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 27. In some embodiments, the degree of conjugation between synthetic SF3a OS and carrier is about 28.[000204] According to another embodiment, for the monovalent SF2a-carrier conjugate, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier (e.g., TT) can be from 1 to 500. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 200. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 100. In one embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 60. In another embodiment, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 5 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 15 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 15 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 10 to 20. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 50. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 30. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 25. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 15. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 10. In some embodiments, the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier can be from 1 to 5.[000205] In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier can be from 5 to 30. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier can be from 10 to 20. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier can be from 15 to 25. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier can be from 20 to 25. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 16-17. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 18-19. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 20-22. In some embodiments, the degree ofconjugation between synthetic SF2a OS and carrier is about 20-24. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 24-28. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 18. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 19. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 20. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 21. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 22. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 23. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 24. In some embodiments, the degree of conjugation between synthetic SF2a OS and carrier is about 25.[000206] In evaluating the efficacy of multivalent Shigella vaccine compositions that can provide the broadest coverage in test subjects, different combinations of bi-, tri, and tetravalent (or quadrivalent) formulations comprising monovalent conjugates of Shigella serotype Sson, SF2a, SF3a, and / or SF6 were evaluated in suitable animal models (e.g., mouse, rabbit), using relative weight ratios of OS equivalents per conjugate. For example, according to one embodiment, in a bivalent formulation (e.g., SF2a: SF3a or Sson: SF2a), the weight ratio between the two conjugates, based in weight of the OS part of each conjugate, can be about 0.25:1, about 0.5:1, about 0.75:1, about 1:1, about 1:1.5, and about 1:2.According to another embodiment, in a trivalent formulation (e.g., SF2a: SF3a: SF6 or SF2a: SF3a: Sson), the weight ratio between the three conjugates, based in weight of the OS part of each conjugate, can be about 1:1:1, about 1:1:1.5, about 1:1:2, about 0.75:1:1, about 0.75:1:1.5, about 0.75:1:2, about 0.5:1:1, about 0.5:0.75:1, about 0.5:1:1.5, about 0.5:0.75:1.5; about 0.5:1:2; and about 0.5:0.75:2. According to another embodiment, in a quadrivalent formulation (e.g., SF2a: SF3a: SF6: Sson), the weight ratio between the four conjugates, based in weight of the OS part of each conjugate, can be about 1: 1: 1: 1, about 1: 1: 1:0.5, about 1:1:1:0.75, about 1:1:0.5:1, about 1:1:0.75:1, about 1:1:0.5:0.5, about 1: 1:0.75:0.75. According to one embodiment of a quadrivalent formulation comprising Sson-TT (e.g., Sson-TT8), SF3a-TT (e.g., SF3a-TT15), SF6-TT (e.g., SF6-TT12), and SF2a-TT8 (e.g., SF2a-TT15) conjugates in a weight ratio of 1:1: 1:1, robust immunogenicity (IgG titers) and functional responses (SB A) were observed in mice and rabbits. According toanother embodiment, a quadrivalent formulation comprising SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT conjugates in a weight ratio of 1: 1: 1:0.5 is provided.[000207] Dose Selection[000208] Licensed conjugate vaccines used for infant population usually include about 1-10 pg of bacterial carbohydrates per vaccine dose, with the dosage varying based on the age of vaccination in infants. The immune system of the pediatric population is not fully developed and therefore immunogenicity is more difficult to elicit than in adults, especially in the youngest infants. Since the multivalent (e.g., quadrivalent) vaccine of the present disclosure is intended to be used in infants starting at 6 months, and previous studies with other vaccine candidates showed that the immunogenicity to certain serotypes was reduced in infants and the efficacy was null, the dose range for the multivalent (e.g., quadrivalent) vaccine of the present disclosure needed to provide safety and a dose that gives maximum immunogenicity in young infants. According to one embodiment, the monovalent component of SF2a included in the multivalent (e.g., quadrivalent) vaccine of the present disclosure was tested in infants at 2 pg and 10 pg, with and without A1H as an adjuvant, and both doses reached different levels of immunogenicity. However, having a multivalent or quadrivalent vaccine with different serotypes in which immune response could be different, higher doses needed to be tested. The use of an adjuvant and / or higher doses could also be helpful in reducing the number of injections needed on the primary series or even the time to boost. According to some embodiments, a dose selection of 2, 10, and 30 pg of OS / serotype can be used.However, any dose selection is possible depending on the level of immunogenicity needed. The dose selection should take into account the carrier (e.g., TT) content, including, e.g., OS: TT molar loading, in order to avoid including high concentrations of carrier as TT is a commonly used protein carrier for other licensed pediatric vaccines.Multivalent Shigella Vaccine Formulations[000209] Adjuvants[000210] Adjuvants are common components of licensed vaccines that help increase the immune response (e.g., antibody titers and / or cell mediated immunity) in relation to magnitude, breadth, and durability. Common active ingredients of adjuvants used in vaccine formulations include, for example, aluminum salts (including, e.g., aluminum oxyhydroxide, usually referred to as aluminum hydroxide (Al(0H)3) (referred to herein as “A1H” or “H” or“A10H” or “ALOH” or “alum”), and aluminum hydroxyphosphate, usually referred to as aluminum phosphate (AIPO4) (referred to herein as “P”, “A1P” or “A1P0” or “ALPO”), double mutant heat-labile toxin (dmLT), TLR agonists (e.g., TLR4, TLR 7 / 8, TLR9), oligonucleotides (CPG), saponins, squalenes, and viral membranes. In addition, vaccine formulations can also include other components, such as, e.g., vitamin E, with formulations comprising, e.g., liposome, oil-in-water emulsions, etc.[000211] In one embodiment, the adjuvant is selected from at least one of MATRIX M®, CPGs, QS-21, amorphous aluminum hydroxyphosphate sulfate (AAHS), potassium aluminum sulfate, aluminum phosphate, aluminum hydroxide, double mutant heat-labile toxin (dmLT), or combinations thereof.[000212] The multivalent Shigella vaccine compositions disclosed herein may or may not comprise any adjuvant. In some embodiments, the multivalent Shigella vaccine compositions disclosed herein comprise an alum salt. In some embodiments, the multivalent Shigella vaccine compositions disclosed herein comprise aluminum phosphate (e.g., ADJUPHOS®). In some embodiments, the multivalent Shigella vaccine compositions disclosed herein comprise aluminum hydroxide (e.g., ALHYDROGEL®). In some embodiments, the multivalent Shigella vaccine compositions disclosed herein comprise Matrix M®.[000213] According to some embodiments, the multivalent Shigella vaccine formulation includes an adjuvant in an amount that is both safe and effective at increasing the immune response of the vaccine. For example, according to one embodiment, the adjuvant (e.g., alum salt) is provided in an amount of 150 to 600 pg / dose. According to another embodiment, in the vaccine formulations and compositions of the present invention, there is between 200 and 500 pg of adjuvant per dose of vaccine. According to another embodiment, in the vaccine formulations and compositions of the present invention, there is between 250 and 500 pg of adjuvant per dose of vaccine. In alternative embodiments of the vaccine formulations and compositions of the present invention, there is between 300 and 500 pg adjuvant per dose of vaccine.[000214] Other Excipients[000215] According to some embodiments, the multivalent Shigella vaccine formulation includes additional components and / or excipients, including, but not limited to, pharmaceutically acceptable excipients, additives, diluents, buffers (e.g., histidine, citrate,Tris, phosphates, etc.), sugars, amino acids (such as, e.g., glycine, glutamine, asparagine, arginine or lysine), chelating agents, surfactants, polyols, bulking agents, stabilizers, lyoprotectants, cryoprotectants, solubilizers, emulsifiers, adjuvants, tonicity modifiers and / or salts, or enhancing agents (such as, e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol), delivery vehicles, and / or preservatives.[000216] According to some embodiments, a surfactant is provided in the multivalent Shigella vaccine formulation, with the surfactant comprising, e.g., nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Polysorbates, sold under the trade name Tween®), including Polysorbate-20 (polyoxyethylene sorbitan monolaurate), Polysorbate-40 (polyoxyethylene sorbitan monopalmitate), Polysorbate-60 (polyoxyethylene sorbitan monostearate), and Polysorbate-80 (polyoxyethylene sorbitan monooleate); polyoxyethylene alkyl ethers, poloxamers (e.g., poloxamer 188); Triton® X-100 and Triton® X-114;NP40; Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; copolymers of ethylene and propylene glycol (e.g., the Pluronic® series of nonionic surfactants such as Pluronic® F68, Pluronic® 10R5, Pluronic® F108, Pluronic® F127, Pluronic® F38, Pluronic® L44, Pluronic® L62; and sodium dodecyl sulfate (SDS).[000217] According to one embodiment, the multivalent Shigella vaccine formulation comprises a surfactant (such as, e.g., polysorbate-80 (PS80)) in a concentration of from about 0.001% to about 0.5% (wt / vol). In some embodiments of this aspect of the invention, the surfactant is present in the formulation in an amount from about 0.005% to about 0.4%; in other embodiments, the surfactant is present in an amount from about 0.01% to about 0.3%; in other embodiments, the surfactant is present in an amount from about 0.01% to about 0.2%; in other embodiments, the surfactant is present in an amount from about 0.01% to about 0.1%; in other embodiments, the surfactant is present in an amount from about 0.01% to about 0.05%. In some embodiments, the surfactant is present in an amount of about 0.02%. In some embodiments, the surfactant is present in an amount of about 0.01%.[000218] According to some embodiments, the multivalent Shigella vaccine formulation comprises a buffer (such as, e.g., Tris) in an amount of about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 75 mM, about 5 mM to about 60 mM, about 5 mM to about 50 mM, about 10 mM to about 90 mM, about 10 mM to about 75 mM, about 10 to about 60 mM, about 10 mM to about 50 mM, about 20 mM to about 90 about, about 20mM to about 75 mM, about 20 to about 60 mM, or about 20 mM to about 50 mM. In alternative embodiments, the vaccine composition comprises about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM of buffer.[000219] According to some embodiments, the multivalent Shigella vaccine formulation comprises a salt and / or a tonicity modifier (e.g., NaCl, KC1, Na₂SO₄, (NH₄)₂SO₄, sodium phosphate, sodium citrate, glycerin, boric acid, calcium chloride, or dextrose) in a concentration of from about 100 mM to 1000 mM. According to another embodiment, the multivalent Shigella vaccine formulation comprises a salt in a concentration from about 100 mM to about 500 mM; according to some embodiments, a salt is provided in a concentration from about 150 mM to 300 mM. In alternative embodiments of the invention, the multivalent Shigella vaccine formulations do not comprise salt.[000220] According to some embodiments, the multivalent Shigella vaccine formulation comprises a preservative (e.g., 2 -phenoxy ethanol (2 -PE), thimerosal, m-cresol, phenol, benzyl alcohol, etc.) in a concentration of 0.01% wt / wt to 10% wt / wt. In one embodiment, the multivalent Shigella vaccine formulation comprises a preservative in a concentration of 0.5% wt / wt to 5% wt / wt. In one embodiment, the multivalent Shigella vaccine formulation comprises a preservative in a concentration of 0.5% wt / wt to 1% wt / wt. In one embodiment, the multivalent Shigella vaccine formulation comprises a preservative in a concentration of 1% wt / wt.[000221] According to one embodiment, a high-dose quadrivalent Shigella vaccine formulation comprises 60 pg / mL each of SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT (based on saccharide concentration), 50 mM Tris, 150 mM NaCl, 1% wt / wt 2-phenoxyethanol (2-PE), 0.02% (wt / v) polysorbate-80 (PS80), and 250-500 pg / mL Al salt at pH 7.0.[000222] According to one embodiment, a mid-dose quadrivalent Shigella vaccine formulation comprises 20 pg / mL each of SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT (based on saccharide concentration), 50 mM Tris, 150 mM NaCl, 1% wt / wt 2-phenoxyethanol (2-PE), 0.02% (wt / v) PS80, and 250-500 pg / mL Al salt at pH 7.0.[000223] According to one embodiment, a low-dose quadrivalent Shigella vaccine formulation comprises 4 pg / mL each of SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT (based on saccharide concentration), 50 mM Tris, 150 mM NaCl, 1% wt / wt 2-phenoxyethanol (2 -PE), 0.02% (wt / v) PS80, and 250-500 pg / mL Al salt at pH 7.0.Exemplary Vaccine Compositions[000224] Specific embodiments of the invention will now be demonstrated by reference to the following examples. It should be understood that these examples are disclosed by way of illustrating the invention and should not be taken in any way to limit the scope of the present invention.[000225] A SF2a / Sson bivalent vaccine candidate[000226] A bivalent Shigella SF2a / Sson vaccine candidate based on the combination of a monovalent SF2a vaccine candidate and a monovalent Sson vaccine candidate was prepared. For example, according to one embodiment, the bivalent Shigella SF2a / Sson vaccine candidate was prepared by combining SF2a-TT (including, e.g., SF2a-TT15) and Sson-TT (including, e.g., Sson-TT8) in a 1:1 molar ratio, with SF2a-TT having an OS to carrier (TT) (OS: TT) molar ratio of 17, and Sson-TT having an OS: TT molar ratio of 12.[000227] To determine whether there was any significant loss of immunogenicity between the monovalent vaccine formulation (SF2a-TT or Sson-TT alone) and the bivalent vaccine formulation (SF2a-TT combined with Sson-TT), the following experiment was conducted, which compared IgG titers of mice that received injections of a monovalent SF2a-TT vaccine conjugate and a monovalent Sson-TT vaccine conjugate separately or combined in a 1:1 ratio to achieve 2.0 pg of OS each per dose, adjuvanted with aluminum hydroxide. Bleeding of the mice for the IgG titers was at three weeks after the 2ndinjection (see Figure 1A) and after the 3rdinjection (see Figure IB), respectively. As shown in Figures 1A and IB, the X-axis illustrates the relevant monovalent and bivalent glycoconjugate formulations, while the Y- axis illustrates the anti-SF2a LPS IgG titer and the anti-Sson LPS IgG titer.[000228] As shown in Figures 1A and IB, for SF2a, after three injections, no statistically significant differences were observed between the monovalent and bivalent formulations, which was independent of the type of adjuvant. Similarly, for Sson, after two and three injections, no statistically significant differences were observed between the monovalent and bivalent formulations. For the two serotypes, the differences in anti-LPS IgG titers induced by the monovalent conjugates and the bivalent combinations were within one log and above 103. Thus, data available for the SF2a-TT / Sson-TT bivalent vaccine combinations showed the absence of any significant loss of immunogenicity in mice for each one of the SF2a andSson glycoconjugates, when combined in equal OS wt:wt amounts (see, e.g., Figures 1A and IB).[000229] A SF2a / SF3a bivalent vaccine candidate[000230] A bivalent Shigella SF2a / SF3a vaccine candidate based on the combination of a monovalent SF2a vaccine candidate and a monovalent SF3a vaccine candidate was prepared. For example, according to one embodiment, the bivalent Shigella SF2a / SF3a vaccine candidate was prepared by combining SF2a-TT (including, e.g., SF2a-TT15) with SF3a-TT (including, e.g., SF3a-TT15) in a 1:1 ratio, with SF2a-TT having an OS to carrier (TT) (OS: TT) molar ratio of 23, and SF3a-TT having an OS: TT molar ratio of 21.[000231] To determine whether use of an adjuvant (e.g., alum) impacted the immunogenicity in mice between the monovalent vaccine formulation (SF2a or SF3a alone) and the bivalent vaccine formulation (SF2a combined with SF3a), the experiment shown in Figures 2A and 2B was conducted. As shown in this experiment of Figures 2A and 2B, there was not any significant loss of immunogenicity in mice for each one of the SF2a and SF3a glycoconjugates, when combined in equal OS wt:wt amounts with or without alum (i.e., adjuvant). This figure also shows the added value of alum (or adjuvant), when using a low amount of OS per dose.[000232] To determine whether there was any significant loss of immunogenicity between the monovalent vaccine formulation (SF2a or SF3a alone) and the bivalent vaccine formulation (SF2a combined with SF3a), an experiment was conducted, which compared anti-SF2a LPS and anti-SF3a LPS IgG titers of mice that received injections of a monovalent SF2a vaccine conjugate and a monovalent SF3a vaccine conjugate separately or combined in a 1:1 ratio to achieve 1.0 pg of OS each per dose, adjuvanted with alum. The data available for the SF2a / SF3a bivalent vaccine combinations showed the absence of any significant loss of immunogenicity in mice for each one of the SF2a and SF3a glycoconjugates, when combined in equal OS wt:wt amounts (see, e.g., Figure 3).[000233] A SF2a / SF3a / SF6 trivalent vaccine candidate[000234] A trivalent Shigella SF2a / SF3a / SF6 vaccine candidate based on the combination of a monovalent SF2a vaccine candidate, a monovalent SF3a vaccine candidate, and a monovalent SF6 vaccine candidate was prepared. For example, according to one embodiment, the trivalent Shigella SF2a / SF3a / SF6 vaccine candidate was prepared bycombining SF2a-TT (including, e.g., SF2a-TT15, featuring a synthetic 15mer SF2a OS hapten (3 RUs)), SF3a-TT (including, e.g., SF3a-TT15, featuring a synthetic 15mer SF3a OS hapten (3 RUs)), and SF6-TT (including, e.g., SF6-TT12, featuring a synthetic 12mer Sson OS hapten (4 RUs)) in a 1:1:1 wt:wt:wt ratio based on OS content per valence, with SF2a-TT having an OS to carrier (TT) (OS: TT) molar ratio of 14, SF3a-TT having an OS: TT molar ratio of 19, and SF6-TT having an OS-TT molar ratio of 13 (see, e.g., Table 1 below, Exp 2).[000235] As shown in Figures 5A-5C, the prepared trivalent Shigella vaccine formulations comprising SF2a-TT, SF3a-TT, and SF6-TT were found to be immunogenic, inducing high IgG antibody titers against all serotypes present in the trivalent formulation. Still, there was a significant reduction in the anti-LPS IgG titers induced after three injections by the trivalent formulation in comparison to the monovalent formulation for both the SF3a and SF6 conjugates. Yet, available data show the absence of any major interference between serotypes, as the measured anti-LPS IgG titers remained in a one log range when comparing data for each of the monovalent conjugates to those for the trivalent formulation, and titers for the trivalent combination were all above the 103threshold.[000236] A SF2a / SF3a / Sson trivalent vaccine candidate[000237] A trivalent Shigella SF2a / SF3a / Sson vaccine candidate based on the combination of a monovalent SF2a vaccine candidate, a monovalent SF3a vaccine candidate, and a monovalent Sson vaccine candidate was prepared. For example, according to one embodiment, the trivalent Shigella SF2a / SF3a / Sson vaccine candidate was prepared by combining SF2a-TT (including, e.g., SF2a-TT15, featuring a synthetic 15mer SF2a OS hapten (3 RUs)), SF3a-TT (including, e.g., SF3a-TT15, featuring a synthetic 15mer SF3a OS hapten (3 RUs)), and Sson (including, e.g., Sson-TT8, featuring a synthetic 8mer Sson OS hapten) in a 1: 1: 1 wt:wt:wt ratio based on OS content per valence, with SF2a-TT having an OS to carrier (TT) (OS: TT) molar ratio of 14, SF3a-TT having an OS: TT molar ratio of 19, and Sson-TT having an OS-TT molar ratio of 13 (see, e.g., Table 1 below, Exp 1).[000238] As shown in Figures 5A, 5B, and 5D, the prepared trivalent Shigella vaccine formulations comprising SF2a-TT, SF3a-TT, and Sson-TT were found to be immunogenic, inducing high IgG antibody titers against all serotypes present in the trivalent formulation. Still, there was a significant reduction in the anti-LPS IgG titers induced after three injections by the trivalent formulation in comparison to the monovalent formulation for both the SF2aand Sson conjugates. Yet, available data show the absence of any major interference between serotypes, as the measured anti-LPS IgG titers remained in a one log range when comparing data for each of the monovalent conjugates to those for the trivalent formulation, and anti-LPS IgG titers measured for the trivalent combination in mice which had received three injections were all above the 103threshold.[000239] A SF2a / SF3a / SF6 / Sson quadrivalent vaccine candidate[000240] Quadrivalent Shigella vaccine formulations comprising SF2a-TT, SF3a-TT, SF6-TT and Sson-TT were prepared. For example, Figures 4A and 4B illustrate embodiments of a quadrivalent Shigella vaccine composition comprising selected SF2a, SF3a, SF6, and Sson monovalent glycoconjugates as components of the vaccine conjugates, with TT as the carrier. In this regard, Figure 4A illustrates a quadrivalent Shigella vaccine composition having SF2a, SF3a, SF6 and Sson monovalent glycoconjugates as components of the quadrivalent Shigella vaccine formulation, with TT as the carrier and an OS: TT loading of “n” (i.e., exact number of RUs per sOS hapten) and “m” (average number of sOS per carrier), according to one embodiment of the invention. Figure 4B illustrates a quadrivalent Shigella vaccine composition having SF2a, SF3a, SF6 and Sson monovalent glycoconjugates as components of the quadrivalent Shigella vaccine formulation, with TT as the carrier and an OS: TT loading of “n” (i.e., exact number of RUs per sOS hapten) and “m” (average number of sOS per carrier), according to one embodiment of the invention.[000241] According to an embodiment, a quadrivalent Shigella SF2a / SF3a / SF6 / Sson vaccine candidate based on the combination of a monovalent SF2a vaccine candidate, a monovalent SF3a vaccine candidate, a monovalent SF6 vaccine candidate, and a monovalent Sson vaccine candidate was prepared. For example, according to one embodiment, the quadrivalent Shigella SF2a / SF3a / SF6 / Sson vaccine candidate was prepared by combining SF2a-TT (including, e.g., SF2a-TT15, featuring a synthetic 15mer SF2a OS hapten (3 RUs)), SF3a-TT (including, e.g., SF3a-TT15, featuring a synthetic 15mer SF3a OS hapten (3 RUs)), SF6-TT (including, e.g., SF6-TT12, featuring a synthetic 12mer SF6 OS hapten (3 RUs)), and Sson (including, e.g., Sson-TT8, featuring a synthetic 8mer Sson OS hapten (4 RUs)), with SF2a-TT having an average OSto carrier (TT) (OS: TT) molar ratio of 14, 23 or 21, SF3a-TT having an average OS: TT molar ratio of 19, 13, 25, or 20, SF6-TT having anaverage OS: TT molar ratio of 13, 12, 19 or 20, and Sson-TT having an average OS-TT molar ratio of 12, 13, 10, or 20, respectively (see, e.g., Table 1 below).Table 1. Carbohydrate: protein molar ratio for the different monovalent glycoconjugates used in combination in the quadrivalent vaccine formulations.[000242] The prepared quadrivalent Shigella vaccine formulations comprising SF2a-TT, SF3a-TT, SF6-TT and Sson-TT were found to be immunogenic, inducing high IgG antibody titers against all serotypes present in the quadrivalent formulation. Interestingly, compositions covering a broad range of ratios between the different glycoconjugates quantified as OS content wt) were proven promising vaccine candidates. No major interference was observed between serotypes, as shown by the measured anti-LPS IgG titers, which remained in a one log range when comparing data for each of the monovalent conjugates to those for the QSV formulation. The selected monovalent conjugates SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT are combined in amounts according to a l:l:x:x ratio, with one embodiment in a 1: 1: 1: 1 ratio, based on OS relative weight amounts. According to another embodiment, a quadrivalent formulation comprising SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT conjugates in a weight ratio of 1: 1: 1:0.5 is provided. According to another embodiment, a quadrivalent formulation comprising SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT conjugates in a weight ratio of 2:2:2:2; 2-2-2-0.5; 2-2-2-0.2; and / or 2-2-0.5-0.2 is provided.[000243] Influence of a Three vs. Four Valence Combination (Figures 5A-5D; Exp 1 in Table 1 above)[000244] In order to determine the influence of the SF6 valence and the Sson valence, solo or in combination, on the immunogenicity of the SF2a and SF3a conjugates, respectively, the following experiment was conducted in which IgG titers were induced against SF2a, SF3a, SF6 and Sson LPS, respectively, in mice (seven mice per category) that received threeinjections of a trivalent or quadrivalent vaccine composition comprising SF2a-TT, SF3a-TT, SF6-TT and / or Sson-TT glycoconjugates combined in a 1: 1: 1:0, l:l:0:l, and 1: 1: 1: 1 ratio to achieve 2.0 pg of OS each per dose. All preparations were adjuvanted with aluminum hydroxide (Al(0H)3, “H”) or with aluminum phosphate (AIPO4, “P”). For each combination, the induced IgG titers are shown for the same type-specific monovalent conjugate administered at the same OS dose. Bleeding of the mice for the IgG titers was at three weeks after the 1st, 2ndand 3rdinjection, respectively (see, e.g., Figures 5A-5D, and Exp 1 in Table 1 above). As shown in Figures 5A-5D, the X-axis illustrates the relevant monovalent, trivalent, and quadrivalent glycoconjugate formulations (e.g., SF2a-TT, SF3a-TT, SF6-TT and / or Sson-TT), while the Y-axis illustrates the anti-SF2a LPS IgG titer (Figure 5A), the anti-SF3a LPS IgG titer (Figure 5B), the anti-SF6 LPS IgG titer (Figure 5C), or the anti-Sson LPS IgG titer (Figure 5D), respectively.[000245] As shown in Figures 5A and 5C, for SF2a and SF6, after three injections, no statistically significant differences were observed between the monovalent and quadrivalent formulations, independent the adjuvant. As shown in Figures 5B and 5D, for SF3a and Sson, after three injections, the monovalent conjugate was found to be superior to the quadrivalent combination. As further shown in Figures 5A and 5C, for SF2a and SF6, after three injections, the IgG titers induced by the aluminum hydroxide (H) adjuvanted glycoconjugates were significantly higher than that induced by the aluminum phosphate (P) adjuvanted ones. For all serotypes, the differences in anti-LPS IgG titers induced by the monovalent conjugates and the quadrivalent combinations were roughly within one log and always above 103.[000246] As shown in the results of Figures 5A-5D, in all cases, at least two injections of the multivalent formulations were required to observe the induction of an anti-LPS IgG titer above threshold ( 103). As shown in Figure 5A and 5B, independently of the adjuvant used, the third injection was shown to be highly beneficial in the case of SF2a and SF3a, respectively, when administered in the form of tri- and quadrivalent combinations. As shown in Figure 5C and Figure 5D, this positive impact was still visible, albeit less pronounced, in the case of SF6 and Sson, respectively, albeit with exception. Surprisingly, in the case of the aluminum hydroxide adjuvanted SF2a / SF3a / Sson trivalent formulation, the third immunization had a negative impact on the anti-Sson LPS IgG titer, in comparison to otherformulations, resulting in a significant difference between the anti-Sson LPS IgG titers induced by monovalent Sson-TT vaccine conjugate and Sson-TT vaccine conjugate as part of the SF2a / SF3a / Sson trivalent formulation. This observation underlines that combining synthetic carbohydrate-based Shigella monovalent conjugates to achieve a Shigella multivalent vaccine is not straightforward, and that careful consideration of multiple parameters is required.[000247] As shown in Figure 5A-5D, comparison of anti-LPS IgG titer induced by the monovalent formulations versus the multivalent formulations at all stages revealed that each glycoconjugate component adopted a unique behavior. After three injections, the monovalent SF6 and Sson conjugates were found superior when administered solo than when administered as part of the quadrivalent formulations, respectively. In the case of SF2a and SF3a, after three injections, no statistically significant difference was observed between the monovalent and quadrivalent formulations, independent of the adjuvant. Interestingly, significant differences were observed in the anti-SF3a and anti-Sson IgG titers induced by the aluminium hydroxide adjuvanted tri- and quadrivalent combinations after both the second and the third immunizations. Moreover, the adjuvant was shown to influence the immune response in a serotype-specific manner. In particular, for SF2a and SF6, after three injections, the IgG titers induced by the aluminum hydroxide (H) adjuvanted monovalent glycoconjugates were significantly higher than that induced by the aluminum phosphate (P) adjuvanted ones. However, this was not necessarily the case for the quadrivalent formulations. Overall, while differences in anti-LPS IgG titers induced by the monovalent conjugates and the quadrivalent combinations were observed, they were within one log. Lastly, all induced anti-LPS IgG titers were above the 103threshold.[000248] Influence of the Total Amount of Carrier (Figures 6A-6D; Exp 2 in Table 1 above)[000249] Antigenic competition is a well-established phenomenon. When developing multivalent glycoconjugate vaccines, carrier-induced epitope suppression is of special concern, whether occurring following pre-exposure or co-exposure to a certain carrier. The latter is related to the risk of overloading the immune system with the carrier owing to its increasing amount in the vaccine formulation respective to that of each single glycan antigen component, when the number of valences increases, while all vaccine components feature thesame carrier. Herein, we investigated the impact of excess protein carrier (i.e., TT) amounts co-administered with either the aluminum phosphate (P) adjuvanted monovalent glycoconjugate vaccine components or the aluminum phosphate (P) adjuvanted quadrivalent formulation[000250] For this experiment, all SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT vaccine conjugates were administered in amounts corresponding to 2.0 pg OS per valence per dose. TT (carrier) amounts added to complement the monovalent formulations were estimated to mimic the total amount of carrier included in the quadrivalent formulation used at 2.0 pg OS dose per valence (i.e., “TT2”). TT amounts added to complement the quadrivalent formulations were estimated to mimic the total amount of carrier included in the quadrivalent formulation used at 10 pg (i.e., “TT10”) or 30 pg (i.e., “TT30”) OS dose per valence, respectively. In all calculations, the composition of each one of the four conjugates used in the formulation was taken into account (see, e.g., Exp. 2 in Table 1).[000251] Figures 6A-6D illustrate the influence of the TT (carrier) content on IgG titers induced against the SF2a, SF3a, SF6, and Sson LPS, respectively, in mice (seven mice per category) receiving three injections of SF2a-TT, SF3a-TT, SF6-TT and / or Sson-TT glycoconjugates, combined in a 1: 1: 1: 1 ratio, when in the form of the quadrivalent vaccine, to achieve 2.0 pg of OS each per dose and adjuvanted with aluminum phosphate (P). The quadrivalent combinations (4v) are complemented with amounts of TT corresponding to the total amount of TT that would be used for amounts of conjugates corresponding to 10 pg (i.e., “TT10”) and 30 pg (i.e., “TT30”) OS each per dose, respectively. For each combination, the induced IgG titers are shown for the same type-specific monovalent conjugate administered at the same OS dose, complemented or not with the amount of TT necessary to reach the total amount of TT present in the quadrivalent formulation used at 2.0 pg OS each. Bleeding of the mice for the IgG titers was at three weeks after the 1st, 2ndand 3rdinjection, respectively. As shown in Figures 6A-6D, the X-axis illustrates the relevant monovalent and quadrivalent (4v) glycoconjugate formulations (e.g., SF2a with adjuvant (i.e., aluminum phosphate (P) or “SF2a-P” or “SF2a-P 4V”) and with / without TT2 (i.e., “SF2a-P TT2”), TT10 (i.e., “SF2a-P 4v TT10”), or TT30 (i.e., “SF2a-P 4v TT30”); SF3a with adjuvant (i.e., aluminum phosphate (P) or “SF3a-P” or “SF3a-P 4V”) and with / without TT2 (i.e., “SF3a-P TT2”), TT10 (i.e., “SF3a-P 4V TT10”), or TT30 (i.e., “SF3a-P 4vTT30”); SF6 with adjuvant (i.e., aluminum phosphate (P) or “SF6-P” or “SF6-P 4V”) and with / without TT2 (i.e., “SF6-P TT2”), TT10 (i.e., “SF6-P 4V TT10”), or TT30 (i.e., “SF6-P 4v TT30”); or Sson with adjuvant (i.e., aluminum phosphate (P) or “Son-P” or “Son-P 4V”) and with / without TT2 (i.e., “Son-P TT2”), TT10 (i.e., “Son-P 4V TT10”), or TT30 (i.e., “Son-P 4v TT30”)), while the Y-axis illustrates the anti-SF2a LPS IgG titer (Figure 6A), the anti-SF3a LPS IgG titer (Figure 6B), the anti-SF6 LPS IgG titer (Figure 6C), and the anti-Sson LPS IgG titer (Figure 6D), respectively. As shown in Figures 6A-6D, for all serotypes, the differences in anti-LPS IgG titers induced by the monovalent conjugates and the quadrivalent combinations were within one log and above 103. Excess TT had a limited impact as the induced anti-LPS IgG antibody titers remained in a one log range for all formulations.[000252] As further shown in the results of Figures 6A-6D, except for the Sson valence, the third immunization contributed to increasing the anti-homologous LPS IgG titers.However, in the case of SF6 and Sson, two injections were deemed sufficient to induce high anti-LPS IgG antibody titers. There was no significant negative impact of excess carrier observed for any of the monovalent conjugates. In contrast, the anti-SF3a and anti-Sson IgG titers induced by the quadrivalent formulations complemented with the “TT30” excess tetanus toxoid amount were slightly lower than those induced by the formulations containing lower amounts of carrier, suggesting a negative influence of the latter when used in large excess. Except for SF3a, the quadrivalent formulations were slightly less immunogenic than the monovalent ones. The difference was statistically significant for SF2a and Sson. The little divergence observed in this experiment suggests further fine-tuning toward an optimal combination. However, the differences in the observed anti-LPS antibody titers were in a one log range and all IgG titers - with one exception - were above 103after three injections, and therefore judged acceptable.[000253] Influence of the Relative Carbohydrate Content (Figure 7, Figure 8, Figures 9A-9D and Exp 3 in Table 1 above)[000254] Data from the results of Figures 5A-5D and Figures 6A-6D discussed above suggest the superior immunogenicity of SF6-TT and Sson-TT when compared to SF2a-TT and SF3a-TT. Accordingly, this experiment investigated quadrivalent combinations featuring non-identical amounts of OS per valence to determine the influence of carbohydrate contenton the IgG titers. As a first step, the influence of the relative OS content on IgG titers induced against the SF2a LPS was determined in mice (seven mice per category) receiving three injections of SF2a-TT glycoconjugate. The influence of the adjuvant, aluminum hydroxide (“alum” or “H”), was also established. Bleeding of the mice for the IgG titers was at three weeks after the 3rdinjection. Figure 7 illustrates the results of the first part of this experiment, with the X-axis providing the OS dose of SF2a-TT and the adjuvant status (i.e., with or without alum), and the Y-axis providing the anti-SF2a LPS IgG titer. As shown in Figure 7, for all doses, the induced anti-LPS IgG titers were within one log and above 103. According to this experiment, a dose response analysis was able to establish that the alum-adjuvanted SF2a-TT vaccine conjugate still induced measurable anti-SF2a LPS IgG titers when administered three times at three weeks interval in amounts corresponding to 40 ng (see, e.g., Figure 7). Moreover, the non-adjuvanted SF2a-TT was still immunogenic, albeit when used at a dose equivalent to 160 ng OS, suggesting a beneficial effect of alum (adjuvant) that is thought to be somewhat enhanced when doses of antigen are diminished.[000255] In a second part of the experiment, the influence of the relative OS content on IgG titers induced against the SF3a LPS was determined in mice (seven mice per category) receiving three injections of SF3a-TT glycoconjugate. The influence of the adjuvant, aluminum hydroxide (“alum” or “H”), was also established. Bleeding of the mice for the IgG titers was at three weeks after the 3rdinjection. Figure 8 illustrates the results of the second part of this experiment, with the X-axis providing the OS dose of SF3a-TT and the adjuvant status (i.e., with or without alum), and the Y-axis providing the anti-SF3a LPS IgG titer. As shown in Figure 8, for all doses, the induced anti-LPS IgG titers were within one log and above 103. According to this experiment, a dose response analysis was able to establish that the SF3a-TT vaccine conjugate demonstrated that the alum -adj uvanted conjugate still induced measurable anti-SF3a LPS IgG titers when administered three times at three weeks interval in amounts corresponding to 40 ng (see, e.g., Figure 8). Moreover, the non-adjuvanted SF3a-TT was still immunogenic, albeit when used at a dose equivalent to 160 ng OS, suggesting a beneficial effect of alum (adjuvant) that is thought to be somewhat enhanced when doses of antigen are diminished.[000256] In a third part of the experiment, while keeping the amounts of SF2a-TT and SF3a-TT to 2.0 pg OS equivalent per dose, the amounts of SF6-TT and Sson-TT werereduced by a factor 4 and / or 10, to achieve 0.5 pg and 0.2 pg OS per dose, respectively. Figures 9A-9D illustrate the influence of the relative oligosaccharide content on the IgG titers induced against the SF2a, SF3a, SF6 and Sson LPS, respectively, in mice (seven mice per category) receiving three injections of SF2a-TT, SF3a-TT, SF6-TT and Sson-TT glycoconjugates combined in a 1: 1: 1: 1, 1:1:1:0.25, 1: 1: 1:0.1, or 1:1:0.5:0.1 ratio to achieve 2.0 pg of OS per dose for SF2a-TT and SF3a-TT. The influence of adjuvant (i.e., aluminum phosphate (P)) was also investigated in the context of the 1: 1: 1: 1 quadrivalent combination. For each combination, the induced IgG titers are shown for the same type-specific monovalent conjugate administered at the same OS dose. Bleeding of the mice for the IgG titers was at three weeks after the 1st, 2ndand 3rdinjection, respectively. As shown in Figures 9A-9D, the X-axis illustrates the relevant monovalent and quadrivalent glycoconjugate formulations (e.g., SF2a-TT, SF3a-TT, SF6-TT, and / or Sson-TT), while the Y-axis illustrates the anti-SF2a LPS IgG titer (Figure 9A), the anti-SF3a LPS IgG titer (Figure 9B), the anti-SF6 LPS IgG titer (Figure 9C), and the anti-Sson LPS IgG titer (Figure 9D), respectively. For Figure 9C, the X-axis further includes a 2.0 (2-P) and 0.5 pg (0.5-P) OS per dose, respectively, while in Figure 9D, the X-axis further includes at 2.0 (2-P), 0.5 pg (0.5-P) and 0.2 pg (0.2-P) OS per dose, respectively. As shown in Figures 9A-9D, for all serotypes, the differences in anti-LPS IgG titers induced by the monovalent conjugates and the quadrivalent combinations were within one log and above 103.[000257] Interestingly, as shown in the results of Figures 9A-9D, the dose-amount of the monovalent SF6-TT and monovalent Sson-TT did not have a major impact on the homologous IgG antibody response. However, observations differed to some extent for the quadrivalent formulations. In particular, reducing the amount of Sson-TT resulted in a significantly lower anti-Sson LPS IgG titer after three injections. By contrast, a beneficial effect was observed for the anti-SF6 LSP IgG titer, and to a lesser extent on the anti-SF2a and anti-SF3a antibody titers. Reducing the amount of SF6-TT had no influence on the Sson response. Variations were in a one log range. Altogether, the data suggested that modulation of the quadrivalent combination beyond a 1: 1: 1: 1 relative proportion of type-specific OS content is feasible and could have some advantage. In particular, the anti-Sson response induced by the quadrivalent formulations featuring the lower amounts of Sson were still acceptable.[000258] Influence of the Adjuvant on the Performance of the Quadrivalent Combination (Figures 10A-10D, QSV1 = Exp 4 in Table 1 above, QSV2 = Exp 5 in Table 1 above)[000259] In this experiment, the adjuvant effect on quadrivalent Shigella vaccine compositions was investigated using well-established adjuvant formulations: 2% ALHYDROGEL® and 0.5% ADJUPHOS®. Figures 10A-10D illustrate the results of this experiment in which IgG titers were induced against the SF2a, SF3a, SF6, and Sson LPS, respectively, in mice (14 mice per category except placebo which had buffer and 7 mice) receiving three injections of SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT glycoconjugates combined in a 1: 1: 1: 1 ratio, to achieve 2.0 pg of OS each per dose, adjuvanted or not with aluminum hydroxide (“A1H”) or aluminum phosphate (“A1P”). “QSV1” and “QSV2” represent two different quadrivalent glycoconjugate compositions. Bleeding of the mice for the IgG titers was at three weeks after the 1st, 2ndand 3rdinjection, respectively. As shown in Figures 10A-10D, the X-axis illustrates the quadrivalent glycoconjugate formulations, adjuvanted or not, while the Y-axis illustrates the anti-SF2a LPS IgG titer (Figure 10A), the anti-SF3a LPS IgG titer (Figure 10B), the anti-SF6 LPS IgG titer (Figure 10C), and the anti-Sson LPS IgG titer (Figure 10D), respectively. As shown in Figures 10A-10D, for SF6 and Sson, no statistically significant differences were observed between the two adjuvants. As further shown in Figures 10A-10D, for all serotypes, the IgG titers induced by the adjuvanted glycoconjugate combinations were significantly higher than that induced by the non-adjuvanted ones.[000260] According to this experiment, the adjuvanted combinations were found to be more immunogenic than the non-adjuvanted ones. Interestingly, there was no statistical difference in the immunogenicity observed for the two aluminum phosphate (“A1P”) adjuvanted quadrivalent formulations when administered in amounts corresponding to 2.0 pg OS each per valence per dose. Moreover, there was no statistical divergence observed for the anti-SF6 and anti-Sson LPS IgG antibody responses induced by the aluminum hydroxide (“A1H”) and A1P formulations. By contrast, slight differences were observed for the corresponding anti-SF2a and anti-SF3a LPS IgG antibody responses. Still, variations were within a one log range paving the way to multiple valuable combinations.[000261] Extension to Rabbits[000262] In one example, the immunogenicity of the quadrivalent Shigella vaccine formulation (“QSV”) was tested in rabbits to assess IgG titers to the corresponding LPS serotype and a serum bactericidal assay (SB A) as a functional assay to determine the quality of the response. In addition, aluminum hydroxide (“A10H”), aluminum phosphate (“A1PO”), and Matrix-M® were tested in two independent rabbit studies to determine whether an adjuvant could boost the immune response of the quadrivalent formulation.[000263] Rabbit study #1: This study compared the immunogenicity of the quadrivalent Shigella vaccine (“QSV”) of the instant disclosure with or without aluminum hydroxide (“ALOH”) or aluminum phosphate (“ALPO”) as adjuvants, after 3 injections at days 1, 29 and 57 (once every 4 weeks). Immunogenicity was assessed at Predose (Day 1) and Days 29 and 57 (prior to dosing), and Day 84 (4 weeks after the last dose). The study design for this example is shown in the Table illustrated in Figure 11. The results of this study are illustrated in Figures 12A-12C.[000264] Rabbit study #2: This study compared the immunogenicity of the quadrivalent Shigella vaccine (“QSV”) of the instant disclosure with or without Matrix-M® (“MM”) or aluminum phosphate (“ALPO”) as adjuvants, after 3 injections at days 1, 29 and 57 (once every 4 weeks). Immunogenicity was assessed at Predose (Day 1) and Days 29 and 57 (prior to dosing), and Day 84 (4 weeks after the last dose). The study design for this example is shown in the Table illustrated in Figure 13. The results of this study are illustrated in Figures 14A-14C.[000265] Overall, immunogenicity (IgG and SBA) data from both rabbit experiments showed similar results. In particular, these studies provided the following results: immunogenicity (both IgG and SBA) was elicited and was higher than baseline; immunogenicity trended towards a dose response; titers trended to be higher after the second injection; and all adjuvants boosted the response, with aluminum hydroxide (“Al OH”) having similar results to aluminum phosphate (“A1PO”) and Matrix-M (“MM”) trending to higher titers than A1PO.[000266] Exemplary Manufacturing Process of Monovalent Shigella OS-Carrier Conjugates[000267] When using TT as the carrier, the overall manufacturing process of SF2a-TT, SF3a-TT, SF6-TT, and Sson-TT are the same, which involves four sub-processes: (1)activation of TT with the heterobifunctional linker TV-y-maleimidobutyryl-oxysuccinimide ester (GMBS); (2) removal of 2-thiopyridine protecting group on the linker-equipped oligosaccharide OS-SPDP; (3) conjugation reaction between modified TT and ready-for- conjugation OS; and (4) capping of unreacted maleimide groups on TT. Purification andbuffer exchange unit operations are done via tangential flow filtration (TFF). An overview of the activation / conjugation / capping (or quenching) process is illustrated in Figure 15.[000268] Process parameters used for the initial purification of TT and modification of TT with GMBS are the same for all four drug substances. For conjugation reaction of TT-GMBS with deprotected OS-SPDP, there are some differences in the conjugation reactionparameters due to the different chemical structures and physicochemical properties of each deprotected OS-SPDP. Thus, the conjugation reaction parameters are optimized to maximize the conjugation efficiency (e.g., molar ratio of OS: TT and overall saccharide yield for each of the monovalent drug substances). The differences in the conjugation process parameters are summarized in Table 2 below.TABLE 2 - Summary of key conjugation reaction parameters for the manufacture of monovalent drug substances for serotypes SF2a, SF3a, SF6, and Sson[000269] General Procedures to Prepare Monovalent Shigella sOS based OS-TetanusToxoid Conjugates using SF2a-TT as an Example[000270] Step 1: Preparation of SF2a-SPDP Stock Solution[000271] Approximately 589.0 mg of lyophilized SF2a-SPDP solid was dissolved to atarget concentration of 20 mM in 100 mM Sodium Phosphate, 5mM EDTA pH 6.3 buffer, to prepare an approximately 50-60 mg / mL (corresponding to approximately 20-25 mM) OS- SPDP stock solution. A sample of this stock solution was tested by the Anthrone assay (discussed below) to confirm the saccharide concentration and was stored at -80 °C until use.[000272] Step 2: TT Buffer Exchange and Concentration[000273] TT was obtained from commercial sources and buffer exchanged into 100 mM HEPES pH 7.8 buffer using tangential flow filtration (TFF). Briefly, approximately 2000 mg (based on BCA assay) of TT was concentrated to a target concentration of about 15 mg / mL using a Sartocon Slice 200 ECO Hydrosart Cassette (30 kDa MWCO) for 7 diavolumes. The target flow rate was 100 mL / min (300 LMH), average feed line pressure was 1.694 bar and the average TMP equaled 1.053 bar. The buffer-exchanged TT protein was analyzed by the BCA assay (discussed below) to determine the protein concentration. The TFF process showed quantitative protein mass recovery and the buffer-exchanged TT was stored at 2-8 °C until use.[000274] Buffer exchange and concentration was performed using spin filters (Millipore, Amicon ultra 4 and 15) with a 10 or 30 kDa MWCO (15 min, 5,000 x g, rt, at least 4 cycles) or TFF using Sartorius Sartocon Slice 200 ECO Hydrosart MWCO 30 kDa cassettes.[000275] Step 3: TT Modification of GMBS[000276] A-y-maleimidobutyryl-oxysuccinimide ester (GMBS) was dissolved in DMSO to a target concentration of about 72 mg / mL. This stock solution was added to the buffer-exchanged TT along with additional diluent (100 mM HEPES pH 7.8) to result in 160 stoichiometric equivalents of GMBS to TT, and an overall reaction concentration of 10 mg / mL of TT. The reaction was allowed to proceed for 1 hour at ambient temperature with continuous mixing using a rocker.[000277] Step 4: TT-GMBS Buffer exchange and Concentration[000278] After the modification reaction, the TT-GMBS was concentrated using a Sartocon Slice 200 ECO Hydrosart Cassette (30 kDa MWCO) to a target concentration of about 15 mg / mL and buffer exchanged for 20 diavolumes into 100 mM Sodium Phosphate, 5 mM EDTA pH 6.3. The target flow rate was 100 mL / min (300 LMH), the average feed line pressure was about 1.746 bar and the average TMP was about 1.081 bar. A sample of the TT-GMBS was tested by the BCA assay to determine the protein concentration, where quantitative recovery (>90%) of protein mass was observed. The TT-GMBS was stored at 2-8 °C and used in the next step (conjugation with reduced OS-SPDP) within 4 hours of the completion of the TFF procedures.[000279] Step 5: Reduction of SF2a-SPDP Stock Solution[000280] The SF2a-SPDP stock solution prepared previously was thawed at ambient temperature. A 30 mM stock solution of TCEP-HC1 in 100 mM Sodium Phosphate, 5mM EDTA pH 6.3 buffer was prepared. The 30 mM TCEP solution was then added to the SF2a-SPDP stock solution along with additional diluent (100 mM Sodium Phosphate, 5 mM EDTA pH 6.3) to result in 1.1 equivalents of TCEP to TT-GMBS, and an overall reaction concentration of 10 mM SF2a-SPDP. The TCEP reduction reaction was run for 1 hour at ambient temperature with continuous mixing using a rocker.[000281] Step 6: SF2a-TT Conjugation and Capping of Unreacted Maleimide using Cysteamine[000282] The entirety of the reduced SF2a-SPDP (238.7 pmol) was combined with TT-GMBS (9.5 pmol) and diluent (100 mM Sodium Phosphate, 5 mM EDTA pH 6.3) to result in 25 equivalents of SF2a to TT-GMBS and an overall reaction concentration of 5.85 mg / mL of TT (reaction volume 243.6 mL). The reaction was allowed to proceed for 16-17 hours at ambient temperature with continuous mixing on a rocker. Cysteamine was dissolved to a target concentration of 19 mg / mL in 100 mM Sodium Phosphate, 5 mM EDTA pH 6.3 buffer. The cysteamine capping solution was added to the conjugation reaction to result in 160 stoichiometric equivalents of cysteamine to TT-GMBS. This quench reaction was run for 1 hour at ambient temperature with continuous mixing on a rocker.[000283] Step 7: Purification of SF2a-TT by TFF and Sterile Filtration[000284] Upon the completion of the cysteamine capping reaction, the crude SF2a-TT conjugate mixture was diluted to a target concentration of about 1 mg / mL (based on saccharide concentration). The diluted SF2a-TT conjugate mixture was then purified by TFF using a Sartocon Slice 200 ECO Hydrosart Cassette (30 kDa MWCO) for 10 diavolumes and buffer exchanged into 50 mM Tris, pH 7.0 buffer. The target flow rate of TFF was 100 mL / min (300 LMH), the average feed line pressure was about 1.660 bar and the average TMP was about 1.063 bar. The purified SF2a-TT conjugate was then sterile-filtered in a biosafety cabinet using aseptic technique and a Sartopore Platinum Sterile Capsule Size 4. The saccharide and protein concentrations of the sterile-filtered SF2a-TT conjugate were determined by the Anthrone and BCA assays, respectively. The purified conjugates are stored at 2-8 °C.[000285] Analytical Assays for Characterization of Synthetic Shigella OS-TT Conjugates and Multivalent Shigella Vaccine Formulations[000286] Protein Concentration Determination[000287] The total protein concentration of monovalent OS-TT conjugates is measured by UV detection (X = 280 nm) for tetanus toxoid conjugates (s(TT) = 8 = 189,460 M'hcrn'1, mw(TT): 150 kDa) or the BCA colorimetric assay using bovine serum albumin (BSA) as quantitation standard.[000288] OS Concentration Determination[000289] For SF2a-TT, SF3a-TT, and SF6-TT conjugates: Anthrone Assay[000290] The concentration of the saccharide components of the SF2a-TT, SF3a-TT, and SF6-TT conjugates were determined using the Anthrone assay. Briefly, a calibration curve, ranging from 10 to 100 nmol, was constructed using a mixture of monosaccharide components reflective of the RUs of each of the hapten. For example, for SF2a and SF3a haptens, a mixture of 1:3:1 (mol:mol:mol) of D-glucose: L-rhamnose: A-acetyl-D-glucosamine is used to generate the quantitation standard curve. For SF6 hapten, a mixture of 1: 1:2 (mol:mol:mol) of D-galacturonic acid: A- acetyl-D-galactosamine: L-rhamnose is used to generate the quantitation standard curve. All samples and standards were cooled on ice before the Anthrone reagent (100 pL, 0.2% w / v Anthrone in sulfuric acid 95-97%) was added. Upon addition of the Anthrone reagent, all samples / standards were equilibrated to room temperature and incubated at 95 °C for 15 min, then cooled on ice. The absorbance was measured at 625 nm. The concentration of the carbohydrate was calculated from the standard curve.[000291] For Sson-TT conjugate: MALDI-MS Analysis[000292] Since the monosaccharide components present in the Sson hapten RU do not react with Anthrone reagent, saccharide concentration cannot be determined by the Anthrone assay as for the other Shigella-T conjugates. For Sson-TT conjugates, the degree of conjugation (molar ratio of Sson OS: TT protein) is estimated by MALDI-MS analysis, which is then used to calculate the saccharide concentration. Prior to MALDI-MS analysis, 15 pL of the Sson-TT conjugate samples (diluted to approximately 1 mg / mL, based on protein concentration determined by BCA assay) was passed through a ZipTip C4 and eluted on a MTP 384 ground steel target plate (Bruker-Daltonics, Germany) with 2 pL of 20 mg / mL a-cyano-4-hydroxycinnamic acid (HCCA) in a 3:2 of ACN and 0.1% aq. TFA as the matrix solution. Samples were air-dried for 15 min. MALDI-MS data were acquired on a Bruker UltrafleXtrem instrument, using the Flexcontrol software (Bruker-Daltonics, Germany). 10,000 shots were recorded in the positive ion linear mode in the m / z range of 30-210 kDa. Unmodified TT and TT-GMBS conjugates were used as controls in MALDI-MS analysis. OS concentration was calculated based on the estimated average loading and measured TT concentration.[000293] Exemplary Procedure To Prepare Multivalent Shigella Vaccine Formulation: Quadrivalent 1: 1: 1: 1 (SF2a-TT: SF3a-TT: SF6-TT: Sson-TT) (Figure 10)[000294] Multivalent Shigella vaccine formulations are prepared based on the weight ratio of saccharide of each monovalent conjugate. In formulations that contain an adjuvant (A1P, A1H, or Matrix M®), a quadrivalent Shigella vaccine concentrate at appropriate saccharide concentration was first prepared and mixed with an appropriate amount of adjuvant prior to injection into animals. All formulation preparation procedures were carried out in a biosafety cabinet, using aseptic transfer techniques and pre-sterilized materials.[000295] Each monovalent conjugate was first diluted to approximately 200 pg / mL with 50 mM Tris pH 7.0 buffer to yield a 200 pg / mL quadrivalent Shigella vaccine (“QSV”) concentrate, as outlined in Table 3 below:TABLE 3N / A: Not Applicable[000296] A high-dose quadrivalent Shigella vaccine formulation (target concentration of 75 pg / mL per each serotype) was prepared by mixing together 75 mL of the 200 pg / mL quadrivalent Shigella vaccine concentrate prepared above, and 125 mL of 50 mM Tris, 240 mM NaCl, 1.6% w / v 2-PE, 0.032% w / v PS-80 pH 7.0 (Dilution Buffer 1) in a 250-mLHDPE bottle, measured volumetrically, using sterile serological pipets for transfer in a biosafety cabinet. The components were mixed by manually inverting the bottle 5-10 times. The bulk quadrivalent Shigella vaccine high-dose formulation (6.4 mL) was filled into 10R Type 1 Borosilicate glass vials by hand, using an automatic pipettor. The filled vials were sealed with rubber stoppers and metal seals and stored at 2-8 °C prior to use.[000297] A low-dose quadrivalent Shigella vaccine formulation (target concentration of 5 pg / mL per each serotype) was prepared by mixing together 12.5 mL of the 75 pg / mL quadrivalent Shigella vaccine high-dose formulation prepared above, and 175 mL of 50 mM Tris, 150 mM NaCl, 1.0% w / v 2-PE, 0.02% w / v PS-80 pH 7.0 (Dilution Buffer 2) in a 250-mL HDPE bottle, measured volumetrically, using sterile serological pipets for transfer in a biosafety cabinet. The components were mixed by manually inverting the bottle 5-10 times. The bulk quadrivalent Shigella vaccine low-dose formulation (6.4 mL) was filled into 10R Type 1 Borosilicate glass vials by hand, using an automatic pipettor. The filled vials were stoppered, sealed, and crimped, and stored at 2-8 °C prior to use.[000298] Diluted ADJU-PHOS® (Adjuvant) Vials:[000299] 0.5% ADJU-PHOS® (adjuvant) was resuspended by inverting the bottle manually 10 times, and visually confirmed to be homogeneous prior to sampling. Upon resuspension, 20 mL of the resuspended 0.5% ADJU-PHOS® was transferred to a sterile 60 mL HDPE bottle and diluted with 20 mL of water-for-inj ection. The diluted ADJU-PHOS® was mixed thoroughly by manual inversion of 5-10 times and filled into 10R Type 1 Borosilicate glass vials (1.6 mL fill volume) by hand, using an automatic pipettor. The filled vials were stoppered, sealed, and crimped, and stored at 2-8 °C prior to use.[000300] General Procedures for Admixing of QSV + ADJU-PHOS® Adjuvant Prior to Rabbit Immunization[000301] Prior to mixing with adjuvant (e.g., ADJU-PHOS®), vials containing the quadrivalent Shigella vaccine (“QSV”) formulations described above were removed from 2-8 °C storage and equilibrated to ambient temperature for ~ 20-30 minutes. A U inch needle was inserted into the rubber stopper of the adjuvant (e.g., ADJU-PHOS®) vial as a vent, and the entire contents (6.4 mL) of the high-dose or low-dose quadrivalent Shigella vaccine formulation vial was transferred into the adjuvant (e.g., ADJU-PHOS®) vial using a hypodermic syringe and needle, the total volume after mixing was about 8.0 mL. The ventingneedle was removed, and the admixed quadrivalent Shigella vaccine formulation + ADJU- PHOS® vials were mixed by inverting the vial 10 times by hand. The admixed vials were incubated at ambient temperature for minimum of 1 hour on an orbital shaker set at 40-50 rpm and administered to animals within 4 hours.General Methods for Immunogenicity Analysis[000302] Mice Immunization[000303] Non-adjuvanted formulation. For each of the selected conjugates, seven week-old Balb / c female mice (Janvier Labs, France) were immunized intramuscularly (i.m.) with amounts of conjugates corresponding to 0.5, 1.0, 2.0, 2.5 or 10 pg equivalent of OS per dose, respectively. The formulated glycoconjugates (200 pL) per immunization per mouse were injected at two sites (100 pL at each site). The conjugates were administered three times at 3 week-interval. Blood samples were recovered on the day before conjugate administration 2 and 3 whenever needed, as well as one week after the third injection, respectively. Seven mice were used per group.[000304] Aluminum hydroxide-adjuvanted formulation. Aluminum hydroxide (A1H, ALHYDROGEL®, Brenntag, Denmark) was used at a concentration of 1.4 mg / mL in Tris pH 7.2, 20 mM, and mixed v / v with the conjugates, resulting to a dose of 143 pg per mouse / per injection, independently of the amount of carbohydrate per dose. After 5 min incubation at room temperature, the adjuvanted conjugates were used as described above.[000305] Aluminum phosphate-adjuvanted formulation. The conjugates were diluted at a concentration of 20 mg OS / mL using the 10 mM histidine, 150 mM NaCl buffer. Aluminum phosphate (A1P) (ADJU-PHOS® adjuvant, 4.9 mg Aluminum / mL, Invivo Gen) was diluted at a concentration of 1.4 mg / mL using the same buffer. The diluted conjugates and the adjuvant (A1P) were mixed v / v. After pipetting up and down for at least 5 min to allow effective adsorption of the antigen, the adjuvanted conjugates were used as described above.[000306] Anti-LPS IgG Response[000307] ELISA Assay[000308] The glycoconjugate-induced anti-LPS IgG response specific for a given LPS was measured by ELISA using LPS purified from a homologous strain as previously described. Briefly, 2.5 pg of purified homologous LPS was coated per ELISA plate well in PBS and incubated at 4 °C overnight. After washing the wells with PBS-Tween 200.01%, saturationwas performed by incubating the plate for 30 min at 37 °C with PBS-BSA 1%. Then, serial dilutions of mouse sera in PBS-BSA 1% were incubated for 1 h at 37 °C. After washing with PBS-Tween 200.01%, anti-mouse IgG peroxidase-labeled conjugate (Sigma-Aldrich) was used as secondary antibody at a dilution of 1 / 5,000. The IgG titer was defined as the last dilution of serum giving rise to twice the OD value obtained with similarly diluted pre-immune serum.[000309] SF2a LPS was purified from strain SF2a strain 454 (from Institut Pasteur, Centre National de Reference des Enterobacteries, Paris, France). SF3a LPS was purified from strain IP 6865. SF6 LPS was purified from SF6 strain Sc544 (bacteria were a gift from Nils Carlin, Etvax, Sweden). Sson LPS was purified from the IP reference strain (CIP 106347).[000310] FACS Analysis[000311] The SF3a clinical isolates were obtained from the French National Reference Center for Escherichia coli. Shigella and Salmonella (Institut Pasteur, Paris, France). They were isolated and characterized from stools of individuals developing diarrhea when back from travelling to diverse countries After isolation on Congo red plates, one single colony for each clinical isolate was grown overnight in TCS (Trypto-Casein-Soy) medium at 37 °C with shaking. Then, 5 mL of each bacterial culture was centrifuged at 5,000 rpm for 5 min and the pellet was suspended in PBS / BSA 0.5% to a concentration of 107CFU / mL. After a washing step using PBS / BSA 0.5%, bacteria resuspended in 1 mL of PBS / BSA 0.5% were incubated with 200 pL of mouse serum samples diluted 1:20. After 30 min incubation at 37 °C, two PBS / BSA 0.5%-washings were performed, and the bacterial pellet resuspended in 1 mL of the same buffer was incubated with anti-mouse IgG Alexa Fluor 488-labeled secondary antibody (Thermo Fisher) at a dilution of 1:2,000. After 30 min incubation at 37 °C in the dark, and two PBS washings, bacteria resuspended in PBS (1.0 mL) were analyzed by flow cytometry (Attune, Thermo Fisher). Controls using AF 488-only labelled bacteria, and bacteria incubated with pre-immune sera, were used to account for background fluorescence and non-specific binding of mouse IgG, respectively. Results were analyzed using FlowJo 10.3.[000312] Serum Bactericidal Assay[000313] Serum from immunized rabbits was tested for functional antibody activity in a serum bactericidal assay. Serial dilutions of test sera (8 dilutions / sample) were prepared induplicate, in assay buffer (SAB) and 20 microliters of each diluted sample was added to a well of a 96 well plate. Shigella strains expressing SF2a, SF3a, SF6, and Sson LPS were used as target bacterial strains for the assay. Frozen aliquots of the four target Shigella strains were washed, diluted in SAB, and 10 pL was added to each well of diluted serum samples, followed by addition of 50 pL / well of diluted baby rabbit complement (BRC). After incubation at 37 °C for 2 hours, plates were kept on ice for 20 minutes. An aliquot (10 pL) of the final reaction mixture from all wells was spotted onto four agar plates, with each agar plate containing a selective antibiotic that allowed growth of only one target Shigella strain.[000314] After overnight incubation, the number of surviving colonies of target bacterial strain on the agar plates was determined and compared to control wells (containing bacteria and BRC, but not test serum). Colony counts (average of the duplicates) were converted to killing indexes (KIs) using validated templates, with a KI defined as the interpolated dilution of serum that kills 50% of the target bacteria.[000315] Serum Luminex Assay[000316] The magnitude of antibody response in rabbits was assessed using a multiplex bead-based assay constructed of bead sets covalently conjugated to purified LPS from Sson, SF2a, SF3a, SF6, and TT. Serial dilutions of test sera (3 dilutions / sample), standard sera (8 dilutions), and QC sera (2 dilutions / sample) were prepared in SLA buffer. Purified Shigella lipopolysaccharides (LPS) were obtained from WRAIR. A bead mixture consisting of LUMINEX® beads coated with SF LPS serotypes 2a (SF2a), 3a (SF3a), and 6 (SF6), Sson LPS, and TT was prepared. Equal volumes of diluted sera and bead mixture (50 pL of each) were mixed in opaque flat-bottomed microtiter plates. Plates were incubated at room temperature (RT) with shaking for 60 minutes. After incubation, plates were washed twice with SLA buffer. After the final wash, diluted Phycoerythrin (PE)-labeled goat anti-rabbit IgG antibody was added to all wells. Plates were incubated at RT with shaking for 60 minutes. After incubation, plates were washed twice with SLA buffer. After the final wash, beads were resuspended in SLA buffer and plates were analyzed with a BIO-PLEX 200 (BioRad, Hercules, CA) to assess the levels of anti-LPS antibodies. For each target, the average median fluorescent values were calculated for each sample for each dilution, and a standard curve was constructed using linear regression. Test and QC sample antibody concentrations were interpolated from the standard and are expressed as units / ml.[000317] Although the invention has been described in certain specific exemplary embodiments, many additional modifications and variations would be apparent to those skilled in the art in light of this disclosure. It is, therefore, to be understood that this invention may be practiced otherwise than as specifically described. Thus, the exemplary embodiments of the invention should be considered in all respects to be illustrative and not restrictive, and the scope of the invention to be determined by any claims supportable by this application and the equivalents thereof, rather than by the foregoing description.

Claims

IN THE CLAIMS1. A multivalent Shigella vaccine composition comprising two or more different monovalent synthetic oligosaccharides (sOS) conjugated to a carrier, wherein the two or more different monovalent sOSs comprise (a) a sOS that comprises one or more O-antigen (O-Ag) repeating units (RUs) for S.flexneri 2a (SF2a), (b) a sOS that comprises one or more O-Ag RUs from S.flexneri 3a (SF3a), (c) a sOS that comprises one or more O-Ag RUs from S.flexneri 6 (SF6), (d) a sOS that comprises one or more O-Ag RUs from S. sonnei (Sson), or (e) a combination of the sOSs of (a), (b), (c), and / or (d).

2. The multivalent Shigella vaccine composition according to claim 1, wherein the carrier comprises tetanus toxoid (TT), tetanus toxin C fragment (TTc), recombinant TT, cholera toxin b subunit (CTB), diphtheria toxoid (DT), non-toxic diphtheria toxin mutant, P. aeruginosa exotoxin A (EP A), recombinant Exoprotein A (rEPA), C. jejuni Acriflavine resistance protein A (Cj AcrA), E. coli Acriflavine resistance protein A (EcAcrA), heat labile enterotoxigenic Escherichia coli (ETEC) subunit B (LTB), QP, outer membrane vesicles (OMVs), glycoengineered proteins, virus-like particles (VLPs), nanocarriers or nanoparticles, recombinant TT with certain mutations, Generalized Modules for Membrane Antigens (GMMA), P. aeruginosa PcrV (PcrV), Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), the recombinant fragment of tetanus toxin heavy chain (rTTHc), a cross-reactive Shigella Ipa protein, a mutated IpaB protein, or T-helper peptides (with natural or not peptide sequence), all in a mono- or multivalent fashion.

3. The multivalent Shigella vaccine composition according to claim 1, wherein the carrier comprises tetanus toxoid or CRM197.

4. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via one or more points of attachment.

5. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via a single point of attachment.

6. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via chemoselective chemistry.

7. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via Michael reaction.

8. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via thiol chemistry.

9. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via a linker.

10. The multivalent Shigella vaccine composition according to claim 1, wherein the one or more O-Ag RUs from SF2a comprise the following:

11. The multivalent Shigella vaccine composition according to claim 10, wherein at least one of Ri, R2, or R3 is acetyl.

12. The multivalent Shigella vaccine composition according to claim 10, wherein Ri or R2 is acetyl.

13. The multivalent Shigella vaccine composition according to claim 10, wherein at least one of Ri, R2, and R3 is H.

14. The multivalent Shigella vaccine composition according to claim 10, wherein Ri, R2, and R3 are H.

15. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF2a.

16. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF2a.

17. The multivalent Shigella vaccine composition according to claim 1, wherein the sOS that comprises one or more O-Ag RUs from SF2a is conjugated to a carrier with a linker.

18. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following structure:

19. The multivalent Shigella vaccine composition according to claim 18, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following structure:

20. The multivalent Shigella vaccine composition according to claim 18 or claim 19, wherein the carrier is TT.

21. The multivalent Shigella vaccine composition according to claim 1, wherein the one or more O-Ag RUs from SF3a comprise the following:

22. The multivalent Shigella vaccine composition according to claim 21, wherein at least one of R4 or R5 is acetyl.

23. The multivalent Shigella vaccine composition according to claim 21, wherein R4 or R5 is acetyl.

24. The multivalent Shigella vaccine composition according to claim 21, wherein at least one of R4 or R5 is H.

25. The multivalent Shigella vaccine composition according to claim 21, wherein R4 is H.

26. The multivalent Shigella vaccine composition according to claim 21, wherein R5 is acetyl.

27. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-antigen RUs from SF3a.

28. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF3a.

29. The multivalent Shigella vaccine composition according to claim 1, wherein the sOS that comprises one or more O-Ag RUs from SF3a is conjugated to a carrier with a linker.

30. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

31. The multivalent Shigella vaccine composition according to claim 30, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

32. The multivalent Shigella vaccine composition according to claim 30 or claim 31, wherein the carrier is TT.

33. The multivalent Shigella vaccine composition according to claim 1, wherein the one or more O-Ag RUs from SF6 comprise the following:

34. The multivalent Shigella vaccine composition according to claim 33, wherein at least one of Re or R7 is acetyl.

35. The multivalent Shigella vaccine composition according to claim 33, wherein Re or R7 is acetyl.

36. The multivalent Shigella vaccine composition according to claim 33, wherein at least one of Re or R7 is H.

37. The multivalent Shigella vaccine composition according to claim 33, wherein Re and R7 are H.

38. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF6.

39. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF6.

40. The multivalent Shigella vaccine composition according to claim 1, wherein the sOS that comprises one or more O-Ag RUs from SF6 is conjugated to a carrier with a linker.

41. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

42. The multivalent Shigella vaccine composition according to claim 41, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

43. The multivalent Shigella vaccine composition according to claim 41 or claim 42, wherein the carrier is TT.

44. The multivalent Shigella vaccine composition according to claim 1, wherein the one or more O-Ag RUs from Sson comprise the following:

45. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from Sson.

46. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 4 O-Ag RUs from Sson.

47. The multivalent Shigella vaccine composition according to claim 1, wherein the sOS that comprises one or more O-Ag RUs from Sson is conjugated to a carrier with a linker.

48. The multivalent Shigella vaccine composition according to claim 1, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the following structure:

49. The multivalent Shigella vaccine composition according to claim 48, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the following structure:

50. The multivalent Shigella vaccine composition according to claim 48 or claim 49, wherein the carrier is TT.

51. The multivalent Shigella vaccine composition according to claim 1, wherein the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier is from 1 to 30.

52. The multivalent Shigella vaccine composition according to claim 1, wherein the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier is from 1 to 30.

53. The multivalent Shigella vaccine composition according to claim 1, wherein the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier is from 1 to 30.

54. The multivalent Shigella vaccine composition according to claim 1, wherein the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier is from 1 to 30.

55. A quadrivalent Shigella vaccine composition comprising four different monovalent sOSs conjugated to a carrier, wherein the four different monovalent sOSs comprise (a) a sOS that comprises one or more O-Ag RUs from SF2a, (b) a sOS that comprises one or more O-Ag RUs from SF3a, (c) a sOS that comprises one or more O-Ag RUs from SF6, and (d) a sOS that comprises one or more O-Ag RUs from Sson.

56. The quadrivalent Shigella vaccine composition according to claim 55, wherein the carrier comprises tetanus toxoid (TT), tetanus toxin C fragment (TTc), recombinant TT, cholera toxin b subunit (CTB), diphtheria toxoid (DT), non-toxic diphtheria toxin mutant, P. aeruginosa exotoxin A (EP A), recombinant Exoprotein A (rEPA), C. jejuni Acriflavine resistance protein A (Cj AcrA), E. coli Acriflavine resistance protein A (EcAcrA), heat labile enterotoxigenic Escherichia coli (ETEC) subunit B (LTB), QP, outer membrane vesicles (OMVs), glycoengineered proteins, virus-like particles (VLPs), nanocarriers or nanoparticles, recombinant TT with certain mutations, Generalized Modules for Membrane Antigens (GMMA), P. aeruginosa PcrV (PcrV), Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), the recombinant fragment of tetanus toxin heavy chain (rTTHc), a cross-reactive Shigella Ipa protein, a mutated IpaB protein, or T-helper peptides (with natural or not peptide sequence), all in a mono- or multivalent fashion.

57. The quadrivalent Shigella vaccine composition according to claim 55, wherein the carrier comprises tetanus toxoid or CRM197.

58. The quadrivalent Shigella vaccine composition according to claim 55, wherein the four different monovalent sOSs are each independently conjugated to a carrier via one or more points of attachment.

59. The quadrivalent Shigella vaccine composition according to claim 55, wherein the four different monovalent sOSs are each independently conjugated to a carrier via a single point of attachment.

60. The quadrivalent Shigella vaccine composition according to claim 55, wherein the four different monovalent sOSs are each independently conjugated to a carrier via a linker.

61. The quadrivalent Shigella vaccine composition according to claim 55, wherein the one or more O-Ag RUs from SF2a comprise the following:

62. The quadrivalent Shigella vaccine composition according to claim 61, wherein at least one of Ri, R2, or R3 is acetyl.

63. The quadrivalent Shigella vaccine composition according to claim 61, wherein Ri or R2 is acetyl.

64. The quadrivalent Shigella vaccine composition according to claim 61, wherein at least one of Ri, R2, and R3 is H.

65. The quadrivalent Shigella vaccine composition according to claim 61, wherein Ri, R2, and R3 are H.

66. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF2a comprises 2 to 10 O-Ag RUs from SF2a.

67. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF2a comprises 3 O-Ag RUs from SF2a.

68. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF2a is conjugated to a carrier with a linker.

69. The quadrivalent Shigella vaccine composition according to claim 68, wherein the sOS that comprises one or more O-Ag RUs from SF2a conjugated to a carrier with a linker has the following structure:

70. The quadrivalent Shigella vaccine composition according to claim 69, wherein the sOS that comprises one or more O-Ag RUs from SF2a conjugated to a carrier with a linker has the following structure:

71. The quadrivalent Shigella vaccine composition according to claim 69 or claim 70, wherein the carrier is TT.

72. The quadrivalent Shigella vaccine composition according to claim 55, wherein the one or more O-Ag RUs from SF3a comprise the following:

73. The quadrivalent Shigella vaccine composition according to claim 72, wherein at least one of R4 or R5 is acetyl.

74. The quadrivalent Shigella vaccine composition according to claim 72, wherein R4 or R5 is acetyl.

75. The quadrivalent Shigella vaccine composition according to claim 72, wherein at least one of R4 or R5 is H.

76. The quadrivalent Shigella vaccine composition according to claim 72, wherein R4 isH.

77. The quadrivalent Shigella vaccine composition according to claim 72, wherein R5 is acetyl.

78. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF3a comprises 2 to 10 O-Ag RUs from SF3a.

79. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF3a comprises 3 O-Ag RUs from SF3a.

80. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF3a is conjugated to a carrier with a linker.

81. The quadrivalent Shigella vaccine composition according to claim 80, wherein the sOS that comprises one or more O-Ag RUs from SF3a conjugated to a carrier with a linker has the following structure:

82. The quadrivalent Shigella vaccine composition according to claim 81, wherein the sOS that comprises one or more O-Ag RUs from SF3a conjugated to a carrier with a linker has the following structure:

83. The quadrivalent Shigella vaccine composition according to claim 81 or claim 82, wherein the carrier is TT.

84. The quadrivalent Shigella vaccine composition according to claim 55, wherein the one or more O-Ag RUs from SF6 comprise the following:

85. The quadrivalent Shigella vaccine composition according to claim 84, wherein at least one of Re or R7 is acetyl.

86. The quadrivalent Shigella vaccine composition according to claim 84, wherein Re or R7 is acetyl.

87. The quadrivalent Shigella vaccine composition according to claim 84, wherein at least one of Re or R7 is H.

88. The quadrivalent Shigella vaccine composition according to claim 84, wherein Re and R7 is H.

89. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF6 comprises 2 to 10 O-Ag RUs from SF6.

90. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF6 comprises 3 O-Ag RUs from SF6.

91. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from SF6 is conjugated to a carrier with a linker.

92. The quadrivalent Shigella vaccine composition according to claim 91, wherein the sOS that comprises one or more O-Ag RUs from SF6 conjugated to a carrier with a linker has the following structure:

93. The quadrivalent Shigella vaccine composition according to claim 92, wherein the sOS that comprises one or more O-Ag RUs from SF6 conjugated to a carrier with a linker has the following structure:

94. The quadrivalent Shigella vaccine composition according to claim 92 or claim 93, wherein the carrier is TT.

95. The quadrivalent Shigella vaccine composition according to claim 55, wherein the one or more O-Ag RUs from Sson comprise the following:

96. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from Sson comprises 2 to 10 O-Ag RUs from Sson.

97. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from Sson comprises 4 O-Ag RUs from Sson.

98. The quadrivalent Shigella vaccine composition according to claim 55, wherein the sOS that comprises one or more O-Ag RUs from Sson is conjugated to a carrier with a linker.

99. The quadrivalent Shigella vaccine composition according to claim 98, wherein the sOS that comprises one or more O-Ag RUs from Sson conjugated to a carrier with a linker has the following structure:

100. The quadrivalent Shigella vaccine composition according to claim 99, wherein the sOS that comprises one or more O-Ag RUs from Sson conjugated to a carrier with a linker has the following structure:

101. The quadrivalent Shigella vaccine composition according to claim 99 or claim 100, wherein the carrier is TT.

102. The quadrivalent Shigella vaccine composition according to claim 55, wherein the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier is from 1 to 30.

103. The quadrivalent Shigella vaccine composition according to claim 55, wherein the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier is from 1 to 30.

104. The quadrivalent Shigella vaccine composition according to claim 55, wherein the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier is from 1 to 30.

105. The quadrivalent Shigella vaccine composition according to claim 55, wherein the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier is from 1 to 30.

106. A vaccine formulation comprising:(a) a multivalent Shigella vaccine composition comprising two or more different monovalent sOSs conjugated to a carrier; and(b) an adjuvant,wherein the two or more different monovalent sOSs conjugated to a carrier comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6,(iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).

107. The vaccine formulation according to claim 106, wherein the carrier comprises tetanus toxoid (TT), tetanus toxin C fragment (TTc), recombinant TT, cholera toxin b subunit (CTB), diphtheria toxoid (DT), non-toxic diphtheria toxin mutant, P. aeruginosa exotoxin A (EP A), recombinant Exoprotein A (rEPA), C. jejuni Acriflavine resistance protein A (CjAcrA), E. coli Acriflavine resistance protein A (EcAcrA), heat labile enterotoxigenic Escherichia coli (ETEC) subunit B (LTB), QP, outer membrane vesicles (OMVs), glycoengineered proteins, virus-like particles (VLPs), nanocarriers or nanoparticles, recombinant TT with certain mutations, Generalized Modules for Membrane Antigens (GMMA), P. aeruginosa PcrV (PcrV), Haemophilus influenzae protein D (PD), the outer membrane protein complex of serogroup B meningococcus (OMPC), the recombinant fragment of tetanus toxin heavy chain (rTTHc), a cross-reactive Shigella Ipa protein, a mutated IpaB protein, or T-helper peptides (with natural or not peptide sequence), all in a mono- or multivalent fashion.

108. The vaccine formulation according to claim 106, wherein the carrier comprises tetanus toxoid or CRM197.

109. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via one or more points of attachment.

110. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs are each independently conjugated to a carrier via a single point of attachment.

111. The vaccine formulation according to claim 106, wherein the adjuvant is selected from at least one of MATRIX M®, CPGs, QS-21, aluminum phosphate, aluminum hydroxide, dmLT, or combinations thereof.

112. The vaccine formulation according to claim 106, wherein the vaccine formulation is free of an adjuvant.

113. The vaccine formulation according to claim 106, wherein the adjuvant is aluminum phosphate.

114. The vaccine formulation according to claim 106, wherein the adjuvant is aluminum hydroxide.

115. The vaccine formulation according to claim 106, wherein the vaccine formulation further comprises a preservative.

116. The vaccine formulation according to claim 115, wherein the preservative is at least one of 2-phenoxyethanol (2 -PE), thimerosal, m-cresol, phenol, benzyl alcohol, or combinations thereof.

117. The vaccine formulation according to claim 115, wherein the preservative is 2-phenoxy ethanol (2 -PE).

118. The vaccine formulation according to claim 106, wherein the vaccine formulation further comprises one or more excipients.

119. The vaccine formulation according to claim 118, wherein the one or more excipients include at least one of a surfactant, a buffer, a tonicity modifier, a preservative, or combinations thereof.

120. The vaccine formulation according to claim 119, wherein the one or more excipients comprise polysorbate-80 (PS-80), Tris (tromethamine), sodium chloride, 2-phenoxyethanol (2 -PE), or combinations thereof.

121. The vaccine formulation according to claim 106, wherein the one or more O-Ag RUs from SF2a comprise the following:

122. The vaccine formulation according to claim 121, wherein at least one of Ri, R2, or R3 is acetyl.

123. The vaccine formulation according to claim 121, wherein Ri or R2 is acetyl.

124. The vaccine formulation according to claim 121, wherein at least one of Ri, R2, and R3 is H.

125. The vaccine formulation according to claim 121, wherein Ri, R2, and R3 are H.

126. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF2a.

127. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF2a.

128. The vaccine formulation according to claim 106, wherein the sOS that comprises one or more O-Ag RUs from SF2a is conjugated to a carrier with a linker.

129. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following structure:

130. The vaccine formulation according to claim 129, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF2a having the following structure:

131. The vaccine formulation according to claim 129 or claim 130, wherein the carrier is TT.

132. The vaccine formulation according to claim 106, wherein the one or more O-Ag RUs from SF3a comprise the following:

133. The vaccine formulation according to claim 132, wherein at least one of R4 or R5 is acetyl.

134. The vaccine formulation according to claim 132, wherein R4 or R5 is acetyl.

135. The vaccine formulation according to claim 132, wherein at least one of R4 or R5 isH.

136. The vaccine formulation according to claim 132, wherein R4 is H.

137. The vaccine formulation according to claim 132, wherein R5 is acetyl.

138. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF3a.

139. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF3a140. The vaccine formulation according to claim 106, wherein the sOS that comprises one or more O-Ag RUs from SF3a is conjugated to a carrier with a linker.

141. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

142. The vaccine formulation according to claim 141, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF3a having the following structure:

143. The vaccine formulation according to claim 141 or claim 142, wherein the carrier is TT.

144. The vaccine formulation according to claim 106, wherein the one or more O-Ag RUs from SF6 comprise the following:

145. The vaccine formulation according to claim 144, wherein at least one of Re or R? is acetyl.

146. The vaccine formulation according to claim 144, wherein Re or R? is acetyl.

147. The vaccine formulation according to claim 144, wherein at least one of Re or R? is H.

148. The vaccine formulation according to claim 144, wherein Re and R? is H.

149. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from SF6.

150. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 3 O-Ag RUs from SF6.

151. The vaccine formulation according to claim 106, wherein the sOS that comprises one or more O-Ag RUs from SF6 is conjugated to a carrier with a linker.

152. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

153. The vaccine formulation according to claim 152, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from SF6 having the following structure:

154. The vaccine formulation according to claim 152 or claim 153, wherein the carrier is TT.

155. The vaccine formulation according to claim 106, wherein the one or more O-Ag RUs from Sson comprise the following:

156. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 2 to 10 O-Ag RUs from Sson.

157. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier comprise a sOS that comprises 4 O-Ag RUs from Sson.

158. The vaccine formulation according to claim 106, wherein the sOS that comprises one or more O-Ag RUs from Sson is conjugated to a carrier with a linker.

159. The vaccine formulation according to claim 106, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the following structure:

160. The vaccine formulation according to claim 159, wherein the two or more different monovalent sOSs conjugated to a carrier include a sOS that comprises one or more O-Ag RUs from Sson having the following structure:

161. The vaccine formulation according to claim 159 or claim 160, wherein the carrier is TT.

162. The vaccine formulation according to claim 106, wherein the degree of conjugation between the one or more O-Ag RUs from SF2a and the carrier is from 1 to 30.

163. The vaccine formulation according to claim 106, wherein the degree of conjugation between the one or more O-Ag RUs from SF3a and the carrier is from 1 to 30.

164. The vaccine formulation according to claim 106, wherein the degree of conjugation between the one or more O-Ag RUs from SF6 and the carrier is from 1 to 30.

165. The vaccine formulation according to claim 106, wherein the degree of conjugation between the one or more O-Ag RUs from Sson and the carrier is from 1 to 30.

166. A method of making a multivalent Shigella vaccine composition, the method comprising:obtaining two or more different monovalent sOSs;conjugating each of the two or more different monovalent sOSs to a carrier to prepare two or more different monovalent sOS carrier conjugates, wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, and / or (iv) a sOS that comprises one or more O-Ag RUs from Sson; and combining the two or more different monovalent sOS carrier conjugates.

167. The method according to claim 166, further comprising mixing the two or more different monovalent sOS carrier conjugates with an adjuvant.

168. A multivalent composition comprising two or more different monovalent sOSs conjugated to a carrier for its use as a vaccine against Shigella,sOS wherein the two or more different monovalent sOSs comprise (i) a sOS that comprises one or more O-Ag RUs from SF2a, (ii) a sOS that comprises one or more O-Ag RUs from SF3a, (iii) a sOS that comprises one or more O-Ag RUs from SF6, (iv) a sOS that comprises one or more O-Ag RUs from Sson, or (v) a combination of the sOSs of (i), (ii), (iii), and / or (iv).

169. The composition for its use according to claim 168, wherein the multivalent Shigella vaccine composition further comprises an adjuvant.

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