The preparation contains the VirG protein and the nucleic acid sequence encoding this VirG protein.

Abstract

The invention relates to a vaccine preparation useful for the prevention and protection against infection based on Shigella.

Description

SHIGELLA VACCINE COMPOSITIONSGOVERNMENT INTEREST

[0001] This invention was made with government support under Grant Numbers AI125841 and AI161839 awarded by the National Institutes of Health. The Government has certain rights in the invention.TECHNICAL FIELD

[0002] This invention relates generally to vaccine compositions useful in preventing and protecting against Shigella-based infection.BACKGROUND

[0003] Diarrheal diseases caused by Shigella species are associated with mortality and life-long disability globally and disproportionally affect children living in poor areas and those lacking clean water and proper sanitation. The annual global death toll is ~ 200,000, one-third being children younger than 5 years of age. Repeated infections lead to growth faltering, impaired cognitive development, and reduced life expectancy. Although treatable by antibiotics and oral rehydration therapy, the rapid and widespread rise of antibiotic-resistant strains and the highly contagious nature of the disease surmise shigellosis as a major global public health concern for which there is no vaccine available.

[0004] Various Shigella vaccine candidates, i.e. , orally delivered live attenuated or killed Shigella vaccines and parenterally administered subunit vaccines, have been evaluated for safety and immunogenicity in humans. Shigella O-polysaccharide (OPS)-based conjugates, the most advanced, are being investigated in controlled human infection models (CHIM) (NCT04078022) and field efficacy trials (NCT04602975). An early Shigella OPS-recombinant Pseudomonas aeruginosa exotoxin A (rEPA) conjugate, although effective in adults and older children, failed to prevent shigellosis in children younger than 3 years of age - the most affected group. A newer version of Shigella flexneri (S. flexneri) 2a OPS-rEPA was moderately protective against only severedisease in a CHIM study. These vaccine candidates rely mainly on immunity that is specific to the bacterial lipopolysaccharide (LPS) and like most others, their coverage is restricted to the immunizing serotype, while there are more than 50 disease-causing Shigella serotypes. OPS-based multivalent formulations that would cover the four most prevalent serotypes are being studied (NCT05156528 and NCT04056117). A multivalent outer membrane vesicle-based vaccine is also being evaluated in adults, 2- to 5-year-old children, and infants 9 months of age in Kenya (NCT05073003), although its monovalent predecessor failed to elicit meaningful protection in a recent adult CHIM study. Still, important obstacles of the multivalent vaccine approach include difficult clinical evaluation and potential interaction / interference among vaccine components that may reduce vaccine effectiveness and increase manufacturing cost.

[0005] In addition to OPS-based vaccines, highly conserved protein components of the Shigella type III secretion system (T3SS) have been proposed as vaccine candidates. Inventors have reported robust preclinical immunogenicity and protective efficacy of the invasion plasmid antigens (Ipa) B, alone or combined with other antigens, against infection with multiple Shigella serotypes. Invaplex, a macromolecular Shigella subunit vaccine containing Ipa proteins and LPS, elicited high levels of protection against S. flexne 2a and S. sonnei in preclinical studies, and was well-tolerated and immunogenic in humans. However, the native Invaplex vaccine failed to prevent disease in an adult CHIM. Accordingly, there is a need in the art for improved Shigella vaccines.SUMMARY

[0006] Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes.

[0007] Inventors have recognized a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. Disclosed herein are compositions that take advantage of the immunogenicity and protective capacity of VirG, the surface-exposed alpha (a) domain of VirG (VirGa), and corresponding fragments as a subunit vaccine in mice. VirGa was produced as arecombinant protein. Surprisingly, this region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGa elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGa given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGa-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells.

[0008] Additional experiments and disclosures related to VirGa fragments also showed promise in eliciting an immune response and preventing Shigella infection in vivo. The VirGa fragments disclosed herein may also be combined in a composition with other components such as IpaB and IpaH antigen polypeptides where in some instances, the IpaB polypeptide can be a conjugate containing non-natural amino acids at one or more positions.

[0009] The inventors have shown that, surprisingly, VirGa and fragments thereof are a promising cross-protective vaccine candidate to prevent Shigella infection. Provided herein is a composition comprising a VirG protein comprising an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 2 or a fragment comprising about 100-400 amino acids thereof. The fragment may comprise a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 3 or 4. The fragment may comprise a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 5.

[0010] The VirG protein may comprise a tag at an N- or C-terminus of the VirG protein. The tag may comprise a His-tag and a protease cleavage site. The protease cleavage site may comprise a Tobacco Etch Virus (TEV) protease cleavage site. The TEV protease cleavage site may comprise the sequence set forth in SEQ ID NO: 6 followed by any amino acid except for proline or tryptophan. The tag may comprise the sequence set forth in SEQ ID NO: 7. The VirG protein may comprise the sequence set forth in SEQ ID NO: 8 or 9.

[0011] The VirG protein may be synthesized using a cell-free system. The composition may comprise an adjuvant. The composition may further comprise one or more Shigella invasion plasmid antigen (Ipa) polypeptides, which may comprise one or more of an IpaB polypeptide and an IpaH polypeptide. The IpaB polypeptide may be an IpaBpolypeptide conjugate comprising one or more non-natural amino acids (nnAA). The IpaB polypeptide may comprise a sequence at least 90% identical to the sequence set forth in any one of SEQ ID NOs: 10-16. The one or more Ipa polypeptides may be synthesized using a cell-free system. The composition may independently comprise about 5-100 pg of each of the VirG protein, and optionally each of the Ipa polypeptides.

[0012] Further provided is a nucleic acid encoding the Shigella VirG protein.

[0013] Provided herein is a method of eliciting an immune response against Shigella in a mammalian subject in need thereof, immunizing a mammalian subject in need thereof against Shigella, or reducing or preventing a Shigella infection in a mammalian subject in need thereof, comprising administering the composition. Also provided is the composition for eliciting an immune response against Shigella in a mammalian subject, immunizing a mammalian subject against Shigella, or reducing or preventing a Shigella infection in a mammalian subject. Further provided is use of the in the manufacture of a medicament for eliciting an immune response against Shigella in a mammalian subject, immunizing a mammalian subject against Shigella, or reducing or preventing a Shigella infection in a mammalian subject. The composition may be delivered or may be intended to be delivered intranasally, transdermally, or intramuscularly.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A-H show cloning, expression, and purification of Shigella VirGa protein. FIG. 1A shows a drawing showing full-length VirG and the a-domain region, which was cloned. VirGa aa sequences across the four Shigella species demonstrate the conserved nature of VirGa Shigella serogroups; FIG. 1 B-D show SDS-PAGE gels and western blot analyses of purified VirGa; Protein expression by positively transformed E. coli BL21 (DE3) pLysS competent cells induced with 1 mM IPTG for 2 h (lane 3) or 3 h (lane 4), the latter achieving higher expression of the 76 kDa VirGa (lane 4, red box). Protein molecular weight marker (MWM; lane 1 ) and uninduced cells (lane 2) included as controls (FIG. 1 B); FIG. 1 C shows purified VirGa after concentration; protein MWM (lane 1 ), eluted VirGa protein (lanes 2-5) and concentrated VirGa (lanes 6-7); FIG. 1 D shows confirmation of 76 kDa VirGa protein (lane 2) by western blot with anti-His tag antibody (left panel) and anti- VirGa immune sera (right panel). MWM (lane 1 ) and ELISA data showing immunereactivity of VirGa compared to full length VirG protein using pooled sera from mice immunized with full length VirG. Serially diluted sera added to plates coated with each protein and replicate samples were performed in the same conditions using the same pooled sera and serial dilutions, and (FIG. 1 E) data represent mean OD450 of replicate wells (R2, r, and p value shown) and linear concordance across dilutions. FIG. 1 F-H show cloning data of VirGa.

[0015] FIG. 2A-D show immunogenicity of Shigella VirGa. FIG. 2A is a schematic illustration of immunization and challenge in adult mice; (b) the kinetics of VirGa-specific serum IgG titers measured by ELISA (n = 15 / group); arrows indicate immunization. Data represent mean of individual titers ± SEM; **** p < 0.0001 vs. PBS or ADJUPHOS® by t-test (FIG. 2B); and (FIG. 2C) VirGa-specific IgG titers achieved before and after boost with escalating doses of VirGa (n = 15 / group).

[0016] FIG. 3A-E show protective efficacy of Shigella VirGa. Mice were immunized as described in FIG. 2 and challenged IN with a lethal dose of S. flexneri 2a 2457T (5 x 106CFU / dose) or S. sonnei Moseley (5.2 x 106CFU / dose) on day 57 post-vaccination, where (FIG. 3A and B) show survival curves from 15-20 mice / group. p values compared to PBS control were determined by log-rank (Mantel-Cox) test; (FIG. 3C and D) show VirGa-specific serum IgG titers at the time of challenge (day 55) in VirGa vaccinated mice that were protected or succumbed to S. flexneri 2a (n = 45) or S. sonnei (n = 20) infection. Data represents mean serum IgG titers ± SEM, * p < 0.05 and ** p < 0.01 by t- test; and (FIG. 3E) shows ROC analysis of VirGa-specific serum IgG levels in VirGa vaccinated survivors and those that succumbed to Shigella lethal infection (n = 65 combining both challenges) and identification of a threshold of protection. Red arrow denotes threshold to predictor of survival with ~ 80% sensitivity and specificity. AUROC = 0.7089 (95% Cl: 0.63 - 0.90), p = 0.0005.

[0017] FIG. 4A-C show immunogenicity and protective efficacy of mucosally administered VirGa in mice. Adult mice (10 per group) immunized IN with 2.5, 5, or 10 pg of VirGa protein admixed to dmLT (2.5 pg) on days 0, 14, and 28 (black arrows) where negative control mice received PBS or dmLT. Mice immunized IN with live attenuated S. flexneri 2s strain CVD1208s on days 0 and 21 (gray arrows) wereincluded as positive controls for S. flexneri 2a challenge: (FIG. 4A) Kinetics of VirGa- specific serum IgG titers measured by ELISA. Mean of individual titers ± SEM; * p < 0.05, **** p < 0.0001 vs. PBS by Mann-Whitney-test; (FIG. 4B) Kinetics of dmLT-specific serum IgG measured by ELISA. Mean of individual titers ± SEM; **** p < 0.0001 compared to PBS control by f-test. FIG. 4C Immunized mice were challenged IN with a lethal dose (107CFU / dose) of S. flexneri 2a on day 56 post-vaccination. Data represents survival curves from 10 mice per group. **** p < 0.0001 compared to PBS control as determined by log-rank (Mantel-Cox) test; and (FIG. 4C) shows survival rates over days for different dosages of a VirGa composition as compared to positive and negative controls.

[0018] FIG. 5A-B show VirGa-specific and total ASC. Frequencies of total and VirGa- specific antibody secreting cells (ASC) were measured in spleen (FIG. 5A) and bone marrow (FIG. 5B) from mice immunized IM with 20 pg of VirGa (IM,) or IN with S. flexneri 2a (si Sf2a-IN) that survived S. flexneri 2a lethal challenge.

[0019] FIG. 6 presents microscopy images of S. flexneri 2a that show immune recognition of VirG on live Shigella by VirGa-induced antibodies. A suspension of S. flexneri 2a was incubated with sera from VirGa-vaccinated mice (top panel) or mice that received ADJUPHOS® (bottom panel). Bound antibodies were detected using AF555 labeled anti-mouse antibodies.

[0020] FIG. 7A-D show inhibition of Shigella adherence and invasion to human epithelial cells by murine VirGa-antibodies. FIG. 7A shows a schematic representation of the Shigella human colonoid infection and inhibition model. S. flexneri 2a (5 x 108CFU / mL) grown in the absence or presence of deoxycholate (DOC) were incubated with murine VirGa mouse antisera and added immediately to the basolateral side of human colonoid monolayers. Relative adhesion (FIG. 7B) or invasion (FIG. 7C) rates of the S. flexneri 2a (grown with and without DOC in the presence of VirGa antibodies or absence (ADJUPHOS® negative control). ADJUPHOS FIG. 7D shows data for VirGa antibody impairment of adherence of S. sonnei to human colonoid monolayers.

[0021] FIG. 8A-D shows titers of various antigen-specific IgG. FIG. 8A shows Ag- specific IgG titers elicited by VirGa, VirG R1 , and VirG R2 when administered to mice; (FIG. 8B) shows VirGa specific IgG titers elicited by VirGa-, VirG R1-, and VirG R2-vaccinated mice; (FIG. 8C) shows VirG R1 specific IgG titers elicited by VirGa-, VirG R1 and VirG R2-vaccinated mice; and (FIG. 8D) shows VirG R2 specific IgG titers for VirGa, VirG R1 , and VirG R2.

[0022] FIG. 9 compares percent survival of mice immunized with VirGa, VirG R1 , VirG R2, and controls (i.e. sublethal dose of S. flexneri 2a (positive) and PBS (negative) after lethal challenge with S. flexneri.

[0023] FIG. 10 shows the reduction in aggregation upon substituting the cysteine at position 130 of VirG R1 with serine.DETAILED DESCRIPTIONDefinitions

[0024] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0026] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0027] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1 %, ± 0.5%, or even ± 0.1 % of the specified amount.

[0028] As used herein, the transitional phrase “consisting essentially of” is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0029] The term “consists essentially of” (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide orpolypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and / or 3’ or N-terminal and / or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0030] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1 .25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold.

[0031] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0032] A “therapeutically effective” or “treatment effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” or “treatment effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of raising an immune response, activating or increasing the number of immune cells known to produce an immune response). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0033] By the term “treat,” “treating,” or “treatment of” (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of thesubject’s condition and / or to alleviate, mitigate or decrease in at least one clinical symptom and / or to delay the progression of the condition.

[0034] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompass any type of prophylactic treatment to reduce the incidence of the condition or delay the onset of the condition.

[0035] A “prevention effective” amount as used herein is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder and / or clinical symptoms in a subject and / or to reduce and / or delay the severity of the onset of a disease, disorder and / or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0036] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0037] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention. In some embodiments, the fragment may also have a length of about 100- 400, 150-350, 200-350, or 250-350 consecutive amino acids of a reference polypeptide or amino acid sequence.

[0038] As used herein, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0039] As used herein, the term “modified,” as applied to a polypeptide sequence, refers to a sequence that differs from a wildtype sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0040] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991 ).

[0041] As used herein, the term “substantially identical” or “corresponding to” means that two nucleic acid or polypeptide sequences have at least 60%, 70%, 80% or 90% sequence identity. In some embodiments, the two nucleic acid or polypeptide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

[0042] An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e. , the entire reference sequence or a smaller defined part of the reference sequence.

[0043] The term “vaccinate” and “immunize” are used interchangeably herein to mean administering a compound and / or composition (e.g., a vaccine) in a prevention effective amount to a subject. Thus, a vaccine may prevent, delay, and / or reduce the severity of a disease, disorder and / or clinical symptoms in a subject after administration to said subject relative to what would occur in the absence of the methods of the invention.

[0044] The term “adjuvant” is used herein to mean a compound and / or substance that is added to a composition to increase the efficacy and / or speed of action of an immunogen (e.g., an antigen) when said composition is administered to a subject. In some embodiments, increasing the efficacy of an immunogen will increase the immuneresponse in said subject. In some embodiments, the adjuvant may increase the efficacy of the immunogen by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000-fold or more. In some embodiments, the adjuvant may reduce the amount of immunogen required to produce an immune response in the subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the adjuvant may prolong the time over which the immunogen produces an immune response in the subject by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50-fold or more. Those skilled in the art will appreciate that the adjuvant may work through any mechanism to produce the effects listed above, including, but not limited to, stabilizing the immunogen against degradation, increasing the binding affinity of the immunogen to an immune cell, activating other components of the immune system (e.g., cytokine production, interferon production, etc.), and / or altering inflammatory responses.

[0045] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, i.e. , the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science; 21 st ed. 2005). Exemplary pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), sterile pyrogen-free water, and other sterile pyrogen- free physiological saline solutions.

[0046] The term “administering” or “administration” of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function (e.g., for raising an immune response in a subject or for vaccinating a subject).

[0047] A “subject” of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a mammal, a reptile, a bird, anamphibian, or a fish. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pigtailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human.

[0048] A “subject in need” of the methods of the invention can be any subject known or suspected of having increased risk of developing an infection as described herein to which raising an immune response and / or vaccinating against said infection may provide beneficial health effects.

[0049] A “sample”, “biological sample”, and / or “ex vivo sample” of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a tissue, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.Compositions

[0050] Disclosed herein is a composition, which may be immunogenic, comprising a VirG protein. The VirG protein may comprise an alpha (a) domain of VirG (VirGa), a fragment of the VirGa, a protein substantially identical thereto, or a fragment of the foregoing. Also provided are nucleic acids encoding the VirG protein. In some embodiments, the VirG protein is from Shigella. The VirG protein may also known as IcsA (Intra-cellular spread gene A). VirG is integrally involved in bacterial pathogenesis. It is essential for Shigella adherence and actin-based motility within the colonic epithelium. Disruption or deletion of VirG largely attenuates Shigella virulence in humans and in animal models. The protein is composed of two main domains: a functionally active surface exposed a-domain (VirGa) and a transporter transmembrane [3-domain. Importantly, VirG is highly conserved among Shigella species and independent of the T3SS. The VirG protein may be from S. flexneri and may comprise the sequence set forth below.SEQ ID NO: 1MNQIHKFFCNMTQCSQGGAGELPTVKEKTCKLSFSPFWGASLLLGGPIAFATPLSGT QELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINSDIMISAG HGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGGEAVTGD NLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNYGEGDG GNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNGAILGGN GGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGSVITGDV QVNNSSILKIINNDYTGTTPTIEGDLCAGDCTTVSLSGNKFTVSGDVSFGENSSLNLAGI SSLEASGNMSFGNNVKVEAIINNWAQKDYKLLSADKGITGFSVSNISIINPLLTTGAIDYT KSYISDQNKLIYGLSWNDTDGDSHGEFNLKENAELTVSTILADNLSHHNINSWDGKSLT KSGEGTLILAEKNTYSGFTNINAGILKMGTVEAMTRTAGVIVNKGATLNFSGMNQTVNT LLNSGTVLINNINAPFLPDPVIVTGNMTLEKNGHVILNNSSSNVGQTYVQKGNWHGKG GILSLGAVLGNDNSKTDRLEIAGHASGITYVAVTNEGGSGDKTLEGVQIISTDSSDKNAF IQKGRIVAGSYDYRLKQGTVSGLNTNKWYLTSQMDNQESKQMSNQESTQMSSRRAS SQLVSSLNLGEGSIHTWRPEAGSYIANLIAMNTMFSPSLYDRHGSTIVDPTTGQLSETT MWIRTVGGHNEHNLADRQLKTTANRMVYQIGGDILKTNFTDHDGLHVGIMGAYGYQD SKTHNKYTSYSSRGTVSGYTAGLYSSWFQDEKERTGLYMDAWLQYSWFNNTVKGDG LTGEKYSSKGITGALEAGYIYPTIRWTAHNNIDNALYLNPQVQITRHGVKANDYIEHNGT MVTSSGGNNIQAKLGLRTSLISQSCIDKETLRKFEPFLEVNWKWSSKQYGVIMNGMSN HQIGNRNVIELKTGVGGRLADNLSIWGNVSQQLGNNSYRDTQGILGVKYTF

[0051] The VirGa may comprise amino acids 53-758 relative to SEQ ID NO: 1 . The VirGa may comprise the following sequence. SEQ ID NO: 2TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINS DIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGG EAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNY GEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNG AILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGS VITGDVQVNNSSILKIINNDYTGTTPTIEGDLCAGDCTTVSLSGNKFTVSGDVSFGENSS LNLAGISSLEASGNMSFGNNVKVEAIINNWAQKDYKLLSADKGITGFSVSNISIINPLLTT GAIDYTKSYISDQNKLIYGLSWNDTDGDSHGEFNLKENAELTVSTILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINAGILKMGTVEAMTRTAGVIVNKGATLNFSGM NQTVNTLLNSGTVLINNINAPFLPDPVIVTGNMTLEKNGHVILNNSSSNVGQTYVQKGN WHGKGGILSLGAVLGNDNSKTDRLEIAGHASGITYVAVTNEGGSGDKTLEGVQIISTDS SDKNAFIQKGRIVAGSYDYRLKQGTASGLNTNKWYLTSQMDNQESKQMSNQESTQM SSR

[0052] In one example, the VirG protein comprises a fragment of the VirGa comprising amino acids 53-353 or 53-352 relative to SEQ ID NO: 1 , referred to herein as VirG R1 . The VirG R1 may comprise the sequence set forth in SEQ ID NO: 3 or 4. The VirG protein may comprise a sequence substantially identical to SEQ ID NO: 3 or 4, or a sequence at least 90% identical (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence set forth in SEQ ID NO: 3 or 4. In some embodiments, the VirG protein consists of or consists essentially of the sequence set forth in SEQ ID NO: 3 or 4. In some embodiments, the present invention comprises a nucleic acid sequence that encodes the VirG protein .SEQ ID NO: 3TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINS DIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGG EAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNY GEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNG AILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGS VITGDVQVNNS

[0053] In another example the VirG R1 may comprise the following sequence.SEQ ID NO: 4TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINS DIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGG EAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNY GEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNG AILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGS VITGDVQVNN

[0054] In another example, the VirG protein comprises a VirGa fragment comprising amino acids 500-758 relative to SEQ ID NO: 1 , referred to herein as VirG R2. The VirGR2 may comprise the sequence set forth in SEQ ID NO: 5. The VirG protein may comprise a sequence substantially identical to SEQ ID NO: 5, or a sequence at least 90% identical (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence set forth in SEQ ID NO: 5. In some embodiments, the VirG protein consists of or consists essentially of the sequence set forth in SEQ ID NO: 5. In some embodiments, the present invention comprises a nucleic acid sequence that encodes the VirG protein.SEQ ID NO: 5ENAELTVSTILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINAGILKMGTV EAMTRTAGVIVNKGATLNFSGMNQTVNTLLNSGTVLINNINAPFLPDPVIVTGNMTLEK NGHVILNNSSSNVGQTYVQKGNWHGKGGILSLGAVLGNDNSKTDRLEIAGHASGITYV AVTNEGGSGDKTLEGVQIISTDSSDKNAFIQKGRIVAGSYDYRLKQGTVSGLNTNKWY LTSQMDNQESKQMSNQESTQMSSR

[0055] The VirG protein may comprise one or more tags, each of which may comprise one or more of a purification tag, a protease cleavage site, and a signal peptide. Purification tags, also known as affinity tags or fusion tags, are short amino acid sequences that are genetically fused to a target protein. These tags are used to facilitate the purification and detection of the tagged protein. Non-limiting examples include His-tag, Glutathione S-transferase (GST), Streptavidin-binding peptide (SBP or Strep-tag), Maltose-binding protein (MBP), Flag-tag, HA-tag, Calmodulin-binding peptide (CBP), and SUMO (Small Ubiquitin-like Modifier) tag. Each of such tags may be located at the N- or C-terminus of the VirG protein.

[0056] The VirG protein may comprise a His-tag at its N- or C-terminus. In one example, the His-tag comprises six consecutive histidines (6xHis). The tag may comprise one or more protease cleavage sites. Non-limiting examples of protease cleavage sites include enterokinase (EK) cleavage site, thrombin cleavage site, Tobacco Etch Virus (TEV) protease cleavage site, factor Xa cleavage site, proteinase k cleavage site, precision protease cleavage site, or caspase cleavage site. In one example, the tag comprises a TEV protease cleavage site, which may comprise the sequence ENLYFQ (SEQ ID NO: 6) followed by any amino acid except for proline or tryptophan. The protease cleavage site may be located at the N- or C-terminus of the VirG protein.

[0057] The protease cleavage site may be present between the purification tag and the polypeptide, to facilitate purification and function. The VirG protein may comprise a His- tag and a TEV protease cleavage site. The TEV cleavage site may be located after or before the His-tag. In one example, the TEV cleavage site is located after the His-tag, which may be a 6xHis tag. In a more specific example, the VirG protein comprises the sequence HHHHHHGGSENLYFQ (SEQ ID NO: 7), which may be located at the N- terminus of the VirG protein.

[0058] In one example, the tagged VirG protein comprises VirG R1 . The VirG protein may comprise the following sequence.SEQ ID NO. 8HHHHHHGSGENLYFQGTPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYI TSSNPSHASRTKLRINSDIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMIL GGSGGSGADHNGDGGEAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNL PIINKGTISGGNGGNNYGEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDG YGGNAITGDNLSVINNGAILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAG NAIHITGGNNSLILHEGSVITGDVQVNN

[0059] In another example, the tagged VirG protein comprises VirG R2. The VirG protein may comprise the following sequence.SEQ ID NO. 9HHHHHHGSGENLYFQGILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINA GILKMGTVEAMTRTAGVIVNKGATLNFSGMNQTVNTLLNSGTVLINNINAPFLPDPVIVT GNMTLEKNGHVILNNSSSNVGQTYVQKGNWHGKGGILSLGAVLGNDNSKTDRLEIAG HASGITYVAVTNEGGSGDKTLEGVQIISTDSSDKNAFIQKGRIVAGSYDYRLKQGTASG LNTNKWYLTSQMDNQESKQMSNQESTQMSSR

[0060] In some aspects, the composition comprises the VirG protein and one or more Shigella invasion plasmid antigen polypeptides. In some embodiments, the composition further comprises an invasion plasmid antigen B (IpaB) polypeptide or a conjugate thereof. In some embodiments, the IpaB polypeptide conjugate comprises at least one non-natural amino acid (nnAA), wherein the nnAA is selected from the group consisting of 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyri din-3 -yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino- 3-(4-(azidomethyl)phenyl)propanoic acid, and any combination. In one example, the nnAA is pAMF. The IpaB polypeptide conjugate may be as described in W02020205584 (the contents of which are incorporated here by reference in its entirety).

[0061] The IpaB polypeptide conjugate may be disclosed in Table 1 of W02020205584. The IpaB polypeptide conjugate may comprise a sequence disclosed in Table 1 below. It should be noted that the IpaB polypeptide conjugate sequences shown below have different sequence numbering than the W02020205584 application. Further, the “X” in the below sequences indicate amino acid positions where substitution by non-natural amino acids can occur as described in W02020205584.Table 1: Exemplary IpaB Polypeptide Conjugates

[0062] In some embodiments, the composition further comprises an invasion plasmid antigen H (IpaH) polypeptide. In some embodiments, the composition comprises the VirG protein, the IpaB polypeptide or conjugate thereof, and the IpaH polypeptide. In some embodiments, the composition may comprise one or more of invasion plasmid antigen C and invasion plasmid antigen D polypeptides.

[0063] In some embodiments, the composition comprises a therapeutically effective amount of the VirG protein and optionally the IpaB polypeptide or conjugate thereof and optionally the IpaH polypeptide. The composition may comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 pg of one or more of the VirG protein, the IpaB polypeptide or conjugate thereof, and the IpaH polypeptide, or a range of two of the foregoing amounts. The composition may comprise about 5-100 pg or about 20-100 pg or about 20-90 pg of the VirG protein, and optionally the IpaB polypeptide or conjugate thereof, and optionally the IpaH polypeptide.

[0064] In some embodiments, the composition further comprises one or more adjuvants. The one or more adjuvants may be selected from the group consisting of alum, ADJUPHOS®, aluminum hydroxide, TLR4 agonsists (MPL and MPL-like), TLR9 agonists (CpG), and STING agonists (cyclic diAMP). ... In some embodiments, the adjuvant comprises ADJUPHOS® (2% AIPO4).

[0065] The composition may be suitable for administration to a subject. In one example, the composition is suitable for mucosal administration. The mucosal administration may be intranasal. In another example, the composition is suitable for intradermal or intramuscular administration.Methods of Use

[0066] Another aspect of this invention relates to eliciting an immune response in a subject against various Shigella serotypes using a composition disclosed herein. A further aspect relates to use of the composition for immunizing a subject against Shigella, or reducing or preventing a Shigella infection in a subject. Also provided are methods of eliciting an immune response against Shigella, immunizing a subject against Shigella, or reducing or preventing a Shigella infection, comprising administering a composition disclosed herein to a subject in need thereof. Compositions disclosed herein may also be used in the manufacture of a medicament for eliciting an immune response against Shigella, immunizing a subject against Shigella, or reducing or preventing a Shigella infection. The subject may be a mammal. The mammal may be a human. The Shigella may be S. flexneri or S. sonnei.In some embodiments, the composition is delivered to a mammalian subject mucosally, which may be intranasally or intradermally (IN).EXAMPLES

[0067] Below are examples and further details surrounding the disclosed invention to help with understanding and teaching the described invention. The additional information provided below is for illustration purposes only and is not intended to limit the scope of the invention in any way.Methods of Bacterial Growth

[0068] S. flexneri 2a 2457T was grown in Luria-Bertani (LB) media (Athena Environmental Sciences, MD) at 37°C, and the invasion plasmid was extracted using a plasmid purification kit (Qiagen). E. coli DH5a and BL21 (DE3) pLysS cells were cultured in LB media at 37°C. S. flexneri 2a 2457T and S. sonnei Moseley were prepared as previously described. Briefly, the strains were streaked on tryptic soy agar (TSA) containing 0.02% Congo red and grown overnight at 37°C. A total of 20-25 colonies were picked and grown in 125 mL LB or tryptic soy broth (TSB) at 37°C with agitation (180 rpm) for 2-3 h or until an ODeoo of 0.8 to 1 .3 was achieved. Bacterial cultures were centrifuged, and pellets were resuspended in PBS. CFU were determined by plating serial dilutions of bacteria suspension on TSA and / or Congo red agar plates.Cloning, expression, and purification of recombinant VirGa protein

[0069] The gene for expression of VirGa (amino acid [aa], GenBank accession number AF386526.1 from S. flexneri 2a) was synthesized and subcloned in pRSETA vector. VirGa gene (2.1 kb) was amplified by PCR using as template the 220 kb S. flexneri 2a 2457T virulence plasmid. The amplified VirGa gene was purified using the gel extraction kit (Qiagen). pRSETA, a high-level prokaryotic expression vector was used to clone VirGa gene with a cleavable poly-histidine (6x His) tag which enables rapid purification with nickel resin and detection with an anti-His antibody. The purified VirGa gene and pRSETA expression vector was digested with restriction enzymes BamHI and EcoRI, followed by gel elution and purification. The digested VirGa and pRSETA vectors were ligated andtransformed into E. coli DH5a competent cells. After transformation, the colonies bearing VirGa-encoding plasmids were screened by PCR and ran on 1 % agarose gel. The plasmids containing the VirGo gene were extracted using plasmid isolation kit (Qiagen). Restriction enzyme digestion with BamHI and EcoRI was performed to further confirm VirGa gene cloning. The plasmid-bearing strains were stored in 15% glycerol stock at - 80°C.

[0070] Plasmids bearing VirGa were transformed into E. coli BL21 (DE3) pLysS cells for protein expression. E. coli cells were grown in 8 L of Terrific broth (Invitrogen) with 30 pg / mL chloramphenicol and 50 pg / mL ampicillin at 37°C, 200 rpm, until reaching an ODeoo of 4.0-5.0. Protein expression was induced with 1 mM isopropyl 1 -thio-|3-D- galactopyranoside (IPTG) (Teknova) for 3 h. After induction, cells were centrifuged at 7,800 rpm for 30 min at 4°C. The bacterial pellet was collected and resuspended in B- PER (Thermo Scientific) extraction buffer with Protease inhibitor cocktail (Sigma-Aldrich), Lysozyme 1 mg / mL, Benzonase 25 U / mL, and 0.5% TritonX-100 and incubated for 1 h at RT. The suspension was then centrifuged at 10,000 xg for 40 min, 4°C. The pellet containing inclusion bodies was washed and solubilized in a solution containing 20 mM Tris, 0.5 M NaCI, 20 mM Imidazole, and 8 M Urea. The expressed recombinant VirGa protein was purified using a chelating nickel column (Cytiva) by affinity chromatography. The bound protein was washed and then refolded within the column using urea gradient starting at 6 M urea in elution buffer containing 20 mM Tris, 0.5 M NaCI, 20 mM Imidazole, pH 8.0, and ending at 0 M urea in the same buffer. The refolded protein was collected in elution buffer and urea was removed by dialysis. The purified protein was then concentrated using Am icon filters (Millipore). Protein concentration was determined using Bicinchoninic Acid (BCA) protein assay (Pierce™ Protein Assay Kit; Thermo Scientific™) with bovine serum albumin (BSA) as standard. Aliquots were stored at - 80°C. The purity of VirGa was determined based on relative intensity and size of the bands by Image Lab™ software (Bio-Rad). The endotoxin level was analyzed by limulus amoebocyte lysate (LAL) cartridges (Charles River Laboratories) according to manufacturer’s instruction. Residual host-cell proteins were determined based on density of non-specific bands in SDS-PAGE with Coomassie staining. VirGa aa sequences of S. flexneri (NCBI reference sequence: WP_17250739.2), S. sonnei (WP_001071793.1 ), S. dysenteriae(WP_13480816.1 ) and S. boydii (WP_148722086.1 ) were used for determination of multiple sequence alignment.SDS-PAGE and Western blot analysis

[0071] For SDS-PAGE, proteins were prepared in 2x-Laemmli sample buffer (Bio-Rad) added with 5% [3-mercaptoethanol and denatured by heating at 95°C for 5 min. The denatured protein samples (20 pl) were loaded onto 12% precasted polyacrylamide gels (Bio-Rad). For western blot analysis, SDS-PAGE-run samples were then transferred into the nitrocellulose membrane using Trans-Blot Turbo Transfer Packs (BioRad). Then the membrane was blocked in 3% BSA containing TBST (TBS, 0.05% (v / v) Tween-20) and incubated with anti-His tag antibodies or murine anti-VirGa immune sera (sera collected from the mice immunized with VirGa protein) overnight at 4°C. The membrane was washed with TBST and incubated with Horseradish peroxidase (HRP)-labeled goat antimouse IgG (KPL SeraCare, Gaithersburg, MD) for 1 h at RT. The membrane was again washed with TBST, added with Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma) and analyzed using Gel Doc imaging system (Bio-Rad).Mice, immunizations, and experimental infection with virulent organisms

[0072] Adult female BALB / c mice (6 to 8 weeks old) were purchased from Charles River Laboratories. For IM vaccination, mice (15 to 20 per group for each challenge strain) were immunized IM on days 0 and 28 with 5, 10, or 20 pg of VirGa adsorbed to ADJUPHOS® (4.8% v / v; InvivoGene) administered in a 100 pL volume (50 pL per leg). Negative control groups received IM ADJUPHOS® or PBS alone. Positive control groups were immunized IN on days 0 and 28 with sublethal doses (1 .0x105CFU / dose) of S. flexneri 2a 2457T or S. sonnei Moseley. For IN vaccination, BALB / c mice were immunized on days 0, 14, and 28 with 2.5, 5, or 10 pg of VirGa in the presence of 2.5 pg of the E. coli dmLT. Negative control groups received IN 2.5 pg of dmLT or PBS alone. Positive control mice were immunized IN with S. flexneri 2a live attenuated strain CVD1208S on days 0 and 21 . Four weeks after the last immunization, mice were challenged IN with 5x106- 1x107CFU / dose of S. flexneri 2a 2457T or 5.2x106CFU / dose of S. sonnei Moseley (corresponding to 3.5- 7 or 2 50% lethal doses [LDso], respectively). IN challenge was performed as previouslydescribed, and mice were monitored daily for 15 days after the challenge. Blood was collected at day -1 (pre-vaccination) and days 13, 27, 42, and 55 (post-vaccination). Spleen and bone marrow cells were obtained from survivors on day 73 (day 16 postchallenge).Antibodies and ASC

[0073] VirGa-specific antibodies were measured in serum by ELISA as previously described. Immulon 2HB plates (Thermo Scientific) were coated with VirGa (2 pg / mL) or full-length VirG (2 pg / mL), and serum samples were tested in two-fold serial dilutions, in duplicate. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (KPL SeraCare, Gaithersburg, MD) was used as detection antibody. Endpoint titers were calculated by interpolation of absorbance values of samples in the linear regression curve of a calibrated in-house standard and reported as EU / mL. An EU corresponds to the reciprocal serum dilution resulting in an absorbance value of 0.2 at 450 nm above the background.

[0074] For ASC measurements, single cell suspensions were prepared from spleen and bone marrow of individual mice (8 per group). Spleen was disrupted with a sterile syringe plunger, and femurs and tibias were flushed by centrifugation using adaptor tubes. Red blood cells were removed with lysis buffer (BIOLEGEND®), and cell suspensions were filtered through a 70-pm cell strainer. The frequencies of total and VirGa-specific IgG- and IgA-secreting cells were determined by mouse IgA / IgG double-color ELISpot assay kit [IMMUNOSPOT® ELISpot Kit, Cellular Technology Limited (CTL)] according to manufacturer’s instructions. Polyvinylidene Fluoride (PVDF) membrane ELISpot plates were pre-wet with 70% ethanol, coated with either anti-mouse IgK / A capture antibodies or 2 pg / mL of VirGa protein overnight at 4°C and blocked with complete RPMI1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin, 8 mM HEPES, and 50 pM 2-mercaptoethanol for 1 h at RT. PBS- coated wells served as negative controls. Spleen and bone marrow cell suspensions from individual mice within each group were pooled, added to plates, and incubated for 6 h at 37°C. After washing, anti-mouse IgA / IgG detection antibody was added, and plates were incubated in the dark at RT for 2 h. After washing, plates were again incubated for 1 hwith Tertiary solution. CTL-TrueBlue and -TrueRed substrate solutions were then added sequentially for color development. Spots were counted using a CTL ImmunoSpot® Analyzer along with ImmunoSpot® Software (ImmunoSpot®, CTL) and results were expressed as the number of spot-forming cells (SFC) per 106cells from quadruplicate wells.Confocal analysis of Shigella VirG antibody recognition

[0075] S. flexneri 2a were grown as described above, and a 100 pL suspension (107CFU / mL) was added to microscope chamber slides. Bacteria were fixed in aqueous 4% paraformaldehyde for 45 min at RT and then washed with 1X PBS. After washing, fixed bacteria were permeabilized and blocked for 1 h at 4°C with PBS containing 15% FBS, 2% BSA, and 0.1% Saponin (all from Sigma-Aldrich), then rinsed with PBS and incubated overnight at 4°C with sera from mice immunized IM with VirGa or ADJUPHOS® diluted 1 :100 in PBS containing 15% FBS and 0.2% BSA. Stained bacteria were washed twice with PBS and incubated with goat anti-mouse IgG AF555-labeled conjugate secondary antibody (Thermo Fisher Scientific) for 1 h at RT in the dark. After washing with PBS, bacteria were mounted in Prolong Gold antifade reagent with 4,6-diamidino-2- phenylindole (DAPI) (Cell Signaling Technology) for DNA staining at 4°C for at least 24 h. Confocal images were conducted using the Leica MICA microscope. Images were captured with a 60X water immersion objective, and settings were adjusted to optimize the signal. Images were collated using FIJI / lmageJ (NIH). Signal processing was applied equally across the entire image.Adherence and invasion inhibition assay in enteroid monolayers

[0076] Shigella adherence and invasion inhibition assays were performed using a human colonoid infection assay. Colonoid fragments seeded on human collagen IV-coated Transwell inserts (3.0 pm pore-size) were grown as monolayer until confluency confirmed by transepithelial electrical resistance with an epithelial volt-ohm meter (EVOM2; World Precision Instruments, USA). Monolayers were differentiated in differentiation medium (DFM) without antibiotics for 5 days. Differentiated colonoid monolayers were inverted and placed in an empty 12-well tissue culture plate. S. flexneri 2a or S. sonnei was grownas described above to an ODeoo of 0.6-0.8 in TSB in the absence or presence of 2.5 mM bile salt deoxycholate (DOC) and a bacterial suspension containing 5 x 108CFU / mL was prepared in DFM. A 50 pL inoculum (2.5x107CFU / 50pL) was combined with 25 pL of heat-inactivated serum from mice immunized with VirGo or ADJUPHOS® and added to the basolateral side of colonoid monolayers. To determine adhesion inhibition, the bacteria-immune sera were incubated with monolayers for 15 min at 37°C with 5% CO2. For invasion inhibition, bacteria grown in the presence of DOC were mixed with mouse immune sera, added onto the monolayers, and incubated for 1.5 h, followed by 30 min incubation with 50 pg / mL gentamicin to kill extracellular bacteria. For both adherence and invasion assays, following incubation, monolayers were washed three times with 1X PBS, and lysed with 0.1 % Triton X-100 for 20 min. Recovered bacteria were serially diluted and plated onto TSA plates for CFU enumeration.Statistical analysis

[0077] All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA). Normality tests were performed on continuous data using D’Agostino and Pearson test. Continuous normally distributed data were compared by either unpaired f-test or one-way ANOVA with Tukey’s multiple comparisons test. Mann- Whitney test and Kruskal-Wallis with Dunn’s multiple comparisons test was used for nonparametric data. Survival curves were compared using a log rank (Mantel-Cox) test. VE was calculated as [(% death in PBS control animals - % death in vaccinated animals) I % death in PBS control animals] x 100. Simple linear regression and Pearson correlation test were used to determine associations between groups. ROC-AUC analysis was used to identify antibody threshold level that would predict protective efficacy of a vaccine. Two- side p values were determined, and p < 0.05 were considered statistically significant. Data presented are either combined or representative of at least two experiments with similar results, and each graph shows measurements from individual samples. VirGa amino acid sequence alignments and percent (%) of homology were determined using NCBI multiple sequence alignment viewer 1 .24.0.Production of recombinant VirGa protein

[0078] A schematic representation of VirG regions is shown in FIG. 1a. Full-length VirG expressed poorly as a recombinant protein, likely due to its large size (120 kDa). Due to the poor expression of full-length VirG, the surface exposed region, VirGa (aa 53 - 758), was cloned, expressed, and produced as a recombinant protein in an E. co / / -based system. VirGa aa sequence across the four Shigella species were determined using NCBI multiple sequence alignment viewer. Identity (%) represents homology of VirGa sequences and non-identical aa residues (red bars) in S. sonnei, S. dysente ae and S. boydii compared to S. flexneri. The homology of the VirGa amino acid sequences among the most prevalent Shigella serotypes is > 99% (FIG. 1 a). The VirGa gene (2.1 kb) was amplified and inserted into a pRSETA plasmid and transformed into E. coli DH5a competent cells (Supplementary FIG. 1a, b). Restriction enzyme digestion with BamHI and EcoRI was performed to confirm successful cloning (Supplementary FIG. 1 c). VirGa- bearing plasmids were transformed into E. coli BL21 (DE3) pLysS competent cells; bacteria were grown, and protein expression induced. High levels of recombinant VirGa (VirGa, 76 kDa) were produced after 3 h of induction. Protein expression by positively transformed E. coli BL21 (DE3) pLysS competent cells induced with 1 mM IPTG for 2 h (lane 3) or 3 h (lane 4), the latter achieving higher expression of the 76 kDa VirGa (lane 4, red box). Protein molecular weight marker (MWM; lane 1 ) and uninduced cells (lane 2) were included as controls (FIG. 1 b). VirGa was purified using a chelating nickel column and refolded within the column. The protein was eluted, dialyzed, and concentrated, resulting in a 76 kDa band in the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel. Purified VirGa after concentration; protein MWM (lane 1 ), eluted VirGa protein (lanes 2-5) and concentrated VirGa (lanes 6-7) (FIG. 1c). Identity was confirmed by western blot analysis using anti-His tag antibody (FIG. 1 d, left panel) and VirGa-reactive mouse antisera (FIG. 1 d, right panel). Confirmation of 76 kDa VirGa protein (lane 2) by western blot with anti-His tag antibody (left panel) and anti-VirGa immune sera (right panel). MWM (lane 1 ). FIG. 1 e shows immune reactivity of VirGa compared to full length VirG protein measured by ELISA against pooled sera from mice immunized with full length VirG. Serially diluted mouse sera added to plates coated with each protein, i.e., VirGa and full length VirG, at the same concentration. The assays were performed in the exact same conditions, testing the same pooled mouse sera againsteach protein, in replicate wells. Data represent mean OD450 of replicate wells (R2, r, and p value shown) and linear concordance across dilutions.

[0079] E. co / / -based production of VirGa was efficient, with a recovery yield of 6.2 mg / L culture, > 95% purity, < 1 % residual host-cell proteins, and < 20 endotoxin units / mg (detection limit); these results were reproduced in subsequent batches.

[0080] The immune reactivity of VirGa was compared with that of full-length VirG by testing recognition of both antigens by pooled sera from mice immunized with full-length VirG in an indirect ELISA (and run in identical conditions). Four-parameter logistic (4PL) dose-response curves of OD450 values for full-length VirG (R2= 0.9998) and VirGa (R2= 0.9995) were produced using a serially diluted immune serum (Fig 1e, left panel). The OD450 values for full-length VirG and VirGa were positively and significantly correlated with Hill slope of 1 (R2= 0.9975 and r = 0.9998, p < 0.0001 ) (FIG. 1 e, right panel). These results indicate that the immune reactive epitopes present on the full-length VirG protein are vastly contained in the protein’s a-domain region.Parenteral immunization with VirGa elicits robust and dose-dependent antibody responses

[0081] To investigate the immunogenicity of VirGa, adult mice were immunized intramuscularly (IM) with escalating doses (5, 10, or 20 pg) of VirGa admixed with ADJUPHOS® on two occasions (day 0 and 28) as illustrated in FIG. 2a. Negative control groups received ADJUPHOS® or phosphate-buffered saline (PBS). Mice immunized intranasally (IN) with sublethal doses (105CFU / dose) of S. flexneri 2a or S. sonnei were included as positive controls of serotype-specific protection. Parenteral immunization with VirGa elicited high levels of VirGa-specific serum IgG in all vaccinated groups compared to PBS or ADJUPHOS® control (FIG. 2b). Antibody responses increased after the second immunization, attaining peak levels 2 weeks after the boost in all groups (FIG. 2b). VirGa- specific IgG responses exhibited a dose-response pattern, i.e. , the magnitude of serum IgG was higher in groups that received larger amounts of VirGa at all timepoints postvaccination (FIG. 2c). The best responders were mice that received the 20 pg dose; mean serum IgG titers at day 27 (before boost) were 1.3x104, 2.1x104, and 5.6x104ELISA Unit (EU) / mL in groups that received 5, 10, and 20 pg, respectively, and 9.6x105, 1 .4x106, and2.1x106EU / mL at day 55 (after boost and around the time of challenge) (FIG. 2b, c). Mean fold increases in serum IgG titers over baseline at day 55 amounted to 153,600, 224,000, and 336,000 EU / ml for the 5, 10, and 20 pg dosage levels, respectively (FIG. 2b, c). An unexpected finding was the low (negligible) VirGa-specific serum IgG responses in mice immunized IN with sublethal doses of S. flexneri 2a and S. sonnei (FIG. 2b), while these controls developed robust serum IgG responses to the OPS of the respective immunizing strain. VirGa-specific IgA responses were detected neither in serum or feces of mice immunized with VirGa via IM, nor in those that received sublethal IN doses of S. flexneri 2a and S. sonnei (data not provided).VirGa protects mice from S. flexneri 2a and S. sonnei lethal infection

[0082] Mice immunized IM with increasing doses of VirGa (as described above) were subjected to lethal IN challenge with virulent S. flexneri 2a 2457T (5x106CFU / dose) or S. sonnei Moseley (5.2x106CFU / dose) on day 57 post vaccination (FIG. 2a). Parenteral immunization with VirGa adjuvanted with ADJUPHOS® afforded high levels of protection against S. flexneri 2a; 83% vaccine efficacy (VE) was achieved with the 20 pg dose, followed by 61 % and 56% VE with 10 and 5 pg, respectively (FIG. 3a and Table 1 ). The VE was significantly higher in all vaccinated groups as compared to the negative control (p < 0.001 ) but did not reach statistical significance when compared among VirGa vaccinated groups. Importantly, VirGa conferred statistically significant cross-protection against S. sonnei lethal infection; the 20 pg and 10 pg doses achieved 57% and 46% VE, respectively (FIG. 3b and Table 1 ). The lower VE of the 20 pg and 10 pg dose of VirGa against S. sonnei is attributed to a more severe infection produced by S. sonnei in the described model (mice succumbed sooner). Mice immunized IN with sublethal doses of virulent S. flexneri 2a or S. sonnei afforded the highest levels of homologous protection although was not significantly different than those that received VirGa (20 pg dose). Virtually all unvaccinated control mice succumbed to lethal pulmonary Shigella challenges (FIG. 3a, b).

[0083] Based on the associations between antibody levels and reduced infection observed in humans, studies were performed to see whether the level of serum IgG elicited by vaccination with VirGa was associated with mouse survival post-infection.VirGa immunized mice that were protected from S. flexneri 2a or S. sonnei challenge had significantly higher levels of VirGa-specific serum IgG at the time of challenge (at day 55) as compared to those that did not survive (non-protected) (FIG. 3c, d). By applying receiver operating characteristics (ROC) curve and area under curve (AUC) analyses, a threshold level (1.26 x 106EU / mL) was identified that allows prediction of protective outcome with ~ 80% sensitivity and specificity (FIG. 3e).

[0084] The immunogenicity and protective efficacy of VirGa were also investigated using a mucosal route and a clinically advanced mucosal adjuvant, the E. coli dmLT. Mice were immunized IN with 2.5, 5, or 10 pg of VirGa admixed with dmLT on days 0, 14, and 28. VirGa-specific serum IgG responses after the 1stand 2ndvaccinations were modest but increased dramatically after the 3rdimmunization. The 10-pg dosage achieved the highest titers (FIG. 4a) and afforded a VE of 90% against S. flexneri 2a; the VE values for the 5 or 2.5 pg immunizing doses were negligible (FIG. 4c and Table 2), indicating that a certain dosage level of the protein might be needed to induce robust vaccine efficacy after IN administration. High levels of dmLT-specific IgG titers were observed, that were almost identical among dmLT recipient groups, indicating proper vaccine preparation and immunization (FIG. 4b).VirGa-specific antibody secreting cells (ASC) in systemic tissues

[0085] The frequencies of IgG and IgA ASC were measured in spleen and bone marrow from mice immunized IM with 20 pg of VirGa (the most efficacious dose) and from mice inoculated IN with sublethal doses of S. flexneri 2a (Sf2a-IN). This analysis was conducted in animals that survived the S. flexneri 2a lethal infection to discern vaccine- elicited immunity evoked by infection. VirGa-alum IM immunization was chosen for this and subsequent studies as this mode of vaccination is the most likely to be used in humans.

[0086] High levels of VirGa-specific IgG ASC were detected both in spleen and bone marrow of VirGa vaccinated mice (FIG. 5a, b). In contrast, VirGa-specific IgG ASC were not detected in mice that received Sf2a-IN. The absence of VirGa-specific IgG ASC in the Sf2a-IN mice is consistent with their negligible antibody responses to VirGa (FIG. 2b). All groups exhibited high frequencies of total (vaccine-agnostic) IgG in the spleen and bonemarrow. In fact, mice that received Sf2a-IN had total IgG ASC levels even higher than the VirGa-immunized mice ruling out a technical error (FIG. 5a, b). Neither mice that received VirGa nor the positive control immunized with sublethal Sf2a-IN developed VirGa-specific IgA ASC (FIG. 5a, b); meanwhile, total IgA ASC were detected in spleen and bone marrow from both groups. The absence of VirGa IgA ASC in these groups is consistent with the lack of serum and fecal IgA responses described above.Anti-microbial function of VirGa-specific antibodies

[0087] The association between serum antibody levels and protection prompted us to examine the anti-microbial mechanism of VirGa antibodies. To this end, we first investigated the capacity of VirGa mouse immune sera to recognize VirG expressed on live S. flexneri 2a by confocal immunofluorescence microscopy. Vaccine-induced VirGa- specific antibodies, but not ADJUPHOS® antisera, recognized VirG expressed by S. flexneri 2a in its typical unipolar pattern, as shown by confocal microscopy images (FIG. 6).

[0088] The capacity of immune sera from VirGa vaccinated mice to block Shigella adherence to intestinal epithelial cells was interrogated using primary human colonoid monolayers. S. flexneri 2a admixed with VirGa-specific murine antisera (or sera from mice that received ADJUPHOS® as negative control) were added to the basolateral side of human colonoid monolayers (FIG. 7a). VirGa-specific immune sera inhibited Shigella adherence to human colonoids; a statistically significant ~ 50% reduction in bacterial adhesion was observed (FIG. 7b). The inhibition of adherence by VirGa-specific antibodies was more pronounced (only 26% of organisms remained attached) when the bacteria were grown in the presence of deoxycholate (DOC) (FIG. 7b), a bile salt that enhances the ability of Shigella to attach to and invade epithelial cells and activates VirG- dependent S. flexneri adhesion. In addition, VirGa-induced serum antibodies significantly impaired the adherence of DOC-stimulated S. sonnei (only 55% bacterial recovery) as compared to control ADJUPHOS sera (FIG. 2D). Using the same approach, the capacity of VirGa-specific mouse antisera to reduce colonocyte invasion by DOC-stimulated S. flexneri 2a was demonstrated; only 65% of the original bacterial load was recovered intracellularly (FIG. 7c). These results demonstrate the capacity of vaccine-inducedVirGa-specific IgG to recognize the native antigen in living organisms and to deploy antimicrobial functions impairing bacterial adhesion and invasion of human colonocytes.

[0089] Described herein are compositions and methods related to using Shigella VirGa or Shigella VirGa fragments as a new subunit-based vaccine candidates. VirGa exhibited robust immunogenicity and broad protective capacity. Animal models of shigellosis are limited and contrived; Shigella does not infect rodent via oral route. Thus, the protective capacity of VirGa was investigated in a mouse model of pulmonary lethal infection which recreates elements of human disease that results in death. Parenteral immunization of mice with VirGa adjuvanted with alum elicited vigorous serum antibody responses and afforded > 80% protection against S. flexneri 2a and 57% against S. sonnei lethal infection. Similarly, mucosal (IN) immunization with VirGa’s admixed with E. coli dmLT conferred 90% protection against S. flexneri 2a infection. VirGa serum IgG produced by parenteral immunization was associated with vaccine effectiveness. A threshold level was identified as a predictor of protective efficacy in the experimental setting; a similar value was reported for IpaB. The concordance between VirG-specific immunity and protection in mice is consistent with the association we had observed between the magnitude of serum IgG ( lgG1 ) against VirG and reduced disease severity in volunteers experimentally challenged and re-challenged with S. flexneri 2a. It is also in agreement with the high levels of VirG-specific antibodies we found in adults living in Shigella endemic areas, who are less susceptible to infection than children, due to natural immunity acquired through repeated exposure. Similarly, we found VirG-specific IgG and IgA in stool from 0- to 5- year-old children infected with Shigella, and these antibodies were more abundant in children who did not develop dysentery as compared with those who had dysentery or diarrhea from other causes. Taken together, these observations point to VirG as an important target of protective immunity, and VirG-specific antibodies as putative antimicrobial effectors.

[0090] It was also confirmed that VirGa antibodies from vaccinated mice recognized VirG expressed on the bacterial outer surface and demonstrated their capacity to prevent Shigella infection by blocking bacterial adhesion and invasion in human colonoid monolayers. These results indicate that VirG antibodies display critical anti-microbial functions that can prevent Shigella infection in the mucosal interface, averting tissuedamage and inflammation. In the context of interrogating bacterial pathogenesis, purified VirG-specific antibodies were shown to block attachment of a hyper adhesive AlpaD Shigella mutant to mammalian cells,33which is consistent with the current findings. Recently, the immunoglobulin class and subclass distribution and Fc receptor binding profile of VirGa-specific antibodies were probed, as well as their capacity to engage innate immune cells using sera obtained from individuals who participated in a CHIM study. VirGa-specific IgG and IgA, Fc receptor binding, as well as VirG-specific antibodydependent complement deposition (ADCD) and antibody-dependent cellular monocyte phagocytosis (ADCP) were identified in individuals who experienced minimal disease.

[0091] In addition to systemic antibodies, IM delivered VirGa elicited antigen-specific IgG ASC that were detected in the spleen and bone marrow. Vaccine-induced ASC, along with memory B cells, provide long term humoral immunity to infection. Spleen ASC derived from germinal center B cells egress to blood and replenish circulating antibody levels. They can also migrate to bone marrow. The ASC in bone marrow represents long- lived plasma cells that maintain humoral immune memory. VirGa-specific humoral and ASC responses were IgG-rich, which is expected for subunit vaccines given parenterally in the presence of alum. A predominant IgG response is also elicited by subunit vaccines in routine immunization programs. The negligible ASC and antibody responses to VirGa in mice dosed IN with live Shigella (either a live attenuated strain or wild-type organisms) was surprising and intriguing, considering the prevalence of VirG-reactive antibodies in individuals infected with Shigella. It could be attributed to limited exposure - insufficient to adequately stimulate immune responses, or to differences in host susceptibility and immunity between species.VirGa Fragments

[0092] VirGa fragments were synthesized using a cell-free expression system. The cell- free expression system used can be found in US Patent No. 9040253, the contents of which are incorporated herein by reference in its entirety. One benefit of using a cell-free system for polypeptide synthesis is that the overall yield of polypeptide is on the order of 200 mg / L, an approximately forty times greater value as compared to conventional methods of polypeptide synthesis.

[0093] As mentioned earlier, the compositions disclosed herein for protecting against various Shigella serotypes may be fragments of c shown in FIG. 8a-d. Here Ag-specific serum IgG titers of VirGa and two fragments (VirG R1 and VirG R2) were compared 42 days post-vaccination with 20 pg dosing each, where VirG R1 corresponds to aa53-353 and VirG R2 corresponds to aa500-758 of the alpha region of the VirG protein, respectively. Here, sub-lethal (dose) Sf2a-IN and PBS (phosphate-buffered saline) were used as positive and negative controls, respectively. As FIG. 8a shows the Ag-specific serum IgG titer for VirG R1 was greater than that of VirGa while VirG R2 was less. This was also the case for VirGa-specific IgG responses (FIG. 8b).

[0094] FIG. 9 shows mouse survival rate over days for VirGa (20 pg), VirG R1 (20 pg ), VirG R2 ( 20 pg), si Sf2a-IN, and PBS. As can be seen, the survival rates for VirGa (53- 758)-20pg and VirG-R1 (53-353)-20pg provided better survival rates than VirG-R2 (500- 758)-20pg. Table 2 below summarizes the information.

[0095] In some examples of the disclosed Shigella compositions, certain amino acids may be substituted with other amino acids to achieve a better outcome. In one nonlimiting example, the cysteine at 130 in VirGa or VirG R1 can be replaced with a serine. In this example, the replacement reduced the amount of aggregation as can be seen in FIG. 10.

[0096] Table 4. Vaccine efficacy of VirGa and its fragments administered IM against Shigella infection

[0097] In other aspects of the disclosed invention, VirGa or VirGa fragments could either operate as a stand-alone vaccine or could be combined with other conjugate vaccine candidates (e.g. IpaB, OPS-lpaB) to maximize efficacy. Inventors recently reported the development of a new Shigella OPS-lpaB conjugate vaccine candidate capable of eliciting cross-protective immunity in mice. IpaB is an appealing vaccine target because of its immunogenic properties and association with protective immunity in humans. In some embodiments, VirGa or VirGa fragments can be combined with IpaB to further increase efficacy. A vaccine based on broad-spectrum safe and effective proteins would be simpler and superior to existing candidates in their greater breadth of coverage, ability to prevent infection caused by eventually all serotypes, ease of manufacturing and quality control, straightforward clinical evaluation, and practical implementation. These advantages will reduce cost and make the vaccine more affordable and appealing to limited resource countries. Based on the excellent safety profile of subunit vaccines in routine schedules, a protein-based Shigella vaccine is expected to be well tolerated by infants and young children, who experience the highest burden of disease. The remarkable immunogenicity of VirGa / VirGa fragments (the same for IpaB) is propitious to generate robust and long lasting protective immunity in those under three years of age for whom Shigella OPS-rEPA and orally delivered live attenuated vaccines have failed. The uncomplicated production of a subunit vaccine will increase affordability. Implementation of such a vaccine, even in poor-resource areas, is achievable by incorporation into existing schedules. The overall disease prevention strategy using broad protective proteins is simple and anticipated to be cost effective. As such, a VirGa orVirGa + IpaB vaccine meet the WHO preferred product features outlined for a vaccine against Shigella.

[0098] The cross protective advantage of conserved proteins must be emphasized as circulation of disease-causing serotypes in the environment is cyclical, and the emergence of strains not contained in O-antigen-based vaccines would require product reconfiguration. The landscape of Shigella vaccines will likely change in the next 5 years as refined concepts and new candidates advance to human clinical trials and CHIM.

Claims

CLAIMS1 . A composition comprising a VirG protein comprising an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO: 2 or a fragment comprising about 100-400 amino acids thereof.

2. The composition of claim 1 , wherein the fragment comprises a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 3 or 4.

3. The composition of claim 1 , wherein the fragment comprises a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 5.

4. The composition of any one of claims 1 -3, wherein the VirG protein comprises a tag at an N- or C-terminus of the VirG protein.

5. The composition of claim 4, wherein the tag is at the N-terminus of the VirG protein.

6. The composition of claim 4 or 5, wherein the tag comprises a His-tag and a protease cleavage site.

7. The composition of claim 6, wherein the protease cleavage site comprises a Tobacco Etch Virus (TEV) protease cleavage site.

8. The composition of claim 7, wherein the TEV protease cleavage site comprises the sequence set forth in SEQ ID NO: 6 followed by any amino acid except for proline or tryptophan.

9. The composition of claim 8, wherein the tag comprises the sequence set forth in SEQ ID NO: 7.

10. The composition of claim 9, wherein the VirG protein comprises the sequence set forth in SEQ ID NO: 8.11 . The composition of claim 9, wherein the VirG protein comprises the sequence set forth in SEQ ID NO: 9.

12. The composition of any one of claims 1 -11 , wherein the VirG protein is synthesized using a cell-free system.

13. The composition of any one of claims 1 -12, further comprising an adjuvant.

14. The composition of any one of claims 1 -13, further comprising one or more Shigella invasion plasmid antigen (Ipa) polypeptides.

15. The composition of claim 14, wherein the one or more Ipa polypeptides comprise one or more of an IpaB polypeptide and an IpaH polypeptide.

16. The composition of claim 15, wherein the IpaB polypeptide is an IpaB polypeptide conjugate comprising one or more non-natural amino acids (nnAA).

17. The composition of claim 15 or 16, wherein the IpaB polypeptide comprises a sequence at least 90% identical to the sequence set forth in any one of SEQ IDNOs: 10-16.

18. The composition of any one of claims 14-17, wherein the one or more Ipa polypeptides are synthesized using a cell-free system.

19. The composition of any one of claims 1 -18, comprising about 5-100 pg of the VirG protein.

20. The composition of any one of claims 14-19, independently comprising about 5-100 pg of each Ipa polypeptide.21 .A method of eliciting an immune response against Shigella in a mammalian subject in need thereof, immunizing a mammalian subject in need thereof against Shigella, or reducing or preventing a Shigella infection in a mammalian subject in need thereof, comprising administering the composition of any of claims 1-20 to the subject.

22. The method of claim 21 , wherein the composition is delivered intranasally, intramuscularly, or transdermally.

23. The composition of any one of claims 1 -20 for eliciting an immune response against Shigella in a mammalian subject, immunizing a mammalian subject against Shigella, or reducing or preventing a Shigella infection in a mammalian subject.

24. The composition of claim 24, wherein the composition is intended for intranasal, intramuscular, or transdermal delivery.

25. Use of the composition of claim 24 in the manufacture of a medicament for eliciting an immune response against Shigella in a mammalian subject, immunizing a mammalian subject against Shigella, or reducing or preventing a Shigella infection in a mammalian subject.

26. The use of claim 25, wherein the medicament is intended for intranasal, intramuscular, or transdermal delivery.

27. A nucleic acid sequence encoding the VirG protein of any one of claims 1 -11.

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