Shigella vaccine composition

Abstract

Provided herein are vaccine compositions for preventing and combating infection by Shigella. In particular, the present disclosure provides a composition comprising a Shigella virulence factor VirG (or IcsA) protein or a fragment thereof comprising about 100-400 amino acids of the VirG protein having an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 2.

Description

[0001] Government interests This application was completed with grants from the National Institutes of Health (NIH) under grant numbers AI125841 and AI161839. The government holds certain rights to the invention described in this application. Technical Field

[0002] This application generally relates to vaccine compositions for the prevention and protection against infections caused by Shigella. Background Technology

[0003] Shigella-induced diarrheal diseases cause death and lifelong disability worldwide, with particularly severe impacts on children in impoverished areas and those lacking access to clean water and adequate sanitation. Approximately 200,000 people die globally each year from this disease, one-third of whom are children under the age of five. Recurrent infections can lead to growth retardation, impaired cognitive development, and shortened life expectancy. Although treatable with antibiotics and oral rehydration therapy, the rapid and widespread spread of drug-resistant strains and the disease's high transmissibility make shigella infection a significant global public health concern, and there is currently no available vaccine.

[0004] Various Shigella vaccine candidates (i.e., oral attenuated or inactivated Shigella vaccines and intramuscular subunit vaccines) have been evaluated for safety and immunogenicity in humans. Shigella O-type polysaccharide (OPS)-based conjugate vaccines, currently the most advanced vaccines, are being studied in a controlled human infection model (CHIM) (NCT04078022) and a field efficacy trial (NCT04602975). An earlier Shigella OPS-recombinant Pseudomonas aeruginosa exotoxin A (rEPA) conjugate vaccine, while effective in adults and older children, failed to prevent Shigella infection in children under 3 years of age (the most severely affected group). In the CHIM study, a more novel Shigella flexneri (… S. flexneriType 2a OPS-rEPA offers only moderate protection against severe illness. These candidate vaccines primarily rely on specific immunity against bacterial lipopolysaccharide (LPS), and like most other vaccines, their protection is limited to the serotype targeted at the time of vaccination, despite the existence of over 50 pathogenic Shigella serotypes. Multivalent OPS-based formulations are currently under investigation to cover the four most common serotypes (NCT05156528 and NCT04056117). In Kenya, a multivalent outer membrane vesicle-based vaccine is being evaluated in adults, children aged 2 to 5 years, and infants as young as 9 months (NCT05073003), although an early monovalent version failed to produce significant protection in a recent CHIM study in adults. Nevertheless, multivalent vaccine development faces significant hurdles, including the difficulty of clinical evaluation and potential interactions / interferences between vaccine components, which could reduce vaccine efficacy and increase production costs.

[0005] In addition to OPS-based vaccines, highly conserved protein components of the Shigella type III secretion system (T3SS) have also been proposed as vaccine candidates. Researchers report that the invading plasmid antigen (Ipa) B, used alone or in combination with other antigens, exhibits potent preclinical immunogenicity and protective efficacy against multiple Shigella serotypes. Invaplex, a large-molecule Shigella subunit vaccine containing Ipa protein and LPS, has shown promise in preclinical studies against Shigella flexneri 2a and Shigella soxehii serotypes. S. sonnei The natural invaplex vaccine exhibits high levels of protective efficacy, is well-tolerated in humans, and is immunogenic. However, the natural invaplex vaccine failed to prevent disease in an adult CHIM model. Therefore, there is an urgent need in the field for an improved Shigella vaccine. Summary of the Invention

[0006] Shigella-induced diarrhea remains a significant cause of high morbidity and mortality among young children worldwide. Currently, there is no approved vaccine, and those vaccines with more mature clinical development have limited coverage because they only induce serotype-specific immunity, while the disease is caused by multiple prevalent serotypes.

[0007] The inventors have discovered a close association between serum antibodies against the Shigella virulence factor VirG (or IcsA) and clinical protective effects in infected individuals. This application discloses compositions utilizing the immunogenicity and protective capabilities of VirG, the surface-exposed α-domain of VirG (VirGα), and corresponding fragments as mouse subunit vaccines. VirGα is prepared in the form of a recombinant protein. Surprisingly, this region exhibits almost identical immunoreactivity to full-length VirG. Intramuscular injection of VirGα with aluminum hydroxide elicits a strong immune response and provides high protective efficacy against Shigella flexneri 2a and Shigella soxehii. Intranasal administration of Escherichia coli double-mutant heat-labile toxin (dmLT) provides near-complete protection. VirGα-specific antibodies recognize VirG expressed on the surface of live Shigella bacteria and block Shigella adhesion and invasion of human colon cells.

[0008] Other experiments and disclosures related to the VirGα fragment have also demonstrated its potential in evoking immune responses and preventing Shigella infection in vivo. The VirGα fragment disclosed in this application can also be combined with other components such as IpaB and IpaH antigenic peptides to form compositions, wherein in some cases, the IpaB peptide can be a conjugate containing non-natural amino acids at one or more positions.

[0009] The inventors have demonstrated, surprisingly, that VirGα and its fragments are promising cross-protective vaccine candidates for the prevention of Shigella infection. This application discloses a composition comprising VirG protein or a fragment containing about 100-400 amino acids of said VirG protein, the amino acid sequence of said VirG protein being at least 90% identical to the sequence shown in SEQ ID NO: 2. The fragment may contain at least 90% of the sequence shown in SEQ ID NO: 3 or 4. The fragment may contain at least 90% of the sequence shown in SEQ ID NO: 5.

[0010] The VirG protein may contain a tag at its N-terminus or C-terminus. The tag includes a His tag and a protease cleavage site. The protease cleavage site may include the tobacco etched virus (TEV) protease cleavage site. The TEV protease cleavage site may contain the sequence shown in SEQ ID NO: 6, followed by any amino acid except proline or tryptophan. The tag contains the sequence shown in SEQ ID NO: 7. The VirG protein may contain the sequence shown in SEQ ID NO: 8 or 9.

[0011] VirG proteins can be synthesized using a cell-free system. The composition may contain an adjuvant. The composition may further contain one or more Shigella invasion plasmid antigen (Ipa) peptides, which may include one or more of IpaB and IpaH peptides. The IpaB peptide may be an IpaB peptide conjugate containing one or more non-natural amino acids (nnAA). The IpaB peptide may contain at least 90% sequence identity with the sequence shown in any one of SEQ ID NOs: 10-16. One or more Ipa peptides can be synthesized using a cell-free system. The composition may independently contain about 5-100 μg of each VirG protein and optionally each Ipa peptide.

[0012] This application also provides nucleic acids encoding the Shigella VirG protein.

[0013] This application provides a method for inducing an immune response against Shigella in a mammalian subject with a need for this, inducing immunity against Shigella in a mammalian subject with a need for this, or alleviating or preventing Shigella infection in a mammalian subject with a need for this, comprising administering the composition to the subject. This application also provides a composition for inducing an immune response against Shigella in a mammalian subject, inducing immunity against Shigella in a mammalian subject, or alleviating or preventing Shigella infection in a mammalian subject. This application further provides the use of the composition in the preparation of a medicament for inducing an immune response against Shigella in a mammalian subject, inducing immunity against Shigella in a mammalian subject, or alleviating or preventing Shigella infection in a mammalian subject. The composition may or may be intended to be delivered intranasally, intramuscularly, or percutaneously. Attached Figure Description

[0014] Figure 1A -H indicates the cloning, expression, and purification of Shigella VirGα protein. Figure 1A A schematic diagram is shown illustrating the full-length VirG and its cloned α-domain regions. The VirGα aa sequence spanning four Shigella species demonstrates the conserved nature of the VirGα Shigella serogroup. Figure 1B -D shows SDS-PAGE gel and Western blot analysis of purified VirGα; protein expression was performed as follows: positively transformed E. coli BL21(DE3) pLysS competent cells were induced with 1 mM IPTG for 2 h (lane 3) or 3 h (lane 4), the latter achieving a higher expression level of 76 kDa VirGα (lane 4, red box). Protein molecular weight markers (MWM; lane 1) and uninduced cells (lane 2) were added as controls. Figure 1B ); Figure 1CThe image shows the concentrated purified VirGα; protein MWM (lane 1), eluted VirGα protein (lanes 2-5), and concentrated VirGα (lanes 6-7). Figure 1D The results of Western blotting validation of 76 kDa VirGα protein (lane 2) using anti-His-tagged antibody (left panel) and anti-VirGα immune serum (right panel) are shown. MWM (lane 1) and ELISA data indicate that VirGα has higher immunoreactivity compared to full-length VirG protein. This experiment used pooled serum from mice immunized with full-length VirG. Serially diluted serum was added to plates coated with each protein, and repeated sample testing was performed under the same conditions using the same pooled serum and serial dilutions. Figure 1E The data represents the average OD of the repeating holes. 450 Value (displays R) 2 r and p The linear consistency between the values ​​and dilutions. Figure 1F -H displays the clone data for VirGα.

[0015] Figure 2A -D indicates the immunogenicity of Shigella VirGα. Figure 2A (a) Schematic diagram of immunization and challenge experiments in adult mice; (b) Kinetic curve of VirGα-specific serum IgG titers measured by ELISA (n = 15 / group); arrows indicate immunization. Data represent the mean of individual titers ± SEM; **** p <0.0001, compared with PBS or AdjuPhos®, using t test( Figure 2B );and( Figure 2C The values ​​shown are VirGα-specific IgG titers obtained before and after boosting with escalating doses of VirGα (n = 15 / group).

[0016] Figure 3A -E indicates the protective efficacy of Shigella VirGα. Mice were immunized as described in Figure 2, and 57 days post-immunization, they were intranasally (IN) injected with a lethal dose of Shigella flexneri 2a 2457T (5 x 10⁻⁶). 6 CFU / dose) or Shigella Moseleyi (5.2 x 10⁻⁶) 6 CFU / dose) for immune challenge, of which ( Figure 3A (A) and (B) show survival curves for 15-20 mice / group. Compared to the PBS control group... p The value was determined using a log-rank (Mantel-Cox) test; Figure 3C(D) Shows VirGα-specific serum IgG titers in mice vaccinated with the VirGα vaccine at infection challenge (day 55), who were either protected against or resistant to Shigella flexneri 2a (n = 45) or Shigella somatoformis (n = 20) infection. Data represent mean serum IgG titer ± SEM, * p <0.05 and ** p <0.01, passed t Test and determination; and ( Figure 3E This study used ROC analysis to show VirGα-specific serum IgG levels in survivors of the VirGα vaccine and those who died from fatal Shigella infections (n=65 in total from the two challenge trials), and determined the protective threshold. The red arrows indicate the survival prediction thresholds, with a sensitivity and specificity of approximately 80%. AUROC = 0.7089 (95% CI: 0.63 – 0.90). p = 0.0005.

[0017] Figure 4A -C shows the immunogenicity and protective efficacy of VirGα administered via mucosal administration in mice. Adult mice (n=10 per group) were intranasally inoculated with 2.5, 5, or 10 μg of VirGα protein (mixed with 2.5 μg of dmLT) on days 0, 14, and 28 (black arrows), with negative control mice receiving either PBS or dmLT. Mice intranasally inoculated with attenuated live Shigella flexneri 2s strain CVD1208s on days 0 and 21 were introduced as positive control groups for Shigella flexneri 2a infection challenge (grey arrows): Figure 4A The kinetic curve of VirGα-specific serum IgG titers as measured by ELISA is shown. Individual titer mean ± SEM; * p < 0.05, **** p < 0.0001, compared with the PBS control group using the Mann-Whitney test; Figure 4B The kinetic curve of dmLT-specific serum IgG measured by ELISA is shown. Individual titer mean ± SEM; **** p<0.0001, compared with the PBS control group, using t-test. Figure 4C The study showed that on day 56 post-vaccination, immunized mice were administered an oral (IN) lethal dose (10... 7 Infection challenge with Shigella flexneri strain 2a (CFU / dose). Data represent survival curves for 10 mice per group. ****Compared with the PBS control group, p < 0.0001, determined by log-rank (Mantel-Cox) test; and ( Figure 4C The results show the day-by-day survival rates of different doses of VirGα composition compared to positive and negative control groups.

[0018] Figure 5A -B shows VirGα-specific ASCs and total ASCs. The spleens of mice immunized with 20 µg VirGα (IM) or nasal injection (IN) of Shigella flexneri 2a (sl Sf2a-IN) and surviving a lethal challenge with Shigella flexneri 2a were measured. Figure 5A ) and bone marrow ( Figure 5B The frequency of total antibody-secreting cells (ASC) and VirGα-specific antibody-secreting cells (ASC) in the study.

[0019] Figure 6 Microscopic images of Shigella flexneri 2a are shown, demonstrating the immune recognition of VirG on live Shigella bacteria by VirGα-induced antibody responses. A suspension of Shigella flexneri 2a was co-incubated with serum from mice inoculated with VirGα (top panel) or mice receiving AdjuPhos® (bottom panel). Antibody binding was detected using AF555-labeled anti-mouse antibodies.

[0020] Figure 7A -D indicates the inhibitory effect of mouse VirGα antibody on Shigella adhesion and invasion of human epithelial cells. Figure 7A Schematic diagram showing Shigella infection of human colonic organoids and inhibition models. Shigella flexneri 2a (5 x 10⁻⁶) grown under conditions with or without deoxycholic acid (DOC). 8 CFU / mL) was incubated with mouse VirGα antiserum and then immediately added to the basal side of human colonic organoid monolayers. The relative adhesion rate of Shigella flexneri 2a (grown in the presence or absence of DOC in the presence or absence of VirGα antibody) was measured. Figure 7B ) or intrusion rate ( Figure 7C (AdjuPhos® negative control group). Figure 7D Data showing the inhibitory effect of VirGα antibody on Shigella Solomonella adhesion to human colonic monolayer cells.

[0021] Figure 8A -D indicates the titer of various antigen-specific IgGs. Figure 8A The results show the Ag-specific IgG titers induced after administration of VirGα, VirG R1, and VirG R2 to mice; Figure 8B This shows the VirGα-specific IgG titers induced in mice vaccinated with VirGα, VirG R1, and VirG R2; Figure 8C The results show the VirG R1-specific IgG titers induced in mice inoculated with VirGα, VirG R1, and VirG R2; and ( Figure 8D This indicates the VirG R2-specific IgG titers of VirGα, VirG R1, and VirG R2.

[0022] Figure 9 The percentage survival of mice immunized with VirGα, VirG R1, VirG R2, and control groups (i.e., sublethal doses of Shigella flexneri 2a (positive control group) and PBS (negative control group) after a lethal challenge with Shigella flexneri) was compared.

[0023] Figure 10 The results showed that replacing the cysteine ​​at position 130 of VirG R1 with serine reduced the aggregation phenomenon. Detailed Implementation

[0024] definition As used in this application specification and appended claims, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” are intended to include their plural forms as well.

[0025] As used in this application, “and / or” means or covers any and all possible combinations of one or more of the relevant listed items, as well as cases where no combination is made when interpreted in alternatives (“or”).

[0026] Furthermore, this application also covers situations in some embodiments where any feature or combination of features described in this application may be excluded or omitted.

[0027] Furthermore, the term “about” used in this application when referring to measurable values ​​(such as the content, dosage, time, temperature, etc. of the compounds or reagents of this application) is intended to include a range of deviations from the specified value of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%.

[0028] As used in this application, the transitional phrase "consistently of..." should be interpreted to encompass the described materials or steps as well as the basic and novel features that do not materially affect the content of the invention. Therefore, the term "consistently of..." as used in this application should not be interpreted as equivalent to "comprising".

[0029] The term "substantially composed of" (and its grammatical variations), when applied to the polynucleotide or polypeptide sequence of this application, means a polynucleotide or polypeptide composed of both: the described sequence (e.g., SEQ ID NO); and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids located at the 5' and / or 3' end or the N-terminus and / or C-terminus of the described sequence, or at both ends (e.g., between domains), such that the function of the polynucleotide or polypeptide is not substantially altered. A total of 10 or fewer nucleotides or amino acids includes the total number of all additional nucleotides or amino acids added together. The term "substantially altered," when applied to the polynucleotide of this application, means an increase or decrease of at least about 50% or more in the ability to express the encoded polypeptide compared to the expression level of the polynucleotide composed of the described sequence. The term "substantially altered," when applied to the polypeptide of this application, means an increase or decrease of at least about 50% or more in biological activity compared to the biological activity of the polypeptide composed of the described sequence.

[0030] The terms “enhancement” or “increase” refer to an increase in a specified parameter by at least approximately 1.25 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 12 times, or even 15 times.

[0031] As used in this application, the terms “inhibition” or “reduction” or their grammatical variations refer to a reduction or decrease in a specified level or activity by an amount of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or more. In certain embodiments, this inhibition or reduction results in minimal or substantially undetectable activity (at most a non-significant amount, e.g., less than about 10% or even 5%).

[0032] The term "therapeuticly effective" or "treatment-effective" as used in this application refers to an amount that brings some improvement or benefit to the subject. In other words, a "therapeuticly effective" or "treatment-effective" amount is an amount that can alleviate, reduce, or decrease at least one clinical symptom in the subject to some degree (e.g., in the case of inducing an immune response, activating or increasing the number of immune cells known to produce an immune response). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as it brings some benefit to the subject.

[0033] The term “treatment / management” (or a grammatically equivalent term) is intended to reduce or at least partially improve or alleviate the severity of a subject’s condition, and / or alleviate, reduce or decrease at least one clinical symptom, and / or delay the progression of the condition.

[0034] As used in this application, the term "prevention" (and its grammatically equivalents) refers to delaying or suppressing the occurrence of a disease. These terms do not require the complete elimination of a disease, but rather cover any type of preventive treatment aimed at reducing the incidence of the disease or delaying its occurrence.

[0035] The term "preventive effectiveness" as used in this application refers to an amount sufficient to prevent and / or delay the onset of disease, symptoms, and / or clinical symptoms in a subject, and / or to reduce and / or delay the severity of such onset compared to situations where the method of this application is not used. Those skilled in the art will understand that complete prevention is not necessary as long as it provides some benefit to the subject.

[0036] As used in this application, unless otherwise stated, the terms "protein" and "polypeptide" are used interchangeably and include both peptides and proteins.

[0037] The term "fragment," when applied to a polypeptide, should be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence, and comprising, substantially comprising, and / or comprising an amino acid sequence that is identical or substantially identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to this application are incorporated, where appropriate, into larger polypeptides in which they are integral components. In some embodiments, such fragments may comprise, substantially comprising, and / or comprising a peptide 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 the polypeptide or amino acid sequence of this application. In some embodiments, the fragment may also be about 100-400, 150-350, 200-350, or 250-350 consecutive amino acids of the reference polypeptide or amino acid sequence.

[0038] As used in this application, "isolated" polypeptide means a polypeptide that has been isolated from at least some other components of a naturally occurring organism or virus, or that is substantially free of at least some other components of a naturally occurring organism or virus (e.g., structural components of a cell or virus, or other polypeptides or nucleic acids typically associated with the polypeptide).

[0039] As used in this application, the term "modified" when applied to a polypeptide sequence means a sequence that is different from the wild-type sequence due to one or more deletions, insertions, substitutions, or any combination thereof.

[0040] As used in this application, “sequence identity” refers to the degree to which two optimized aligned polynucleotide or polypeptide sequences remain unchanged within a component (e.g., nucleotide or amino acid) alignment window. “Identity” can be readily calculated by known methods, including but not limited to those described in the following literature: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, 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 in this application, the terms "substantially identical" or "corresponding to" mean that two nucleic acid or polypeptide sequences have at least 60%, 70%, 80%, or 90% sequence identity. In some embodiments, two nucleic acid or polypeptide sequences may have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0042] The “identity score” of the aligned fragments of the test sequence or reference sequence is the number of common components shared by the two aligned sequences divided by the total number of components in the reference sequence fragment; that is, the entire reference sequence or a smaller, defined portion of the reference sequence.

[0043] The terms “vaccination” and “immunization” are used interchangeably in this application, referring to the administration of a preventive dose of a compound and / or composition (e.g., a vaccine) to a subject. Therefore, administration of a vaccine to a subject can prevent, delay, and / or reduce the severity of any disease, condition, and / or clinical symptom that would occur if the method of this application were not employed.

[0044] As used in this application, the term "adjuvant" refers to compounds and / or substances added to a composition to enhance the potency and / or rate of action of an immunogen (e.g., an antigen) when the composition is administered to a subject. In some embodiments, enhancing the potency of the immunogen will enhance the immune response of the subject. In some embodiments, the adjuvant can enhance the potency 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 times or more. In some embodiments, the adjuvant can reduce the amount of immunogen required to generate 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 can prolong the time it takes for the immunogen to generate an immune response in the subject by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 times or more. Those skilled in the art will understand that the adjuvant can act through any mechanism to produce the effects listed above, including but not limited to: stabilizing the immunogen to prevent its degradation, increasing the binding affinity of the immunogen to immune cells, activating other components of the immune system (e.g., cytokine production, interferon production, etc.), and / or altering the inflammatory response.

[0045] As used in this application, "pharmaceutically acceptable" means a material that has no adverse effects on a biological or other basis; that is, the material can be administered to an individual with the composition of this application without causing significant harmful biological effects or harmful interactions with any other component in the composition containing it. Materials can be naturally selected to minimize any degradation of the active ingredient and to minimize any adverse side effects that may occur to the subject, as is known to those skilled in the art (e.g., see Remington's Pharmaceutical Science; 21st edition, 2005). Exemplary pharmaceutically acceptable carriers for the compositions of this application include, but are not limited to, phosphate-buffered saline (PBS), sterile pyrogen-free water, and other sterile pyrogen-free saline solutions.

[0046] The term “administer” to a subject includes any route by which the composition of this application is introduced or delivered to the subject to perform its intended function (e.g., to induce an immune response in the subject or to administer a vaccine to the subject).

[0047] The term "subject" in this application can include any animal for which it is desired. In some embodiments, the subject can be, for example, a mammal, reptile, bird, amphibian, or fish. Mammal subjects can include, but are not limited to, laboratory animals (e.g., rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cattle, pigs, horses, goats, donkeys, sheep, etc.), or domesticated animals (e.g., cats, dogs, ferrets, gerbils, hamsters, etc.). In some embodiments, mammalian subjects can be primates or non-human primates (e.g., chimpanzees, baboons, macaques (e.g., rhesus monkeys, cynomolgus monkeys, macaques, pig-tailed macaques), monkeys (e.g., squirrel monkeys, owl monkeys, etc.), marmosets, gorillas, etc.). In some embodiments, mammalian subjects can be humans.

[0048] The "subject in need" of the method of this application can be any subject who is known or suspected of having an increased risk of developing the infection described in this application specification, for whom inducing an immune response and / or vaccination against the infection may produce beneficial health effects.

[0049] The “sample,” “biological sample,” and / or “ex vivo sample” in this application can be any biological material, such as biological fluids, cell extracts, extracellular matrix isolated from tissues, cells (in solution or attached to a solid carrier), tissues, tissue homogenates, and similar materials known in the art.

[0050] Composition This application discloses a potentially immunogenic composition comprising the VirG protein. The VirG protein may include: the α-domain of VirG (VirGα), a fragment of VirGα, a substantially identical protein, or a fragment thereof. This application also provides a nucleic acid encoding the VirG protein. In some embodiments, the VirG protein is derived from Shigella. The VirG protein may also be referred to as IcsA (intracellular propagation gene A). VirG is fully involved in bacterial pathogenicity. It is crucial for Shigella adhesion within the colonic epithelium and for actin-based motility. Disruption or absence of VirG significantly attenuates the virulence of Shigella in both human and animal models. The protein consists of two main domains: a functionally active, surface-exposed α-domain (VirGα) and a transmembrane β-domain for transport proteins. Importantly, VirG is highly conserved within the Shigella genus and is independent of T3SS. The VirG protein may be derived from Shigella flexneri and may contain the sequence shown below.

[0051] SEQ ID NO: 1 VirGα may contain amino acids located at positions 53 - 758 relative to SEQ ID NO: 1. VirGα may contain the following sequence.

[0052] SEQ ID NO: 2 TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINSDIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGGEAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNYGEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNGAILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGSVITGDVQVNNSSILKIINNDYTGTTPTIEGDLCAGDCTTVSLSGNKFTVSGDVSFGENSSLNLAGISSLEASGNMSFGNNVKVEAIINNWAQKDYKLLSADKGITGFSVSNISIINPLLTTGAIDYTKSYISDQNKLIYGLSWNDTDGDSHGEFNLKENAELTVSTILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINAGILKMGTVEAMTRTAGVIVNKGATLNFSGMNQTVNTLLNSGTVLINNINAPFLPDPVIVTGNMTLEKNGHVILNNSSSNVGQTYVQKGNWHGKGGILSLGAVLGNDNSKTDRLEIAGHASGITYVAVTNEGGSGDKTLEGVQIISTDSSDKNAFIQKGRIVAGSYDYRLKQGTASGLNTNKWYLTSQMDNQESKQMSNQESTQMSSR In one example, the VirG protein comprises a fragment of VirGα containing amino acids located at positions 53-353 or 53-352 relative to SEQ ID NO: 1, referred to herein as VirG R1. VirG R1 may comprise the sequence shown 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 that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence shown in SEQ ID NO: 3 or 4. In some embodiments, the VirG protein consists of or is substantially composed of the sequence shown in SEQ ID NO: 3 or 4. In some embodiments, this application includes a nucleic acid sequence encoding the VirG protein.

[0053] SEQ ID NO: 3 TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINSDIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGGEAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGD AVTGVNLPIINKGTISGGNGGNNYGEGDGGNGGDAITGGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNGAILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGSVITGDVQVNNS In another instance, VirG R1 can contain the following sequence.

[0054] SEQ ID NO: 4 TPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINSDIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGGEAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGD AVTGVNLPIINKGTISGGNGGNNYGEGDGGNGGDAITGSSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNGAILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGSVITGDVQVNN In another example, the VirG protein comprises a VirGα fragment containing amino acids located at positions 500-758 relative to SEQ ID NO: 1, referred to herein as VirG R2. VirG R2 may comprise the sequence shown in SEQ ID NO: 5. The VirG protein may comprise a sequence substantially identical to SEQ ID NO: 5, or a sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence shown in SEQ ID NO: 5. In some embodiments, the VirG protein consists of or is substantially composed of the sequence shown in SEQ ID NO: 5. In some embodiments, this application includes a nucleic acid sequence encoding the VirG protein.

[0055] SEQ ID NO: 5 ENAELTVSTILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINAGILKMGTVEAMTRTAGVIVNKGATLNFSGMNQTVNTLLNSGTVLINNINAPFLPDPVIVITGNMTLEKNGHVILNNSS SNVGQTYVQKGNWHGKGGILSLGAVLGNDNSKTDRLEIAGHASGITYVAVTNEGGSGDKTLEGVQIISTDSSDKNAFIQKGRIVAGSYDYRLKQGTVSGLNTNKWYLTSQMDNQESKQMSNQESTQMSSR VirG proteins can contain one or more tags, each of which can include one or more of the following: purification tags, protease cleavage sites, and signal peptides. Purification tags, also known as affinity tags or fusion tags, are short amino acid sequences fused to the target protein via genetic means. These tags are used to facilitate the purification and detection of tagged proteins. Non-limiting examples include His tags, glutathione S-transferase (GST), streptavidin-binding peptide (SBP or Strep tag), maltose-binding protein (MBP), Flag tags, HA tags, calmodulin-binding peptide (CBP), and SUMO (small ubiquitin-like modified protein) tags. Each such tag can be located at the N-terminus or C-terminus of the VirG protein.

[0056] VirG proteins may contain a His tag at their N-terminus or C-terminus. In one instance, the His tag contains six consecutive histidine residues (6xHis). The tag may contain one or more protease cleavage sites. Non-limiting examples of protease cleavage sites include enterokinase (EK) cleavage sites, thrombin cleavage sites, tobacco etch virus (TEV) protease cleavage sites, factor Xa cleavage sites, proteinase K cleavage sites, precision protease cleavage sites, or caspase cleavage sites. In one instance, the tag contains a TEV protease cleavage site, which may contain the sequence ENLYFQ (SEQ ID NO: 6), followed by any amino acid except proline or tryptophan. The protease cleavage site may be located at the N-terminus or C-terminus of the VirG protein.

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

[0058] In one instance, the tagged VirG protein contains VirG R1. VirG proteins may contain the following sequences.

[0059] SEQ ID NO. 8 HHHHHHGSGENLYFQGTPLSGTQELHFSEDNYEKLLTPVDGLSPLGAGEDGMDAWYITSSNPSHASRTKLRINSDIMISAGHGGAGDNNDGNSCGGNGGDSITGSDLSIINQGMILGGSGGSGADHNGDGGEAVTGDNLFIINGEIISGGHGGDSYSDSDGGNGGDAVTGVNLPIINKGTISGGNGGNNYGEGDGGNGGDAITGSSLSVINKGTFAGGNGGAAYGYGYDGYGGNAITGDNLSVINNGAILGGNGGHWGDAINGSNMTIANSGYIISGKEDDGTQNVAGNAIHITGGNNSLILHEGSVITGDVQVNN In another instance, the tagged VirG protein comprises VirG R2. The VirG protein can comprise the following sequence.

[0060] SEQ ID NO. 9 HHHHHHGSGENLYFQGILADNLSHHNINSWDGKSLTKSGEGTLILAEKNTYSGFTNINAGILKMGTVEAMTRTAGVIVNKGATLNFSGMNQTVNTLLNSGTVLINNINAPFLPDPVIVTGNMTLEKNGHVILNNSSSNVGQTYVQKGNWHGKGGILSLGAVLGNDNSKTDRLEIAGHASGITYVAVTNEGGSGDKTLEGVQIISTDSSDKNAFIQKGRIVAGSYDYRLKQGTASGLNTNKWYLTSQMDNQESKQMSNQESTQMSSR In some aspects, the composition comprises VirG protein and one or more Shigella invasion plasmid antigen peptides. In some embodiments, the composition further comprises an invasion plasmid antigen B (IpaB) peptide or a conjugate thereof. In some embodiments, the IpaB peptide conjugate comprises at least one non-natural amino acid (nnAA), wherein nnAA is selected from 2-amino-3-(4-azidophenyl)propionic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propionic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propionic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propionic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propionic acid, and any combination thereof. In one example, nnAA is pAMF. IpaB polypeptide conjugates can be as described in WO2020205584 (the contents of which are incorporated herein by reference in their entirety).

[0061] IpaB peptide conjugates are disclosed in Table 1 of WO2020205584. IpaB peptide conjugates may comprise the sequences disclosed in Table 1 below. It should be noted that the IpaB peptide conjugate sequences shown below have different sequence numbers than those in WO2020205584 application. Furthermore, the “…” in the following sequences… X "Indicates the position of an amino acid that has been replaced by a non-natural amino acid, as described in WO2020205584."

[0062] Table 1: Exemplary IpaB peptide conjugates

[0063] In some embodiments, the composition further comprises an invading plasmid antigen H (IpaH) polypeptide. In some embodiments, the composition comprises VirG protein, IpaB polypeptide or a conjugate thereof, and IpaH polypeptide. In some embodiments, the composition may comprise one or more of invading plasmid antigen C and invading plasmid antigen D polypeptides.

[0064] In some embodiments, the composition comprises a therapeutically effective amount of VirG protein and optionally an IpaB peptide or a conjugate thereof and optionally an IpaH peptide. The composition may comprise one or more of the following: about 5 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 35 µg, 40 µg, 45 µg, 50 µg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 80 µg, 85 µg, 90 µg, 95 µg, or 100 µg: VirG protein, IpaB peptide or a conjugate thereof, and IpaH peptide, or a range defined by both of the foregoing amounts. The composition may comprise about 5-100 μg, about 20-100 μg, or about 20-90 μg of VirG protein, and optionally an IpaB peptide or a conjugate thereof, and optionally an IpaH peptide.

[0065] In some embodiments, the composition further comprises one or more adjuvants. The one or more adjuvants may be selected from: aluminum hydroxide, ADJUPHOS®, aluminum hydroxide, TLR4 agonists (MPL and MPL-like molecules), TLR9 agonists (CpG), and STING agonists (cyclodiAMP). In some embodiments, the adjuvant comprises AdjuPhos® (2% AlPO4).

[0066] The composition may be suitable for administration to a subject. In one example, the composition is suitable for mucosal application. Mucosal application may be intranasal. In another example, the composition is suitable for intradermal or intramuscular application.

[0067] How to use Another aspect of this application relates to inducing an immune response against various Shigella serotypes in a subject using the compositions disclosed herein. A further aspect relates to using the compositions to induce immunity against Shigella in a subject, or to reduce or prevent Shigella infection in a subject. This application also provides a method for inducing an immune response against Shigella, inducing immunity against Shigella in a subject, or reducing or preventing Shigella infection, the method comprising administering the compositions disclosed herein to a subject in need. The compositions disclosed herein can also be used to prepare medicaments for inducing an immune response against Shigella in mammalian subjects, inducing immunity against Shigella in mammalian subjects, or reducing or preventing Shigella infection in mammalian subjects. The subject may be a mammal. The mammal may be a human. The Shigella may be Shigella flexneri or Shigella soxehni.

[0068] In some embodiments, the composition is delivered to a mammalian subject via a mucosal route, which may be intranasal (IN) or intradermal.

[0069] Example The following are embodiments and further details regarding the disclosure of this application, intended to aid in understanding and illustrating the invention. The additional information provided below is for illustrative purposes only and is not intended to limit the scope of this application in any way.

[0070] Methods of bacterial growth Shigella flexneri 2a 2457T was grown in Luria-Bertani (LB) medium (Athena Environmental Sciences, MD) at 37°C, and the invading plasmid was extracted using a plasmid purification kit (Qiagen). Escherichia coli DH5α and BL21 (DE3) pLysS cells were cultured in LB medium at 37°C. Shigella flexneri 2a 2457T and Shigella soxehii Moseley were prepared as described above. In short, the strains were inoculated onto tryptone soybean agar (TSA) plates containing 0.02% Congo red and grown overnight at 37°C. 20-25 colonies were picked and grown in 125 mL LB or tryptone soybean broth (TSB) at 37°C with stirring (180 rpm) for 2-3 hours or until OD. 600 Achieve a concentration of 0.8 to 1.3. Centrifuge the bacterial culture and resuspend the precipitate in PBS. Assay CFU as follows: Inoculate serial dilutions of the bacterial suspension onto TSA and / or Congo red agar plates.

[0071] Cloning, expression and purification of recombinant VirGα protein A gene for expressing VirGα (amino acid [aa], derived from Shigella flexneri 2a, GenBank accession number AF386526.1) was synthesized and subcloned into the pRSETA vector. The VirGα gene (2.1 kb) was amplified by PCR using a 220 kb pathogenic plasmid of Shigella flexneri 2a 2457T as a template. The amplified VirGα gene was purified using a gel extraction kit (Qiagen). The high-level prokaryotic expression vector pRSETA was used to clone the VirGα gene with a cleavable polyhistidine (6xHis) tag, which could be rapidly purified using nickel resin and detected with an anti-His antibody. The purified VirGα gene and pRSETA expression vector were digested with restriction enzymes BamHI and EcoRI, followed by gel elution and purification. The digested VirGα and pRSETA vector were ligated and transformed into E. coli DH5α competent cells. After transformation, colonies carrying plasmids encoding VirGα were screened by PCR and run on 1% agarose gels. The plasmid containing the VirGα gene was extracted using a plasmid extraction kit (Qiagen). Restriction enzyme digestion with BamHI and EcoRI was performed to further confirm the VirGα gene clone. The plasmid-carrying strains were stored in 15% glycerol stock solution at -80°C.

[0072] The plasmid carrying VirGα was transformed into *E. coli* BL21(DE3) pLysS cells for protein expression. *E. coli* cells were grown in 8 L of Invitrogen TB medium containing 30 µg / mL chloramphenicol and 50 µg / mL ampicillin at 37°C and 200 rpm until an OD value of 4.0–5.0 was reached. 600 Protein expression was induced for 3 hours with 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) (Teknova). Following induction, cells were centrifuged at 7,800 rpm at 4°C for 30 min. The bacterial pellet was collected and resuspended in B-PER (Thermo Scientific) extraction buffer, containing a protease inhibitor mixture (Sigma-Aldrich), 1 mg / mL lysozyme, 25 U / mL benzamide enzyme, and 0.5% Triton X-100, and incubated at room temperature for 1 hour. The suspension was then incubated at 4°C with a 10,000 mL / min incubation solution. xgCentrifuge for 40 min. Wash the precipitate containing inclusion bodies and dissolve it in a solution containing 20 mM Tris, 0.5 M NaCl, 20 mM imidazole, and 8 M urea. Purify the expressed recombinant VirGα protein using affinity chromatography on a chelated nickel column (Cytiva). Wash the bound protein and then refold it in the column using a urea gradient, starting with 6 M urea in elution buffer (containing 20 mM Tris, 0.5 M NaCl, 20 mM imidazole, pH 8.0) and ending with 0 M urea in the same buffer. Collect the refolded protein in the elution buffer and remove urea by dialysis. Then concentrate the purified protein using an Amicon (Millipore) filter. Protein concentration was determined using the biuret acid (BCA) protein assay (Pierce™ Protein Assay Kit; Thermo Scientific™), with bovine serum albumin (BSA) as a standard. Store aliquots at -80°C. VirGα purity was determined based on the relative intensity and size of the bands via Image Labs. TM Software (Bio-Rad) was used for assays. Endotoxin levels were analyzed using the Limulus amebocyte lysate (LAL) kit (Charles River Laboratories) according to the manufacturer's instructions. Residual host cell proteins were determined based on the density of nonspecific bands after SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining. VirGα aa sequences of Shigella flexneri (NCBI reference sequence: WP_17250739.2), Shigella somnifera (WP_001071793.1), Shigella dysenteriae (WP_13480816.1), and Shigella boydii (WP_148722086.1) were used to determine multiple sequence alignments.

[0073] SDS-PAGE and Western blot analysis For SDS-PAGE, proteins were prepared in 2x-Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol and denatured by heating at 95°C for 5 min. Denatured protein samples (20 µl) were loaded onto 12% pre-prepared polyacrylamide gels (Bio-Rad). For Western blot analysis, the SDS-PAGE-coated samples were then transferred to nitrocellulose membranes using a Trans-BlotTurbo transfer pack (Bio-Rad). The membranes were then blocked in 3% BSA containing TBST (TBS, 0.05% (v / v) Tween-20) and incubated overnight at 4°C with either anti-His-tagged antibody or mouse anti-VirGα immune serum (serum collected from mice immunized with VirGα protein). The membranes were washed with TBST and incubated at room temperature for 1 h with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (KPL SeraCare, Gaithersburg, MD). The membrane was washed again with TBST, Immobilon Western Chemiluminescent HRP substrate (Millipore Sigma) was added, and analysis was performed using the Gel Doc imaging system (Bio-Rad).

[0074] Mice, immunization, and experimental infections with pathogenic microorganisms Adult female BALB / c mice (6 to 8 weeks old) were purchased from Charles River Laboratories. For intramuscular immunization, mice (n=15 to 20 per group) were immunized intramuscularly on days 0 and 28 with 5, 10, or 20 µg of VirGα adsorbed onto AdjuPhos® (4.8% v / v; InvivoGene), administered in 100 μL volumes (50 μL per hind limb). The negative control group received only intramuscular administration of AdjuPhos® or PBS. The positive control group received sublethal doses (1.0 x 10⁻⁶) intranasally on days 0 and 28. 5 CFU / dose) of Shigella flexneri 2a 2457T or Shigella soxehii Moseley. For intranasal vaccination, BALB / c mice were immunized with 2.5, 5, or 10 µg of VirGα on days 0, 14, and 28 in the presence of 2.5 µg of Escherichia coli dmLT. The negative control group received only 2.5 µg of dmLT or PBS intranasally. The positive control group mice were immunized intranasally with a live attenuated strain of Shigella flexneri 2a CVD1208S on days 0 and 21, respectively. Four weeks after the last immunization, mice were challenged intranasally with 5 x 10 6 – 1x10 7CFU / dose of Shigella flexneri 2a 2457T or 5.2 x 10⁻⁶ 6 CFU / dose of Shigella Moseleyi (equivalent to 3.5–7 or 2 50% lethal doses respectively) 50 Intranasal challenge was performed as described above, and mice were monitored daily for 15 days after the challenge. Blood samples were collected on day -1 (before vaccination) and on days 13, 27, 42, and 55 (post-vaccination). Spleen and bone marrow cells were obtained from surviving mice on day 73 (day 16 post-challenge).

[0075] Antibodies and ASCs As previously described, VirGα-specific antibodies were detected in serum using an ELISA method. Immulon 2HB plates (Thermo Scientific) were plated with either VirGα (2 µg / mL) or full-length VirG (2 µg / mL), and serum samples were tested in duplicate with serially diluted two-fold serial dilutions. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (KPLSeraCare, Gaithersburg, MD) was used as the detection antibody. The endpoint titer was calculated by interpolating the sample absorbance from a linear regression curve of calibrated internal standards and reported in EU / mL. 1 EU is equivalent to the reciprocal of the serum dilution that results in an absorbance value at 450 nm that is 0.2 above the background value.

[0076] To perform ASC measurements, single-cell suspensions were prepared from the spleen and bone marrow of individual mice (8 mice per group). The spleen was ruptured using a sterile syringe plunger, and the femur and tibia were rinsed by centrifugation using an adapter tube. Red blood cells were removed using lysis buffer (Biolegend®), and the cell suspension was filtered through a 70µm cell filter. The frequencies of total IgG and IgA-secreting cells, as well as VirGα-specific IgG and IgA-secreting cells, were determined using the Mouse IgA / IgG Dual-Color ELISpot Assay Kit [ImmunoSpot® ELISpot Kit, Cellular Technology Limited (CTL)] according to the manufacturer's instructions. ELISpot plates made of polyvinylidene fluoride (PVDF) were pre-wetted with 70% ethanol, then coated overnight at 4°C with anti-mouse Igκ / λ capture antibody or 2 µg / mL VirGα protein, and blocked for 1 hour with complete RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / mL penicillin, 100 µg / mL streptomycin, 8 mM HEPES, and 50 µM 2-mercaptoethanol. Wells coated with PBS served as negative controls. Spleen and bone marrow cell suspensions from individual mice within each group were collected, added to the culture plates, and incubated at 37°C for 6 hours. After washing, anti-mouse IgA / IgG detection antibodies were added, and the plates were incubated at room temperature in the dark for 2 hours. After washing, the plates were incubated again with tertiary solution for 1 hour. Then, CTL-TrueBlue and CTL-TrueRed chromogenic substrates were added sequentially for color development. Spots were counted using a CTL ImmunoSpot® analyzer with ImmunoSpot® software (ImmunoSpot®, CTL), and the results were expressed as per 10 6 The number of cells forming a spot (SFC) was determined from replicate data from four wells.

[0077] Confocal analysis of Shigella VirG antibody recognition To grow Shigella flexneri 2a as described above, 100 µL of suspension (10 7 CFU / mL) was added to the microscope slide. Bacteria were fixed in 4% paraformaldehyde aqueous solution at room temperature for 45 minutes, then washed with 1X PBS. After washing, the fixed bacteria were infiltrated and blocked at 4°C with PBS containing 15% FBS, 2% BSA, and 0.1% saponin (all purchased from Sigma-Aldrich) for 1 hour, then rinsed with PBS and incubated at 4°C. 0C. Incubate overnight with serum derived from mice immunized intramuscularly with VirGα or AdjuPhos® diluted 1:100 in PBS containing 15% FBS and 0.2% BSA. Wash the stained bacteria twice with PBS and incubate for 1 hour at room temperature in the dark with a goat anti-mouse IgG AF555-conjugated secondary antibody (Thermo Fisher Scientific). After washing with PBS, stain the bacteria for DNA at 4°C for at least 24 hours in ProLong Gold anti-fading reagent (CellSignaling Technology) containing 4,6-diamidinyl-2-phenylindole (DAPI). Confocal imaging was performed using a Leica MICA microscope. Images were acquired through a 60x water immersion objective, and parameters were adjusted to optimize signal strength. Images were processed using FIJI / ImageJ (NIH). The same signal processing was performed on all images.

[0078] Assay for adhesion and invasion inhibition in intestinal organoid monolayers Shigella adhesion and invasion inhibition assays were performed using human colonic organoid infection assays. Colonic organoid fragments inoculated onto human type IV collagen-coated Transwell inserts (3.0 μm pore size) were grown as a monolayer until the assay was performed using an epithelial voltammometer (EVOM). 2 Fusion was confirmed by transepithelial electrical resistance (TEI) at World Precision Instruments (USA). Monolayers of cells were differentiated for 5 days in antibiotic-free differentiation medium (DFM). Differentiated colonic monolayers were inverted and placed in an empty 12-well tissue culture plate. *Shigella flexneri* 2a or *Shigella soxehni* were grown to OD in TSB medium with or without 2.5 mM bile salt deoxycholic acid (DOC), as described above. 600 To achieve a pH of 0.6–0.8, and to prepare in DFM containing 5 x 10 8 A bacterial suspension of CFU / mL. Add 50 µL of inoculum (2.5 x 10⁻⁶ CFU / mL). 7A mixture of 50 µL (CFU / 50 µL) and 25 µL of heat-inactivated serum from mice immunized with VirGα or AdjuPhos® was added to the basal side of colonic monolayer cells. To assess adhesion inhibition, the bacterial immune serum was incubated with the monolayer cells at 37°C and 5% CO2 for 15 min. For inhibition of invasion, bacteria grown in the presence of DOC were mixed with mouse immune serum, added to the monolayer cells, and incubated for 1.5 h, followed by incubation with 50 µg / mL gentamicin for 30 min to kill extracellular bacteria. For adhesion and invasion assays, after incubation, the monolayer cells were washed three times with 1X PBS and lysed with 0.1% Triton X-100 for 20 min. The recovered bacteria were serially diluted and then plated onto TSA plates for CFU counting.

[0079] Statistical analysis All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad software, La Jolla, CA). The D'Agostino and Pearson tests were used to test the normality of continuous data. Continuously normally distributed data were analyzed using unpaired... t Comparisons were performed using either one-way ANOVA or Tukey's multiple comparison test. The Mann-Whitney test and Kruskal-Wallis test combined with Dunn's multiple comparison test were used for nonparametric data. The log-rank (Mantel-Cox) test was used to compare survival curves. VE was calculated as [(percentage of death in the PBS control group – percentage of death in the vaccinated group) / percentage of death in the PBS control group] x 100. Simple linear regression and Pearson correlation tests were used to determine associations between groups. ROC-AUC analysis was used to determine the antibody threshold level that could predict vaccine efficacy. Two-tailed analyses were determined. p value, p <0.05 was considered statistically significant. The data presented are either pooled data or represent at least two experiments with similar results, and each figure shows the measurement results for a single sample. VirGα amino acid sequence alignment and percentage of homology (%) were determined using NCBI Multiple Sequence Alignment Viewer 1.24.0.

[0080] Preparation of recombinant VirGα protein A schematic diagram of the VirG region is shown in Figure 1a. The full-length VirG region exhibits low expression efficiency as a recombinant protein, likely due to its large molecular weight (120 kDa). Given the low expression efficiency of the full-length VirG region, the surface-exposed region VirGα (aa 53–758) was cloned, expressed in an E. coli-based system, and used to produce a recombinant protein. The VirGα aa sequence in four Shigella species was determined using the NCBI multiple sequence alignment viewer. Identity (%) indicates the homology of the VirGα sequence and non-identical aa residues (red bars) in Shigella flexneri, Shigella dysenteriae, and Shigella boydii compared to Shigella flexneri. Homology of the VirGα amino acid sequence was >99% in the most common Shigella serotypes (Figure 1a). The VirGα gene (2.1 kb) was amplified, inserted into the pRSETA plasmid, and transformed into E. coli DH5α competent cells (Supplementary Figures 1a, b). Restriction endonuclease digestion with BamHI and EcoRI was performed to confirm successful cloning (Supplementary Fig. 1c). The plasmid carrying VirGα was transformed into *E. coli* BL21 (DE3) pLysS competent cells; bacterial growth was induced, and protein expression was generated. High levels of recombinant VirGα (VirGα, 76 kDa) were produced after 3 hours of induction. Positively transformed *E. coli* BL21 (DE3) pLysS competent cells were induced with 1 mM IPTG for 2 hours (lane 3) or 3 hours (lane 4) for protein expression, with the latter achieving higher expression of 76 kDa VirGα (lane 4, red box). Protein molecular weight markers (MWM; lane 1) and uninduced cells (lane 2) were included as controls (Fig. 1b). VirGα was purified using a chelated nickel column and refolded within the column. The protein was eluted, dialyzed, and concentrated, yielding a 76 kDa band on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The concentrated purified VirGα; protein MWM (lane 1), eluted VirGα protein (lanes 2-5), and concentrated VirGα (lanes 6-7) (Fig. 1c). Identification was confirmed by Western blotting using an anti-His-tagged antibody (Fig. 1d, left panel) and VirGα-reactive mouse antiserum (Fig. 1d, right panel). The 76 kDa VirGα protein (lane 2) was confirmed by Western blotting using an anti-His-tagged antibody (left panel) and anti-VirGα immune serum (right panel). MWM (lane 1). Fig. 1e shows the immunoreactivity of VirGα compared to the full-length VirG protein, measured by ELISA control from pooled serum from mice immunized with full-length VirG. Serially diluted mouse serum was added at the same concentration to culture plates coated with each protein (i.e., VirGα and full-length VirG).These assays were performed under identical conditions, using the same pooled mouse serum to test for each protein, and were repeated in multiple wells. Data represent the mean OD of the replicate wells. 450 (Display R) 2 , r, and p Linear consistency between the value and each dilution.

[0081] The production efficiency of VirGα based on Escherichia coli was high, with a recovery rate of 6.2 mg / L of culture medium, a purity of >95%, residual host cell protein of <1%, and endotoxin content of <20 endotoxin units / mg (limit of detection); these results were reproduced in subsequent batches.

[0082] The immunoreactivity of VirGα is compared with that of full-length VirG as follows: The test assesses whether both antigens can be recognized in an indirect ELISA (run under identical conditions) using pooled serum from mice immunized with full-length VirG. Full-length VirG (R...) 2 = 0.9998) and VirGα (R 2 OD = 0.9995 450 Four-parameter logistic (4PL) dose-response curves were generated using serially diluted immune serum (Fig. 1e, left panel). OD values ​​for full-length VirG and VirGα were also analyzed. 450 The values ​​showed a significant positive correlation, with the slope of Hill being 1 (R²). 2 = 0.9975 and r = 0.9998, p <0.0001) (Figure 1e, right side). These results indicate that the immunoreactive epitopes present on the full-length VirG protein are mainly contained in the α-domain region of the protein.

[0083] VirGα was used to induce potent and dose-dependent antibody responses via parenteral immunization. To investigate the immunogenicity of VirGα, adult mice were immunized intramuscularly (IM) twice (day 0 and day 28) with escalating doses (5, 10, or 20 µg) of VirGα mixed with AdjuPhos®, as shown in Figure 2a. The negative control group received either AdjuPhos® or phosphate-buffered saline (PBS). This included sublethal doses (10 µg, 10 µg, 2 ... 5Mice immunized intranasally (IN) with Shigella flexneri 2a or Shigella soxehnifolia (CFU / dose) served as positive controls for serotype-specific protection. Parenteral immunization with VirGα induced high levels of VirGα-specific serum IgG in all immunization groups, compared to the PBS or AdjuPhos® control groups (Fig. 2b). Antibody responses increased after secondary immunization, peaking at 2 weeks post-boost immunization in all groups (Fig. 2b). VirGα-specific IgG responses exhibited a dose-response pattern, with higher serum IgG levels in groups receiving larger doses of VirGα at all time points post-immunization (Fig. 2c). The best responders were mice receiving a 20 µg dose; the mean serum IgG titers (before booster immunization) at day 27 were 1.3 x 10⁻⁶ in the 5 µg, 10 µg, and 20 µg dose groups, respectively. 4 2.1x10 4 and 5.6x10 4 The ELISA units (EU) / mL at day 55 (after booster immunization and close to the challenge time) were 9.6 x 10⁻⁶. 5 1.4x10 6 and 2.1x10 6 EU / mL (Fig. 2b, c). On day 55, serum IgG titers increased by a mean fold relative to baseline of 153,600 EU / mL, 224,000 EU / mL, and 336,000 EU / mL at dose levels of 5, 10, and 20 µg, respectively (Fig. 2b, c). An unexpected finding was the low (negligible) VirGα-specific serum IgG response in mice immunized intranasally with sublethal doses of Shigella flexneri 2a and Shigella sogii (Fig. 2b), whereas these controls produced strong serum IgG responses to the corresponding immunized strains' OPS. No VirGα-specific IgA response was detected in the serum or feces of mice immunized intramuscularly with VirGα, or in the serum or feces of mice receiving sublethal intranasal doses of Shigella flexneri 2a and Shigella sogii (data not provided).

[0084] VirGα protects mice from lethal infections by Shigella flexneri 2a and Shigella solanacearum. On day 57 post-vaccination, mice immunized intramuscularly with an increased dose of VirGα (as described above) were subjected to pathogenic Shigella flexneri 2a 2457T (5 x 10⁻⁶). 6 CFU / dose) or Shigella Moseleyi (5.2 x 10⁻⁶) 6Intranasal infection challenge at a lethal dose (CFU / dose) (Figure 2a). Parenteral immunization with VirGα containing AdjuPhos® adjuvant provided high levels of protection against Shigella flexneri 2a; 83% vaccine potency (VE) was achieved with a 20 µg dose, followed by 61% and 56% VE with 10 µg and 5 µg doses, respectively (Figure 3a and Table 1). VE was significantly higher in all vaccination groups compared to the negative control group (Figure 3a and Table 1). p <0.001), but the difference did not reach statistical significance when compared between VirGα vaccination groups. Importantly, VirGα provided statistically significant cross-protection against lethal Shigella infection; 20 µg and 10 µg doses achieved 57% and 46% VE, respectively (Figure 3b and Table 1). The lower VE against Shigella with 20 µg and 10 µg doses of VirGα is attributed to the more severe Shigella infection (earlier mouse death) in the described model. Mice immunized intranasally with sublethal doses of lethal Shigella flexneri 2a or Shigella flexneri showed the highest level of homologous protection, but this level of protection was not significantly different from that of mice receiving VirGα (20 µg dose). Almost all unvaccinated control mice died from lethal pulmonary Shigella infection challenge (Figure 3a, b).

[0085] Based on the association between antibody levels and reduced infection risk observed in human studies, these studies aimed to investigate whether VirGα-induced serum IgG levels were associated with survival rates after infection in mice. Mice immunized with VirGα developed protection upon challenge with Shigella flexneri 2a or Shigella sophorae, exhibiting significantly higher levels of VirGα-specific serum IgG at challenge time (day 55) compared to mice that did not survive (unprotected) (Figures 3c, d). A threshold (1.26 x 10⁻⁶) was determined using receiver operating characteristic (ROC) and area under the curve (AUC) analyses. 6 EU / mL), this threshold can predict protective results, with both sensitivity and specificity of about 80% (Figure 3e).

[0086] The immunogenicity and protective efficacy of VirGα were also investigated using the mucosal route and the clinically proven mucosal adjuvant *E. coli* dmLT. Mice were intranasally immunized with 2.5, 5, or 10 µg of VirGα mixed with dmLT on days 0, 14, and 28. VirGα-specific serum IgG responses were moderate after the first and second vaccinations, but significantly enhanced after the third immunization. The 10 µg dose achieved the highest titers (Fig. 4a) and provided 90% of the VE against *Shigella flexneri* 2a; the VE values ​​produced by the 5 or 2.5 µg immunization doses were negligible (Fig. 4c and Table 2), suggesting that a certain protein dose level may be required after intranasal administration to induce potent vaccine efficacy. High levels of dmLT-specific IgG titers were observed, with nearly identical titers across the dmLT recipient groups, indicating appropriate vaccine preparation and immunization (Fig. 4b).

[0087] VirGα-specific antibody-secreting cells (ASCs) in systemic tissues The frequencies of IgG and IgA ASCs were measured in the spleen and bone marrow of mice immunized intramuscularly with 20 µg of VirGα (the most effective dose) and mice intranasally inoculated with a sublethal dose of Shigella flexneri 2a (Sf2a-IN). This analysis was performed in animals that survived lethal Shigella flexneri 2a infection to distinguish vaccine-induced immunity from infection. Intramuscular immunization with VirGα-aluminum hydroxide was chosen for this study and future studies because this vaccination modality is most likely to be applicable to humans.

[0088] High levels of VirGα-specific IgG ASCs were detected in the spleen and bone marrow of mice vaccinated with the VirGα vaccine (Fig. 5a, b). In contrast, VirGα-specific IgG ASCs were not detected in mice receiving Sf2a-IN. The lack of VirGα-specific IgG ASCs in Sf2a-IN mice is consistent with their negligible antibody response to VirGα (Fig. 2b). High frequencies of total (non-vaccine-agnostic) IgG were observed in the spleen and bone marrow of all groups. In fact, the total IgG ASC levels in Sf2a-IN mice were even higher than those in mice immunized with VirGα, ruling out the possibility of technical error (Fig. 5a, b). VirGα-specific IgA ASCs were not produced in either the VirGα-vaccinated mice or the positive controls immunized with sublethal Sf2a-IN (Fig. 5a, b); meanwhile, total IgA ASCs were detected in the spleen and bone marrow of both groups of mice. The absence of VirGα IgA ASCs in these groups is consistent with the lack of IgA response in serum and feces described above.

[0089] Antibacterial function of VirGα specific antibodies The association between serum antibody levels and protective efficacy prompted us to investigate the antibacterial mechanism of VirGα antibodies. To this end, we first used confocal immunofluorescence microscopy to investigate the ability of VirGα mouse immune serum to recognize VirG expressed on live Shigella flexneri 2a. Vaccine-induced VirGα-specific antibodies, rather than AdjuPhos® antiserum, were able to recognize VirG expressed by Shigella flexneri 2a in its typical unipolar pattern, as shown in the confocal microscopy images. Figure 6 ).

[0090] We investigated the ability of immune serum from VirGα-vaccinated mice to block Shigella adhesion to intestinal epithelial cells using primary human colonic monolayers. Shigella flexneri 2a was mixed with VirGα-specific mouse antiserum (or serum from mice receiving AdjuPhos® as a negative control) and added to the basal side of human colonic monolayers (Fig. 7a). VirGα-specific immune serum inhibited Shigella adhesion to the human colon; statistically, a significant reduction of approximately 50% in bacterial adhesion was observed (Fig. 7b). The inhibitory effect of VirGα-specific antibody on adhesion was even more pronounced when bacteria were grown in the presence of deoxycholic acid (DOC) (only 26% of the organisms remained adherent) (Fig. 7b). DOC enhances Shigella adhesion and invasion of epithelial cells and activates VirGα-dependent Shigella flexneri adhesion. Furthermore, compared to the control group AdjuPhos serum, VirGα-induced serum antibodies significantly impaired the adhesion of DOC-stimulated Shigella flexneri (only 55% bacterial recovery) (Figure 2D). Using the same method, the ability of VirGα-specific mouse antiserum to reduce the invasion of DOC-stimulated Shigella flexneri 2a into colon cells was demonstrated; only 65% ​​of the original bacterial load was recovered intracellularly (Figure 7c). These results indicate the ability of vaccine-induced VirGα-specific IgG to recognize in vivo natural antigens and deploy antimicrobial functions against bacteria that impair adhesion and invasion in the human colon.

[0091] This application describes compositions and methods related to using Shigella VirGα or fragments of Shigella VirGα as novel subunit vaccine candidates. VirGα exhibits strong immunogenicity and broad protective efficacy. Animal models of Shigella are limited and artificial; Shigella infects rodents via oral administration. Therefore, the protective efficacy of VirGα was investigated in a mouse model of lethal lung infection that reproduces the characteristics of the fatal human disease. Parenteral immunization of mice with VirGα containing aluminum hydroxide adjuvant induced a strong serum antibody response, providing >80% protection against Shigella flexneri 2a and 57% protection against lethal Shigella soxetella infection. Similarly, intranasal (IN) immunization with VirGα mixed with Escherichia coli dmLT provided 90% protection against Shigella flexneri 2a infection. Serum IgG generated by parenteral immunization with VirGα was correlated with vaccine efficacy. Threshold levels have been identified as predictive of protective efficacy in experimental settings; similar values ​​have been reported for IpaB. The consistency between VirG-specific immune responses and protective effects in mice is consistent with our observations in volunteers: volunteers who underwent experimental and subsequent Shigella flexneri 2a infections showed higher serum VirG-specific IgG (IgG1) titers and lower disease severity. This finding also aligns with the high levels of VirG-specific antibodies we detected in adults in Shigella-endemic regions, who are less susceptible to infection than children due to natural immunity acquired from repeated exposure. Similarly, we detected VirG-specific IgG and IgA in the feces of Shigella-infected children aged 0-5 years, with higher levels of these antibodies in children without dysentery compared to children with dysentery or diarrhea from other causes. Taken together, these observations suggest that VirG is an important target for protective immunity, and VirG-specific antibodies represent potential antimicrobial effector factors.

[0092] The study also confirmed that the VirGα antibodies produced by vaccinated mice could recognize VirG expressed on the outer surface of bacteria, and demonstrated their ability to prevent Shigella infection by blocking bacterial adhesion and invasion into human colonic monolayer cells. These results indicate that VirG antibodies exhibit key antibacterial functions, preventing Shigella from initiating infection at the mucosal interface, thereby avoiding tissue damage and inflammation. In the context of exploring bacterial pathogenic mechanisms, this study shows that purified VirG-specific antibodies can block a highly adhesive... Attachment of IpaD Shigella mutant to mammalian cells 33This is consistent with current findings. Recently, using serum obtained from participants in the CHIM study, the immunoglobulin classes and subclasses of VirGα-specific antibodies, their Fc receptor binding characteristics, and their ability to bind to innate immune cells were investigated. In individuals with mild symptoms, VirGα-specific IgG and IgA, Fc receptor binding, and VirGα-specific antibody-dependent complement deposition (ADCD) and antibody-dependent mononuclear cell phagocytosis (ADCP) were detected.

[0093] In addition to systemic antibodies, intramuscularly delivered VirGα elicited antigen-specific IgG ASCs, which have been detected in the spleen and bone marrow. Vaccine-induced ASCs, along with memory B cells, provide long-term humoral immunity against infection. Splenic ASCs, derived from germinal center B cells, enter the bloodstream and replenish circulating antibody levels. They can also migrate to the bone marrow. Bone marrow ASCs represent long-lived plasma cells that maintain humoral immune memory. The VirGα-specific humoral and ASC responses are rich in IgG, consistent with the expectation of parenteral administration of subunit vaccines in the presence of aluminum hydroxide. Subunit vaccines also elicit a major IgG response in routine immunization programs. Given the prevalent presence of VirG-reactive antibodies in individuals infected with Shigella, the negligible ASC and antibody responses to VirGα in mice intranasally inoculated with live Shigella (whether attenuated or wild-type) are surprising and intriguing. This could be attributed to limited exposure – insufficient to adequately stimulate an immune response – or to differences in host susceptibility and immunity between species.

[0094] VirGα fragments The VirGα fragment was synthesized using a cell-free expression system. The cell-free expression system used is described in US Patent 9040253, the entire contents of which are incorporated herein by reference. One advantage of using a cell-free system for peptide synthesis is that the overall peptide yield can reach the order of 200 mg / L, which is approximately forty times higher than that of conventional peptide synthesis methods.

[0095] As previously stated, the composition disclosed in this application for the prevention of various Shigella serotypes can be fragment c shown in Figures 8a-d. Here, 42 days after vaccination, the Ag-specific serum IgG titers of VirGα and the two fragments (VirG R1 and VirG R2) were compared, with each being administered at a dose of 20 μg. VirG R1 corresponds to aa53-353 of the VirG protein α region, and VirG R2 corresponds to aa500-758 of the VirG protein α region. Sublethal doses of Sf2a–IN and PBS (phosphate-buffered saline) were used as positive and negative controls, respectively. As shown in Figure 8a, the Ag-specific serum IgG titer of VirG R1 was greater than that of VirGα, while the titer of VirG R2 was less than that of VirGα. The VirGα-specific IgG response also showed the same trend (Figure 8b).

[0096] Figure 9 The changes in mouse survival rates over time are shown for VirGα (20 μg), VirG R1 (20 μg), VirG R2 (20 μg), sl Sf2a-IN, and PBS. It can be seen that the survival rates of VirGα (53-758)-20µg and VirG-R1 (53-353)-20µg are superior to those of VirG-R2 (500-758)-20µg. Table 2 below summarizes the information.

[0097] In some examples of disclosed Shigella compositions, certain amino acids can be substituted with other amino acids to achieve better results. In one non-limiting example, the cysteine ​​at position 130 in VirGα or VirG R1 can be substituted with serine. In this example, this substitution reduces the amount of aggregation, as seen from... Figure 10 As can be seen in the text.

[0098] Table 4. Vaccine efficacy of intramuscularly administered VirGα and its fragments against Shigella infection.

[0099] In other aspects of the published application, VirGα or VirGα fragments can be used either as a standalone vaccine or in combination with other conjugate vaccine candidates (e.g., IpaB, OPS-IpaB) to maximize efficacy. The inventors recently reported the development of a novel Shigella OPS-IpaB conjugate vaccine candidate that induces cross-protective immunity in mice. IpaB is a highly attractive vaccine target due to its immunogenicity and its association with human protective immunity. In some embodiments, VirGα or VirGα fragments can be combined with IpaB to further enhance efficacy. A vaccine based on a broad-spectrum, safe, and effective protein would be simpler and more advantageous than existing vaccine candidates in terms of broader coverage, prevention of infections ultimately caused by all serotypes, ease of production and quality control, intuitive clinical evaluation, and practical application. These advantages would reduce costs, making the vaccine more affordable and attractive to resource-constrained countries. Based on the excellent safety profile of subunit vaccines in routine immunization programs, this protein-based Shigella vaccine is expected to be well-tolerated by infants and young children, who are the most vulnerable to the disease. The VirGα / VirGα fragment (and IpaB) exhibit significant immunogenicity, which helps generate potent and durable protective immunity in children under 3 years of age, for whom Shigella OPS-rEPA vaccines and oral live attenuated vaccines have failed. The simple manufacturing process of subunit vaccines will improve their affordability. Even in resource-limited areas, successful implementation through integration into existing immunization programs is possible. The holistic disease prevention strategy utilizing broad-spectrum protective proteins is simple, easy to implement, and expected to be cost-effective. Therefore, VirGα or VirGα + IpaB vaccines meet the preferred product characteristics listed by the WHO for Shigella vaccines.

[0100] It must be emphasized that the cross-protective advantage of conserved proteins is due to the cyclical spread of pathogenic serotypes in the environment, and the emergence of strains not covered by O antigen-based vaccines necessitates product redesign. As concepts continue to refine and new candidate vaccines enter human clinical trials and CHIM stages, the overall profile of Shigella vaccines is likely to change significantly within the next five years.

Claims

1. A composition comprising a VirG protein or a fragment of the VirG protein containing about 100-400 amino acids, wherein the amino acid sequence of the VirG protein is at least 90% identical to the sequence shown in SEQ ID NO:

2.

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

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

5.

4. The composition according to any one of claims 1-3, wherein the VirG protein contains a tag at its N-terminus or C-terminus.

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

6. The composition according to 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 shown in SEQ ID NO: 6, followed by any amino acid other than proline or tryptophan.

9. The composition of claim 8, wherein the label comprises the sequence shown in SEQ ID NO:

7.

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

8.

11. The composition of claim 9, wherein the VirG protein comprises the sequence shown in SEQ ID NO:

9.

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

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

14. The composition according to 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 comprises one or more of IpaB polypeptide and IpaH polypeptide.

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

17. The composition according to claim 15 or 16, wherein the IpaB polypeptide comprises at least 90% identical sequences to those shown in any one of SEQ ID NO: 10-16.

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

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

20. The composition according to any one of claims 14-19, wherein it independently comprises about 5-100 μg of each Ipa polypeptide.

21. A method for inducing an immune response against Shigella in a mammalian subject in need, eliciting immunity against Shigella in a mammalian subject in need, or mitigating or preventing Shigella infection in a mammalian subject in need, comprising administering to the subject the composition according to any one of claims 1 to 20.

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

23. The composition according to any one of claims 1-20, used to induce an immune response against Shigella in a mammalian subject, to induce immunity against Shigella in a mammalian subject, or to reduce or prevent Shigella infection in a mammalian subject.

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

25. Use of the composition of claim 24 in the preparation of a medicament for inducing an immune response against Shigella in a mammalian subject, eliciting immunity against Shigella in a mammalian subject, or mitigating or preventing Shigella infection in a mammalian subject.

26. The use according to claim 25, wherein the drug is intended for intranasal, intramuscular, or percutaneous delivery.

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

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