Nitrated invasion plasmid antigen d and derivatives for enhancing immune responses

Abstract

The present invention provides an antigenic nitrated recombinant polypeptide derived from invasion plasmid antigen D (IpaD) of Shigella flexneri. Also provided is a vaccine composition comprising the antigenic nitrated recombinant polypeptide and preparation thereof. Further provided is a method for inducing an immune response in a subject, comprising administering the vaccine composition to the subject.

Description

[0001]2101715-001306 -1- NITRATED INVASION PLASMID ANTIGEN D AND DERIVATIVES FOR ENHANCING IMMUNE RESPONSES CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to United States Provisional Application No. 63 / 656,559, filed June 5, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes. REFERENCE TO U.S. GOVERNMENT SUPPORT This invention was made with government support under grant numbers CBET- 2032243 awarded by the National Science Foundation The United States has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2101715- 001306_SequenceListing.xml, created June 4, 2025, which is 46 KB in size. The information in XML file format of the Sequence Listing is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The invention relates to nitrated antigens derived from pathogens as vaccines for eliciting immune response to the pathogens. BACKGROUND OF THE INVENTION Vaccines are one of the most vital medical contributions in history and have resulted in dramatic increases in public health and life expectancy. However, effective, long-lasting and broadly protective vaccines remain elusive for many pathogens. One persistent limitation in developing broad protection results from antigenic variation between serotypes and the inability to direct immune responses towards desired sequences of antigens that are conserved and / or neutralizing. With the rise of antibiotic resistance and the risk of the development of pandemic strains of disease, vaccines with broader cross-strain protection and increased neutralizing responses are needed. With advancements in synthetic biology, novel amino acids distinct from the standard 20 amino acids can be incorporated into proteins. Previous research has shown that the inclusion of nonstandard amino acids (nsAA), most notably para-nitro- L-phenylalanine (pN-Phe), in protein antigens can be used to break immune self- tolerance and increase the immunogenicity of self-proteins such as TNF-a, RBP4, RANKL, and C5a. In these demonstrations, the introduction of pN-Phe generated novel immunogenic epitopes into antigens, resulting in the induction of cross-reactive, sustained systemic antibody responses toward the antigen, lasting over 40 weeks. 2101715-001306 -2- These demonstrations are based on several previous examples of the impact nitroaromatic molecules have on the immune system including the use of nitroaromatic compounds such as dinitrophenol as haptens to generate cross-reactive immune responses and the presence of 3-nitrotyrosine modifications in autoimmune conditions. Additionally, it has been shown that para-nitro-L-phenylalanine (pN-Phe) can be used to modify a universal helper T cell epitope to enhance the immunogenicity of cancer antigens for cancer vaccine design. In these examples, the effect of pN-Phe in improving immune responses has been dependent on specific site of mutation and the MHC-II background of the subject the vaccine was delivered to and highly dependent on the site of introduction. The applications of pN-Phe or other nsAAs to enhance immune response have been found limited to increases in antibody responses to self-proteins. For example, Dulin et al. (Microbiology Spectrum 10, e01902-22 (2022)) interrogated the effect of the introduction of nitrated nsAAs at random sites within a foreign antigen, the hemagglutinin (HA) antigen from influenza, and found out that immunization with the randomly nitrated HA antigen exhibited lower virus-specific antibody responses compared to a non-nitrated variant. Shigella, a genus of bacteria, causes diseases in primates, for example, human shigellosis. During infection, it typically causes dysentery. Shigella is a leading cause of bacterial diarrhea worldwide, with an estimated 80–165 million annual cases and 74,000 to 600,000 deaths. Shigella species are classified by three serogroups and one serotype: Serogroup A (S. dysenteriae) with 15 serotypes, Serogroup B (S. flexneri) with 9 serotypes, Serogroup C (S. boydii) with 19 serotypes, and Serogroup D (S. sonnei) with one serotype. Given the high burden posed by Shigella infections, there is a public health demand for a safe and effective vaccine, and in particular for a vaccine that can stimulate strong neutralizing responses across serotypes of the pathogen. Invasion plasmid antigen D (IpaD) is an essential component of the type III secretion system in Shigella, which enables the invasion and pathogenesis of the bacterium, and IpaD is highly conserved across all Shigella serotypes. Previous studies have demonstrated that anti-IpaD antibodies can neutralize Shigella invasion and B lymphocyte hemolysis, and mouse studies have shown that immunization with IpaD- containing vaccines can provide cross strain protection in challenge studies. Vaccines have been developed against Shigella, but no clinically approved Shigella vaccine is currently available commercially and previously developed vaccine formulations lack broad protection. Vaccine formulations containing IpaD or other type III secretion system antigens have often required potent adjuvants due to the poor immunogenicity 2101715-001306 -3- of these antigens alone. Thus, there remains a need for antigens capable of eliciting strong and protective immune response against Shigella. SUMMARY OF THE INVENTION The present invention relates to antigenic nitrated recombinant polypeptides and uses thereof for inducing an immune response. The inventors have surprisingly discovered that an antigenic nitrated recombinant polypeptide comprising a truncated invasion plasmid antigen D (IpaD) of Shigella flexneri with substitution of tyrosine at position 104 of the full-length native IpaD (SEQ ID NO: 1) with nonstandard amino acid para-nitro-L-phenylalanine (pN-Phe) induced greater immune response and antibody shifting in a subject as compared with that induced by a control polypeptide in a control subject. The present invention provides an antigenic nitrated recombinant polypeptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, wherein an amino acid in SEQ ID NO: 4 or a fragment thereof is substituted with a nitrated amino acid, and wherein the antigenic nitrated recombinant polypeptide comprises a nitrated amino acid corresponding to the nitrated amino acid in the SEQ ID NO: 4 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 6. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence selected from SEQ ID NOS: 48-50. The fragment may consist of 12-18 amino acids. The fragment may consist of the amino acid sequence selected from the group consisting of SEQ ID NOS: 7-15. The substituted amino acid may be selected from the group consisting of Y91, W122, Y136, Y140, Y147, Y151, F154, W164, Y193, W213, Y263, W266, F270, Y288, and F295 in SEQ ID NO: 4. The nitrated amino acid may be para-nitro-L-phenylalanine (pN-Phe). A vaccine composition is provided. The vaccine composition comprises an effective amount of the antigenic nitrated recombinant polypeptide and a pharmaceutically acceptable adjuvant, carrier and / or excipient. A method for inducing an immune response in a target subject is provided. The method comprises administering to a target subject an effective amount of the vaccine composition, whereby a target immune response is induced in the target subject. The target immune response may be greater than a control immune response induced by a 2101715-001306 -4- control polypeptide in a control subject. The target induced immune response may comprise production of one or more target antibodies against the antigenic nitrated recombinant polypeptide, wherein the one or more target antibodies cross-react against a control polypeptide, and wherein the one or more target antibodies comprise IgG, IgA, IgG1, IgG2b, IgG2c, or a combination thereof. The one or more target antibodies may bind a first region in the antigenic nitrated recombinant polypeptide and a second region in the control polypeptide, wherein the first region and the second region correspond to different regions of SEQ IN NO: 4. A method for producing the vaccine composition is provided. The method comprises admixing the antigenic nitrated recombinant polypeptide with the pharmaceutically acceptable adjuvant, carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows circular dichroism of purified IpaD (SEQ ID NO: 4) with pN-Phe substitution at position Y91. FIGS. 2A-F show antibody titers for mice immunized (prime and boost) with one of the following: PBS, wild-type IpaD (WT) (SEQ ID NO: 4), or nitrated IpaD (Mut) (SEQ ID NO: 6) from day 28 after initial dosage. (A) Serum IgG titers. (B) Serum IgG1 titers. (C) Serum IgG2b titers. (D) Serum IgG2c titers. (E) BALF IgG titers. (F) BALF IgA titers. Serum and BALF dilution factors for indirect ELISA: IgG and IgG subtypes: 1:1000; IgA: 1:4). Significance was determined via parametric or non-parametric t- test depending on data set normality. P < 0.05 was considered significant. FIGS. 3A-D show (A) B cell epitope profiling was performed using a library of linear peptides spanning the length of the sequence of IpaD (SEQ ID NO: 4). (B) The library was developed with each peptide of length 15 amino acids synthesized and then a 5 amino acid offset was used so that each subsequent peptide has 10 amino acids of overlap with the previous for a total of 32 peptides, including Peptide 1 (GFSPNNTNGSSTET) (SEQ ID NO: 16) and Peptide 2 (NTNGSSTETVNSDIK) (SEQ ID NO: 17). (C) Additional peptides corresponding to the region of nitration, corresponding to Peptide 17* (QLLDILSRHEpNPINK) (SEQ ID NO: 48), Peptide 18* (RHEpNPINKDAREL) (SEQ ID NO: 49), and Peptide 19* (pNPINKDARELLHSAP) (SEQ ID NO: 50), were synthesized with pN-Phe substitution at the Y90 site. (D) Peptide ELISAs were performed with animals immunized with serum IgG from animals immunized with PBS with CpG (PBS), wild-type IpaD (SEQ ID NO: 3) with CpG (WT), and IpaD nitrated at site Y90 (SEQ ID NO: 6) with CpG (Y90) for the wild-type peptide library and the nitrated peptide variants. Peptides 1-32 (SEQ ID NO: 16-47) and peptides 17* (SEQ ID NO: 48), 18* (SEQ ID NO: 49), and 19* (SEQ ID NO: 50) were used to coat plates for the ELISA. * indicates that peptide is nitrated. 2101715-001306 -5- FIGS. 4A-D show nitrated IpaD (SEQ ID NO: 6) elicits heightened neutrophil presence at site of immunization and costimulatory molecule expression on APCs. Whole murine lungs were digested after 18h in vivo immunization to form a single cell suspension for flow cytometry analysis. (A) Timeline of short-term study and post- study sample processing. (B) Cell type and abundance in whole lung cell suspensions from groups including PBS, wild-type IpaD (WT, SEQ ID NO: 4). (C) Antigen presentation (CD83 and MHC-II) surface marker analysis of four APC types. (D) Costimulatory molecule (CD80 and CD86) surface marker analysis. Significance determined via two-way ANOVA and Tukey’s test. FIGS. 5A-C show quantification of cytokine response in BALF samples. (A) Mice were euthanized 18 h post-immunization with PBS, wild-type IpaD (SEQ ID NO: 4) spiked with LPS to levels equivalent to nitrated IpaD (WT (S)), WT IpaD (SEQ ID NO: 4) without LPS spiking (WT (NS)), and nitrated IpaD (SEQ ID NO: 6) (Mut) and BALF samples were submitted to UMD Cytokine Core Laboratory for multiplex cytokine ELISA analysis to identify and measure the following cytokines: IFN-γ, IL-1β, KC, TNF-ɑ, and BAFF. Of these five, BAFF and TNF-ɑ were quantifiable in at least one of the immunization groups. (B) The increase in BAFF titers for nitrated IpaD-immunized mice were statistically significant relative to both spiked and unspiked wild-type IpaD. (C) TNF-ɑ was detected in BALF of mice immunized with spiked wild-type and nitrated IpaD. However, it was only quantifiable for mice immunized with nitrated IpaD with the titers in the spiked wild-type IpaD immunization group being below the quantification range of 2-500 pg / mL. BAFF responses were compared across immunization groups using one-way ANOVA with Tukey’s multiple comparisons test, and P < 0.05 was considered significant. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antigenic nitrated recombinant polypeptides derived invasion plasmid antigen D (IpaD) or a fragment thereof. The invention is based on the inventors’ surprising discovery that incorporation of nonstandard amino acid (nsAA) para-nitro-L-phenylalanine (pN-Phe) into the IpaD amino acid sequence resulted in enhancement of immune response to Shigella and antibody shifting in a subject infected by Shigella. Also provided by the present invention are methods for inducing an immune response in a subject and methods for producing the antigenic nitrated recombinant polypeptides. The inventors have successfully introduced nsAA pN-Phe into model pathogen- derived antigen IpaD for Shigella to generate nitrated IpaD (SEQ ID NO: 6) by substitution of a tyrosine at position 90 of a truncated IpaD (SEQ ID NO: 2) with the 14 N-terminal amino acids removed from the full length native IpaD (SEQ ID NO: 1) of 2101715-001306 -6- Shigella flexneri. The nitrated IpaD increased humoral immune response in a mouse model and enhanced responses to epitopes surrounding the site of nitration, potentially enabling the targeting of regions in the IpaD for pathogen neutralization that are conserved across variants. The inventors have also discovered sites in the IpaD for incorporation of pN-Phe or other nsAAs for enhancement of immune responses to foreign antigen IpaD in a subject. Immunization of the subject with nitrated IpaD or derivatives thereof may enhance antibody titers in the subject following immunization and potentially limit the need for adjuvant to elicit strong immune responses against Shigella. In addition, the introduction of pN-Phe or other nsAAs could elicit a distinct immune response and direct antibody titers to particular regions of the antigens, for example, epitopes of interest, in addition to, or instead of, a net increase in production of antibodies. The term “polypeptide” as used herein refers to a single linear chain of at least 12 amino acids. The polypeptide may have about 12-18, 12-50, 12-100, 12-300, 12- 500, 12-1000, 300-400 or 300-500 amino acids. The polypeptide may comprise standard amino acids. The polypeptide may further comprise one or more nonstandard amino acids. The term “antigenic” as used herein refers to a molecule, for example, a polypeptide, capable of inducing or eliciting an immune response in a subject exposed to a foreign substance. The foreign substance may a pathogen (e.g., bacterium) or a pathogenic agent, for example, a protein of the pathogen. The term “standard amino acid” as used herein refers to alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), or valine (Val or V). Tyrosine, phenylalanine, and tryptophan are aromatic amino acids. The term “nonstandard amino acids (nsAA)” as used herein refers to an amino acid that is not a standard amino acid. Examples of the nsAAs include para-nitro-L- phenylalanine (pN-Phe), o-methyltyrosine, para-azido-L-phenylalanine, pyrrolysine, 3- nitro-L-tyrosine, N(6)-(2-(2,4-dinitrophenyl)acetyl)lysine, sulfotyrosine, and Nε-(tert- butoxycarbonyl)-L-lysine. The term “nitrated amino acid” as used herein refers to an amino acid containing a nitro functional group (-NO2). Examples of nitrated amino acids include para-nitro-L-phenylalanine (pN-Phe), 3-nitrotyrosine, and N(6)-(2-(2,4- dinitrophenyl)acetyl)lysine. 2101715-001306 -7- The term “nitrated polypeptide” as used herein refers to a polypeptide comprising one or more nitrated amino acids. A control polypeptide of a nitrated polypeptide have the same amino acid sequence except that the control polypeptide does not comprise the one or more nitrated amino acids. The term “derivative” as used herein refers to a polypeptide comprising an amino acid sequence identical or homologous to a naturally occurring, also known as native, polypeptide. The term “homolog” as used herein refers to a protein having an amino acid sequence similar to another protein. The similarity or homology may be at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100%. The term “effective amount” as used herein refers to an amount effective for achieving an intended goal, for example, inducing an immune response in a subject to which the vaccine is administered. The effective amount may depend on the nature of the antigenic polypeptide, the physical condition of the subject, and the intended goal. The effective amount may be determined by a physician. The term “adjuvant” as used herein refers to a substance that enhances an immune response to an antigen in a subject. Exemplary adjuvants include aluminum hydroxide, TLR9 agonists such as CpG, and TLR4 agonists such as monophosphoryl lipid A. The term “carrier” as used herein refers to a substance that improves delivery, effectiveness, specificity or safety of an active ingredient, for example, a drug. The carrier may be a protein bound to the active ingredient. Exemplary carriers include virus-like particles, lipid nanoparticles, and metal organic framework-based nanoparticles. The term “excipient” as used herein refers to an inactive ingredient added intentionally to a pharmaceutical or diagnostic product, but not intended to exert a therapeutic effect. The excipient may be a media component. The excipient may improve the delivery of the product. Exemplary excipients include polysorbate 80, sucrose, and sodium chloride. The term “subject” as used herein refers to an animal, for example, a mammal. The subject may be selected from the group consisting of humans, monkeys, mice, rats, pigs, cows, chickens, cage birds, aviary birds, reptiles, and amphibians. For example, the subject may be a human. A target subject may be a test subject while a 2101715-001306 -8- control subject may be in the same genus and the same species as its corresponding target subject. The present invention provides an antigenic recombinant polypeptide. The antigenic recombinant polypeptide comprises an amino acid sequence homologous to the amino acid sequence of invasion plasmid antigen D (IpaD) or a fragment thereof, and an amino acid in IpaD or a fragment thereof is substituted with a nonstandard amino acid. The antigenic recombinant polypeptide comprises a nonstandard amino acid corresponding to the nonstandard amino acid in the IpaD or a fragment thereof. In other words, the nonstandard amino acid in the antigenic recombinant polypeptide and the nitrated amino acid in the IpaD or a fragment thereof have the same surrounding amino acids. The nonstandard amino acid may be para-nitro-L-phenylalanine (pN-Phe), o- methyltyrosine, para-azido-L-phenylalanine, pyrrolysine, 3-nitro-L-tyrosine, N(6)-(2- (2,4-dinitrophenyl)acetyl)lysine, sulfotyrosine, and Nε-(tert-butoxycarbonyl)-L-lysine. The nonstandard amino acid may be a nitrated amino acid. The nitrated amino acid may be para-nitro-L-phenylalanine (pN-Phe), 3-nitro-L-tyrosine, and N(6)-(2-(2,4- dinitrophenyl)acetyl)lysine. The substituted amino acid may be an aromatic amino acid, for example, tyrosine (Tyr or Y), phenylalanine (Phe or F) or tryptophan (Try or W). The antigenic recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99- 100% homologous to the IpaD or a fragment thereof. The IpaD may be a full-length native IpaD of Shigella flexneri (SEQ ID NO: 1), a truncated IpaD, or a modified IpaD. The truncated IpaD may have one or more amino acids removed from the N-terminal and / or C-terminal of the full-length native IpaD. For example, the truncated IpaD may consist of the amino acid sequence of SEQ ID NO: 2, which does not have the 14 N-terminal amino acids of the full-length native IpaD. The modified IpaD may comprise one or more amino acids in addition to the full-length native IpaD or the truncated IpaD. For example, the modified IpaD fragment may consist of the amino acid sequence of SEQ ID NO: 4. The IpaD fragment comprises an amino acid sequence overlapping with a part of the full-length IpaD, truncated IpaD or modified IpaD. The IpaD fragment may have about 12-15, 12-18, 12-50, 12-100, 12-300, 12-310, 12-320, or 12-330 amino acids. The IpaD fragment may comprise an amino acid sequence selected from the group 2101715-001306 -9- consisting of SEQ ID NO: 7-15. The IpaD fragment may consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 7-15. Where the IpaD consists of the amino acid sequence of SEQ ID NO: 1, the antigenic recombinant polypeptide may be a nitrated polypeptide comprising an amino acid sequence homologous to SEQ ID NO: 1 or a fragment thereof, an amino acid in SEQ ID NO: 1 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 1. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to SEQ ID NO: 1 or a fragment thereof, an amino acid in SEQ ID NO: 1 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 1. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 1 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 1 or a fragment thereof, an amino acid in SEQ ID NO: 1 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 1. The nitrated amino acid may be pN-Phe. The substituted amino acid may be Y104, W135, Y149, Y153, Y160, Y164, F167, W177, Y206, W226, Y276, W279, F283, Y301 or F308 in SEQ ID NO: 1 or a fragment thereof. Where the IpaD consists of the amino acid sequence of SEQ ID NO: 2, the antigenic recombinant polypeptide may be a nitrated polypeptide comprising an amino acid sequence homologous to SEQ ID NO: 2 or a fragment thereof, an amino acid in SEQ ID NO: 2 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 2. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to SEQ ID NO: 2 or a fragment thereof, an amino acid in SEQ ID NO: 2 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 2. The antigenic nitrated recombinant polypeptide may comprise an 2101715-001306 -10- amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 2 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 2 or a fragment thereof, an amino acid in SEQ ID NO: 2 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 2. The nitrated amino acid may be pN-Phe. The substituted amino acid may be Y90, W121, Y135, Y139, Y146, Y150, F153, W163, Y192, W212, Y262, W265, F269, Y287 or F294 in SEQ ID NO: 2. Where the IpaD consists of the amino acid sequence of SEQ ID NO: 4, the antigenic recombinant polypeptide may be a nitrated polypeptide comprising an amino acid sequence homologous to SEQ ID NO: 4 or a fragment thereof, an amino acid in SEQ ID NO: 4 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 4. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to SEQ ID NO: 4 or a fragment thereof, an amino acid in SEQ ID NO: 4 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 4. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 4 or a fragment thereof. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 4 or a fragment thereof, an amino acid in SEQ ID NO: 4 or a fragment thereof may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 4. The nitrated amino acid may be pN-Phe. The substituted amino acid may be Y91, W122, Y136, Y140, Y147, Y151, F154, W164, Y193, W213, Y263, 2101715-001306 -11- W266, F270, Y288 or F295 in SEQ ID NO: 4. For example, the antigenic nitrated recombinant polypeptide may consist of the amino acid sequence of SEQ ID NO: 6. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of an IpaD fragment, an amino acid in the IpaD fragment may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in the IpaD fragment. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of an IpaD fragment, an amino acid in the IpaD fragment may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in the IpaD fragment. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40- 100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70- 100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to the amino acid sequence of an IpaD fragment. The antigenic nitrated recombinant polypeptide may consist of the amino acid sequence of an IpaD fragment, an amino acid in the IpaD fragment may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in the IpaD fragment. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of DILSRHEYPINKDAR (SEQ ID NO: 7), Y in SEQ ID NO: 7 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 7. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of DILSRHEYPINKDAR (SEQ ID NO: 7), Y in SEQ ID NO: 7 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 7. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40- 100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60- 80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70- 2101715-001306 -12- 100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 7. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 7, wherein Y may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of MISHRELWAKIANSI (SEQ ID NO: 8), W in SEQ ID NO: 8 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 8. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of MISHRELWAKIANSI (SEQ ID NO: 8), W in SEQ ID NO: 8 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 8. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 8. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 8, wherein W may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of DINEQYLKVYEHAVS (SEQ ID NO: 9), either or both Ys in SEQ ID NO: 9 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 9. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of DINEQYLKVYEHAVS (SEQ ID NO: 9), either or both Ys in SEQ ID NO: 9 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 9. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40- 90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50- 99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70- 90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90- 2101715-001306 -13- 99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 9. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 9, wherein either or both Y may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of HAVSSYTQMYQDFSA (SEQ ID NO: 10), one or more of the two Ys and F in SEQ ID NO: 10 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 10. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of HAVSSYTQMYQDFSA (SEQ ID NO: 10), one or more of the two Ys and F in SEQ ID NO: 10 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 10. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40- 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60- 95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80- 95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99- 100% homologous to SEQ ID NO: 10. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 10, wherein one or more of the two Ys and F may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of SSLAGWISPGGNDGNSV (SEQ ID NO: 11), W in SEQ ID NO: 11 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 11. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 11. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 11, wherein W may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. 2101715-001306 -14- The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of EELKEKYKDKPLYPA (SEQ ID NO: 12), Y in SEQ ID NO: 12 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 12. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of EELKEKYKDKPLYPA (SEQ ID NO: 12), Y in SEQ ID NO: 12 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 12. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 12. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 12, wherein Y may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of QANKWLTELGGTIGK (SEQ ID NO: 13), W in SEQ ID NO: 13 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 13. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of QANKWLTELGGTIGK (SEQ ID NO: 13), W in SEQ ID NO: 13 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 13. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40- 99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60- 70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70- 99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 13. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 13, wherein W may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. 2101715-001306 -15- The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of NAKYQAWNAGFSAED (SEQ ID NO: 14), one or more of Y, W and F in SEQ ID NO: 14 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 14. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of NAKYQAWNAGFSAED (SEQ ID NO: 14), one or more of Y, W and F in SEQ ID NO: 14 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 14. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 14. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 14, wherein one or more of Y, W and F may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of LVQKYSNANSIFDNL (SEQ ID NO: 15), either or both Y and F in SEQ ID NO: 15 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 15. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of LVQKYSNANSIFDNL (SEQ ID NO: 15), either or both Y and F in SEQ ID NO: 15 may be substituted with a nitrated amino acid, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the nitrated amino acid in SEQ ID NO: 15. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99-100% homologous to SEQ ID NO: 15. The antigenic nitrated recombinant polypeptide may consist of SEQ 2101715-001306 -16- ID NO: 15, wherein either or both Y and F may be substituted with a nitrated amino acid. The nitrated amino acid may be pN-Phe. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of QLLDILSRHEpNPINK (SEQ ID NO: 48), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 48. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of QLLDILSRHEpNPINK (SEQ ID NO: 48), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 16. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40- 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60- 95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80- 95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99- 100% homologous to SEQ ID NO: 48. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 48. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of LSRHEpNPINKDAREL (SEQ ID NO: 49), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 49. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of LSRHEpNPINKDAREL (SEQ ID NO: 49), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 49. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40- 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60- 95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80- 95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99- 100% homologous to SEQ ID NO: 49. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 49. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence homologous to the amino acid sequence of pNPINKDARELLHSAP (SEQ ID NO: 50), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may 2101715-001306 -17- comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 18. The antigenic nitrated recombinant polypeptide may consist of an amino acid sequence homologous to the amino acid sequence of pNPINKDARELLHSAP (SEQ ID NO: 50), wherein pN is pN-Phe, and the antigenic nitrated recombinant polypeptide may comprise a nitrated amino acid corresponding to the pN-Phe in SEQ ID NO: 50. The antigenic nitrated recombinant polypeptide may comprise an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, or about 40- 50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-99%, 40-100%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-95%, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60- 95%, 60-99%, 60-100%, 70-80%, 70-90%, 70-95%, 70-99%, 70-100%, 80-90%, 80- 95%, 80-99%, 80-100%, 90-95%, 90-99%, 90-100%, 95-99%, 95-100%, or 99- 100% homologous to SEQ ID NO: 50. The antigenic nitrated recombinant polypeptide may consist of SEQ ID NO: 50. The present invention also provides a vaccine composition. The vaccine composition comprises an effective amount of one or more of the antigenic nitrated recombinant polypeptides of the present invention. The vaccine composition may further comprise a pharmaceutically acceptable adjuvant, carrier and / or excipient. In one embodiment, the vaccine composition does not comprise a pharmaceutically acceptable adjuvant. The present invention further provides a method for inducing an immune response in a target subject. The immunization method may comprise administering to the target subject an effective amount of a vaccine composition. As a result, a target immune response may be induced in the target subject. The target subject may be selected from the group consisting of humans, monkeys, mice, rats, pigs, cows, chickens, cage birds, aviary birds, reptiles and amphibians. In one embodiment, the target subject is a human. The target subject may be in need of the induced immune response. According to the immunization method, the vaccine composition comprises an effective amount of one or more of the antigenic nitrated recombinant polypeptides of the present invention. The vaccine composition may further comprise a pharmaceutically acceptable adjuvant, carrier and / or excipient. In one embodiment, the vaccine composition does not comprise a pharmaceutically acceptable adjuvant. The immunological response may comprise production of one or more antibodies against the one or more antigenic nitrated recombinant polypeptides. The immunological response may comprise a B-cell mediated response, a T-cell mediated response, or a combination thereof. 2101715-001306 -18- The target immune response may be greater than a control immune response induced by a control polypeptide in a control subject by, for example, at least about 10%, 50%, 100%, 200% or 500%. While the antigenic nitrated recombinant peptide comprises a nitrated amino acid, the control polypeptide has the same amino acid sequence as the antigenic nitrated recombinant peptide except that the control polypeptide does not comprise the nitrated amino acid. The control subject and the target subject are in the same genus and the same species. The target induced immune response may comprise production of one or more target antibodies against the one or more antigenic recombinant polypeptides. The one or more target antibodies may cross-react against a control polypeptide. The one or more target antibodies may comprise IgG, IgA, IgG1, IgG2b, IgG2c, or a combination thereof. The one or more target antibodies may bind a first region in the antigenic recombinant polypeptide and a second region in the control polypeptide. The first region and the second region may correspond to different regions of the IpaD or a fragment thereof. The first region and the second region may correspond to the same region of the IpaD or a fragment thereof. For each vaccine composition of the present invention, a method for producing the vaccine composition is provided. The production method comprises admixing one or more antigenic recombinant polypeptides of the present invention with the pharmaceutically acceptable adjuvant, carrier or excipient. The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate. Example 1. Antigenic nitrated recombinant IpaD polypeptides The conserved antigen from the bacterial genus Shigella known as IpaD was chosen as a model antigen for this approach due to the current lack of a clinically approved Shigella vaccine on the market today and the lack of broad protection demonstrated in previous vaccine formulations. Here, a truncated IpaD of Shigella flexneri (SEQ ID NO: 2) was prepared by truncating the 14 N-terminal amino acids to remove the invasion activity of the full length native IpaD (SEQ ID NO: 1). Amber codon suppression was used to mutate the codon for a native tyrosine residue at position 90 in the amino acid sequence of the truncated IpaD (SEQ ID NO: 2) to a TAG codon in the DNA sequence. Using this construct, pN-Phe was incorporated at the site of mutation using an engineered M. jannaschii aminoacyl-tRNA synthetase. We then expressed wild-type and nitrated antigens in a recoded strain of E. coli that has been optimized for nsAA incorporation via amber codon suppression. All 2101715-001306 -19- antigens were then purified using two rounds of Ni-NTA chromatography followed by anion exchange chromatography. After, proteins were further processed using poly- lysine resin to remove E. coli endotoxin, and endotoxin was quantified via mammalian toll-like receptor 4 (TLR4) activation assay. As a result, the wild-type IpaD antigen (WT) was the modified IpaD (SEQ ID NO: 4), in which residue 91 was tyrosine (Y) while the nitrated IpaD antigen (Y90) was the modified IpaD (SEQ ID NO: 4), in which residue 91 was pN-Phe. In vivo immunization studies were carried out to assess the immunogenicity of the nitrated IpaD antigen (SEQ ID NO: 6) relative to the wild-type IpaD antigen (SEQ ID NO: 4) and the PBS control (PBS). Studies lasted 28 days, and groups of n = 5 C57BL / 6 mice were immunized via orotracheal instillation on days 1 and 14 with 10 mg of antigen. On day 28, mice were euthanized and serum and bronchoalveolar lavage fluid (BALF) were collected for analysis. Indirect ELISA was used to characterize antibody response to antigens, and ELISA plates were coated with wild-type antigen at a concentration of 1 mg / mL. In addition, indirect ELISA was used to measure serum IgG and BALF IgA, which are representative of systemic and mucosal immune responses, respectively, as well as to analyze serum IgG subclass antibodies: IgG1, IgG2b, IgG2c, and IgG3. Additional B cell epitope profiling was performed. Materials & Methods: Strain Construction. E. coli strains and plasmids used in this study are listed in Table 1. Molecular cloning and vector propagation were performed in E. coli DH5α. PCR-based DNA replication was performed using KOD XTREME Hot Start Polymerase. Cloning was performed using Gibson Assembly with constructs and oligos for PCR amplification shown in Table 2. The sequence for truncated S. flexneri IpaD (Uniprot P18013) (SEQ ID NO: 2) was designed with the 14 amino acids at the N terminus omitted from the full length native IpaD (SEQ ID NO: 1) and modified to include an N- terminal hexahistidine purification tag followed by the TEV protease consensus sequence after the start codon. The modified gene encoding wild-type IpaD was optimized for E. coli K12 using the IDT Codon Optimization Tool and purchased as a gene fragment from Twist Bioscience. The sequence for H1N1 All plasmid sequences were confirmed via Sanger sequencing. Endotoxin testing for antigens expressed in E. coli species was performed using the HEK-Blue™-4 strain of engineered HEK293 cells from the HEK-Blue™ LPS Detection Kit 2 (Invivogen). Antigen Expression and Purification. Nitrated IpaD (SEQ ID NO: 5) was expressed using an engineered strain of E. coli C321.ΔA optimized for improved growth and stabilization of aldehyde molecules (RecRARE) harboring both a pEVOL plasmid containing orthogonal translation machinery and a pZE plasmid encoding expression of 2101715-001306 -20- IpaD with the tyrosine residue at full-length position 104 mutated to a UAG stop codon (SEQ ID NO: 5). 5 mL of LB broth containing kanamycin (50 µg mL-1) and chloramphenicol (34 µg mL-1) was inoculated from a frozen stock and grown to confluence overnight. Confluent overnight cultures were used to inoculate 500-1000 mL of Terrific broth (TB) supplemented with kanamycin, chloramphenicol, and 2 mM para- nitro-L-phenylalanine at a 100x dilution. Orthogonal translation machinery was induced at inoculation via addition of 0.2% (v / v) L-arabinose. Expression cultures were grown at 34^°C in a shaking incubator at 250 RPM until the culture reached mid-exponential phase (OD600 of 0.4-0.6). IpaD expression was induced by addition of 0.2 nM anhydrotetracycline. After induction, cultures were grown for an additional 12-16 h at 34°C, 250 RPM. Wild-type IpaD antigen (SEQ ID NO: 4) was expressed in three different strains of E. coli. For the first study, a strain of E. coli BL21 (DE3) harboring a pZE plasmid encoding expression of wild-type IpaD with an N-terminal hexahistidine tag followed by TEV protease consensus cleavage sequence was used. Expression was conducted at 37^°C in TB media with kanamycin. For the additional studies, expression was performed in a strain of E. coli C321.ΔA in an attempt to equalize levels of endotoxin in wild-type and nitrated antigens. Expression cultures were grown at the same scale as nitrated antigen in a shaking incubator at their strain-dependent temperature and 250 RPM until OD600 of 0.4-0.6 was reached. IpaD expression was induced by addition of 0.2 nM anhydrotetracycline. After induction, cultures were grown for an additional 12- 16 h at their strain-dependent temperature, 250 RPM. Cultures were centrifuged at 4°C at 7,197g for 5 min to pellet cells in 50 mL conical tubes such that each tube contained roughly 250 mL of culture. Pellets were either purified immediately or stored at -80°C until time of purification. For antigen preparation for initial animal studies, cell pellets were resuspended in 8 mL lysis buffer containing 25^mM HEPES, 10^mM imidazole, 300^mM NaCl, 10% glycerol, and titrated to pH 7.4 and lysed via sonication using a QSonica Q125 sonicator with cycles of 5s at 90% amplitude and 10s off for 5^min for 3 to 4 total cycles. For subsequent studies, cell pellets were resuspended in a detergent-based, enzymatic cell lysis buffer optimized for intracellular recombinant protein extraction consisting of 0.5% Triton X-100, 0.05% Tween-20, 50 mM Tris-HCl, 25 mM NaCl, 2 mM EDTA, 1 mg / mL lysozyme and titrated to pH 8.0. Detergent-treated cell pellets were incubated at 37°C for 30 min, then subjected to sonication as mentioned previously. Lysate was then transferred to Eppendorf tubes, and centrifuged at 4°C at 18,213g for 1 h to pellet cell debris, and soluble antigen-containing fractions were collected, sterile filtered, and loaded into a HisTrap Ni-NTA column using an ÄKTA Pure 2101715-001306 -21- GE fast protein liquid chromatography (FPLC) system. Protein was washed with three column volumes (CVs) at 60^mM imidazole and four CVs at 90^mM imidazole. IpaD antigen was eluted in 250^mM imidazole in 1.5^ml fractions. Elution fractions were pooled and dialyzed into roughly 25 mL pre-TEV buffer (50 mM Tris-HCl, pH 8.0) using Amicon Ultra-1510K MWCO filter tubes (MilliporeSigma) until total volume was under 5mL. A small portion of dialyzed protein was denatured in Lamelli SDS reducing sample buffer (62.5^mM Tris–HCl, 1.5% SDS, 8.3% glycerol, 1.5% β-mercaptoethanol and 0.005% bromophenol blue) for 10^min at 95^°C and subsequently run on an SDS–PAGE gel with a Thermo Fisher Scientific Spectra Broad Range Protein Ladder to confirm purified protein size. Gels were imaged using an Azure c280 imaging system. After SDS-PAGE verification of correct protein mass (~36 kDa), antigen concentration was determined via Bradford assay using 0.125-2.0 mg / mL BSA as a standard. Recombinant antigen was next treated with tobacco etch virus (TEV) protease to remove the N-terminal hexahistidine affinity purification tag from the antigen. Reactions were carried out at 1mL total volume with 0.5-1.0 mg of total protein per tube along with polyhistidine-tagged TEV protease (GenScript) and TEV reaction buffer (50 mM Tris-HCl, 1 mM DTT, 0.5 mM EDTA). Reaction tubes were then mixed and incubated overnight at 4°C for 16-18h, and incubated at 30°C for 2h the following day. Reactions were then pooled and once again filtered and loaded on a HisTrap Ni-NTA column and subjected to affinity-based chromatography via ÄKTA Pure FPLC system. Following this step, wild-type protein (SEQ ID NO: 4) and nitrated IpaD (SEQ ID NO: 6) with sequence corresponding to that used for downstream immunization studies was obtained. Column flow through elution fractions were collected at 0mM imidazole to collect only the antigen with no affinity tag. Fractions were then dialyzed into at least 25mL salt-free start buffer (20 mM Tris-HCl, pH 8) for anion exchange chromatography via Amicon Ultra-1510K MWCO filter tubes. Final antigen sample volume was then adjusted to 10mL using the previously mentioned start buffer, and samples were loaded onto a HiTrap Q anion exchange chromatography (AEX) column using the ÄKTA Pure FPLC system. Samples were eluted over a linear gradient of increasing salt concentration using an elution buffer (20 mM Tris-HCl, 1 M NaCl, pH 8). After AEX, elution fractions were pooled and dialyzed again into 25 mL sterile 1X PBS using previously described methods and stored at either 4°C or -80°C until endotoxin removal. MS Analysis and Circular Dichroism Spectroscopy. Purified antigens were diluted in ultrapure water to a concentration of 0.1 mg / mL and volume of 100 µL. Samples were then submitted for intact protein MS analysis to confirm correct molecular weight and mass shift between wild-type and nitrated antigens. MS spectra was deconvoluted 2101715-001306 -22- using MassLynx software and the evaluated mass range was 30,000 Da to 40,000 Da for identification of wild-type (SEQ ID NO: 4) and nitrated IpaD (SEQ ID NO: 6). After MS analysis, samples were submitted for circular dichroism spectroscopy. Circular dichroism spectroscopy data was acquired via JASCO 1500 Spectrometer at 20 nm / min with a resolution of 0.5 nm and averaged 3 times. To verify the structure and folding of our nitrated antigen with that of wild-type in literature, temperature dependence of the molar ellipticity at 222 nm of nitrated antigen was monitored from 10°C to 90°C at 2.5°C intervals at a rate of 15°C / h as done in previous literature. Endotoxin removal. For the preliminary animal study, no endotoxin removal was performed. For all subsequent studies, endotoxins were removed from recombinant antigens after AEX purification via Pierce High Capacity Endotoxin Removal Spin Columns. To remove endotoxin, 1 mL spin columns were first equilibrated overnight at room temperature with 0.2 M NaOH per manufacturer’s protocol. The following day, the columns were rinsed with the following and centrifuged at 500g for 1 min: once with 2 M NaCl, once with endotoxin-free water, then three times with sterile 1X PBS. After equilibration, samples were diluted to 5 mL and applied to the spin columns. Spin columns were first incubated at 4°C with end-over-end mixing for 2 h. Incubation time was later increased up to 16 h in order to maximize endotoxin removal, as shorter column incubation times were found to be insufficient for removal of tightly-bound endotoxin. After incubation, spin columns were placed in collection tubes, and centrifuged as previously mentioned. Antigens were then concentrated to a total volume less than 1 mL, and final concentrations were measured via Bradford assay. Aliquots were taken of each protein for subsequent endotoxin quantification, and antigens were flash frozen and stored at -80°C until later use. Endotoxin quantification via LAL and TLR4 assays. Endotoxin levels in recombinant antigen were quantified via two different colorimetric assays. Endotoxin levels were first quantified after our preliminary animal study via ToxinSensor Chromogenic LAL Assay kit (GenScript) which utilizes Limulus amebocyte lysate to detect and quantify endotoxin in samples. Due to the inability of the LAL assay to discern between biologically active and inactive endotoxin, we moved quantification to a toll-like receptor 4-based assay (TLR4). For subsequent studies, endotoxin was quantified prior to immunization via HEK-Blue LPS Detection Kit (Invivogen). HEK-Blue hTLR4 cells (InvivoGen) were cultured in Dulbecco’s modified eagle medium, high glucose / L-glutamine supplemented with 10% fetal bovine serum, penicillin (50 U / mL), streptomycin (50 µg / mL), and Normocin (100 µg / mL) at 37°C and 5% CO2 in a humidified incubator. Cells were subcultured at least twice prior to being used for endotoxin measurement. 2101715-001306 -23- After reaching roughly 80% confluency, cells were harvested and diluted to a concentration of 2 x 105cells / mL in medium and plated on 96-well Costar tissue culture plates (Corning). E. coli 055:B5 LPS (Invivogen) was used as a positive control and a two-fold serial dilution was prepared to generate a standard curve. Antigen samples were initially diluted to the same protein concentration, then diluted again via three- fold serial dilution and added to cell suspensions. Blank wells, in which endotoxin-free water was added to cells, were used as a negative control. Cells were incubated at 37°C and 5% CO2 for 18-24 h. Supernatant from the cell suspension was added to pre- warmed QUANTI-Blue (Invivogen) in a new 96-well plate and incubated for an additional 2 h at 37°C and 5% CO2. After, absorbance was measured via plate reader at 655 nm, and the twofold serial dilution of endotoxin standards was used to generate a standard curve that was linear for concentrations ranging from 0.004-0.125 EU / mL. Endotoxin concentrations in antigen dilutions were then calculated. Mouse immunizations. For all immunization studies, female 6-week old C57BL / 6 mice (Jackson Laboratories) were immunized via orotracheal instillation with a dosing volume of 50 μL. Doses were administered via pipette after mice were anesthetized with isoflurane. Full-length immunization studies were 28 days with prime and boost immunizations on days 0 and 14. For each immunization, experimental groups (n = 5 mice per group) received 10 μg of wild-type (SEQ ID NO: 4) or nitrated IpaD (SEQ ID NO: 6), and control groups were immunized with sterile PBS. For all studies, prior to immunization, wild-type IpaD (SEQ ID NO: 4) was spiked with E. coli 055:B5 lipopolysaccharide (Invivogen) to equalize the endotoxin concentration of wild-type (SEQ ID NO: 4) and nitrated IpaD (SEQ ID NO: 6) antigens using the TLR4 assay results. Antigens were administered without an adjuvant. For the short-term immunization study designed to assess inflammatory response and antigen presentation, mice were euthanized after 18 h and experimental groups immunized with either spiked wild-type IpaD (SEQ ID NO: 4), unspiked wild-type IpaD (SEQ ID NO: 4), nitrated IpaD (SEQ ID NO: 6), or PBS. Animals were routinely monitored by University of Delaware LSRF veterinary technicians. All immunizations were performed under isoflurane anesthesia. Mice were euthanized by CO2asphyxiation in accordance with the University of Delaware Institutional Animal Care and Use Committee. After euthanasia, bronchoalveolar lavage fluid (BALF) and serum were collected from mice for antibody quantification in all studies. For the short-term study, whole lung was harvested for phagocyte cell count, antigen-presenting cell surface marker analysis and cytokine quantification. Antibody quantification. Antibodies in serum and BALF were quantified via indirect enzyme-linked immunosorbent assay (ELISA). For all assays, Immulon 2HB 2101715-001306 -24- ELISA plates (Thermo Scientific) were coated overnight at 4°C with wild-type IpaD diluted in 50 mM sodium carbonate / bicarbonate buffer (pH 9.6) to a concentration of 1 µg / mL. Plates were then washed three times with PBS-Tween (PBST), and blocked for 2 h at room temperature using 5% dry milk solution. Serum and BALF samples containing IgG and IgA antibodies were diluted (IgG and isotypes in serum and BALF: 1:1000; IgA in BALF: 1:4) in 10% FACS buffer (10% fetal bovine serum in PBS) and added to plates. Plates were sealed and incubated at 37°C for 2 h. Antibodies in serum and BALF samples were detected using the following horseradish peroxidase (HRP)- conjugated secondary antibodies (SouthernBiotech): goat anti-mouse IgG (H+L), IgG1, IgG2b, IgG2c, IgG3, and IgA. Secondary antibodies were diluted 1:4000 in 10% FACS buffer, and added to ELISA plates. Plates were sealed and incubated again at 37°C for 2 h. Afterwards, plates were washed five times with PBST, and 3,3′,5,5′- tetramethylbenzidine (TMB) substrate was added to each plate and allowed to develop in the dark at room temperature for 15 min. Reactions were quenched with 2 N H2SO4 and absorbance was measured at 450 nm and 570 nm via plate reader. Background absorbance (OD570) was subtracted from OD450 and the resulting absorbance was plotted for each immunization group. B cell epitope peptide ELISA. To assess serum IgG recognition and binding to IpaD B cell epitopes, a peptide library was designed and purchased (GenScript). 15mer peptides were designed to span the entire sequence of wild-type IpaD with an offset of 5 amino acids. The library consisted of 65 total peptides, with 3 containing pN-Phe. Peptides were diluted in 50 mM sodium carbonate / bicarbonate coating buffer (pH 9.6) to a concentration of 0.1 mg / mL and added to Nunc Immobilizer Amino ELISA plates (ThermoFisher) overnight at 4oC with each well containing a single peptide. The following day, peptide ELISAs were performed as previously mentioned using sera from immunized mice. Absorbance was measured at both 450 and 570 nm. Whole lung digestion and flow cytometry staining. After BALF collection for 18 h short-term study, whole lungs were removed from antigen and PBS-immunized mice. Lungs were mechanically disrupted via manual homogenization and then digested in 1.5 mL of 5 mg / mL Collagenase IV (Gibco). After 30 min, an additional 500 µL of Collagenase IV was added and pipette mixed, and after 2 h of enzymatic digestion, a single cell suspension was generated. The cells were then filtered through a 70 um cell strainer and resuspended in 1 mL of RBC lysis buffer (Fisher Scientific) to remove residual red blood cells. Samples were then pipetted for 30 s, centrifuged for 5 min at 500 RPM, and washed twice in 1X PBS. Samples were next stained for flow cytometric analysis, beginning with Zombie Yellow viability stain (Biolegend) for 15 min at room temperature. Samples were then washed with 1X PBS, followed by an additional wash 2101715-001306 -25- with 2% FACS buffer (2% fetal bovine serum in 1X PBS). After viability staining, total lung digest volumes for each sample were split in half for further antibody staining, with one half used to assess APC antigen presentation markers and the other to assess APC inflammatory response. Each staining group received a different combined antibody cocktail solution diluted in 2% FACS buffer, and samples were incubated on ice for 30 min after antibody addition. After, samples were washed in 2% FACS buffer followed by an additional wash in 1X PBS. Samples were fixed for 15 min using 4% paraformaldehyde in 1X PBS followed by additional washes in 1X PBS and FACS buffer. Samples were then resuspended in 200 µL of 2% FACS buffer and ran on Novocyte Flow Cytometer (Agilent Technologies). Cytokine analysis. BALF samples collected from 18 h short-term study were submitted to the Cytokine Core Laboratory at University of Maryland for subsequent analysis. BALF samples (200 µL) were analyzed via multiplex cytokine quantification of five cytokines using the Luminex™ 100 Multi-analyte System (ThermoFisher). Bio-Plex Manager Software (Bio-Rad) was used to collect and analyze data. Additional cytokine analysis of BALF samples from the 18 h study was performed in-house. IL-4 and IL-23 were quantified via sandwich ELISA using ELISA MAXTMDeluxe Set Mouse IL-4 and IL- 23 kits (Biolegend). In vitro and ex vivo APC analysis. In vitro and ex vivo APC responses to nitrated and wild-type IpaD were assessed at 24 and 72 h timepoints using the following cell lines: RAW264.7 murine macrophage cells, JAWSII murine dendritic cells, bone marrow-derived macrophages (BMMs), and bone marrow-derived dendritic cells (BMDCs). For studies using immortalized cells, cells were seeded in 96-well flat-bottom plates (Corning) at 5x104cells / well 18-24 h prior to antigen dosing. Wild-type and nitrated IpaD antigens were dosed at 15 μg / mL. E. coli LPS and complete cell culture medium were used as positive and negative controls, respectively. Antigen dose was determined using in vitro pilot studies. To capture the effects of pN-Phe on antigen presentation, macrophage polarization, and cytokine response, in vitro studies ranged from 24-72 h. After the desired timepoint was reached, culture medium supernatant was removed and stored at -20oC for later cytokine analysis and cells were then washed twice with 1X PBS. Cells were then detached from the plate using 0.05% Trypsin-EDTA and incubated for 5 min, then quenched using an equal volume of complete medium. Cells were then washed twice with 1X PBS, and primary cell lines were then stained with Zombie Yellow cell viability stain (Biolegend) at a 1:500 dilution for 15 min at room temperature. Immortalized cell lines were not stained with Zombie Yellow. All cells were then washed with 1X PBS and 2% FACS buffer, stained with an antibody cocktail, and analyzed via flow cytometry to assess the following: antigen 2101715-001306 -26- presentation (CD83 and MHC-II), costimulatory molecule expression (CD80 and CD86), M1 / M2 macrophage polarization (MHC-IIhiand CD206+populations), and dendritic cell phenotype (CD11b+and CD103+populations). Cytokines in cell culture supernatant were analyzed in-house using ELISA MAXTMDeluxe Set Mouse cytokine sandwich ELISA kits (Biolegend). Statistics. GraphPad Prism 10 (GraphPad Software Inc.) was used for all statistical analyses and quantitative figure design. Numerical data is shown as mean ± standard deviation unless otherwise noted. Cytokine, serum, and BALF ELISA data sets were first analyzed for normality. Statistical significance was determined by either parametric or nonparametric ANOVA or Student’s t-test depending on data set and distribution. Flow cytometry data consisting of whole lung suspension cell count and surface marker expression level was analyzed via two-way ANOVA. Significance thresholds and post hoc analyses are reported in corresponding figure captions. Results: Identification of a model antigen, residues for mutagenesis, and orthogonal translation system. To test whether foreign antigen nitration was capable of eliciting a heightened in vivo immune response, Shigella Invasion Plasmid Antigen D (IpaD) was selected as our model antigen (SEQ ID NO: 1). IpaD plays a key role in Shigella pathogenesis due to its role in the type III secretion system (T3SS) and conservation across all Shigella serotypes. To select sites for nitration in the IpaD antigen, initial steps were taken to choose a site that would minimally perturb the structure of the protein following introduction of pN-Phe. Initially, using available crystal structure data and the predictive protein structure software PredictProtein, surface-exposed residues in the IpaD sequence were identified. From this set of residues, the residue Y90 was selected to retain the aromatic character of the site and minimize perturbation. To enable site-selective incorporation of pN-Phe at the Y90 site, an engineered tyrosyl-tRNA synthetase / tRNATyrCUA pair, TetRS-C11 AARS / MjtRNATyrCUA, derived from the archaeal species M. jannaschii expressed on the pEVOL plasmid was co- transformed into strains of interest with a pZE plasmid with anhydrotetracycline inducible production of IpaD (Y90(pN-Phe)) (SEQ ID NO: 5). Initial testing with this construct was performed using the genetically engineered commercial E. coli strain ClearColi to alleviate downstream endotoxin removal and tested pN-Phe incorporation at concentrations ranging from 1-5 mM. Western blot analysis showed consistent expression at all tested concentrations. However, cell growth appeared to decrease with increasing pN-Phe concentration and subsequent MS analysis of ClearColi- expressed nitrated IpaD (SEQ ID NO: 6) showed a mixture of nitrated and wild-type mass peaks. To combat this, we switched our expression host to the recoded E. coli 2101715-001306 -27- strain C321.ΔA which was optimized for amber codon suppression. After expression, recombinant antigens were purified via a series of chromatography steps. First, antigens were purified via IMAC chromatography. Then, the N-terminal region was removed via TEV protease cleavage followed by IMAC chromatography to produce a variant of the antigen for both the wild-type (SEQ ID NO: 4) and nitrated (SEQ ID NO: 6) variant for use in downstream immunization studies. Purified antigens were then submitted for MS analysis to confirm antigen mass. Nitrated IpaD (SEQ ID NO: 6) spectra indicated a mass shift of 28 Da relative to wild-type, confirming mutation to pN-Phe. Further structural characterization was performed and CD spectroscopic analysis of our nitrated IpaD (SEQ ID NO: 6) variant indicated that pN-Phe incorporation at Y104does not alter protein secondary structure relative to wild-type IpaD (SEQ ID NO: 4) (Fig. 1). After confirming desired mass and structure, samples were subjected to anion exchange chromatography to remove residual host cell proteins followed by an endotoxin removal step. Endotoxin was removed from both antigens via 16-18 h incubation on poly- lysine resin. TLR4 activation assays were performed using HEK-Blue cells to quantify the remaining endotoxin in antigen samples. Following these steps, the nitrated IpaD (SEQ ID NO: 6) consistently had higher concentrations of residual LPS than wild-type (SEQ ID NO: 4). Using the endotoxin concentrations measured from the TLR4 activation assays, LPS was added to wild-type IpaD (SEQ ID NO: 4) to equalize endotoxin concentrations between samples prior to in vivo immunization. This was done to reduce the possibility of endotoxin contamination increasing the immune response elicited by nitrated IpaD (SEQ ID NO: 6) and allow for proper comparison between nitrated and wild-type antigen. For all studies, antigen was administered such that endotoxin concentrations were below the recommended preclinical threshold for subunit vaccines of 20 EU / mL. Nitrated and LPS-spiked wild-type antigens were stored after each immunization study for later analysis to confirm that LPS spiking equalized concentrations. Quantification of in vivo systemic and mucosal antibody response. To characterize the change in vivo immune response elicited by nitrated IpaD (SEQ ID NO: 6) relative to wild-type, multiple animal studies were conducted in which groups of five 6-week old female C57BL / 6 mice were immunized via orotracheal instillation. Studies lasted 28 days, and consisted of prime and boost immunizations on days 0 and 14, respectively, with either 10 μg of wild-type (SEQ ID NO: 4) or nitrated IpaD (SEQ ID NO: 6) or PBS. Our results indicate that mice immunized with nitrated IpaD (SEQ ID NO: 6) exhibited significantly elevated serum IgG and that antibodies raised against nitrated IpaD (SEQ ID NO: 6) were cross-reactive with wild-type (SEQ ID NO: 4) (FIG. 2101715-001306 -28- 2). In addition, IgG1, IgG2b, and IgG2c were all elevated in nitrated IpaD (SEQ ID NO: 6)-immunized mice and statistically significant relative to the wild-type. IgG3 response was insignificant in all groups. IgG isotype analysis can be used to approximate the immune response bias towards a Th1 or Th2 phenotype. IgG2c and IgG3 are commonly associated with Th1 phenotype while IgG1 and IgG2b are associated with Th2 bias. Mice immunized with nitrated IpaD (SEQ ID NO: 6) had significantly increased Th1- associated IgG2c and Th2-associated IgG1 and IgG2b while wild-type IpaD resulted in negligible IgG isotype response. Overall, pN-Phe incorporation played a key role in boosting the systemic immune response and IgG isotype analysis suggests a balanced Th1 / Th2 response. Previous research has shown the importance of mucosal immunity for defense against and prevention of Shigella infection. To assess mucosal immune responses, bronchoalveolar lavage fluid (BALF) was collected from immunized mice on day 28 and used to quantify mucosal IgG and IgA antibody responses. We found that nitrated IpaD elicited statistically significant BALF IgG and IgA titers relative to wild-type. Mice immunized with wild-type IpaD (SEQ ID NO: 4) had negligible BALF IgG and IgA responses. The substantial increase in BALF IgG and IgA titers in mice immunized with nitrated IpaD (SEQ ID NO: 6) suggests that nitration via pN-Phe incorporation promotes: (1) a heightened local immune response at the immunization site, and (2) immunoglobulin class-switching to the mucosal antibody IgA. B Cell Epitope study. A peptide library with 15mers designed to span the entire sequence of wild-type IpaD (SEQ ID NO: 4) with an offset of 5 amino acids was synthesized (SEQ ID NO: 16-47). In addition, nitrated peptide variants were created to assess binding to the nitrated regions as well (SEQ ID NO: 48-50). Binding to the individual peptides was measured using an ELISA with serum derived from animals immunized with PBS and 10 µg CpG, wild-type IpaD (SEQ ID NO: 4) with CpG, and nitrated IpaD at site Y91 (SEQ ID NO: 6) with CpG (FIG. 3). Based on these results, we see a uniform increase in binding antibodies from serum of animals immunized with the Y91 variant. In addition, a significant increase was observed in regions containing and bordering the region of nitration bordering the regions of nitration using the wild-type peptides. This is likely due to improved responses toward the region of nitration which is seen from the results for the nitrated peptide binding. In vivo cytokine quantification in bronchoalveolar lavage fluid. To further characterize differences in the immune response elicited in wild-type (SEQ ID NO: 4) and nitrated IpaD (SEQ ID NO: 6), cytokine analysis was performed. To assess inflammatory response and Th1 / Th2 immune response bias to pulmonary delivery of nitrated IpaD (SEQ ID NO: 6), five cytokines and chemokines were quantified in BALF 2101715-001306 -29- samples from the 18 h in vivo study at University of Maryland’s Cytokine Core Laboratory. The proteins quantified via multiplex were IFN-g, TNF-a, IL-1b, KC (CXCL1), and B cell-activating factor (BAFF). IFN-g, TNF-a, and IL-1b are key Th1- associated cytokines responsible for initiating pro-inflammatory responses upon immunization. We further probed the extent of neutrophil activity at the site of immunization via cytokine analysis. The murine IL-8 homolog KC is a key chemokine responsible for neutrophil localization at the site of immunization and was analyzed to explain the increased neutrophil presence in response to nitrated IpaD (SEQ ID NO: 6). BAFF is secreted by neutrophils and promotes B cell differentiation to antibody- secreting plasma cells, and was quantified to further characterize the increased humoral response seen in mice immunized with nitrated IpaD (SEQ ID NO: 6). Of the five cytokines measured, only TNF-ɑ and BAFF were detectable in BALF samples. IFN-γ, IL-1^, and KC were undetectable in all samples. TNF-ɑ was only detectable in mice immunized with wild-type IpaD (SEQ ID NO: 4) spiked with endotoxin to equalize concentration and mice immunized with nitrated IpaD (SEQ ID NO: 6), and mice immunized with nitrated IpaD (SEQ ID NO: 6) had the only quantifiable TNF-ɑ response (19.59 ± 7.996 pg / mL). This finding suggests that nitrated IpaD (SEQ ID NO: 6) may elicit a pro-inflammatory response relative to wild-type IpaD (SEQ ID NO: 4) in the absence of adjuvant when endotoxin concentration is controlled for. Interestingly, BAFF was the only cytokine accurately quantified in all four immunization groups. BAFF levels in PBS-, spiked wild-type IpaD (SEQ ID NO: 4)-, unspiked wild-type IpaD (SEQ ID NO: 4)-, and nitrated IpaD (SEQ ID NO: 6)- immunized mice were 1367 ± 216.4 pg / mL, 1634 ± 521.4 pg / mL, 1663 ± 279.3 pg / mL, and 2573 ± 463.5 pg / mL, respectively. Neutrophil-driven BAFF secretion at the site of immunization has been linked to increased B cell response, as BAFF is directly involved in the activation of B cells and amplification of antibody responses. The increased neutrophil presence seen in mice immunized with nitrated IpaD (SEQ ID NO: 6) plays a role in heightened BAFF response, and this phenomenon may play a key role in increased humoral immune response. Since all antigens were administered without adjuvants, we believe that pN- Phe and its effect on MHC-II antigen presentation may induce a local pro-inflammatory response at the site of immunization. 18 h in vivo analysis of immunophenotype via phagocyte surface marker expression. C57 / BL6 mice were immunized orotracheally with either 10 μg of wild-type IpaD (SEQ ID NO: 4), wild-type IpaD (SEQ ID NO: 4) spiked with E. coli endotoxin to equalize concentration between wild-type and nitrated IpaD antigens, nitrated IpaD (SEQ ID NO: 6), or PBS. After 18 h, whole lungs were collected from immunized mice 2101715-001306 -30- to generate single-cell suspensions for analysis of phagocytic cells. We developed a flow cytometric gating scheme of whole lung digests to identify the following cell types: alveolar macrophages, neutrophils, CD11b+ dendritic cells, and CD11b- dendritic cells. Here, mice immunized with nitrated IpaD had significantly elevated neutrophil count relative to all other immunization groups and nitrated IpaD (SEQ ID NO: 6)- immunized mice had significantly higher neutrophil count relative to alveolar macrophages, whereas all other groups exhibited the opposite trend. The increased neutrophil presence may play a role in the increased antibody response seen in nitrated IpaD (SEQ ID NO: 6)-immunized mice due to their role in the initiation of adaptive immune responses and proinflammatory nature upon recruitment to the site of immunization. It was additionally observed that CD11b+ dendritic cells from nitrated IpaD (SEQ ID NO: 6)-immunized mice showed significantly increased CD80 expression relative to wild-type (SEQ ID NO: 4), and interestingly, CD11b- dendritic cells from nitrated IpaD (SEQ ID NO: 6)-immunized mice had significantly increased CD80 and CD86 expression (FIG. 4). Altogether, these findings suggest that nitration via pN-Phe incorporation plays an important role in the generation of a strong adaptive immune response due to increased expression of the costimulatory surface markers, CD80 and CD86, which play a direct role in the activation of T cells. Thus, nitration may enhance T cell activation and increase downstream effector cell activity, which may explain some of the increased systemic and mucosal antibody responses seen in nitrated IpaD (SEQ ID NO: 6)-immunized mice. This coupled with increased crosstalk between the adaptive and innate immune systems via increased neutrophil presence at the site of immunization support our hypothesis that nitration of foreign bacterial antigens may be a suitable method for increasing the immunogenicity of a protein antigen. In vivo cytokine quantification in bronchoalveolar lavage fluid. To further characterize differences in the immune response elicited in wild-type (SEQ ID NO: 4) and nitrated IpaD (SEQ ID NO: 6), cytokine analysis was performed. To assess inflammatory response and Th1 / Th2 immune response bias to pulmonary delivery of nitrated IpaD (SEQ ID NO: 6), five cytokines and chemokines were quantified in BALF samples from the 18 h in vivo study at University of Maryland’s Cytokine Core Laboratory. The proteins quantified via multiplex were IFN-g, TNF-a, IL-1b, KC (CXCL1), and B cell-activating factor (BAFF). IFN-g, TNF-a, and IL-1b are key Th1- associated cytokines responsible for initiating pro-inflammatory responses upon immunization. We further probed the extent of neutrophil activity at the site of immunization via cytokine analysis. The murine IL-8 homolog KC is a key chemokine responsible for neutrophil localization at the site of immunization and was analyzed to 2101715-001306 -31- explain the increased neutrophil presence in response to nitrated IpaD (SEQ ID NO: 6). BAFF is secreted by neutrophils and promotes B cell differentiation to antibody- secreting plasma cells and was quantified to further characterize the increased humoral response seen in mice immunized with nitrated IpaD (SEQ ID NO: 6). Of the five cytokines measured, only TNF-ɑ and BAFF were detectable in BALF samples. IFN-γ, IL-1^, and KC were undetectable in all samples. TNF-ɑ was only detectable in mice immunized with wild-type IpaD (SEQ ID NO: 4) spiked with endotoxin to equalize concentration and mice immunized with nitrated IpaD (SEQ ID NO: 6), and mice immunized with nitrated IpaD (SEQ ID NO: 6) had the only quantifiable TNF-ɑ response (19.59 ± 7.996 pg / mL). This finding suggests that nitrated IpaD (SEQ ID NO: 6) may elicit a pro-inflammatory response relative to wild-type IpaD (SEQ ID NO: 4) in the absence of adjuvant when endotoxin concentration is controlled for. Interestingly, BAFF was the only cytokine accurately quantified in all four immunization groups. BAFF levels in PBS-, spiked wild-type IpaD- (SEQ ID NO: 4), unspiked wild-type IpaD- (SEQ ID NO: 4), and nitrated IpaD (SEQ ID NO: 6)- immunized mice were 1367 ± 216.4 pg / mL, 1634 ± 521.4 pg / mL, 1663 ± 279.3 pg / mL, and 2573 ± 463.5 pg / mL, respectively (FIG. 5). Neutrophil-driven BAFF secretion at the site of immunization has been linked to increased B cell response, as BAFF is directly involved in the activation of B cells and amplification of antibody responses. The increased neutrophil presence seen in mice immunized with nitrated IpaD (SEQ ID NO: 6) plays a role in heightened BAFF response, and this phenomenon may play a key role in increased humoral immune response. Since all antigens were administered without adjuvants, we believe that pN- Phe and its effect on MHC-II antigen presentation may induce a local pro-inflammatory response at the site of immunization. Conclusions: Indirect ELISA results using serum and BALF from animal studies show statistically significant increases in the following antibodies in mice immunized with nitrated Shigella antigen (SEQ ID NO: 6) relative to both wild-type antigen (SEQ ID NO: 4) and PBS control: IgG, IgA, IgG1, IgG2b, and IgG2c. Antibody responses were confirmed across multiple studies using n = 5-6 mice per group. Since many bacterial pathogens without existing vaccines, such as Shigella, are growing increasingly problematic on a global scale with the continued emergence of antimicrobial-resistant strains, there is a need for strategies to enhance the immunogenicity of conserved foreign antigens. These studies using a nitrated bacterial antigen elaborate on previous findings of the Peter Schultz Lab, which were limited to self-proteins that were already native to the immunized mice. These previous findings 2101715-001306 -32- showed that site-specific mutagenesis of a native standard amino acid to the nsAA para-nitro-L-phenylalanine resulted in the breaking of self-tolerance and a sustained, cross-reactive antibody response. The results in this study suggest for the first time that these findings are not limited to self-proteins and can increase the humoral immune response elicited by a foreign antigen. Additional preliminary B cell epitope analysis indicates that regions surrounding the site of nitration observe a significant increase in antibodies that bind to that region as well. Use of this strategy could enable reductions in the use of adjuvants in future vaccine formulations for Shigella or enhance titers targeting regions for neutralization. Table 1. Sequences SEQ ID Name Species Sequence NO MNITTLTNSISTSSFSPNNTNGSSTETVNSDIKTTTSS HPVSSLTMLNDTLHNIRTTNQALKKELSQKTLTKTSLE EIALHSSQISMDVNKSAQLLDILSRNEYPINKDARELL Full- HSAPKEAELDGDQMISHRELWAKIANSINDINEQYLK length Shigella 1 VYEHAVSSYTQMYQDFSAVLSSLAGWISPGGNDGNS native flexneri VKLQVNSLKKALEELKEKYKDKPLYPANNTVSQEQAN IpaD KWLTELGGTIGKVSQKNGGYVVSINMTPIDNMLKSLD NLGGNGEVVLDNAKYQAWNAGFSAEDETMKNNLQTL VQKYSNANSIFDNLVKVLSSTISSCTDTDKLFLHF FSPNNTNGSSTETVNSDIKTTTSSHPVSSLTMLNDTLH NIRTTNQALKKELSQKTLTKTSLEEIALHSSQISMDVN KSAQLLDILSRHEYPINKDARELLHSAPKEAELDGDQM ISHRELWAKIANSINDINEQYLKVYEHAVSSYTQMYQ Truncated Shigella 2 DFSAVLSSLAGWISPGGNDGNSVKLQVNSLKKALEEL IpaD flexneri KEKYKDKPLYPANNTVSQEQANKWLTELGGTIGKVSQ KNGGYVVSINMTPIDNMLKSLDNLGGNGEVVLDNAKY QAWNAGFSAEDETMKNNLQTLVQKYSNANSIFDNLV KVLSSTISSCTDTDKLFLHF MGSSHHHHHHENLYFQGFSPNNTNGSSTETVNSDIK IpaD for TTTSSHPVSSLTMLNDTLHNIRTTNQALKKELSQKTLT expressio KTSLEEIALHSSQISMDVNKSAQLLDILSRHEYPINKD 3 Synthetic n in E. ARELLHSAPKEAELDGDQMISHRELWAKIANSINDINE coli QYLKVYEHAVSSYTQMYQDFSAVLSSLAGWISPGGND GNSVKLQVNSLKKALEELKEKYKDKPLYPANNTVSQE 2101715-001306 -33- SEQ ID Name Species Sequence NO QANKWLTELGGTIGKVSQKNGGYVVSINMTPIDNMLK SLDNLGGNGEVVLDNAKYQAWNAGFSAEDETMKNNL QTLVQKYSNANSIFDNLVKVLSSTISSCTDTDKLFLHF GFSPNNTNGSSTETVNSDIKTTTSSHPVSSLTMLNDTL HNIRTTNQALKKELSQKTLTKTSLEEIALHSSQISMDV NKSAQLLDILSRHEYPINKDARELLHSAPKEAELDGDQ Modified MISHRELWAKIANSINDINEQYLKVYEHAVSSYTQMY 4 IpaD Synthetic QDFSAVLSSLAGWISPGGNDGNSVKLQVNSLKKALEE (FIG. 3A) LKEKYKDKPLYPANNTVSQEQANKWLTELGGTIGKVS QKNGGYVVSINMTPIDNMLKSLDNLGGNGEVVLDNA KYQAWNAGFSAEDETMKNNLQTLVQKYSNANSIFDN LVKVLSSTISSCTDTDKLFLHF MGSSHHHHHHENLYFQGFSPNNTNGSSTETVNSDIK Nitrated TTTSSHPVSSLTMLNDTLHNIRTTNQALKKELSQKTLT IpaD with KTSLEEIALHSSQISMDVNKSAQLLDILSRHEpNPINK Y107 DARELLHSAPKEAELDGDQMISHRELWAKIANSINDI substitute NEQYLKVYEHAVSSYTQMYQDFSAVLSSLAGWISPGG 5 d with Synthetic NDGNSVKLQVNSLKKALEELKEKYKDKPLYPANNTVS pN-Phe QEQANKWLTELGGTIGKVSQKNGGYVVSINMTPIDN expressio MLKSLDNLGGNGEVVLDNAKYQAWNAGFSAEDETMK n in E. NNLQTLVQKYSNANSIFDNLVKVLSSTISSCTDTDKLF coli LHF GFSPNNTNGSSTETVNSDIKTTTSSHPVSSLTMLNDTL HNIRTTNQALKKELSQKTLTKTSLEEIALHSSQISMDV Nitrated NKSAQLLDILSRHEpNPINKDARELLHSAPKEAELDGD IpaD with QMISHRELWAKIANSINDINEQYLKVYEHAVSSYTQM Y91 6 Synthetic YQDFSAVLSSLAGWISPGGNDGNSVKLQVNSLKKALE substitute ELKEKYKDKPLYPANNTVSQEQANKWLTELGGTIGKV d with SQKNGGYVVSINMTPIDNMLKSLDNLGGNGEVVLDN pN-Phe AKYQAWNAGFSAEDETMKNNLQTLVQKYSNANSIFD NLVKVLSSTISSCTDTDKLFLHF IpaD 7 Synthetic DILSRHEYPINKDAR region 1 2101715-001306 -34- SEQ ID Name Species Sequence NO IpaD 8 Synthetic MISHRELWAKIANSI region 2 IpaD 9 Synthetic DINEQYLKVYEHAVS region 3 IpaD 10 Synthetic HAVSSYTQMYQDFSA region 4 IpaD 11 Synthetic SSLAGWISPGGNDGNSV region 5 IpaD 12 Synthetic EELKEKYKDKPLYPA region 6 IpaD 13 Synthetic QANKWLTELGGTIGK region 7 IpaD 14 Synthetic NAKYQAWNAGFSAED region 8 IpaD 15 Synthetic LVQKYSNANSIFDNL region 9 peptide-1 16 (FIG. Synthetic GFSPNNTNGSSTETV 3A / 3D) peptide-2 17 (FIG. Synthetic NTNGSSTETVNSDIK 3A / 3D) peptide-3 18 Synthetic STETVNSDIKTTTSS (FIG. 3D) peptide-4 19 Synthetic NSDIKTTTSSHPVSS (FIG. 3D) peptide-5 20 Synthetic TTTSSHPVSSLTMLN (FIG. 3D) peptide-6 21 Synthetic HPVSSLTMLNDTLHN (FIG. 3D) peptide-7 22 Synthetic LTMLNDTLHNIRTTN (FIG. 3D) peptide-8 23 Synthetic DTLHNIRTTNQALKK (FIG. 3D) 2101715-001306 -35- SEQ ID Name Species Sequence NO peptide-9 24 Synthetic IRTTNQALKKELSQK (FIG. 3D) peptide- 25 10 (FIG. Synthetic QALKKELSQKTLTKT 3D) peptide- 26 11 (FIG. Synthetic ELSQKTLTKTSLEEI 3D) peptide- 27 12 (FIG. Synthetic TLTKTSLEEIALHSS 3D) peptide- 28 13 (FIG. Synthetic SLEEIALHSSQISMD 3D) peptide- 29 14 (FIG. Synthetic ALHSSQISMDVNKSA 3D) peptide- 30 15 (FIG. Synthetic QISMDVNKSAQLLDI 3D) peptide- 31 16 (FIG. Synthetic VNKSAQLLDILSRHE 3D) peptide- 32 17 (FIG. Synthetic QLLDILSRHEYPINK 3D) peptide- 33 18 (FIG. Synthetic LSRHEYPINKDAREL 3D) peptide- 34 19 (FIG. Synthetic YPINKDARELLHSAP 3D) 2101715-001306 -36- SEQ ID Name Species Sequence NO peptide- 35 20 (FIG. Synthetic DARELLHSAPKEAEL 3D) peptide- 36 21 (FIG. Synthetic LHSAPKEAELDGDQM 3D) peptide- 37 22 (FIG. Synthetic KEAELDGDQMISHRE 3D) peptide- 38 23 (FIG. Synthetic DGDQMISHRELWAKI 3D) peptide- 39 24 (FIG. Synthetic ISHRELWAKIANSIN 3D) peptide- 40 25 (FIG. Synthetic LWAKIANSINDINEQ 3D) peptide- 41 26 (FIG. Synthetic ANSINDINEQYLKVY 3D) peptide- 42 27 (FIG. Synthetic DINEQYLKVYEHAVS 3D) peptide- 43 28 (FIG. Synthetic YLKVYEHAVSSYTQM 3D) peptide- 44 29 (FIG. Synthetic EHAVSSYTQMYQDFS 3D) peptide- 45 30 (FIG. Synthetic SYTQMYQDFSAVLSS 3D) 2101715-001306 -37- SEQ ID Name Species Sequence NO peptide- 46 31 (FIG. Synthetic YQDFSAVLSSLAGWI 3D) peptide- 47 32 (FIG. Synthetic AVLSSLAGWISPGGN 3D) Peptide 17* 48 Synthetic QLLDILSRHEpNPINK (FIG. 3C / 3D) Peptide 18* 49 Synthetic LSRHEpNPINKDAREL (FIG. 3C / 3D) Peptide 19* 50 Synthetic pNPINKDARELLHSAP (FIG. 3C / 3D) 1. pN is pN-Phe. 2. Underlined amino acids may be substituted by a nitrated amino acid, for example, pN-Phe. All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

2101715-001306 -38- WHAT IS CLAIMED:

1. An antigenic nitrated recombinant polypeptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, wherein an amino acid in SEQ ID NO: 4 or a fragment thereof is substituted with a nitrated amino acid, and wherein the antigenic nitrated recombinant polypeptide comprises a nitrated amino acid corresponding to the nitrated amino acid in the SEQ ID NO: 4 or a fragment thereof.

2. The antigenic nitrated recombinant polypeptide of claim 1, wherein the antigenic nitrated recombinant polypeptide consists of an amino acid sequence at least 90% homologous to the amino acid sequence of SEQ ID NO: 4 or a fragment thereof.

3. The antigenic nitrated recombinant polypeptide of claim 1, wherein the antigenic nitrated recombinant polypeptide consists of the amino acid sequence of SEQ ID NO: 4 or a fragment thereof.

4. The antigenic nitrated recombinant polypeptide of claim 1, wherein the antigenic nitrated recombinant polypeptide consists of SEQ ID NO:

6.

5. The antigenic nitrated recombinant polypeptide of claim 1, wherein the antigenic nitrated recombinant polypeptide consists of an amino acid sequence selected from SEQ ID NOS: 48-50.

6. The antigenic nitrated recombinant polypeptide of any one of claims 1-5, wherein the fragment consists of 12-18 amino acids.

7. The antigenic nitrated recombinant polypeptide of any one of claims 1-5, wherein the fragment consists of the amino acid sequence selected from the group consisting of SEQ ID NOS: 7-15.

8. The antigenic nitrated recombinant polypeptide of any one of claims 1-5, wherein the substituted amino acid is selected from the group consisting of Y91, W122, Y136, Y140, Y147, Y151, F154, W164, Y193, W213, Y263, W266, F270, Y288, and F295 in SEQ ID NO:

4.

9. The antigenic nitrated recombinant polypeptide of any one of claims 1-5, wherein the nitrated amino acid is para-nitro-L-phenylalanine (pN-Phe).

10. A vaccine composition comprising an effective amount of the antigenic nitrated recombinant polypeptide of any one of claims 1-5 and a pharmaceutically acceptable adjuvant, carrier and / or excipient.

11. A method for inducing an immune response in a target subject, comprising administering to a target subject an effective amount of the vaccine composition of claim 10, whereby a target immune response is induced in the target subject.2101715-001306 -39- 12. The method of claim 11, wherein the target immune response is greater than a control immune response induced by a control polypeptide in a control subject.

13. The method of claim 12, wherein the target induced immune response comprises production of one or more target antibodies against the antigenic nitrated recombinant polypeptide, wherein the one or more target antibodies cross-react against a control polypeptide, and wherein the one or more target antibodies comprise IgG, IgA, IgG1, IgG2b, IgG2c, or a combination thereof, 14. The method of claim 13, wherein the one or more target antibodies bind a first region in the nitrated antigenic recombinant polypeptide and a second region in the control polypeptide, wherein the first region and the second region correspond to different regions of SEQ IN NO:

4.

15. A method for producing the vaccine composition of claim 10, comprising admixing the nitrated antigenic recombinant polypeptide of claim 1 with the pharmaceutically acceptable adjuvant, carrier or excipient.

Images