DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication

DNA vaccines and recombinant Listeria monocytogenes that express and secrete SIV Gag and Env antigens were combined in a nonhuman primate prime-boost immunogenicity study followed by a challenge with SIV239. We report that recombinant DNA vaccine delivered intramuscularly, and recombinant L. monocytogenes delivered orally each individually have the ability to induce CD8+ and CD4+ T cell immune responses in a nonhuman primate. Four rhesus monkeys were immunized at weeks 0, 4, 8, and 12 with the pCSIVgag and pCSIVenv DNA plasmids and boosted with SIV expressing L. monocytogenes vaccines at weeks 16, 20, and 28. Four rhesus monkeys received only the L. monocytogenes vaccines at weeks 16, 20, and 28. A final group of monkeys served as a control group. Blood samples were taken before vaccination and 2 weeks post each injection and analyzed by ELISPOT for CD4+ and CD8+ T cell responses. Moderate vaccine induced SIV-specific cellular immune responses were observed following immunization with either DNA or L. monocytogenes vectors. However, the SIV antigen-specific immune responses were significantly increased when Rhesus macaques were primed with SIV DNA vaccines and boosted with the SIV expressing L. monocytogenes vectors. In addition, the combined vaccine was able to impact SIV239 viral replication following an intrarectal challenge. This study demonstrates for the first time that oral L. monocytogenes can induce a cellular immune response in a nonhuman primate and is able to enhance the efficacy of a DNA vaccine as well as provide modest protection against SIV239 challenge.     

Immune responses to the oral administration of recombinant Bacillus subtilis expressing multi-epitopes of foot-and-mouth disease virus and a cholera toxin B subunit

Bacillus subtilis has been engineered successfully to express heterologous antigens for use as a vaccine vehicle that can elicit mucosal and systemic immunity response. In this study, a recombinant B. subtilis expressing the B subunit of cholera toxin (CT-B) and an epitope box constituted with antigen sites from foot-and-mouth disease virus (FMDV) type Asia 1 was constructed and named 1A751/CTB-TEpiAs. Its capability to induce mucosal, humoral, and cellular responses in mice and guinea pigs was evaluated after oral administration with vegetative cells of 1A751/CTB-TEpiAs. In addition, its capability to protect guinea pigs against homologous virus challenge was examined. All animals were given booster vaccination at day 21 after initial inoculation and guinea pigs were challenged 3 weeks after booster vaccination. The control groups were inoculated with a commercial vaccine or administered orally with 1A751/pBC38C or an oral buffer. All animals vaccinated with 1A751/CTB-TEpiAs developed specific anti-FMDV IgA in lung and gut lavage fluid, serum ELISA antibody, neutralizing antibody as well as T lymphocyte proliferation, and IFN-γ secretory responses. Three of the five guinea pigs vaccinated with 1A751/CTB-TEpiAs were protected completely from the viral challenge. The results demonstrate the potential viability of a B. subtilis-based recombinant vaccine for the control and prevention of FMDV infections.     

Evaluation of recombinant Kunjin replicon SIV vaccines for protective efficacy in macaques

Persistent gag-specific T cell immunity would be a useful component of an effective HIV vaccine. The Flavivirus Kunjin replicon was previously engineered to persistently express HIV gag and was shown to induce protective responses in mice. We evaluated Kunjin replicon virus-like-particles expressing SIVgag-pol in pigtail macaques. Kunjin-specific antibodies were induced, but no SIV-specific T cell immunity were detected. Following SIVmac251 challenge, there was no difference in SIV viremia or retention of CD4 T cells between Kunjin-SIVgag-pol vaccine immunized animals and controls. An amnestic SIV gag-specific CD8 T cell response associated with control of viremia was observed in 1 of 6 immunized animals. Refinements of this vector system and optimization of the immunization doses, routes, and schedules are required prior to clinical trials.     

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.     

Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vectors Prime for Strong Cellular Responses to Simian Immunodeficiency Virus Gag in Rhesus Macaques

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV)gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIVgag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.     

含密码子优化的SIV gag基因的DNA疫苗与rAd5疫苗构建及免疫原性评价

目的 构建含有SIVgag基因的DNA疫苗和重组腺病毒疫苗,为后期在SIV感染的猴模型中进行多载体疫苗联合免疫策略的治疗效果评价奠定基础.方法 将SIV gag基因按照哺乳动物偏嗜密码子进行优化并构建至pVR载体,作为DNA疫苗.以Western Blot方法比较优化前后gag基因表达水平.将优化后的gag基因插入重组腺病毒载体,构建rAd5-SIVgag疫苗.在BALB/c小鼠中分别比较DNA疫苗及rAd5-SIV.gag疫苗单独及联合免疫的效果.结果 密码子优化的SIVgag基因的表达水平远高于野生型SIVgag基因.重组腺病毒疫苗免疫一次或两次诱导的细胞免疫反水平分别高于DNA疫苗免疫一次或两次.两种疫苗联合免疫的反应水平与腺病毒疫苗免疫两次的水平相当,高于DNA疫苗单独免疫及腺病毒疫苗单独免疫一次的结果.结论 成功优化了SIV gag基因,使其不依赖Rev高水平表达;成功构建了表达优化后SIV gag基因的DNA疫苗和rAd5疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.     

A novel dendritic cell-targeted lentiviral vector,encoding Ag85A-ESAT6 fusion gene of Mycobacterium tuberculosis,could elicit potent cell-mediated immune responses in mice

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to high mortality worldwide. It is well-established that cellular immunity plays a critical role to control Mtb infection. Dendritic Cells (DCs) are potent antigen presenting cells, which play an important role to prime cell-mediated immune responses. In vivo targeting of DCs has been shown to induce both strong cellular immunity and protection against tumor challenges. The aim of the present study was not only to assess the immunizing potential of a novel DC-targeted recombinant lentivirus expressing fusion antigen Ag85A-ESAT6 of Mtb, but also to compare it with a recombinant lentivirus with broad cellular tropism expressing the same antigen in mice. The findings demonstrated that our novel recombinant DC-targeted lentivector was able to successfully transduce and express the fusion antigen Ag85A-E6 in vitro and in vivo. Moreover, a single footpad injection of targeted lentivectors could elicit strong T-helper 1 (Th1) immunity against the above mentioned antigen, as indicated by the specific high-level production of IFN-γ and IL-2 using spleen lymphocytes and lymphoproliferative responses. Despite of these promising results, more attempts are required to elucidate the protective and therapeutic efficacy of this approach in future.     

Intradermal Delivery of Recombinant Vaccinia Virus Vector DIs Induces Gut‐Mucosal Immunity

Antigen‐specific mucosal immunity is generally induced by the stimulation of inductive mucosal sites. In this study, we found that the replication‐deficient vaccinia virus vector, DIs, generates antigen‐specific mucosal immunity and systemic responses. Following intradermal injection of recombinant DIs expressing simian immunodeficiency virus gag (rDIsSIVgag), we observed increased levels of SIV p27‐specific IgA and IgG antibodies in faecal extracts and plasma samples, and antibody‐forming cells in the intestinal mucosa and spleen of C57BL/6 mice. Antibodies against p27 were not detected in nasal washes, saliva, and vaginal washes. The enhanced mucosal and systemic immunity persisted for 1 year of observation. Induction of Gag‐specific IFN‐γ spot‐forming CD8⁺ T cells in the spleen, small intestinal intraepithelial lymphocytes, and submandibular lymph nodes was observed in the intradermally injected mice. Heat‐inactivated rDIsSIVgag rarely induced antigen‐specific humoral and T‐helper immunity. Moreover, rDIsSIVgag was detected in MHC class II IA antigen‐positive (IA⁺) cells at the injection site. Consequently, intradermal delivery of rDIs effectively induces antigen‐specific humoral and cellular immunity in gut‐mucosal tissues of mice. Our data suggest that intradermal injection of an rDIs vaccine may be useful against mucosally transmitted pathogens.     

Intradermal and oral immunization with recombinant Mycobacterium bovis BCG expressing the simian immunodeficiency virus Gag protein induces long-lasting, antigen-specific immune responses in guinea pigs

To develop a new recombinant BCG (rBCG) vaccine, we constructed rBCG that expresses the full-length Gag protein of simian immunodeficiency virus (rBCG-SIVGag) at a level of 0.5 ng/mg after 3 weeks of bacterial cell culture. Intradermal (i.d.) inoculation of guinea pigs with 0.1 mg of rBCG-SIVGag resulted in the induction of delayed-type hypersensitivity (DTH) responses to both purified protein derivative (PPD) of tuberculin and SIV Gag p27 protein; responses that were maintained for the duration of the 50-week study. In contrast, guinea pigs orally vaccinated with 160 mg of the same antigen exhibited a long-lasting DTH response to the SIV Gag p27 protein, but mounted no response to PPD. Proliferative responses to SIV Gag p27 and PPD antigens were detected in both i.d. and orally immunized animals; however, the levels of PPD-specific responses were significantly higher in guinea pigs immunized by the i.d. than the oral route. A significant increase in the level of PPD- and SIV Gag p27-specific IFNgamma mRNA expression was also detected in both immunization groups receiving rBCG-SIVGag. In addition, both i.d. and oral immunization with rBCG-SIVGag induced PPD- and SIV Gag p27-specific serum IgG responses. Insertion of the SIV gag gene into BCG did not appear to change the ability of rBCG-immunized animals to elicit PPD-specific immune responses. These results indicate that rBCG-SIVGag has the ability to effectively induce long-lasting, cell-mediated and humoral immunity against both viral and bacterial antigens in guinea pigs, suggesting that rBCG-Gag has the potential to elicit immunities specific not only for tuberculosis but also for HIV at human doses.     

Mucosal prior to systemic application of recombinant adenovirus boosting is more immunogenic than systemic application twice but confers similar protection against SIV-challenge in DNA vaccine-primed macaques

We investigated the immunogenicity and efficacy of a bimodal prime/boost vaccine regimen given by various routes in the Simian immunodeficiency virus (SIV) rhesus monkey model for AIDS. Twelve animals were immunized with SIV DNA-vectors followed by the application of a recombinant adenovirus (rAd5) expressing the same genes either intramuscularly (i.m.) or by oropharyngeal spray. The second rAd5-application was given i.m. All vaccinees plus six controls were challenged orally with SIVmac239 12 weeks post-final immunization.Both immunization strategies induced strong SIV Gag-specific IFN-γ and T-cell proliferation responses and mediated a conservation of CD4⁺ memory T-cells and a reduction of viral load during peak viremia following infection. Interestingly, the mucosal group was superior to the systemic group regarding breadth and strength of SIV-specific T-cell responses and exhibited lower vector specific immune responses. Therefore, our data warrant the inclusion of mucosal vector application in a vaccination regimen which makes it less invasive and easier to apply.     

Loss of mucosal CD103+ DCs and IL-17+ and IL-22+ lymphocytes is associated with mucosal damage in SIV infection

Human immunodeficiency virus (HIV) and Simian immunodeficiency virus (SIV) disease progression is associated with multifocal damage to the gastrointestinal tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation, but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of interleukin (IL)-17-producing lymphocytes, cells that microarray analysis showed expressed genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ dendritic cells (DCs) after SIV infection, which associated with the loss of IL-17- and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and coculture of CD103+ DCs and naïve T cells led to increased IL17A and RORc expression in differentiating T cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis.     

Immune responses and protection efficacy of a recombinant swinepox virus expressing HA1 against swine H3N2 influenza virus in mice and pigs

Swine influenza virus (SIV) is not only an important respiratory pathogen in pigs but also a potent threat to human health. Even though immunization with recombinant vaccinia poxviruses expressing protective antigens as a vaccination strategy has been widely used for many infectious diseases, development of recombinant swinepox virus (rSPV) vector for this purpose has been less successful. Here, we report the construction of a recombinant swinepox virus (rSPV) expressing hemagglutinin (HA1) of H3N2 SIV (rSPV-H3). Immune responses and protection efficacy of the vaccination vector were assessed in both mouse and pig models. Prime and boost inoculations of rSPV-H3 yielded neutralization antibody against SIV and elicited potent H3N2 SIV-specific INF-γ response from T-lymphocytes. Complete protection of pigs against H3N2 SIV challenge was achieved. No pigs showed severe systemic and local reactions and no SIV was found shed from the pigs vaccinated with rSPV-H3 after challenge. The data suggest that the SPV-based recombinant vector expressing HA1 of H3N2 SIV might serve as a promising SIV vaccine for protection against SIV infection.     

Characterisation of simian immunodeficiency virus-infected cells in pigtail macaques

Defining which cells become infected with simian immunodeficiency virus (SIV) in vivo should assist in unravelling the pathogenesis of human immunodeficiency virus (HIV)/SIV infection. HIV/SIV infection of CD4⁺ T cells resulted in down-regulation of CD3 and CD4 surface molecules in vitro, however this phenomenon is poorly characterised in vivo. Intracellular SIV p27 was studied by flow cytometry in serial blood samples and lymph node samples during acute infection of 17 SIVmac-infected pigtail macaques. Two weeks after infection, a mean of 56±6.8% the p27⁺ cells were lymphocytes negative for surface CD4 and CD3, and indeed the highest proportion of SIV infected cells were found in the small subset of CD3^LoCD4⁻CD8⁻ lymphocytes, indicating that infection has lead to down-regulation of these markers in vivo. Furthermore, the relative amount of SIV p27 within lymphocytes (based of mean fluorescence intensity) was higher in CD3^LoCD4⁻ and CD3⁻ infected cells than in CD3⁺ or CD4⁺ p27⁺ populations, consistent with greater viral production in CD4⁺ T cells down-regulating CD3 and CD4 molecules. The CD3⁻CD4⁻ infected cells expressed T cell markers CD2 and CD5 and were negative for monocyte, NK and B cell markers. The majority of infected cells were CD28⁺CD95⁺ central memory T cells. Surprisingly, p27⁺ blood lymphocytes were mostly negative for activation markers CD25 and CD69, but most of the infected lymph nodes cells were activated. Our results characterise productively-infected macaque lymphocytes in vivo. The high proportion of SIV-infected lymphocytes that are CD3⁻CD4⁻ has important implications for the in vivo study of pathogenesis of SIV/HIV infections.     

Human Dendritic Cell Interactions with Whole Recombinant Yeast: Implications for HIV-1 Vaccine Development

Defects in number and function of dendritic cells (DCs) have been observed during HIV-1 infection, so therapeutic HIV-1 vaccine approaches that target or activate DCs may improve vaccine immunogenicity. To determine the potential of recombinant Saccharomyces cerevisiae yeast as an HIV-1 vaccine, we investigated interactions between yeast and human DCs. Yeast induced direct phenotypic maturation of monocyte-derived DCs (MDDCs) and enriched blood myeloid DCs (mDCs), but only indirectly matured blood plasmacytoid DCs (pDCs). Yeast-pulsed MDDCs and blood mDCs produced inflammatory cytokines and stimulated strong allo-reactive T cell proliferation. Both blood DC subsets internalized yeast, and when pulsed with yeast recombinant for HIV-1 Gag protein, both stimulated in vitro expansion of Gag-specific CD8⁺ memory T cells. These results suggest that S. cerevisiae yeast have potent adjuvant effects on human DCs. Furthermore, recombinant yeast-derived antigens are processed by human blood DCs for MHC class-I cross-presentation. These DC-targeting characteristics of yeast suggest that it may be an effective vaccine vector for induction of HIV-1-specific cellular immune responses.     

Role of D-alanylation of Streptococcus gordonii lipoteichoic acid in innate and adaptive immunity

In recent years, there has been considerable interest in using the oral commensal gram-positive bacterium Streptococcus gordonii as a live vaccine vector. The present study investigated the role of d-alanylation of lipoteichoic acid (LTA) in the interaction of S. gordonii with the host innate and adaptive immune responses. A mutant strain defective in d-alanylation was generated by inactivation of the dltA gene in a recombinant strain of S. gordonii (PM14) expressing a fragment of the S1 subunit of pertussis toxin. The mutant strain was found to be more susceptible to killing by polymyxin B, nisin, magainin II, and human beta defensins than the parent strain. When it was examined for binding to murine bone marrow-derived dendritic cells (DCs), the dltA mutant exhibited 200- to 400-fold less binding than the parent but similar levels of binding were shown for Toll-like receptor 2 (TLR2) knockout DCs and HEp-2 cells. In a mouse oral colonization study, the mutant showed a colonization ability similar to that of the parent and was not able to induce a significant immune response. The mutant induced significantly less interleukin 12p70 (IL-12p70) and IL-10 than the parent from DCs. LTA purified from the bacteria induced tumor necrosis factor-alpha and IL-6 production from wild-type DCs but not from TLR2 knockout DCs, and the mutant LTA induced a significantly smaller amount of these two cytokines. These results show that d-alanylation of LTA in S. gordonii plays a role in the interaction with the host immune system by contributing to the relative resistance to host defense peptides and by modulating cytokine production by DCs.     

Immunoprotection against influenza H5N1 virus by oral administration of enteric-coated recombinant Lactococcus lactis mini-capsules

Edible vaccines that can be made widely available and easily administered could bring great benefit to the worldwide battle against pandemic viral infections. They can be used not only for the vaccination of humans and domesticated animals, but also for wild herds and live stock which are otherwise difficult to vaccinate. In this study, we report the development of an edible mini-capsule form of live, non-persisting, recombinant Lactococcus lactis (L. lactis) vaccine against the highly virulent influenza H5N1 strain. Recombinant L. lactis-based H5N1 HA antigen expression constructs were made and shown to be able to induce higher levels of HA-specific serum IgG and fecal IgA antibody production after oral administration. The vectors were then formulated into a mini-capsule dosage form and fed to mouse. Four doses of oral administration rendered complete protection of the mouse against lethal challenges of H5N1 virus.     

Immunogenic and neutralization efficacy of recombinant perfringolysin O of Clostridium perfringens and its C-terminal receptor-binding domain in a murine model

Clostridium perfringens is a Gram-positive anaerobe ubiquitously present in different environments, including the gut of humans and animals. C. perfringens have been classified in the seven toxinotypes based on the secreted toxins that cause different diseases in humans and animals. Perfringolysin O (PFO), a cholesterol-dependent pore-forming cytolysin, is one of the potent toxins secreted by almost all C. perfringens isolates. The PFO acts in synergy with α-toxin in the progression of gas gangrene in humans and necrohemorrhagic enteritis in the calves.

C. perfringens infections spread very fast, and the animals die within a few hours of the onset of infection. This necessitates the use of vaccines to control clostridial infections. Though the vaccine potential of other toxins has been reported, PFO has remained unexplored. The present study describes the immunogenic and protective potential of native recombinant PFO (WTrPFO). Since the PFO is toxic to the host cells, the non-toxic C-terminal domain of PFO (rPFOC-ter) was also assessed for its immunogenicity and protective efficacy. Immunization of mice with the purified soluble recombinant histidine-tagged WTrPFO and rPFOC-ter, expressed in E. coli, generated robust mixed immune response and T cell memory. Pre-incubation of the WTrPFO with anti-WTrPFO and rPFOC-ter antisera negated its hemolytic activity in mice RBCs, as well as its cytotoxic effect in mice peritoneal macrophages in vitro. Thus, immunization with the WTrPFO and its non-toxic C-terminal domain generated neutralizing antibodies, suggesting their vaccine potential against the PFO. Thus, the non-toxic C-terminal domain of PFO could serve as an alternative to PFO as a vaccine candidate.

     

Protection of sheep against caseous lymphadenitis by use of a single oral dose of live recombinant Corynebacterium pseudotuberculosis.

An inactive form of the Corynebacterium pseudotuberculosis phospholipase D (PLD) gene was constructed and expressed in a PLD-negative strain (designated Toxminus) of C. pseudotuberculosis. Antibody responses specific to Toxminus and both Toxminus and PLD proteins were detected in sheep following oral administration of Toxminus or Toxminus expressing the PLD toxoid, respectively. However, only those sheep vaccinated with Toxminus expressing PLD toxoid were protected against wild-type challenge. These results confirm the importance of PLD as a protective antigen and demonstrate both the potential for developing an oral caseous lymphadenitis vaccine and C. pseudotuberculosis Toxminus as a live vaccine vector.     

Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response.Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals; the former include gas gangrene, food poisoning, necrotizing enterocolitis of infants, and enteritis necroticans (28, 37, 45). C. perfringens is an obligately anaerobic rod-shaped bacterium commonly found in the gastrointestinal tracts of both animals and humans and widely distributed in soil and sewage. The ability of C. perfringens to cause disease is associated with the production of a variety of extracellular toxins (13 different toxins have been reported so far). On the basis of differential production of toxins, the strains of C. perfringens can be divided into five types, A through E (35), of which type A and type C strains are implicated in human diseases while other types are of veterinary importance. Type A strains cause gas gangrene, the most destructive of all clostridial diseases, which is characterized by rapid destruction of tissue with production of gas (4, 42). The incidence of disease ranged from 1% or less of wounded personnel during World War II to 10% of wounded personnel during World War I (27). Hundreds of thousands of soldiers died of gas gangrene as a result of battlefield injuries, and C. perfringens was widely recognized as the most important causal organism of the disease. Besides gas gangrene, type A strains also cause gastrointestinal diseases in humans (food poisoning, antibiotic-associated diarrhea, sporadic diarrhea, sudden infant death syndrome) and animals (diarrhea in foals and pigs, etc.). C. perfringens type C strains cause necrotic enteritis in humans and animals, in addition to enterotoxemia in sheep. Moreover, C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; therefore, efforts to develop strategies for detection and protection are warranted.Interest in a vaccine against gas gangrene has been intermittent; most efforts were made during World Wars I and II and were devoted to the therapeutic use of antisera. Such antisera, raised against toxoids of all five species of clostridia associated with gas gangrene, were shown to have benefits if they were given soon after trauma (20). Active immunization against the disease has received little attention until a few years ago (32, 43, 44). A number of clinical studies of other pathogenic bacteria, including Clostridium difficile, have highlighted the importance of nontoxin protein antigens in disease expression, especially in colonization by the bacterium (9, 12, 16, 26).Despite a sudden spurt of activity in the proteomic characterization of bacterial pathogens, for reasons unknown, clostridia have been largely ignored. Clostridium difficile is the only clostridial species whose proteome has been analyzed to some extent (34). Proteomic methodology has been used to elucidate proteins regulated by the VirR/VirS system in Clostridium perfringens (40).To invade, multiply in, and colonize host tissues, a pathogen must be able to evade the host immune system and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance for the understanding of microbial pathogenesis. The exoproteins of Gram-positive bacteria are likely to contain some of these key factors. The term “secretome” refers to and takes into account both the protein secretion systems and the secreted proteins; in monoderm bacteria (Gram-positive cell envelope architecture), these proteins can also be found in the membrane and/or cell wall. The proteins found in the extracellular milieu of Gram-positive bacteria are hence extracellular proteins, or exoproteins, which form the exoproteome; these exoproteins are not necessarily secreted by known secretion systems (15). This terminology is used for the subproteome described in this study.Further, the correct identification of C. perfringens pathovars is critical for epidemiological studies and for the development of effective preventive measures, including vaccination. It is not well understood why C. perfringens produces illnesses that differ so greatly in severity. Specific illnesses may reflect the particular type of tissue that high numbers of the organism are able to invade. Clearly, the presence of certain toxins is one explanation, but it falls short in many instances. For example, strains that cause food poisoning may differ from those that cause gas gangrene only by the presence of an enterotoxin gene in the former, yet food-poisoning strains have never been found to cause gas gangrene. Elucidation of the exoproteins of this bacterium is likely to reveal factors responsible for the host specificity of the different C. perfringens pathovars.The present investigation was carried out with the following objectives: (i) identification of dominant exoproteins from the C. perfringens type A strain ATCC 13124 (a gas gangrene isolate and the species type strain) and comparison with those of type C strains, (ii) elucidation of immunogenic components of the exoproteome, (iii) in silico analysis of the identified proteins with respect to localization, predicted function, and likely potential as surface markers for detection and as vaccine candidates, and (iv) validation of selected candidates with respect to vaccine potential in an experimental mouse model of gas gangrene. To the best of our knowledge, ours is the first proteomic elucidation of the C. perfringens exoproteome. The identification of extracellular proteins of C. perfringens type A and type C strains in the present investigation advances our understanding of the pathogenesis of the disease and opens significant opportunities for the identification of diagnostic markers and the development of vaccines against gas gangrene in humans.