An attenuated Salmonella-vectored vaccine elicits protective immunity against Mycobacterium tuberculosis

There is a need to develop protective vaccines against tuberculosis (TB) that elicit full immune responses including mucosal immunity. Here, a live attenuated Salmonellatyphimurium aroA SL7207 vector TB vaccine, namely SL(E6-85B), harboring the Mycobacterium tuberculosis (M. tb) H37Rv ESAT6-Ag85B fusion gene was developed. The experimental data demonstrated that this SL(E6-85B) vaccine, or when it is combined with BCG vaccination, induced the strongest TB Ag-specific mucosal, humoral, and cellular immune responses comprised of increased proliferation of T cells, IFN-gamma expression, granzyme B production, as well as the greatest IFN-gamma production of effector-memory T (T_EM) or effector CD8⁺ T cell responses and exerted high protective efficacy in mice against virulent M. tb H37Rv challenge compared to the other vaccinated groups (mice immunized with SL(Ag85B), a DNA vaccine or BCG only). This strategy may represent a novel promising mucosal vaccine candidate for the prevention of TB which are inexpensive to produce, efficacious, and able to be given orally rather than by injection.     

鼠伤寒沙门菌生物学分型方法在流行病学调查和监测中的应用

为探索用简便有效的手段对鼠伤寒沙门菌感染进行调查监测，采用英国学者Ｄｕｇｕｉｄ的鼠伤寒沙门菌生物学分型方法，应用于医院天津监测、病例追踪、食物中毒调查。结果：从医院感染患儿分离菌株的主要生物型与医院环境污染、医务人员携带菌株的生物型分布相似；截瘫患者尿中菌株与从其家庭环境和妻子手上分离菌株生物型别一致；从食物中毒者和乘彩中分离菌株的生物型别与鸭子携带菌株主要生物型别一致。结论：患儿医院感染是由于病     

Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4⁺ T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4⁺ T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.     

CD40 triggering induces strong cytotoxic T lymphocyte responses to heat-killed Staphylococcus aureus immunization in mice: a new vaccine strategy for staphylococcal mastitis

Staphylococcus (S.) aureus is a major pathogen involved in chronic bovine mastitis. Staphylococcal mastitis is difficult to control due to the ability of S. aureus to invade and survive within host cells. We therefore postulated that induction of CD8(+) cytotoxic T lymphocyte (CTL) responses leading to destruction of infected cells could help in the control of S. aureus mastitis. We demonstrate that immunization of mice with heat-killed S. aureus together with agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of reducing the severity of subsequent staphylococcal mastitis. Our study shows promise for CTL-dependent vaccination against S. aureus mastitis.     

Platycodin D is a potent adjuvant of specific cellular and humoral immune responses against recombinant hepatitis B antigen

The ideal adjuvants for hepatitis B vaccines should be capable of eliciting both strong humoral and cellular immune responses, especially Th1 cell and cytotoxic T lymphocyte (CTL) responses. However, Alum used as adjuvants in the hepatitis B vaccines currently commercialized offers limitation in inducing cell-mediated response. Therefore, a less hemolytic saponin platycodin D (PD) from the root of Platycodon grandiflorum has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with Alum, antigen-specific cellular and humoral immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with PD and Alum in mice. The Con A-, LPS-, and HBsAg-induced splenocyte proliferation and the serum HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the HBsAg-immunized mice were significantly enhanced by PD (P < 0.05, P < 0.01 or P < 0.001). PD also significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines and up-regulated the mRNA expression of Th1 cytokines (IL-2 and IFN-γ) in splenocytes from the mice immunized with HBsAg (P < 0.001). Besides, PD remarkably increased the killing activities of natural killer (NK) cells and CTLs from splenocytes in the HBsAg-immunized mice (P < 0.001), which may have important implications for vaccination against hepatitis B virus. The results indicated that PD has strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response against HBsAg, and that PD may be the candidates as adjuvants for use in prophylactic and therapeutic hepatitis B vaccine.     

A novel approach for construction of an inactivated typhoid vaccine candidate that effectively augments both humoral and cellular immune responses

Salmonella enterica serovar Typhi ghost was constructed as a vaccine candidate against typhoid fever. An asd⁺ plasmid pJHL187 harboring a ghost cassette comprised of PhiX 174 E lysis gene stringently controlled under the convergent promotor components and was transformed into the asd gene-deleted mutant S. Typhi Ty21a strain (STG). Twenty female BALB/c mice randomly assigned into two groups were subcutaneously vaccinated at 5 weeks of age to assess immunogenic characteristics of the constructed STG. The level of serum IgG in the immunized mice was significantly increased during the observational period (P < 0.001) as the mice showed the significant elevation of secretory IgA at week 6 compared to those in the non-immunized mice (P < 0.05). The CD3⁺CD4⁺ T cell subpopulation in the primed splenocytes showed approximately twofold increase in the immunized group. Further, the gene expression of various immunomodulatory cytokines associated with Th-1, Th-2 and Th-17 immunity was observed in in vitro restimulated splenocytes isolated from the immunized mice. Serum Bactericidal activity of antibodies produced in the rabbits immunized with STG was proved by the elimination of almost all of wild-type S. Typhi in the presence of exogenous complement over an hour at week 6 after the first immunization. The immuno-stimulatory traits of STG demonstrated that the construct effectively enhanced the immunological responses, providing a potential of STG as the vaccine candidate against typhoid fever.     

Long-lasting humoral and cellular immune responses elicited by immunization with recombinant chimeras of the Plasmodium vivax circumsporozoite protein

The circumsporozoite protein (CSP), the most abundant surface antigen of sporozoites, has been extensively studied in different expression platforms as a vaccine candidate. Clinical trials have shown the necessity of broad and highly avid humoral immune responses together with high numbers of CSP-specific T_CD4+ and T_CD8+ cells, especially those producing IFN-γ, to induce protection. To this aim, we designed two distinct recombinant immunogens based on previously-described antigenic fragments of Plasmodium vivax CSP (PvCSP) to be used as vaccine candidates. The first one is a virus-like particle (VLP) comprising the repeat region of PvCSP (B and T_CD4+ epitopes) within the loop of the hepatitis B virus core antigen (HBcAgPvCSP). The second one is a PvCSP multi-epitope polypeptide, rPvCSP-ME, designed based on antigenic regions of PvCSP recognized by lymphocytes of individuals from endemic areas.     

Evaluation of humoral and cellular immune responses to BP26 and OMP31 epitopes in the attenuated Brucella melitensis vaccinated sheep

In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P = 0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.     

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C,a host defense peptide and polyphosphazine,elicits strong and long lasting cellular and humoral immune responses

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.     

Therapeutic vaccination with Salmonella-delivered codon-optimized outer inflammatory protein DNA vaccine enhances protection in Helicobacter pylori infected mice

Vaccination had demonstrated as an alternative way to combat Helicobacter pylori challenge. In the present study, codon-optimized outer inflammatory protein gene (oipA) for Mus species codon usage, the inclusion of optimal Kozak sequence, and modified of GC content was applied to construct a novel DNA construct. The Salmonella-delivered wild type oipA construct (SL7207/poipA) and the Salmonella-delivered codon-optimized oipA construct (SL7207/poipA-opt) were prepared and their therapeutic efficacy was evaluated in H. pylori-infected mice. The codon-optimized oipA construct (poipA-opt) expressed almost six-fold higher protein than that of wild type construct (poipA) as normalized to the β-actin expression in AGS cells. Oral therapeutic immunization with SL7207/poipA-opt significantly eliminated H. pylori colonization in the stomach; and protection was related to a robust Th1/Th2 immune response. Therefore, our results suggested that fine therapeutic efficacy was related to sufficient expression of the antigen. It is supposed that codon-optimized oipA gene improves protein expression and consequently enhances the immunogenicity of DNA vaccine, which resulted in a significant reduction of bacterial loads in H. pylori infected mice. The Salmonella-delivered codon-optimized DNA construct could be a candidate vaccine against H. pylori for the clinical application.     

A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis

An abscess in a gum pocket, resulting from bacterial infection, is a common source of chronic halitosis. Although antibiotics are generally prescribed for abscesses, they require multiple treatments with risks of creating resistant bacterial strains. Here we develop a novel vaccine using ultraviolet-inactivated Fusobacterium nucleatum (F. nucleatum), a representative oral bacterium for halitosis. A gum pocket model, established by continuous inoculation of F. nucleatum, was employed to validate the vaccine potency. Mice immunized with inactivated F. nucleatum effectively minimized the progression of abscesses, measured by swollen tissues of gum pockets. Most notably, the immunized mice were capable of eliciting neutralizing antibodies against the production of volatile sulfur compounds of F. nucleatum. The novel vaccine inducing protective immunity provides an alternative option to conventional antibiotic treatments for chronic halitosis associated with abscesses.     

Priming with a DNA vaccine delivered by attenuated Salmonella typhimurium and boosting with a killed vaccine confers protection of chickens against infection with the H9 subtype of avian influenza virus

Control of the circulation of H9 low-pathogenic avian influenza virus (LPAIV) is a major concern for both animal and public health. To improve vaccine efficacy against H9 LPAIV, we have utilized a novel prime–boost vaccination strategy. Specific-pathogen free (SPF) chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella typhimurium, followed by boosting with a killed avian influenza (AI) vaccine. Chickens in this combined vaccination group were completely protected against both oropharyngeal and cloacal virus shedding after intranasal challenge with H9N2 AIV, while viruses were detected from these sites in other vaccination groups. Prior to challenge, chickens in the prime–boost group also had higher (P < 0.05) serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA ELISA titers against AIV, and higher lymphoproliferation stimulation indices than those from other groups. Thus, we have demonstrated the efficacy of a novel prime–boost vaccination strategy against H9N2 avian influenza virus, which could be also applied for the development of vaccines against other mucosally infectious pathogens.     

Immunization with the conjugate vaccine Vi-CRM(197) against Salmonella Typhi induces Vi-specific mucosal and systemic immune responses in mice

Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM(197), composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM(197), is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM(197). The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM(197) was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM(197) and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM(197) as a promising candidate vaccine against Salmonella Typhi.     

HLA class II transgenic mice develop a safe and long lasting immune response against StreptInCor, an anti-group A streptococcus vaccine candidate

Streptococcus pyogenes infections remain a health problem in several countries because of post-streptococcal sequelae, such as rheumatic fever and rheumatic heart disease. We developed a vaccine epitope (StreptInCor) composed of 55 amino acid residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. Recently, by using human blood samples, we showed that the StreptInCor epitope is able to bind to different HLA class II molecules and that it could be considered a universal vaccine epitope. In the present work, we evaluated the immune response of HLA class II transgenic mice against aluminum hydroxide-absorbed StreptInCor. After a period of one year, several organs were analyzed histologically to verify the safety of the candidate vaccine epitope. Our results showed that StreptInCor is able to induce robust and safe and long lasting immune response without deleterious reactions in several organs. In conclusion, the results presented here indicate that StreptInCor could be considered a safe vaccine against severe streptococcus-induced diseases.     

Evaluation of the adjuvant properties of Astragalus membranaceus and Scutellaria baicalensis GEORGI in the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models

Human vaccines are not available and current anti-toxoplasma treatment is disappointing. To investigate the possible adjuvant effect of aqueous extracts obtained from medicinal herbs of Astragalus membranaceus (Am) and Scutellaria baicalensis GEORGI (Sb) on the immune response to Toxoplasma gondii in the mouse models induced by ultraviolet (UV)-attenuated T. gondii, this paper studies the possible vaccination strategies to help combat infections with Toxoplasma and looking towards developing new vaccine and approaches. We used UV-attenuated T. gondii (UV-T.g) of RH strain as a vaccine and the extracts of Am (AmE) and Sb (SbE) as adjuvant. Mice were infected by intraperitoneal (i.p.) injection of 10² RH tachyzoites alone (infected controls), infected and treated with AmE (T.g + AmE) and SbE (T.g + SbE), respectively; and mice immunized i.p. with UV-T.g alone, UV-T.g co-administrated with AmE (UV-T.g + AmE) or SbE (UV-T.g + SbE), and then challenged with T.g, respectively. The animal survival time, parasite burden in peritoneal lavage fluids, liver histopathological analysis, and levels of serum antibodies among the groups were compared after either infection or challenge. The results showed that, compared to infected controls, infected mice treated with AmE or SbE, or vaccinated mice and then challenged, had significantly prolonged survival time, decreased parasite burden, improved liver histopathological score, and increased Th1-type cellular immune response; furthermore, vaccinated mice co-administrated with AmE or SbE had even longer survival, lower parasite burden, lower liver histopathological score, and higher Th1 response after challenge. Our data demonstrated that the protective immunity of UV-attenuated T. gondii could be markedly enhanced by AmE or SbE co-administration, which suggests that both AmE and SbE may have the potential to be used as effective vaccine adjuvant.     

Immunostimulatory and antigen delivery properties of liposomes made up of total polar lipids from non-pathogenic bacteria leads to efficient induction of both innate and adaptive immune responses

Novel liposomes prepared from total polar lipids of non-pathogenic bacteria, viz. Leptospira biflexa serovar Potac (designated leptosomes) and Mycobacterium smegmatis (designated smegmosomes) were evaluated for their adjuvant effects with various antigen presenting cells (APCs), viz. murine macrophage cell line, J774A.1 and bone marrow derived dendritic cells (BMDCs). These liposomes induced strong membrane fusion as evident from resonance energy transfer (RET) assays and effectively transferred the fluorescent probe to the membrane of these APCs. Moreover, both vesicles caused significant activation of APCs as revealed by release of proinflammatory cytokines (IL-6, IL-12, TNF-α) and enhanced expression of co-stimulatory signals and maturation markers (CD80, CD86, MHCII), which was significantly higher for smegmosomes as compared to leptosomes. Additionally, activation of APCs by liposomes correlated with their ability to stimulate allospecific T cell proliferation and IFN-γ release. In contrast, conventional PC/chol liposomes failed to fuse and induced only a very low level of APC activation. Interestingly, the stimulatory activity of these lipid vesicles was restricted to APCs as they did not cause any significant activation or mitogenic effect on lymphocytes (B and T cells) in vitro. Overall, the activation of APCs by both leptosomes and smegmosomes correlated with activation of strong humoral and cell mediated immune responses in C57/BL6 mice to entrapped ovalbumin (OVA) and was significantly higher than those induced by conventional liposomes and alum, which failed to activate cytotoxic T lymphocytes (CTLs). Taken together these results demonstrate the adjuvant potential of these novel lipid vesicles that may simultaneously induce both innate and adaptive immune responses due to their immune stimulatory and antigen delivery properties.     

A novel hepatitis B vaccine containing Advax™, a polysaccharide adjuvant derived from delta inulin,induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing

Although current HBV vaccines have an outstanding record of safety and efficacy, reduced immunogenicity is a problem in those of older age, or having renal impairment or diabetes mellitus. In this study, we tested the ability of Advax™ adjuvant, a novel polysaccharide adjuvant based on delta inulin, to enhance the immunogenicity of hepatitis B surface antigen (HBs) in mice and guinea pigs by comparison to the traditional alum adjuvant. Advax™ provided antigen-sparing, significantly enhanced both anti-HBs antibody titers, and anti-HBs CD4 and CD8 T-cells, with increases in Th1, Th2 and Th17 cytokine responses. Unlike alum, the adjuvant effect of Advax™ was seen even when injected 24 h before the HBs antigen. Advax™ adjuvant similarly enhanced humoral and cellular immune responses in guinea pigs to a third generation preS-HBs antigen. Advax™ adjuvant when combined with HBs antigen could provide enhanced protection over current generation HBV vaccines for immunization of low responder populations.     

Genotypic screening of atypical Staphylococcus aureus strains isolated from clinical samples for presence of selected adhesin genes

Objective

The aim of this study was to screen Staphylococcus aureus negative for production of coagulase or clumping factor and for presence of selected adhesin genes.

Methods

Sixty coagulase-negative and 20 clumping factor-negative S. aureus strains were studied. Detection of methicillin resistance was performed using the agar screen technique with 6 mg/L of oxacillin and was confirmed by amplification mec A gene. The presence of bone binding protein (bbp), collagen binding protein (cna), fibronectin A binding protein (fnbA), fibronectin B binding protein (fnbB) and clumping factor A (clfA) genes was detected by multiplex PCR.

Results

Almost all (98%) of the strains were positive for clfA gene. There were fnbA and fnbB in 85%, cna in 54% and bbp in 5% of strains found. No correlation between presence of the particular genes and clinical samples was observed. The prevalence of fnbA, fnbB and cna was statistically higher in coagulase-negative than in clumping factor-negative strains (89, 89, 66 and 70, 70, 15%, respectively). Similarly, all of these genes were more often observed in MRSA than in MSSA atypical strains. The cna was detected only in coagulase-negative MRSA.     

Response of MUTZ-3 dendritic cells to the different components of the Haemophilus influenzae type B conjugate vaccine: towards an in vitro assay for vaccine immunogenicity

Potency testing is mandatory for vaccine registration and batch release. Due to various limitations to in vivo potency testing, there is need for relevant in vitro alternatives. These alternative tests should preferably comprise cells from the target (human) species. The whole suite of immune responses to vaccination that occur in vivo in humans cannot be tested in vitro using a single cell type. Even so, dendritic cells (DC) form an important candidate cell type since they are pivotal in inducing and orchestrating immune responses. Cell lines are preferred over ex vivo cells for reasons of safety, accessibility, and reproducibility. In this first feasibility study we used the human cell line MUTZ-3, because it most closely resembles ex vivo human DC, and compared its response to monocyte-derived DC (moDC).Haemophilus influenzae type B (HiB) vaccine was chosen because its components exert different effects in vivo: while the HiB antigen, polyribosyl ribitol phosphate (PRP) fails to induce sufficient protection in children below 2 years of age, conjugation of this polysaccharide antigen to outer membrane protein (OMP) of Neisseria meningitides, results in sufficient protection. Effects of PRP, OMP, conjugated PRP-OMP, and adjuvanted vaccine (PedVax HiB), on cytokine production and surface marker expression were established. PRP induced no effects on cytokine production and the effect on surface marker expression was limited to a minor decrease in CD209 (DC-SIGN). In both MUTZ-3 and moDC, OMP induced the strongest response both in cytokine production and surface marker expression. Compared to OMP alone conjugated PRP-OMP generally induced a weaker response in cytokine production and surface marker expression. The effects of PedVax HiB were comparable to conjugated PRP-OMP. While moDC showed a larger dynamic range than MUTZ-3 DC, these cells also showed considerable variability between donors, with MUTZ-3 DC showing a consistent response between the replicate assays. In our view, this makes MUTZ-3 DC the cells of choice. In conclusion, our results demonstrate that the MUTZ-3 DC assay allows discrimination between compounds with different immunogenicity. The potential of this cell line as (part of) an in vitro immunogenicity assay should be further explored.     

A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models

Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans.