Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model

Molecular Immunology 65 (2015) 51–57

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Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model Das Shreya, Siva R. Uppalapati, Joseph J. Kingston ∗ , Murali H. Sripathy, Harsh V. Batra Division of Microbiology, Defence Food Research Laboratory, Siddartha Nagar, Mysore 570011, Karnataka, India

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Article history: Received 6 November 2014 Received in revised form 7 January 2015 Accepted 8 January 2015 Keywords: Chimeric protein r-Cpae Clostridium perfringens type A Alpha toxin Enterotoxin Active protection Passive protection

a b s t r a c t Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (␣C) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of ␣C and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (␣C) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5 × LD100 doses of ␣C (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and ␣C and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91–100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2–3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against ␣C and CPE of C. perfringens type A toxemia. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Clostridium perfringens is an anaerobic, Gram positive, spore forming pathogen responsible for many serious nosocomial and community-acquired infections in humans and animals (Hiscox et al., 2014). The organism is classified into 5 different toxinotypes based on the variety of the toxins it produces (type A—␣ toxin; type B—␣ + ␤ + ␧ toxins; type C—␣ + ␤ toxins; type D—␣ + ␧ toxins and type E—␣ + ␫ toxins). Enterotoxin (CPE) may be produced by any toxinotype (Sakurai et al., 1997). C. perfringens type A is the most common among all the toxinotypes encountered in foods and soil and is responsible for majority of the wound and enteric infections (Gurjar et al., 2008). The pathogen infects the host through two main routes; cut or open wounds (generally non-enterotoxigenic C. perfringens type A) and intra-gastric route (enterotoxigenic

∗ Corresponding author. Tel.: +91 821 2579435; fax: +91 821 2473468. E-mail address: [email protected] (J.J. Kingston). http://dx.doi.org/10.1016/j.molimm.2015.01.005 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

C. perfringens type A). When the infection is set through open wounds, C. perfringens damages connective and muscle tissues and causes acute soft tissue infections like cutaneous abscesses, necrotizing muscular infections and gas gangrene (Stevens, 2000). Alternatively, during the oral route of infection, the pathogen destroys intestinal mucosa resulting in diarrhea, food poisoning, enteritis, etc. (Meer et al., 1997; Brynestad and Granum, 2002). Two toxins play a major role in setting up the aforementioned disease conditions during C. perfringens type A infections; alpha toxin (␣C), encoded by chromosomal cpa gene and CPE encoded by either a chromosomal or plasmid borne cpe gene. ␣C is the major virulence factor responsible for gas gangrene in humans and animals (Niilo, 1980; Gurjar et al., 2008) and is considered a marker toxin for C. perfringens (Titball et al., 1999). This 42 kDa protein is the first bacterial toxin demonstrated to be an enzyme with an Nterminal catalytic domain and a C-terminal binding domain (Naylor et al., 1998). It belongs to the zinc-metallophospholipase super family and possesses haemolytic, dermonecrotic, sphingomyelinase, myonecrotic and lecithinase activities (Rood and Cole, 1991).

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CPE, produced only during sporulation, is the major virulence factor in enterotoxigenic C. perfringens type A food poisoning. CPE has also been implicated in other gastrointestinal illnesses like antibiotic-associated diarrhea, chronic diarrhea and infantile diarrhea (Brynestad and Granum, 2002). This 35 kDa toxin elicits cell death by binding to claudins and forming pores on the intestinal epithelial and endothelial tight junctions. The pores cause paracellular permeability changes triggering influx of Ca2+ ions ultimately leading to abdominal cramps, epithelial desquamation and severe diarrhea (McClane, 2001). Currently, there are no licensed vaccines or toxoids against C. perfringens type A infections like gas gangrene or food poisoning approved for human use (Titball, 2009). The only accepted treatment regimen includes antimicrobials/antibiotics and anti-inflammatory drugs along with fluid therapy or surgical debridation of damaged tissue (Headley, 2003). Many efforts have been made in the development of an effective vaccine against C. perfringens gas gangrene (␣C) however, reports demonstrating in vivo protective efficacy of any candidate vaccine molecule against CPE challenge is still scarce. Traditionally, formaldehyde toxoids from crude culture filtrates of C. perfringens type A had been shown to induce protection against experimental gas gangrene (Roberston and Keppie, 1943; Owen-Smith and Matheson, 1968; Kameyama et al., 1975) however these vaccines exhibited varied efficacy (Nagahama et al., 1997). Many non-toxic recombinant ␣C variants with point mutations at critical amino acids were developed alternatively to combat gas gangrene (Guillouard et al., 1996; Nagahama et al., 1997). Sub-unit vaccines utilizing the Cdomain of ␣C were proven to protect animals against experimental gas gangrene (Williamson and Titball, 1993; Stevens et al., 2004; Thompson et al., 2006; Zekarias et al., 2008; Kulkarni et al., 2010; Uppalapati et al., 2014). On the other hand, Mietzner et al. (1992) established that the C-terminal binding region of CPE (C-CPE) is capable of generating antibodies that neutralize CPE toxicity in vitro while its in vivo protective capacity has not yet been proved. Considering the indispensable role of ␣C and CPE in the pathogenesis and the rapid emergence of multi-drug resistance patterns, if an effective multivalent candidate vaccine were to be developed against C. perfringens type A, it should incorporate the immunodominant epitopes from both the toxins. The immense potential of structural biology in designing and developing effective vaccines has led to the emergence of a new branch of science; structural vaccinology, which aims at identification of protective domains/epitopes in the immunogenic proteins of a particular pathogen or multiple pathogens to rationally design and construct synthetic protein chimeras comprising two or more such domains (Dormitzer et al., 2008; Rinaudo et al., 2009; Nuccitelli et al., 2011). The major drawback of differential immunogenicity due to competition between the cocktail components in conventional multivalent vaccines can be effectively overcome by this strategy. (Singh et al., 2014). In the present study, following structural vaccinology strategy, an attempt was made to develop a candidate bivalent sub-unit vaccine against C. perfringens type A toxemia by splicing the immunodominant binding regions of C. perfringens cpa and cpe genes through glycine linker. The chimeric gene was directionally cloned into a prokaryotic expression system and the fusion protein generated was evaluated for its protective efficacy.

2. Materials and methods 2.1. Animals Specific-Pathogen-Free (SPF) 4–6-week-old female BALB/c mice were procured from Central Animal Facility, Defence Food Research

Laboratory (D.F.R.L.), Mysore, India. The mice were acclimatized to laboratory conditions for one week before the experiments and provided with food and water ad libitum. All animal experiments in this study have been approved and performed in accordance with guidelines of Institutional Animal Ethical Committee, D.F.R.L., Mysore.

2.2. Toxins Native wild type ␣C ((P7633) was procured commercially (Sigma, Bangalore, India) and crude CPE was extracted according to Granum and Whitaker (1980). Wild type ␣C and purified CPE proteins were sterilized by 0.2 ␮m filtration and stored in aliquots at −20 ◦ C before their use in in vitro and in vivo studies. 2.3. Cell lines and media Caco-2 cell line was procured from National Centre for Cell Science, Pune, India. Cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% FBS, 50 units/ml penicillin, and 50 ␮g/ml streptomycin. The cells were maintained at 37 ◦ C in 5% CO2 . All the cell culture media, reagents and chemicals were purchased from Sigma-Aldrich (India), unless mentioned otherwise.

2.4. Construction of cpae chimeric gene All the gene sequences were retrieved from NCBI database and primers were custom synthesized from Eurofins, Bangalore. Chimeric gene cpae was constructed, cloned, expressed and purified as soluble protein under non-denaturing conditions as mentioned in Supplementary File.1.

2.5. Immunization protocol Two groups of 12 mice each received a primary sub-cutaneous (s.c.) injection of 30 ␮g r-Cpae antigen (in 0.1 ml volume) with Freund’s complete adjuvant in 1:1 (v/v) ratio. Subsequently, on days 7, 21 and 35, all the animals received booster s.c. doses with similar concentrations of r-Cpae antigen in Freund’s incomplete adjuvant. Two control groups, each with 12 mice were sham-immunized with a similar volume of 1× sterile PBS (pH 7.4 ± 0.2) in adjuvant. One group each of r-Cpae immunized and sham immunized mice was maintained for ␣C challenge while the remaining groups of mice were kept for CPE challenge. Blood samples were drawn periodically (days 0, 14, 28, 42) through retro-orbital plexus and the sera were collected and stored in −20 ◦ C until further use. 2.6. Antibody titers The r-Cpae specific serum IgG titers were measured using two fold serial dilutions of anti-r-Cpae sera (from 6 randomly selected r-Cpae immunized mice) by indirect ELISA as described earlier (Uppalapati et al., 2012). End-point titres were determined as the maximum antibody dilution whose mean O.D. was twice more than the mean O.D. value of control sera. The antigenic competition by each of the components in r-Cpae was also evaluated by indirect ELISA, where 10 ␮M each of ␣C, CPE and r-Cpae were coated onto microtitre plate and probed with 1:1000 dilution of pooled 42nd day anti-r-Cpae sera. Absorbance was measured thrice at a wavelength of 492 nm in 1 min intervals (Infinite M200 PRO; Tecan, Grodig, Austria) and the mean O.D. ± S.D. values were plotted on a graph.

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2.7. In vitro toxin neutralization assay (TNA) Sera collected at 42nd day from 6 randomly selected r-Cpae immunized and sham immunized mice were pooled, two-fold diluted in saline (pH 7.4) and heated at 56 ◦ C for 20 min for complement inactivation prior TNA. The ␣C and CPE neutralization capacity of anti-r-Cpae-sera was evaluated as a function of toxin induced cytoxicity on mouse RBCs (mRBCs) (hemolytic units, H.U.) and Caco-2 cells (cytotoxic units, C.U.), respectively. One toxicity unit was considered to be the amount of toxin required to either increase the absorbance of the supernatant of 50% mRBCs by one H.U. or increase the absorbance of the MTT reagent in a completely confluent well by one C.U. ␣C neutralization assay was performed as described previously (Uppalapati et al., 2012). For CPE neutralization assay, Caco-2 cells (106 cells/well) were exposed to different concentrations of CPE (in 100 ␮l volume) pre-incubated (at 37 ◦ C for 1 h) with equal volumes of complement inactivated anti-r-Cpae or sham immune sera and incubated at 37 ◦ C for 4 h in 5% CO2. The percentage cell viability in each treatment was measured by MTT based in vitro toxicology assay kit (Sigma, Bangalore, India), following manufacturer’s instructions and plotted against CPE concentrations. Caco-2 cells and mRBCs treated with PBS were maintained as negative control.

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of mice (n = 12) received toxins incubated in the sera from shamimmunized mice. The animals in both active and passive challenge experiments were monitored for morbidity and mortality for 15 days post challenge and the survival percentage in each group was represented by Kaplan-Meier Graph. The LD100 value was defined as the smallest dilution of toxin in 0.5 ml volume causing 100% lethality of mice (n = 10) within 24 h post injection. In our study, one LD100 value was found equivalent to 2.5 × 103 H.U./kg and 5 × 104 C.U./kg mouse weight for ␣C and CPE, respectively. 2.9. Detection of intestinal mucosal secretary IgA (s-IgA) The intestinal mucosal s-IgA was extracted as per Zeng et al. (2011). Briefly, post removal of the intestinal contents, the ileal mucous of r-cpae immunized and sham immunized was washed with 1.0 ml of Protease inhibitor tablet and vortexed for 30 s followed by centrifugation for 10 min to remove the debris. The resulting extract was further filtered by passing through 0.45 ␮m filter and amount of total protein present was quantified by Bradford assay using known standards. The r-Cpae specific s-IgA was assayed by mouse isotyping kit as per antigen mediated ELISA procedure described by the manufacturer (Sigma-Aldrich, India). The experiment was performed in triplicate and the mean O.D. ± S.D. values were plotted on a graph.

2.8. Toxin challenge studies

2.10. Histopathology

On the 45th day of the immunization schedule, mice were challenged either with 0.2 ml of 5 × LD100 doses of ␣C (diluted in sterile PBS, pH 7.4) or 0.5 ml of 5 × LD100 doses of CPE (diluted in 0.25 M carbonate buffer, pH 9.5) through intra-muscular (i.m.) and intra-gastric gavage (i.g.) routes, respectively. For passive transfer studies, 5 × LD100 dose of ␣C was made to 0.25 ml in PBS and incubated for 1 h at 37 ◦ C with equal amounts of two-fold diluted complement inactivated anti-r-Cpae sera as prepared above. Alternatively, 0.1 ml 5 × LD100 dose of CPE was mixed with equal volume of anti-r-Cpae sera and 0.25 ml of 0.25 M carbonate buffer, pH 9.5, to help protect the toxin and antibodies against acid degradation in the stomach. The ␣C mixtures were administered i.m. and the CPE mixtures were administered i.g. to 2 different groups of naïve female BALB/c mice (n = 12 per group). Control groups

Before necropsy, representative animals from each group were humanely sacrificed for histopathology analysis. Muscles tissues and iliac regions of small intestine were fixed in 10% formaldehyde solution. The specimens were paraffin embedded, sectioned and stained with haematoxyline–eosine. Tissues were observed with a Nikon Eclipse Ni-E upright bright-field microscope under 40× objective. Images were captured using NIS-Elements imaging software (Nikon, India). 2.11. Statistical analysis The data were presented as mean ± S.D. Mantel-Cox (logrank) test was used to compare the survival curves and Student’s ttest was used for other statistical comparisons. All graphical

Fig. 1. Design and expression of r-Cpae. (A) Schematic representation of the design of chimeric protein r-Cpae. Crystal structures of C. perfringens ␣C (PDB ID:1CA1) and CPE (PDB ID:2XH6) were drawn by Cn3D software. Both the domains are connected by G4 S linkers. (B) 12% SDS-PAGE of whole cell lysates of uninduced and IPTG induced r-Cpae recombinant E. coli clones stained with Coomassie Blue. Arrow mark indicates the expressed recombinant protein. UI, Uninduced clone; I, induced clone; P, purified protein; M, marker.

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illustrations were constructed. Graph Pad Prism 5 software. Significance (P) value summary: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ****P ≤ 0.0001. 3. Results 3.1. Characterization of r-Cpae The r-Cpae chimeric protein, encompassing non-toxic immunodominant C-terminal regions of ␣C and CPE was designed using structural vaccinology approach (Fig. 1A). The r-Cpae chimeric gene (731 bp) was synthesized by interlinking the two regions through a G4 S spacer by SOE-PCR strategy, cloned and expressed in Escherichia coli BL21(DE3) and the integrity of cloned gene in recombinant plasmid pRSET A-r-Cpae was authenticated by sequencing. The expression of 27 kDa 6 × Histidine tagged r-Cpae fusion protein in E. coli BL21(DE3) host was detected on the SDS-PAGE gel stained with Coomassie blue (Fig. 1B). A total of ∼20 mg r-Cpae per gram of bacterial pellet was obtained after purification by immobilized metal affinity chromatography under non-denaturing conditions.

Fig. 3. Western blot analysis of r-Cpae protein (lane 1), CPE (lane 2) and ␣C (lane 3) proteins using anti-r-Cpae sera.

A substantial and progressive induction of anti-r-Cpae antibodies was noted in the sera of s.c. immunized BALB/c mice (Fig. 2A). After 3 subsequent boosters with the chimera, the average titer levels reached to a final of 1: 64,000 (antilog 4.712 ± 0.3098) with almost equivalent IgG serum titers against ␣C and CPE regions (Fig. 2B) implying minimal antigenic competition. The control sera, in contrast, showed no background levels of r-Cpae specific immunoglobulins. Also, hyperimmune antiserum against r-Cpae specifically reacted with the wild-type toxins demonstrating lucid bands at 42 and 35 kDa in Western blot analysis (Fig. 3).

mice (12/12) died due to severe toxemia within 2 days post challenge (Fig. 4A). The difference in the survival percentages between r-Cpae immunized and control mice was statistically significant (P**). Necropsy of muscle tissue and intestine from control animals revealed acute muscular ischemia and extremely weak and friable intestine, respectively. Histopathological analysis revealed muscle tearing, interfibrillar oedema and neutrophil extravasion from adjacent vasculatures denoting acute myonecrosis in the thigh muscle, destruction of normal architecture of intestinal villi, detachment of the epithelial layer from the lamina propria and lesions in the small intestine of the control mice. In contrast, tissues from surviving r-Cpae immunized mice exhibited inconspicuous damage on gross or histopathological analysis (Fig. 4B).

3.3. Immunization with r-Cpae protects mice against ˛C and CPE induced toxaemia

3.4. Immunization with r-Cpae elicits intestinal mucosal s-IgA production

Groups of mice immunized with r-Cpae or PBS were challenged with 5 × LD100 doses of ␣C or CPE. Amongst the r-Cpae immunized mice; 12/12 and 9/12 survived ␣C and CPE challenge respectively during 15 day observation time, whereas all the sham immunized

Owing to the pivotal role played by gastro-intestinal s-IgA in mucosal immune protection against enteric toxins, we explored whether immunization with r-Cpae was capable of increasing intestinal mucosal s-Ig A levels in mice. Ileal mucus of r-Cpae

3.2. Immunogenicity of r-Cpae in BALB/c mice

Fig. 2. Determination of r-Cpae induced anti-r-Cpae antibodies in immunized BALB/c mice. (A) Sera was collected on 0th, 14th, 28th and 42nd day from 6 randomly selected r-Cpae immunized mice and total anti-r-Cpae antibodies evoked by r-Cpae immunization were assessed by indirect ELISA and end point titers were plotted. (B) Determination of ␣C and CPE specific serum IgG titers in the 42nd day (1:1000th dilution) anti-r-Cpae sera. The experiments were performed in triplicates and the data are represented in mean ± S.D.

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Fig. 4. Percentage survival, gross pathology and histopathology analysis of mice challenged with ␣C or CPE. (A) Groups of sham immunized and r-Cpae immunized female BALB/c mice (n = 12) were injected either with 5 × LD100 units of ␣C i.m. or CPE i.g. 10 days post final booster and survival was monitored for 15 days. The protective efficacy of r-Cpae sub unit vaccine was calculated by Kaplan-Meier’s method to compare percentage survival curves. (B) Gross pathological and histopathological analysis of muscle and intestines of challenged animals. Sections were stained with hematoxylin and eosin. (A, B, G, H) Gross pathological changes in the muscles and intestine of r-Cpae immunized and sham immunized mice upon toxin challenge. Acute muscular inflammation (B) and intestinal blackening (H) can be observed in sham immunized group. (D, E, J, K) Histopathological changes in muscles and intestinal tissue of the sham immunized and r-Cpae immunized mice upon challenge. Necrosis (E) and intestinal disintegration (K) can be observed in sham immunized group. Recombinant Cpae immunized group (D,J) did not show any pathological characters and are comparable to naïve animal tissues (F, L).

immunized mice exhibited significant increase in r-Cpae specific s-IgA levels (P***), whereas the sham immunized mice exhibited negligible amounts or background levels of r-Cpae specific intestinal mucosal s-IgA (Fig. 5). 3.5. Anti-r-Cpae specific antibodies neutralize ˛C and CPE in vitro and in vivo. The ␣C and CPE neutralizing ability of the anti-r-Cpae sera was evaluated as a function of toxin induced cell death. Mouse RBCs

incubated with control serum and ␣C (6.0 H.U.) resulted in 99.9% cell death within 2 h whereas those incubated with anti-r-Cpae sera and toxin showed only 18.8% cell death (Fig. 6A). Alternatively, only 10% of Caco-2 cell monolayers treated with control sera were found viable after 2 h exposure to CPE (4.0 C.U.) while the viability was 89.5% in case of anti-r-Cpae polysera treated Caco-2 cells (Fig. 6B). The in vivo correlates of the ␣C and CPE neutralization by anti-rCpae antibodies were analyzed by passive immunization studies. Majority of the animals injected with ␣C (12/12) or CPE (11/12) along with anti-r-Cpae sera survived the challenge up to 15 days, whereas all the control mice (12/12) injected with ␣C or CPE along with sham immune serum succumbed to toxin challenge within 48 h (Fig. 7). The difference in the survival percentages between r-Cpae immunized and control mice was statistically significant (P**). These observations ascertained that anti-r-Cpae sera effectively neutralize wild-type ␣C and CPE under in vitro and in vivo conditions.

4. Discussion

Fig. 5. Determination of r-Cpae specific intestinal mucosal s-IgA antibodies in immunized BALB/c mice. (A) Total intestinal mucosal protein was collected on 0th, 14th, 28th and 42nd day from 6 randomly selected r-Cpae immunized and sham immunized mice and total r-Cpae specific intestinal mucosal s-IgA antibodies were assessed by mouse isotyping kit as per antigen mediated ELISA procedure described by the manufacturer (Sigma, Bangalore, India). The experiment was performed in triplicate and the mean O.D. ± S.D. of the absorbance values were plotted on a graph.

Designing multivalent candidate vaccines involving rationally chosen two or more sub-units representing the virulence associated antigenic repertoire of the targeted pathogen has the potential to provide protective immunity against multiple-diseases and hence would be convenient for administration, cost-effective and amenable for stockpiling (Hamad, 2011). The splicing of individual subunits to form a single functional antigen with protective immune response in hosts is made possible by the structural vaccinology approach where in multiple immunodominant regions are combined via a flexible spacer to avert conformational steric hindrances from the individual domains (Nuccitelli et al., 2011; Singh et al., 2014; Uppalapati et al., 2014). In the present study, we

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Fig. 6. Anti-cytolytic property of r-Cpae hyperimmune antisera demonstrated by toxin neutralization assay. (A) Different concentrations of native ␣C (H.U.) were incubated in equal volumes of complement inactivated 2 fold diluted anti-r-Cpae sera or sham sera in saline (pH 7.4) or PBS for 1 h. The dilutions were mixed with 2% mRBC suspension and after 1 h incubation at 37 ◦ C; the extent of haemolysis was measured by recording absorbance of supernatants at 544 nm. The mean percentage death of mRBCs ± S.D. was plotted against toxin concentration (H.U.). The toxicity inhibition was also demonstrated on Blood agar plates. (B) Different concentrations of CPE (C.U.) were incubated in equal volumes of 2 fold diluted anti-r-Cpae sera or sham sera in saline (pH 7.4) or PBS for 1 h. All dilutions were added to 1 × 105 Caco-2 cells/well and after 4 h incubation, cell viability was determined by MTT assay. The mean percentage viability of mRBCs ± S.D. was plotted against toxin concentration (C.U.). Microscopic images showing CPE induced cell death inhibition by anti-r-Cpae sera were shown.

designed a novel bivalent r-Cpae structural vaccine and established its protective efficacy against type A C. perfringens toxemia. The r-Cpae molecule was constructed by rationally choosing the host cell membrane binding ‘C’ terminal regions of C. perfringens ␣C and CPE (Sakurai et al., 2004; Nagahama et al., 2002; Van Itallie et al., 2008). The C-domain of ␣C has been reported to provide active and passive protection in immunized animals (Williamson and Titball, 1993; Stevens et al., 2004; Thompson et al., 2006; Zekarias et al., 2008; Kulkarni et al., 2010; Uppalapati et al., 2014) while, the region between 171 and 309 amino acids of CPE have been proven to neutralize CPE toxicity in vitro on Vero cell line and rabbit

Fig. 7. Passive protection of mice against ␣C and CPE toxemia. Two groups of SPF 4–6-week-old naïve female BALB/c mice (n = 12 per group) were injected i.m. and i.g. with ␣C and CPE mixtures in anti-r-Cpae sera as described in Materials and Methods. Control groups of mice received similar mixtures in polysera from sham immunized mice. The animals were observed for the next 15 days for their mortality rates. Protective efficacy of r-Cpae subunit vaccine was calculated using Kaplan-Meier’s method to compare percentage survival curve.

intestinal brush border membranes by blocking the binding of CPE to its receptors (Mietzner et al., 1992). The strategy of employing C-domains of ␣C and CPEs in vaccine design has two major advantages; firstly the recombinant vaccine molecule would be non-toxic as the catalytic and the pore forming domains of ␣C and CPE (Naylor et al., 1998; Mietzner et al., 1992; Kokai-Kun and McClane, 1997), respectively, are excluded and secondly, the chimera in its native form can bind to host cells thereby increasing the exposure time of antigen to the host immune system. Recombinant Cpae chimera did not show any toxicity to mouse erythrocytes or Caco-2 cells although it bound to Caco-2 cell membranes (data not shown). The chimeric protein was found to be immunogenic with r-Cpae immunizations generating high titers of circulating antibodies that reacted lucidly with both ␣C and CPE in western blot. This further confirmed that fusion of the truncated ␣C and CPE through G4S linker prevented steric hindrances between the two components and possibly the native immunodominant epitopes from either of the components were maintained in the chimera (Singh et al., 2014; Kolli et al., 2006). Moreover, both the ␣C and CPE regions of r-Cpae generated equivalent amounts of specific antibodies (Fig. 2B) setting aside the possibility for antigenic competition seen in the case of cocktail vaccines elsewhere (Hunt et al., 1994; Singh et al., 2014) and implying that immunization with r-Cpae would generate equal protection against both the toxins. C. perfringens type A toxemia in mammalian hosts proceeds either through wounds or via oro-gastric route depending on the toxins produced. Therefore for complete protection against type A toxemia, a vaccine molecule capable of generating both systemic and mucosal immunity is ideally required to neutralize the soft tissue and intestinal damages mediated via ␣C and CPE, respectively. Active immunization of mice with r-Cpae generated serum IgG (systemic), substantially increased the intestinal mucosal s-IgA antibody titres and provided significant protection to the immunized animals with petite or no tissue morbidity when challenged with either of the toxins. The upsurge of intestinal s-IgA antibodies from a parentral route of immunization is in accordance with (Zeng et al., 2011) wherein s.c. immunization with C. perfringens toxoids

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was able to significantly increase the levels of intestinal mucosal s-IgA. The anti r-Cpae sera effectively neutralized wild type ␣C and CPE in both in vitro and in vivo assays. Mouse RBCs and Caco2 cells exposed with concoction of anti-r-Cpae antibodies and ␣C and CPE, respectively, resulted in significantly higher protection against the respective toxins while mice administered with a similar mixture exhibited reduced signs of distress and majority of them survived the observation period (Fig. 7). Thus, anti r-Cpae antibodies qualify as an effective systemic antitoxin providing protection against both ␣C and CPE toxemia. Taken together, the protection due to active immunization of r-Cpae and induction of neutralizing antibodies demonstrate the exceptional efficacy of this novel chimera in conferring protection against ␣C and CPE and paves the way for further research to be undertaken in terms of cellular immune responses and memory responses elicited by r-Cpae. Moreover, the fact that C-terminal of r-Cpae encompasses CCpe a proven mucosal adjuvant (Suzuki et al., 2010; Kakutani et al., 2010) holds promise for further research in terms of development of r-Cpae as a mucosal vaccine. To the best of our knowledge, this is the first report of a candidate vaccine eliciting protective immune response against CPE (a potent food poisoning agent) along with alpha toxin, the two most potent C. perfringens type A toxins. Acknowledgements S.D. is funded by Junior Research Fellowship from Defence Research and Development Organization, India. S.R.U. is supported by Senior Research Fellowship of Council for Scientific and Industrial Research, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molimm. 2015.01.005. References Brynestad, S., Granum, P.E., 2002. Clostridium perfringens and foodborne infections. Int. J. Food Microbiol. 74 (3), 195–202. Dormitzer, P.R., Ulmer, J.B., Rappuoli, R., 2008. Structure-based antigen design: a strategy for next generation vaccines. Trends Biotechnol. 26, 659–667. Granum, P.E., Whitaker, J.R., 1980. Improved method for purification of enterotoxin from Clostridium perfringens Type A. Appl. Environ. Microbiol. 39, 1120–1122. Guillouard, I., Garnier, T., Cole, S.T., 1996. Use of site-directed mutagenesis to probe structure-function relationships of alpha toxin from Clostridium perfringens. Infect. Immun. 64, 2440–2444. Gurjar, A.A., Hegde, N.V., Love, B.C., Jayarao, B.M., 2008. Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle. Mol. Cell Probes 22, 90–95. Hamad, M., 2011. Universal vaccines: shifting to one for many or shooting too high too soon! APMIS 119, 565–573. Headley, A.J., 2003. Necrotizing soft tissue infections: a primary care review. Am. Fam. Phys. 68, 323–328. Hiscox, T.J., Ohtani, K., Shimizu, T., Cheung, J.K., Rood, J.I., 2014. Identification of a two-component signal transduction system that regulates maltose genes in Clostridium perfringens. Anaerobe 30, 199–204. Hunt, J.D., Jackson, D.C., Brown, L.E., Wood, P.R., Stewart, D.J., 1994. Antigenic competition in a multivalent foot rot vaccine. Vaccine 125, 457–464. Kakutani, H., Kondoh, M., Fukasaka, M., Suzuki, H., Hamakubo, T., Yagi, K., 2010. Mucosal vaccination using claudin-4-targeting. Biomaterials 31, 5463–5471. Kameyama, S., Sato, H., Murata, R., 1975. The role of ˛-toxin of Clostridium perfringens in experimental gas gangrene in guinea pigs. Jpn. J. Med. Sci. Biol. 25, 200. Kokai-Kun, J.F., McClane, B.A., 1997. Deletion analysis of the Clostridium perfringens enterotoxin. Infect. Immun. 65, 1014–1022.

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