key: cord-0025828-1rgm36kt
authors: Singh, Ankita; Rawat, Prashant; Choudhury, Devapriya; Dixit, Aparna
title: Immunogenic and neutralization efficacy of recombinant perfringolysin O of Clostridium perfringens and its C-terminal receptor-binding domain in a murine model
date: 2022-01-15
journal: Immunol Res
DOI: 10.1007/s12026-021-09254-9
sha: 1cca57f2628a1104a071508e64f80c273874e077
doc_id: 25828
cord_uid: 1rgm36kt

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12026-021-09254-9.

Clostridium perfringens is a fast-growing, spore-forming, Gram-positive, rod-shaped anaerobe of the Bacillaceae family [1, 2] . It is widely present in different habitats such as soil, food, sewage, and aquatic ecosystems (marine, estuarine, and freshwater) and colonizes the gastrointestinal tract of animals and humans [3, 4] . Its ubiquitous presence in the environment is responsible for causing many histotoxic and enteric diseases in humans and animals [5] . This bacterium secretes more than 20 extracellular enzymes and toxins that are believed to be potent lethal factors behind pathogenicity [6] . Massive secretion of these toxins results in an outbreak of a broad range of diseases in animals and humans, such as gas gangrene, necrotic enteritis, food poisoning, and numerous enterotoxaemia [7, 8] . Further, it is believed to be responsible for local and systemic tissue damage and the rapid death of bovines [6, 9] .

C. perfringens strains are classified into seven toxinotypes (types A, B, C, D E, F, and G) based on the production of different toxins, namely α, β, ε, ί, CPE, and NetB [10] . Perfringolysin O (PFO) is secreted by all the strains, and it is believed to assist other toxins of C. perfringens in the onset and progression of various Clostridia-associated diseases [11] . PFO acts in synergy with α-toxin that causes gas gangrene and bovine necrohemorrhagic enteritis [12] [13] [14] . It has also been reported to augment the virulence effect of ε-toxin in causing type D enterotoxemia in a mouse model [12] [13] [14] . Despite the role that the PFO plays in clostridial diseases, it has not been explored for its immunogenic and vaccine potential. It has rightly been referred to as "the underrated Clostridium perfringens toxin" due to lack of attention from the research community [15] . After exposure to C. perfringens without any apparent premonitory clinical signs of necrohemorrhagic enteritis, the quick death of animals demands a safe and efficient preventive measure such as vaccination to control clostridial infections [1, 16] . In the past decades, efficient genetically engineered and denatured toxoid-based vaccines have been commercially developed that could confer protective immunity to economically important livestock, including sheep, goats, and bovines, against C. perfringens-associated infections [17, 18] . However, the protective effect of formaldehyde denatured toxoidbased antibodies generated in calves was diminished in the intestinal loop model for bovine necrohemorrhagic enteritis compared to antibodies against native toxins [19] . Since active toxins cannot be considered safe for vaccine development, alternative non-toxic variants of the native toxins need to be assessed for the development of safe and efficient vaccines. Genetically modified toxoids with reduced toxic activity or immunologically active fragments of the essential toxins in this regard could be a good choice. Though recombinant ε, β, and α toxins have been assessed for their vaccine potential [20] [21] [22] [23] [24] , their use is restricted against the specific toxinotypes of C. perfringens. The PFO, secreted by all toxinotypes of C. perfringens, has been reported as the second most immunogenic toxin of C. perfringens type A after the α-toxin and thus highlights the importance of PFO as a potential candidate for vaccine development against C. perfringens [19] .

The crystal structure of the PFO monomer revealed that it could be divided into four domains, domain 1 (D1), domain 2 (D2), domain 3 (D3), and domain 4 (D4) or C-terminal domain predominantly made up of β-strands. Domain 1 encompasses amino acid residues 37-53, 90-178, 229-274, and 350-373; domain 2 encompasses amino acid residues 54-89 and 374-390, and domain 3 encompasses amino acid residues 179-228 and 275-349; thus, these are not in a continuous way. However, in the C-terminal domain or domain 4, the residues are continuous (391-500) [25] . The C-terminal domain (domain 4) is the smallest functional unit of PFO, responsible for the cholesterol recognition and binding of the toxin to cholesterol-containing membranes [26, 27] . The isolated C-terminal domain was reported to be non-toxic and could recognize and bind with membrane cholesterol [28] .

The current study was therefore undertaken to produce soluble recombinant PFO using heterologous expression system and evaluate its immunogenicity in the mice model. Furthermore, since the whole toxin has been reported to be toxic to the host cell, we also assessed the immunogenicity of its receptor-binding domain, i.e., C-terminal domain produced using recombinant routes in parallel with WTrPFO. Further, the antisera generated against the full-length mature PFO and the C-terminal domain was assessed for its neutralizing potential in vitro.

All chemicals used in the study were of molecular biology grade. The chemicals and the bacterial growth medium used in the study are acquired from Sisco Research Laboratories Pvt. Ltd. (SRL), India, and Sigma-Aldrich Chemical Co., USA. PageRuler Protein molecular weight markers were from New England Biolab, USA. Ni 2+ -NTA agarose and the bicinchoninic acid (BCA) protein estimation kit were purchased from G-Biosciences, USA. Monoclonal anti-polyhistidine, anti-mouse-IgG (Fc specific)-alkaline phosphataseconjugated antibody produced in goat was procured from Sigma-Aldrich Chemical Co., USA. Cell proliferation kit (XTT based) and Pierce® LDH cytotoxicity assay kit were purchased from Biological Industries and Thermo Fisher Scientific, USA, respectively.

E. coli strains DH5α and BL21(λDE3)pLysS cells used to propagate the plasmid DNA and recombinant protein expression, respectively, were procured from Novagen, USA.

The Institutional Animal Ethics Committee of the University approved the use of animals for the immunization study (project code IAEC # 03/2019). All procedures were performed as per the guidelines and recommendations of the committee. The Inbred Swiss albino mice (female, aged 4-6 weeks old, 18±2 g) were obtained from the Central Laboratory Animal Resources (CLAR), Jawaharlal Nehru University, New Delhi. The mice were housed (n= 5 mice/ group/cage) in fixed light and dark conditions and were kept on drinking water (RO) and food ad libitum.

Synthetic gene constructs harboring the mature full-length pfo of C. perfringens (without the signal sequence) and its deletion variant harboring only the C-terminal domain comprising 390 to 499 (PFO C-ter ) residues were designed based on the sequence information available for C. perfringens pfo gene (GenBank Accession number: M36704). The respective synthetic genes cloned into expression vector pET22b(+) at the NdeI and XhoI restriction sites were procured from GenScript, USA, and designated pET22.PFO wt and pET22.PFO C-ter , respectively. The recombinant proteins WTrPFO and rPFO C-ter encoded from the two constructs would be 482 aa (~54 kDa) and 119 aa (~14 kDa) residues long (including the amino acids contributed from the vector), respectively.

Expression analysis of the recombinant proteins (WTrPFO and rPFO C-ter ) was carried out essentially as described earlier [29] . Briefly, secondary cultures (prepared with 1% of overnight culture) of the E. coli BL21 (λDE3)pLysS harboring the plasmids pET22.PFO wt and pET22.PFO C-ter for recombinant expression, respectively, were induced with 0.8 mM IPTG for 8 h. An aliquot (1 ml) of the uninduced culture was taken out prior to induction. The cells were harvested (6000 rpm, 4 °C, 10 min; Eppendorf centrifuge 5415R, UK), and the cell lysates were analyzed on 12% SDS-PAGE.

Localization of expression analysis was carried out as described earlier [30] . Briefly, the induced cell lysates of the E. coli BL21(λDE3)pLysS cells expressing WTrPFO and rPFOC-ter were centrifuged at 13000 rpm (Eppendorf centrifuge 5415R, UK) for 30 min at 4 °C to prepare the soluble (supernatant) and insoluble (pellet) fractions. The pellet was treated with 8 M urea buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 8 M urea) to solubilize the proteins. The fractions were analyzed by SDS-PAGE (12%), followed by Coomassie Brilliant Blue (CBB-R250) staining.

Optimization of induction time for maximum expression was performed by inducing the cultures with 0. 8 

Large-scale purification of the two recombinant proteins (WTrPFO and rPFO C-ter ) from the soluble fraction was done using Ni 2+ -NTA affinity chromatography essentially as described earlier [30] . The soluble fraction of the cell lysates was prepared from the induced cell cultures (1 mM IPTG at 16 °C for 22 h). The soluble fraction was incubated with the Ni 2+ -NTA agarose resin. After removing the non-specifically bound proteins by thoroughly washing with wash buffer (20 mM imidazole, 20-mM potassium phosphate buffer pH 8.0, 500 mM NaCl, 1 mM PMSF), specifically bound recombinant proteins were eluted using 5 ml aliquots of wash buffer containing different imidazole concentrations. The eluted fractions along with flow-through were analyzed on 12% SDS-PAGE. The fractions showing pure proteins were pooled and dialyzed against 20-mM potassium phosphate buffer (pH 8) and stored in small aliquots at −80 °C for further use.

Western blotting was carried out as described earlier [31] . The cell lysates prepared from the uninduced and induced cultures or purified proteins were resolved by 12% SDS-PAGE. The proteins were transferred to the nitrocellulose membrane at constant voltage (50 V for 2 h at 4 °C) using 1× transfer buffer [25 mM Tris-HCl, pH 8.3; 192 mM glycine; and 20% (v/v) methanol]. The membrane was then incubated in blocking buffer [2% BSA in 1× PBST solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4, and 0.05 % Tween 20) overnight at 4 °C followed by three 1× PBST washes of 10 min each. The membrane was then incubated with the primary antibody (1:10,000 in PBS) for 1 h at RT. After thoroughly washing with 1× PBST, the membrane was incubated with secondary antibody [1:10000; alkaline phosphatase (AP)-conjugated goat anti-mouse/rabbit antibody] at RT for 1 h, followed by 1× PBST washes as before. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP)/ nitroblue tetrazolium (NBT)] substrate was added to develop the color. The image was captured by Gel-doc (Bio-Rad Laboratories, Inc., USA).

Pre-immune sera were collected from the mice before immunization. Mice (n=5 per group) were immunized with purified recombinant proteins (30 µg) emulsified in complete Freund adjuvant (1:1) through the intraperitoneal route (I.P.). Mice immunized with 1×PBS-complete Freund adjuvant emulsion were included as controls. Subsequent boosters with the same protein concentrations emulsified in incomplete Freund adjuvant were given on days 14, 28, and 42 after primary immunization. Mice were bled through retroorbital plexus before immunization (day 0) for collection of pre-immune (PI) serum, on day 14 post-immunization, and a week after each booster administration (day 21, day 35, and day 49). The collected blood was kept at 25 °C for 1 h, followed by centrifugation at 8000 rpm (Eppendorf centrifuge 5415R, UK), 4 °C for 10 min [32] . The sera were stored at −20 °C in small aliquots till further use.

ELISA was performed for antibody titer determination and antibody isotyping of the antiserum generated in immunized mice [32] . Briefly, proteins (500 ng/100 µl in each well, in triplicates) were coated in flat-bottom 96-well ELISA plates overnight at 4 °C. The unbound proteins were removed by washing three times with 1× PBST, followed by blocking with 2% BSA prepared in 1 × PBST at 37 °C for 1 h. After rewashing the wells with 1× PBST as before, different dilutions (1:10,000 to 1: 160,000) of the antisera or isotypespecific antibodies (1:10,000) were added to the well and incubated at 37 °C for 1 h. The plates were washed with 1× PBST, as stated before. Secondary antibody (AP-conjugated anti-mouse antibody/anti-rabbit antibody (1:10,000, 100 μl/ well) was added and incubated at 37 °C for 1 h, followed by three 1× PBST washes. The color was developed by adding the substrate (p-nitrophenyl phosphate substrate, 1 mg/ ml) and incubating at room temperature for 20 min. The absorbance was measured at 420 nm. The endpoint titers were determined as the highest dilution of the sera that gave a positive absorbance over and above that obtained with preimmune serum [33] .

Levels of different isotypes of IgG (IgG1, IgG2a, IgG2b) in the antisera were determined by indirect ELISA using isotype-specific horseradish peroxidase (HRP)-conjugated secondary antibodies from BD Biosciences, USA, as per the manufacturer's instructions.

For the isolation of splenocytes, immunized mice were sacrificed after 1 week of the second booster, i.e., on day 35 of primary immunization, and the spleen was surgically removed under aseptic conditions. The splenocytes were isolated as described earlier [31] . Splenocytes from the recombinant protein immunized mice and PBS-immunized mice (control) were seeded (1×10 6 cells/100 µl/well, in triplicates) in a 96-well culture plate and stimulated with respective protein (20 µg in 100 µl PBS). PBS-stimulated cells were included as a control. The stimulated cells were cultured at 37°C for 24 h, 48 h, and 72 h, under 5% CO2 humidified atmosphere. The culture supernatant was collected at different time intervals, and the cell proliferation was assayed using XTT assay as per the manufacturer's direction. The absorbance was measured at 450 nm using an ELISA plate reader (Tecan, USA).

The immune response generated (CD4 + T helper cell proliferation) in the culture supernatants of the protein-and PBS-stimulated splenocytes was evaluated by determining IL-4 and IFN-γ levels using cytokine specific ELISA kit (Becton-Dickinson Pharmingen, USA).

Hemolytic activity of the purified WTrPFO was measured using mice blood by the method of Tweten [34] . Mice RBCs were separated from the blood as described by Hanson et al., with minor modifications [35] . Briefly, mice blood (1 ml) was collected through the retro-orbital plexus vein in EDTA (1.5 mg/ml) to prevent coagulation and centrifuged at 500×g (Eppendorf microcentrifuge 5415R) at 4 °C for 5 min. The supernatant (plasma) was discarded, and the pellet was washed three times with 1× PBS, pH 7.4 at 4 °C. The washed blood cell pellet was suspended in 10 volumes of 1× PBS to pellet volume. The RBCs (2.5×10 6 in 50 µl) were treated with different concentrations of the WTrPFO (0.4 µg/ ml to 2.0 µg/ml) in a final volume of 150 µl and incubated at 37 °C for 1 h. The samples were centrifuged at 1000 rpm for 5 min at 4 °C to separate the cell debris. Hemoglobin present in the supernatant was measured at 540 nm. The cells treated with PBS and 1% SDS were included as negative and positive controls, respectively. The SDS-treated cells were considered to have undergone 100 % lysis. Therefore, the percentage of hemolysis in the treated samples is determined with respect to SDS-treated cells. One hemolytic activity (HU) unit was defined as the amount of WTrPFO required to cause 50 % hemolysis.

For assessing the ability of the anti-WTrPFO and anti-rPFO C-ter antisera to inhibit the hemolytic activity of the WTrPFO, pooled antisera collected from all mice of each group on day 35 post-immunization were used. A range of dilutions of both antisera (50-5%) was prepared in 1× PBS. The WTrPFO (50 µl, 0.2 mg/ml) that caused 80% hemolysis was incubated with an equal volume of the diluted antiserum (50 µl) and neat antiserum at 37 °C for 30 min. The proteinantisera mixture was then added to RBCs (2.5×10 6 RBCs in 50 µl) and allowed to incubate at 37 °C for 1 h. The cells were then centrifuged at 1000 rpm for 5 min (Eppendorf microcentrifuge), and the supernatant was collected carefully. The absorbance at 540 nm was measured to assess the inhibition of hemolysis by the antisera.

Cytotoxic activity of the recombinant proteins was evaluated using phagocytic macrophages employing LDH cytotoxicity assay kit as described earlier [29] . Mice peritoneal macrophages (5×10 4 cells/100μl/well) isolated by the method described by Ray and Dittel [36] were treated with different concentrations of the proteins (10-100 µg/ ml, in triplicates) and incubated at 37 °C for 24 h in a CO 2 (5 %) incubator. The culture supernatants of the treated cells were subjected to LDH coupled activity assay as per the manufacturer's directions. The absorbance measured at 490 nm was directly proportional to the released LDH and cytotoxicity of the recombinant protein(s).

The antisera against the WTrPFO and rPFO C-ter collected on day 35 post-immunization were evaluated for their neutralization capacity against the WTrPFO toxicity in mice peritoneal macrophages. The WTrPFO (100 µg/ml, 50 µl) was pre-incubated with an equal volume of different dilutions of the antisera (1:10-1:10,000) for 37 °C for 30 min. Mice peritoneal macrophages were seeded in a 96-well plate (5×10 4 macrophages/100 µl/well, in triplicates) and treated with the WTrPFO pre-incubated with different dilutions of antiserum at 37 °C for 24 h in 5 % CO 2 atmosphere. The cells treated with WTrPFO alone were included as positive control and pre-incubated with the pre-immune serum as a negative control. Percentage neutralization of cytotoxicity in the samples pre-incubated with different antisera was calculated with respect to cells treated only with the WTrPFO (100 % cytotoxicity).

The data represent the mean±S.D. of two independent experiments performed in triplicates. Statistical significance of the difference between the experimental and control was determined using one-way ANOVA or two-tailed Student's t-test as applicable using Prism 8.0 (Graphpad, CA, USA).

The expression of WTrPFO and rPFO C-ter was achieved in the induced cell lysates of E. coli BL21(λDE3)pLysS harboring the pET22.PFO wt and pET22. PFO C-ter , respectively, upon induction with 1 mM IPTG (Fig. 1a) . A strong band of ~14 kDa and ~54 kDa was exclusively observed in the induced cell lysates (lanes I) of E. coli BL21(λDE3)pLysS cells expressing rPFO C-ter (C-ter) and WTrPFO (rPFO), respectively. No bands were detected in the uninduced cell lysates (lanes UI) of the respective cells harboring pET22. PFO C-ter and pET22. FO wt . Western blot analysis, using the monoclonal anti-histidine tag antibody, confirmed the authenticity of the expressed proteins as distinct bands at the expected position of WTrPFO (~ 54 kDa) and rPFO C-ter (~14kDa) were observed only in the respective induced cell lysates (Fig. 1b , lanes I). Inducer concentration optimization showed the expression of WTrPFO and rPFO C-ter to occur at a concentration as low as 0.1 mM (Supplementary Fig. 1a and b, respectively). The expression of WTrPFO increased slightly with the increase in IPTG concentration, with maximum expression at 0.8 mM. Unlike the WTrPFO, the rPFO C-ter expression did not significantly increase with increasing inducer concentration. Time kinetics of WTrPFO expression showed an increase in expression with induction time till 8 h, beyond which there is no further increase in expression with the increase in induction time (Supplementary Fig. 2a) . The rPFO C-ter expression was found to increase Fig. 2b) . The expression analysis of both the proteins (WTrPFO and rPFO C-ter ) at different temperatures (16 °C, 25°C, and 37 °C) for 8 h showed the presence of a dominant band at the expected positions ( Fig. 2a and b) . Both the proteins showed maximum expression at 37 °C. At 16 °C, the expression of both the proteins was slightly lower. Therefore, the induction at 16 °C was performed for 22 h (overnight) to obtain higher amounts of the protein for further analysis.

Analysis of the soluble (lane S) and insoluble (lane P) fractions of the induced cell lysates cultured at 25 °C and 37 °C for 8 h showed WTrPFO (Fig. 3a ) and rPFO C-ter (Fig. 3b) to be exclusively present in the insoluble fraction (lane P). However, when the expression of the WTrPFO and rPFO C-ter was induced at 16 °C for 22 h, the recombinant proteins were predominantly present in the soluble fractions (lane S in both the panels). Therefore, for large-scale purification, the cultures were induced at 16 °C for 22 h, and the WTrPFO and rPFO C-ter were purified from the soluble fraction using Ni +2 -NTA agarose chromatography. The specifically bound proteins were eluted with 100-mM imidazole (Fig. 4) . Both the WTrPFO and rPFO C-ter could be purified near homogeneity (>99 %), as evident from a single band of the purified WTrPFO (Fig. 4a, lane 1 ) and rPFO C-ter (Fig. 4b, lane 1) at their expected positions (~54 kDa and ~14 kDa, respectively) which was detected on SDS-PAGE. The yield of the purified WTrPFO and rPFO C-ter was determined to be 70 mg and 75 mg, respectively, from the 1L culture at shake flask level. 

In order to see if the histidine-tagged WTrPFO retained its biological activity, it was evaluated for its hemolytic and cytotoxic activities using mice RBCs and mice peritoneal macrophages, respectively. As shown in Fig. 5a , hemolysis (~ 20 %) could be seen at a concentration of 0.8 µg/ml of WTrPFO with respect to 1 % SDS (included as a positive control and considered to cause 100 % lysis). A dosedependent increase in the hemolysis was observed, and at the 2 µg/ml of WTrPFO, ~80 % lysis was observed. Hemolytic activity of purified WTrPFO from the soluble fraction was determined as 10 5 HU/mg of protein.

The purified WTrPFO was biologically active, as evident from its cytotoxicity towards mice peritoneal macrophages evaluated by the LDH release assay (Fig. 5b) . Like the hemolytic activity, an increase in percentage cytotoxicity was observed with an increase in WTrPFO concentration, and ~ 84 % cytotoxicity was observed at 100 µg/ml concentration with respect to 10× lysis buffer, included as positive control and considered to cause 100 % cell death.

Since the study was targeted to carry out a comparative analysis of the immunogenic and neutralizing potential of the WTrPFO and rPFO C-ter , immunization studies were performed with the two proteins. The immunoglobulin (IgG) levels in antisera of both WTrPFO (Fig. 6a ) and rPFO C-ter (Fig. 6b ) immunized mice were found to be higher on day 14 after immunization and increased further on day 21, day 35, and day 49 after the administration of the boosters. Although the IgG level against the rPFOC-ter was lower than that observed with the WTrPFO (indicated by relatively lower absorbance), the IgG levels remained significantly higher than that of pre-immune serum. The endpoint titers of the anti-WTrPFO antisera drawn on day 14, day 21, and day 35 and day 49 post-immunization were determined to be much greater than 1:20,000 (day 14), 1:80,000 (day 21), and 1:160,000 (day 35 and day 49) as the absorbance remained significantly higher (p≤0.001) than that obtained with preimmune serum. On the other hand, the endpoint titers for anti-rPFOC-ter were determined to be ˃1: 10,000˃1: 20,000, ˃1: 80,000, and ˃1: 160,000 on day 14, day 21, day 35, and day 49 post-immunization, respectively. Percentage hemolysis was determined with respect to 1% SDS included as positive control and considered as 100% hemolysis. b Cytotoxic activity of WTrPFO. Mice peritoneal macrophages (5 × 10 4 cells/100 µl/well) were treated with dif-ferent concentrations (10-100 μg /ml) of WTrPFO (in triplicates) at 37 °C for 24 h in 5% CO2 humidified atmosphere. LDH release was taken as a measure of cytotoxicity, and the absorbance at 490 nm was considered as an indicator of lysis. Percentage cytotoxicity was calculated with respect to lysis caused by lysis buffer included as a positive control (PC) and considered to cause 100% lysis. Data represent mean ± SD of three independent experiments performed in triplicates

Immunoblot analysis of the induced cell lysates of the E. coli BL21(λDE3)plysS cells harboring pET22.PFO wt and pET22. PFO C-ter showed that the anti-WTrPFO antiserum collected on day 35 (a week after the second booster) detected a single band corresponding to the recombinant protein in the induced cell lysates (Fig. 7a, lane I, rPFO) . The absence of the band in the uninduced cell lysate indicates the specificity of the anti-WTrPFO antibodies (Fig. 7a, lane UI, rPFO) . Surprisingly, the anti-WTrPFO antiserum could not detect the rPFO C-ter protein (Fig. 7a, lane I, C-ter) in the cell lysates of E. coli BL21(λDE3) plysS expressing rPFO C-ter . Likewise, the antigen specificity and the cross-reactivity analysis of anti-rPFO C-ter antiserum were analyzed by immunoblotting the induced cell lysates of the E. coli BL21(λDE3)plysS expressing WTrPFO. Detection of a single band at ~54 kDa position revealed that the anti-rPFO C-ter antiserum (raised against only the C-terminal of the PFO) could specifically cross-react with the full-length protein (Fig. 7b , lane I, rPFO). Detection of a band at ~14 kDa position in the cells Fig. 6 Antibody titers of the anti-WTrPFO and anti-rPFO C-ter antisera. Different dilutions of the antisera drawn on different days post-immunization (D.P.I.) from mice immunized with WTrPFO (a) or rPFO C-ter (b) were subjected to ELISA for the determination of antigen-specific antibody titers. Anti-mouse Fc-specific alkaline phosphatase-conjugated antibody was used as a secondary antibody. Endpoint titers of the anti-WTrPFO antisera were determined to be ≥ 1:20,000 on day14, ≥ 1:80,000 on days 21and 35, and ≥ 160,000 on day 49 post-immunization. Endpoint titers for anti-rPFO C-ter antisera were determined to be ˃1: 10,000, ˃1: 20,000, ˃1: 80,000, and ˃1: 160,000 on days 14, 21, 35, and 49 post-immunization, respectively. Absorbance at 420 nm (A420) depicts mean ± S.D. of pooled antisera samples (n = 5 mice per group) analyzed in triplicates. One-way ANOVA was used to determine the significant difference between PI and different antisera dilutions anti-rPFO C-ter antisera was determined by immunoblot analysis. Ten micrograms of the uninduced (UI) and induced (I) cell lysates of the E. coli BL21(λDE3)pLysS cells expressing WTrPFO (rPFO) or rPFO C-ter (C-ter) were resolved on SDS-PAGE and immunoblotted either with anti-WTrPFO antiserum (anti-rPFO, panel a) or anti-rPFO C-ter antisera (anti-C-ter, panel b) at 1:10,000 dilution. Alkaline phosphatase (AP)-conjugated goat anti-mouse/ anti-rabbit antibody (1:10,000) dilution was used as a secondary antibody. In both the panels, the image on the left shows the parallelly run SDS-PAGE of the same lysates stained with Coomassie blue stain. Lane M denotes the pre-stained protein molecular weight (kDa) marker expressing the rPFO C-ter revealed the antigen specificity of the anti-rPFO C-ter antiserum (Fig. 7b, lane I, C-ter).

Different IgG isotypes, namely, IgG1, IgG2a, and IgG2b, in the anti-WTrPFO and anti-rPFO C-ter antisera collected on day 21, day 35, and day 49 post-immunization were assayed to determine the type of immune response generated against the test proteins (Fig. 8) . Significantly elevated levels of all the three isotypes were observed in the antisera generated against WTrPFO (Fig. 8a) and rPFOC-ter (Fig. 8b) compared to the respective pre-immune sera. The ratios of IgG1/IgG2a and IgG1/IgG2b in the anti-WTrPFO collected on day 21, day 35, and day 49 were >1, reflecting the Th2-biased immune response. The ratio of IgG1/IgG2a in the anti-rPFO C-ter antisera was determined to be much ˃1 at all study intervals (8.67, 7 .89, and 8.64 on day 21, day 35, and day 49, respectively), whereas the ratio of IgG1/IgG2b was determined to be slightly <1 (0.962, 0.867, and 0.925 on day 21, day 35, and day 49, respectively), suggesting predominantly Th2-biased mixed immune response.

In vitro proliferation of the splenocytes isolated from the mice immunized with the WTrPFO and rPFO C-ter and stimulated with the respective proteins resulted in significantly higher proliferation than that stimulated with PBS at all study intervals ( Fig. 9a and b) . No increase in the proliferation was noted when the splenocytes isolated from the PBS-immunized mice were stimulated with the test proteins. The significant increase in proliferation with WTrPFO stimulation of the respective splenocytes was determined to be at p ≤ 0.05, p≤0.01, and p≤ 0.001 at 24 h, 48 h, and 72 h, respectively (Fig. 9a) . The significance levels of change with rPFO C-ter were found to be (p ≤0.01) at all the three intervals post-stimulation (Fig. 9b) . The stimulation index of the WTrPFO-and rPFO C-ter -stimulated splenocytes was determined to be 1.4 and 2.08, respectively, which was noticeably higher than that of the control splenocytes (~1.15), indicating significant T cell activation and proliferation in response to antigens.

The type of T cell immune response (evaluated by antibody isotype analysis) was further confirmed by assessing IL-4 (Th2 marker) and IFN-γ (Th1 marker) levels in the splenocyte culture supernatants post-stimulation of splenocytes isolated from mice immunized with the proteins. A significant increase ( p≤ 0.05-0.01) in both IL-4 and IFN-γ levels in splenocytes isolated from WTrPFO ( Fig. 10a and c) and rPFO C-ter -( Fig. 10b and d) immunized mice stimulated with the respective proteins demonstrated that both proteins are able to generate mixed T cell immune response at all the time points. It was noted that at 48 h, the increase in IL-4 levels was more prominently indicative of Th2 immune response; in the WTrPFO-stimulated splenocytes isolated from the immunized mice, at 72 h, there was a shift to Th1 response as IFN-γ levels increased further with a concomitant decrease in IL-4 levels in the splenocytes isolated from the WTrPFO (Fig. 10a and c) and rPFO C-ter (Fig. 10b and  d) immunized mice, suggesting a shift to Th1-biased mixed immune response at later intervals.

Pre-incubation of WTrPFO at 2 µg/ml (that caused 80 % hemolysis) with different dilutions (50-5%) of both the Fig. 8 Determination of antibody isotypes. The anti-WTrPFO (a) and anti-PFO C-ter (b) antisera, collected on days 21, 35, and 49 postimmunization, were analyzed for different IgG isotype levels by ELISA using respective HRP-conjugated isotype-specific secondary antibodies. Pre-immune (PI) serum was included as control. Data represent mean ± S.D. of absorbance (A 420 ) of analyses performed in triplicates. The significance of the difference in the different isotypes in the antisera collected on different days with respect to PI was determined using a one-way ANOVA and is denoted as p ≤ 0.05(*), p ≤ 0.01(**), and p ≤ 0.001 (***) anti-WTrPFO and anti-rPFO C-ter antisera prior to addition to mice RBCs (2.5×10 6 , 50 µl) resulted in inhibition of hemolytic activity to a great extent, indicating the excellent inhibitory capacity of both antisera ( Fig. 11a and b) . Inhibition of the hemolytic activity of the WTrPFO was maximum (more than 90 %) at the dilution of 25 % of both the antisera. Incubation of the toxin (WTrPFO) with other dilutions of the antisera (50 %, 10 %, and 5%) also resulted in more than 80 % inhibition of hemolytic activity. No significant inhibition (~4.56 %) was observed when the WTrPFO was pre-incubated with neat pre-immune serum.

Pre-incubation of the WTrPFO with the antisera generated against both the WTrPFO and rPFO C-ter , at all the dilutions (1:10-1: 10,000), was able to neutralize the cytotoxicity of the purified WTrPFO (100 µg/ml) significantly towards the mice peritoneal macrophages (Fig. 11c and  d) . The neutralization capacity of the antisera was determined as the percentage of reduced cytotoxicity of the purified WTrPFO pre-incubated with different dilutions (1:10-1:10,000) of each antiserum, with respect to the cells incubated with the WTrPFO alone (considered as 100 %). Interestingly, the neutralization capacity of neat anti-WTrPFO and anti-rPFO C-ter was slightly lower ~40 % and ~49 % compared to other dilutions of the antisera (1:10-1: 10,000; ≥80 %) with constant WTrPFO concentration (100 µg/ml). The anti-rPFO C-ter antiserum also efficiently neutralized the WTrPFO cytotoxicity (≥ 80%) at all the dilutions. Maximum neutralization (96 %) was achieved when the WTrPFO was pre-incubated with the anti-rPFO C-ter antiserum at a dilution of 1:500 that indicated its equivalence zone.

Different types of C. perfringens strains cause various diseases in humans and animals, attributed to single or multiple toxins secreted by different strains. The bacterium is ubiquitously present in the environment and is highly pathogenic. It has a high proliferation rate and results in the death of animals either within hours of the appearance of symptoms or without any clinical symptoms. Though it is susceptible to several antibiotics, including penicillin and clindamycin, its rapid proliferation rate results in death without a premonitory sign making antibiotics ineffective [16] . This endorses the development of vaccines against the toxins involved in the pathogenesis of C. perfringens to counteract the infection. Though attempts have been made to develop vaccines against different toxins secreted by a specific type, these can be used against a specific type of C. perfringens. Therefore, a toxin secreted by all types is likely to assist in protecting the animals from all types of C. perfringens. PFO is one of the toxins of C. perfringens that contributes to the pathogenesis of C. perfringens and is believed to be produced by all the isolates [11] . Although PFO is not the main diseasecausing toxin, it acts synergistically with α-toxin in calves, causing myonecrosis and necrohemorrhagic enteritis [12, 13] . PFO has also been reported to augment the toxicity of ɛ-toxin in a mouse model for enterotoxaemia caused by C. perfringens type D, a disease of goat and sheep. These data highlight the importance of PFO in supporting the outbreak of different C. perfringens-associated Fig. 9 Assessment of in vitro T cell proliferation response. Splenocytes (1 × 10 5 cell/well in 100 µl) isolated from mice immunized with WTrPFO (a) and rPFO C-ter (b) (ip; 30 µg/mice) were stimulated with the respective protein (20 µg/ml, stimulated) and vehicle (1 × PBS, unstimulated). "Ctrl" in both the panels indicates splenocytes isolated from mice immunized with 1 × PBS (control) and stimulated with either WTrPFO (panel a) or rPFO C-ter (panel b) or PBS (unstimu-lated). Splenocyte proliferation was measured by XTT assay at different time intervals post-stimulation. Data represent the mean ± S.D. of 3 independent experiments performed in triplicates. Statistical significance (p value) was calculated with respect to PBS-stimulated splenocytes of the same group using the Student's two-tailed t-test. p ≤ 0.05(*), p ≤ 0.01(**), and p ≤ 0.001 (***) diseases [14] , making it an attractive vaccine candidate that could be used in combination with the existing vaccine preparation against the toxin secreted by a specific type. Earlier reports by Goossen et al. demonstrated that the α-toxin and PFO are the most immunogenic proteins in vaccine preparations, and immunization with the native toxin(s) or toxoids confers protection against the toxininduced necrotic lesions in the intestinal loop model [19] . In vitro neutralizing capacity of a PFO derivative with a single amino acid substitution (PFO L491D ) against the hemolytic activity and cytotoxic effect of PFO in horse red blood cells and bovine endothelial cells has been reported [37] . However, detailed in vivo immune response analysis against these proteins has not been reported. Since the type of immune response generated against an antigen is critical in inducing a long-lasting protective immune response, we carried out the present study to assess the vaccine potential of soluble recombinant PFO (WTrPFO). The native toxins are generally not considered safe for vaccine development, as these can cause local tissue damage at the administration site. Therefore, the vaccination dose needs to be closely monitored not to cause adverse effects on the organism. Other approaches, including genetically modified toxins by site-directed mutagenesis or by using a non-toxic immunogenic fragment of the toxin, could be used to generate an effective and protective immune response that has been reported with the C-terminal domain of α-toxin [20, [38] [39] [40] . In the present study, we targeted the receptor-binding C-terminal domain of the PFO, which is crucial for initiating oligomer formation in the membrane by recognizing and binding the toxin to the host cell membrane [41] . The two proteins were produced through recombinant routes, and a comparative analysis of the immune response generated against these proteins was carried out. We expressed the WTrPFO and rPFO C-ter using heterologous host E. coli for analyzing their immunogenic and neutralizing potential. A tight and intense band of WTrPFO and rPFOC-ter only in the induced cell lysates at the expected positions (~54 kDa and ~14 kDa, respectively) established the tight regulation of anti-rPFO C-ter (panel d) for 1 h at 37 °C. The cells were then incubated at 37 °C for 24 h in 5% CO2 atmosphere, followed by LDH release. WTrPFO pre-incubated with PI (neat) was considered as a negative control. Percentage neutralization was calculated with respect to LDH released by the cells treated with WTrPFO incubated with 1 × PBS, taken as 100% cytotoxicity. Data represent mean ± SD of neutralization percentage of experiments performed in triplicates. Ordinary one-way ANOVA was used to determine the significance of neutralization ability of the antiserum with respect to control cells treated with WTrPFO incubated with PI. p ≤ 0.05(*), p ≤ 0.001(**), p ≤ 0.001(***) the T7 expression system. Initial attempts to express the proteins at 25 °C and 37 °C resulted in the expression of the two proteins as insoluble proteins. This is expected as higher expression of recombinant protein(s) without the signal sequence results in misfolding of the protein in the cytoplasm and leads to aggregation to form inclusion bodies. Another recombinant cytolysin, such as the C-terminal domain of streptolysin O, has also been reported to express as inclusion bodies [42] . For biologically active protein, it is important to express the protein as a soluble protein (native form). Therefore, optimization of expression conditions was carried out to direct the expression to soluble fraction. Induction of expression at a lower temperature for a prolonged period led to the expression of these two proteins predominantly in soluble fraction. Significantly higher yields of the purified recombinant proteins (70 mg/l and 75 mg/l for WTrPFO and rPFO C-ter , respectively) were obtained compared to 15 mg/l reported by Tweten, who purified the protein from a clone identified from a C. perfringens genome library in bacteriophage λ [34] . Tweten's PFO clone from the genomic library contained the signal sequence and directed the expression of rPFO in the periplasm, resulting in a significantly lower yield [34] . In the present study, the protein was expressed without the signal sequence and with a histidine tag which facilitated a single-step purification, thus improving the yield. This is important as higher production of the protein is desirable for the assessment of its biological activity as well as vaccine potential.

The WTrPFO purified from the soluble fraction was biologically active, and its hemolytic activity (10 5 HU/mg) was comparable with that reported by Tweten [34] . Furthermore, sufficient cytotoxicity (close to SDS, included as positive control) observed with WTrPFO in mice peritoneal macrophages confirmed that histidine tag did not affect its biological activity, and the structural conformation of the soluble WTrPFO was similar to that of native PFO. On the other hand, as expected, the rPFO C-ter did neither cause any hemolysis of the mice RBCs nor was it found to be cytotoxic to mice peritoneal macrophages, confirming its non-toxic nature (data not shown).

Immunization of mice with the WTrPFO and non-toxic rPFO C-ter emulsified with Freund adjuvant resulted in robust immune response, evidenced by very high antigenspecific antibody titers in the antisera generated against the two proteins. Although the endpoint titers of the rPFO C-ter antiserum appeared to be slightly lower than the fulllength WTrPFO, the antigen-specific immune response (determined by immunoblotting) of the anti-rPFO C-ter was as strong as the anti-WTrPFO. Effective and specific cross-reactivity of antiserum raised against the nontoxic rPFO C-ter with the WTrPFO toxin suggested that the antibodies present in the anti-rPFO C-ter could neutralize WTrPFO toxicity and thus can serve as a non-toxic vaccine candidate.

Further, for a vaccine candidate to act effectively, the generation of immune memory is a desirable criterion so that the immune cells can get activated upon exposure to the respective pathogen. The stimulation of lymphocytes isolated from WTrPFO and rPFO C-ter immunized mice with the respective proteins resulted in enhanced proliferation of splenocytes, indicating that both the proteins could generate T cell memory in mice after immunization. An increase in the IgG1, IgG2a, and IgG2b with a ratio of IgG1/IgG2a more than 1 and IgG1/IgG2b almost close to 1 indicated a Th2-biased immune response. The cytokine ELISA of the culture supernatants of the splenocytes isolated from the WTrPFO and rPFO C-ter immunized mice stimulated with the respective protein also confirmed the Th2-biased immune response as a significant increase in both IFN-γ (a marker of Th1-type immune response) and IL-4 (a marker of Th2type immune response) was noted which again confirmed mixed immune response. A vaccine that generates a mixed immune or Th1 response is considered a better candidate than that stimulates Th2-type response [43, 44] . Thus, both the WTrPFO and rPFO C-ter that generated mixed immune response are likely to prove promising vaccine candidates against the PFO toxicity of all types of C. perfringens strains.

PFO, known to cause hemolysis, is also responsible for C. perfringens-dependent macrophages cytotoxicity by resisting the C. perfringens to be killed by phagocytosis of macrophages [45] . Therefore, these two assays were used for assessing the toxin neutralization ability of the antisera. The ability of the anti-WTrPFO antiserum and anti-rPFOC-ter antiserum to neutralize the hemolytic activity and cytotoxicity of WTrPFO confirmed that the antibodies present in these antisera are neutralizing type. Surprisingly, the hemolytic inhibition by neat and antisera diluted two folds (50 %) appeared less than the 4 folds diluted antiserum (25 %), despite higher concentrations of antibodies in these compared to 25 % diluted antisera dilutions. Similarly, the neutralization capacity of neat anti-WTrPFO and anti-rPFO C-ter antisera against the cytotoxic effect of WTrPFO was found to be lower (~40 % and ~49 %, respectively), when compared to other dilutions of the antisera (1:10-1:10,000; ≥80 %) keeping the amount of the WTrPFO constant (100 µg/ml). The neutralization percentage of anti-WTrPFO was ≥ 80% at the dilutions of 1:250-1:10,000 and attained maximum neutralization (99%) at the dilution fraction 1:500 that indicated the equivalence zone of antigen-antibody interaction. Higher neutralizing capacity of the diluted antiserum compared to neat antiserum could be attributed to the Lattice theory that explained the precipitation reactions in fluid media in which antigen-binding sites in concentrated polyclonal antibodies became saturated, reducing their antigen-binding ability [46] .

Comparable neutralization (~90 %) of the WTrPFO cytotoxicity by both the anti-WTrPFO and anti-rPFO C-ter antisera in mice peritoneal macrophages demonstrates the effectiveness of non-toxic rPFO C-ter in negating the PFO toxicity. The same can be employed as a promising alternative to native PFO toxin in combination with the nontoxic C-terminal domain of alpha-toxin or another nontoxic alternative of main virulent disease-causing toxins of different types of C. perfringens to generate a protective immune response against the bacterium. The present study thus reports a comparative analysis of the immune response generated against the WTrPFO and non-toxic C-terminal domain of PFO. Despite relatively lower IgG levels in the anti-rPFO C-ter antiserum, it could specifically recognize the full-length PFO and neutralize the hemolytic and cytotoxic activity of the PFO in vitro.

Thus, the present study reports recombinant expression and purification of the full-length PFO of C. perfringens and its C-terminal receptor-binding domain as soluble proteins. Comparative analysis of the immunogenicity and antigenicity of both the WTrPFO and its deletion variant (i.e., C-terminal receptor-binding domain, rPFO C-ter ) showed that both could generate a robust immune system response. However, the IgG levels in the antiserum generated against the C-terminal domain were relatively lower than full-length PFO. Both the proteins generated T cell memory and mixed immune response, which is a characteristic of a promising vaccine candidate as both the arms of immune response play an important role in conferring protection against the pathogen/toxin. The antisera generated against WTrPFO and the C-terminal domain could specifically recognize the full-length PFO and neutralize the hemolytic and cytotoxic activity of the PFO in vitro. The antiserum's specific and robust crossreactivity against the non-toxic C-terminal domain with the full-length PFO and its ability to neutralize its toxicity clearly demonstrate that the C-terminal domain is nontoxic and has the potential to neutralize the toxicity of the PFO which is secreted by all clostridial toxino types. Since PFO has been reported to act in synergy with other main toxins of different clostridial strains, further in vivo immunization and protective efficacy studies of the PFO need to be carried out in combination with these toxins. Both WTrPFO and rPFO C-ter could also be used as an adjuvant vaccine together with other clostridial toxins as PFO has been reported to aid in the action of other clostridial toxins. Also, since the structure of PFO secreted by C. perfringens strains shares similarities with other cholesteroldependent cytolysins of other genera such as Streptococcus, Bacillus, and Listeria [8] , the non-toxic variant of PFO reported in the present study could be assessed as a candidate for potentiating the protective immune responses against other bacterial infections as well.

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s12026-021-09254-9.

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Acknowledgements AS thanks the Jawaharlal Nehru University, New Delhi, for the research fellowship. The authors acknowledge intramural support from the Jawaharlal Nehru University, New Delhi.Author contribution Ankita Singh: Conceptualization, formal execution, analysis of all experiments, data curation, writing-original draft preparation.Prashant Rawat: Initial investigations on recombinant protein expression Aparna Dixit: Conceptualization, project administration, supervision, and writing-review and editing Devapriya Choudhury: Writing, reviewing, and editing

Conflict of interest The authors declare that they have no conflict of interest.

The Institutional Animal Ethics Committee of the university approved the use of animals for the immunization study (project code IAEC # 03/2019). All procedures were performed as per the guidelines and recommendations of the committee.