Immunization with a novel Clostridium perfringens epsilon toxin mutant rETXY196E-C confers strong protection in mice

Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETXY196E-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:105 in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETXY196E-C is a potential vaccine candidate for future applied researches.

Immunization with rETX Y196E -C. Six-week-old female BALB/c mice (about 18-22 g), purchased from Laboratory Animal Center of The Academy of Military Medical Sciences, were divided into seven groups (twenty mice of each group) randomly. These groups of mice were immunized by two immune pathways: subcutaneous injection and intraperitoneal injection, and different concentrations of rETX Y196E -C: 5 μg/mouse, 10 μg/mouse, 15 μg/mouse and 0.01M phosphate-buffered saline solution (PBS). Protein rETX Y196E -C was diluted with PBS and mixed with the same volume of Freund's adjuvant. The mice were immunized three times and each vaccination was performed at an interval of two weeks. Serum was collected from the tail vein of mice one week after each immunization. The survival status and weight changes were recorded daily.

Measurement of antibody titers.
Enzyme-linked immunosorbent assay (ELISA) was used to measure the sera antibody titers of immunized BALB/c mice. The 96-well plate was coated with 100 μL of 10 μg/mL rETX in 0.05 M carbonate-buffered saline solution overnight, and then the plate was blocked with 200 μL of 5% bovine serum albumin (BSA) for 2 h at 37 °C. After being incubated with 100 μL of 10-fold serial diluted serum for 1 h at 37 °C and washed three times by PBST (0.01M PBS, 0.05% Tween-20), the plate was incubated with HRP-coupled goat anti-mouse IgG (1:50000) at 37 °C for 1 h. Then the plate was washed three times with PBST and added the TMB substrate solution (TIANGEN Biotechnology) to each plate, and finally 2 M H 2 SO 4 was used to stop the reaction. The absorbance was determined at 450 nm by a microplate reader (Molecular Device).
Assessment of antibody affinity. ELISA was used to assess the affinity between rETX Y196E -C-induced antibody and rETX or rETX Y196E -C according to previous studies 21 . The proteins rETX or rETX Y196E -C (10 μg/mL) were coated into the 96-well plate overnight at 4 °C and washed three times with PBS. The plate was blocked with 5% BSA diluted in 0.01M PBS for 1 h at 37 °C. The 2-fold diluted rETX Y196E -C immunized sera (come from 15 μg/ mouse subcutaneous immunized mice), PBS immunized sera (1:100) or mouse anti-ETX monoclonal antibody (developed by our laboratory) were added to the plate and incubated at 37 °C for 1 h. After being washed three times with PBST, the plate was incubated with NH 4 SCN diluted in PBS at 37 °C for 15 min at the following concentrations of 8.0 M, 4.0 M, 2.0 M, 1.0 M, and 0.5 M. The plate was washed three times and the HRP-coupled goat anti-mouse IgG (1:50000) was added to the plate and incubated at 37 °C for 1 h. The absorbance was obtained by a microplate reader.
Challenge with rETX. One week after the third immunization, mice immunized by rETX Y196E -C were challenged with 1000 × LD 50 , 500 × LD 50 , and 100 × LD 50 of rETX and mice immunized with PBS were challenged with 10 × LD 50 of rETX intraperitoneally, the value of LD 50 (180 ng/mouse) comes from our previous paper 15 . The survival status of all mice was daily recorded for 5 days.
In vitro neutralization assay. rETX (50 μL) at doses of 1 × CT 50 (440 ng/mL), 2 × CT 50 , 4 × CT 50 , 8 × CT 50 , 16 × CT 50 and 32 × CT 50 was incubated with an equal volume of mouse polyclonal antiserum from mice of 15 μg/ mouse subcutaneous groups or from immunized mice with PBS for 30 min at 37 °C. The toxin-antiserum mixtures were added to the 96-well plate seeded with 2-3 × 10 4 MDCK cells and incubated at 37 °C for 24 h. After the plate was washed three times with PBS, 100 μL of culture medium containing 20 μL MTS was added to each well and incubated for 3 h. The absorbance was measured at 490 nm, which reflected the number of living cells.
The sera samples (500 μL) from 15 μg/mouse subcutaneous immunized mice were mixed with an equal volume of the doses of 10 × LD 50 , 100 × LD 50 or 500 × LD 50 rETX and incubated for 30 min at 37 °C. Groups of five female BALB/c mice were challenged intraperitoneally with 500 μL of the mixed solution. The survival status was observed daily for 5 days.
Histopathologic alterations. Previous study indicated that ETX mainly accumulated in brains and kidneys of sensitive animals and caused sudden death 22 . Mice subcutaneously immunized three times with PBS and Scientific RepoRts | 6:24162 | DOI: 10.1038/srep24162 15 μg of rETX Y196E -C were challenged with 1000 × LD 50 of rETX. The brains and kidneys from challenged mice were separated for histopathologic study.

Result
Thermal stability of recombinant toxins. In this assay, the fluorescent dye could bind to exposed hydrophobic regions of the unfolded proteins and the fluorescent signal had a significant increase. The structure of protein changed with temperature, and the hydrophobic regions were exposed. Thus, the melting temperature, which is a characteristic of protein stability, can be automatically monitored by detecting the change in fluorescent signals. As shown in Table 1, the T m of the rETX Y196E -C and rETX were 93.41 ± 0.23 °C and 88.60 ± 3.86 °C respectively. The results indicated that the stability of the mutant was similar to the wild toxin, and they were structurally similar. Specific antibody response induced by rETX Y196E -C. One week after each vaccination, the antibody titers were measured by ELISA. The data are shown in Fig. 1. The results showed that the antibody titers were 1:10 2 after the initial immunization, but the antibody titers increased to 1:10 4 after the second immunization. One week after the third vaccination, the mean IgG titer against protein rETX Y196E -C of the six groups could reach about 1:10 5 and each group revealed similar titers and had no statistical differences (P > 0.05), which indicated that vaccination of mice with recombinant toxin rETX Y196E -C induced a specific antibody response. Antibody affinity. The results of antibody affinity assay were obtained and shown in Fig. 2. The affinity indexes between antiserum and rETX ( Fig. 2A) or rETX Y196E -C (Fig. 2B) were defined as the concentration of NH 4 SCN when the absorbance reached the half of the highest absorbance. Figure 2 indicated that the two affinity   indexes were 4.28 ± 0.77 M and 3.31 ± 0.75 M respectively, and had no significant difference (P > 0.05), but they were higher than those of PBS immune group and anti-ETX monoclonal antibody group.
Protective effect in mice immunized with rETX Y196E -C. Mice immunized with different doses and routes of rETX Y196E -C were challenged with 100, 500 and 1000 × LD 50 of active rETX per mouse. The result showed that mutant rETX Y196E -C stimulated strong protection against enterotoxemia in mice. All mice survived in the six rETX Y196E -C immunized groups which were challenged with 100 × LD 50 dose (Table 2), and all mice survived in the three rETX Y196E -C subcutaneously immunized groups which were challenged with 500 × LD 50 dose (Table 2). Furthermore, 80% of the mice in the 15 μg/mouse subcutaneous group survived when the challenge dose increased to 1000 × LD 50 of rETX (Table 2). However, Mice immunized with PBS died when challenged with 10 × LD 50 dose of rETX (Table 2). In addition, those results indicated that immunized pathway and dose contributed greatly to protective effect. Subcutaneously immunized pathway was better than intraperitoneally immunized pathway, and 15 μg rETX Y196E -C was the most efficient dose to induce a specific antibody response and protect mice from ETX challenge. The body weights of mice in each group were recorded daily for 30 days, and the weight changes are shown in Fig. 3. After the initial immunization, the mean body weight of mouse in 15 μg/mouse lost about 20% of their body weight (Fig. 3C). Similarly, mice in 5 μg/mouse intraperitoneal group and 10 μg/mouse intraperitoneal group lost less than 20% of their body weight. However, the mean body weights of mouse in subcutaneous groups had slight or no changes (Fig. 3A,B). Those data showed that intraperitoneally immunized pathway was more toxic for mice than subcutaneously immunized pathway during the first vaccination.
In vitro neutralization assay. Toxin neutralization assay in MDCK cells. Antiserum in this assay was collected from mice in 15 μg/mouse subcutaneous group. The MDCK cells were incubated with toxin-antiserum mixtures, and the viability was obtained. The ETX neutralization effects occurred only in the sera from the rETX Y196E -C vaccinated mice. As shown in Fig. 4A, the serum was able to protect MDCK cells against wild type ETX. However, the sera from PBS vaccinated mice did not inhibit ETX induced cytotoxicity and the cell viabilities were significantly lower than those of rETX Y196E -C vaccinated groups (P < 0.05).

Toxin neutralization assay in mouse.
To test the ETX neutralization effect in vivo, mice were injected with mixtures of activated rETX and antiserum, and the survival results are shown in Fig. 4B. All mice survived when challenged with mixtures of 10 × LD 50 rETX and antiserum. The results indicated that the anti-rETX Y196E -C antibodies could neutralize 10 × LD 50 dose of rETX and protect mice against ETX challenge, however, the mice died when the concentration increased to 100 × LD 50 dose of rETX.
Histopathologic analysis. After being challenged with 1000 × LD 50 dose of rETX, kidneys and brains of mice in PBS immunized group showed obvious pathological alterations including serious hemorrhage, cytoplasmic vacuolation and mild edema compared to that of the control group (Fig. 5). However, there were no phanerous pathological alterations in rETX Y196E -C immunized group (Fig. 5). The results showed that rETX Y196E -C immunization could protect the sensitive organs against harm induced by ETX.

Discussion
Previous studies have reported that site-directed mutants of ETX could significantly reduce the toxicity and be considered vaccine candidates 1,9,20 . The mutant H106P has previously been reported to non-toxic to mice and the immunized mice could survive at the 1000 × LD 50 dose of wild type ETX 9 . Recombinant epsilon toxoid vaccines could lead to a rise in serum potency in animals and as potential vaccine candidates to protect against enterotoxemia, but it took a long time to inactivate recombinant toxins 23,24 . Also, our team developed a mutant F199E, which showed a 2572-fold decrease in toxicity compared with that of wild type ETX and it can protect the immunized mice against a challenge of 100 × LD 50 dose of rETX 20 . However, mutant F199E was unstable and was more likely to precipitate. Meanwhile, the expression level of the mutant F199E in the soluble form was relatively low. It was previously reported that the mutant Y196E showed a slight deficiency in toxicity, and one of our previous studies concerning the mutant Y196E had achieved similar results. However, we also found that the C terminal peptide and amino acid Y196E mutation synergistically alleviated the toxicity of ETX, the cytotoxicity of rETX Y196E -C (13,110 ng/mL) was more significantly reduced than that of rETX (440 ng/mL) and the LD 50 of rETX Y196E -C (1,770,110 ng/kg) was reduced more than 200-fold compared with rETX (7910 ng/kg) 15 . In addition,  and PBS immune groups of mice were recorded every two days after the first immunization and the weights were recorded for 30 days (the arrows pointing the time of each immunization). Each point represents the current total weight relative to original total weight. mutant rETX Y196E -C showed a higher level of expression in the soluble form and a robust stability, which makes it easy to develop and store antigens. Therefore, rETX Y196E -C is more likely to be a promising vaccine candidate.
Recently, mutant with two mutations Y30A-Y196A showed that it could markedly reduce cytotoxic activities in MDCK cells and was considered a recombinant vaccine candidate against enterotoxemia. However, the data of challenge of the immunized animal were not provided 1 .
Immunizing doses and inoculation routes were optimized to obtain a better protective effect. Although varied immunizing doses and inoculation routes have achieved similar antibody titers, the dose of 15 μg per mouse and the subcutaneous immunization have provided the best protection of mice. High dose inoculation and subcutaneous immunization are likely to raise the proportion of effective antibodies. Even if the dose of 15 μg per mouse may not be the best and a higher immunizing dose may strengthen the protection, it may also exhaust the immune system and do some harm to immunized mice. Besides, we found that subcutaneous immunization was a safer way than intraperitoneal immunization, since mice in three intraperitoneally immunized groups lost about 20% of their original body weight after the first vaccination (Fig. 3).
Toxoid vaccines for use in domesticated sheep and goats are widely available commercially and have been used extensively over the past decades 25 . Although toxoid vaccines are effective in preventing enterotoxaemia in animals, inflammatory responses following vaccination have been reported to lead to reduced feed consumption 26 . However, the mutant rETX Y196E -C showed a predominant safety. For one thing, rETX Y196E -C has poor toxicity to MDCK cells or mice. For another, the weight of subcutaneously immunized mice in vaccination period keeps increasing rather than decreasing, as in control groups, which suggested that administration of rETX Y196E -C did not interrupt the growth of the mice.
The mutant rETX Y196E -C possessed high immunogenicity. The mean IgG titers could reach about 1:10 5 after the third vaccination. Besides, the affinity between antibodies and ETX was relatively high (Fig. 2). Accordingly, the mutant rETX Y196E -C could provide an excellent protective effect. Under the optimal condition, rETX Y196E -C could completely protect mice against challenge of 500 × LD 50 dose of wild type rETX and protect 80% of mice against a dose of 1000 × LD 50 of rETX. This protective effect is better than that of mutant F199E and may be better than that of mutant H106P, since our previous studies showed that H106P could provide similar protective effect as F199E. Previous studies suggested H106P could form the basis of a vaccine against ETX 9 . Therefore, rETX Y196E -C could be another promising vaccine candidate. Besides, immune sera stimulated by rETX Y196E -C could be considered an immunotherapeutic drug. The in vitro neutralization assay showed that 50 μL of immune sera was able to significantly reduce the cytotoxicity of 32 × CT 50 dose of rETX to MDCK cells (P < 0.05) (Fig. 4), while mice could be completely protected against 10 × LD 50 dose of rETX by 250 μL of immune sera without showing any clinical symptoms.
In conclusion, all these results in the current study demonstrate that rETX Y196E -C is a potential recombinant vaccine candidate against animal enterotoxemia. Further studies should be performed to determine whether rETX Y196E -C is able to protect susceptible animals, such as sheep, goats and calves, from enterotoxemia induced by epsilon toxin.
Statistical analysis. Treatment group differences were analyzed using analysis of variance (ANOVA) and student's paired t-test. *P < 0.05 represents statistical significance between the two groups. In this graph, control group represents the normal mice, PBS group represents the PBS immunized mice challenged with 1000 × LD 50 of rETX and 15 μg/mouse subcutaneous group represents the mice immunized subcutaneously with 15 μg of rETX Y196E -C challenged with 1000 × LD 50 of rETX. In PBS group, obvious hemorrhage (Upper arrow) was occurred in kidney, and vacuolation and mild edema (Lower arrow) were occurred in brain. The situation was significantly improved in 15 μg/mouse subcutaneous group mice.