CPA is coded by the chromosomic gene
plc. This gene is present in every toxinotype and is expressed at the highest levels in toxinotype A [
28]. The active toxin CPA is a 370-residue long, zinc-dependent, phospholipase C (PLC) with sphingomyelinase and lectinase activity and approximately 42.528 kDa [
29,
30]. The LD
50 for CPA has been calculated as 3 µg/kg in mice [
11]. This toxin is divided into the
N-terminal (1–246) and the
C-terminal (247–370) domains. The
N-terminal domain comprises the catalytic core of the toxin, while the
C-terminal domain and the central loop (55–93) are responsible for binding host phospholipids and GM1a ganglioside respectively (for review, see Oda et al. [
31]). Two zinc ions (Zn
2+) are strongly bound to CPA. One ion is bound to His148 and Glu152 and is crucial for enzymatic activity. The remaining ion is bound to His11 and Asp130 and has structural function [
32,
33]. Histidine residues 68, 126, and 136 also bind calcium and help the toxin bind phospholipids in the host cell membrane.
Recombinant CPA Production Strategies and Animal Model Immunizations
The first studies involving the cloning and expression of the gene encoding CPA determined the nucleotide sequence and protein molecular weight of CPA [
35,
36,
37,
38,
39]. This way, a 28-amino acid
N-terminal signal peptide was identified, as well as some biochemical properties of the toxin were described. Many strategies have been employed in an attempt to obtain a non-toxic version of CPA for vaccination, the most common of which are site-directed mutagenesis, isolation of the strains that naturally produce non-toxic CPA, expression of only the
N- or
C-terminal domain, expression of chimeric toxins, and expression on the surface of
Bacillus subtilis spores (
Table 2) [
32,
40,
41,
42,
43,
44,
45].
Site-directed mutagenesis studies initially aimed to characterize essential residues for CPA toxicity and became the base for the production of genetically modified, non-toxic, immunogenic, recombinant CPA. It has been reported that the H68G, H148G/L, D56G, and E152Q mutations are capable of obliterating the toxicity of CPA [
32,
33]. In one study, mutation of D56N was able to reduce platelet aggregation and PLC activity, thereby increasing LD
50 from 0.5 to 100 μg/kg in mice [
49]. The researchers found that the mutation T272P reduced CPA toxicity by 35% [
50]. Site-directed mutagenesis of D336N, Y275N, D269N, Y331L, Y331F, Y307F, and Y275F reduced hemolytic activity by 11%, 11%, 19%, 30%, 36%, 38%, and 73%, respectively [
51]. Shoepe et al. [
45] identified a naturally occurring non-toxic variant of CPA (CPA-121A/91) with M13V, A174N, T177A, H212R, P295Q, S335P, I345V, and W360G mutations. This variant presented no hemolytic, PLC, or sphingomyelinase activity. In an alternative study, vaccinations with CPA-121A/91 were able to extend the lifespan of challenged mice, but could not prevent death. Interestingly, reversion of the H212R mutation was able to protect 76% (17/21) of the vaccinated mice [
52]. These results provide some insights into the possible epitopes and crucial sites for the toxin to act, and might prove useful for the development of both protective and therapeutic antibodies.
Recombinant, non-mutated CPA may present residual toxicity with dermonecrotic activity and might not be suitable for vaccination [
53]. Formaldehyde is extensively used to detoxify native CPA, although it also reduces immunogenicity [
19,
54,
55]. Alternatively, site-direct mutagenized recombinant or naturally occurring non-toxic CPA could be used for vaccination; however, in such cases, the immune response will be against the whole toxin, not solely the protective epitopes. Immunization of mice with
N- and
C-terminal domains of CPA (CPA-N
(1–246) and CPA-C
(247–370), respectively), or with the latter fused to GST (GST-CPA-C
(247–370)) expressed in
E. coli demonstrated that CPA-C
(247–370) (19 kDa) alone is capable of conferring immunity against challenge with 50 µg of CPA or 10
9 C. perfringens cells [
44,
48]. Animals inoculated with CPA-N
(1−246) were not protected against CPA. Taken together, these results indicate that blocking CPA binding to host cell is a necessary and sufficient method of conferring immunity against this toxin. It negates the need to neutralize its enzymatic activity and renders the
C-terminal domain as the main vaccine candidate against CPA. Furthermore, in one study, mice vaccinated with rCPA-C
(247–370) were protected against PLCs from
Clostridium absonum (CAA) and
Clostridium bifermentans (Cpb), which share 60% and 50% identity respectively with
C. perfringens CPA [
46].
Structural vaccinology is a branch of structural biology that studies the epitopes responsible for conferring immunity. It is possible to design chimeras that consist only of protective epitopes of different toxins and to exclude the domains that do not confer immunity. This approach simplifies the production process because only one process is required to produce a chimera that can confer immunity to a range of toxins as opposed to many processes being executed for different toxins [
56]. Considering this, modifying the whole rCPA molecule excluding unnecessary domains would be a useful approach. In fact, the rCPA-C
(247–370) domain is being used to replace the whole rCPA as vaccine component, allowing the construction of novel chimeras for experimental vaccines against
C. perfringens. For example, a recombinant chimera (rCPAE) comprising CPA-C
(284–398), fused to the
C-terminal portion of
C. perfringens Enterotoxin (CPE-C
(197–312)), was found to protect 100% (12/12) of mice challenged with CPA, and 75% (9/12) of mice challenged with CPE [
43]. The protection induced rCPAE face the challenge with both CPA and CPE toxins has not been evaluated. CPA-C
(284–398) fused to the
N-terminal domain of
Staphylococcus aureus Alpha-hemolysin (SAA
(36–221)) protected 100% (6/6) of mice challenged with either CPA or SAA, and 81.3% (5/6) of mice challenged with both toxins (
Figure 1) [
41].
Zeng et al. [
26] evaluated four vaccine formulations against
C. perfringens toxins: (1) rCPA; (2) bivalent recombinant chimera comprised of CPB and CPB2—rCPB2B1; (3) co-administration of rCPB2B1 and rCPA; and (4) trivalent recombinant chimera comprised CPA, CPB, and CPB2—rCPAB2B1. The recombinant antigens were expressed and used as inclusion bodies in immunizations. Mice vaccinated with rCPA presented 80% protection (24/30) when challenged with 1 × LD
100 of
C. perfringens toxinotype C culture supernatant. Group 3 was 100% (30/30) protected against twice the challenge dose of Group 1. Group 4 was 93% (28/30) protected against the same challenge. The authors argued that the lower protection observed in Group 4 in comparison to the groups that received co-administered antigens was due to an alteration in conformational epitopes that resulted from many antigens joining together in only one polypeptide chain. Goossens et al. [
53] demonstrated that animals inoculated with GST-CPA-C
(247–370) were less protected than animals inoculated with CPA-C
(247–370) alone, suggesting that the presence of GST disrupts the protective potential of the
C-terminal domain of CPA. Williamson and Titball [
44] previously obtained similar results when mice vaccinated with CPA-C
(247–370) produced two times as many neutralizing antibodies as mice vaccinated with GST-CPA-C
(247–370). Surprisingly, the GST-CPA-C
(247–370) chimera expressed on the surface of
B. subtilis spores elicited the production of both systemic IgG and sIgA in the saliva, feces, and lung samples of the vaccinated animals. Mice immunized with 2 × 10
9 or 5 × 10
10 orally or intranasally respectively, were 100% (6/6) protected against 12 × LD
50 [
40]. These results suggest that it is not just vaccine composition and antigen design that are essential to the generation of immunity, but also the fashion in which antigens are presented to the immune system is crucial to achieving immunity against high doses of challenge. We strongly suggest all these aspects are taken into account when designing and testing novel vaccines, not only for clostridial toxins but also for all pathogens.
E. coli is by far the most used expression system for the expression of rCPA. Two kinds of plasmid vectors are frequently employed for this purpose: pT7, and pET. Both vectors contain the T7 promoter, an antibiotic resistance gene, and a copy of the
lacI gene for the regulation of the expression. The
E. coli strain BL21 (DE3), which contains the coding gene for the T7 DNA polymerase in its genome under the control of lac operon, is the most commonly used strain. Lactose or similar molecules, such as alollactose or the synthetic derivate of galactose isopropyl-β-1-
d-galactopiranoside (IPTG), which cannot be metabolically degraded by
E. coli, can induce the lac operon. For the expression of rCPA, concentrations of 0.3–1 mM of IPTG are described in the literature as successful, and, most interestingly, only one work has described the attainment of an insoluble protein, although the culture conditions (i.e., medium, temperature, and induction time) were very similar to other works that described soluble rCPA. The expression of insoluble antigens is always perceived to be a problem for recombinant vaccine development since many protective epitopes can be lost due to erroneous protein folding. Thus, the optimization of expression conditions (medium, inductor concentration, pre-induction, and induction time, etc.) is often indicated. However, Zeng et al. [
26] described how the inclusion bodies of rCPA can be successfully used for animal vaccination without the need for denaturation, refolding, or even purification.