1. Introduction
Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients [
1,
2]. The bacterium has two toxins, A and B, that play a pivotal role in the pathogenesis of the disease [
3]. Up to 35% of patients have recurrent
C. difficile infection (CDI) [
4,
5].
C. difficile can cause asymptomatic colonization, as well as disease with various degrees of severity from mild diarrhea to fulminant colitis and death [
6,
7,
8]. Such variability in the outcome of
C. difficile infection can be attributed to the patient’s characteristics, including advanced age, the severity of underlying comorbidities, and the systemic humoral immune response against
C. difficile toxins [
9,
10,
11,
12].
Epidemiological observational studies have demonstrated the important role of the systemic humoral immune response directed against
C. difficile toxin A (TcdA) and B (TcdB) in the risk of primary and recurrent CDI [
10,
11,
12,
13,
14,
15] and mortality following CDI [
9]. Kyne et al. have demonstrated high levels of serum immunoglobulin G (IgG) directed against TcdA among asymptomatic carriers of
C. difficile compared to persons who developed diarrhea [
12]. They also showed an increased risk for recurrent CDI in persons with low serum IgG antibodies against TcdA compared to those with a high antibody level [
11]. Solomon et al. showed that low anti-TcdA IgG titer was significantly associated with 30-day all-cause mortality in patients with severe CDI [
9]. Such an association was not found for TcdB IgG antibodies [
9]. Others, including recent studies, on the other hand, have highlighted the role of serum IgG or IgA against TcdB, rather than TcdA, in CDI outcome [
10,
13,
14,
15,
16,
17]. Recent clinical trials (MODIFY I and MODIFY II) with the human monoclonal antibodies actoxumab and bezlotoxumab against TcdA and TcdB, respectively, showed that among participants receiving antibiotic treatment for primary or recurrent CDI, bezlotoxumab was associated with a lower rate of recurrent infection than placebo, while the addition of actoxumab did not improve efficacy [
18]. Secondary analysis of the placebo group in these trials showed an inverse association between high endogenous antibody titers against TcdB but not TcdA and recurrent CDI [
19]. Despite this evidence, there are unresolved questions, namely the relationship of the humoral immune responses against
C. difficile toxins with the disease severity of CDI and the relative importance of serum antibodies against toxins TcdA and TcdB. The main aim of this study was to examine the differences in serum IgG and IgA levels against TcdA and TcdB between CDI and control patients and according to CDI severity. Our hypothesis was that patients with mild CDI might have higher humoral responses compared to patients with severe CDI. The evaluation of the humoral immune responses according to the severity of CDI is novel, compared to previous studies that mostly focused on the prevention and recurrence of CDI.
4. Discussion
We found significantly higher levels of serum IgG antibodies against TcdA and TcdB in patients with CDI compared to the control group. This finding might be somewhat surprising, given the compelling evidence on the protective role of circulating IgG antibodies against
C. difficile toxins in preventing colonization and primary and recurrent CDI [
10,
11,
12,
15,
19]. However, unlike our study, previous studies [
10,
11,
12,
15,
19] have assessed the levels of pre-existing serum antibodies and the subsequent outcome of
C. difficile infection. Sera obtained from the CDI patients in our study mainly represent the early and late convalescent phase of disease that typically shows increases in serum antibodies over the acute phase levels [
14,
27,
28,
29]. Our finding of a higher systemic humoral response against
C. difficile toxins than the control group is thus reasonable and compatible with findings from studies that employed a similar study design [
28,
29,
30].
The main findings of this study are the differences in the humoral immune response against TcdA and TcdB in relation to CDI severity. We found a higher level of serum IgA antibodies against TcdB among CDI patients with mild disease compared to patients with severe CDI. About 7–14 days after CDI diagnosis, the levels of serum IgG levels against TcdA and TcdB were significantly higher in patients with mild vs. severe CDI. These findings suggest that serum antibodies against TcdA and TcdB likely moderate the severity of CDI. These results add new supportive evidence regarding the importance of circulating serum antibodies against both toxins in moderating the disease severity. While earlier studies have emphasized anti-TcdA serum antibodies in preventing CDI and its recurrence [
11,
12], later studies have highlighted the significance of antibodies directed against TcdB [
10,
18]. However, the dissection of the specific and independent role of antibodies against TcdA and TcdB is complex given the significant correlations between them, as evident in our findings. Another interesting point is the relative importance of serum IgA vs. IgG antibodies.
C. difficile causes a mucosal disease with the colon being the target organ, thus the involvement of IgA, which usually correlates with mucosal immunity, seems intuitive. However, the involvement of circulating IgG antibodies in CDI severity is unique and, as mentioned above, corroborates with existing knowledge [
10,
11,
12]. Our factor analysis supports the notion that both serum IgA and IgG levels against both TcdA and TcdB play significant roles in moderating CDI severity.
The mechanism that can explain the protective effect of serum IgA and IgG antibodies directed towards
C. difficile toxins is still not fully clear. However, these circulating antibodies were shown to correlate with neutralizing capabilities [
27], although such correlations were not always demonstrated [
13,
27]. Interestingly the neutralizing capabilities were demonstrated in convalescent sera [
27]. This supports the observed inverse association that we report between serum IgG antibody levels and disease severity 7–14 days after the diagnosis of CDI (
Table 2), which accompanies the increased IgG levels against both toxins in paired sera (
Table 3). Collectively, ours and others’ findings [
10,
11,
12] re-establish the importance of circulating serum IgG and IgA levels against both TcdA and TcdB in the prevention and control of CDI.
Our studies on other bacterial pathogens, namely
Shigella spp., that target the colon and cause dysentery also show the importance of circulating IgG antibodies in the prevention of shigellosis [
31]. The pre-existing serum IgG antibody level against
Shigella lipopolysaccharides (LPSs) was shown to be protective against natural infection caused by
Shigella sonnei [
32]. Clinical trials with conjugate
S. sonnei vaccines (based on the LPS linked with carrier protein) administered intramuscularly resulted in significant protection against the disease in young adults [
33]. The vaccine also resulted in increases in serum IgG level against
S. sonnei LPS [
33] and it has been proposed as the correlate of protection [
31]. Similarly, serum IgG antibodies against
C. difficile toxins might serve as a correlate of protection in clinical trials with active vaccines targeting
C. difficile. Currently there is no licensed
C. difficile-active vaccine but several are in clinical development [
34,
35,
36]. Based on the current evidence from observational studies [
9,
10,
11,
12,
19] and our new findings, the concept of presenting antigens that can prime or boost the immune system towards the production of antitoxin circulating antibodies seems a sensible approach for developing preventive and therapeutic vaccines and technologies for CDI.
The additional implications of our findings relate the use of monoclonal antibodies for the prevention of CDI. Recent clinical trials with the human monoclonal antibodies, bezlotoxumab against TcdB, demonstrated a significant efficacy in preventing recurrent CDI, while the addition of TcdA monoclonal antibody actoxumab [
18] did not improve the efficacy of preventing recurrence. Bezlotoxumab was approved for the prevention of recurrent CDI by the regulatory agencies in US and Europe [
37]. Our finding suggests that antibodies against TcdA might be needed for moderating the severity of CDI. Moreover, our findings suggest the need to expand the assessment of efficacy of bezlotoxumab and actoxumab in reducing CDI severity and complications in future large-scale studies. Altogether, ours and others’ findings provide supportive evidence for accelerating the development and evaluation of active and passive immunotherapies for the management of CDI, as an alternative or supplement to existing antibiotic treatments or fecal microbiota transplants.
The strong points of this study include the well-defined prospective case-control study of CDI risk factors [
20,
21], in which patients’ characteristics and samples were collected according to standardized protocols, the comprehensive assessment of both serum IgA and IgG levels against both
C. difficile toxins, and the analytical approach that signifies the importance of all these markers in CDI severity. Our study has limitations—namely, the small sample size, especially the number of participants in the subgroups of disease severity, in addition to the lack of pre-existing sera. The selection of hospital controls might be a limitation since they do not necessarily represent the general population. However, immunosuppression conditions that might negatively affect the immune system, such as cancer and chemotherapy, were more common among the CDI cases than the controls [
20,
21].