Clostridioides difficile is a Gram positive, anaerobic, spore-forming bacterium, and the major cause of infectious nosocomial diarrhea. Toxin production is considered as the main pathogenic factor for disease development. Toxigenic C. difficile strains harbor two toxins (TcdA and TcdB). In addition, some “hypervirulent” strains, such as ribotype 027 (RT027), express the binary toxin (CDT) of still unresolved clinical importance. RT027 and other “hypervirulent” strains exhibit higher levels of toxin production causing more severe course of disease (Warny et al., 2005).

The spectrum of CDI-related symptoms ranges from mild to severe courses of diseases (Napolitano and Edmiston, 2017; McDonald et al., 2018) with recurrence occurring in ≤30% of cases (McDonald et al., 2018). Risk factors for disease development and recurrence include among others antibiotic treatment, advanced age (≥ 65 years), or immunosuppression (Loo et al., 2011; Cózar-Llistó et al., 2016; Napolitano and Edmiston, 2017). However, intestinal dysbiosis with limited neutralization capacity of toxins seems to be crucial for disease development (Kyne et al., 2001; McDonald et al., 2018).

Oral antibiotic therapy of C. difficile is currently considered the mainstay of treatment for CDI (McDonald et al., 2018). However, these therapies pose the risk of recurrence.

While usage of toxin absorbing substances (Tolevamer) was effective in animal models but not in humans (Johnson et al., 2014), there are few studies suggesting a correlation between CDI-associated antibodies and a reduced risk of recurrence (Gupta et al., 2016; Kelly et al., 2019). Hence, promising approaches for new preventive therapies, such as monoclonal antibodies (Johnson and Gerding, 2019) or vaccines (Bézay et al., 2016; Kitchin et al., 2020), are currently developed. The first monoclonal antibody (Bezlotoxumab) was approved in 2017 for prevention of recurrences by neutralizing C. difficile toxin B (TcdB; Johnson and Gerding, 2019).

For further development of therapeutic strategies, the natural immune response seems to be of utmost importance. Likewise, antibodies against toxins and other C. difficile-specific targets may elaborate protective effects of humoral immune response. The common C. difficile antigen [glutamate dehydrogenase (GDH)] and cell wall-associated proteins (surface-layer proteins, SLP) may be involved. CWP84 is a paralogue of SLPs responsible for cleavage of SLP precursors into high molecular weight (HMW) and low molecular weight (LMW) subunits (de la Riva et al., 2011). Although cited very often in major textbooks, the association between occurrence of antibodies and clinical outcome is still debated (Gilbert et al., 2021), as data on antibody formation and their ability to neutralize toxins in the acute phase are limited.

Therefore, the aim of this prospective single-center study was to investigate the presence and dynamics of natural antibody response during the acute phase of CDI (anti-TcdA, anti-TcdB, anti-GDH, anti-CWP84, and neutralizing antibodies) and to assess the corresponding clinical course of disease.

Humoral immunity is crucial for treatment and prevention of a variety of toxin-related diseases, including CDI. Despite the frequently cited importance of antibodies in CDI, knowledge about natural antibody response is still limited (Viscidi et al., 1983; Kyne et al., 2001; Wullt et al., 2012; Gupta et al., 2016; von Eichel-Streiber et al., 2016; Kelly et al., 2019; Gilbert et al., 2021).

Only a low prevalence of anti-TcdA and of anti-TcdB antibodies were detected in our CDI patients, which is in line with previous studies (Kelly et al., 2019), and rates were only slightly higher in studies with prolonged follow-up (Bacon and Fekety, 1994; von Eichel-Streiber et al., 2016).

During the first week after CDI diagnosis, antibody titers remained stable and booster effects were not found, similarly to Wullt et al. (2012) which showed no significant anti-TcdA increase and only a moderate increase for anti-TcdB during prolonged follow-up. Kyne et al. (2001) could demonstrate a slight increase of anti-TcdA-IgG for patients with first episodes of CDI, but also stable antibody titers in patients with recurrence. However, in the previous aforementioned study, the dynamics of antibody response were marginal and gradual differences compared to our study might be related to a prolonged observation period, which might have captured a delayed antibody response.

Antibody detection during the first days of disease will very likely represent the preformed B-cell memory rather than a primary immune response, as antibody prevalence in studies with healthy control groups (Bacon and Fekety, 1994; von Eichel-Streiber et al., 2016) were comparable to those observed in the present study. Small differences could be related to methods but also to the fact that their patient cohort consisted of younger patients with no underlying diseases in contrast to our older patients with a history of numerous hospitalizations associated with an increased risk of C. difficile colonization.

Few studies focused on antibodies to toxin-unrelated antigens, such as GDH or CWP84. Overall, a study of Péchiné et al. showed a similar prevalence and stable titers of anti-CWP84 (Péchiné et al., 2005).

To our best knowledge, this is the first study investigating the prevalence and course of anti-GDH antibodies. In fact, seroprevalence of anti-GDH was high compared to other C. difficile-specific antibodies indicating prior C. difficile contact. Surprisingly, only 30% of our small healthy control group were anti-GDH positive. Increased anti-GDH seroprevalence of CDI patients could be explained by extended exposure of chronically ill patients to C. difficile contaminants in the healthcare system. Furthermore, antibodies could be elicited by other clostridial species (e.g., C. clostridioforme) due to cross-reactivity. Similar to other C. difficile antibodies, anti-GDH titers remained stable without significant booster effects.

GDH is the common C. difficile antigen secreted with high abundance following infection with C. difficile strains, irrespective of being toxigenic or non-toxigenic (Girinathan et al., 2014) and almost all individuals have repeated and regular C. difficile exposure since early infancy (Sánchez-Hurtado et al., 2008; Kelly and Kyne, 2011). It is tempting to speculate that anti-GDH might be used as a diagnostic parameter to estimate the overall C. difficile seroprevalence in the population. However, we found an anti-GDH prevalence ≤85% in CDI patients as well as heathy controls, which suggests that this parameter is not well suited to be used as a general indicator of past C. difficile history.

In the present study, 26% of CDI patients had neutralizing antibodies against RT027 toxins and only 11% against both toxins (RT027 and RT014). Seroprevalence was also low for neutralizing antibodies of heathy controls (30%). Before, Viscidi et al. (1983) described also low seroprevalence of neutralizing antibodies against TcdB (22%) as well as TcdA (9%). Furthermore, in recent vaccination studies, the seroprevalence and also quantitative titers of neutralizing antibodies were low in unvaccinated controls (Kitchin et al., 2020). Low or absent neutralizing antibody titers at onset of disease in combination with missing dynamics of antibody response in the early phase of CDI may explain why natural antibodies to C. difficile and its toxins did not influence the clinical CDI course (von Eichel-Streiber et al., 2016). No correlation could be found between the presence of neutralizing antibodies and disease severity, which is consistent with previous studies (von Eichel-Streiber et al., 2016; Kelly et al., 2019).

In our study, the presence of anti-TcdB was correlated with the presence of neutralizing antibodies against RT014 and RT027 toxins. Viscidi et al. (1983) suggested that anti-TcdB and anti-TcdA correlated with neutralization. In the present study, a correlation with anti-TcdA was ruled out. Discrepant results might have occurred prior due to cross-reactivity of diagnostic antigen preparations while highly purified toxins were now available for the present study.

High relapse rates were also found among anti-TcdB positive (38%) and NT positive patients (25%). One other recent study also failed to demonstrate an association between the presence of anti-TcdB and the risk of recurrence (Gilbert et al., 2021).

However, anti-TcdB titers can be obtained by vaccines which is different to regular antibody production after natural intestinal contact (Kitchin et al., 2020) and are required for preventing CDI recurrence (Gupta et al., 2016; Kelly et al., 2019) demonstrated by successful passive immunization with Bezlotoxumab and active toxoid vaccination (Kitchin et al., 2020).

While TcdB is accepted to be the major toxin associated with CDI, the clinical role of TcdA is still a matter of debate. In this study, anti-TcdB but not anti-TcdA was correlated to toxin neutralization confirms the fact that TcdB is the predominant toxin related to cytopathic effects and presumably also to toxin-related disease.

Interestingly, neutralization capacity of RT014 and RT027 toxins was different for a number of patients. Twelve patients had neutralizing antibodies to RT027 toxins and five of them to RT014 toxins also. No single sample could neutralize RT014 alone.

Thus, neutralizing antibodies were always active against RT027 toxins while antibodies directed against RT014 toxins only were not present in our study. This main finding confirms the assumption that toxigenic C. difficile strains express conserved but also strain-specific neutralizing TcdB epitopes (Supplementary Figure 2; Hernandez et al., 2015). Antibodies against conserved epitopes potentially bind and neutralize TcdB of different RTs, which was demonstrated here for a minority of patients with the capacity of RT014 and RT027 toxin neutralization. In contrast, antibodies against epitopes in variable regions would only be able to neutralize toxins of specific strains.

This single-center study has several limitations. This is a pilot study with a small cohort of patients and controls. A substantial loss of long-term follow-up is intrinsic to almost all studies with C. difficile dealing with an elderly multimorbid cohort. These limitations for statistical analysis may be overcome by larger, prospective multi-center studies. Early booster effects after CDI could be ruled out for all C. difficile antibodies investigated. However, we cannot exclude a delayed antibody response because samples after prolonged period of times were missing. This remains to be focused in future studies, especially to collect an antibody status before any recurrence. Furthermore, neutralizing antibodies should also be tested against conditioned media of other endemic and epidemic ribotypes (e.g., RT001) in comparison to distinguish strain-specific from broad-spectrum epitopes of antibodies.

The natural C. difficile antibody response was low. Booster effects with seroconversion or increased antibody production were not evident within the first week after CDI. Presence of specific antibodies was not associated with patients’ characteristics, clinical symptoms, or disease severity. Anti-TcdB was directly correlated with toxin neutralization. Samples with broad-range and with strain-restricted neutralization capacity were found, which might be related to different neutralizing epitopes.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Saarland Medical Council No. 20/14. The patients/participants provided their written informed consent to participate in this study.

LvM: conceptualization and supervision. LvM, SR, AN, and JB: methodology. SR and JB: investigation. SR: data curation and writing-original draft preparation. LM, JB, SLB, FB, PJ, and AM: writing-review and editing. SR and PJ: visualization. All authors contributed to the article and approved the submitted version.

The present work was supported by an unrestricted grant from TechLab, Inc., Blacksburg, Virginia and by the German National Reference Center for Clostridioides (Clostridium) difficile supported with an unrestricted grant from the Robert Koch Institute, Germany.

JB is employee at TechLab Research and Development, FB received consultant fees from MSD and Pfizer.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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