Anti-toxin Immune Responses Research Articles

Development of a new serological assay for the diagnosis of Clostridium difficile infections with prognostic value

PurposeThe most common hospital-acquired enteral infection is caused by Clostridium difficile. Unfortunately, Clostridium difficile infections (CDI) are of high risk to recur and little is known about how to predict recurrences. Previous findings have shown that high risk for recurrence correlates with low levels of C. difficile toxin-A and -B specific antibodies suggesting the protective role of humoral immunity against bacterial virulence factors. Therefore, the aim of this study was to develop an immunoassay, which specifically measures C.difficile toxin-specific antibodies in the serum that might be correlated with the risk of recurrence. MethodsWe developed a simple ELISA to measure the quantity of toxin-A and -B-specific antibodies in human serum. The assay was then used to test anti-toxin immune response in healthy controls, in patients with primary CDI and patients with CDI recurrence. ResultsThe developed assay is simple, reproducible and fast. When using this test in a small clinical trial our results showed a trend toward a higher antibody level in those patients with only one episode of CDI, whereas patients with recurrent CDI had less anti-toxin A or B-specific antibodies in their serum indicating inadequate C. difficile anti-toxin immunity may facilitate recurrent infections. ConclusionsIt has already been observed that low antibody levels are associated with recurrent CDI (Bauer et al., 2014). The findings of our clinical trial show a similar trend. Our developed ELISA test could help to conduct further research and it might be helpful in clinical use to detect patients of high risk for CDI recurrence.

Nanotoxoid vaccines

To improve innate defense against diseases, vaccine formulations are routinely administered to mount immune responses against disease-causing organisms or their associated toxins. These formulations are typically prepared with weakened forms of microbes, their surface proteins, or their virulence factors, which can train the immune system to recognize and neutralize similar infectious threats in later exposures. Owing to many unique properties of nanoparticles in enhancing vaccine potency, nanoscale carriers are drawing increasing interest as a platform for developing safer and more effective vaccine formulations. Notably, a nanoparticle-based strategy was recently demonstrated to safely deliver intact, non-denatured protein toxins to mount a potent anti-toxin immune response. A biomimetic nanoparticle cloaked in biological membranes was used to sequester membrane-active toxins. Upon interaction with the nanoparticles, the toxins become retrained and lose their toxicity as they are precluded from interacting with cellular targets. The resulting particle/toxin complex adopts a nanoparticulate morphology that facilitates the toxins' intracellular delivery. This sequestration approach has immense immunological implications owing to its ability in enabling structurally preserved toxins for immune processing. This technique offers opportunities in novel toxoid vaccine designs that promise more effective anti-toxin immune responses and contrasts the existing paradigm in toxoid preparation, in which toxins are antigenically altered to ensure virulence removal. The potent nanotoxoid formulations provide a viable anti-virulence measure in combating microbial infections that involve membrane-damaging toxins, including methicillin-resistant Staphylococcus aureus (MRSA) and Group A streptococcal infections.

Mortality in patients with Clostridium difficile infection correlates with host pro-inflammatory and humoral immune responses

Host anti-toxin immune responses play important roles in Clostridium difficile disease and outcome. The relationship between host immune and inflammatory responses during severe C. difficile infection (CDI) and the risk of mortality has yet to be defined. We aimed to investigate the host systemic IgG anti-toxin immune responses, the in vitro cytotoxicity of the infecting C. difficile ribotyped strain, and the host inflammatory markers and their relationship to CDI disease severity and risk of mortality. Inflammatory markers, co-morbidities and CDI outcomes were recorded in a prospective cohort of 150 CDI cases. Serum anti-cytotoxin A (TcdA) and anti-TcdB IgG titres were measured by ELISA and the infecting C. difficile isolate was ribotyped and the in vitro cytotoxin titre assessed. A low median anti-TcdA IgG titre was significantly associated with 30-day all-cause mortality (P<0.05). Ribotype 027 isolates were significantly more toxinogenic than other ribotypes (P<0.00001). High cytotoxin titres correlated with increased inflammatory markers but also higher anti-TcdA and -TcdB (P<0.05) IgG responses resulting in a lower risk of mortality. On multivariate analysis, predictors of mortality were peak white cell count >20 × 10(9) l(-1) [odds ratio (OR) 11.53; 95 % confidence interval (CI) 2.38-55.92], creatinine concentration >133 µmol l(-1) (OR 6.54; 95 % CI 1.47-29.07), Horn's index >3 (OR 4.09; 95 % CI 0.76-22.18) and low anti-TcdA IgG (OR 0.97; 95 % CI 0.95-0.99), but not ribotype, cytotoxin titre or anti-TcdB IgG. Thus, host pro-inflammatory and humoral responses correlate with the cytotoxin titre of the infecting strain and effective anti-toxin immune responses reduce the risk of mortality.

Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002–2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (μg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6–11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5–15.4% and 6.3–13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log10 transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r2=0.980–0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.

The host immune response to Clostridium difficile

Clostridium difficile is the most common cause of nosocomial bacterial diarrhoea in the Western world. Diarrhoea and colitis are caused by the actions of toxins A and B released by pathogenic strains of C. difficile. Adaptive immune responses to these toxins influence the outcomes of C. difficile infection (CDI). Symptomless carriers of toxinogenic C. difficile and those with a single episode of CDI without recurrence show more robust antitoxin immune responses than those with symptomatic and recurrent disease. Immune-based approaches to CDI therapy and prevention have been developed using active vaccination or passive immunotherapy targeting C. difficile toxins. Innate immune responses to C. difficile and its toxins are also central to the pathophysiology of CDI. An acute intestinal inflammatory response with prominent neutrophil infiltration and associated tissue injury is characteristic of CDI. Furthermore, inhibiting this acute inflammatory response can protect against the intestinal injury that results from exposure to C. difficile toxins in animal models. Studies examining host risk factors for CDI have led to validated clinical prediction tools for risk of primary and of recurrent disease. Risk factors associated with severe CDI with poor clinical outcomes have also been identified and include marked elevation of the peripheral white blood cell count and elevated creatinine. However, further work is needed in this area to guide the clinical application of new approaches to disease prevention and treatment including new antimicrobials as well as passive and active immunization.

Molecular recognition of botulinum neurotoxin B heavy chain by human antibodies from cervical dystonia patients that develop immunoresistance to toxin treatment

We determined the entire profile of the continuous antigenic regions recognized by blocking antibodies (Abs) in sera from 30BoNT/B-treated cervical dystonia (CD) patients who developed unresponsiveness to treatment. The sera protected mice against a lethal dose of BoNT/B. We analyzed Ab binding to a panel of 60 synthetic 19-residue peptides (peptide C31 was 24 residues) that overlapped consecutively by 5 residues and encompassed the entire BoNT/B heavy (H) chain (residues 442-1291). Most Abs recognized a limited set of peptides but the pattern and Ab levels bound varied with the patient, consistent with genetic control of immune responses and with responses to each epitope being separately controlled. Abs were bound by peptides (in decreasing order): C1 (residues 848-866), C10 (974-992), C16 (1058-1076), C14 (1030-1048), N15 (638-656), N21/N22 (722-740/736-754), N24/N25 (764-782/778-796) and N29 (834-852). Peptides N3/N4 (470-488/484-502), N27 (806-824), C2 (862-880), C4 (890-908), C6/C7 (918-936/932-950), C17 (1072-1090), C24 (1170-1188), C29 (1240-1258) and C31 (1268-1291) exhibited low Ab binding. The remaining peptides bound little or no Abs. Of the 30 antisera, 28 (93.3%) had Abs that bound to peptides C1, C10, C14 or C16, and 27 (90.0%) bound to peptide N22. No peptide was recognized by all the antisera, but peptide combinations N24+C1, N22+N24+C1, N24+C1+C10, C10+C14+C16, N22+N24+C1+C10, C1+C10+C14+C16 or N22+N24+C1+C10+C14 bound blocking Abs in 30 (100%) antisera. BoNT/B-treated CD patients had higher Ab levels and bound to more epitopes (at least 11) than did BoNT/A-treated patients (5 regions). The regions recognized by anti-BoNT/B Abs occupied surface areas that displayed no correlation to surface electrostatic potential, hydrophilicity, hydrophobicity, or temperature factor. These regions afford candidates for epitope-specific manipulation of anti-toxin immune responses.

Transcutaneous immunization with the heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC): Protective efficacy in a double-blind, placebo-controlled challenge study

Background An enterotoxigenic Escherichia coli (ETEC) vaccine could reduce diarrhea among children in developing countries and travelers to these countries. The heat-labile toxin (LT) of ETEC is immunogenic but too toxic for oral or nasal vaccines. Methods In a double-blind, placebo-controlled trial, 59 adults were randomized to receive 50 μg of LT or placebo in a patch applied to alternating arms on days 0, 21, and 42. On day 56, 27 vaccinees and 20 controls were challenged orally with 6 × 10 8 cfu of LT +/ST + ETEC. Results 100 and 97% of vaccinees had 4-fold increases in anti-LT IgG and IgA, and 100 and 90% developed IgG- and IgA-antibody-secreting cell responses. The study did not meet the primary endpoint: 82% of vaccinees and 75% of controls had moderate to severe ETEC illness. However, vaccinees with ETEC illness had lower numbers (6.8 versus 9.7, p = 0.04) and weights of loose stools (840 g versus 1147 g, p < 0.05), a decreased need for intravenous fluids (14% versus 40%, p = 0.03) and a delayed onset of diarrhea (30 h versus 22 h, p = 0.01). Conclusions Transcutaneous LT vaccination induced anti-toxin immune responses that did not prevent but mitigated illness following a high-dose challenge with a virulent LT +/ST + ETEC strain.

Clostridium difficile

Clostridium difficile is a major cause of antibiotic-associated diarrhea and colitis. The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics. Pathogenic strains of C. difficile produce two protein exotoxins, toxin A and toxin B, that cause colonic mucosal injury and inflammation. Many patients who are colonized are asymptomatic, and recent evidence indicates that diarrhea and colitis occur in those individuals who lack a protective antitoxin immune response. In patients who do develop symptoms, the spectrum of C. difficile disease ranges from mild diarrhea to fulminant pseudomembranous colitis. Prevention of nosocomial C. difficile infection involves judicious use of antibiotics and multidisciplinary infection control measures to reduce environmental contamination and patient cross-infection. Ultimately, active or passive immunization against C. difficile may be an effective means of controlling the growing problem of nosocomial C. difficile diarrhea and colitis.

Oral administration of a bacterial immunomodulator enhances the immune response to cholera toxin

Attempts to achieve IgA responses in the intestine by oral immunization with non replicating antigens have been characterized by ineffective responses of short duration unless long term dosages are administered. Cholera toxin (CT) is an exception in that it is able to produce a high secretory and systemic immune response. We study the effects of a bacterial immunomodulator [3 × 10 10 Propionibacterium granulosum ml −1 and lipopolysaccharide (LPS) 5 mg ml −1] on the immune response to CT orally administered to Wistar rats. The immunomodulator was orally administered as follows: in schedule 1 during 7 days prior to the first dose of CT; and in schedule 2, 2 days before, together, and 3 days after the first dose of CT. Schedules 1 and 2 were effective in increasing the specific IgA in the intestinal fluid and specific IgG in serum ( P<0.001) when compared to controls. Besides, schedule 2 was more effective than schedule 1 when the levels of specific IgG in serum or specific IgA in intestinal fluid was measured ( P<0.05). Total IgA in the intestinal fluid was increased in rats receiving the immunomodulator ( P<0.01). However, the ratio of specific IgA per total IgA was higher in rats receiving treatment 1 or 2 when compared to controls ( P<0.01). The number of antitoxin antibody producing cells was not increased in the Peyer patches, but a significant increase was observed in the mesenteric lymph nodes and spleen when compared to controls ( P<0.05). The administration of LPS alone produced an increase in the antitoxin immune response when compared to controls, but it was lower than those produced by the administration of the immunomodulator. These results indicate that this immunomodulator is an effective adjuvant of the mucosal and systemic immune response to CT. The mechanisms of action possibly involve nonespecific and specific modulations of the immune response.

Serum antibacterial and antitoxin responses in clinical cholera caused by Vibrio cholerae O139 Bengal and evaluation of their importance in protection

Vibrio cholerae O139 Bengal strain was the causative agent of the recent epidemics of cholera in India and Bangladesh. We studied antibacterial and antitoxin immune responses in acute and convalescent phase paired sera collected from seven of these cholera patients. Significant rise in the levels of both antibacterial and antitoxin antibodies was demonstrable in the sera of convalescent cholera patients. Antibacterial antibodies, directed primarily against O139 lipopolysaccharides (LPS), belonged to IgM class, while antitoxin antibodies were of IgG and IgA class and neutralized cholera toxin. The convalescent sera, however, showed no increase in the reactivity towards V. cholerae O1 whole cells or their LPS preparation. Immunoblotting experiments revealed that the convalescent, but not the acute, phase serum recognized the truncated form of LPS characteristics of O139 strains. Convalescent serum also induced definite protection against O139, but not O1, challenge in experimental animal model. Further studies showed that such protection was probably mediated by antibodies inhibiting intestinal colonization of O139 organisms. These results suggest that critical difference(s) exists between the immunogenic somatic components of V. cholerae O1 and O139 organisms that are of considerable importance in protection against cholera.

Host defense against cholera toxin is strongly CD4+ T cell dependent

This study investigates the role of CD4+ T cells in host defense against cholera enterotoxin-induced diarrhea. Antitoxin immunoglobulin A formation and gut protection against cholera toxin (CT) following oral immunizations with CT were evaluated in normal mice and mice that had been depleted of CD4+ T cells by in vivo treatment with specific anti-CD4 monoclonal antibodies. Flow cytometer analysis demonstrated that anti-CD4 monoclonal antibody effectively eliminated CD4+ T cells in the spleen, mesenteric lymph nodes, and Peyer's patches. In contrast, lamina propria lymphocytes demonstrated only some decrease in CD4+ T-cell numbers following antibody treatment. However, CD4 expression of individual lamina propria lymphocytes was strongly down-regulated. Depletion of CD4+ T cells performed prior to oral immunization with CT completely inhibited the ability to respond to CT. No antitoxin production, as detected at the single-cell level by the ELISPOT technique, was found in the spleen, mesenteric lymph nodes, or Peyer's patches, nor did we observe serum antitoxin responses in these mice. Control mice demonstrated strong antitoxin responses in all locations following oral immunization with CT. Anti-CD4 antibody treatment also effectively inhibited the antitoxin immunoglobulin A response in the lamina propria to CT as well as blocked the ability to develop gut protection against CT challenge of ligated intestinal loops after oral CT immunization. Thus, in vivo CD4+ T-cell depletion rendered these mice unable to develop protective immunity in the gut following oral immunization with CT. Moreover, CD4+ T-cell depletion effectively inhibited the antitoxin immune response in the gut lamina propria, mesenteric lymph nodes, Peyer's patches, and spleen when performed prior to both priming and booster immunizations with CT. This study clearly demonstrates the requirement of functional CD4+ T cells in the gut immune system for the development of host defense against CT-induced disease. Our data also reinforce the concept of a strong association between gut protection against CT and local production of neutralizing immunoglobulin A antitoxin.