Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers

doi:10.1016/j.vaccine.2005.07.022

Vaccine

Volume 24, Issue 18, 1 May 2006, Pages 3709-3718

https://doi.org/10.1016/j.vaccine.2005.07.022 Get rights and content

Abstract

In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 × 10⁸ or 4 × 10⁹ cfu of ETEC strain E24377A (LT⁺, ST⁺, CS1⁺, CS3⁺) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 × 10⁹ cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75–95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p < 0.001) and 0.82 (p < 0.001), respectively after challenge and 0.78 (p < 0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p < 0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

Introduction

Enterotoxigenic Escherichia coli is among the most common causes of diarrhea among infants and children in developing countries, and is also the primary etiologic agent of Traveler's diarrhea among international visitors to the developing world [1]. A vaccine to prevent ETEC diarrhea would be a great public health benefit in reducing morbidity and mortality among children in developing countries and would also help reduce lost work hours and vacation time among business and holiday travelers [1], [2].

Antigen-specific intestinal secretory IgA (sIgA) responses are considered to play an important role in recovery from infectious diarrhea and in mediating vaccine or infection-induced protection against subsequent disease [3]. Direct measurement of intestinal sIgA responses to pathogen- or vaccine-specific antigens by lavage, endoscopy or aspiration is not practical on a large scale and suffers from quality control issues resulting from proteolytic degradation that can occur in fecal samples. Simpler quantitative methods are needed to support enteric vaccine development efforts and address regulatory agency interest in methods for evaluation of mucosal immune responses that can be validated and correlate with development of protective immunity. Chang and Sack [4] and Qadri et al. [5] have recently presented encouraging evidence indicating that the ELISPOT and a new in vitro assay, the antibody secreted in lymphocyte supernatants (ALS assay), are comparable as surrogate measures of mucosal responses to Vibrio cholerae antigens following natural infection or vaccination. The ALS assay was initially employed by B. Forrest who demonstrated its utility in measuring LPS-specific IgA responses to attenuated S. typhi vaccines [6]. The ALS assay has also been used to assess the mucosal immunogenicity of live attenuated S. typhimurium strains expressing Helicobactor pylori urease [7], and to identify patients with active cases of tuberculosis [8]. These positive results suggest that the ALS assay may also be suitable for use as a surrogate measure of mucosal immunity to ETEC antigens in subjects infected with this enteric pathogen or immunized with vaccine candidates. However, the ELISPOT and ALS procedures have yet to be directly compared in this regard.

In this study we evaluated the ALS assay as a surrogate measure of mucosal immunity to ETEC antigens by characterizing the ALS response to the CS3 and CS1 coli surface antigens and LT toxin in subjects experimentally infected with ETEC strain E24377A in a controlled clinical setting and in subjects immunized with a new candidate oral, live attenuated ETEC vaccine designated PTL-003. The primary goal of this study was to compare the ALS and ELISPOT procedures in their ability to measure the frequency and magnitude of recent mucosal immune responses to ETEC infection or vaccine immunization. A second aim was to compare ELISA and time-resolved fluorescence (TRF) as detection methods for measuring antigen-specific IgA antibody levels in ALS supernatants. As part of this secondary evaluation, frozen ALS supernatants were shared between laboratories to determine the degree of inter-laboratory concordance between the two potential ALS assay methods and also to assess the reliability of the ALS assay as a predictor of mucosal immunization when quantified using two different technologies. The results of this extensive comparative analysis indicate that ALS is a sensitive indicator of local immune responses to ETEC toxins and colonization factor antigens and is comparable to the more commonly used ELISPOT procedure. The successful inter-laboratory comparison of ALS detection methods using cryopreserved supernatants also serves to further highlight the greater flexibility and reliability of this technique and its greater potential for validation compared to the ELISPOT assay.

Section snippets

Safety and immunogenicity trial of two live attenuated ETEC vaccines in adult volunteers

Two live attenuated prototype vaccines, designated PTL-002 and PTL-003 were prepared from ETEC strain E1392/75-2A, a spontaneous toxin negative mutant (both LT and ST negative), which expresses coli surface antigens CS1 and CS3 [9]. The specific genetic deletions, final characterization, and preliminary safety and immunogenicity profiles of these vaccine constructs has been reported elsewhere [9]. These vaccines were further evaluated for safety and immunogenicity in a randomized, double-blind

Summary of clinical outcomes and immunological responses in subjects challenged with ETEC strain E24377A or immunized with the PTL-002 or PTL-003 vaccine constructs

Both attenuated vaccine constructs were well tolerated by immunized subjects and both induced anti-CFA/II specific mucosal immune responses after vaccination (Bourgeois AL, McKenzie R, Engstrom F et al. Abstract E-71. 101st Annual meeting of the American Society for Microbiology, Orlando, Fla, 20–24 May 2001). The incidences of general or gastrointestinal symptoms were not significantly different among recipients of either vaccine compared to placebo (Fisher's exact test for sparse outcomes,

Discussion

The present study is the first to compare the ALS and ELISPOT assays as detection methods for measuring mucosal immune responses to ETEC antigens among subjects challenged with a wild-type ETEC strain or orally immunized with a live attenuated candidate vaccine. The ALS assay has several advantages over the ELISPOT that might serve to make it a more practical assay for supporting ETEC vaccine development if it yields comparable results. Our results from the subjects infected with E24377A

Acknowledgements

This work was presented in part at the Vaccines for Enteric Diseases Conferences held in Tampere, Finland, September 2001 and in Montego Bay, Jamaica April 2004. The authors thank the GCRC, Osler 5 Research Ward staff, Lucy Gibson, Jennifer Kyle, and LaNisha Burke for their assistance in conducting this research and in preparing this manuscript. This work was supported in part by funds from Acambis Research, Ltd., Cambridge, UK, and by the Johns Hopkins University School of Medicine General

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Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial
2020, Vaccine
Citation Excerpt :
Interestingly, the trend of lower magnitudes of response to fractional booster doses was more pronounced for serum antibody responses than ALS antibody responses. In the case of non-invasive enteric pathogens such as V. cholerae and ETEC, results from the ALS assay, which reflects the mucosal immune response [11,15–19], are more biologically relevant than serum antibodies, as vaccine-specific antibodies are required in the gut lumen to neutralise the bacteria and associated toxins. Nevertheless, the relative importance of antibody concentrations and antibody avidity for vaccine-induced protection against cholera is not known.
Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.
To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).
We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.
Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.
ISRCTN registry identifier 11806026.
Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses
2019, Vaccine
Citation Excerpt :
After infection or vaccination in the intestinal mucosa, activated intestinal lymphocytes transiently migrate to the circulation before homing back to the mucosa. Therefore, ASCs present among peripheral blood mononuclear cells (PBMCs) are suitable surrogate markers of mucosal immunity [13,20–24], particularly if blood samples are collected at optimal time points after lymphocyte activation [16,18,25,26]. ASC responses can be analyzed using ELISPOT or by ALS, which is commonly based on ELISA techniques for detection of secreted antibodies in the culture medium, and results from the two assays correlate very well with each other [15,24,27,28].
The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology.
Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA.
ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87–100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62–93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens.
The promising results in adults supported testing ETVAX in descending age groups of children.
ClinicalTrials.gov Identifier: NCT02531802.
Cholera immunity and development and use of oral cholera vaccines for disease control
2019, Mucosal Vaccines: Innovation for Preventing Infectious Diseases
The development of oral cholera vaccines (OCVs) is a good example of how basic research can generate new knowledge that can be translated into a life-saving medical product.
The key mechanisms of disease and immunity in cholera, which allowed outlining the principles for the first effective OCV, the inactivated whole cell/B subunit (WC-BS) OCV, later to be licensed as Dukoral, were defined already in the 1970s. Cholera was identified as an archetype for noninvasive, enterotoxin-mediated infectious diarrheal diseases in which immune protection is mediated largely by locally produced secretory immunoglobulin A (SIgA) antibodies interfering with bacterial attachment and colonization and/or preventing toxin binding and action in the intestine. These antibodies are directed primarily against the cell wall lipopolysaccharide (LPS) and cholera toxin B subunit (CTB), respectively. Since the intestinal SIgA responses in cholera lasts for only approximately 6–9 months, the continued protection for over 2–3 more years seen after infection or vaccination depends on mucosal immunological memory with a duration of more than 10 years, allowing a rapid SIgA antibacterial and antitoxic anamnestic response upon renewed pathogen exposure. Serum vibriocidal antibodies, although often correlating with adaptive immune protection, are only a proxy for the intestinal SIgA immune status.
After Dukoral, which was internationally licensed in 1993, both inactivated and live, genetically attenuated OCVs have been developed. Inactivated OCVs have had the greatest success in achieving licensure and international acceptance. Three WHO-prequalified OCVs—Dukoral, Shanchol, and Euvichol—are currently available and differ mainly in that, in addition to several killed whole cell components shared with the other OCVs, the Dukoral OCV contains CTB. More than 15 large field trials have consistently shown that the described inactivated OCVs are quite safe and provide 60%–70% specific immune protection for at least 3 years. In addition, they confer significant “herd protection,” which further increases their total impact for reducing (or even eliminating) cholera in vaccinated communities.
OCVs are now a cornerstone of a global initiative, “Ending Cholera—A Global Roadmap to 2030,” which was launched in October 2017 by the World Health Organization’s Global Task Force for Cholera Control together with more than 50 other signatories, with the stated goal of reducing cholera deaths by at least 90% by 2030.
Kinetics of antibody-secreting cell and fecal IgA responses after oral cholera vaccination in different age groups in a cholera endemic country
2017, Vaccine
Immune responses to oral enteric vaccines in children and infants may be influenced by factors such as age, previous priming with related microorganisms and breast feeding. In this study, we aimed to determine optimal time points to assess immune responses to oral enteric vaccines in different clinical specimens. This was done by investigating antibody secreting cell (ASC) and fecal antibody responses on different days after vaccination using the licensed oral cholera vaccine Dukoral, containing cholera toxin B-subunit (rCTB) and inactivated Vibrio cholerae bacteria, as a model vaccine.
Two vaccine doses were given 2 weeks apart to infants (6–11 months), young children (12–18 months), toddlers (19 months–5 years) and adults in a cholera endemic country (Bangladesh). IgA ASC responses, as determined by the antibodies in lymphocyte supernatant (ALS) assay, plasma IgA and IgG responses and secretory IgA (SIgA) responses in extracts of fecal samples were evaluated 4/5 and 7 days after each vaccination.
After the first vaccine dose, anti-CTB ALS IgA responses in adults and toddlers were high and comparable on day 5 and 7, while responses were low and infrequent in young children. After the second dose, highest ALS responses were detected on day 5 among the time points studied in all age groups and the responses declined until day 7. In contrast, plasma IgA and IgG anti-CTB responses were high both on day 5 and 7 after the second dose. Fecal SIgA responses in young children and infants were highest on day 7 after the second dose.
Our results suggest that ASC/ALS responses to two doses of the oral cholera vaccine Dukoral and related oral vaccines should be analyzed earlier than previously recommended (day 7) at all ages. Fecal antibody responses should preferably be analyzed later than ASC/ALS responses to detect the highest antibody responses.
Induction of long term mucosal immunological memory in humans by an oral inactivated multivalent enterotoxigenic Escherichia coli vaccine
2016, Vaccine
We have evaluated the capacity of an oral multivalent enterotoxigenic Escherichia coli (ETEC) vaccine (MEV) to induce mucosal immunological memory. MEV consists of four inactivated E. coli strains over-expressing the major colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the LTB-related toxoid LCTBA. Memory responses were analyzed by comparing the magnitudes and kinetics of intestine-derived antibody-secreting cell responses to a single dose of MEV in three groups of adult Swedish volunteers (n = 16–19 subjects per group) in a Phase I trial: non-immunized controls (I) and subjects who in a previous Phase I trial 13–23 months earlier had received two biweekly doses of MEV (II) or MEV + double mutant LT (dmLT) adjuvant (III). Responses against CFs and LTB were analyzed in antibodies in lymphocyte secretions (ALS) of blood mononuclear cells collected before (day 0) and 4/5 and 7 days after immunization. Specific circulating memory B cells present at the time of the single dose vaccination were also studied to determine if such cells may reflect mucosal memory.
Considerably higher and significantly more frequent IgA ALS responses against all CFs and LTB were induced by the single vaccine dose in the previously immunized than in non-immunized volunteers. Furthermore, peak IgA ALS responses against all antigens were observed on days 4/5 in most of the previously immunized subjects whereas only a few previously non-vaccinated individuals responded before day 7. Priming with adjuvant did not influence memory responses. Circulating vaccine specific IgA memory B cells were not detected, whereas anti-toxin IgG memory B cells were identified 13–23 months after priming vaccination.
We conclude that MEV induces functional mucosal immunological memory which remains at least 1–2 years. Furthermore, our results support that analysis of antibody-secreting cell responses after booster vaccination may be a useful approach to evaluate longstanding mucosal immunological memory in humans.
Clinical trials registration: ISRCTN27096290.
Different kinetics of circulating antibody-secreting cell responses after primary and booster oral immunizations: A tool for assessing immunological memory
2013, Vaccine
We show that the kinetics of circulating IgA as well as IgG antibody-secreting cell (ASC) responses differs considerably after primary and booster vaccination with the oral cholera vaccine Dukoral^®, as determined by the antibody in lymphocyte supernatant (ALS) as well as ELISPOT methods. Thus, whereas the antitoxin ASC responses did not peak until 7–9 days after primary vaccination, peak responses to a second dose given after two weeks, or a single booster dose given 6 months to 14 years later, were recorded already after 4–5 days and then rapidly declined. Our results indicate that many previous studies reporting ASC results 7–10 days after repeated immunization may have substantially underestimated the magnitudes of the responses. The results also suggest that detection of peak ASC responses at an early time point after booster immunization can be used as a simple tool to assess immunological memory.

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Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers

Abstract

Introduction

Section snippets

Safety and immunogenicity trial of two live attenuated ETEC vaccines in adult volunteers

Summary of clinical outcomes and immunological responses in subjects challenged with ETEC strain E24377A or immunized with the PTL-002 or PTL-003 vaccine constructs

Discussion

Acknowledgements

Vaccine

Immunobiol

Vaccine

Anal Biochem

J Immunol Methods

J Immunol Methods

Vaccine

Vaccine

Vaccines against enterotoxigenic Escherichia coli infections. Part II. Live oral vaccines and subunit (purified fimbriae and toxin subunit) vaccines

Development of a novel in vitro assay (ALS assay) for evaluation of vaccine-induced antibody secretion from circulating lymphocytes

Clin Diagn Lab Immunol

Antigen-specific immunoglobulin A antibodies secreted from circulating B cells are an effective marker for recent local immune responses in patients with cholera: comparison to antibody-secreting cell responses and other immunological markers

Infect Immun

Indirect measurement of intestinal immune responses to an orally administered attenuated bacterial vaccine

Infect Immun

Pilot study of phoP/phoQ-deleted Salmonella enterica serovar typhimurium expressing Helicobacter pylori urease in adult volunteers

Infect Immun

Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions

J Infect Dis