Abstract
Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.
Highlights
Vaccines confer safety and are the most effective means of protection against infectious disease as demonstrated by the success of vaccines against polio, smallpox, measles, diphtheria, and tetanus among others
Quantitative Tetanus toxoid (TT) response differs depending on the manufacturer To further understand the precision of the assay, we studied the anti-TT IgG responses to bulk antigen from different manufacturers
Use of ELISpot is well established for detection of antigen-specific memory B cells and determining the frequency of pathogen and vaccine-specific B cells[25,27,28,32,33,34,35]
Summary
Vaccines confer safety and are the most effective means of protection against infectious disease as demonstrated by the success of vaccines against polio, smallpox, measles, diphtheria, and tetanus among others. Introduction of infant immunization against these pathogens or their disease-provoking toxins has led to an impressive reduction of global disease burden and improved survival of neonates and children worldwide. The bacterial toxins affect the nervous system and infection leads to painful muscle spasms and to death. Booster vaccinations are required to acquire long-term protection against tetanus and to maintain immunity over a lifetime. Having established immunity in childhood, adolescents, and adults are recommended to receive booster vaccinations every 10 years at the latest[5,6]. In Germany, >75% of adults have been vaccinated within last 10 years[3,7]
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