A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen

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.

Determination of DTaP vaccine potency by multiplex immunogenicity testing using electrochemiluminescence

“…Nevertheless, this does not exclude that continued efforts might abolish in vivo testing in accordance with the consistency approach that indirectly proves potency and safety through demonstration of preserved critical attributes of the vaccine formulation (Dierick et al 2022). This will foster development and acceptance of cell-based assays for testing of functional integrity of vaccine antigens via induction of vaccine antigen-specific immune response (Ticha et al 2021;Vandebriel et al 2022), ELISA-based methods detecting and quantifying immunogenic epitopes of antigens in vaccine formulations with well-characterized specific monoclonal antibodies (Szeto et al 2023;Winsnes et al 2004;Riches-Duit et al 2021a;Riches-Duit et al 2021b;Vermeulen et al 2023;Agnolon et al 2016;Westdijk et al 2017), or even non-invasive physical technologies such as Raman microspectroscopy (Silge et al 2018).…”

Development of an In Vitro Test Method to Replace an Animal-Based Potency Test for Pertactin Antigen in Multivalent Vaccines

“…Efforts to better characterize candidate mAbs and/or to assess in vitro assays for vaccine testing (including DTaP vaccines) have been undertaken by different groups [ 18 , 19 , 20 ], including the Vaccine to Vaccine (VAC2VAC) project managed by the Innovative Medicines Initiative 2 (IMI2) [ 21 ]. The key objective of the VAC2VAC was to provide proof of concept of the consistency approach for batch release testing of established vaccines through the development of in vitro analytical methods [ 21 ].…”