Catpagavalli Asokanathan

An in vitro assay system as a potential replacement for the histamine sensitisation test for acellular pertussis based combination vaccines

The histamine sensitisation test (HIST) for pertussis toxin is currently an official batch release test for acellular pertussis containing combination vaccines in Europe and North America. However, HIST, being a lethal endpoint assay, often leads to repeated tests due to large variations in test performance. Although a more precise HIST test based on measurement of temperature reduction after the histamine challenge is used in Asian countries, this test still uses animals. An in vitro test system based on a combination of enzyme coupled-HPLC and carbohydrate-binding assays with results analysed by a mathematical formula showed a good agreement with the in vivo HIST results based on measurement of temperature reduction after histamine challenge. The new in vitro test system was shown to be a potential alternative to the current in vivo HIST.

Evaluation of an in vitro assay system as a potential alternative to current histamine sensitization test for acellular pertussis vaccines

The histamine sensitization test (HIST) is a lethal test for batch release of acellular pertussis or its combination vaccines (ACV). Large numbers of animals have been used and it is difficult to standardize. Therefore there is an urgent need to develop an in vitro alternative to HIST. An in vitro test system has been developed as a potential alternative to HIST, to examine both the functional domains of PT based on a combination of enzyme coupled-HPLC (E-HPLC) and carbohydrate binding assays. We describe here an international collaborative study, which involved sixteen laboratories from 9 countries to assess the methodology transferability of the in vitro test system and its suitability for the testing of three different types of ACV products that are currently used worldwide. This study also evaluated further the relationship between the in vivo activity by HIST and the in vitro assay system. The results showed that the methodology of the E-HPLC and carbohydrate binding assays are transferable between laboratories worldwide and is suitable for the three types of ACV products included in the study. Although direct correlation between the in vitro assay system and the in vivo HIST (temperature reduction assay) for each individual vaccine lot cannot be established due to the large variation in the HIST results, the observation that the mean estimates of the in vitro and in vivo activities gave the same rank order of the three vaccine types included in the study is encouraging. The in vitro systems provide reproducible product specific profiles which supports their use as a potential alternative to the HIST.

Investigation in a murine model of possible mechanisms of enhanced local reactions to post-primary diphtheria-tetanus toxoid boosters in recipients of acellular pertussis-diphtheria-tetanus vaccine

In recipients primed with acellular pertussis diphtheria-tetanus combined vaccine (DTaP) an increased incidence of severe local reactions with extensive redness/swelling has been reported for each subsequent dose of diphtheria-tetanus based combination vaccine given as a booster. This has been attributed to residual active pertussis toxin (PT) in the primary vaccine. In this study, we investigated the possible contribution of the A-subunit enzymatic activity and the B-oligomer carbohydrate binding activity of residual PT in DTaP to local reactions in a murine model using Japanese DTaP batches produced before and after the introduction of a test for reversion of pertussis toxoid to toxin. Residual PT activity was correlated with the B-oligomer carbohydrate binding activity. The in vivo mouse footpad swelling model assay indicated that the B-oligomer carbohydrate binding activity and possibly other factors were associated with intensified sensitization to local reaction following diphtheria toxoid booster.

Confocal microscopy study of pertussis toxin and toxoids on CHO-cells

Pertussis toxin in its detoxified form is a major component of all current acellular pertussis vaccines. Here we report the membrane translocation and internalization activities of pertussis toxin and various pertussis toxoids using Chinese hamster ovary cells and confocal microscopy based on indirect immunofluorescence labeling. Chemically detoxified pertussis toxoids were able to translocate/internalize into cells at the concentration about 1,000 times higher than the native toxin. Pertussis toxoids detoxified with different procedures (glutaraldehyde, glutaraldehyde plus formaldehyde, hydrogen peroxide or genetic mutation) showed differences in fluorescence intensity under the same condition, indicating toxoids from different detoxification methods may have different translocation/internalization activities on cells.

Relationship of immunogenicity to protective potency in acellular pertussis vaccines

Comparison of the immunogenicity response and resistance to challenge in the modified intracerebral challenge assay induced by various acellular pertussis vaccines showed that these were not closely linked. The immunogenicity assay was effective for confirming the presence of specific antigenic components and was invaluable for detecting minor components present in co-purified vaccines. However, the magnitude of antibody responses was not consistently related to antigen concentration nor did it correlate with protection in the modified intracerebral challenge assay. The immunogenicity assay detected degradation of pertussis toxin and pertactin components but not of filamentous haemagglutinin or fimbriae 2 and 3 in denatured acellular pertussis vaccines. The modified intracerebral challenge assay was effective in detecting antigen degradation in all types of acellular pertussis vaccines including those of European/North American origin but was dominated by the response to pertussis toxin. Aerosol challenge was more sensitive in detecting denaturation of filamentous haemagglutinin or fimbriae. The modified intracerebral challenge assay was the only assay that provided a quantitative indication of protective activity. Both immunogenicity and challenge assays provided useful data on acellular pertussis vaccine properties but were complementary and not alternatives.

PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model

Poly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response.

Characterization of co-purified acellular pertussis vaccines

Whole-cell pertussis vaccines (WPVs) have been completely replaced by the co-purified acellular vaccines (APVs) in China. To date few laboratory studies were reported for co-purified APVs in terms of their antigenic composition and protective immune responses. To further understand the antigenic composition in co-purified APVs, in the present study 2-dimensional gel electrophoresis-based proteomic technology was used to analyze the composition of co-purified APVs. The results showed that besides the main antigens pertussis toxin (PT) and filamentous hemagglutinin (FHA), co-purified APVs also contained pertactin (PRN), fimbriae (FIM) 2and3 and other minor protein antigens. Of the 9 proteins identified, 3 were differentially presented in products from manufacturer 1 and manufacturer 2. Compared with WPVs and purified APVs, co-purified APVs induced a mixed Th1/Th2 immune response with more toward to a Th1 response than the purified APVs in this study. These results hint that different immune mechanisms might be involved in protection induced by co-purified and purified APVs.

Effect of different detoxification procedures on the residual pertussis toxin activities in vaccines

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis and its detoxified form is one of the major protective antigens in vaccines against whooping cough. Ideally, PTx in the vaccine should be completely detoxified while still preserving immunogenicity. However, this may not always be the case. Due to multilevel reaction mechanisms of chemical detoxification that act on different molecular sites and with different production processes, it is difficult to define a molecular characteristic of a pertussis toxoid. PTx has two functional distinctive domains: the ADP-ribosyltransferase enzymatic subunit S1 (A-protomer) and the host cell binding carbohydrate-binding subunits S2-5 (B-oligomer); and in this study, we investigated the effect of different detoxification processes on these two functional activities of the residual PTx in toxoids and vaccines currently marketed worldwide using a recently developed in vitro biochemical assay system. The patho-physiological activities in these samples were also estimated using the in vivo official histamine sensitisation tests. Different types of vaccines, detoxified by formaldehyde, glutaraldehyde or by both, have different residual functional and individual baseline activities. Of the vaccines tested, PT toxoid detoxified by formaldehyde had the lowest residual PTx ADP-ribosyltransferase activity. The carbohydrate binding results detected by anti-PTx polyclonal (pAb) and anti-PTx subunits monoclonal antibodies (mAb) showed specific binding profiles for toxoids and vaccines produced from different detoxification methods. In addition, we also demonstrated that using pAb or mAb S2/3 as detection antibodies would give a better differential difference between these vaccine lots than using mAbs S1 or S4. In summary, we showed for the first time that by measuring the activities of the two functional domains of PTx, we could characterise pertussis toxoids prepared from different chemical detoxification methods and this study also highlights the potential use of this in vitro biochemical assay system for in-process control.