Johanna Watkins

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.

Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T-cell subsets as Th1, Th2 or Th0

In studies of the mechanism of immunity to Bordetella pertussis in a murine respiratory infection model, we have previously demonstrated that natural infection of immunization with a whole cell vaccine induces a potent protective immune response, which is mediated by T‐helper type‐1 (Th1) cells. In contrast an acellular vaccine generates Th2 cells and is associated with delayed bacterial clearance following respiratory challenge. In the present study we have investigated the apparent Th1/Th2 cell dichotomy in acquired immunity and have examined the factors that affect their induction or detection. The cytokine profiles of B. pertussis‐specific T cells in immune animals were determined using antigen‐stimulated ex vivo spleen cells or CD4+ T‐cell lines and clones established in the presence of interleukin‐2 (IL‐2) or IL‐4. Antigen‐specific T cells derived from mice immunized with the acellular vaccine were almost exclusively of the Th2 cell type. In contrast, T‐cell lines and clones established following respiratory infection or immunization with the whole cell vaccine were predominantly of the Th1 type. However, a proportion of T cells from convalescent mice, especially when cultured in the presence of IL‐4, secreted IL‐4 and IL‐5 with or without detectable IL‐2 and interferon‐γ (IFN‐γ), suggesting that Th0 or Th2 cells were also primed during natural infection in vivo. Furthermore, when mice were assessed 6 months after infection, spleen cells produced significant levels of IL‐4 and IL‐5, which were not evident at 6 weeks. The route of immunization and the genetic background of the mice were also found to influence the preferential priming of Th1 cells, and this was directly related to the level of protection against respiratory or intracerebral (i.c.) challenge. Our findings underline the critical role of CD4+ Th1 cells in immunity to B. pertussis, but also demonstrate that a number of factors in the in vivo priming and in vitro restimulation can skew the apparent dominance of one Th cell type over another.

Interaction of Haemophilus influenzae type b conjugate vaccines with diphtheria-tetanus-pertussis vaccine in control tests

The effects of combining three Haemophilus influenzae type b (Hib) capsular polysaccharide vaccines, conjugated to different proteins, with DTP vaccine on the subsequent control testing were examined. The addition of the Hib vaccines had little effect on the reactogenicity or the potency of the whole-cell pertussis component. The potency of, and antibody responses to, the diphtheria component were also unaffected in all three combinations. However, combination with the Hib vaccine comprising polysaccharide conjugated to tetanus toxoid resulted in a fivefold potentiation of the tetanus potency and large increases in the antibody responses to tetanus toxin and toxoid and Hib polysaccharide. These results have implications for the control testing of combined vaccines containing a whole-cell pertussis component and Hib polysaccharide-tetanus protein conjugate vaccine.