Combination of DTP and Haemophilus influenzae type b conjugate vaccines can affect laboratory evaluation of potency and immunogenicity.

A commonly used Diphtheria-Tetanus-Pertussis (whole-cell) vaccine was combined with each of three different Haemophilus influenzae type b (Hib) capsular polysaccharide vaccines. Each Hib vaccine incorporated one of three different protein conjugates: tetanus toxoid, diphtheria CRM197 toxoid or group B Neisseria meningitidis outer membrane vesicles. The effects of these combinations on the subsequent laboratory control testing were examined. The addition of the Hib vaccines had no significant 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 had dramatic effects on tetanus potency and immunogenicity when assayed in mice. This combination resulted in a five-fold potentiation of the tetanus potency and a similarly large increase in the antibody responses to tetanus toxin and toxoid. The level of the antibody response to the Hib polysaccharide in this vaccine was also elevated, more than 20-fold, as a result of the combination. Such phenomena were not evident with combinations involving the other two Hib vaccines. These results have implications for the control testing of combined vaccines containing a whole-cell pertussis component and Hib polysaccharide-tetanus protein conjugate vaccine.

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.

In vitro assays for detection of diphtheria toxin.

Subunit-specific antipeptide antibodies have been used to develop a dot blot immunoassay for detection of diphtheria toxin (DT) produced by toxigenic strains of Corynebacterium diphtheriae. A detection limit of 0.1-1.0 ng DT/ml was obtained. DT is cytotoxic to cultured Vero cells and cell viability if conveniently assessed using a tetrazolium dye, MTT. This has been used as the basis of a cytotoxicity assay for DT, which has a detection limit of 20 pg/ml of biologically active toxin. Sixty isolates of C. diphtheriae were assayed for toxigenicity. There was complete concordance between the Vero cell cytotoxicity assay and guinea pig intradermal assay in vivo, demonstrating the utility of the cytotoxicity assay.

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses.

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.