Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b.

A competitive ELISA method is described for the measurement of total antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HibCPS) in human sera. The competitive method showed an excellent correlation to the radioantigen binding assay (RABA, or Farr assay) and improved correlation of sera with low titers with respect to the more conventional noncompetitive method. Overestimation of samples in the low concentration range was no longer observed with the competitive ELISA method. The free HibCPS competition allowed us to eliminate the day-to-day background variation typical of some sera; thus, only values representing the true anti-HibCPS response were determined. The use of precoated microplates, which could be stored up to 8 months, greatly improved the speed of the procedure. An overall correlation coefficient of 0. 9660 was found when 407 serum samples with a wide variety of anti-HibCPS antibody levels were tested with the competitive ELISA and RABA. The regression line was very close to the ideal line, with a slope of 1.0045 and an intercept of -0.1996. A subset of 96 serum samples representative of all pre- and postimmunization samples was used to compare the competitive ELISA with a previously described ELISA method. The competitive method performed in two laboratories in different countries showed a better correlation with the RABA. The correlation factors were 0.9770 and 0.9816, respectively, while a factor of 0.9547 was found with the previously described noncompetitive procedure, which was better for this method than previously reported (r = 0.917). Therefore, the competitive ELISA is proposed for the assay of anti-HibCPS titers in sera from vaccinated subjects.