Further Studies on the Immunogenicity of Haemophilus influenzae Type b and Pneumococcal Type 6A Polysaccharide-Protein Conjugates

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Abstract
Conjugates were prepared by carbodiimide-mediated coupling of adipic acid hydrazide derivatives of H. influenzae type b (Hib), Escherichia coli K100 and pneumococcal 6A (Pn6A) polysaccharides with tetanus toxoid (TT), as an example of a useful carrier, and horseshoe crab hemocyanin (HCH), as an example of a nonsense carrier. These conjugates were injected into NIH mice; their serum antibody responses to the polysaccharides and proteins were characterized. Hib conjugates increased the immunogenicity of the capsular polysaccharide and elicited greater than the estimated protective levels of anti-Hib antibodies in most recipients after 1 injection and in all after the 3rd injection. Both Hib conjugates induced similar anti-Hib responses. The K100-HCH conjugate was more immunogenic than the K100-TT conjugate and elicited anti-Hib responses similar to the Hib conjugates after the 3rd injection. Simultaneous injection of the K100 and the Hib conjugates did not enhance the anti-Hib response. The Pn6A-TT conjugate induced low levels of anti-Hib antibodies; when injected simultaneously with the Hib conjugates, the anti-Hib response was enhanced, as all mice responded after the 1st injection and with higher levels of anti-Hib than observed with the Hib conjugates alone (P < 0.05). The Pn6A conjugates were not as immunogenic as the Hib conjugates. Pn6A-TT was more effective than was Pn6A-HCH; it elicited anti-Pn6A (> 100 ng of antibody N per ml) in 6 of 10 mice after the 3rd injection. The addition of the Hib-HCH conjugate to the Pn6A-TT conjugate increased the anti-Pn6A response with a higher geometric mean antibody titer; 9 of 10 mice responded after the 3rd injection. A preparation of diptheria toxoid, TT and pertussis vaccine increased the anti-Hib antibody levels after the 1st injection only in mice receiving Hib-TT, but not in mice receiving Hib-HCH, suggesting that additional carrier protein (TT) enhanced the anti-polysaccharide response. Simultaneous injection of Hib and Pn6A conjugates with the same or different carriers resulted in an enhanced serum antibody response to each polysaccharide. The anti-tetanus toxin response reached protective levels (> 0.01 .+-./ml) in most mice after the 1st injection and in all mice after the 2nd and 3rd injections of TT conjugates. A progressive increase in the anti-HCH response with each additional injection was noted in animals receiving HCH conjugates. Animals receiving the diphtheria toxoid-TT-pertussis vaccine preparation responded with a greater increase in anti-carrier antibody than those receiving the conjugates alone. This method of synthesis provided conjugates capable of inducing protective levels of antibodies to both the polysaccharides and carrier proteins.