ANTIGENIC SIMILARITIES BETWEEN BRAIN COMPONENTS AND BACTERIA CAUSING MENINGITIS: Implications for Vaccine Development and Pathogenesis

Considerations for formulating the second-generation pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups.

A system for the production of monoclonal antibodies, particularly of the IgG type, against weakly immunogenic bacterial polysaccharide antigens is described. This system, which is based on the autoimmune NZB mouse strain, has been used to produce a monoclonal IgG2a antibody against the meningococcus group B and Escherichia coli K1 polysaccharides, identical homopolymers of alpha (2----8)-linked units of N-acetylneuraminic acid that are extremely poor immunogens. Comparison of the humoral immune responses of normal BALB/c mice and autoimmune NZB mice to hyperimmunization with group A, B, and C meningococci showed that, although both strains mounted a weak meningococcal B polysaccharide-specific IgM response, only the NZB strain mounted an IgG response. Similarly, NZB mice mounted a stronger IgG response to the more immunogenic group C meningococcal polysaccharide than did BALB/c mice, although this difference was less pronounced than that observed with meningococcal B polysaccharide. No difference between the two strains of mice was demonstrable with the strongly antigenic group A meningococcal polysaccharide. These results indicate that the NZB system may be generally useful for the production of monoclonal antibodies against weakly antigenic bacterial determinants.

https://www.pnas.org/content/pnas/82/4/1194.full.pdf

NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci.

https://www.ccrc.uga.edu/~rcarlson/Wein.pdf

Immunology of bacterial polysaccharide antigens.

Conjugates were prepared by carbodiimide-mediated coupling of adipic acid hydrazide derivatives of Haemophilus 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, and their serum antibody responses to the polysaccharides and proteins were characterized. As originally reported, 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 one injection and in all after the third injection (Schneerson et al., J. Exp. Med. 152:361-376, 1980). 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 third 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 first injection and with higher levels of anti-Hib than observed with the Hib conjugates alone (P 100 ng of antibody nitrogen per ml) in 6 of 10 mice after the third 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, and 9 of 10 mice responded after the third injection. A preparation of diphtheria toxoid, TT, and pertussis vaccine increased the anti-Hib antibody levels after the first 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 U/ml) in most mice after the first injection and in all mice after the second and third 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.

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

The successful coupling of the meningococcal groups A, B, and C polysaccharides to tetanus toxoid to yield water soluble conjugates is described. Reactive aldehyde groups were strategically introduced into the terminal residues of the polysaccharides by the controlled periodate oxidation of the native groups B and C polysaccharides and of the group A polysaccharide previously modified by the reduction of its terminal reducing N-acetyl-mannosamine residue. This produced essentially monovalent polysaccharide molecules, which were subsequently covalently linked to tetanus toxoid by means of reductive amination. Although the groups A and C polysaccharides proved to be poor immunogens in rabbits and mice, their tetanus toxoid conjugates produced high levels of polysaccharide-specific antibodies in both animals. By contrast, even in the form of its tetanus toxoid conjugate, the group B polysaccharide failed to elicit homologous polysaccharide-specific antibodies in either animal; a major proportion of the antibodies actually produced had a specificity for the linkage area of the conjugate. This evidence is compatible with the hypothesis of the poor immunogenicity of the group B polysaccharide being structure related. Hyperimmunization of mice with the groups A and C polysaccharide-tetanus toxoid conjugates produced antisera with good bactericidal activity against their respective homologous organisms, and indicated the potential of these conjugates as potential human vaccines.

C Lugowski

Papers

Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates.