ALL RABBITS IMMUNIZED WITH TYPE-A INFLUENZA VIRIONS HAVE A SERUM HEMAGGLUTINATION-INHIBITION ANTIBODY-RESPONSE BIASED TO A SINGLE EPITOPE IN ANTIGENIC SITE-B

Nine rabbits were immunized with type A influenza virions and the epit ope specificities of the secondary serum haemagglutination-inhibition (HI) antibody response were analysed with a panel of neutralizing mono clonal (MAb) antibody double escape mutants. Each of the latter was ma de by sequential selection using a MAb directed to an epitope of a dis crete antigenic site, site A, site B or site D, of the haemagglutinin (HA). Thus the epitope reactivity of the escape mutants was represente d as A(+)B(-)D(-), A(-)B(+)D(-) and A(-)B(-)D(+). The HI antibody resp onse of all antisera was biased to the site B epitope. In 9/12 antiser a, obtained from seven rabbits immunized with whole virions, the site B epitope was predominant, representing 65-82% of the total HI antibod y. The restriction of HI antibody was unaffected by strain of rabbit, route of inoculation (intravenous or subcutaneous), use of Freund's ad juvant, and up to four immunizing injections. In 3/7 rabbits immunized with whole virus, there was a HI antibody response to the HC2 (site A ) or HC10 (site D) epitope, but not both, of equal magnitude to the si te B epitope. The HI antibody response in one of the rabbits (#40) bec ame more biased to the site B epitope between the third and fourth imm unizing doses. Two further rabbits were immunized with virions which h ad been partially digested with bromelain and then purified from free HA. Both of these made equal HI antibody responses to the site B epito pe and the site D epitope, possibly because their remaining HA spikes were better exposed. Overall, these data demonstrate an unexpected deg ree of restriction in the production of biologically relevant antibody , such that some rabbits (e.g. #45) mount an HI antibody response whic h is essentially epitope-specific. Implications for epitope specificit y of HI antibody stimulated by human influenza vaccines, and also for the generation of antigenic drift variants are discussed. The reason f or the non-responsiveness of the immune system to the many other HI ep itopes of the HA is not known.