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All rabbits immunized with type A influenza virions have a serum haemagglutination-inhibition antibody response biased to a single epitope in antigenic site B

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

Nine rabbits were immunized with type A influenza virions and the epitope specificities of the secondary serum haemagglutination-inhibition (HI) antibody response were analysed with a panel of neutralizing monoclonal (MAb) antibody double escape mutants. Each of the latter was made by sequential selection using a MAb directed to an epitope of a discrete antigenic site, site A, site B or site D, of the haemagglutinin (HA). Thus the epitope reactivity of the escape mutants was represented as A⁺B^-D^-, A^-B⁺D^- and A^-B^-D⁺. The HI antibody response of all antisera was biased to the site B epitope. In 9/12 antisera, obtained from seven rabbits immunized with whole virions, the site B epitope was predominant, representing 65–82% of the total HI antibody. The restriction of HI antibody was unaffected by strain of rabbit, route of inoculation (intravenous or subcutaneous), use of Freund's adjuvant, 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 site B epitope. The HI antibody response in one of the rabbits (#40) became more biased to the site B epitope between the third and fourth immunizing doses. Two further rabbits were immunized with virions which had been partially digested with bromelain and then purified from free HA. Both of these made equal HI antibody responses to the site B epitope and the site D epitope, possibly because their remaining HA spikes were better exposed. Overall, these data demonstrate an unexpected degree of restriction in the production of biologically relevant antibody, such that some rabbits (e.g. #45) mount an HI antibody response which is essentially epitope-specific. Implications for epitope specificity of HI antibody stimulated by human influenza vaccines, and also for the generation of antigenic drift variants are discussed. The reason for the non-responsiveness of the immune system to the many other HI epitopes of the HA is not known.

^* Author for correspondence. Fax +44 203 523568. e-mail nd@dna.bio.warwick.ac.uk

Received 7 October 1994; accepted 30 November 1994.

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