No abstract

The recurrence of influenza virus infection in man is attributed primarily to changes occurring in the antigenic structure of the viral surface glycoproteins, especially of the haemagglutinin (HA) molecule. Comparative antigenic analysis of epidemic influenza virus strains has allowed the description of 'strain-specific' and 'cross-reactive' antigenic determinants. However, the interpretation of these findings remained ambiguous, because the specificity of the applied antisera was insufficiently defined and because the antigenic differences among the HA molecules of various epidemic virus strains resulted presumably from a large number of amino acid substitutions. Thus, in characterizing the antigenic structure of the HA molecule, our approach has been (1) to generate a panel of monoclonal anti-HA hybridoma antibodies, (2) to use some of these antibodies to select mutants of the influenza A/PR/8/34 (PR8) virus expressing antigenically altered HA molecules, and (3) to construct an operational antigenic map of the HA molecule by comparative antigenic analysis of the mutant viruses with the monoclonal antibodies. As we report here, analysis of the 34 mutant viruses selected has enabled us to define four antigenic sites on the HA molecule. Our observation that these sites have undergone antigenic drift to a different extent in nature implies that the mechanisms responsible for antigenic drift act selectively on distinct structures of the HA molecule.

We have examined the amino-terminal sequence of the Kc light chains of a set of monoclonal antibodies specific for one of the major antigenic determinants (Sb) on the influenza virus PR8[A/PR/8/34(HlNl)J hemagglutinin molecule. This set was believed to be structurally related from earlier serological analysis that typed these K chains as members of the variable (V) region VK21 group [Staudt, L. M. & Gerhard, W. (1983) J. Exp. Med. 157,. Our sequence analysis confirms and extends this conclusion; all examples of this set belong to a subgroup of the VK21 group, V,,21C. A special feature of this set of K light chains is that all examples were derived from the same mouse (designated H36). This sequence analysis along with the characterization of gene rearrangements at the K light chain loci of these hybridomas is consistent with the idea that certain members of this set are the progeny of one or two lymphocytes. Because of this potential clonal relationship, we can reach several conclusions about the diversity observed among these K light chains: (') the diversity is due to somatic mutation, (it) somatic mutations occur sequentially and accumulate in the first complementarity-determining region, and (iii) the extent of somatic variation in this sample is high, suggesting a somatic mutation rate of about 10-3 per base pair per generation.Antibody diversity arises from several sources. Individuals inherit multiple variable (V) region gene segments for both heavy (VH) and light (VK, VA) chains, joining (J) gene segments (JH, J,, Jh), and diversity (D) gene segments (DH).The initial antibody repertoire of an individual is a product of the combinatorial joining of these gene segments, i.e., V4s with JKS or different VH, DH, and JH combinations, that form complete V1 or VH genes. Errors committed during the process of joining contribute additional diversity to this repertoire (reviewed in ref. 1). Finally, that somatic mutation further amplifies this germ-line repertoire seems to be established (2). The original evidence for somatic mutation favored a model by which point mutations accumulate sequentially during cell division (3). Other models link somatic mutation with specific events during lymphocyte differentiation (4, 5) and propose cataclysmic mechanisms of mutagenesis that introduce multiple amino acid substitutions in one step (6, 7). These models are based on the comparison of V region sequences of independently induced plasmacytoma and hybridoma antibodies to their putative germ-line counterparts. Hence, little can be concluded about the time course of somatic mutation.A better understanding of the nature of somatic mutation can be reached by comparisons of the V genes of a cell lineage. Scharff and colleague have analyzed certain mutants and revertants of the cell line S107 and conclude that the in vitro rate of mutation at the VH gene expressed in this plasmacytoma is significantly higher than that of nonimmunoglobulin genes (8). A possible in vivo analogy is described here: we have initiated a structu...

Recovery from influenza virus infection has long been known to require an intact T-cell compartment. More recent studies revealed that CD8 and CD4 T cells can promote recovery through independent mechanisms. The CD4 T-cell-dependent recovery process appears to operate primarily through promotion of the T-dependent antibody response as B-cell-deficient microMT mice cannot recover from infection if they have been depleted of CD8 T cells. The potential therapeutic activity of the B-cell response was further studied by transfer of antibodies into infected SCID mice. At the dose of 200 micrograms/mouse, most antibodies (of IgG2a isotype) to the viral transmembrane protein HA cured the infection, while those to the transmembrane proteins NA and M2 suppressed virus titers in the lung but failed to clear the infection. The ability of passive antibody to resolve the infection was closely related to its prophylactic activity, suggesting that neutralization of progeny virus (VN) played an important role in the process of virus clearance in vivo, while reaction of antibodies with infected host cells contributed to but was insufficient, on its own, for cure. HA-specific antibodies of IgM and IgA isotypes were therapeutically ineffective against pulmonary infection, presumably because of a preferential delivery into the upper respiratory tract, while IgG exhibited highest activity against pulmonary and minimal activity against nasal infection. B cells appear to be of similar importance for recovery from primary infection as CD8 T cells.

Specificity of cytotoxic T-cell function was investigated for a range of different influenza viruses. T cells from mice immunized with A or B strain influenza viruses, or with vaccinia virus, showed reciprocal exclusion of cytotoxicity. Extensive cross-reactivity was, however, found for lymphocyte populations from mice infected with a variety of serologically distinct influenza A viruses, though serum antibodies did not cross-react when tested in a radioimmunoassay using comparable target cells as immunoadsorbents. This apparent lack of T-cell specificity was recognized for immune spleen cells generated after intraperitoneal inoculation of high titers of virus, and for mediastinal lymph node populations from mice with pneumonia due to infection with much less virus. The phenomenon could not be explained on the basis of exposure to the chicken host component, which is common to A and B strain viruses. However, not all of the virus-immune T-cell clones are cross-reactive. Competitive-inhibition experiments indicate that a considerable proportion of the lymphocyte response is restricted to the immunizing virus. Even so, the less specific component is significant. Also, exposure to one type A virus was found to prime for an enhanced cell-mediated immunity response after challenge with a second, serologically different A strain virus.