Structural analysis of the neutralising antibody repertoire to influenza virus Haemagglutinin.

Laeeq, Sabahat (1997). Structural analysis of the neutralising antibody repertoire to influenza virus Haemagglutinin. PhD thesis The Open University.

Abstract

Protective immunity to influenza virus correlates with levels of neutralising antibodies directed against haemagglutinin (HA), the major membrane glycoprotein of the virus. The antibodies exert selective pressure resulting in the recurrent and annual emergence of new variant viruses.

Although all five antigenic sites are implicated in antibody recognition, immunodominance is evident in the secondary antibody response, following natural infection of inbred mice as has been shown in this laboratory; BALB/c (H-2d) mAbs predominantly recognise HA1 198, and CBA/Ca (H-2k) mAbs, HA1 158, as deduced by sequencing the HA genes of X31 laboratory variants. Such restriction in the antibody response raises several questions concerning the mechanisms involved in repertoire selection and the structural basis of the selection process, which this investigation attempted to address using transgenic mice expressing human Ig μ chains.

The purpose of examining the memory repertoire for influenza HA in transgenic mice was to determine whether restricted VH region gene usage and/or inability to class switch (lgM->lgG) with a resultant lack of affinity maturation would restrict the potential antibody repertoire. I have demonstrated, by structural analysis of variant virus HA genes, selected with mAbs expressing human μ chains, that there is a preferential selection for mutations within conserved residues that constitute part of the receptor binding site (HA1 225, HA1 226) that do not occur in previously documented laboratory variants. Also the majority of these variant viruses have amino acid substitutions at twp different positions: (HA1 145, 226) or (HA1 135, 225) or (HA1 135, 158) or (HA1 145, 158).

In the second part of this study, I have examined the influence of concommitant immunity to X31 on re-challenge with an X31 variant virus in an attempt to mimic the human situation of recurrent infection: CBA/Ca mice were infected intranasally with X31 and their neutralising antibody repertoire investigated by hemisplenectomy, mAb production, X31 variant selection and sequencing of their HA genes. Following a rest period, mice were re-challenged with a laboratory variant, A43, differing from X31 at known antigenic sites (HA1 145, 158, del 224-230) and which was not recognised by the majority of mAbs from the primary challenge. The mAbs, established after re-infection with A43, showed heteroclitic reactivity forX31 and selected laboratory variants thereof, with substitutions at both conserved residues within the receptor binding site and at known antigenic sites (HA1 193,198, 226) or HA1 (198, 223).

I have demonstrated in two different systems (a) human IgH transgenic mice, or (b) recurrent infection with a variant virus, that there is preferential selection of laboratory mutants containing multiple substitutions, including changes in conserved residues that constitute part of the receptor-binding pocket (HA1 225 or 226). This has led me to conclude that antibody affinity might play a determinant role in the selection of mutations that affect receptor binding function. The majority of X31 variants that I have cloned, and characterised, do indeed have altered receptor-binding specificity as shown by their resistance to horse serum inhibition of haemagglutination and/or altered binding to neoglycpproteins containing terminal a 2,3 or a 2,6 sialyl residues.

Reduced affinity of the neutralising Ab response during infection - such as in the immunocompromised, or the very young or very old, or due to previous exposure to a related virus might skew the antibody repertoire to selection of receptor-binding variants and therefore be a further determinant of antigenic drift.

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