Peet, Nicola Mary; (1996) Towards an epitope specific vaccine: Murine immune responses to wildtype and mutant HIV-1 gp120. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The envelope glycoproteins, gp120 and gp41 are the target of the main neutralising antibody response to HIV-1, however this tends to be type-specific and a high level of antigenic variation enables viral escape. The third variable loop (V3) of the external envelope glycoprotein (gp120) contains a dominant B cell epitope which may powerfully compete with other epitopes, in particular, with conserved epitopes, to enhance viral escape. The experiments described in this thesis are concerned with reducing the immunogenicity of V3 with the aim of testing this hypothesis of epitope competition and possibly to define a mutant gp120 construct which may have application as part of a subunit vaccine for HIV-1. gp120 mutants were made with blocks of serine substitutions in one or both halves of V3. These constructs were used to establish stable protein expression in Chinese hamster ovary cell lines using glutamine synthetase as a selectable and amplifiable marker. Analysis of the secreted mutant proteins, using monoclonal antibodies, revealed that mutation of the C-terminal half of V3 produced detectable conformation changes. Mice were immunised with the wildtype (unmutated) gp120 by conventional protein-plus-adjuvant immunisation and by nucleic acid immunisation (NAI). Protein immunisation generated ten-fold higher titres of gp120-specific IgGl but NAI resulted in significantly higher levels of peptide reactivity in the immune sera. Following NAI of mice with the mutant gp120 genes it was found that certain mutations of V3 did result in reduction of V3 immunogenicity and that V3 is probably immunodominant in these mice. Wildtype DNA sequences were required on the N-terminal side of V3 if the resulting antisera were to recognise wildtype V3 peptides. No concomitant increase in immunogenicity of constant regions of the mutated gp120 was recorded in the experiments presented here but this issue is still open.

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