Expression and characterization of HIV-1 envelope protein

We postulated that gp120 and CD4 interaction might expose cryptic epitopes on gp120 that could be immunogenic and so widen the immunogenic response. A fusion protein of a C clade strain of HIV-1 (HIVCN54) gp120 and full-length human CD4 was constructed and expressed using a recombinant baculovirus system. The protein was purified, characterized and used to immunize rabbits. The antiserum generated had an expected anti-gp120 activity and demonstrated a higher capacity than a control serum raised to gp120 alone to block b12 binding, a marker of neutralization. A formal neutralization assay however did not detect neutralizing activity in the CD4-gp120CN54 antiserum. To enhance overall immunogenicity, a gp120CN54-FPV168 fusion protein was also expressed. FPV168 is a fowlpox virus protein. Although designed to elicit a stronger host immune response, the antiserum generated to gp120CN54-FPV168 had a weaker binding activity to gp120 than the antiserum generated to non-fused gp120CN54. A possible reason was protein instability associated with fusion to the N-terminus of FPV168. To ameliorate this problem, three gp120 fusion proteins with N-terminal truncated FPV168 were also constructed. The stability of these proteins was vastly improved but their performance as immunogens continued to be poorer than immunization with gp120 alone. Lastly, experiments describing an alternative strategy of immune enhancement based on targeting of gp120 or fragments of gp120 to antigen presenting cells via use of the human immunoglobulin Fc domain are presented. These were more successful and indicate a future direction that could yet produce an HIV-1 gp120 based immunogen capable of raising the antibody responses required as part of a successful vaccine.EThOS - Electronic Theses Online ServiceGBUnited Kingdo