An immunodominant neutralization epitope on the ‘thumb’ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies

Abstract

An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the ‘helix clump’) of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.