Induction of antibody-mediated neutralization in SIVmac239 by a naturally acquired V3 mutation

Abstract

Achieving humoral immunity against human immunodeficiency virus (HIV) is a major obstacle in AIDS vaccine development. Despite eliciting robust humoral responses to HIV, exposed hosts rarely produce broadly neutralizing antibodies. The present study utilizes simian immunodeficiency virus (SIV) to identify viral epitopes that conferred antibody neutralization to clone SIV/17E-CL, an in vivo variant derived from neutralization resistant SIVmac239. Neutralization assays using rhesus macaque monoclonal antibodies were performed on viruses engineered to express single or multiple amino acid mutations. Results identified a single amino acid mutation, P334R, in the carboxy-terminal half of the V3 loop as a critical residue that induced neutralization while retaining normal glycoprotein expression on the surface of the virus. Furthermore, the R334 residue yielded neutralization sensitivity by antibodies recognizing diverse conformational and linear epitopes of gp120, suggesting that neutralization phenotype was a consequence of global structural changes of the envelope protein rather than a specific site epitope.

Figure 1

Design and characterization of SIV mutant viruses. Mutations in SIV/17E-CL are distributed throughout the gp120 env gene and cluster around the V1/V2 and V3 regions. The cloning strategy utilizes the Bpu10I restriction site, 5’ to the start codon of gp120, and the Nhe1 restriction site on the 3’ end of the ectodomain of gp41. All mutant constructs created infectious virus in vitro that activated LTR-luc expression in the TZM-bl. Viral titer stocks ranged from 10²–10⁵ TCID₅₀ per ml.

Figure 2

Analysis of gp120 expression in mutant viruses. (a) Virus supernatants were pelleted and denatured viral proteins were immunodetected for gp120 env (MAb 3.11H) and structural protein p27gag (MAb 2F12). (b) Band densities were determined and the gp120/ p27gag ratio of each mutant was calculated. The data reported is the percent change in mean gp120/ p27gag ratio (±S.D.) compared to SIVmac239 from two independent experiments.