Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

Abstract

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.

FIG. 1.

Sequence conservation of the HIV-1 gp120 V3 loop (A) and the membrane-proximal gp41 ectodomain (C). Mutations were introduced in the V3 loop (B) and the membrane-proximal gp41 ectodomain (D) of SIV239 in order to generate the HIV-1 epitope-engrafted mutants.

FIG. 2.

Comparative infectivity of SIV239, SIV316, SIV239-2F5, SIV239-4E10, SIV239-447D, SIV239-2F5-4E10, and SIV239-2F5-4E10-447D. Virus stocks were obtained from the transfection of 293T cells. Stocks were normalized to the amount of p27 and used to infect C8166-45 SIV-SEAP cells. (A) SEAP activity in supernatant was measured by using a chemiluminescent assay (see Materials and Methods) at 3 days postinfection. (B) SEAP activity normalized to the amount of p27. Neg., negative.

FIG. 3.

Replication kinetics of viruses with the engrafted 2F5 and 4E10 HIV-1 epitopes in PBMCs obtained from two different rhesus macaques. (A) 419.91. (B) 382.90 d.p.i., days postinfection.

FIG. 4.

Envelope incorporation into virions. Virions were pelleted, lysed, separated by SDS-PAGE, and visualized by Western blotting. (A) Western blot of viral pellets with anti-gp120 MAb and anti-p27 (capsid) MAb. (B) Relative Env incorporation into virions. The ratios of gp120 to p27 were calculated by using phosphorimaging analysis. Data are presented relative to the ratios found for SIV239 virions (Rn/R239).

FIG. 5.

Comparative neutralization of SIV239, SIV239-2F5, and SIV239-4E10 by 4E10 HIV-1 monoclonal antibody (A) and 2F5 HIV-1 monoclonal antibody (B).

FIG. 6.

Comparative neutralization of SIV239, SIV239-2F5, and SIV239-4E10 by pooled plasma from six SIVmac-positive rhesus macaques (top row), different rhesus anti-gp120 MAbs (middle row), and a mouse anti-gp41 MAb (bottom row). Neg., negative.

FIG. 7.

Peptide competition. The possible correlation between 4E10-specific antibodies and neutralization by LTNP2 plasma was assessed by competition with peptide 94-1 (A). The ability of the 94-1 peptide to bind to the antigen-binding site of 4E10 antibody was also corroborated in a competition neutralization assay (B).

FIG. 8.

Neutralization of 239-4E10 by LTNP2 plasma (A) and IgG and IgA isolated from LTNP2 plasma (B).