Background:  Only a small number of monoclonal antibodies with broad, neutralizing activity against HIV-1 have been isolated to date, giving rise to the assumption that such antibodies are rarely induced during infection. However, adequate techniques for defining the frequency of such antibodies in plasma have been lacking. Although routine assays can readily measure neutralizing activity in patient plasma, such assays do not reveal which among the various specificities in a polyclonal sample contribute to the observed activity. We have introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV) to create an epitope-specific neutralization assay.

Methods:  Recognition sequences for HIV-1-specific, neutralizing monoclonal antibodies 2F5, 4E10, and 447-52D were introduced into homologous regions of SIV₂₃₉ by site-directed mutagenesis. Virus stocks were produced by transient transfection and assayed for infectivity and replication using a reporter cell line (C8166-SEAP) and rhesus peripheral blood mononuclear cells (PBMC). Quantitative neutralization assays were conducted using C8166-SEAP cells.   

Results:  The infectivity of the SIV_239-447-52D variant was too low for further study. The SIV_239-2F5 and SIV_239-4E10 variants were specifically neutralized by the 2F5 and 4E10 monoclonal antibodies, respectively, with IC₅₀ values similar to what has been reported previously for HIV-1 primary isolates. Both epitope-engrafted variants were then used to screen a panel of more than 100 HIV⁺ patient plasma samples. None of the samples tested exhibited significant neutralization of the SIV_239-2F5 variant. One sample exhibited >90% neutralization of SIV_239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in purified, concentrated IgG or IgA fractions. 

Conclusions:  We have confirmed by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1⁺ individuals or, if present, represent a small fraction of the total neutralizing activity in a given plasma sample. The parental SIV₂₃₉ and neutralization-sensitive SIV₃₁₆ control viruses were also not neutralized, indicating that significant cross-reactive neutralizing activity is not present in HIV⁺ patient plasma. Our results also suggest that the Env spikes of SIV₂₃₉ and HIV-1 are structurally similar, and that resistance of SIV₂₃₉ and HIV-1 primary isolates to antibody-mediated neutralization arises from similar mechanisms.