Identification of gp120 regions targeted by a highly potent neutralizing antiserum elicited in a chimpanzee inoculated with a primary human immunodeficiency virus type 1 isolate

Abstract

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1(DH12)) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1(DH12), nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1-V2 and V4 regions of HIV-1(DH12) could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.

FIG. 1

Schematic diagram of the parental and chimeric HIV-1 envelope glycoproteins and results of neutralization assay with chimpanzee 1206 antiserum in human PBMC. The regions encoding the signal peptide (SP), surface glycoprotein gp120, transmembrane glycoprotein gp41, hypervariable regions (V1 to V5), and cleavage sites (▾) are indicated. The individual vertical lines represent amino acid residues that are different between HIV-1_DH12 and HIV-1_AD8. Neutralization assays were performed using anti-CD3 and -CD28 antibody-activated PBMC, as previously described (16). Neutralization is reported as positive (calculated V_n/V_o = 0.1, serum titers > 1:1,600) or negative (calculated V_n/V_o = 0.1, serum titers < 1:100). ND, neutralization could not be determined using PBMC as target cells. HIV-1_AD8 was previously reported to be neutralization resistant (50).

FIG. 2

Neutralization of the parental and chimeric viruses using the HOS-CD4.CCR5 cell line for target cells. (a) Replication of each virus in the presence of chimpanzee 1206 antiserum is shown as a percentage of that for the no-serum control. Each virus was incubated with or without the antiserum (1:300 dilution) prior to infecting cells. Culture medium was replaced at 1 day postinfection and culture supernatant was analyzed for RT activity at 4 days postinfection. (b) The parental HIV-1_DH12 and two chimeric viruses (AD8-DH120G and -I) were incubated with various dilutions of the antiserum to determine relative sensitivities to the antiserum.

FIG. 3

Immunoprecipitation of parental and chimeric gp120s with chimpanzee 1206 antiserum. (a) gp120 from HIV-1_AD8 (AD), HIV-1_DH12 (DH), or HIV-1_IIIB (IIIB) was immunoprecipitated with serum from either an uninfected chimpanzee or chimpanzee 1206, which had been previously inoculated with HIV-1_DH12. (b) The parental HIV-1_AD8 (AD) and HIV-1_DH12 (DH) gp120s or chimeric gp120s were subjected to SDS-PAGE followed by Western blot analysis using rabbit anti-gp160 antiserum. (c) The same parental and chimeric gp120s were first immunoprecipitated (using 10 times the amount of protein that was directly loaded on the gel in panel b) with the antiserum from chimpanzee 1206. The immunoprecipitated proteins were subsequently subjected to SDS-PAGE followed by Western blotting using rabbit anti-gp160 antiserum. Numbers, on the left are molecular masses, in kilodaltons.