Crosslinked HIV-1 envelope-CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques

Abstract

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine development. In this study, we evaluated one such structure, expressed as either a gp120-CD4 or a gp140-CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (shCD4) and HIV-1(IIIB) envelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIV(BaL) gp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.

Figure 1

Analyses of macaque immune sera by ELISA using HIV Env and CD4 proteins. Macaques were immunized and sera were collected as described in Materials and Methods. Serum samples designated bleed 9 were analyzed by ELISA with HIV_IIIB gp120–CD4 complexes (□), denatured HIV_IIIB gp120 (), or nondenatured HIV_IIIB gp120 (■) (A) or by ELISAs performed with D1D4-soluble rhesus CD4 (□), D1D2 shCD4 (), or D1D4 shCD4 (■) (B) as described in Materials and Methods. Serum from a naïve animal was tested as a control. Reciprocal end-point binding titers reflect serum dilutions giving absorbance values equal to background ± 3 standard deviations. Mean reciprocal end-point titers for duplicate assays are shown. The data are from a representative experiment repeated several times with similar results.

Figure 2

Fractionation of neutralizing Ig by affinity chromatography on SCBaL/M9. (A) Sera from the four Env–CD4-immunized animals (492, 493, 497, and 498) were pooled and passed over a SCBaL/M9 affinity column or a mock column prepared with BSA. A flow-through (FT) fraction containing unbound material was collected from each column. Serial dilutions of the flow-throughs and serum pool were analyzed in parallel in ELISAs with HIV-1_BaL gp120 (□) and SCBaL/M9 (■) captured via sheep polyclonal anti-gp120 Ig (37, 38), in shCD4 ELISAs (), and in neutralization assays using U373 MAGI cells and a primary X4 virus, HIV-1₂₀₄₄ (). To compare the assay curves, the data obtained with the flow-through fractions were corrected for the dilution factor introduced by the chromatography procedure. The ELISA assay values represent the corrected reciprocal end-point serum dilution that produced an absorbance value equal to background ± 3 standard deviations. For neutralization assays, the assay value represents the corrected reciprocal serum dilution that produced 50% neutralization (ID₅₀) of macaque Ig in the sample. The percentage neutralization was calculated versus naïve macaque serum tested in parallel. Infection in wells with naïve sera showed no difference to those without macaque serum. All ELISAs were carried out in triplicate; the bars indicate standard deviation. Neutralization assays were carried out in triplicate or quadruplicate; the mean values from three neutralization experiments are shown. Bars indicate standard errors of the mean. (B) Ig eluted from the SCBaL/M9 affinity matrix was tested in the U373 MAGI cell assay (see Materials and Methods) by using the TCLA HIV-1_SF2 (○) and primary strains HIV-1_BaL (▵), HIV-1₂₀₄₄ (), HIV-1_89.6 (□), HIV-1_SF162 (⋄), HIV-1_92UG021 (■), HIV-1_22069–05 (▴), and HIV-1₂₀₇₅ (●). Matching serial concentrations of naïve macaque Ig were used as controls. Percentage neutralization by the SCBaL/M9 affinity-purified Ig was calculated relative to matching concentrations of naive Ig, which did not inhibit infection at any concentration tested. The average values obtained from triplicate assays are shown. (C) Ig recovered from either SCBaL/M9 (425 μg/ml) or HIV_BaL gp 120 (400 μg/ml) affinity matrices was analyzed in ELISAs with HIV_BaL gp120 (□) or SCBaL/M9 () captured via sheep polyclonal anti-gp120 Ig (37, 38) The Ig was also tested in ELISAs with shCD4 either directly adsorbed to the solid phase () or captured with mAb Q4120 (). Neutralizing activity was assessed in U373 MAGI cell assays with the primary X4 virus, HIV-1₂₀₄₄ (■). The ELISA assay values represent the reciprocal end-point concentration of macaque Ig (μg/ml) that produced an absorbance value equal to background ± 3 standard deviations. For neutralization assays, the assay value represents the concentration of macaque Ig (μg/ml) that produced 90% neutralization (IC₉₀). All ELISAs represent single experiments performed in duplicate, and neutralization assays represent single experiments in triplicate. Mean values of all assays are shown. Error bars indicate standard deviation in the ELISAs and standard errors of the mean in the neutralization assays.