Antigenic conservation and immunogenicity of the HIV coreceptor binding site

Abstract

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1-infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1-infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

Figure 1.

CD4-dependent neutralization of HIV-2 by HIV-1 antibodies. Neutralization of HIV-2_7312A (A and B) and HIV-2_7312A/V434M (C) infectivity in JC53BL-13 cells (reference 3) was mediated by plasma from patients with HIV-1 clade A (6X4F), B (CUCY2236), C (49M), or D (KAMW) infection or by the HIV-1 CD4i monoclonal antibodies 21c, 19e, or 17b. sCD4 concentrations correspond to the IC₅₀ values specific for each virus.

Figure 2.

Blocking of biotinylated 19e binding to HIV-1 and HIV-2 gp120-sCD4 complexes by human plasma samples from either normal uninfected donors (sample nos. 1–5) or HIV-1–infected subjects (sample nos. 6–16). Unlabeled 19e effectively competed with biotinylated (B*) 19e for binding to all gp120-sCD4 complexes and served as a positive control.

Figure 3.

Screening of CD4i monoclonal antibodies for binding to HIV-2_7312A (A) and to additional HIV and SIV (B) gp120-sCD4 complexes. 1.7A is a human HIV-2 gp120-specific monoclonal antibody, whereas all other monoclonal antibodies are CD4i antibodies derived from HIV-1–infected humans.

Figure 4.

Envelope gp120 alignments for HIV-2 (7312A and UC1), SIV (Mac239 and Ver-Tyo1), and HIV-1 (YU2 and HXB2). Bridging sheet, variable loops, amino acid identities, and site-directed mutations (H419R, Q422L, and V434M) are indicated. The signal peptide-gp120 cleavage position for HIV-1 is shown. Variable loops (V1/V2, V3, and V4) have conventionally been defined by disulfide-linked cysteine residues at their bases as depicted. However, the actual limits of variable loops have been resolved structurally in the HXB2–CD4–17b crystal complex (reference 8), and these sequences are indicated by green bars. It is possible that structural details diverge in the more distantly related HIV/SIV sequences. The amino acids contributing to the bridging sheet are highlighted in yellow. Blue dots indicate residues contributing to chemokine coreceptor binding based on site-directed mutagenesis studies (references 29, 30). Additional amino acids within the stem of V3, including 298R, 301N, 303T, 323I, 325N, 326M, and 327R, may contribute to gp120 interaction with CCR5 (reference 76). Red dots indicate HIV-1 contact residues for CD4 based on crystal structure analyses (reference 8). Asterisks below the sequence indicate conservation of amino acid identity across all five virus strains. Overall gp120 sequence identity was calculated based on amino acid residues exclusive of the initiator methionine of the (cleaved) signal peptide and a gap-stripped alignment of the sequences shown. Except for SIVverTYO1, sequences were obtained from the HIV Sequence Compendium 2002 (reference 18). We determined experimentally the nucleotide sequence of the SIVverTYO1 clone used in our studies (λ phage SAH12) and found that it differed from the reported sequence of the same clone in the Compendium at positions 171 (-), 172 (N), 402 (D), 418 (C), and 427 (W). Numbering is according to the HXB2 sequence.

Figure 5.

Neutralization of S736-68 and S736-68m/TI infectivity in JC53BL-13 cells (reference 3) by sCD4 (A), anti-CD4 monoclonal antibody RPA-T4 (B), CD4i monoclonal antibody 17b (C), and autologous patient plasma from day 278 after acute infection by HIV-1 (D).