Although much is known about the HIV-1 envelope glycoprotein (7
), the present study provides new insight into its immunogenicity and antigenic conservation. Previous studies suggested that the conformationally dependent coreceptor binding surface on HIV-1 was only weakly immunogenic and CD4i antibodies were relatively uncommon (31
). This paper indicates quite the opposite to be the case. We find the vast majority (94%) of HIV-1–infected patients infected by any 1 out of 10 different clades or CRFs harbor HIV-specific CD4i Nabs with IC50
titers ranging from 1:20 to >1:100,000. The mean CD4i Nab titer against HIV-27312A/V434M
among 189 subjects was 1:250 and the median titer was 1:2,500. 114 subjects had Nab titers ≥1:1,000, the highest reaching 1:143,000. In a related study, we found that 8 out of 10 healthy, uninfected human volunteers immunized with ALVAC vCP1452 HIV-1 gp140 alone or in combination with soluble monomeric HIV-1 gp120 (AIDSVAX B/B) developed HIV-1 CD4i-neutralizing antibodies against HIV-27312A
, compared with 0 out of 5 control subjects who were vaccinated with placebo (unpublished data). To explain the elicitation of CD4i Nabs by soluble HIV-1 gp120 or expressed gp140, we suspect that envelope glycoprotein is bound to cell surface–associated CD4, undergoes conformational change, and elicits a CD4i antibody response.
The observation that CD4i antibodies elicited by HIV-1 infection potently neutralized multiple strains of HIV-2 came as a surprise. Although most primary human and simian lentiviruses use CCR5 as a coreceptor for cell attachment and entry (23
), functionally important amino acids in the HIV-1 envelope coreceptor binding region identified by mutagenesis experiments (8
) are only partially conserved in HIV-2, SIVmac, and SIVagm (). Moreover, conserved receptor binding would not necessarily be expected to be reflected in conserved receptor antigenicity (43
). Thus, the finding that HIV-1 CD4i monoclonal antibodies such as 19e and 21c could bind viral glycoproteins as divergent as those from HIV-1, HIV-2, SIVsm, SIVmac, and SIVmne in a CD4-dependent fashion (), and that monoclonal and polyclonal antibodies from HIV-1–infected humans routinely neutralized sCD4-triggered HIV-2 ( and ), was quite unexpected. We even found that sCD4-treated SIVverTyo1 from African green monkey () is susceptible to CD4i neutralization by some HIV-1–infected patient samples in titers as high as 1:1,000 (Fig. S2). Together, these observations highlight the extraordinary degree of antigenic conservation linked to coreceptor binding exhibited by diverse HIV-1 and HIV-2 lineages, and at the same time, an ability of the human humoral immune system to exploit these constraints.
It is of interest to consider the cooperative interactions that may be occurring among sCD4, the HIV-2 envelope glycoprotein, and CD4i antibody that result in potent virus neutralization. We have ruled out the possibility that HIV-1–elicited CD4i antibodies neutralize HIV-2 by binding directly to CD4 because a scorpion toxin-based CD4 mimetic that differs substantially in amino acid sequence from CD4 also results in conformational changes in HIV-2 gp120 leading to binding and neutralization by different monoclonal and polyclonal CD4i antibodies (reference 46
and unpublished data). If sCD4 does not interact directly with CD4i antibodies, then it must enhance the susceptibility of virus to neutralization by inducing conformational change and exposure of CD4i epitopes, but in a cooperative manner because the magnitude of HIV-2 neutralization we observe is far greater than would be expected on the basis of additive stoichiometry. Of note, Berger et al. (47
) have demonstrated cooperative interactions between different gp120 protomers within a trimer complex of HIV-1.
A role for CD4i antibodies in natural HIV-1 infection may become apparent. Our data, together with other results (37
), suggest that HIV-1 variants with exposed coreceptor binding surfaces and varying degrees of CD4 independence, are generated spontaneously in vivo where they are almost certainly neutralized by CD4i or other HIV-1–specific antibodies. In fact, four studies have now shown that single amino acid substitutions in the HIV-1 glycoprotein, either at the base of V1/V2 (3
) or in the V3 loop (the present text and reference 41
), are sufficient to confer on the virus varying degrees of CD4 independence, spontaneous exposure of the coreceptor binding site, and enhanced susceptibility to CD4i Nabs. Principles of viral dynamics suggest that such mutations must be occurring in vivo on a virtually continuous basis, as has been documented for comparable mutations leading to antiretroviral drug resistance (49
). Thus, CD4i antibodies may influence HIV-1 natural history and pathogenesis to a greater extent than is currently recognized by constraining virus to CD4 dependence. Consistent with this interpretation, Gabuzda et al. have shown that HIV-1 virus within the central nervous system (where circulating antibodies are relatively excluded) has less dependence on cell surface–bound CD4 for attachment and entry (50
). CD4i antibodies could also influence the frequency of R5/X4 coreceptor switching (51
) and target viruses with short or otherwise constrained envelope variable loop sequences (52
The discovery that sCD4-triggered HIV-2 is susceptible to binding and neutralization by HIV-1–elicited CD4i antibodies has practical application in studies of HIV-1 natural history and vaccine assessment. A number of investigative groups have attempted to stabilize the HIV-1 envelope glycoprotein in a CD4-bound configuration to use it as an immunogen designed to elicit antibodies against viral receptor surfaces or other intermediate envelope structures (53
). But methods to selectively identify and titer Nabs specific for such epitopes have been limited. Here, we show that neutralization of sCD4-treated HIV-2 represents an extremely sensitive and specific assay to detect HIV-1–elicited CD4i antibodies. Investigators have also targeted the membrane-proximal external region (MPER) of HIV-1 gp41 for vaccine development (56
) because conserved epitopes in this region are capable of eliciting broadly reactive Nabs in natural infection (56
). But again, neutralization assays are lacking that allow for the sensitive and specific detection of MPER epitope-specific Nabs (17
). We considered the possibility that HIV-2 could act as a “molecular scaffold” on which to present these and other HIV-1 epitope-specific antigens in the context of a functional envelope glycoprotein that does not otherwise cross-react with HIV-1–neutralizing antibodies. In recent studies, we have identified and modified by site-directed mutagenesis HIV-2 strains that can be used to detect and titer neutralization by the HIV-1 gp41 MPER-specific human monoclonal antibodies 4E10 and 2F5 with high sensitivity and specificity (unpublished data). Thus, the strategy described in this paper of using HIV-2 envelope glycoproteins in the context of infectious virions or as isolated proteins to detect HIV-1 epitope-specific neutralizing antibodies may find application in the assessment of candidate vaccines and in studies of HIV-1 natural history.