Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis.

Infection of mononuclear cells by human immunodeficiency virus (HIV) begins with binding of the viral envelope glycoprotein, gp120, to its receptor, CD4. CD4 contains four extracellular immunoglobulin-like domains, the first of which (V1) is sufficient for HIV binding. V1 contains three sequences homologous to the antigen-complementarity-determining regions (CDR1 to -3) of immunoglobulin variable domains. While all three immunoglobulin CDRs are involved in antigen binding, only amino acids within and flanking the CDR2-like region of CD4 have been shown previously to be involved in gp120 binding. To investigate whether other regions in V1 take part in gp120 binding, we substituted alanine for each of 64 amino acids, including all of the hydrophilic residues in this domain. Mutations at four locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) markedly affected gp120 binding, but not the overall structure of V1 as probed with eight conformationally sensitive monoclonal antibodies. Thus, the gp120-binding site of CD4 is not limited to the CDR2-like sequence and consists of several discontinuous segments. Several amino acids were identified that are critical for the conformation of V1; the importance of these residues suggests some differences in the folding of this domain compared to immunoglobulin variable domains. Three amino acid substitutions were found that increase the affinity for gp120 significantly (1.7- to 2-fold individually and 4.2-fold when combined), suggesting that it may be possible to improve the HIV-blocking ability of CD4-based molecules by increasing their gp120 binding affinity.     

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.     

Identification and structural analysis of residues in the V1 region of CD4 involved in interaction with human immunodeficiency virus envelope glycoprotein gp120 and class II major histocompatibility complex molecules.

The human CD4 molecule binds both human immunodeficiency virus envelope protein gp120 and class II major histocompatibility complex (MHC) molecules. We have studied a series of mutants in the region of amino acids 42-49 of CD4 for their ability to bind gp120, to interact with class II MHC, to enhance T-cell activation, and to bind a panel of anti-CD4 antibodies. The mutation Q40P (Gln40----Pro) and the deletion d42-49 were found to disrupt most antibody epitopes in the V1 domain of CD4, suggesting major conformational changes, whereas mutants F43L, G47R, and P48S retained the binding of most of the anti-CD4 antibodies tested. The mutants d42-49, Q40P, F43L, and G47R lost both gp120 and class II MHC binding as well as the ability to enhance T-cell activation. In contrast, the mutation P48S affected neither gp120 binding, nor class II MHC binding, nor T-cell activation. We conclude that within this region the binding sites for gp120 and for class II MHC molecules overlap and that amino acids Phe43 and Gly47 comprise an intimate part of both binding sites. These observations are consistent with a three-dimensional model of the V1 domain of CD4 that was developed in order to understand the structural basis for binding to CD4.     

Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly.

CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.     

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.     

Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens

Epitopes located in and around the coreceptor binding site of HIV-1 envelope glycoprotein (gp120) exhibit enhanced exposure after attachment to the CD4 receptor and comprise some of the most conserved and functionally important residues on the viral envelope. Therefore, antibody responses to these epitopes [designated as CD4-induced (CD4i)] should be highly cross-reactive and potentially useful for HIV vaccine development. To address this question, rhesus macaques were vaccinated with subunit immunogens designed to raise humoral responses against CD4i epitopes and challenged rectally with SHIV(162P3), which encodes a heterologous envelope versus the immunogen. We found that animals vaccinated with a rhesus full-length single-chain (rhFLSC) complex exhibited significantly accelerated clearance of plasma viremia and an absence of long-term tissue viremia compared with unvaccinated control animals. Such control of infection correlated with stronger responses to CD4i epitopes in the rhFLSC-vaccinated animals, compared with macaques immunized with gp120, cross-linked gp120-CD4 complexes, or soluble CD4 alone. These responses were strongly boosted in the rhFLSC-vaccinated animals by SHIV(162P3) infection. The control of infection was not associated with anti-CD4 responses, overall anti-gp120-binding titers, or neutralizing activity measured in conventional assays. Vaccine-naive animals also developed anti-CD4i epitope responses after simian/ human immunodeficiency virus (SHIV) challenge, which appeared later than the overall anti-gp120 responses and in concert with the decline of viremia to a low set point. Collectively, these data suggest that antibodies to CD4i epitopes may play a role in controlling SHIV infection and provide insights for HIV vaccine development.     

Synergistic neutralization of HIV-1 by human monoclonal antibodies against the V3 loop and the CD4-binding site of gp120.

Two distinct regions or epitope clusters of human immunodeficiency virus type 1 (HIV-1) gp120 have been shown to elicit neutralizing antibodies: the V3 loop and the CD4-binding site. We have isolated neutralizing human monoclonal antibodies (HuMAbs) against conserved epitopes in both of these regions. In this study, we demonstrate that an equimolar mixture of two of these HuMAbs, one directed against the V3 loop and the other against the CD4-binding site, neutralizes HIV-1 at much lower concentrations than does either of the individual HuMAbs. Mathematical analysis of this effect suggests cooperative neutralization of HIV-1 by the two HuMAbs and demonstrates a high level of synergy, with combination indices (CIs) of 0.07 and 0.16 for 90% neutralization of the MN and SF-2 strains, respectively. The dose reduction indices (DRIs) for each of the two HuMAbs at 99% neutralization range approximately from 10 to 150. A possible mechanism for this synergism is suggested by binding studies with recombinant gp160 of the MN strain; these show enhanced binding of the anti-CD4 binding site HuMAb in the presence of the anti-V3 loop HuMAb. These results demonstrate the advantage of including both V3 loop and CD4-binding site epitopes in a vaccine against HIV-1 and indicate that combinations of HuMAbs against these two sites may be particularly effective in passive immunotherapy against the virus.     

Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain.

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.     

Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1.

This study reports on the direct effect of the envelope glycoprotein (gp120) of the human immunodeficiency virus type 1 (HIV-1) on human monocyte function. Addition of preparations of purified gp120 from the HIV-1 to human monocytes resulted in the production of interleukin 1 (IL-1) and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways. Quantification of prostaglandin E2 (PGE2) and IL-1 revealed an increase in both mediators with 50 ng of gp120 per ml and an increase of 12- and 30- to 40-fold with 200-400 ng of gp120 per ml, respectively. Unlike native gp120, the recombinant nonglycosylated gp120 fragments PB1-RF and PB1-IIIB, as well as one of the core structural proteins of HIV-1, p24, did not increase arachidonic acid metabolism or IL-1 activity. Cytofluorometric analysis revealed that gp120 blocked the binding of OKT4A to the CD4 on monocytes, whereas OKT4 binding was unaffected. Involvement of the CD4 in signal transduction was further demonstrated by the ability of OKT4 and OKT4A monoclonal antibodies to increase monocyte PGE2, IL-1 activity, and nanogram amounts of IL-1 beta.     

A soluble recombinant polypeptide comprising the amino-terminal half of the extracellular region of the CD4 molecule contains an active binding site for human immunodeficiency virus.

Infection of helper T lymphocytes by human immunodeficiency virus is initiated by a specific interaction of the viral envelope glycoprotein with CD4, an integral membrane glycoprotein of the target cell. We have adapted a vaccinia virus-based mammalian cell expression system to produce variants of the CD4 molecule for structure-function studies. In this report we demonstrate that a truncated 180-amino acid fragment representing approximately the N-terminal half of the extracellular region of CD4 is found primarily in soluble form in the extracellular medium. Epitope analysis with a panel of anti-CD4 murine monoclonal antibodies indicates that the fragment reacts with those antibodies known to block the interaction between CD4 and the human immunodeficiency virus envelope glycoprotein but reacts poorly or not at all with those antibodies that do not block this interaction. We also show that the fragment forms a specific complex with a soluble form of gp120, the CD4-binding subunit of the viral envelope glycoprotein. These results indicate that this soluble CD4 fragment contains an active binding site for human immunodeficiency virus.     

Polyionic compounds selectively alter availability of CD4 receptors for HIV coat protein rgp120

We studied the ability of several polyionic compounds, previously shown to have activity in vitro against human immunodeficiency virus (anti-HIV) to block binding of anti-CD4 and recombinant HIV gp120 to the CD4 receptor on human lymphocytes. We found that Evans blue and aurin tricarboxylic acid could completely inhibit binding of anti-CD4 (Leu3a) and rgp120 and have selectivity for the CD4 receptor. A number of other compounds, including dextran sulfate and heparin had no effect on binding of rgp120 and were shown to be nonspecific for inhibition of binding of monoclonal antibodies to different T-cell receptors. Studies using a number of membrane-active drugs showed that changes in membrane potential or ion fluxes were not involved in the inhibition of binding of rgp120 by Evans blue or aurin tricarboxylic acid.     

Localized conformational changes in the N-terminal domain of CD4 identified in competitive binding assay of monoclonal antibodies and HIV-1 envelope glycoprotein.

The CD4 antigen is established as a major cellular receptor for the human immunodeficiency virus (HIV). Previous studies have suggested that certain anti-CD4 monoclonal antibodies (MAbs) can inhibit or enhance the binding of the viral envelope glycoprotein gp120 to CD4 by allosteric effects. In the study reported here, 17 anti-CD4 MAbs were tested for their ability to influence the binding of each other to recombinant soluble CD4 in a solid-phase radioimmunoassay. Marked enhancement of binding between specific pairs of MAbs was seen, as well as inhibition or lack of interaction. Enhancement was seen less often when CD4+ cells were used as the target antigen. Information on patterns of enhancement and inhibition permitted grouping of MAbs on the basis of epitope specificity, and this grouping was in agreement with published findings based on X-ray crystallographic studies. These results demonstrate connectivity between epitopes in the first domain of recombinant CD4 and suggest a high degree of flexibility of surface structure. These findings may be of physiological significance both in the normal function of CD4 and in the interaction of CD4 with HIV. The data have implications for research or therapeutic strategies based on recombinant CD4 or CD4 mutants and highlight the problems of interpreting experimental findings based on abrogation of MAb binding.     

Characterization of CD4-induced epitopes on the HIV type 1 gp120 envelope glycoprotein recognized by neutralizing human monoclonal antibodies

The entry of human immunodeficiency virus (HIV-1) into target cells typically requires the sequential binding of the viral exterior envelope glycoprotein, gp120, to CD4 and a chemokine receptor. CD4 binding exposes gp120 epitopes recognized by CD4-induced (CD4i) antibodies, which can block virus binding to the chemokine receptor. We identified three new CD4i antibodies from an HIV-1-infected individual and localized their epitopes. These epitopes include a highly conserved gp120 beta-strand encompassing residues 419-424, which is also important for binding to the CCR5 chemokine receptor. All of the CD4i antibodies inhibited the binding of gp120-CD4 complexes to CCR5. CD4i antibodies and CD4 reciprocally induced each other's binding, suggesting that these ligands recognize a similar gp120 conformation. The CD4i antibodies neutralized laboratory-adapted HIV-1 isolates; primary isolates were more resistant to neutralization by these antibodies. Thus, all known CD4i antibodies recognize a common, conserved gp120 element overlapping the binding site for the CCR5 chemokine receptor.     

N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding.

Syu et al. recently reported that deletion of residues Ile-108 to Leu-116 from the amino terminus of gp120 abolished CD4 binding. The authors have investigated the role of this region using a monospecific antipeptide antibody. As assessed by a microtiter plate-based radioimmunoassay, the antibody, raised in sheep against a synthetic peptide encompassing this deleted region, does not inhibit the gp120-CD4 association. The reported loss of CD4 binding ability, resulting from the deletion in this region of gp120, is likely to be due to indirect structural changes in gp120 rather than representing an integral part of the CD4 binding domain.     

A defined conformational epitope from the C4 domain of HIV type 1 glycoprotein 120: anti-cyclic C4 antibodies from HIV-positive donors magnify glycoprotein 120 suppression of interleukin 2 produced by T cells

The C4 domain of HIV gp120 plays a functionally vital role in the binding of gp120 to CD4 receptors on target cells. Antibodies to an 11-amino acid cyclic C4 peptide were obtained from immunized rabbits and from the serum of an HIV-positive human and were found to recognize gp120 bound to CD4. Anti-cyclic C4 antibodies magnified gp120-induced suppression of IL-2 produced by T cells in vitro. Rabbit antibodies to the 11-amino acid linear C4 peptide did not recognize gp120 in the free state or when bound to CD4. These results indicate that a conformationally defined, highly conserved epitope in the gp120 C4 region remains exposed on CD4 binding. Naturally occurring antibodies to this epitope can augment gp120-induced immunosuppression and may contribute to disease progression.     

HIV-1 neutralizing monoclonal antibodies induced by a synthetic peptide.

We have developed a series of murine monoclonal antibodies to a region of the 120 kD envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1). This region has previously been implicated as a site for virus neutralization by antisera raised to recombinant proteins and by antibodies made to full-length gp120 purified from virus. The antigen employed was a synthetic peptide containing 15 amino acids, representing amino acid residues 308-322, RIQRGPGRAFVTIGK, of env gp120 (HTLV-IIIB isolate). Five of the monoclonal antibodies raised to this antigen have reactivity with gp120 from divergent strains of HIV-1 in Western blot assays. The two of these five which were tested with live cells infected with the divergent HIV-1 isolates IIIB, MN, and RF were specifically reactive by fluorescence analyses with cells infected with the MN and IIIB isolates. Four of the five monoclonal antibodies blocked the fusion of IIIB-infected cells with uninfected MOLT-4 target cells. The monoclonal antibody most reactive with MN-infected cells by fluorescence, #5025A, blocked the fusion of MN-infected cells with uninfected MOLT-4 cells. Four of the five monoclonal antibodies neutralized the IIIB isolate of HIV-1 in vitro, but none neutralized the MN or RF isolates at the levels of antibody tested (less than or equal to 50 micrograms/ml). Taken together these data indicate that monoclonal antibodies to the immunodominant neutralizing domain of HIV-1 gp120 display different levels of group reactivity depending on the assay system being examined.     

Peptides selected from a phage display library with an HIV-neutralizing antibody elicit antibodies to HIV gp120 in rabbits, but not to the same epitope

Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.     

The N-terminal region of the human immunodeficiency virus envelope glycoprotein gp120 contains potential binding sites for CD4.

Human immunodeficiency virus (HIV) vaccines targeted at blocking HIV-CD4 interactions are expected to be less affected by the sequence heterogeneity of HIV than those targeted at variable regions of the envelope outercoat glycoprotein, gp120. All potential CD4 binding sites identified thus far in HIV are localized in the C-terminal region of gp120. In this study we demonstrate that the N-terminal region of gp120 also contains conserved residues critical for binding to CD4 and that gp120-CD4 interactions can be blocked by an antiserum with binding specificity to an N-terminal region of gp120. These results suggest that not all potential CD4 binding sites are present in the C-terminal region of gp120 and that an alternative HIV vaccine development strategy may have to include the N-terminal gp120 region as a component to raise effective CD4-blocking antibodies.     

Detection of IgA inhibiting the interaction between gp120 and soluble CD4 receptor in serum and saliva of HIV-1-infected patients

OBJECTIVE: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. METHODS: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120-soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. RESULTS: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120-sCD4 interaction by approximately 50%. The gp120-sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. CONCLUSION: Immunogens capable of eliciting IgA antibodies that inhibit gp120-CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-1.     

Monoclonal antibodies to the C4 region of human immunodeficiency virus type 1 gp120: use in topological analysis of a CD4 binding site.

We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.