HIV-1 gp120: a novel viral B cell superantigen

The envelope glycoprotein of the human immunodeficiency virus (HIV-1), gp120, has recently been characterized as a novel immunoglobulin superantigen (Ig-SAg) [1,2]. Analogous to the interaction of SAgs with T cells, gp120 binds to an unusually large proportion of immunoglobulins (lgs) from HIV-uninfected individuals; most, if not all of these Igs are members of the VH3 family [3]. Functionally, gp120 preferentially stimulates VH3 B cells in vitro. This stimulation correlates with an in vivo VH3 activation during HIV infection. Curiously, this initial activation is followed by a subsequent depletion of VH3-expressing B cells as individuals progress to AIDS. In this article we will review our current understanding of the superantigenic properties of HIV gp120. Specifically we will focus on structural aspects of the binding interaction. on the ontological development of these superantigen-binding antibodies, and on potential roles that this unconventional Ig-pathogen interaction might play in the pathogenesis of HIV-induced disease.     

B cell superantigens: potential modifiers of the normal human B cell repertoire

Staphylococcal protein A (SPA), HIV gp120, and staphylococcal enterotoxins (SE) are B cell superantigens that induce VH specific B cell responses. In addition, the red blood cell antigens, i/I, have some features of a B cell superantigen. Binding of SPA, SE and HIV gp120 are VH family specific, whereas binding of i/I is VH gene specific. SPA and HIV gp120 function by stimulating VH3-expressing B cells, whereas SE appear to function by enhancing survival of the appropriate VH-expressing B cells. Moreover, HIV gp120 has been shown to delete VH3-expressing B cells. In this review, we describe evidence that shows how these superantigens may play a role in shaping the normal B cell repertoire.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.     

DNA vaccination using expression vectors carrying FIV structural genes induces immune response against feline immunodeficiency virus

Following inactivated virus vaccination trials, the surface glycoprotein gp120 of the feline immunodeficiency virus (FIV) was considered as one of the determinants for protection. However, several vaccination trials using recombinant Env protein or some peptides failed to induce protection. To understand the role of the gp120 protein in vivo, we vaccinated cats with naked DNA coding for FIV structural proteins gp120 and p10. We analyzed the ability of these vaccinations to induce immune protection and to influence the onset of infection. Injection in cat muscles of expression vectors coding for the FIV gp120 protein induced a humoral response. Cats immunized twice with the gp120 gene showed different patterns after challenge. Two cats were, like the control cats, infected from the second week after infection onwards. The two others maintained a low proviral load with no modification of their antibody pattern. The immune response induced by gp120 DNA injection could control the level of viral replication. This protective-like immune response was not correlated to the humoral response. All the cats immunized with the gp120 gene followed by the p10 gene were infected, like the control cats, from the second week but they developed a complete humoral response against viral proteins after challenge. Furthermore, they showed a sudden but transient drop of the proviral load at 4 weeks after infection. Under these conditions, one injection of the p10 gene after one injection of the gp120 gene was not sufficient to stimulate protection. On the contrary, after a period, it seems to facilitate virus replication.     

B and T cell function parameters during zidovudine treatment of human immunodeficiency virus-infected patients

The aim of this study was to assess the effects of zidovudine on B cell dysregulation in human immunodeficiency virus (HIV)-infected patients and the phenomenon of gp 120/anti-gp 120 antibody complex adhesion to CD4+ cells. Compared with pretherapy figures, zidovudine treatment was not associated with a change in spontaneous in vitro synthesis of anti-HIV antibodies but was related to restoration of lymphocyte ability to produce Epstein-Barr virus-specific antibodies in 43% of previously unresponsive patients. After 30 days of therapy, the percentage of circulating CD4+/IgG+ lymphocytes decreased; the number of available CD4 receptors per cell increased, and antibodies to gp 120, evident in CD4+ cell eluates from most untreated patients, were no longer detectable. These results indicate that zidovudine partly restores in vitro humoral responsiveness but does not substantially influence the overall activation of the B cell compartment. The findings also suggest that zidovudine may down-regulate some immunopathologic phenomena that amplify direct viral damage.     

Experimental study of the possible implications of some apoptosis mechanisms in the pathogenesis of HIV infection. 1. In vitro apoptotic death of peripheral blood lymphocytes

The human immunodeficiency virus (HIV) infection produces a gradual depletion of T-helper CD4+ lymphocytes as, surprisingly, a consequence of apoptosis of the uninfected lymphocytes. We suggested that this is the result of the action exerted by HIV inductors of apoptosis (for example, gp 120) in the absence of viral apoptosis suppressor, which confers protection to the infected cell. We intended to demonstrate this hypothesis within the framework of a complex study regarding the apoptosis mechanisms in HIV infection. We started this study by setting up an apoptosis model on HIV-negative peripheral blood lymphocytes (PBLs)* cultivated in vitro in the presence of dexamethasone (Dex). In this work we characterize this model morphologically and biochemically. Three unreported morphological changes observed by us--namely: I) fringing of nucleus with advancement of fringes up to the plasma membrane; II) segmentation and peripheral migration of condensed chromatin through a rotation movement; III) "flowering of the cell" consisting in the radial separation of the lymphocyte into centrally united "petals" with the tendency to form apically multiple apoptotic bodies--completed the classical morphology of the apoptosis phenomenon. The apoptotic death was confirmed by the oligonucleosomal (multiples of 200 bp) and mononucleosomal fragmentation of DNA isolated from lymphocytes.     

Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA

The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.     

Molecular mimicry accompanying HIV-1 infection: human monoclonal antibodies that bind to gp41 and to astrocytes

Monoclonal antibodies that bound to HIV gp41 and cross-reacted with astrocytes were recovered from the blood of three patients infected with HIV-1. Mapping of the specificity of these monoclonal antibodies, using synthetic gp41 peptides, located their epitope to amino acids 644-663 and established their conformation dependence. Six other human monoclonal anti-HIV antibodies were found to bind to HIV gp41 or gp120 but not to reactive astrocytes in brain tissue. Sharing of linear or conformational protein determinants between disparate viral and host proteins is termed molecular mimicry. The consequences of such mimicry by anti-viral antibodies interacting with astrocytes may play a role in the dementia of AIDS patients since a major function of astrocytes is to maintain the appropriate milieu for neuronal function. The finding of such cross-reactive antibodies adds to the evidence for a possible autoimmune pathogenesis in some of the disease manifestations accompanying HIV infection.     

CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4(+) T cells

AIDS is characterized by a progressive decrease of CD4(+) helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56(lck) and Gialpha. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4(+) but not in CD8(+) T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.     

Viral superantigen-induced hyporesponsiveness of T cells and polyclonal B cell activation in HIV-1 infection

The mechanisms of CD4 depletion and hyporesponsiveness during human immunodeficiency virus (HIV) infection are still unknown. Given the ability of superantigens to stimulate a higher number of lymphocytes than conventional antigens, they may play a major role in this process. Recently, a novel superantigen, the rabies virus nucleocapsid (NC), was described in humans. In the present work, we tested the responses of peripheral blood lymphocytes from asymptomatic HIV-infected patients to this superantigen. In contrast to its effect in normal controls, NC failed to expand T cells from HIV-infected individuals expressing the V beta 8 family, and induced a strong decrease in the response to CD3 activation. This absence of response was not the consequence of programmed cell death, and was explained by an anergic state induced by the superantigen. NC superantigen was also able to induce polyclonal activation of B cells, as measured by the secretion of anti-HIV antibodies and autoantibodies. Moreover, V beta 8 depletion experiments showed that induction of autoantibody secretion was V beta 8 dependent, whereas secretion of HIV-1 antibody was not. Interleukin secretion studies showed that NC was able to induce high levels of interleukin-4 and interleukin-10. Taken together, our results suggest a role for exogenous viral superantigens such as NC in the induction of T cell hyporesponsiveness and polyclonal B cell activation during HIV infection. The induction of a Th2 response and the role of these superantigens in the immunopathogenesis of acquired immunodeficiency syndrome are discussed.     

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.     

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.     

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.     

Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination.     

Spectral analysis of a thalamus-to-cortex seizure pathway

CD4 ligation of HIV envelope gp120 results in conformational changes in gp120 that lead to exposure of the gp41 fusogenic domain and fusion with the host cell membrane. One determinant at or near the CD4-binding site exposed on gp120 subsequent to CD4 binding is defined by two human MAbs termed 17b and 48d. These MAbs do not block CD4 binding to gp120; rather, their binding to gp120 is upregulated following CD4 binding. To determine if synthetic peptide mimetopes could be found that reflect conformational determinants on the surface of gp120, synthetic gp120 peptides from 10 divergent HIV isolates were screened for their ability to bind to 17b and 48d in ELISAs. Although MAb 48d binds to HIV IIIB recombinant gp120 protein, in our studies 48d selectively bound only to the HIV Can0A V3 peptide and not to HIV IIIB V3 peptide, whereas MAb 17b bound none of the peptides tested. Monoclonal antibody 48d bound to the HIV Can0A V3 peptide both in solid-phase ELISA and in solution in a competitive ELISA, but could not bind to HIV Can0A V3 peptide bound to human T cells. The HIV Can0A V3 peptide induced anti-HIV antibodies in rhesus monkeys that neutralized the laboratory-adapted HIV MN strain but did not induce antibodies that neutralized HIV IIIB/LAI, HIV SF-2, or HIV RF isolates, or that neutralized HIV primary isolates. These data suggested that the primary sequence of the HIV Can0A V3 loop exists in a conformer that mimicks a non-V3 determinant of native gp120 exposed subsequent to CD4 binding on the surface of gp120 of laboratory-adapted HIV strains. Structural studies of the Can0A V3 peptide and/or the 48d MAb may provide important information regarding the nature of gp120 conformational changes that occur following gp120 ligation by CD4.     

Comparative analysis of humoral immune responses to HIV type 1 envelope glycoproteins in mice immunized with a DNA vaccine, recombinant Semliki Forest virus RNA, or recombinant Semliki Forest virus particles

The Semliki Forest virus (SFV) system seems to be a useful new approach for generating effective immune responses against HIV-1 in animal models. We evaluated this system by comparing the humoral immune responses raised in mice immunized against the HIV-1 envelope with the SFV system, a DNA vaccine, and a recombinant Env glycoprotein. gp160 ELISA antibody titers (204,800) were highest in the sera from mice immunized with recombinant Semliki Forest virus particles. These sera contained antibodies to the CD4-binding site and recognized linear epitopes on gp120 and gp41 that were also recognized by a pool of sera from HIV1-infected individuals. This demonstrates that the HIV-1 envelope produced in vivo by the SFV system does not fold aberrantly. A low level of neutralizing antibodies against the HIV-1LAI strain was also detected in the serum of one mouse immunized with recombinant SFV particles, suggesting that booster injections should be given to achieve a more effective immune response. SFV recombinant particles induced the strongest humoral responses to the HIV-1 envelope of all the potential HIV env vaccines tested.     

HIV preventive vaccines. Progress to date

The major conceptual problem for HIV vaccine development has been the lack of information on immune responses known to correlate with protection against HIV infection in humans. In this regard, studies on the natural history of HIV infection and AIDS, especially of people with apparent resistance to HIV infection and of patients with HIV infection who have long term survival without disease progression, may provide important information for vaccine development. In addition, a major concern for the development of broadly effective vaccines has been the extensive genetic variability which is characteristic of HIV. In spite of these unknowns, the first generation of HIV candidate vaccines has been developed and evaluated. HIV candidate vaccines based on the subunit recombinant envelope concept (gp120 or gp160) have been shown to protect chimpanzees from HIV infection on challenge, and have now been evaluated in humans in phase I and phase II trials. These products are well tolerated, and capable of inducing neutralising antibodies, but not cytotoxic T lymphocytes. A second vaccine concept, currently in phase I trials, is based on live recombinant vectors, especially using poxvirus vectors followed by boosting with subunit recombinant envelope vaccines. This concept is theoretically very attractive because preliminary data suggest that these vaccines induce both humoral and cell-mediated immunity. However, no published information is available on the ability of live recombinant vector vaccines to protect chimpanzees from HIV infection. The next step in HIV vaccine development is to proceed carefully to expanded phase II and phase III trials to assess the protective efficacy of these candidate vaccines in humans. These trials will be extremely complex from the logistical, scientific and ethical points of view, and will require close collaboration between clinical, basic science and behavioural researchers, national and international organisations, and the pharmaceutical industry.     

Induction of lymphomonocyte activation by HIV-1 glycoprotein gp120. Possible role in AIDS pathogenesis

Dysfunction of cytokine secretion pattern has been suggested to play a central role in the immunopathogenesis of HIV infection. In fact a shift of T helper cell functions from a Th1-type to TH0- or TH2-type has been observed in HIV-1 infected subjects undergoing disease progression. The inhalance of cytokine network is accompanied by persistent activation of the immune system, impaired ability to mount a proper activation response (anergy), and priming to apoptosis. Extensive investigation during the last decade has been conducted on the influence of HIV-1 gp120 or of its precursor gp160 on several lymphocyte and monocyte functions. Gp120 is able to rise intracellular calcium concentration and to induce the formation of inositol triphosphate, can block mitogen- or antigen-driven T cell activation, can induce altered cytokine production by activated PBMC subpopulations, determines impaired cytotoxicity and chemotactic response to antigens, interferes with the activity of antigen presenting cells, enhances or induces apoptosis, stimulates polyclonal B cell activation and induces or up-modulates a number of cytokines, including IL-6. TNF, IL-1-alpha and -beta, IL-10 and IL-8. Furthermore, both IFN-alpha and -gamma, as well as several markers of IFN activity, such as beta 2-microglobulin and neopterin, are induced in gp120-stimulated PBMC. However, neither IL-4 (Th2-type) nor IL-2 (Th1-type), nor DNA synthesis are activated by gp120. On the other hand gp120-stimulated PBMC express increased IL-2 receptors, and can be induced by exogenous IL-2 to proliferate, suggesting that they are in a state of at least partial activation. According to this hypothesis, other activation markers, both early (such as CD69), and late (such as CD45RO and CD71), are induced by gp120, but this even partial activation does not lead to the ability of PBMC to support productive infection by HIV-1, unless in the presence of exogenous IL-2. The HIV-induced cytokines can influence HIV infection either directly, by up- or down-modulating virus replication, or indirectly, by modulating the expression of cellular molecules. In fact, during the budding process, the HIV envelope captures a number of cell membrane proteins, including cytokine receptors such as IL-2R, adhesion molecules such as LFA-1, ICAM-1, -2, HLA Class I and II, as well as cell lineage markers. Gp120-induced cytokines, particularly IFN-gamma, upmodulate the cellular expression of intercellular adhesion molecules, such as ICAM-1. We have shown that the IFN-gamma-driven increase of the expression of ICAM-1 by cells chronically infected with HIV-1 can be transmitted to the virus progeny, resulting in phenotypic alteration of the virus, and leading to the expansion of its host cell spectrum to CD4-negative cells expressing the appropriate ligands, i.e. LFA-1. Intercellular adhesion molecules are also involved in the cell-mediated transmission of HIV infection, and the increased ICAM-1 expression induced by IFN-gamma determines a stimulation of the transmission of HIV from abortively infected endothelial cells to permissive CD4 lymphocytes. On the whole, these data indicate that HIV, or its soluble products such as gp120, can modify several PBMC functions, by inducing a number of cytokines and a partial state of immune activation. It is possible that the gp120-driven changes of PBMC functions are not only an epiphenomenon of HIV infection, but rather, it is likely that they can participate in the immunopathological events responsible for disease progression.     

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.