Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor.

The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 microM in various T4+ cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment.     

CD4 is a critical component of the receptor for human herpesvirus 7: interference with human immunodeficiency virus.

In this study, we demonstrate that the glycoprotein CD4, a member of the immunoglobulin superfamily, is a critical component of the receptor for human herpesvirus 7 (HHV-7), a recently discovered T-lymphotropic human herpesvirus. A selective and progressive downregulation of the surface membrane expression of CD4 was observed in human CD4+ T cells in the course of HHV-7 infection. Various murine monoclonal antibodies to CD4 and the recombinant soluble form of human CD4 caused a dose-dependent inhibition of HHV-7 infection in primary CD4+ T lymphocytes. Moreover, radiolabeled HHV-7 specifically bound to cervical carcinoma cells (HeLa) expressing human CD4. A marked carcinoma cells (HeLa) expressing human CD4. A marked reciprocal interference was observed between HHV-7 and human immunodeficiency virus (HIV), the retrovirus that causes the acquired immunodeficiency syndrome and also uses CD4 as a receptor. Previous exposure of CD4+ T cells to HHV-7 dramatically interfered with infection by both primary and in vitro-passaged HIV-1 isolates. Reciprocally, persistent infection with HIV-1 or treatment with the soluble form of gp120, the CD4-binding envelope glycoprotein of HIV-1, rendered CD4+ T cells resistant to HHV-7 infection. These data indicate that CD4 is critically involved in the receptor mechanism for HHV-7. The antagonistic effect between HHV-7 and HIV could be exploited to devise therapeutic approaches to AIDS.     

Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication.

Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). We show that human rsCD4 does not efficiently inhibit SIVmac replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIVmac activity in vitro. Plasma of these animals efficiently blocks SIVmac replication in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIVmac-infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4+ but not CD8+ concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals.     

CD4+ lymphocyte function with early human immunodeficiency virus infection.

The pathogenesis of cellular immune deficiency following human immunodeficiency virus (HIV) infection could result from quantitative and/or qualitative dysfunction of the CD4+ lymphocyte population. To better characterize the T-cell response to soluble antigen with HIV infection, we have isolated peripheral blood lymphocytes and purified populations of CD4+ lymphocytes from healthy HIV antibody-positive subjects, patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC), and healthy HIV antibody-negative controls. T-lymphocyte function was determined by proliferative response to lectin (phytohemagglutinin), phorbol 12-myristate 13-acetate (PMA), calcium ionophore, purified recombinant HIV envelope gp120, tetanus toxoid antigen, and tetanus toxoid antigen in the presence of recombinant gp120 or purified recombinant soluble CD4. PBLs and CD4+ lymphocytes from asymptomatic HIV-infected subjects responded equally well to lectin, PMA, and/or calcium ionophore and to tetanus toxoid as cells from uninfected control subjects. The cells that proliferated in response to a soluble antigenic stimulus did not respond to gp120. Cells from subjects with ARC had a selective antigen recognition defect independent of the number of CD4+ lymphocytes. Recombinant gp120 inhibited CD4+ lymphocyte proliferation to antigenic stimulus by 30-40%. Recombinant soluble CD4, a proposed therapeutic for HIV, had no effect on T-cell response to antigen. A selective antigen recognition response was not compromised early in HIV infection but was compromised in subjects with ARC. Inhibition of proliferation to tetanus toxoid by gp120 suggests that HIV may affect major histocompatibility complex II restricted antigen recognition independent of CD4+ cell loss.     

Chimpanzees immunized with recombinant soluble CD4 develop anti-self CD4 antibody responses with anti-human immunodeficiency virus activity.

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.     

Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells.

Certain human immunodeficiency virus (HIV) strains were shown to infect human fibroblastoid cells. Replication was demonstrated only by coculturing normal peripheral blood mononuclear cells or human T-lymphotropic virus type 1-transformed T-cell lines with the infected human cells. This infection of human fibroblastoid cells did not involve the CD4 molecule and did not have the properties of endocytosis. Human sera could be distinguished by their ability to neutralize HIV infection of the fibroblastoid versus human T-cell lines. These observations demonstrate further that other mechanisms for viral entry, besides CD4 binding, must be considered for HIV. They also indicate the wide cellular host range and heterogeneity of HIV strains. The possibility that fibroblastoid cells serve as a reservoir for the AIDS virus and are involved in connective tissue disorders of infected individuals merits attention.     

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.     

Naturally occurring soluble CD4 in patients with human immunodeficiency virus infection.

To investigate its use as a marker of disease severity, serum soluble CD4 (sCD4) was measured by ELISA in patients with human immunodeficiency virus (HIV) infection. Levels of sCD4 were higher in patients than in controls (P less than .001) but did not increase with disease severity. sCD4 release per CD4 lymphocyte showed a linear increase with disease severity and performed as well as beta 2-microglobulin, a widely used marker. To study the role of sCD4 in the pathogenesis of HIV infection, an ELISA to detect sCD4 complexed with glycoprotein 120 (gp120) HIV envelope protein was developed. Preformed sCD4-gp120 complexes were not detectable in patient serum, but addition of recombinant gp120 showed that circulating sCD4 is capable of binding HIV envelope proteins. This study indicates that the sCD4-to-CD4 lymphocyte ratio increases linearly with disease severity and may be a useful marker of CD4 lymphocyte damage. In addition, serum sCD4 can bind viral particles, which may have implications for the use of recombinant sCD4 as a therapy in HIV infection.     

95- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor.

125I-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extent with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25-kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.     

Antibodies to CD4 in individuals infected with human immunodeficiency virus type 1.

The attachment of human immunodeficiency virus type 1 (HIV-1) to target cells is mediated by a specific interaction between the viral envelope glycoprotein (gp120) and the CD4 receptor. Here we report that approximately 10% of HIV-1-infected individuals produce antibodies that recognize the extracellular portion of the CD4 molecule. Carboxyl-terminal deletions of CD4 that do not affect HIV-1 gp120 binding eliminate recognition of CD4 by patient antisera. In contrast, mutations in the amino-terminal domain of CD4 that attenuate HIV-1 gp120 binding do not diminish CD4 recognition by patient antisera. These results suggest that HIV-1 infection can generate antibodies directed against a region of the viral receptor distinct from the virus-binding domain.     

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.     

Chemokine receptor polymorphisms and human immunodeficiency virus disease progression.

The role of polymorphisms in genes encoding chemokines and their receptors (CCR2B, SDF-1, and the promoter region of CCR5) in human immunodeficiency virus (HIV) disease progression was studied in 132 white HIV type 1 (HIV-1)-infected participants from a United Kingdom cohort study. Genotyping was done by use of amplification refractory mutation system-polymerase chain reaction with sequence-specific primers, and Cox proportional hazards models were used to examine the impact of polymorphisms on time to a CD4 cell count <200x106/L and to CDC stage IV disease. The results confirm a significant association of the CCR2B-64I mutant genotype with slower progression to a CD4 count <200 (hazards ratio [HR], 0.39; 95% confidence interval [CI], 0.17-0.91) but not with the SDF-1alpha 3' UTR homozygous mutation. The effects of the CCR5 and CCR2 mutations were genetically independent and similar in the magnitude of their protective effect on progression to a CD4 count <200 cells. A novel finding was an association of borderline significance between homozygosity for C at nucleotide position 59353 in the CCR5 promoter region and a slower rate of CD4 cell decline to <200x106/L (HR, 0. 58; 95% CI, 0.34-0.996).     

Envelope proteins from clinical isolates of human immunodeficiency virus type 1 that are refractory to neutralization by soluble CD4 possess high affinity for the CD4 receptor.

Recent evidence indicates that primary clinical isolates of human immunodeficiency virus type 1 (HIV-1) require significantly more soluble CD4 (sCD4) to block infection than the prototypic laboratory strain HTLV-IIIB. The currently accepted explanation for these observations is that the envelope glycoproteins from primary clinical isolates possess lower affinities for CD4 than laboratory strains. This observation has far reaching implications for the clinical effectiveness of sCD4. To test whether the resistance of clinical isolates to sCD4 neutralization correlates with low-affinity binding to gp120, we have compared gp120 glycoproteins derived from the clinical isolates HIV-1 JR-CSF and JR-FL with those derived from the prototypic strain HIV-1 BH10 in quantitative sCD4 binding studies. Surprisingly, our results demonstrate that gp120 derived from HIV-1 JR-CSF and JR-FL possess sCD4 binding affinities of equal or greater magnitude than gp120 derived from HIV-1 BH10. Thus primary clinical HIV-1 isolates can and do possess gp120 with high affinity for CD4, and sensitivity to neutralization by sCD4 is dependent upon factors other than the intrinsic affinity of gp120 for CD4.     

Expression of human CD4 in transgenic mice does not confer sensitivity to human immunodeficiency virus infection.

Transfection of the human CD4 molecule into mouse cells does not confer susceptibility to human immunodeficiency virus type 1 (HIV-1) infection. Expression of the human CD4 molecule in transgenic mice was seen to offer some new possibilities. However, transgenic mouse T cells expressing either the human CD4 receptor, or a hybrid human/mouse CD4 receptor alone or in conjunction with human major histocompatibility complex class I molecules, were refractory to in vitro HIV-1 infection. In addition, no infection was observed after in vivo HIV inoculation to mice of these various transgenic lines. Injection of recombinant gp160 viral protein to the transgenic mice did not alter their T and B cell populations. The existence of a dominant block in mouse cells that prevents HIV entry is discussed.     

CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals.

PCR was used to clone isolates of the human immunodeficiency virus type 1 (HIV-1) nef gene directly from peripheral blood leukocytes of HIV-1-infected individuals. A transient expression system with human CEM T cells was used to assess the effect of nef on CD4 antigen expression on the cell surface. We show that CD4 down-regulation is a frequent property of primary HIV-1 nef alleles. Mutations in conserved amino acid motifs of Nef disrupted CD4 down-regulation. Our observations strongly suggest that CD4 down-regulation reflects a conserved function of nef, which is selected in vivo in human HIV-1 infection. Methodology described here provides quantitative assays to establish whether alterations in nef correlate with the dynamics of disease progression in human AIDS.     

Soluble CD4 enhances the efficacy of immunotoxins directed against gp41 of the human immunodeficiency virus.

Monoclonal antibodies specific for the gp120 or gp41 portions of the human immunodeficiency virus (HIV) envelope protein gp160 were conjugated to ricin A chain, and their immunotoxic activities against HIV-infected cells were evaluated in the presence or absence of soluble CD4 (sCD4). Immunotoxin activity was measured in vitro as cytotoxicity and inhibition of secretion of infectious HIV. The efficacy of anti-gp41 immunotoxins was enhanced at least 30-fold in the presence of sCD4. This effect was specific for HIV-infected cells, but not for uninfected cells, and was seen at concentrations of sCD4 as low as 0.1 micrograms/ml. Anti-gp120 immunotoxins were marginally inhibited at higher concentrations of sCD4. Flow cytometry analyses showed that sCD4 increased the expression of gp41 on the surface of infected cells and increased internalization of gp120 and gp41. These data suggest that sCD4 alters the cellular trafficking of HIV envelope proteins. These findings also have important implications for the therapeutic use of anti-HIV immunotoxins and may be generalizable to other immunotoxins as well.     

Isotypic restriction of the antibody response to human immunodeficiency virus

HIV-infected individuals progress toward AIDS despite the early elicitation of a specific immune response. Analysis of the isotypic distribution of HIV-specific antibodies appears of special interest for two reasons: first, isotypic diversity is partly under the control of antigen-specific T-helper cells, the very cells infected by HIV; second, isotype determines antibody functions, effector (neutralization, antibody-dependent complement, or cell-mediated cytotoxicity) as well as blocking functions. We have investigated by Western blot analysis the isotypic profile of the antibody response to HIV structural proteins (env, gag, pol) and to the nonstructural protein F (3' orf), which is absent from the virion and might primarily target infected cells. In 115 asymptomatic individuals, infected by sexual contact (homosexual men) or intravenously (hemophiliacs), the response to gag-products was polyisotypic, including IgM, IgG1, IgG3 and IgA; the response to F was more restricted (IgM, IgG1, IgA) and the response to env strikingly restricted to the IgG1 isotype, suggesting different regulatory mechanisms in the B-cell response to these proteins. The isotypic distribution was also influenced by the route of infection, IgG4 and IgE (gag-specific) being exclusively elicited in the hemophiliac group. Finally, observations of potential diagnostic interest were made in a limited number of at-risk individuals; these included the presence of gag- and pol-specific IgM or IgA in the absence of any HIV-specific IgG isotypes; and the presence of gag- and F-specific antibodies in the absence of env-specific antibodies, suggesting the early occurrence of both isotypic and antigenic selection mechanisms during the course of HIV infection.     

Monoclonal antibody to chicken oviduct progesterone receptor.

Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Ag14 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the 125I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [3H]progesterone or [3H]ORG 2058 (a high-affinity synthetic progestin). Kd for the 8-9S progesterone receptor was approximately equal to 1 nM. Progesterone receptor-monoclonal antibody complexes were labeled with radioactive progesterone, suggesting that antibody does not prevent hormone binding. By using a [35S]methionine-labeled antibody, we were able to detect the progesterone receptor independently of its characteristic function of binding radioactive hormone. No crossreaction with human progesterone receptor was detected.     

Feline immunodeficiency virus targets activated CD4+ T cells by using CD134 as a binding receptor

The major surface glycoprotein of feline immunodeficiency virus (FIV) specifically binds to a 43-kDa glycoprotein expressed on the surface of a subset of T cells in peripheral blood mononuclear cells and IL-2-dependent T cell lines. Binding to this molecule, in conjunction with CXC chemokine receptor (CXCR) 4, is required for productive infection of these cells by primary isolates of FIV. Here, we demonstrate that the 43-kDa molecule is CD134, a receptor for FIV recently identified independently [Shimojima, M., et al. (2004) Science 303, 1192-1195]. Furthermore, we show that CD134 is specifically up-regulated on CD4+ T cells that have been activated by treatment with IL-2 and Con A. CD8+ T cells remained negative for CD134 expression regardless of the activation state. Binding of the FIV major surface glycoprotein on activated CD4+ T cells was observed through direct interaction with CD134 whereas, on activated CD8+ T cells, the binding was CD134-independent and mediated by CXCR4 and, to a lesser extent, heparan sulfate proteoglycans. However, this CD134-independent interaction was not sufficient to render CD8+ T cells permissive to FIV infection, as FIV replicated primarily in activated CD4+ T cells and not in cells negative for CD134 expression. Altogether, our results substantiate that CD134 acts as a primary binding receptor for FIV and explain the specific targeting and depletion of the CD4+ T cell population observed during the course of infection independent of the use of CD4 as a binding receptor/coreceptor.