Bispecific antibody targeting of human immunodeficiency virus type 1 (HIV-1) glycoprotein 41 to human macrophages through the Fc IgG receptor I mediates neutralizing effects in HIV-1 infection.

Human monocyte-derived macrophages that express the CD4 molecule and the Fc receptor for IgG (Fc gamma R) play a major role in the pathogenesis of human immunodeficiency virus (HIV) infection. To explore this possibility further, human monoclonal antibody to glycoprotein 41 (gp41) was produced, and a heterobifunctional antibody composed of F(ab') x F(ab')2 fragments of monoclonal anti-gp41 and anti-Fc gamma RI 22.2 were constructed. Both antibodies were analyzed for neutralizing effects, and the role of the CD4 molecule in HIV infection was studied with human monocyte-derived macrophages. The bispecific antibody exhibited strong neutralizing properties, in contrast to the monoclonal anti-gp41 antibody. Moreover, in the presence of monoclonal anti-Leu-3a antibody, viral production was completely inhibited. These findings demonstrate the necessity of the CD4 molecule in HIV infection of human macrophages and emphasize the usefulness of such heterobifunctional antibody directed to virus and monocyte-derived macrophage Fc receptors in prevention of HIV infection.     

Dexamethasone inhibits CD4 T cell deletion mediated by macrophages from human immunodeficiency virus-infected persons.

Prednisolone slows the loss of CD4 T cells in individuals with human immunodeficiency virus (HIV) disease and inhibits antigen-induced apoptosis of recently HIV-infected CD4 cells in vitro. This study investigated whether dexamethasone inhibits the ability of macrophages to delete CD4 T cells via anti-CD4 antibody or immune-complexed HIV envelope protein gp120. Peripheral blood mononuclear cells from HIV-negative persons were incubated with CD4-reactive ch412 monoclonal antibody or with gp120/IgG immune complexes and resident macrophages, with and without dexamethasone. Dexamethasone inhibited CD4 cell deletion in a dose-dependent manner. The deletion of normal CD4 cells by macrophages from HIV-infected patients also was inhibited by dexamethasone. Furthermore, up-regulation of CD95 expression on T cells exposed to anti-CD4 and gp120/IgG, which predisposes T cells to CD95-mediated apoptosis, is inhibited by dexamethasone in a dose-dependent fashion. Dexamethasone inhibits the macrophage-mediated deletion of CD4 lymphocytes in HIV-infected persons.     

Pathogen-specific induction of CD154 is impaired in CD4+ T cells from human immunodeficiency virus-infected patients

The pathogenesis of immunodeficiency associated with human immunodeficiency virus (HIV) infection remains incompletely understood. CD154, a molecule that is expressed primarily on activated CD4(+) T cells, is pivotal for regulation of cell-mediated and humoral immunity and is crucial for control of many opportunistic infections. We investigated whether CD4(+) T cells from HIV-infected patients exhibit defective induction of CD154 in response to opportunistic pathogens. Incubation of purified human CD4(+) T cells with monocytes plus antigenic preparations of either Candida albicans, cytomegalovirus, or Toxoplasma gondii resulted in induction of CD154. Expression of CD154 in response to these pathogens was impaired in CD4(+) T cells from HIV-infected patients. This defect correlated with decreased production of interleukin (IL)-12 and interferon (IFN)-gamma in response to T. gondii. Recombinant CD154 partially restored secretion of IL-12 and IFN-gamma in response to T. gondii in cells from HIV-infected patients. Together, defective induction of CD154 is likely to contribute to impaired cell-mediated immunity against opportunistic pathogens in HIV-infected patients.     

Anti-Fas monoclonal antibody is cytocidal to human immunodeficiency virus-infected cells without augmenting viral replication.

A cytotoxic monoclonal antibody (anti-Fas mAb) against the 200-kDa cell surface Fas antigen, which is associated with the tumor necrosis factor (TNF) receptor, was examined for its in vitro activity on human immunodeficiency virus (HIV)-infected cells. It was found that both TNF and anti-Fas mAb selectively killed the chronically HIV-infected cells. Uninfected cells were less sensitive to the antibody than those infected with HIV. When the cells were cultured in the presence of anti-Fas mAb immediately after the HIV infection, the spread of HIV-infected cells was suppressed by the antibody. TNF augmented both the synthesis of HIV-specific mRNA in HIV-infected cells and formation of multinucleated giant cells. In contrast, the anti-Fas mAb did not augment HIV replication or enhance the HIV-induced formation of syncytia. The results indicated that anti-Fas mAb mimicks the cytocidal action of TNF but does not augment HIV replication.     

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.     

Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies.

The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270-kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria.     

Full-length recombinant CD4 and recombinant gp120 inhibit fusion between HIV infected macrophages and uninfected CD4-expressing T-lymphoblastoid cells

Human immunodeficiency virus-(HIV) infected monocyte-macrophages may contribute to the pathogenesis of HIV-associated immune deficiency and dysfunction by acting as a target and potential reservoir for the virus in vivo, and by functioning abnormally following infection. We have shown that HIV-infected macrophages fuse with uninfected CD4-expressing lymphoid cells in vitro; this may provide an additional mechanism for CD4 lymphocyte depletion in vivo. We report here the inhibition of syncytium formation between HIV-infected macrophages and uninfected CD4-expressing T-lymphoid cells by monoclonal antibody S3.5, directed against an epitope of CD4 involved in binding HIV gp120, by a recombinant protein that comprises the full-length extracellular domain of the CD4 molecule, and by recombinant full-length HIV envelope glycoprotein, gp120. These results indicate that both molecules (gp120 and CD4) are critical to the fusion process, and suggest that gp120 is expressed on the surface of HIV-infected monocyte-macrophages.     

Idiotype cascades associated with the CD4-HIV glycoprotein 120 interaction: immunization with anti-idiotypic antibodies induces anti-anti-idiotypic responses with anti-CD4 specificity and in vitro neutralizing activity

Anti-idiotypic antibodies (Ab-2), which were generated in baboons against a mouse monoclonal antibody specific for the CD4 molecule expressed on human T cells, were used to produce anti-anti-idiotypic antibodies (Ab-3) in mice. This response induced by Ab-2 immunization of BALB/c mice was classified as anti-anti-idiotype (Ab-3) on the basis of the ability of the mouse Ab-3 to (1) specifically bind the baboon Ab-2 preparation, but not irrelevant baboon IgG preparations, (2) inhibit the binding of the anti-CD4 Ab-1 preparation to the baboon Ab-2, and (3) recognize a second baboon Ab-2, along with a rabbit Ab-2 specific for the monoclonal anti-CD4 Ab-1 preparation. The murine Ab-3 response also recognizes the CD4 molecule expressed on a human CD4+ T cell line, as determined by flow cytometry; recognizes the same epitopes on the CD4 molecule as the Ab-1; inhibits HIV-1 syncytium formation; and neutralizes HIV-1 primary isolates in vitro. These studies suggest that Ab-3 responses can be induced by anti-Id immunization, which serologically mimicks the antigen and Id specificities of the monoclonal anti-CD4 preparation used to generate the anti-Id. Thus, the Ab-3 response exhibits the characteristics of a population that represents the internal image of the Ab-1.     

Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu-peripheral blood leukocyte (PBL)-severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL-severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.     

Chemokine pattern in relation to disease state in human immunodeficiency virus-infected children.

Although beta chemokines can block human immunodeficiency virus (HIV) entry into target cells, their role in HIV disease progression is controversial. To determine the association of RANTES with HIV disease state, we examined constitutive mRNA expression by reverse-transcribed polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMCs) and induction of RANTES secretion by enzyme-linked immunosorbent assay (ELISA) in anti-CD3 monoclonal antibody (MAb)-stimulated cultures of PBMCs, and in CD4+ and CD8+ T cell subsets of 17 HIV-infected children. In comparison with uninfected subjects, PBMCs of HIV-infected children were deficient in both constitutive RANTES mRNA expression as well as in stimulus-induced RANTES production. Children in clinical category C were found to be more deficient than children in clinical category A. Expression of RANTES mRNA in PMBCs was inversely correlated with plasma virus load and correlated directly with CD4+ T cell counts. In T cell subsets, RANTES production was equivalent between CD4+ and CD8+ T cells in patients and controls but CD8+ T cells of children in clinical category A produced higher RANTES levels than those of children in clinical category C. The beta-chemokine RANTES may play an important role in slowing clinical disease progression in HIV-infected children.     

Recombinant glycoprotein 120 of human immunodeficiency virus is a potent interferon inducer.

Cells infected with human immunodeficiency virus (HIV) induce antiviral activity in peripheral blood mononuclear cells (PBMC) from healthy donors. This activity is neutralized by anti-interferon-alpha antibody and partially destroyed at pH 2. Previous studies with enriched cell populations and monoclonal antibodies suggest that B lymphocytes are the main IFN-producing cells, and that both CD4 and HLA class II antigens are essential for IFN induction. Since the initial event of HIV infection of CD4+ cells is the interaction of the virus coat glycoprotein gp120 with CD4 molecule, we investigated whether gp120 is responsible for IFN induction. Using PBMC and recombinant gp120 obtained from a baculovirus expression system, dose-dependent induction of antiviral activity was observed with titers approaching 10(3) IU/ml. This induction was blocked in the presence of antibody to gp120. The antiviral activity was characterized as IFN-alpha by neutralization with IFN alpha-specific antibody. Preincubation of PBMC with anti-CD4 or the presence of soluble CD4 during incubation inhibited IFN induction, indicating that interaction of gp120 with cell-associated CD4 is responsible for this induction. Neither lymphoproliferation nor interleukin-2 (IL-2) production was observed during IFN induction. However, class G immunoglobulin secretion was enhanced by gp120, indicating that B cells are direct or indirect targets of gp120 stimulation in this experimental system. Since gp120 is shed from HIV-infected cells and occurs in the serum of acquired immunodeficiency syndrome (AIDS) patients, our data suggest that this glycoprotein is responsible for the induction of endogenous IFN and the polyclonal activation of B cells both of which are observed in AIDS patients.     

Polyspecific self-reactive antibodies in individuals infected with human immunodeficiency virus facilitate T cell deletion and inhibit costimulatory accessory cell function.

Self-reactive polyspecific IgG antibodies (PSAs) arise in human immunodeficiency virus (HIV)-seropositive subjects before they develop AIDS. Self-reactive PSA levels correlate with the destruction of CD8 T cells in HIV-infected individuals and mediate the antibody-dependent cellular toxicity-based destruction of human T cells in tissue culture. PSAs react across the species barrier and bind to T cell antigens in mice. Such reactivity with mouse lymphocytes was not detected in normal human serum. Injection of human PSA IgG causes massive T cell depletion in the spleen, lymph nodes, and thymus in mice: evidence that PSA IgG facilitates T cell destruction in vivo. In addition to facilitating macrophage cytotoxicity, self-reactive PSA IgG inhibits the macrophage-mediated activation of T cells with antigen receptor-specific monoclonal antibody or with antigen. Exogenous costimulatory stimuli or interleukin (IL)-12 can reverse the inhibition. In contrast, exogenous IL-10 mimics this inhibition. These data implicate PSA IgG as a pathogenic factor in the development of HIV disease.     

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.     

Transforming growth factor beta and noncytopathic mechanisms of immunodeficiency in human immunodeficiency virus infection.

This study examines the contribution of transforming growth factor beta (TGF beta), one of the most potent endogenous immunosuppressive factors, to the development of immunodeficiency in human immunodeficiency virus (HIV) infection. Increased titers of TGF beta were found in supernatants of peripheral blood mononuclear cells (PBMCs) from HIV-infected donors as compared to uninfected controls (P less than 0.001). This correlated closely with defective responses of CD4+ lymphocytes to the recall antigens tuberculin purified protein derivative or tetanus toxoid. The addition of TGF beta-neutralizing antibody to PBMCs partially restored these defective T-cell responses. Furthermore, purified TGF beta or HIV+ PBMC culture supernatants preferentially inhibited proliferation of CD4+ lymphocytes as compared to CD8+ cells. The increased expression of the TGF beta protein was associated with increased TGF beta mRNA as determined by a polymerase chain reaction assay. This increase in TGF beta protein and mRNA was due to a selective upregulation of the TGF beta 1 isoform. These results indicate that overexpression of TGF beta 1 occurs in HIV-infected individuals and that this cytokine can contribute to impaired immune functions and to depletion of CD4+ T lymphocytes.     

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.     

Compromised B cell responses to influenza vaccination in HIV-infected individuals

BACKGROUND: Yearly influenza vaccination, although recommended for human immunodeficiency virus (HIV)-infected individuals, has not received thorough evaluation in the era of antiretroviral therapy. We assessed the impact of HIV disease on B cell responses to influenza vaccination. METHODS: Sixty-four HIV-infected and 17 HIV-negative individuals received the 2003-2004 trivalent inactivated influenza vaccine. Frequencies of influenza-specific antibody-secreting cells (ASCs) were measured by enzyme-linked immunospot (ELISPOT) assay, and antibody responses were measured by hemagglutination-inhibition (HI) assay. Memory responses to influenza were measured by ELISPOT assay after polyclonal activation of B cells in vitro. RESULTS: Prevaccination HI titers were significantly higher in HIV-negative than in HIV-infected individuals. Peak HI titers and influenza-specific ASC frequencies were directly correlated with CD4+ T cell counts in HIV-infected individuals. Influenza-specific memory B cell responses were significantly lower in HIV-infected than in HIV-negative individuals and were directly correlated with CD4+ T cell counts. CONCLUSIONS: HIV infection is associated with a weak antibody response to influenza vaccination that is compounded by a poor memory B cell response. CD4+ T cell count is a critical determinant of responsiveness to influenza vaccination, and the contribution of plasma HIV RNA level is suggestive and warrants further investigation.     

CD4+CD7-CD57+ T cells: a new T-lymphocyte subset expanded during human immunodeficiency virus infection.

CD7 and CD57 are two cell surface molecules related to the differentiation or functional stages of CD4+ T cells. The CD4+CD7- T cells represent a minor subset of CD4+ cells in normal individuals and are considered to contain the normal counterpart of Sézary T cells; the CD4+CD57+ peripheral blood lymphocytes (PBL) are detectable in long-term renal allograft recipients. We compared the cell surface expression of these CD7 and CD57 markers on CD4+ T lymphocytes in peripheral blood and lymphoid organs from normal individuals and human immunodeficiency virus (HIV)-infected patients. Our results indicate that CD4+CD7- T cells in normal PBL do not express CD57 and were poorly responsive to anti-CD3 monoclonal antibody (MoAb), the activation being restored by addition of anti-CD28 MoAb. This CD4+CD7- cell subset is increased in peripheral blood during HIV infection, and its progressive expansion mirrors both the absolute and relative decrease of CD4+ T cells. The lack of CD7 expression is correlated with CD57 acquisition on CD4+ T cells because CD4+CD7-CD57+ cells represent a major component of the CD4+CD7- subset in HIV-infected patients. Our results suggest that the presence and the expansion of CD4+CD7-CD57+ T lymphocytes, which do not behave as previously defined helper subsets, may participate to the immune dysfunction observed during HIV infection.     

Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity.

The differentiation antigen T4, present on the helper/inducer subset of T lymphocytes, is thought to serve as the receptor for the human immunodeficiency virus (HIV). We find that a 60-kDa protein, immunoprecipitable by monoclonal antibody (mAb) OKT4, is present on membranes from human brain as well as human T cells. Furthermore, the radioiodinated HIV envelope glycoprotein [125I-labeled gp120 (125I-gp120)] can be specifically covalently affixed to a molecule present on rat, monkey, and human brain membranes to yield a complex that is indistinguishable from that formed on human T cells. T4 antigen has been studied on unfixed squirrel monkey, rat, and human brain sections by autoradiography using the mAb OKT4. A highly conserved neuroanatomical pattern has been demonstrated, suggesting an analogous organization in these three mammalian brains. Furthermore, the localization of 125I-gp120 receptor binding appears similar to that of T4 and is highly reminiscent of patterns for many previously characterized neuropeptide receptors. A computer-assisted analysis of gp120 suggested that a previously unremarkable octapeptide sequence within the gp120 protein, which we have synthesized and termed "peptide T," may play an important role in HIV attachment. Thus, peptide T and three rationally designed peptide analogs, each with a systematic amino acid substitution, potently inhibit specific 125I-gp120 binding to brain membranes. Additionally, when tested in a viral infectivity assay, these peptides show the same rank order and similar absolute potency to block HIV infection of human T cells. Thus, peptide T may provide a useful pharmacological or immunological basis for the control and treatment of AIDS.     

Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain.

Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.