Structural analysis of the human immunodeficiency virus-binding domain of CD4. Epitope mapping with site-directed mutants and anti-idiotypes.

The CD4 molecule, a differentiation marker expressed primarily by T lymphocytes, plays an important role in lymphocyte activation. CD4 is also the receptor for HIV. A number of recent studies have localized the high affinity binding site of the HIV envelope glycoprotein, gp120, to the NH2-terminal (V1) domain of CD4, a region with sequence and predicted structural homology with Ig kappa chain V domains (V kappa). In this report, we show that V1 bears structural similarities with V kappa regions through detailed epitope mapping of 26 CD4 mAbs. The binding sites of these mAbs were initially defined relative to one another by crossblocking analysis and were then localized to specific domains of CD4 in blocking studies with truncated, soluble CD4 proteins. The epitopes within the V1 domain were mapped in detail with a panel of 17 substitution mutants, and the specificities of several mAbs that appear to recognize very similar epitopes were examined in crossblocking studies with anti-idiotype antibodies. The location of the epitopes is consistent with a V kappa-like structure of V1. Most of the epitopes lie within regions of predicted exposed loops. A number of these epitopes span discontinuous residues in the linear sequence that lies in close proximity in an Ig fold. Alignment of CD4 V1 with the Ig V kappa chains places these epitopes within stretches corresponding to the complimentarity-determining regions. This epitope analysis is relevant for a vaccine strategy for HIV based on anti-idiotype antibodies to CD4 mAbs and for studies with CD4 antibodies on the role of CD4 in T lymphocyte activation.

Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.

Infection with HIV-1 requires expression of CD4 and the chemokine receptors CXCR4 or CCR5 at the target cell surface. Engagement of these receptors by the HIV-1 envelope glycoprotein is essential for membrane fusion, but may additionally activate intracellular signaling pathways. In this study, we demonstrate that chemokines and HIV-1 envelope glycoproteins from both T-tropic and macrophage-tropic strains rapidly induce tyrosine phosphorylation of the protein tyrosine kinase Pyk2. The response requires CXCR4 and CCR5 to be accessible on the cell surface. The results presented here provide the first evidence for activation of an intracellular signaling event that can initiate multiple signaling pathways as a consequence of contact between HIV-1 and chemokine receptors.

Early treatment of SIV+ macaques with an α4ß7 mAb alters virus distribution and preserves CD4+ T cells in later stages of infection.

Integrin α4ß7 mediates the trafficking of leukocytes, including CD4+ T cells, to lymphoid tissues in the gut. Virus mediated damage to the gut is implicated in HIV and SIV mediated chronic immune activation and leads to irreversible damage to the immune system. We employed an immuno-PET/CT imaging technique to evaluate the impact of an anti-integrin α4ß7 mAb alone or in combination with ART, on the distribution of both SIV infected cells and CD4+ cells in rhesus macaques infected with SIV. We determined that α4ß7 mAb reduced viral antigen in an array of tissues of the lung, spleen, axillary, and inguinal lymph nodes. These sites are not directly linked to α4ß7 mediated homing; however, the most pronounced reduction in viral load was observed in the colon. Despite this reduction, α4ß7 mAb treatment did not prevent an apparent depletion of CD4+ T cells in gut in the acute phase of infection that is characteristic of HIV/SIV infection. However, α4ß7 mAb appeared to facilitate the preservation or restoration of CD4+ T cells in gut tissues at later stages of infection. Since damage to the gut is believed to play a central role in HIV pathogenesis, these results support further evaluation of α4ß7 antagonists in the study and treatment of HIV disease.

Heterozygosity for a defective gene for CC chemokine receptor 5 is not the sole determinant for the immunologic and virologic phenotype of HIV-infected long-term nonprogressors.

HIV-1-infected long-term nonprogressors are a heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. CC chemokine receptor 5 (CCR5) has recently been identified as an important coreceptor for HIV-1 entry into CD4+ T cells. A mutant allele of CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals and partial protection against HIV disease progression in heterozygotes. The frequency of CCR5 heterozygotes is increased among HIV-1- infected long-term nonprogressors compared with progressors; however, the host defense mechanisms responsible for nonprogression in CCR5 heterozygotes are unknown. We hypothesized that nonprogressors who were heterozygous for the mutant CCR5 gene might define a subgroup of nonprogressors with higher CD4+ T cell counts and lower viral load compared with CCR5 wild-type nonprogressors. However, in a cohort of 33 HIV-1-infected long-term nonprogressors, those who were heterozygous for the mutant CCR5 gene were indistinguishable from CCR5 wild-type nonprogressors with regard to all measured immunologic and virologic parameters. Although epidemiologic data support a role for the mutant CCR5 allele in the determination of the state of long-term nonprogression in some HIV-1- infected individuals, it is not the only determinant. Furthermore, long-term nonprogressors with the wild-type CCR5 genotype are indistinguishable from heterozygotes from an immunologic and virologic standpoint.

Identification of preferential CD4+ T-cell targets for HIV infection in the cervix.

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)ß(7), or α(4)ß(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)ß(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)ß(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)ß(7) still demonstrated enhanced entry into α(4)ß(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)ß(7) or α(4)ß(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)ß(7) or α(4)ß(1) with HIV envelope.

Bitter-sweet symphony: defining the role of dendritic cell gp120 receptors in HIV infection.

BACKGROUND: Dendritic cells (DC) are believed to be one of the first cell types infected during HIV transmission. Recently a single C-type lectin receptor (CLR), DC-SIGN, has been reported to be the predominant receptor on monocyte derived DC (MDDC) rather than CD4. The role of other CLRs in HIV binding and HIV binding by CLRs on other types of DC in vivo is largely unknown. OBJECTIVES AND STUDY DESIGN: Review HIV binding to DC populations, both in vitro and in vivo, in light of the immense interest of a recently re-identified CLR called DC-SIGN. RESULTS AND CONCLUSIONS: From recent work, it is clear that immature MDDC have a complex pattern of HIV gp120 binding. In contrast to other cell types gp120 has the potential to bind to several receptors on DC including CD4 and several types of C type lectin receptor, not just exclusively DC-SIGN. Given the diverse types of DC in vivo future work will need to focus on defining the receptors for HIV binding to these different cell types. Mucosal transmission of HIV in vivo targets immature sessile DCs, including Langerhans cells which lack DC-SIGN. The role of CLRs and DC-SIGN in such transmission remains to be defined.

The genetic analysis of the HIV envelope binding domain on CD4.

Through mutagenesis, we identified a single high-affinity binding site for gp120 on the human CD4 protein. This site is localized in the V1 domain within residues 41 to 55. The collection of mutants was also used to define the epitopes for 55 anti-CD4 monoclonal antibodies. The locations of these epitopes are consistent with a V kappa-like structure for the V1 domain. In the context of this structure, the gp120 binding site encompasses the small CDR2 loop. Through deletion mutagenesis at the termini of the V1 domain, we further defined the minimal region required to retain high-affinity binding to gp120. Short deletions at both termini disrupt binding to gp120 and recognition by conformation-sensitive anti-CD4 monoclonal antibodies. We conclude that amino acids at both the amino and carboxy termini are critical to the conformation of the V1 domain and, in particular, to the integrity of the gp120 binding site.

Antigen engineering in DNA immunization.

J. A. Wolff and colleagues demonstrated in 1990 (1) that naked, non-infectious DNA plasmids can be transferred into muscle cells of living mice to express the encoded protein in vivo. Further studies have shown that proteins expressed in vivo via such a "DNA inoculation" process can serve as immunogens to effectively stimulate the body's immune system to produce potentially strong, long-lasting, antigen-specific, humoral and cell-mediated immune responses (2-8).

Defective plasmacytoid dendritic cell-NK cell cross-talk in HIV infection.

HIV viremia is associated with a wide range of immune dysfunctions that contribute to the immunocompromised state. HIV viremia has been shown to have a broad effect on several immune cell types and/or their interactions that are vital for mounting an effective immune response. In this study, we investigated the integrity of plasmacytoid dendritic cell (pDC)-NK cell interactions among HIV viremic, aviremic, and seronegative individuals. We describe a critical defect in the ability of pDCs from HIV-infected individuals to secrete IFN-alpha and TNF and subsequently activate NK cells. We also describe an inherent defect on NK cells from HIV-infected individuals to respond to pDC-secreted cytokines. Furthermore, we were able to demonstrate a direct effect of HIV trimeric gp120 on NK cells in vitro similar to that described ex vivo. Finally, we were able to establish that the HIV gp120-mediated suppressive effect on NK cells was a result of its binding to the integrin alpha(4)beta(7) expressed on NK cells. These findings suggest a novel mechanism by which HIV is capable of suppressing an innate immune function in infected individuals.

Chemokines, cytokines and HIV: a complex network of interactions that influence HIV pathogenesis.

The important role of chemokine receptors in HIV pathogenesis is becoming increasingly apparent. The level at which certain chemokine receptors that serve as HIV co-receptors are available influences the susceptibility of a CD4+ cell to viral infection and to certain HIV envelope-induced alterations in cellular function. Numerous pathogens, including HIV, can stimulate the production of chemokines and cytokines from a variety of cell types. Both cytokines and chemokines modulate CCR5 and CXCR4 availability, resulting in differential replication potentials for RS and X4 HIV strains depending on the milieu in the microenvironment. In addition, differential expression of CCR5 and CXCR4 on activated memory T cells appears to play an important role in preferential replication of RS HIV strains in vivo. However, expression of HIV co-receptors and CD4 may not be sufficient for effective HIV entry and replication. Intracellular signaling events, triggered by interaction between chemokine receptors and chemokines or HIV envelope, are important for efficient entry and completion of early replication events. Envelope proteins of different HIV isolates vary in their ability to transduce these signals, a characteristic that may play a role in determining the ability of a virus to productively infect certain cell types. Finally, the interaction between chemokine receptors and chemokines or HIV envelope has significant effects on cellular functions which likely play a role in HIV pathogenesis.

Immunization with soluble murine CD4 induces an anti-self antibody response without causing impairment of immune function.

The T cell surface molecule CD4 (L3T4 in mouse) is important in the T lymphocyte response to Ag presented in association with MHC class II molecules. To examine the role of CD4 in immune function, we expressed a soluble form of murine CD4 by deleting the transmembrane and cytoplasmic regions of the L3T4 gene and transfecting the altered gene into Chinese hamster ovary cells. The recombinant soluble mouse CD4 (smCD4) retained the native conformation of the external portion, as indicated by the binding of L3T4 mAb. In vitro, smCD4 did not inhibit class II-dependent, Ag-specific, T cell proliferation or MLR, even at concentrations 300-fold greater, on a molar basis, than that of anti-CD4 mAb. Immunization of mice with smCD4 induced a strong anti-CD4 response. These antibodies showed some binding to native cell surface CD4, indicating that immunization with smCD4 generated an anti-self response. Analysis of lymphoid cells from spleen, lymph node, and thymus of smCD4-treated mice revealed no alteration in subset phenotypes. Also, Th cell function, as measured by response to soluble Ag, was not compromised. Thus, smCD4 did not inhibit T cell activity in vitro, and the autoimmune response arising from immunization with smCD4 had no apparent consequences for normal immune function.

Promonocytic U937 subclones expressing CD4 and CXCR4 are resistant to infection with and cell-to-cell fusion by T-cell-tropic human immunodeficiency virus type 1.

Different strains of human immunodeficiency virus type 1 (HIV-1) vary markedly in the ability to infect cells of the monocyte/macrophage (M/M) lineage. M/M are generally resistant to infection with T-cell-tropic (T-tropic) strains of HIV-1. Recently, the chemokine receptors CCR5 and CXCR4 were identified as cofactors for fusion/entry of macrophage- and T-tropic strains of HIV-1, respectively. To investigate the mechanisms of resistance of M/M to T-tropic HIV-1 infection, we examined a number of subclones of the U937 promonocytic cell line. We found that certain subclones of U937 (plus clones) could, while others (minus clones) could not, support replication of T-tropic strains of HIV-1. We demonstrate that (i) both minus and plus clones support HIV-1 replication when transfected with an infectious molecular cDNA clone of a T-tropic HIV-1; (ii) minus clones do not, but plus clones do, efficiently support fusion with cells expressing HIV-1 IIIB Env; (iii) both plus and minus clones (with the exception of one clone) express physiologically functional CXCR4 protein as well as CD4 on the cell surface; (iv) introduction of CXCR4 into the CXCR4-negative clone does not restore fusogenicity with or susceptibility to T-tropic HIV-1; and (v) a ligand (stromal cell-derived factor 1) for or a monoclonal antibody (12G5) to CXCR4 does not effectively inhibit HIV-mediated cell-to-cell fusion of U937 cells. These data indicate that resistance to T-tropic HIV-1 infection of U937 minus clones occurs at fusion/ entry events and that expression of functional CXCR4 and CD4 is not a sole determinant for susceptibility to T-tropic HIV-1 infection; furthermore, they suggest that other factors are positively or negatively involved in HIV-mediated cell-to-cell fusion in U937 promonocytic cells.

HIV-1 envelope induces activation of caspase-3 and cleavage of focal adhesion kinase in primary human CD4(+) T cells.

Binding of HIV type 1 (HIV-1) envelope glycoproteins to the surface of a CD4(+) T cell transduces intracellular signals through the primary envelope receptor, CD4, and a coreceptor, either CCR5 or CXCR4. Furthermore, envelope-CD4(+) cell interactions increase rates of apoptosis in peripheral blood mononuclear cells (PBMCs). We demonstrate that in primary T lymphocytes, recombinant HIV-1 envelope proteins induce the activation of caspase-3 and caspase-6, which belong to a family of cysteine proteases that, upon activation, promote programmed cell death. Envelope-mediated activation of caspase-3 and caspase-6 depended on envelope-CD4 receptor interactions; CCR5-utilizing as well as CXCR4-utilizing envelopes elicited this response. Focal adhesion kinase (FAK) is a substrate of both caspase-3 and caspase-6, and inactivation of FAK by these caspases promotes apoptosis. En-velope treatment of lymphocytes led to the cleavage of FAK in a manner consistent with caspase-mediated cleavage.

Antigen-specific mediated suppression of beta cell autoimmunity by plasmid DNA vaccination.

In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4(null) mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit beta cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.

Simian immunodeficiency virus-specific cytotoxic T-lymphocyte induction through DNA vaccination of rhesus monkeys.

In view of the growing evidence that virus-specific cytotoxic T lymphocytes (CTL) play an important role in containing the early spread of human immunodeficiency virus type 1 (HIV-1) in infected individuals, novel vaccine strategies capable of eliciting HIV-1-specific CTL are being pursued in attempts to create an effective AIDS vaccine. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the induction of AIDS virus-specific CTL responses by DNA vaccination. We found that the inoculation of rhesus monkeys with plasmid DNA encoding SIVmac Env and Gag elicited a persisting SIVmac-specific memory CTL response. These CTL were CD8+ and major histocompatibility complex class I restricted. These studies provide evidence for the potential utility of DNA inoculation as an approach to an HIV-1 vaccine.

A soluble form of CD4 (T4) protein inhibits AIDS virus infection.

CD4 (T4) is a glycoprotein of relative molecular mass 55,000 (Mr 55K) on the surface of T lymphocytes which is thought to interact with class II MHC (major histocompatibility complex) molecules, mediating efficient association of helper T cells with antigen-bearing targets. The CD4 protein is also the receptor for HIV, a T-lymphotropic RNA virus responsible for the human acquired immune deficiency syndrome (AIDS) (refs 4-7). To define the mechanisms of interaction of CD4 with the surface of antigen-presenting cells and with HIV, we have isolated the CD4 gene and expressed this gene in several different cellular environments. Here we describe an efficient expression system in which a recombinant, soluble form of CD4 (sCD4) is secreted into tissue culture supernatants. This sCD4 retains the structural and biological properties of CD4 on the cell surface, binds to the envelope glycoprotein (gp110) of HIV and inhibits the binding of virus to CD4+ lymphocytes, resulting in a striking inhibition of virus infectivity.

Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) enter target cells by forming a complex between the viral envelope protein and two cell-surface membrane receptors: CD4 and a 7-span transmembrane chemokine receptor. Isolates of HIV that differ in cellular tropism use different subsets of chemokine receptors as entry cofactors: macrophage-tropic HIVs primarily use CCR5, whereas T-cell-tropic and dual-tropic isolates use CXCR4 receptors. HIV-mediated signal transduction through CCR5 is not required for efficient fusion and entry of HIV in vitro. Here we show that recombinant envelope proteins from macrophage-tropic HIV and SIV induce a signal through CCR5 on CD4+ T cells and that envelope-mediated signal transduction through CCR5 induces chemotaxis of T cells. This chemotactic response may contribute to the pathogenesis of HIV in vivo by chemo-attracting activated CD4+ cells to sites of viral replication. HIV-mediated signalling through CCR5 may also enhance viral replication in vivo by increasing the activation state of target cells. Alternatively, envelope-mediated CCR5 signal transduction may influence viral-associated cytopathicity or apoptosis.

Induction of phosphorylation and intracellular association of CC chemokine receptor 5 and focal adhesion kinase in primary human CD4+ T cells by macrophage-tropic HIV envelope.

Binding of HIV-1 envelope glycoproteins to the surface of a CD4+ cell transduces intracellular signals through the primary envelope receptor, CD4, and/or the envelope coreceptor, a seven-transmembrane chemokine receptor. Macrophage-tropic strains of HIV-1 preferentially use CCR5 as an entry coreceptor, whereas T cell-tropic strains use CXC chemokine receptor-4 for entry. Intracellular signals transduced by HIV-1 envelope may have immunopathogenic consequences, including anergy, syncytium formation, apoptosis, and inappropriate cell trafficking. We demonstrate here that a recombinant envelope protein derived from an M-tropic isolate of HIV-1 can transduce CD4-dependent as well as CCR5-dependent intracellular signals in primary human CD4+ T cells. Novel HIV-induced intracellular signals that were identified include tyrosine phosphorylation of focal adhesion kinase (FAK) and CCR5, which are involved in cell adhesion and chemotaxis, respectively. HIV envelope-induced cellular association of FAK and CCR5 was also demonstrated, suggesting that ligation of CD4 and CCR5 leads to the formation of an activation complex composed of FAK and CCR5. Activation of this signaling pathway by HIV-1 envelope may be an important pathogenic mechanism of dysregulated cellular activation and trafficking during HIV infection.

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV.

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.