Susceptibility of HIV type 2 primary isolates to CCR5 and CXCR4 monoclonal antibodies, ligands, and small molecule inhibitors

Human immunodeficiency virus (HIV) entry into susceptible cells involves the interaction between viral envelope glycoproteins with CD4 and a chemokine receptor (coreceptor), namely CCR5 and CXCR4. This interaction has been studied to enable the discovery of a new class of antiretroviral drugs that targets the envelope glycoprotein-coreceptor interaction. However, very few data exist regarding HIV-2 susceptibility to these coreceptor inhibitors. With this work we aimed to identify this susceptibility in order to assess the potential use of these molecules to treat HIV-2-infected patients and to further understand the molecular basis of HIV-2 envelope glycoprotein interactions with CCR5 and CXCR4. We found that CCR5-using HIV-2 isolates are readily inhibited by maraviroc, TAK-779, and PF-227153, while monoclonal antibody 2D7 shows only residual or no inhibitory effects. The anti-HIV-2 activity of CXCR4-targeted molecules reveals that SDF-1α/CXCL12 inhibited all HIV-2 tested except one, while mAb 12G5 inhibited the replication of only two isolates, showing residual inhibitory effects with all the other CXCR4-using viruses. A major conclusion from our results is that infection by HIV-2 primary isolates is readily blocked in vitro by maraviroc, at concentrations similar to those required for HIV-1. The susceptibility to maraviroc was independent of CD4(+) T cell counts or clinical stage of the patient from which the virus was obtained. These findings indicate that maraviroc could constitute a reliable therapeutic alternative for HIV-2-infected patients, as long as they are infected with CCR5-using variants, and this may have direct implications for the clinical management of HIV-2-infected patients.     

Constitutive cell surface association between CD4 and CCR5.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4-CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4-CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env-CD4-CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors.     

CD4-independent infection of human B cells with HIV type 1: detection of unintegrated viral DNA.

Although B lymphocytes are a major constituent of lymphoid organs and acquire a significantly altered phenotype and function in HIV-infected individuals, it remains unclear whether CD4-negative B cells are a susceptible host for viral entry and long-term productive infection. We screened a number of Epstein-Barr virus (EBV)-positive and-negative Burkitt's lymphoma (BL) B cell lines as well as subpopulations of normal B cells that include tonsillar naive and germinal center/memory B cells for the expression of HIV-1 receptors CD4, CXCR4, and CCR5. Cell lines and resting or activated normal B cells lacked CD4 and CCR5 but expressed CXCR4. We demonstrate HIV-1 infection of a CD4-negative, EBV-negative (BL) cell line, CA46, which remained productively infected yet noncytopathic for more than 36 months in culture. HIV-1 (HTLV-III(B)) infection of CA46 cells was mediated through CXCR4 in a CD4-independent manner and correlated with upregulation of the expression of B cell activation markers CD23 and CD95 (Fas receptor). Despite Fas receptor expression, HIV-1-infected CA46 cells remained resistant to Fas-mediated cell death. CA46-derived, CD4-independent viral isolates were proficient in infecting and causing syncytium formation in Molt4 T cells. The HIV-1 genomic organization in persistently infected CA46 clones was found to be predominantly unintegrated linear and circular DNA. Importantly, naive and germinal center/memory B cells could also be infected by HIV-1 in a CD4-independent manner. Although these B cell subpopulations expressed moderate to high levels of CXCR4, they required activation through CD40 and interleukin 4 receptor for infection. These findings point to B cells as an additional HIV-1 target and suggest a structural evolution of the HIV-1 genome responsible for CD4-independent and noncytopathic infections.     

The susceptibility to X4 and R5 human immunodeficiency virus-1 strains of dendritic cells derived in vitro from CD34(+) hematopoietic progenitor cells is primarily determined by their maturation stage.

Dendritic cells (DC) were sorted on day 8 from cultures of CD34(+) cells with stem cell factor/Flt-3 ligand/ granulocyte-macrophage colony-stimulating factor (GM-CSF)/tumor necrosis factor-alpha (TNF-alpha)/interleukin-4 (IL-4). Exposing immature CCR5(+)CXCR4(lo/-) DC to CCR5-dependent human immunodeficiency virus (HIV)-1Ba-L led to productive and cytopathic infection, whereas only low virus production occurred in CXCR4-dependent HIV-1LAI-exposed DC. PCR analysis of the DC 48 hours postinfection showed efficient entry of HIV-1Ba-L but not of HIV-1LAI. CD40 ligand- or monocyte-conditioned medium-induced maturation of HIV-1Ba-L-infected DC reduced virus production by about 1 Log, while cells became CCR5(-). However, HIV-1Ba-L-exposed mature DC harbored 15-fold more viral DNA than their immature counterparts, ruling out inhibition of virus entry. Simultaneously, CXCR4 upregulation by mature DC coincided with highly efficient entry of HIV-1LAI which, nonetheless, replicated at the same low level in mature as in immature DC. In line with these findings, coculture of HIV-1Ba-L-infected immature DC with CD3 monoclonal antibody-activated autologous CD4(+) T lymphocytes in the presence of AZT decreased virus production by the DC. Finally, whether they originated from CD1a+CD14(-) or CD1a-CD14(+) precursors, DC did not differ as regards permissivity to HIV, although CD1a+CD14(-) precursor-derived immature DC could produce higher HIV-1Ba-L amounts than their CD1a-CD14(+) counterparts. Thus, both DC permissivity to, and capacity to support replication of, HIV is primarily determined by their maturation stage.     

Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.     

HIV type 1 chemokine receptor usage in mother-to-child transmission.

To investigate the role of the HIV-1 phenotype in mother-to-child HIV-1 transmission, we evaluated coreceptor usage and replication kinetics in chemokine receptor-expressing U87MG.CD4 cells of primary isolates from 32 HIV-1-infected mothers of Italian origin, none under preventive antiretroviral therapy, and from their infected infants. Five of 15 mothers of infected children and 2 of 17 mothers of uninfected children harbored viruses able to use CXCR4 as coreceptor. However, all isolates used CCR5, alone or in association with CXCR4. The replicative capacity in coreceptor-expressing cells of the viral isolates did not differ between the two groups of mothers. All mothers with an R5 virus transmitted a virus with the same coreceptor usage, whereas those four with a multitropic virus transmitted such a virus in one case. Although the presence of a mixed viral population was documented in the mothers, we did not observe transmission solely of X4 viruses. Interestingly, the only child infected with a multitropic virus carried a defective CCR5 allele. Analysis of the env V3 region of the provirus from this child revealed infection with multiple viral variants with a predominance of R5-type over X4-type sequences. These findings show that CCR5 usage of a viral isolate is not a discriminating risk factor for vertical transmission. Furthermore, X4 viruses can be transmitted to the newborn, although less frequently. In particular, we document the transmission of multiple viral variants with different coreceptor usage in a Delta32 CCR5 heterozygous child, and demonstrate that the heterozygous genotype per se does not contribute to the restriction of R5-type virus spread.     

Epidemiology and predictive factors for chemokine receptor use in HIV-1 infection

BACKGROUND: CXCR4-using virus is associated with higher viral load and accelerated human immunodeficiency virus (HIV) disease progression. Additionally, CCR5 antagonists may not reduce the HIV-1 RNA load when mixed/dual-tropic or CXCR4-using virus is present. The determination of coreceptor tropism may be required before CCR5 or CXCR4 antagonists are initiated, unless reliable predictive markers of coreceptor use are established. METHODS: Samples from treatment-naive and -experienced HIV-1-positive individuals with date-matched CD4 and CD8 cell counts and HIV-1 RNA, clade, and pol sequences were assessed for coreceptor usage, by use of the ViroLogic PhenoSense assay. RESULTS: Coreceptor use determination was successful in 563 of 861 samples. Non-clade B virus was present in 18.6% of samples. CXCR4 or mixed/dual-tropic CCR5/CXCR4 virus was present in 112 of samples (19.9%). Only 4 samples (0.7%) showed exclusive CXCR4 use. In a multivariate model, higher CD4 cell count, lower viral load, and higher natural killer cell counts were significantly associated with CCR5 usage. No associations with treatment experience, clade, or pol gene mutations were observed. CONCLUSION: The prevalence of CCR5-tropic HIV-1 phenotype declines with decreasing CD4 cell count and increasing viral load. Mixed/dual tropic virus is found in all CD4 cell count and viral load strata. Coreceptor usage is not influenced by viral clade.     

Productive infection of double-negative T cells with HIV in vivo

HIV induces CD4 down-regulation from the surface of infected cells by several independent mechanisms, suggesting an important biological role for this phenomenon. In vitro CD4 down-regulation generates T cells with a double-negative (DN) CD4(-)CD8(-) T cell receptor-alphabeta(+) phenotype. However, evidence that this down-regulation occurs in vivo in HIV-infected subjects is lacking, and viral load or viral production assays invariably focus on CD4(+) T cells. We show here that HIV infection can often be detected in sorted DN cells from peripheral blood and lymph nodes, even when plasma viral load is undetectable. DN T cells infected with HIV represented up to 20% of the cellular viral load in T cells, as determined by DNA PCR. In patients on successful highly active antiretroviral therapy, the viral load decreased in the plasma in CD4(+) and in DN T cells, suggesting that infected DN cells, like CD4(+) cells, contribute to viral production and are sensitive to highly active antiretroviral therapy. Indeed, HIV unspliced and multispliced RNAs were often detectable in DN T cells in spite of the small size of this subset. Infectious virus from DN T cells was transmitted efficiently in coculture experiments with uninfected T cell lymphoblasts, even when viral DNA in the DN cells was barely detectable. We conclude that a discrete population of infected DN T cells exists in HIV-positive subjects, even when the plasma viral load is undetectable. These cells may represent an important source of infectious virus.     

Effects of human immunodeficiency virus type 1 infection on CCR5 and CXCR4 coreceptor expression on CD4 T lymphocyte subsets in infants and adolescents

HIV-1 infection alters expression of CCR5 and CXCR4 on CD4 T cells in adults, although an effect by virus on expression of coreceptor genes in pediatric subjects is unknown. We designed an exploratory study to evaluate surface expression of CXCR4 and CCR5 on CD45RA and CD45RO subsets of CD4 T lymphocytes from 17 HIV-1-infected infants and adolescents and 16 healthy age-matched individuals. While age in the absence of HIV-1 infection was unrelated to coreceptor expression, infection affected coreceptor expression differentially in infants and adolescents. Among infected adolescents, CCR5 and CXCR4 expression was significantly increased on CD4 CD45RO T cells, while CXCR4 was diminished in the CD4 CD45RA subset. Although HIV-1 infection in infants was also associated with increased CXCR4 expression on the CD4 CD45RO subset, in contrast to adolescents, infection in infants had no impact on coreceptor expression within the CD45RA CD4 subset. The proportion of CD4 T cells coexpressing CD45RA and CD45RO was increased by infection in both infants and adolescents. The CD45RA CD45RO subset in culture expressed high levels of CD4, CXCR4, and CD69, an early activation marker, and was highly susceptible to HIV-1 infection and replication. Infection of transitional CD4 T cells coexpressing CD45RA and CD45RO could contribute in part to provirus in either CD45RA or CD45RO subsets. Deleterious effects by HIV-1 infection on CD4 T cell homeostasis were greater in infants then adolescents, indicating that adolescence may be an optimal age group for assessing vaccines to prevent or treat HIV-1 infection.     

Up-regulation of HIV coreceptors CXCR4 and CCR5 on CD4(+) T cells during human endotoxemia and after stimulation with (myco)bacterial antigens: the role of cytokines

Concurrent infections in patients with human immunodeficiency virus (HIV) infection stimulate HIV replication. Chemokine receptors CXCR4 and CCR5 can act as HIV coreceptors. The authors hypothesized that concurrent infection increases the HIV load through up-regulation of CXCR4 and CCR5. Using experimental endotoxemia as a model of infection, changes in HIV coreceptor expression were assessed in 8 subjects injected with lipopolysaccharide (LPS, 4 ng/kg). The expression of CXCR4 and CCR5 on CD4(+) T cells was increased 2- to 4-fold, 4 to 6 hours after LPS injection. In whole blood in vitro, LPS induced a time- and dose-dependent increase in the expression of CXCR4 and CCR5 on CD4(+) T cells. Similar changes were observed after stimulation with cell wall components of Mycobacterium tuberculosis (lipoarabinnomannan) or Staphylococcus aureus (lipoteichoic acid), or with staphylococcal enterotoxin B. LPS increased viral infectivity of CD4-enriched peripheral blood mononuclear cells (PBMCs) with a T-tropic HIV strain. In contrast, M-tropic virus infectivity was reduced, possibly because of elevated levels of the CCR5 ligand cytokines RANTES and MIP-1beta. LPS-stimulated up-regulation of CXCR4 and CCR5 in vitro was inhibited by anti-TNF and anti-IFN gamma. Incubation with recombinant TNF or IFN gamma mimicked the LPS effect. Anti-interleukin 10 (anti-IL-10) reduced CCR5 expression, without influencing CXCR4. In accordance, rIL-10 induced up-regulation of CCR5, but not of CXCR4. Intercurrent infections during HIV infection may up-regulate CXCR4 and CCR5 on CD4(+) T cells, at least in part via the action of cytokines. Such infections may favor selectivity of HIV for CD4(+) T cells expressing CXCR4. (Blood. 2000;96:2649-2654)     

CXCR4-tropic HIV-1 suppresses replication of CCR5-tropic HIV-1 in human lymphoid tissue by selective induction of CC-chemokines

In infected individuals, human immunodeficiency virus type 1 (HIV-1) exist as a "swarm" of quasi species compartmentalized in tissues where individual viral variants may interact locally. We have used human lymphoid tissue, where the critical events of HIV disease occur, to study local interactions in model HIV-1 binary swarms ex vivo. We infected tissue blocks with binary mixtures consisting either of CCR5-dependent and CXCR4-dependent variants or of 2 dual-tropic HIV-1 variants, of which one is skewed to utilization of CXCR4 and the other of CCR5. HIV-1 variants that use CXCR4 suppress replication of CCR5-dependent HIV-1 variants, whereas CCR5-dependent HIV-1 variants do not affect replication of CXCR4-dependent HIV-1. CC-chemokines that inhibit replication of CCR5-dependent HIV-1 variants were up-regulated by CXCR4-dependent HIV-1, thus possibly contributing to this suppression. Tissue-specific chemokine/cytokine network modulations triggered by individual HIV-1 variants may be an important mechanism of local interactions among HIV-1 quasi species in infected tissue.     

Pathogenesis of macrophage tropic HIV-1

Despite numerous studies on the impact of viral diversity, human immunodeficiency virus type 1 (HIV-1)-specific immune responses and host factors on disease progression, we still do not have a firm understanding of the pathogenesis of HIV-1 infection. Rapid depletion of CD4+ T-lymphocytes has been associated with a switch in viral coreceptor usage from CCR5 to CXCR4 in approximately 40 to 50% of infected individuals. However, the majority of infected individuals who progress to AIDS harbor only CCR5-dependent (R5) viral strains. HIV-1 disease progression is associated with an enhanced tropism of R5 viral strains for cells of the monocyte/macrophage lineage (enhanced M-tropism). However, the underlying molecular mechanisms contributing to enhanced M-tropism by R5 HIV-1 strains, and how HIV-1 variants with enhanced M-tropism cause CD4+ T-cell depletion in vivo are unknown. This review examines the relationship between viral coreceptor usage, M-tropism, and pathogenicity of HIV-1. We highlight evidence supporting the hypothesis that enhanced M-tropism of R5 HIV-1 results from adaptive viral evolution, resulting in HIV-1 variants that have increased ability to utilize relatively low levels of CD4 and CCR5 expressed on macrophages. The evidence also suggests that these late-emerging, R5 viral strains have reduced sensitivity to entry inhibitors, and increased ability to cause CD4+ T-lymphocyte loss. These variants are likely to impact HIV-1 disease progression, particularly in patients who persistently harbor only R5 viral strains.     

Human Leydig cells are productively infected by some HIV-2 and SIV strains but not by HIV-1

OBJECTIVES: With the use of highly active antiretroviral therapy, the identification of HIV reservoirs within the body has become an important issue. However, the testis has been largely ignored despite representing a pharmacologic sanctuary which could act as a viral reservoir. DESIGN: Because alterations in testosterone production have frequently been reported in HIV-infected individuals, we investigated whether the testosterone-producing Leydig cells could become directly infected by HIV-1, HIV-2 or SIV. METHODS: Purified Leydig cells were infected with a panel of HIV-1, HIV-2 and SIV strains and examined for expression of HIV/SIV receptors. Additionally, the impact of CD4 transduction on Leydig cell infection was determined. RESULTS: Leydig cells were unable to support productive infection of the seven HIV-1 isolates tested. No CD4, CXCR4 or CCR5 expression was evident on the surface of Leydig cells and transduction with a CD4 expressing adenovirus did not induce HIV-1 infection. In contrast, some primary and laboratory adapted CD4-independent HIV-2 and SIV strains were able to enter and replicate productively in Leydig cells. CONCLUSIONS: Our results suggest that Leydig cells do not represent a target for HIV-1 infection within the testis. In contrast, Leydig cells support HIV-2 and SIV infection and thus represent a potential target for infection. Receptor use and significance of HIV-2/SIV infection of Leydig cells remain to be determined.     

Short communication: Influence of active tuberculosis on chemokine and chemokine receptor expression in HIV-infected persons

Tuberculosis (TB) is the major opportunistic infection of HIV-1-infected patients in developing countries. Concurrent infection with TB results in immune cells having enhanced susceptibility to HIV-1 infection, which facilitates entry and replication of the virus. Cumulative data from earlier studies indicate that TB provides a milieu of continuous cellular activation and irregularities in cytokine and chemokine circuits that favor viral replication and disease progression. To better understand the interaction of the host with HIV-1 during active tuberculosis, we investigated in vivo expression of the HIV-1 coreceptors, CCR5 and CXCR4, and circulating levels of the inhibitory beta-chemokines, macrophage inflammatory protein-1-alpha (MIP-1alpha), macrophage inflammatory protein-1-beta (MIP-1beta), and regulated upon activation T cell expressed and secreted (RANTES), in HIV-positive individuals with and without active pulmonary tuberculosis. We found a significant decrease from normal in the fraction of CD4+ T cells expressing CCR5 and CXCR4 in individuals infected with HIV. However, CCR5 and CXCR4 expression did not differ significantly between HIV patients with and without tuberculosis. Higher amounts of MIP-1alpha, MIP-1beta, and RANTES were detected in plasma of HIV-1-positive individuals, particularly those with dual infection, although the increase was not found to be statistically significant.     

Infection of baboon microglia with SIV-HIV recombinant viruses: role of CD4 and chemokine receptors

Microglia constitute the primary cell type infected with HIV in the brain and play a major role in viral persistence in the CNS and in the development of AIDS dementia. Lack of a suitable animal model and limitations in the availability of human tissues hinder most HIV/AIDS studies investigating the neuropathogenesis of AIDS dementia. The aims of this study were to determine whether baboon microglia can be productively infected with SIV-HIV (SHIV) recombinant viruses in vitro and whether they express HIV-1 receptors and coreceptors. Our results show the presence of mRNA for CD4, CCR5, and CXCR4 chemokine receptors on baboon microglial cells. Microglia lacked mRNA for the CCR3 chemokine receptor. We also show productive infection of baboon microglial cells by two SHIV isolates, SHIV-KU and SHIV-89.6P, and blockade of the infection with soluble CD4 protein, CCR5, and CXCR4 monoclonal antibodies. This study demonstrating the feasibility of infecting baboon microglia with SHIV isolates is an important first step in using the baboon as an alternative nonhuman primate model to study HIV neuropathogenesis.     

Increased turnover of CCR5+ and redistribution of CCR5- CD4 T lymphocytes during primary human immunodeficiency virus type 1 infection

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) type 1 during primary infection. CCR5+ CD4 T lymphocytes were studied in subjects with primary HIV-1 infection (PHI) or acute Epstein-Barr virus (EBV) infection and in HIV-uninfected controls. The early decline of CD4 T lymphocytes during PHI resulted from depletion of CCR5- CD4 T lymphocytes. After antiretroviral therapy, Ki-67- CCR5- CD4 T cell counts rapidly increased in the circulation, which suggests that the initial decrease was due to an alteration in trafficking and/or sequestration. In the CCR5+ subset of CD4 T cells, there was an elevation in the proliferative (Ki-67+) fraction during PHI, yet their total number remained in the normal range. In contrast, in acute EBV infection, proliferating CCR5+ CD4 T cells accumulated to very high levels, suggesting they have an important role in the early antiviral response, which may be impaired in HIV-1 infection.     

The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1

The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)