Entry inhibitor-based microbicides are active in vitro against HIV-1 isolates from multiple genetic subtypes

Inhibitors of viral entry are under consideration as topical microbicides to prevent HIV-1 sexual transmission. Small molecules targeting HIV-1 gp120 (BMS-378806) or CCR5 (CMPD167), and a peptide fusion inhibitor (C52L), each blocks vaginal infection of macaques by a SHIV. A microbicide, however, must be active against multiple HIV-1 variants. We therefore tested BMS-C (a BMS-378806 derivative), CMPD167, C52L and the CXCR4 ligand AMD3465, alone and in combination, against 25 primary R5, 12 X4 and 7 R5X4 isolates from subtypes A-G. At high concentrations (0.1-1 microM), the replication of most R5 isolates in human donor lymphocytes was inhibited by >90%. At lower concentrations, double and triple combinations were more effective than individual inhibitors. Similar results were obtained with X4 viruses when AMD3465 was substituted for CMPD167. The R5X4 viruses were inhibited by combining AMD3465 with CMPD167, or by the coreceptor-independent compounds. Thus, combining entry inhibitors may improve microbicide effectiveness.     

Restricted replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not in Th1 CD4(+) T cells

We have previously reported that CCR5-dependent human immunodeficiency virus type-1 (HIV-1; R5), but not CXCR4-restricted (X4) virus, efficiently replicates in T helper cell type 1 (Th1), Th2, or Th0 polyclonal T cells obtained from human umbilical cord blood (CB lines). The X4 virus restriction was env-dependent but did not occur at the level of viral entry. Here, we describe that in contrast to these monotropic HIVs, primary HIV-1 isolates capable of using CCR5 or CXCR4 indifferently for entry (i.e., R5X4 viruses) efficiently replicated in Th2 but not in Th1 CB lines. Although Th1 cells secreted significantly higher amounts of the three CCR5-binding chemokines in comparison with Th2 cells, this restriction was not explained by a defective infection of Th1 cells. Interferon-gamma (IFN-gamma) down-regulated CCR5 in Th1 cells and inhibited, whereas interleukin-4 (IL-4) up-regulated CXCR4 and enhanced the spreading of R5 and R5X4 viruses in polarized CB lines. However, both cytokines did not rescue the replication of X4 and dualtropic viruses in both types of CB lines or in Th1 cells, respectively, whereas addition of anti-IL-4- or anti-IFN-gamma-neutralizing antibodies did not activate virus expression. These findings together suggest the existence of post-entry restriction pathways influenced by gp120 Env/chemokine coreceptor interaction that may significantly contribute to the superior capacity of R5 and R5X4 HIV-1 strains to spread in vivo in comparison to X4 monotropic viruses.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

Inhibition of R5X4 dualtropic HIV-1 primary isolates by single chemokine co-receptor ligands

The susceptibility of HIV-1 to chemokine-mediated inhibition may be lost as a consequence of the expanded usage of chemokine co-receptors frequently occurring in clade B isolates obtained from individuals with advanced disease. Since chemokine-based immune intervention is under intense investigation, it is crucial to determine its potential effect on primary dualtropic HIV isolates characterized by simultaneous utilization of CCR5 and CXCR4 chemokine co-receptors (R5X4 viruses). In the present study, the CCR5 binding chemokine regulated upon activation normal T cell expressed and secreted (RANTES) strongly inhibited the replication of two of eight primary R5X4 viruses in mitogen-activated primary peripheral blood mononuclear cells (PBMC). The CXCR4 antagonist AMD3100 efficiently suppressed the replication of other two HIV isolates, whereas the remaining four viruses were partially inhibited by treatment with either RANTES or AMD3100. The potency of chemokine-mediated inhibition was influenced by PBMC donor variability, but it was usually independent from the levels of expression of CCR5 or CXCR4. Dual co-receptor usage was maintained by the viruses after two serial passages on U87.CD4 astrocytic cell lines expressing exclusively either CCR5 or CXCR4. The gp120 env variable domains were sequenced before and after passages on U87.CD4 cells. Virus replication into U87.CD4-CXCR4 cells did not result in changes in the V3 region but perturbed the dominant env V4 sequence. Interestingly, double passage onto U87.CD4-CXCR4 cells determined the loss of susceptibility to RANTES inhibition. In conclusion, interference with CCR5 may efficiently inhibit the replication of at least some dualtropic HIV-1 strains, whereas forced CXCR4 usage may result in viral escape from CCR5-dependent inhibitory effects.     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Replicative fitness of CCR5-using and CXCR4-using human immunodeficiency virus type 1 biological clones

CCR5-tropic viruses cause the vast majority of new HIV-1 infections while about half of the individuals infected with HIV-1 manifest a co-receptor switch (CCR5 (R5) to CXCR4 (X4)) prior to accelerated disease progression. The underlying biological mechanisms of X4 outgrowth in AIDS patients are still poorly understood. Although X4 viruses have been associated with increased "virulence" in vivo, in vitro replication and cytopathicity studies of X4 and R5 viruses have led to conflicting conclusions. We studied the replicative fitness of HIV-1 biological clones with different co-receptor tropism, isolated from four AIDS patients. On average, R5 and X4 clones replicated equally well in mitogen-activated T cells. In contrast, X4 variants were transferred more efficiently from dendritic cells to autologous CD4+ T cells. These observations suggest that interaction between X4 viruses, DC and T cells might contribute to the preferential outgrowth of X4 viruses in AIDS patients.     

Asymmetric HIV-1 co-receptor use and replication in CD4(+) T lymphocytes

Susceptibility to infection by the human immunodeficiency virus type-1 (HIV-1), both in vitro and in vivo, requires the interaction between its envelope (Env) glycoprotein gp120 Env and the primary receptor (R), CD4, and Co-R, either CCR5 or CXCR4, members of the chemokine receptor family. CCR5-dependent (R5) viruses are responsible for both inter-individual transmission and for sustaining the viral pandemics, while CXCR4-using viruses, usually dualtropic R5X4, emerge in ca. 50% of individuals only in the late, immunologically suppressed stage of disease. The hypothesis that such a major biological asymmetry is explained exclusively by the availability of cells expressing CCR5 or CXCR4 is challenged by several evidences. In this regard, binding of the HIV-1 gp120 Env to the entry R complex, i.e. CD4 and a chemokine R, leads to two major events: virion-cell membrane fusion and a cascade of cell signaling. While the fusion/entry process has been well defined, the role of R/Co-R signaling in the HIV-1 life cycle has been less characterized. Indeed, depending on the cellular model studied, the capacity of HIV-1 to trigger a flow of events favoring either its own latency or replication remains a debated issue. In this article, we will review the major findings related to the role of HIV R/Co-R signaling in the steps following viral entry and leading to viral spreading in CD4(+) T lymphocytes.     

Selective HIV-1-induced downmodulation of CD4 and coreceptors

Summary.  CD4 and members of the chemokine receptor family are required for infection of host cells, in vitro and in vivo, by the human immunodeficiency virus type-1. Although it is established that HIV-1 gp120 interacts with CD4 and the coreceptors CCR5 or CXCR4 at the plasma membrane during HIV entry, longer-term interactions taking place between these molecules and HIV Env are less well understood. We have measured the cell surface expression of CD4, CCR5 and CXCR4 on a CD4⁺/CXCR4⁺CCR5⁺ T cell line following infection by cell line-adapted X4 and primary X4, X4R5 and R5 viruses. We report a selective downmodulation of CD4 by X4 and R5X4 viruses, but not by R5 viruses. None of the viruses tested significantly reduced CXCR4 expression at any time after infection. CCR5 protein and mRNA expression was eliminated by chronic infection with R5 viruses. These results indicate that chronic HIV-1 infection has distinct effects on CD4 and coreceptor membrane expression that depends on viral origin and coreceptor usage. Accepted October 25, 1999     

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS.

The chemokine receptors CCR5 and CXCR4 are co-receptors together with CD4 for human immunodeficiency virus (HIV)-1 entry into target cells. Macrophage-tropic HIV-1 viruses use CCR5 as a co-receptor, whereas T-cell-line tropic viruses use CXCR4. HIV-1 infects the brain and causes a progressive encephalopathy in 20 to 30% of infected children and adults. Most of the HIV-1-infected cells in the brain are macrophages and microglia. We examined expression of CCR5 and CXCR4 in brain tissue from 20 pediatric acquired immune deficiency syndrome (AIDS) patients in relation to neuropathological consequences of HIV-1 infection. The overall frequency of CCR5-positive perivascular mononuclear cells and macrophages was increased in the brains of children with severe HIV-1 encephalitis (HIVE) compared with children with mild HIVE or non-AIDS controls, whereas the frequency of CXCR4-positive perivascular cells did not correlate with disease severity. CCR5- and CXCR4-positive macrophages and microglia were detected in inflammatory lesions in the brain of children with severe HIVE. In addition, CXCR4 was detected in a subpopulation of neurons in autopsy brain tissue and primary human brain cultures. Similar findings were demonstrated in the brain of adult AIDS patients and controls. These findings suggest that CCR5-positive mononuclear cells, macrophages, and microglia contribute to disease progression in the central nervous system of children and adults with AIDS by serving as targets for virus replication.     

Characterization of a chemokine receptor CCR5-negative T cell line and its use in determining human immunodeficiency virus type 1 phenotype

A human CD4-positive T cell line from a donor homozygous negative for the chemokine receptor CCR5 was established, characterized, and used for determining the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) isolates. Clones of this IL-2 dependent human T-cell lymphotropic virus type 1 (HTLV-I) immortalized cell line, named IsnoR5 clones 1 and 2, are susceptible to infection by HIV-1 isolates that use CXCR4 as a coreceptor but resistant to infection by CCR5 tropic HIV-1 viruses. HIV-1 isolates whose replication is inhibited in IsnoR5 cells in the presence of the bicyclam AMD 3100, a CXCR4 specific inhibitor, utilize a coreceptor distinct from CCR5 and CXCR4. Using a panel of primary HIV-1 isolates we have shown that a single T cell line is sufficient to discriminate between use of CCR5, CXCR4 or an alternative coreceptor. As IsnoR5 clone 1 cells revealed the existence of even minor populations of CXCR4-using virus variants, they could be useful for the early identification of changes in coreceptor usage in HIV infected individuals facilitating the timely introduction of appropriate clinical treatments.     

Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D)

We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry. Neither escape mutant could replicate detectably in peripheral blood mononuclear cells (PBMC) from two donors homozygous for the CCR5-Delta32 allele and both were insensitive to the CXCR4-specific inhibitor, AMD3100. Hence, the SCH-D escape mutants retained the R5 phenotype. One of the resistant isolates was, however, capable of replication in U87.CD4.CXCR4 cells and, after expansion in those cells, was sensitive to AMD3100 in primary CD4+ T-cells. Hence, some X4 variants may be present in this escape mutant swarm. A notable observation was that the SCH-D escape mutants were also cross-resistant to PSC-RANTES and AOP-RANTES, chemokine derivatives that are reported to down-regulate cell surface CCR5 almost completely. However, the extent to which CCR5 is down-regulated was dependent upon the detection MAb. Hence, the escape mutants may be using a CCR5 configuration that is only detected by some anti-CCR5 MAbs. Finally, two SCH-D-resistant clonal viruses revealed no amino acid changes in the gp120 V3 region relative to the parental viruses, in marked contrast to clones resistant to the AD101 small molecule CCR5 inhibitor that possess 4 such sequence changes. Several sequence changes elsewhere in gp120 (V2, C3 and V4) were present in the SCH-D-resistant clones. Their influence on the resistant phenotype remains to be determined.     

IL-13 and TNF-alpha inhibit dual-tropic HIV-1 in primary macrophages by reduction of surface expression of CD4, chemokine receptors CCR5, CXCR4 and post-entry viral gene expression

We show that IL-13 in the presence of TNF-alpha effected an equal or greater antiviral activity against a dual-tropic HIV-1 (R5X4) in macrophages. A temporary or continued exposure of macrophages to both cytokines significantly decreased the infection and replication of R5X4 HIV-1(89.6) (median, 128-fold, n = 9, p = 0.024) in macrophages as compared to untreated controls when analyzed over six decreasing multiplicities of infection. A quantitative flow cytometric assay revealed that IL-13 induced a significant (approximately 50 %) reduction in the number of CD4 and CC chemokine receptor 5 (CCR5) antibody binding sites while completely abrogating surface expression of CXC chemokine receptor 4 (CXCR4). In the presence of IL-13 and TNF-alpha, expression of CCR5 was completely abrogated while the expression of CD4 and CXCR4 remained significantly reduced as compared to untreated controls. A reduction in CD4 and HIV-1 coreceptors was associated with a decrease in reverse-transcribed viral DNA at 24 h post-infection. Quantification of viral gene expression using amphotropic MLV Env pseudotyped luciferase reporter viruses suggested that IL-13 inhibited HIV-1 gene expression within 24 h by up to 90 % in the presence or absence of TNF-alpha. In conclusion, our data suggest that IL-13 is a powerful counter-regulatory agent against TNF-alpha-induced HIV-1 expression while also acting with TNF-alpha in inhibiting de novo infection of macrophages.     

Molecular determinants of HIV-1 subtype C coreceptor transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of a heterologous R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy.     

HIV-1 infection in individuals with the CCR5-Delta32/Delta32 genotype: acquisition of syncytium-inducing virus at seroconversion

Homozygosity for the 32 base-pair deletion (Delta32/Delta32) in the CCR5 coreceptor gene is associated with incomplete HIV-1 resistance. Six HIV-1-infected Delta32/Delta32 patients have been reported. We report 2 additional Delta32/Delta32-infected individuals, among 106 seroconverters in a vaccine preparedness study. Like the previous 6, these individuals experienced rapid CD4 decline. However, taken together, the 8 patients have neither uniformly high virus load nor rapid progression to AIDS. We obtained five virus isolates from 1 patient at 5, 6, 7, 10, and 12 months after the estimated time of infection. The earliest isolate exhibits the syncytium-inducing (SI) phenotype and exclusive use of the CXCR4 coreceptor, suggesting acquisition of HIV-1 through this coreceptor. Of the remaining 104 seroconverters, 8 were CCR5-Delta32/+ and 96 were CCR5-+/+. Three CCR5-+/+ seroconverters who showed the uncommon pattern of early SI virus and rapid CD4 decline had uniformly high viral load and more heterogeneous coreceptor usage. These results further support the conclusion that Delta32-mediated resistance is incomplete and is associated with acquisition of exclusively-X4 variants of HIV-1. The pathogenic potential of these viruses may be different from late-stage X4 virus or early X4 virus acquired by individuals with other CCR5 genotypes.     

Role of nucleotide sequences in the V3 region in efficient replication of CCR5-utilizing human immunodeficiency virus type 1 in macrophages

Macrophages express both CXCR4 and CCR5 coreceptors, but restrict X4 HIV-1 replication unless the Env-V3 region, a major determinant of cell tropism, is exchanged with that of R5 HIV-1. As the V3 exchange concomitantly alters the nucleotide sequences, we introduced silent mutations in the V3 or C2 region of macrophage-tropic R5 JRFL without changing the amino acids. Immunoblot analysis confirmed that viral proteins including Env-gp120 were similarly incorporated in wild-type (wt) and mutant virions. The silent mutants infected CCR5-positive MAGIC5 cells but not CCR5-negative MAGI cells, as productively as wt viruses, indicating that the silent mutations did not alter coreceptor utilization. In contrast, two of three silent V3-mutant viruses failed to replicate efficiently in primary macrophages, whereas other V3- or C2-mutants and wt JRFL infected macrophages productively. Furthermore, synthesis of the full-length viral DNA of the aberrant V3-mutant was largely reduced in macrophages. These results suggest that V3 nucleotide sequences may be one of the postentry factors restricting HIV-1 replication in macrophages.     

R5X4 HIV-1 coreceptor use in primary target cells: implications for coreceptor entry blocking strategies

Entry coreceptor use by HIV-1 plays a pivotal role in viral transmission, pathogenesis and disease progression. In many HIV-1 infected individuals, there is an expansion in coreceptor use from CCR5 to include CXCR4, which is associated with accelerated disease progression. While targeting HIV-1 envelope interactions with coreceptor during viral entry is an appealing approach to combat the virus, the methods of determining coreceptor use and the changes in coreceptor use that can occur during disease progression are important factors that may complicate the use of therapies targeting this stage of HIV-1 replication. Indicator cells are typically used to determine coreceptor use by HIV-1 in vitro, but the coreceptors used on these cells can differ from those used on primary cell targets. V3 based genetic sequence algorithms are another method used to predict coreceptor use by HIV-1 strains. However, these algorithms were developed to predict coreceptor use in cell lines and not primary cells and, furthermore, are not highly accurate for some classes of viruses. This article focuses on R5X4 HIV-1, the earliest CXCR4-using variants, reviewing the pattern of coreceptor use on primary CD4+ lymphocytes and macrophages, the relationship between primary cell coreceptor use and the two principal approaches to coreceptor analysis (genetic prediction and indicator cell phenotyping), and the implications of primary cell coreceptor use by these strains for treatment with a new class of small molecule antagonists that inhibit CCR5-mediated entry. These are important questions to consider given the development of new CCR5 blocking therapies and the prognosis associated with CXCR4 use.     

Inducible-costimulator-mediated suppression of human immunodeficiency virus type 1 replication in CD4(+) T lymphocytes

We investigated the effects of signaling through CD28 family molecules on human immunodeficiency virus type 1 (HIV-1) replication in vitro. A monoclonal antibody (mAb) specific for inducible costimulator (ICOS) suppressed both X4 and R5 HIV-1 replication in CD4(+) peripheral blood mononuclear cells (PBMC). This suppression was not attributable to reduced cell growth or viability. CD28 mAb showed variable effects and also suppressed HIV-1 replication when immobilized. Replication of pseudotype viruses with HIV-1-but not with vesicular stomatitis virus G-envelope was efficiently suppressed in CD4(+) PBMC treated with ICOS or CD28 mAbs. However, CD4, CXCR4, and CCR5 expression on the surface was not down-regulated. Moreover, HIV-1 replication in CD4(+) PBMC was suppressed by a soluble form of human B7-H2, a ligand of ICOS, but was enhanced by soluble B7-1, a ligand for CD28. These findings suggest that natural or artificial ligands for ICOS potentially suppress HIV-1 replication mainly at the entry stages.     

Entry of R5X4 and X4 human immunodeficiency virus type 1 strains is mediated by negatively charged and tyrosine residues in the amino-terminal domain and the second extracellular loop of CXCR4

CXCR4 mediates the fusion and entry of X4 and R5X4 strains of human immunodeficiency virus type 1 (HIV-1). The residues involved in CXCR4 coreceptor function have not all yet been identified, but tyrosine and negatively charged residues in the amino-terminal domain of CCR5 were shown to be indispensable for gp120 binding and entry of R5 and R5X4 strains. We therefore evaluated the role of such residues in CXCR4 coreceptor function by replacing tyrosines (Y), aspartic acids (D), and glutamic acids (E) with alanines (A) and testing the ability of these mutants to mediate the entry of X4 and R5X4 HIV-1 isolates. Our results show that viral entry depends on YDE-rich clusters in both the amino-terminus and the second extracellular loop of CXCR4. Different viral isolates vary in their dependence on residues in one or the other domain. The determinants of CXCR4 coreceptor function are, therefore, more diffuse and isolate-dependent than those of CCR5.