HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES, show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5. However, the way resistance to SCH-D and other small molecule CCR5 inhibitors is manifested depends on the target cell and the nature of the assay (single- vs. multi-cycle). In multi-cycle assays using primary lymphocytes, SCH-D does not inhibit resistant molecular clones, and it can even enhance their infectivity modestly. In contrast, the same viruses (as Env-pseudotypes) are significantly inhibited by SCH-D in single-cycle entry assays using U87-CD4/CCR5 cells, resistance being manifested by incomplete inhibition at high SCH-D concentrations. When a single-cycle, Env-pseudotype entry assay was performed using either U87-CD4/CCR5 cells or PBMC under comparable conditions, entry was inhibited by up to 88% in the former cells but by only 28% in the PBMC. Hence, there are both cell- and assay-dependent influences on how resistance is manifested. We also take this opportunity to correct our previous report that SCH-D-resistant isolates are also substantially cross-resistant to PSC-RANTES [Marozsan, A.J., Kuhmann, S.E., Morgan, T., Herrera, C., Rivera-Troche, E., Xu, S., Baroudy, B.M., Strizki, J., Moore, J.P., 2005. Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 338 (1), 182-199]. A substantial element of this resistance was attributable to the unappreciated carry-over of SCH-D from the selection cultures into analytical assays.     

人PBMCs内表达CCR5Delta32蛋白对HIV-1辅受体抑制作用的研究

目的:在人PBMCs内表达CCR5Delta32蛋白,研究其对细胞表面HIV-1辅受体CCR5和CXCR4的抑制作用。方法:构建pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测目的蛋白的表达。继续培养靶细胞,FACS分析细胞表面CCR5和CXCR4分子的变化。结果:成功构建了pLenti-CCR5Delta32慢病毒载体,包装后产生重组慢病毒。将其转染PBMCs,Western blot检测到目的蛋白的表达。FACS分析表明,靶细胞内目的蛋白的表达对靶细胞表面辅受体CCR5和CXCR4的产生起抑制作用,抑制率在转染后第6天达到高峰(CCR5的抑制率为51.69%,CXCR4的抑制率为61.05%)。结论:靶细胞内目的蛋白的成功表达及其对靶细胞表面HIV-1辅受体CCR5和CXCR4产生的抑制作用,为后续的AIDS基因治疗研究奠定了基础。     

Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs)

Although a number of chemokine receptors display coreceptor activities in vitro, chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) remain the major coreceptors used by the human immunodeficiency virus type 1 (HIV-1). In this study, we used an envelope-mediated fusion assay to demonstrate low CCR4 coreceptor activity with some primary HIV-1 and simian immunodeficiency virus-1 (mac316) isolates in vitro. The coreceptor activity was sensitive to CCR4-specific antibodies and to the CCR4-specific chemokine ligand macrophage-derived chemokine (MDC)/chemokine ligand 22 (CCL22). Treatment of peripheral blood mononuclear cells (PBMCs; which express high levels of CCR4) with CCL22 caused down-modulation of endogenous CCR4 but had no significant effect on HIV-1 entry, suggesting that CCR4 may not be used as an entry coreceptor. Despite expression of other minor coreceptors on PBMCs, CCR5 and CXCR4 are preferentially used by HIV-1 isolates, as shown by chemokine-inhibition data. To determine the factors involved in this selective use, we analyzed CCR4 coreceptor activity and compared it with CCR5 use in PBMCs. We used a quantitative fluorescence-activated cell-sorting assay to estimate the numbers of CCR4 and CCR5 antibody-binding sites (ABS) on PBMCs. Although CCR4 was found on a higher percentage of CD4(+) cells, CCR5 ABS was twofold greater than CCR4 ABS on CD4(+) cells. Confocal microscopy revealed strong cell-surface CD4/CCR5 but weak CD4/CCR4 colocalization in PBMCs. Binding studies demonstrated that soluble gp120 had greater affinity to CCR5 than CCR4. The results suggested that coreceptor density, colocalization with CD4, and affinity of the viral gp120 to the coreceptor may determine preferential coreceptor use by HIV-1.     

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.     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.     

Optimization of ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells for use in anti-HIV immunotherapy and gene therapy strategies

The rhesus macaque model is a useful experimental system to evaluate effects of T-cell autotransfusion and gene therapies for HIV-1 infection and AIDS prior to a clinical trial. To obtain sufficient numbers of primary macaque CD4 T lymphocytes for this purpose, we examined the culture conditions that were needed to optimize ex vivo activation and expansion of macaque primary CD4-enriched peripheral blood mononuclear cells (PBMCs). In this report, we compared the effects of various stimulants on cell expansion, surface expression of CCR5 and CXCR4, and levels of transduction with a Moloney leukemia virus (MoLV) vector encoding the phenotypic selection marker truncated human nerve growth factor receptor (deltaNGFR) alone or with the human anti-HIV-1 tat intrabody sFvhutat2. The use of feeder cells strikingly increased the proliferation rate of macaque CD4-enriched PBMCs in vitro. In the presence of an irradiated rhesus macaque B-lymphoblastoid cell line (BLCL), the highest cell expansion over 21 days was achieved with cells activated by Con A (9648-fold), in turn, from high to low, phytohemagglutinin (PHA) (4855-fold), and anti-CD3/CD28-coated beads (2367-fold). Further studies showed that BLCL feeder cells were more effective than human PBMCs (hPBMCs) in promoting proliferation of macaque CD4-enriched PBMCs activated with Con A and anti-CD3/CD28, respectively. The combined use of both BLCL and hPBMC feeder cells did not further increase cell expansion when compared with the use of BLCL cells alone. In addition, the addition of BLCL-conditioned medium (CM) and hPBMC-CM induced cell growth at a rate higher than did the culture medium alone but not as high as with feeder cells. Con A-activated macaque CD4-enriched PBMCs retained 88% of CXCR4 and 39% of CCR5 expression over 17 days compared with PHA-activated cells (50% for CXCR4, 16% for CCR5) and anti-CD3/CD28-activated cells (34% for CXCR4, 37% for CCR5). Finally, PHA, Con A, and CD3/CD28-coated beads supported comparable levels of MoLV transduction. The results should improve the utility of the rhesus macaque model for the testing of T-cell autotransfusion and gene therapies for HIV-1 infection/AIDS.     

CXCR4 and CCR5 expression on CD4+ T cells in vivo and HIV-1 antigen beta-chemokine production in vitro after treatment with HIV-1 immunogen (REMUNE)

BACKGROUND: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes. STUDY DESIGN/METHODS: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression. RESULTS: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05). CONCLUSIONS: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.     

PRO 140 Monoclonal Antibody to CCR5 Prevents Acute Xenogeneic Graft-versus-Host Disease in NOD-scid IL-2Rynull Mice

Graft-versus-host disease (GVHD) is a prevalent and potentially lethal complication of hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic GVHD are important tools used to study the human immune response in vivo. Here we used NOD-scid IL-2Ry^null mice (NSG) transplanted with human bone marrow stem cells to evaluate the role of immune cell engraftment in the production of acute GVHD. PRO 140, a humanized monoclonal antibody targeting the chemokine receptor, CCR5, was used to evaluate its influence on bone marrow cell engraftment and modulation of acute GVHD. We evaluated the kinetics of engraftment by determining the percentage and absolute numbers of human CD45⁺ cells and CD3⁺ T cells from peripheral blood, spleen, and bone marrow in treated and control mice. With a dosing schedule of 2?mg of test or control antibody administered i.p. twice weekly, PRO 140–treated mice showed no signs of GVHD throughout the 70-day study period and gained weight until they were killed at 70 days for flow cytometry analysis. Control mice started losing weight after 25 days, showed classic signs of GVHD (ruffled fur, lethargy, severe hunching), and all were killed by day 54. The percentage and absolute numbers of human CD45⁺ cells in peripheral blood increased in both groups of mice throughout the 50-day comparison period and was lower in the PRO 140–treated mice at day 50. There was no difference in human CD45⁺ cells detected in bone marrow from control and PRO 140–treated killed mice. At this time point 76.1% and 68.2% of the hematopoietic cells from peripheral blood and from bone marrow, respectively, were of human lineage and 14.9% and 28%, respectively, were of mouse origin. With a schedule using 10-fold less dose of antibody (.2?mg i.p. twice weekly), PRO 140 still significantly modulated acute GVHD in terms of both weight loss and survival times, but no mice from either control or test group survived. By targeting the CCR5 chemokine receptor, PRO 140 modulated acute GVHD in a dose-response fashion in this xenogeneic mouse model without significantly altering engraftment.     

Regulation of CCR5 and CXCR4 Expression by Type 1 and Type 2 Cytokines: CCR5 Expression Is Downregulated by IL-10 in CD4-Positive Lymphocytes

HIV-1 transmission and disease progression is, in general, characterized by initial predominance of macrophage tropic, non-syncytium-inducing strains followed by a switch to T-cell tropic, syncytium-inducing strains. Using sensitive, quantitative kinetic RT-PCR, we examined cytokine regulation of tropism-specific HIV-1 coreceptor expression in PBMCs from HIV-1-seronegative individuals. Proinflammatory (TNF-alpha and IL-12) and type 1 cytokines (IFN-gamma and IL-2) significantly upregulated CCR5 (wt allele) mRNA expression in CCR5 homozygous wild-type (wt/wt) and heterozygous individuals (wt/del) (P < 0.02). CCR5 (wt) mRNA expression in unstimulated PBMCs was significantly increased in wt/wt individuals compared to that of wt/del individuals (P < 0.01). In wt/del individuals, del CCR5 mRNA was expressed at 10-fold greater levels than wt CCR5 mRNA in unstimulated PBMCs from the same individual. Flow cytometry confirmed that upregulated CCR5 mRNA following type 1 cytokine stimulation leads to increased cell surface expression of CCR5 protein. The type 2 cytokine IL-10 downregulated both CCR5 mRNA and protein expression in wt/wt and wt/del individuals. Proinflammatory, type 1, and type 2 cytokines significantly increased CXCR4 mRNA expression in wt/wt, wt/del, and del/del CCR5 genotypes (P < 0.02). These results suggest that changes in the cytokine milieu influence chemokine receptor expression and may explain emergence of tropism-specific strains facilitating HIV transmission and disease progression.     

The GHS-R blocker D-[Lys3] GHRP-6 serves as CCR5 chemokine receptor antagonist

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. This antagonist is one of the most common antagonists utilized in vivo to block GHS-R function and activity. Here, we found that DLS also has the ability to modestly block chemokine function and ligand binding to the chemokine receptor CCR5. The DLS effects on RANTES binding and Erk signaling as well as calcium mobilization appears to be much stronger than its effects on MIP-1α and MIP-1β. CCR5 have been shown to act as major co-receptor for HIV-1 entry into the CD4 positive host cells. To this end, we also found that DLS blocks M-tropic HIV-1 propagation in activated human PBMCs. These data demonstrate that DLS may not be a highly selective GHS-R1a inhibitor and may also effects on other G-protein coupled receptor (GPCR) family members. Moreover, DLS may have some potential clinical applications in blocking HIV infectivity and CCR5-mediated migration and function in various inflammatory disease states.     

Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

Characterization of a simian human immunodeficiency virus encoding the envelope gene from the CCR5-tropic HIV-1 Ba-L

The tat, rev, vpu, and env genes from the monocytotropic CCR5-dependent HIV-1 Ba-L isolate were substituted for homologous simian immunodeficiency virus (SIV) sequences in the SIV genome. The resultant SHIV (SHIV Ba-L) replicated in CCR5-positive PM-1 cells but not in CCR5-negative CEMX174 cells. Infection of HOS cells expressing different co-receptors showed SHIV Ba-L to be strictly CCR5-dependent. Infection of PM-1 cells and rhesus peripheral blood mononuclear cells (PBMCs) was highly sensitive to RANTES but not to SDF-1. Although SHIV Ba-L infected rhesus and pigtail macaques intravenously or rectally, plasma viremia was controlled after 3 weeks. After serial passage through 4 pigtails by blood and bone marrow transfer, virus from pigtail PBMCs had higher in vitro infectious titers on rhesus PBMCs and was efficiently transmitted vaginally in rhesus and cynomolgus macaques. Plasma viremia generally persisted longer than after infection with unpassaged virus but was eventually controlled with no significant decrease in CD4+ T-cell counts in peripheral blood. The envelope gene of SHIV Ba-L revealed a very little genetic drift during in vivo passage. SHIV Ba-L provides a potentially useful model for R5 HIV-1 infection of humans.     

早、中、晚孕期胎盘因子对人外周血淋巴细胞CD4、CCR5和CXCR4表达的影响

目的 通过研究早、中、晚孕期胎盘因子(PF)对人外周血淋巴细胞(PBLs)中CD4、CCR5和CXCR4表达的作用,探讨PF在人免疫缺陷病毒-1(HIV-1)垂直传播中的作用及其机理.方法 制备早、中、晚孕期PF.分离人外周血单个核细胞,并分别与相对浓度为25%的早、中、晚孕期PF作用,培养24 h后收集细胞,荧光抗体标记,流式细胞术检测外周血淋巴细胞(PBLs)中CD4、CCR5和CXCR4表达,以及CD4 T细胞中CCR5 细胞、CXCR4 细胞、CCR5 CXCR4 细胞所占的百分率.结果 各孕期PF均可显著降低PBLs中CCR5的表达,其中早孕期PF的作用明显强于中、晚孕期PF的作用;各孕期PF组CD4 T细胞中CCR5 细胞的百分率均显著低于对照组,早孕期PF组CD4 T细胞中CCR5 细胞的百分率明显低于中、晚孕期PF组;各孕期PF组CD4 T细胞中CCR5 CXCR4 细胞的百分率均显著低于对照组,早孕期PF组CD4 T细胞中CCR5 CXCR4 细胞的百分率显著低于晚孕期PF组.结论 各孕期PF均可显著降低PBLs中CCR5的表达,以及CD4 T细胞中CCR5 细胞和CCR5 CXCR4 细胞的百分率,早孕期PF作用最强,中、晚孕期PF效应相当,PF可能通过抑制R5病毒的人胞而具有抗R5病毒的作用,并可能在阻断HIV-1宫内感染中具有重要作用.     

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.     

Marked differences in CCR5 expression and activation levels in two South African populations

The chemokine receptor CCR5 is pivotal in determining an individual's susceptibility to HIV-1 infection and rate of disease progression. To establish whether population-based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5(+) cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) -cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5(+) CD8(+) T-cell subsets and CCR5(+) NK-cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA-DR expression, than SAC individuals in CD4(+) T-cell subsets (P = 0·002) and CD56(+) NK-cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both cell activation and CCR5 expression, which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression.     

CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells

R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection. However, in PBMCs, only ECL2 CCR5 antibodies HGS004 and HGS101, but not 2D7, inhibited infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not 2D7, restored the antiviral activity of MVC against resistant virus in PBMCs. In flow cytometric studies, CCR5 binding by the HGS mAbs, but not by 2D7, was increased when PBMCs were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Thus, HGS004 and HGS101 have antiviral mechanisms distinct from 2D7 and could help overcome MVC resistance.     

Constrained use of CCR5 on CD4+ lymphocytes by R5X4 HIV-1: Efficiency of Env-CCR5 interactions and low CCR5 expression determine a range of restricted CCR5-mediated entry

R5X4 HIV-1 has impaired utilization of CCR5 on primary CD4+ lymphocytes but the mechanisms responsible are not well defined. Using a panel of diverse R5X4 Envs we identified a spectrum of CCR5 use on CD4+ lymphocytes. Greater lymphocyte CCR5 use correlated with relative resistance to CCR5 mAbs and small molecule antagonists. Increasing CCR5 expression on lymphocytes increased the proportion of entry mediated by CCR5 for all R5X4 isolates except 89.6. In cell lines with regulated CCR5 expression, strains with greater lymphocyte CCR5 use better exploited limiting levels of CCR5. Introduction of an R306S mutation in the 89.6 V3 domain enhanced its utilization of CCR5 at low levels and switched its preference to CCR5 for lymphocyte entry. Thus, the degree to which R5X4 HIV-1 use primary lymphocyte CCR5 is determined by low CCR5 expression coupled with variations in the efficiency of Env-CCR5 interactions, which is in part governed by V3 sequences.