Analysis of Clinical HIV-1 Strains with Resistance to Maraviroc Reveals Strain-Specific Resistance Mutations, Variable Degrees of Resistance, and Minimal Cross-Resistance to Other CCR5 Antagonists.

Maraviroc (MVC) is an allosteric inhibitor of human immunodeficiency virus type 1 (HIV-1) entry, and is the only CCR5 antagonist licensed for use as an anti-HIV-1 therapeutic. It acts by altering the conformation of the CCR5 extracellular loops, rendering CCR5 unrecognizable by the HIV-1 envelope (Env) glycoproteins. This study aimed to understand the mechanisms underlying the development of MVC resistance in HIV-1-infected patients. To do this, we obtained longitudinal plasma samples from eight subjects who experienced treatment failure with phenotypically verified, CCR5-tropic MVC resistance. We then cloned and characterized HIV-1 Envs (n = 77) from plasma of pretreatment (n = 36) and treatment failure (n = 41) samples. Our results showed variation in the magnitude of MVC resistance as measured by reductions in maximal percent inhibition of Env-pseudotyped viruses, which was more pronounced in 293-Affinofile cells compared to other cells with similar levels of CCR5 expression. Amino acid determinants of MVC resistance localized to the V3 Env region and were strain specific. We also observed minimal cross-resistance to other CCR5 antagonists by MVC-resistant strains. We conclude that 293-Affinofile cells are highly sensitive for detecting and measuring MVC resistance through a mechanism that is CCR5-dependent yet independent of CCR5 expression levels. The strain-specific nature of resistance mutations suggests that sequence-based diagnostics and prognostics will need to be more sophisticated than simple position scoring to be useful for managing resistance in subjects taking MVC. Finally, the minimal levels of cross-resistance suggests that recognition of the MVC-modified form of CCR5 does not necessarily lead to recognition of other antagonist-modified forms of CCR5.

Frequency and Env determinants of HIV-1 subtype C strains from antiretroviral therapy-naive subjects that display incomplete inhibition by maraviroc.

BACKGROUND: Entry of human immunodeficiency virus type 1 (HIV-1) into cells involves the interaction of the viral gp120 envelope glycoproteins (Env) with cellular CD4 and a secondary coreceptor, which is typically one of the chemokine receptors CCR5 or CXCR4. CCR5-using (R5) HIV-1 strains that display reduced sensitivity to CCR5 antagonists can use antagonist-bound CCR5 for entry. In this study, we investigated whether naturally occurring gp120 alterations in HIV-1 subtype C (C-HIV) variants exist in antiretroviral therapy (ART)-naïve subjects that may influence their sensitivity to the CCR5 antagonist maraviroc (MVC). RESULTS: Using a longitudinal panel of 244 R5 Envs cloned from 20 ART-naïve subjects with progressive C-HIV infection, we show that 40% of subjects (n = 8) harbored viruses that displayed incomplete inhibition by MVC, as shown by plateau's of reduced maximal percent inhibitions (MPIs). Specifically, when pseudotyped onto luciferase reporter viruses, 16 Envs exhibited MPIs below 98% in NP2-CCR5 cells (range 79.7-97.3%), which were lower still in 293-Affinofile cells that were engineered to express high levels of CCR5 (range 15.8-72.5%). We further show that Envs exhibiting reduced MPIs to MVC utilized MVC-bound CCR5 less efficiently than MVC-free CCR5, which is consistent with the mechanism of resistance to CCR5 antagonists that can occur in patients failing therapy. Mutagenesis studies identified strain-specific mutations in the gp120 V3 loop that contributed to reduced MPIs to MVC. CONCLUSIONS: The results of our study suggest that some ART-naïve subjects with C-HIV infection harbor HIV-1 with reduced MPIs to MVC, and demonstrate that the gp120 V3 loop region contributes to this phenotype.

Molecular Gymnastics: Mechanisms of HIV-1 Resistance to CCR5 Antagonists and Impact on Virus Phenotypes.

Human immunodeficiency virus type 1 (HIV-1) enters host cells through the binding of its envelope glycoproteins (Env) to the host cell receptor CD4 and then subsequent binding to a chemokine coreceptor, either CCR5 or CXCR4. CCR5 antagonists are a relatively recent class addition to the armamentarium of anti-HIV-1 drugs. These compounds act by binding to a hydrophobic pocket formed by the transmembrane helices of CCR5 and altering the conformation of the extracellular domains, such that they are no longer recognized by Env. Maraviroc is the first drug within this class to be licenced for use in HIV-1 therapy regimens. HIV resistance to CCR5 antagonists occurs either through outgrowth of pre-existing CXCR4-using viruses, or through acquisition of the ability of CCR5-using HIV-1 to use the antagonist bound form of CCR5. In the latter scenario, the mechanism underlying resistance is through complex alterations in the way that resistant Envs engage CCR5. These significant changes are unlikely to occur without consequence to the viral entry phenotype and may also open up new avenues to target CCR5 antagonist resistant viruses. This review discusses the mechanism of action of CCR5 antagonists, how HIV resistance to CCR5 antagonists occurs, and the subsequent effects on Env function.

Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

BACKGROUND: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes. RESULTS: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency. CONCLUSIONS: GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

Quantifying susceptibility of CD4+ stem memory T-cells to infection by laboratory adapted and clinical HIV-1 strains.

CD4+ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4+ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4+ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4+ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4+ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs.

Linkages between HIV-1 specificity for CCR5 or CXCR4 and in vitro usage of alternative coreceptors during progressive HIV-1 subtype C infection.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype C (C-HIV) is spreading rapidly and is now responsible for >50% of HIV-1 infections worldwide, and >95% of infections in southern Africa and central Asia. These regions are burdened with the overwhelming majority of HIV-1 infections, yet we know very little about the pathogenesis of C-HIV. In addition to CCR5 and CXCR4, the HIV-1 envelope glycoproteins (Env) may engage a variety of alternative coreceptors for entry into transfected cells. Whilst alternative coreceptors do not appear to have a broad role in mediating the entry of HIV-1 into primary cells, characterizing patterns of alternative coreceptor usage in vitro can provide valuable insights into mechanisms of Env-coreceptor engagement that may be important for HIV-1 pathogenesis. RESULTS: Here, we characterized the ability of luciferase reporter viruses pseudotyped with HIV-1 Envs (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects experiencing progression from chronic to advanced C-HIV infection over an approximately 3-year period, who either exclusively maintained CCR5-using (R5) variants (n = 20 subjects) or who experienced a coreceptor switch to CXCR4-using (X4) variants (n = 1 subject), to utilize alternative coreceptors for entry. At a population level, CCR5 usage by R5 C-HIV Envs was strongly linked to usage of FPRL1, CCR3 and CCR8 as alternative coreceptors, with the linkages to FPRL1 and CCR3 usage becoming statistically more robust as infection progressed from chronic to advanced stages of disease. In contrast, acquisition of an X4 Env phenotype at advanced infection was accompanied by a dramatic loss of FPRL1 usage. Env mutagenesis studies confirmed a direct link between CCR5 and FPRL1 usage, and showed that the V3 loop crown, but not other V3 determinants of CCR5-specificity, was the principal Env determinant governing the ability of R5 C-HIV Envs from one particular subject to engage FPRL1. CONCLUSIONS: Our results suggest that, in the absence of coreceptor switching, the ability of R5 C-HIV viruses to engage certain alternative coreceptors in vitro, in particular FPRL1, may reflect an altered use of CCR5 that is selected for during progressive C-HIV infection, and which may contribute to C-HIV pathogenicity.

Longitudinal Analysis of CCR5 and CXCR4 Usage in a Cohort of Antiretroviral Therapy-Naïve Subjects with Progressive HIV-1 Subtype C Infection.

HIV-1 subtype C (C-HIV) is responsible for most HIV-1 cases worldwide. Although the pathogenesis of C-HIV is thought to predominantly involve CCR5-restricted (R5) strains, we do not have a firm understanding of how frequently CXCR4-using (X4 and R5X4) variants emerge in subjects with progressive C-HIV infection. Nor do we completely understand the molecular determinants of coreceptor switching by C-HIV variants. Here, we characterized a panel of HIV-1 envelope glycoproteins (Envs) (n = 300) cloned sequentially from plasma of 21 antiretroviral therapy (ART)-naïve subjects who experienced progression from chronic to advanced stages of C-HIV infection, and show that CXCR4-using C-HIV variants emerged in only one individual. Mutagenesis studies and structural models suggest that the evolution of R5 to X4 variants in this subject principally involved acquisition of an "Ile-Gly" insertion in the gp120 V3 loop and replacement of the V3 "Gly-Pro-Gly" crown with a "Gly-Arg-Gly" motif, but that the accumulation of additional gp120 "scaffold" mutations was required for these V3 loop changes to confer functional effects. In this context, either of the V3 loop changes could confer possible transitional R5X4 phenotypes, but when present together they completely abolished CCR5 usage and conferred the X4 phenotype. Our results show that the emergence of CXCR4-using strains is rare in this cohort of untreated individuals with advanced C-HIV infection. In the subject where X4 variants did emerge, alterations in the gp120 V3 loop were necessary but not sufficient to confer CXCR4 usage.