Richard M. Gibson

CCR5- and CXCR4-Tropic Subtype C Human Immunodeficiency Virus Type 1 Isolates Have a Lower Level of Pathogenic Fitness than Other Dominant Group M Subtypes: Implications for the Epidemic

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C is the dominant subtype globally, due largely to the incidence of subtype C infections in sub-Saharan Africa and east Asia. We compared the relative replicative fitness (ex vivo) of the major (M) group of HIV-1 subtypes A, B, C, D, and CRF01_AE and group O isolates. To estimate pathogenic fitness, pairwise competitions were performed between CCR5-tropic (R5) or CXCR4-tropic (X4) virus isolates in peripheral blood mononuclear cells (PBMC). A general fitness order was observed among 33 HIV-1 isolates; subtype B and D HIV-1 isolates were slightly more fit than the subtype A and dramatically more fit than the 12 subtype C isolates. All group M isolates were more fit (ex vivo) than the group O isolates. To estimate ex vivo transmission fitness, a subset of primary HIV-1 isolates were examined in primary human explants from penile, cervical, and rectal tissues. Only R5 isolates and no X4 HIV-1 isolates could replicate in these tissues, whereas the spread to PM1 cells was dependent on active replication and passive virus transfer. In tissue competition experiments, subtype C isolates could compete with and, in some cases, even win over subtype A and D isolates. However, when the migratory cells from infected tissues were mixed with a susceptible cell line, the subtype C isolates were outcompeted by other subtypes, as observed in experiments with PBMC. These findings suggest that subtype C HIV-1 isolates might have equal transmission fitness but reduced pathogenic fitness relative to other group M HIV-1 isolates.

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

Sensitive Deep Sequencing-based HIV-1 Genotyping Assay to Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as well as HIV-1 Coreceptor Tropism

ABSTRACT With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals.

A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates

Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinetics, or to develop personalized vaccines. Extreme HIV-1 heterogeneity leaves few restriction enzyme sites for bacterial cloning strategies. In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus species such as hepatitis C virus and influenza virus.

RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells.

We have identified a mutant of the human G-protein Cdc42Hs, R66E, that fails to form a detectable complex with the GDP-dissociation inhibitor RhoGDI in cell-free systems or in intact cells. This point mutant is prenylated, binds guanine nucleotide and interacts with GTPase-activating protein in a manner indistinguishable from wild-type Cdc42Hs. Immunofluorescence localization studies revealed that this RhoGDI-binding-defective mutant is found predominantly in the Golgi apparatus, with a staining pattern similar to that of the wild-type protein. However, unlike wild-type Cdc42Hs, which is distributed in both the microsomal membrane and cytosolic fractions, studies using differential centrifugation show that prenylated R66E Cdc42Hs is found exclusively in association with lipid bilayers. Additionally, whereas the overexpression of RhoGDI results in an apparent translocation of wild-type Cdc42Hs from the Golgi apparatus into the cytosol, identical RhoGDI-overexpression conditions do not alter the Golgi localization of the R66E mutant. Furthermore, overexpression of this RhoGDI-binding-defective mutant of Cdc42Hs seems to activate redistribution of the actin cytoskeleton and filopodia formation in fibroblasts in a manner indistinguishable from the wild-type protein. Taken together, these results suggest that the interaction of Cdc42Hs with RhoGDI is not essential for proper membrane targeting of nascent prenylated Cdc42Hs in mammalian cells; neither is this interaction an essential part of the mechanism by which Cdc42Hs activates filopodia formation. However, it does seem that redistribution of Cdc42Hs to the cytosolic compartment is absolutely dependent on RhoGDI interaction.

An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation.

Past, present, and future of entry inhibitors as HIV microbicides.

Preventing the transmission of human immunodeficiency virus (HIV) is the main goal of numerous studies trying to develop an effective vaccine and microbicide agents. Here we review the use of antiretroviral drugs to inhibit viral entry as potential HIV microbicides. After the failure of nonoxynol-9 microbicide strategies shifted towards the use of compounds creating a physical barrier to virus attachment (e.g., surfactants) or inhibit the virus in the vaginal milieu (e.g., polyanions). These early, non-specific inhibitors showed promise in both in vitro and in vivo(non-human primates) studies but provided only modest protection from HIV transmission in clinical efficacy trials. The next generation of HIV entry microbicides was based on specifically blocking virus from entering host cells by targeting CD4 attachment, gp120 binding, and virus-cell membrane fusion events. Although protection from an SIV-HIV hybrid was evident in non-human primates treated and challenged in the vaginal cavity, none of these compounds have advanced to clinical trials as a microbicide. Here we will discuss the reasons for these failures, including the selection of drug resistant HIV variants, which raises questions as to the future of broadly effective microbicides based on HIV entry inhibitors. The outcome of continued research and potential efficacy trials on the next generation of entry inhibitors might reveal whether or not an effective entry microbicide can be developed.

Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

ABSTRACT CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy

ABSTRACT Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.

Next-Generation Sequencing to Help Monitor Patients Infected with HIV: Ready for Clinical Use?

Given the extreme variability of the human immunodeficiency virus (HIV) and its ability to replicate as complex viral populations, HIV variants with reduced susceptibility to antiretroviral drugs or with specific coreceptor tropism (CCR5 and/or CXCR4) may be present as minority members of the viral quasispecies. The sensitivity of current HIV genotypic or phenotypic assays is limited, and thus, these tests usually fail to detect low-abundance viral variants. Next-generation (deep) sequencing (NGS) produces an enormous amount of information that allows the detection of minority HIV variants at levels unimaginable using standard Sanger sequencing. NGS technologies continue to evolve, opening new and more affordable opportunities to implement this methodology in clinical laboratories, and HIV is not an exception. The ample use of a battery of more effective antiretroviral drugs, together with careful patient monitoring based on HIV resistance testing, has resulted in HIV-infected patients whose disease is usually well-controlled. The vast majority of adherent patients without detectable resistance become virologically suppressed; however, a subset of these patients with undetectable resistance by standard methods may fail antiretroviral therapy, perhaps due to the presence of minority HIV-resistant variants. Novel NGS-based HIV assays with increased sensitivity for identifying low-level drug resistance and/or coreceptor tropism may play an important role in the success of antiretroviral treatments.