Clonal analysis of HIV-1 genotype and function associated with virologic failure in treatment-experienced persons receiving maraviroc: Results from the MOTIVATE phase 3 randomized, placebo-controlled trials.

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated.

Prevalence of Darunavir Resistance in the United States from 2010 to 2017.

The emergence and transmission of antiretroviral drug resistance have been and remain a concern among people living with human immunodeficiency virus (HIV)-1 infection. The protease inhibitor (PI) darunavir has been approved for use in the United States for more than 10 years and has demonstrated a high barrier to resistance. Previous analyses identified significant reductions in the prevalence of samples with darunavir resistance-associated mutations (RAMs) and with phenotypic resistance to darunavir and other PIs between 2006 and 2012.

Detailed Transmission Network Analysis of a Large Opiate-Driven Outbreak of HIV Infection in the United States.

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak.

Geographic Disparities in Access to Syringe Services Programs Among Young Persons With Hepatitis C Virus Infection in the United States.

Using commercial laboratory data, we found 80% of 29382 young persons currently infected with hepatitis C virus lived >10 miles from a syringe services program. The median distance was 37 miles, with greater distances in rural areas and Southern and Midwestern states. Strategies to improve access to preventive services are warranted.

A decade of HIV-1 drug resistance in the United States: trends and characteristics in a large protease/reverse transcriptase and co-receptor tropism database from 2003 to 2012.

Background: Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database.

Methods: Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated.

A case-based reasoning system for genotypic prediction of HIV-1 co-receptor tropism.

Accurate co-receptor tropism (CRT) determination is critical for making treatment decisions in HIV management. We created a genotypic tropism prediction tool by utilizing the case-based reasoning (CBR) technique that attempts to solve new problems through applying the solution from similar past problems. V3 loop sequences from 732 clinical samples with diverse characteristics were used to build a case library.

Prediction of virological response and assessment of resistance emergence to the HIV-1 attachment inhibitor BMS-626529 during 8-day monotherapy with its prodrug BMS-663068.

Background: BMS-663068 is the phosphonooxymethyl prodrug of BMS-626529, a small-molecule attachment inhibitor that targets the HIV-1 envelope glycoprotein gp120 preventing it from binding to CD4 T cells. In vitro investigations have demonstrated considerable variation in susceptibility of different HIV-1 isolates to BMS-626529. BMS-663068 monotherapy in HIV-1-infected subjects produced a mean maximum change from baseline of -1.

Estimating the fitness cost of escape from HLA presentation in HIV-1 protease and reverse transcriptase.

Human immunodeficiency virus (HIV-1) is, like most pathogens, under selective pressure to escape the immune system of its host. In particular, HIV-1 can avoid recognition by cytotoxic T lymphocytes (CTLs) by altering the binding affinity of viral peptides to human leukocyte antigen (HLA) molecules, the role of which is to present those peptides to the immune system. It is generally assumed that HLA escape mutations carry a replicative fitness cost, but these costs have not been quantified.

Exploring the complexity of the HIV-1 fitness landscape.

Although fitness landscapes are central to evolutionary theory, so far no biologically realistic examples for large-scale fitness landscapes have been described. Most currently available biological examples are restricted to very few loci or alleles and therefore do not capture the high dimensionality characteristic of real fitness landscapes. Here we analyze large-scale fitness landscapes that are based on predictive models for in vitro replicative fitness of HIV-1.

Assessing predicted HIV-1 replicative capacity in a clinical setting.

HIV-1 replicative capacity (RC) provides a measure of within-host fitness and is determined in the context of phenotypic drug resistance testing. However it is unclear how these in-vitro measurements relate to in-vivo processes. Here we assess RCs in a clinical setting by combining a previously published machine-learning tool, which predicts RC values from partial pol sequences with genotypic and clinical data from the Swiss HIV Cohort Study.

A systems analysis of mutational effects in HIV-1 protease and reverse transcriptase.

The development of a quantitative understanding of viral evolution and the fitness landscape in HIV-1 drug resistance is a formidable challenge given the large number of available drugs and drug resistance mutations. We analyzed a dataset measuring the in vitro fitness of 70,081 virus samples isolated from HIV-1 subtype B infected individuals undergoing routine drug resistance testing. We assayed virus samples for in vitro replicative capacity in the absence of drugs as well as in the presence of 15 individual drugs.

Principal component analysis of general patterns of HIV-1 replicative fitness in different drug environments.

To detect general patterns and temporal trends of HIV-1 resistance, we apply principal component analysis (PCA) to in vitro fitness data. Twenty-eight thousand virus samples, obtained between 2002 and 2008, were assayed for fitness in 16 to 21 selective environments. Fitness measurements are based on replication capacity (RC), which quantifies in vitro viral replication in a single cycle of infection.

Loss of asparagine-linked glycosylation sites in variable region 5 of human immunodeficiency virus type 1 envelope is associated with resistance to CD4 antibody ibalizumab.

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab.

Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens.

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies.

Mutational pathways and genetic barriers to CXCR4-mediated entry by human immunodeficiency virus type 1.

To examine mutational pathways that lead to CXCR4 use of HIV-1, we analyzed the genotypic and phenotypic characteristics of envelope sequences from a large panel of patient virus populations and individual clones containing different V3 mutations. Basic amino acid substitutions at position 11 were strong determinants of CXCR4-mediated entry but required multiple compensatory mutations to overcome associated reductions in infectivity. In contrast, basic amino acid substitutions at position 25, or substitutions at positions 6-8 resulting in the loss of a potential N-linked glycosylation site, contributed to CXCR4-mediated entry but required additional substitutions acting cooperatively to confer efficient CXCR4 use.

Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN.

Dual-tropic HIV type 1 isolates vary dramatically in their utilization of CCR5 and CXCR4 coreceptors.

Objective(s): Dual HIV-1 utilizes cellular CCR5 and CXCR4 coreceptors to enter host cells. Recent studies indicate that the ability of these viruses to use both coreceptors varies significantly in cell lines expressing CXCR4 or CCR5; however, it is not clear whether differences in coreceptor mediated infection in vitro reflect infection of primary cells in vivo.

Methods: We evaluated coreceptor usage of dual envelope clones from patient viruses using a single-cycle pseudovirus assay conducted in cell lines and a replication-competent assay performed using peripheral blood mononuclear cells.

Characterization of emergent HIV resistance in treatment-naive subjects enrolled in a vicriviroc phase 2 trial.

Background: Vicriviroc is a C-C motif chemokine receptor 5 (CCR5) antagonist that is in clinical development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This study explored the molecular basis for the development of phenotypically resistant virus.

Method: HIV-1 RNA from treatment-naive subjects who experienced virological failure in a phase 2 dose-finding trial was evaluated for coreceptor usage and susceptibility.

Characterization of human immunodeficiency virus type 1 populations containing CXCR4-using variants from recently infected individuals.

We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically.

Vertical transmission of X4-tropic and dual-tropic HIV-1 in five Ugandan mother-infant pairs.

Background: We previously reported the existence of CXCR4-using HIV-1 in 6-14 week-old Ugandan infants. Whether these viruses were transmitted from the mother perinatally or evolved after transmission is not known. In the current study, we investigated the origin of the CXCR4-using viruses in these infants by comparing HIV-1 envelope clones from the infants to those from their mothers at or near the time of delivery.

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody.

Suppression of dualtropic human immunodeficiency virus type 1 by the CXCR4 antagonist AMD3100 is associated with efficiency of CXCR4 use and baseline virus composition.

In a phase I/II evaluation of the CXCR4 antagonist AMD3100, human immunodeficiency virus RNA levels were significantly reduced in a single study subject who harbored CXCR4 (X4)-tropic virus, but not in subjects who harbored either dual/mixed (DM)-tropic or CCR5 (R5)-tropic virus (C. W. Hendrix et al.

Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein.

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants.

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations.

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial).

Virologic characterization of HIV type 1 with a codon 70 deletion in reverse transcriptase.

We identified a deletion at codon 70 (Delta70) of HIV-1 reverse transcriptase (RT) occurring together with L74V and Q151M mutations in a sample from a tenofovir (TFV)- and abacavir (ABC)-treated patient with extensive prior antiretroviral treatment. To investigate the characteristics of this mutant, we studied the drug susceptibility, relative infectivity, and fitness of viruses carrying Delta70 and associated RT mutations. The Delta70, L74V, and Q151M mutations were introduced into Hxb2 RT by site-directed mutagenesis and expressed in HIV-1 recombinants.