Yolanda Lie

A Novel Phenotypic Drug Susceptibility Assay for Human Immunodeficiency Virus Type 1

ABSTRACT Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Design of a Potent d-Peptide HIV-1 Entry Inhibitor with a Strong Barrier to Resistance

ABSTRACT The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d-Peptides (peptides composed of d-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.

Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure

BackgroundClinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naïve patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naïve patients to date.ResultsWe now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication.ConclusionsSince H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug.

A Public Health Model for the Molecular Surveillance of HIV Transmission in San Diego, California

Background:Current public health efforts often use molecular technologies to identify and contain communicable disease networks, but not for HIV. Here, we investigate how molecular epidemiology can be used to identify highly related HIV networks within a population and how voluntary contact tracing of sexual partners can be used to selectively target these networks. Methods:We evaluated the use of HIV-1 pol sequences obtained from participants of a community-recruited cohort (n = 268) and a primary infection research cohort (n = 369) to define highly related transmission clusters and the use of contact tracing to link other individuals (n = 36) within these clusters. The presence of transmitted drug resistance was interpreted from the pol sequences (Calibrated Population Resistance v3.0). Results:Phylogenetic clustering was conservatively defined when the genetic distance between any two pol sequences was less than 1%, which identified 34 distinct transmission clusters within the combined community-recruited and primary infection research cohorts containing 160 individuals. Although sequences from the epidemiologically linked partners represented approximately 5% of the total sequences, they clustered with 60% of the sequences that clustered from the combined cohorts (odds ratio 21.7; P ≤ 0.01). Major resistance to at least one class of antiretroviral medication was found in 19% of clustering sequences. Conclusion:Phylogenetic methods can be used to identify individuals who are within highly related transmission groups, and contact tracing of epidemiologically linked partners of recently infected individuals can be used to link into previously defined transmission groups. These methods could be used to implement selectively targeted prevention interventions.

HLA-DR+ CD38+ CD4+ T Lymphocytes Have Elevated CCR5 Expression and Produce the Majority of R5-Tropic HIV-1 RNA In Vivo

ABSTRACT Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR+ CD38+ (DR+ 38+) CD4+ T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR− 38+ T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5+ cells were elevated in DR+ 38+ CD4+ T cells (median, 36.4%) compared to other CD4+ T-cell subsets (median values of 5.7% for DR− 38− cells, 19.4% for DR+ 38− cells, and 7.6% for DR− 38+ cells; n = 18; P < 0.001). In sorted CD8− lymph node T cells, median HIV-1 RNA copies/105 cells was higher for DR+ 38+ cells (1.8 × 106) than for DR− 38− (0.007 × 106), DR− 38+ (0.064 × 106), and DR+ 38− (0.18 × 106) subsets (n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR+ 38+ cells. Percentages of CCR5+ CD4+ T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8− DR/38 subsets (P < 0.001 for both). Median HIV-1 DNA copies/105 cells was higher in DR+ 38+ cells (5,360) than in the DR− 38− (906), DR− 38+ (814), and DR+ 38− (1,984) subsets (n = 7; P ≤ 0.031). Thus, DR+ 38+ CD4+ T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR+ 38+ CD4+ T cells may substantially inhibit HIV-1 replication.

Prevalence of Primary HIV-1 Drug Resistance among Recently Infected Adolescents: A Multicenter Adolescent Medicine Trials Network for HIV/AIDS Interventions Study

This study examined the prevalence of primary human immunodeficiency type 1 (HIV-1) drug resistance among recently infected youth in the United States. Of the 55 subjects studied, major mutations conferring HIV drug resistance were present in 10 (18%). Eight (15%) had nonnucleoside reverse-transcriptase inhibitor (NNRTI) mutations, with the majority (6) having the K103N mutation; 2 (4%) had nucleoside reverse-transcriptase inhibitor (NRTI) mutations; and 2 (4%) had protease inhibitor (PI) mutations. Phenotypic drug resistance was present in 12 (22%) subjects: 10 (18%) for NNRTIs, 2 (4%) for NRTIs, and 3 (5.5%) for PIs. The prevalence of primary HIV-1 drug resistance, particularly to NNRTIs, in this group of recently infected youth was high.

Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. Continuous-flow ultracentrifugation has been used to concentrate extracellular particles from culture fluid of a recombinant CHO cell subclone for molecular characterization. Particles exhibiting reverse transcriptase activity and associated with mammalian C-type retrovirus structural proteins banded in sucrose gradients at a density characteristic of retroviruses. Examination of gradient-purified particles by electron microscopy revealed morphology and size similar to other retroviruses. Double-gradient-purified particles contained RNA which hybridized to probes for murine leukemia virus, and endogenous Chinese hamster intracisternal A-particle elements. DNA sequence analysis of a cDNA clone isolated from purified particles revealed multiple interruptions of the endonuclease reading frame, providing one possible explanation for the noninfectious nature of the observed particles. Sequences present as RNA in purified particles were also present as conserved, repetitive, provirus sequences in genomic DNA of all CHO cell lines examined and in Chinese hamster liver DNA. The observed particles are therefore likely to be the products of endogenous retroviruslike elements present in the germline of Chinese hamsters.

Phenotypic changes in drug susceptibility associated with failure of human immunodeficiency virus type 1 (HIV-1) triple combination therapy

The emergence of drug-resistant human immunodeficiency virus type 1 is a frequent cause of failure of combination therapies comprising reverse transcriptase and protease inhibitors. Rational design of salvage therapies requires new methods to assess drug susceptibility. A novel phenotypic drug susceptibility assay was developed and used to measure the drug susceptibilities of viruses obtained from 2 patients treated with zidovudine, lamivudine, and nelfinavir. Results showed that phenotypic drug resistance may be detectable before virus load rebound, treatment failure does not always imply viral resistance to all drugs in a treatment regimen, and persons with similar antiviral treatment histories and clinical courses may have different phenotypic drug resistance profiles at the time that treatment fails.

Clinical Utility of HIV Standard Genotyping among Antiretroviral-Naive Individuals with Unknown Duration of Infection

In clinical settings, we have found a high rate of human immunodeficiency virus (HIV) drug resistance among antiretroviral-naive patients for whom the duration of infection was unknown. These high rates were most likely the result of both transmitted resistance and informal antiretroviral use, and they suggest that routine resistance testing among antiretroviral-naive patients would be a cost-effective clinical practice.

Relationship between HIV coreceptor tropism and disease progression in persons with untreated chronic HIV infection.

Objective:To assess the effect of HIV coreceptor tropism (CRT) on the relative risk of progression to a composite outcome of CD4+ count ≤350 cells per microliter, treatment initiation, or death. Methods:CRT assays were performed after study closure in baseline samples obtained from enrollees in a prospectively monitored cohort of treatment-naive adults with ≥450 CD4+ cells per microliter and ≥1000 HIV-1 RNA copies per milliliter. Results:Dual/mixed (D/M) and R5 CRT were detected in 32 and 282 patients, respectively. The baseline CD4+ count (617 versus 694 cells/μL; P = 0.05) differed in patients with D/M versus R5 CRT. Otherwise, baseline laboratory characteristics were similar.The relative risk of progression to the composite end point was 2.15 (P = 0.002) for D/M versus R5 CRT, 2.07 per 1.0 log10 higher viral load (P < 0.001) and 0.87 per 50 cells per microliter higher CD4+ cell count (P < 0.001). The effect of D/M CRT was also significant in separate analyses of time to initiation of antiretroviral therapy or CD4+ cell count ≤350 cells per microliter. Conclusions:Untreated patients with D/M rather than R5 CRT had a faster rate of disease progression, whether assessed by a composite outcome of time to CD4+ count ≤350 cells per microliter, treatment initiation, or death or by separate analyses of time to CD4+ count ≤350 cells per microliter or treatment initiation.