Hepatitis C Virus (HCV) NS3 Sequence Diversity and Antiviral Resistance-Associated Variant Frequency in HCV/HIV Coinfection

ABSTRACT HIV coinfection accelerates disease progression in chronic hepatitis C and reduces sustained antiviral responses (SVR) to interferon-based therapy. New direct-acting antivirals (DAAs) promise higher SVR rates, but the selection of preexisting resistance-associated variants (RAVs) may lead to virologic breakthrough or relapse. Thus, pretreatment frequencies of RAVs are likely determinants of treatment outcome but typically are below levels at which the viral sequence can be accurately resolved. Moreover, it is not known how HIV coinfection influences RAV frequency. We adopted an accurate high-throughput sequencing strategy to compare nucleotide diversity in HCV NS3 protease-coding sequences in 20 monoinfected and 20 coinfected subjects with well-controlled HIV infection. Differences in mean pairwise nucleotide diversity (π), Tajima's D statistic, and Shannon entropy index suggested that the genetic diversity of HCV is reduced in coinfection. Among coinfected subjects, diversity correlated positively with increases in CD4 + T cells on antiretroviral therapy, suggesting T cell responses are important determinants of diversity. At a median sequencing depth of 0.084%, preexisting RAVs were readily identified. Q80K, which negatively impacts clinical responses to simeprevir, was encoded by more than 99% of viral RNAs in 17 of the 40 subjects. RAVs other than Q80K were identified in 39 of 40 subjects, mostly at frequencies near 0.1%. RAV frequency did not differ significantly between monoinfected and coinfected subjects. We conclude that HCV genetic diversity is reduced in patients with well-controlled HIV infection, likely reflecting impaired T cell immunity. However, RAV frequency is not increased and should not adversely influence the outcome of DAA therapy

Cross-sectional characterization of HIV-1 env compartmentalization in cerebrospinal fluid over the full disease course

To characterize HIV-1 env compartmentalization between cerebrospinal fluid (CSF) and peripheral blood plasma over all stages of the HIV-1 disease course, and to determine the relationship between the extent of CSF HIV-1 env compartmentalization and clinical neurologic disease status

HIV-1 Populations in Semen Arise through Multiple Mechanisms

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus

HIV-1 Populations in Semen Arise through Multiple Mechanisms

HIV-1 is present in anatomical compartments and bodily fluids. Most transmissions occur through sexual acts, making virus in semen the proximal source in male donors. We find three distinct relationships in comparing viral RNA populations between blood and semen in men with chronic HIV-1 infection, and we propose that the viral populations in semen arise by multiple mechanisms including: direct import of virus, oligoclonal amplification within the seminal tract, or compartmentalization. In addition, we find significant enrichment of six out of nineteen cytokines and chemokines in semen of both HIV-infected and uninfected men, and another seven further enriched in infected individuals. The enrichment of cytokines involved in innate immunity in the seminal tract, complemented with chemokines in infected men, creates an environment conducive to T cell activation and viral replication. These studies define different relationships between virus in blood and semen that can significantly alter the composition of the viral population at the source that is most proximal to the transmitted virus

Enfuvirtide Resistance Mutations: Impact on Human Immunodeficiency Virus Envelope Function, Entry Inhibitor Sensitivity, and Virus Neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed

HIV-1 Transmission during Early Antiretroviral Therapy: Evaluation of Two HIV-1 Transmission Events in the HPTN 052 Prevention Study

In the HPTN 052 study, transmission between HIV-discordant couples was reduced by 96% when the HIV-infected partner received suppressive antiretroviral therapy (ART). We examined two transmission events where the newly infected partner was diagnosed after the HIV-infected partner (index) initiated therapy. We evaluated the sequence complexity of the viral populations and antibody reactivity in the newly infected partner to estimate the dates of transmission to the newly infected partners. In both cases, transmission most likely occurred significantly before HIV-1 diagnosis of the newly infected partner, and either just before the initiation of therapy or before viral replication was adequately suppressed by therapy of the index. This study further strengthens the conclusion about the efficacy of blocking transmission by treating the infected partner of discordant couples. However, this study does not rule out the potential for HIV-1 transmission to occur shortly after initiation of ART, and this should be recognized when antiretroviral therapy is used for HIV-1 prevention

HIV-1 transmission during early antiretroviral therapy: evaluation of two HIV-1 transmission events in the HPTN 052 prevention study.

In the HPTN 052 study, transmission between HIV-discordant couples was reduced by 96% when the HIV-infected partner received suppressive antiretroviral therapy (ART). We examined two transmission events where the newly infected partner was diagnosed after the HIV-infected partner (index) initiated therapy. We evaluated the sequence complexity of the viral populations and antibody reactivity in the newly infected partner to estimate the dates of transmission to the newly infected partners. In both cases, transmission most likely occurred significantly before HIV-1 diagnosis of the newly infected partner, and either just before the initiation of therapy or before viral replication was adequately suppressed by therapy of the index. This study further strengthens the conclusion about the efficacy of blocking transmission by treating the infected partner of discordant couples. However, this study does not rule out the potential for HIV-1 transmission to occur shortly after initiation of ART, and this should be recognized when antiretroviral therapy is used for HIV-1 prevention

Central Nervous System Compartmentalization of HIV-1 Subtype C Variants Early and Late in Infection in Young Children

<div><p>HIV-1 subtype B replication in the CNS can occur in CD4+ T cells or macrophages/microglia in adults. However, little is known about CNS infection in children or the ability of subtype C HIV-1 to evolve macrophage-tropic variants. In this study, we examined HIV-1 variants in ART-naïve children aged three years or younger to determine viral genotypes and phenotypes associated with HIV-1 subtype C pediatric CNS infection. We examined HIV-1 subtype C populations in blood and CSF of 43 Malawian children with neurodevelopmental delay or acute neurological symptoms. Using single genome amplification (SGA) and phylogenetic analysis of the full-length <em>env</em> gene, we defined four states: equilibrated virus in blood and CSF (n = 20, 47%), intermediate compartmentalization (n = 11, 25%), and two distinct types of compartmentalized CSF virus (n = 12, 28%). Older age and a higher CSF/blood viral load ratio were associated with compartmentalization, consistent with independent replication in the CNS. Cell tropism was assessed using pseudotyped reporter viruses to enter a cell line on which CD4 and CCR5 receptor expression can be differentially induced. In a subset of compartmentalized cases (n = 2, 17%), the CNS virus was able to infect cells with low CD4 surface expression, a hallmark of macrophage-tropic viruses, and intermediate compartmentalization early was associated with an intermediate CD4 entry phenotype. Transmission of multiple variants was observed for 5 children; in several cases, one variant was sequestered within the CNS, consistent with early stochastic colonization of the CNS by virus. Thus we hypothesize two pathways to compartmentalization: early stochastic sequestration in the CNS of one of multiple variants transmitted from mother to child, and emergence of compartmentalized variants later in infection, on average at age 13.5 months, and becoming fully apparent in the CSF by age 18 months. Overall, compartmentalized viral replication in the CNS occurred in half of children by year three.</p> </div

Evidence of multiple transmission events during pediatric HIV-1 subtype C infection.

<p>Phylogenetic and sequence analysis of plasma and CSF HIV-1 populations for subject 3002. (a) Neighbor-joining tree. Sequences from the CSF are labeled with solid blue circles, and plasma sequences (PL) are labeled with solid red triangles. Bootstrap values ≥40 are indicated (<b>*</b>) at the appropriate nodes. Genetic distance is scaled at the bottom of the figure (0.001) and indicates the number of nucleotide substitutions per site between <i>env</i> sequences. The subject's age is noted, as well as the overall TMRCA and the TMRCA of the two transmitted viruses. The CNS sequestered population is represented by an open black circle. (b) Highlighter plot of aligned <i>env</i> plasma and CSF sequences, generated at <a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>. The HXB2 nucleotide position number is indicated on the <i>x</i> axis, and the sequence identifier is indicated on the <i>y</i> axis. Nucleotide changes are indicated by the following ticks on the highlighter plot: A, green; T, red; G, orange; and C, blue. The two transmitted populations are separated by a heavy black line.</p

Older age and a higher CSF/blood viral load ratio are strong determining factors for compartmentalization.

<p>Relationships between clinical characteristics and genetic compartmentalization were assessed using the Mann-Whitney test in GraphPad Prism 4. Horizontal bars represent median values. A <i>P</i> value<0.05 was considered statistically significant. (a) Comparisons between age of the subjects and compartmentalization. (b) Comparisons between the CSF/blood viral load ratio of the subjects and compartmentalization.</p