Dana N. Raugi

Genetic impact of vaccination on breakthrough HIV-1 sequences from the STEP trial

We analyzed HIV-1 genome sequences from 68 newly infected volunteers in the STEP HIV-1 vaccine trial. To determine whether the vaccine exerted selective T cell pressure on breakthrough viruses, we identified potential T cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances to the vaccine sequence for sequences from vaccine recipients than from placebo recipients. The most significant signature site distinguishing vaccine from placebo recipients was Gag amino acid 84, a site encompassed by several epitopes contained in the vaccine and restricted by human leukocyte antigen (HLA) alleles common in the study cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (HIV-1 Gag, Pol and Nef) and not found in other HIV-1 proteins. These results represent what is to our knowledge the first evidence of selective pressure from vaccine-induced T cell responses on HIV-1 infection in humans.

HLA Class I-Driven Evolution of Human Immunodeficiency Virus Type 1 Subtype C Proteome: Immune Escape and Viral Load

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) mutations that confer escape from cytotoxic T-lymphocyte (CTL) recognition can sometimes result in lower viral fitness. These mutations can then revert upon transmission to a new host in the absence of CTL-mediated immune selection pressure restricted by the HLA alleles of the prior host. To identify these potentially critical recognition points on the virus, we assessed HLA-driven viral evolution using three phylogenetic correction methods across full HIV-1 subtype C proteomes from a cohort of 261 South Africans and identified amino acids conferring either susceptibility or resistance to CTLs. A total of 558 CTL-susceptible and -resistant HLA-amino acid associations were identified and organized into 310 immunological sets (groups of individual associations related to a single HLA/epitope combination). Mutations away from seven susceptible residues, including four in Gag, were associated with lower plasma viral-RNA loads (q < 0.2 [where q is the expected false-discovery rate]) in individuals with the corresponding HLA alleles. The ratio of susceptible to resistant residues among those without the corresponding HLA alleles varied in the order Vpr > Gag > Rev > Pol > Nef > Vif > Tat > Env > Vpu (Fishers exact test; P ≤ 0.0009 for each comparison), suggesting the same ranking of fitness costs by genes associated with CTL escape. Significantly more HLA-B (χ2; P = 3.59 × 10−5) and HLA-C (χ2; P = 4.71 × 10−6) alleles were associated with amino acid changes than HLA-A, highlighting their importance in driving viral evolution. In conclusion, specific HIV-1 residues (enriched in Vpr, Gag, and Rev) and HLA alleles (particularly B and C) confer susceptibility to the CTL response and are likely to be important in the development of vaccines targeted to decrease the viral load.

Demographic Processes Affect HIV-1 Evolution in Primary Infection before the Onset of Selective Processes

ABSTRACT HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.

Amino-Acid Co-Variation in HIV-1 Gag Subtype C: HLA-Mediated Selection Pressure and Compensatory Dynamics

Background Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C. Methodology/Principal Findings Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites. Conclusions/Significance Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints.

HIV-2 Integrase Variation in Integrase Inhibitor-Naïve Adults in Senegal, West Africa

Background Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance. Methods We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1. Results No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein. Conclusion Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.

Phenotypic susceptibility of HIV-2 to raltegravir: integrase mutations Q148R and N155H confer raltegravir resistance.

Objectives:Raltegravir is the first integrase strand transfer inhibitor approved for treating HIV-1 infection. Although emerging data suggest that raltegravir may also be useful for HIV-2 treatment, studies addressing the in-vitro susceptibility of HIV-2 to raltegravir are scarce, and the genetic pathways leading to raltegravir resistance in HIV-2 have not been adequately characterized. Our objectives were to directly compare the susceptibilities of HIV-1 and HIV-2 to raltegravir and to examine the role of mutations in HIV-2 integrase in emergent raltegravir resistance. Materials and methods:Single-cycle and spreading infection assays were used to quantify the sensitivities of wild-type HIV-1 and HIV-2 strains to raltegravir. HIV-2 integrase mutants were constructed by site-directed mutagenesis, and the replication capacities and raltegravir susceptibilities of the resultant variants were analyzed in single-cycle assays. Results:Raltegravir showed comparable activity against wild-type HIV-1 and HIV-2 in both single-cycle and spreading infections, with EC50 values in the low nanomolar range. Amino acid changes Q148R and N155H individually conferred resistance to raltegravir (14-fold and seven-fold, respectively), whereas the Y143C replacement had no statistically significant effect on raltegravir sensitivity. The combination of Q148R with N155H resulted in high-level raltegravir resistance (>1000-fold). In addition, all HIV-2 integrase variants tested showed impairments in replication capacity. Conclusion:Our data support clinical studies of raltegravir for treating HIV-2 infection and show that the Q148R and N155H changes alone are sufficient for raltegravir resistance in HIV-2. Further efforts are needed to improve access to HIV-2-active antiretrovirals, including raltegravir, in resource-limited areas where HIV-2 is endemic.

Complex patterns of protease inhibitor resistance among antiretroviral treatment-experienced HIV-2 patients from Senegal: Implications for second-line therapy

ABSTRACT Protease inhibitor (PI)-based antiretroviral therapy (ART) can effectively suppress HIV-2 plasma load and increase CD4 counts; however, not all PIs are equally active against HIV-2, and few data exist to support second-line therapy decisions. To identify therapeutic options for HIV-2 patients failing ART, we evaluated the frequency of PI resistance-associated amino acid changes in HIV-2 sequences from a cohort of 43 Senegalese individuals receiving unboosted indinavir (n = 18 subjects)-, lopinavir/ritonavir (n = 4)-, or indinavir and then lopinavir/ritonavir (n = 21)-containing ART. Common protease substitutions included V10I, V47A, I54M, V71I, I82F, I84V, L90M, and L99F, and most patients harbored viruses containing multiple changes. Based on genotypic data, we constructed a panel of 15 site-directed mutants of HIV-2ROD9 containing single- or multiple-treatment-associated amino acid changes in the protease-encoding region of pol. We then quantified the susceptibilities of the mutants to the HIV-2 “active” PIs saquinavir, lopinavir, and darunavir using a single-cycle assay. Relative to wild-type HIV-2, the V47A mutant was resistant to lopinavir (6.3-fold increase in the mean 50% effective concentration [EC50]), the I54M variant was resistant to darunavir and lopinavir (6.2- and 2.7-fold increases, respectively), and the L90M mutant was resistant to saquinavir (3.6-fold increase). In addition, the triple mutant that included I54M plus I84V plus L90M was resistant to all three PIs (31-, 10-, and 3.8-fold increases in the mean EC50 for darunavir, saquinavir, and lopinavir, respectively). Taken together, our data demonstrate that PI-treated HIV-2 patients frequently harbor viruses that exhibit complex patterns of PI cross-resistance. These findings suggest that sequential PI-based regimens for HIV-2 treatment may be ineffective.

Three Main Mutational Pathways in HIV-2 Lead to High- Level Raltegravir and Elvitegravir Resistance: Implications for Emerging HIV-2 Treatment Regimens

Human immunodeficiency virus type 2 (HIV-2) is intrinsically resistant to non-nucleoside reverse transcriptase inhibitors and exhibits reduced susceptibility to several of the protease inhibitors used for antiretroviral therapy of HIV-1. Thus, there is a pressing need to identify new classes of antiretroviral agents that are active against HIV-2. Although recent data suggest that the integrase strand transfer inhibitors raltegravir and elvitegravir may be beneficial, mutations that are known to confer resistance to these drugs in HIV-1 have been reported in HIV-2 sequences from patients receiving raltegravir-containing regimens. To examine the phenotypic effects of mutations that emerge during raltegravir treatment, we constructed a panel of HIV-2 integrase variants using site-directed mutagenesis and measured the susceptibilities of the mutant strains to raltegravir and elvitegravir in culture. The effects of single and multiple amino acid changes on HIV-2 replication capacity were also evaluated. Our results demonstrate that secondary replacements in the integrase protein play key roles in the development of integrase inhibitor resistance in HIV-2. Collectively, our data define three major mutational pathways to high-level raltegravir and elvitegravir resistance: i) E92Q+Y143C or T97A+Y143C, ii) G140S+Q148R, and iii) E92Q+N155H. These findings preclude the sequential use of raltegravir and elvitegravir (or vice versa) for HIV-2 treatment and provide important information for clinical monitoring of integrase inhibitor resistance in HIV-2–infected individuals.

In vitro activity of dolutegravir against wild-type and integrase inhibitor-resistant HIV-2

BackgroundDolutegravir recently became the third integrase strand transfer inhibitor (INSTI) approved for use in HIV-1–infected individuals. In contrast to the extensive dataset for HIV-1, in vitro studies and clinical reports of dolutegravir for HIV-2 are limited. To evaluate the potential role of dolutegravir in HIV-2 treatment, we compared the susceptibilities of wild-type and INSTI-resistant HIV-1 and HIV-2 strains to the drug using single-cycle assays, spreading infections of immortalized T cells, and site-directed mutagenesis.FindingsHIV-2 group A, HIV-2 group B, and HIV-1 isolates from INSTI-naïve individuals were comparably sensitive to dolutegravir in the single-cycle assay (mean EC50 values = 1.9, 2.6, and 1.3 nM, respectively). Integrase substitutions E92Q, Y143C, E92Q + Y143C, and Q148R conferred relatively low levels of resistance to dolutegravir in HIV-2ROD9 (2- to 6-fold), but Q148K, E92Q + N155H, T97A + N155H and G140S + Q148R resulted in moderate resistance (10- to 46-fold), and the combination of T97A + Y143C in HIV-2ROD9 conferred high-level resistance (>5000-fold). In contrast, HIV-1NL4-3 mutants E92Q + N155H, G140S + Q148R, and T97A + Y143C showed 2-fold, 4-fold, and no increase in EC50, respectively, relative to the parental strain. The resistance phenotypes for E92Q + N155H, and G140S + Q148R HIV-2ROD9 were also confirmed in spreading infections of CEM-ss cells.ConclusionsOur data support the use of dolutegravir in INSTI-naïve HIV-2 patients but suggest that, relative to HIV-1, a broader array of replacements in HIV-2 integrase may enable cross-resistance between dolutegravir and other INSTI. Clinical studies are needed to evaluate the efficacy of dolutegravir in HIV-2–infected individuals, including patients previously treated with raltegravir or elvitegravir.

HIV-1 outcompetes HIV-2 in dually infected Senegalese individuals with low CD4+ cell counts

Objective:Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has implications for transmission, progression, and antiretroviral therapy (ART). Few studies have examined viral dynamics in this setting. Our objective was to directly compare HIV-1 and HIV-2 viral loads and to examine whether this relationship is associated with CD4+ cell count. Study design:This is a retrospective analysis of data from observational cohort studies. Methods:We compared HIV-1 and HIV-2 viral loads from 65 dually infected, ART-naive Senegalese individuals. Participants provided blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. We assessed relationships between HIV-1 and HIV-2 levels using linear regression with generalized estimating equations to account for multiple study visits. Results:After adjusting for CD4+ cell count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen, CVL, and oral fluids. Despite similar peripheral blood mononuclear cell DNA levels among individuals with CD4+ cell counts above 500 cells/&mgr;l, individuals with CD4+ cell counts below 500 cells/&mgr;l had higher HIV-1 and lower HIV-2 DNA levels. Individuals with high CD4+ cell counts had higher mean HIV-1 plasma RNA viral loads than HIV-2, with HIV-1 levels significantly higher and HIV-2 levels trending toward lower mean viral loads among individuals with low CD4+ cell counts. Conclusion:Our data are consistent with the hypothesis that with disease progression, HIV-1 outcompetes HIV-2 in dually infected individuals. This finding helps explain differences in prevalence and outcomes between HIV-1, HIV-2, and HIV-dual infection.