Marcelo A. Soares

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

HIV-1 subtype C dissemination in southern Brazil

Objectives:To describe the molecular and epidemiological profile of HIV-1 in patients followed at the University Hospital of Rio Grande, Brazil. Design and methods:A cross-sectional study was conducted from September to December 2002. Plasma viral RNA of 85 patients was extracted and protease and reverse transcriptase genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined to antiretroviral resistance mutations. Laboratory data and past history of antiretroviral treatment were also collected. Results:Most viruses were either subtype B (42%) or subtype C (45%). No risk behaviour, sexual orientation or laboratory parameter was associated with any specific subtype, but subtype C tended to be more frequently found in women (P = 0.06). The prevalence of subtype C has increased over the HIV/AIDS epidemic, accounting for almost 60% of cases diagnosed in 2002. Intra-subtype genetic distances were smaller in subtype C than in subtype B, suggesting a more recent introduction of the former in the epidemic. Of patients under treatment, 60% had at least one antiretroviral drug resistance mutation, but no mutation was specifically associated with any HIV-1 subtype. Only one resistance mutation each was found in drug-naive patients with subtypes B and C. Conclusion:Despite the fact that subtype C appeared in southern Brazil more recently than subtype B, it is now the predominant strain in Rio Grande. The epidemic spread of subtype C could be taking place in Brazil, and possibly in south America, a phenomenon similar to that seen in other countries where this subtype is now totally dominant.

Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent

ABSTRACT The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.

Analysis of HIV-1 pol sequences using Bayesian Networks: implications for drug resistance

Human Immunodeficiency Virus-1 (HIV-1) antiviral resistance is a major cause of antiviral therapy failure and compromises future treatment options. As a consequence, resistance testing is the standard of care. Because of the high degree of HIV-1 natural variation and complex interactions, the role of resistance mutations is in many cases insufficiently understood. We applied a probabilistic model, Bayesian networks, to analyze direct influences between protein residues and exposure to treatment in clinical HIV-1 protease sequences from diverse subtypes. We can determine the specific role of many resistance mutations against the protease inhibitor nelfinavir, and determine relationships between resistance mutations and polymorphisms. We can show for example that in addition to the well-known major mutations 90M and 30N for nelfinavir resistance, 88S should not be treated as 88D but instead considered as a major mutation and explain the subtype-dependent prevalence of the 30N resistance pathway.

Epidemiologic and molecular characterization of human immunodeficiency virus type 1 in Southern Brazil

HIV subtype C is the most prevalent subtype in the world. Despite its recent expansion in Brazil, HIV-1C already prevails in the southernmost state of Brazil, Rio Grande do Sul. This unique HIV epidemiology has prompted us to characterize that population. Seventy-seven HIV-1–infected subjects attending the largest HIV/AIDS clinic of the state had the protease and reverse transcriptase (RT) genes of their virus subtyped and genotyped. When subtype-specific infections were plotted according to year of diagnosis, the prevalence of subtype C was shown to increase over the last 18 years of the epidemic, along with a concomitant decrease of subtype B. Comparison of subtype C–infected treated and untreated subjects revealed amino acid differences in protease and RT, especially in the RT mutation D/G123S. The overall analysis of drug resistance mutations in viruses from treated subjects has highlighted some associations between subtypes and particular mutations, such as V82A/F/T/S in protease and subtype F1 and M41L and L210W in RT and subtype B. The characterization of this important population, which is one of a few in the developing world where a large number of HIV-1C–infected subjects are under antiretroviral treatment, underscores its potential usefulness in clinical, treatment, and vaccine trials in Brazil.

Human Papillomavirus E6 Triggers Upregulation of the Antiviral and Cancer Genomic DNA Deaminase APOBEC3B

ABSTRACT Several recent studies have converged upon the innate immune DNA cytosine deaminase APOBEC3B (A3B) as a significant source of genomic uracil lesions and mutagenesis in multiple human cancers, including those of the breast, head/neck, cervix, bladder, lung, ovary, and other tissues. A3B is upregulated in these tumor types relative to normal tissues, but the mechanism is unclear. Because A3B also has antiviral activity in multiple systems and is a member of the broader innate immune response, we tested the hypothesis that human papillomavirus (HPV) infection causes A3B upregulation. We found that A3B mRNA expression and enzymatic activity were upregulated following transfection of a high-risk HPV genome and that this effect was abrogated by inactivation of E6. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause A3B upregulation, and a panel of high-risk E6 proteins triggered higher A3B levels than did a panel of low-risk or noncancer E6 proteins. Knockdown experiments in HPV-positive cell lines showed that endogenous E6 is required for A3B upregulation. Analyses of publicly available head/neck cancer data further support this relationship, as A3B levels are higher in HPV-positive cancers than in HPV-negative cancers. Taken together with the established role for high-risk E6 in functional inactivation of TP53 and published positive correlations in breast cancer between A3B upregulation and genetic inactivation of TP53, our studies suggest a model in which high-risk HPV E6, possibly through functional inactivation of TP53, causes derepression of A3B gene transcription. This would lead to a mutator phenotype that explains the observed cytosine mutation biases in HPV-positive head/neck and cervical cancers. IMPORTANCE The innate immune DNA cytosine deaminase APOBEC3B (A3B) accounts for a large proportion of somatic mutations in cervical and head/neck cancers, but nothing is known about the mechanism responsible for its upregulation in these tumor types. Almost all cervical carcinomas and large proportions of head/neck tumors are caused by human papillomavirus (HPV) infection. Here, we establish a mechanistic link between HPV infection and A3B upregulation. The E6 oncoprotein of high-risk, but not low-risk, HPV types triggers A3B upregulation, supporting a model in which TP53 inactivation causes a derepression of A3B gene transcription and elevated A3B enzyme levels. This virus-induced mutator phenotype provides a mechanistic explanation for A3B signature mutations observed in HPV-positive head/neck and cervical carcinomas and may also help to account for the preferential cancer predisposition caused by high-risk HPV isolates. The innate immune DNA cytosine deaminase APOBEC3B (A3B) accounts for a large proportion of somatic mutations in cervical and head/neck cancers, but nothing is known about the mechanism responsible for its upregulation in these tumor types. Almost all cervical carcinomas and large proportions of head/neck tumors are caused by human papillomavirus (HPV) infection. Here, we establish a mechanistic link between HPV infection and A3B upregulation. The E6 oncoprotein of high-risk, but not low-risk, HPV types triggers A3B upregulation, supporting a model in which TP53 inactivation causes a derepression of A3B gene transcription and elevated A3B enzyme levels. This virus-induced mutator phenotype provides a mechanistic explanation for A3B signature mutations observed in HPV-positive head/neck and cervical carcinomas and may also help to account for the preferential cancer predisposition caused by high-risk HPV isolates.

Synonymous Genetic Polymorphisms within Brazilian Human Immunodeficiency Virus Type 1 Subtypes May Influence Mutational Routes to Drug Resistance

BACKGROUND Most published data on antiretroviral-drug resistance is generated from in vitro or in vivo studies of subtype B virus. However, this subtype is associated with <10% of HIV infections worldwide, and it is essential to explore subtype-specific determinants of drug resistance. One potential cause of the differences between subtypes is the synonymous codon usage at key resistance positions. METHODS We investigated the nucleotide sequences at drug resistance-related sites, for all major Brazilian subtypes (B, C, and F1) of human immunodeficiency virus type 1 (HIV-1) group M. RESULTS We identified a change at positions 151 and 210 of the reverse-transcriptase region in subtype F1, such that the emergence of these key nucleoside/nucleotide analogue resistance mutations required an extra nucleotide change in subtype F1, compared with subtypes B and C. The clinical significance of position 210 was confirmed within a large Brazilian database, in which we identified a lower prevalence of the L210W mutation in subtype F1 virus, compared with subtype B virus, in patients matched for thymidine-analogue experience. An inverse relationship between the L210W and K70R mutations was also observed. CONCLUSIONS The findings of the present study illustrate an important mechanism by which a subtype may determine genetic routes to resistance, with implications for treatment strategies for populations infected with HIV-1 subtype F.

Different epidemic potentials of the HIV-1B and C subtypes.

HIV, the cause of AIDS in humans, is characterized by great genetic heterogeneity. In particular, HIV-1 group M subtypes are responsible for most of the infections worldwide. We investigate the demographic history of HIV-1B and HIV-1C subtypes in South Africa and Brazil using both a parametric and a nonparametric approach based on coalescent theory. Our results show that although both subtypes are spreading exponentially in Brazil, the HIV-1C growth rate is about twice that of Brazilian HIV-1B or South African HIV-1C, providing evidence, for the first time, of a different epidemic potential between two HIV-1 subtypes. The present study not only may have important consequences for devising future vaccination and therapeutic strategies, but also offers additional evidence that skyline plots are indeed a simple and powerful tool for monitoring and predicting the behavior of viral epidemics.

Differential Drug Resistance Acquisition in HIV-1 of Subtypes B and C

Background Subtype C is the most prevalent HIV-1 subtype in the world, mainly in countries with the highest HIV prevalence. However, few studies have evaluated the impact of antiretroviral therapy on this subtype. In southern Brazil, the first developing country to offer free and universal treatment, subtypes B and C co-circulate with equal prevalence, allowing for an extensive evaluation of this issue. Methods and Findings Viral RNA of 160 HIV-1+ patients was extracted, and the protease and reverse transcriptase genes were sequenced, subtyped and analyzed for ARV mutations. Sequences were grouped by subtype, and matched to type (PI, NRTI and NNRTI) and time of ARV exposure. Statistical analyses were performed to compare differences in the frequency of ARV-associated mutations. There were no significant differences in time of treatment between subtypes B and C groups, although they showed distinct proportions of resistant strains at different intervals for two of three ARV classes. For PI, 26% of subtype B strains were resistant, compared to only 8% in subtype C (p = 0.0288, Fishers exact test). For NRTI, 54% of subtype B strains were resistant versus 23% of subtype C (p = 0.0012). Differences were significant from 4 years of exposure, and remained so until the last time point analyzed. The differences observed between both subtypes were independent of time under rebound viremia in cases of virologic failure and of the number of HAART regimens used by treated patients. Conclusions Our results pointed out to a lower rate of accumulation of mutations conferring resistance to ARV in subtype C than in subtype B. These findings are of crucial importance for current initiatives of ARV therapy roll-out in developing countries, where subtype is C prevalent.

Conservation Patterns of HIV-1 RT Connection and RNase H Domains: Identification of New Mutations in NRTI-Treated Patients

Background Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fishers exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.