Dario A. Dilernia

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.

Drug resistance testing provides evidence of the globalization of HIV type 1: a new circulating recombinant form.

To monitor HIV-1 diversity in Argentina, a phylogenetic-based analysis of HIV-1 partial pol sequences obtained for resistance testing in 587 treatment failure patients was performed in Buenos Aires city between 2001 and 2003. HIV-1 RNA was isolated from plasma samples and partial pol fragments amplified by RT-PCR. Sequences were obtained by automated sequencing. Phylogenetic analysis was performed and recombination patterns characterized. A total of 299 sequences grouped into clade B (50.94%) and 284 were B/F recombinants (48.38%). Four sequences were grouped into clades A, C, and F (0.68%). The clade C sample, 96105, was found to be a BC recombinant and samples 103396 and 104575 showed the same mosaic pattern with Kisii5009 from Kenya and 97KR004 from Korea, previously described as A2D recombinants. With the presence of two full-length genomes, one from Kenya and one from Korea, and now two partial genomes from Argentina, this recombinant is designated CRF16_A2D. Its presence on three continents shows that CRF16_A2D has a global distribution.

Drug-resistance surveillance among newly HIV-1 diagnosed individuals in Buenos Aires, Argentina

Objective:Our objective was to estimate primary resistance in an urban setting in a developing country with a long history of antiretroviral delivery and high coverage levels. Design:We carried out a resistance surveillance study according to WHO HIV-Resistance Guidelines. Methods:Blood samples were collected from 323 drug-naive HIV-1 infected individuals diagnosed at two HIV voluntary counselling and testing centers in Buenos Aires. Viral-load, CD4 cell counts and detuned assays were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbor-joining trees and bootscanning analysis. Results:We found that 12 (4.2%) of the 284 samples sequenced harbored primary resistance mutations, of which K103N, M41L and V108I were most prevalent. Phylogenetic analysis revealed evidence for the transmission of the K103N mutation among the drug-naive population. The proportion of recent infections identified by the detuned assay was 10.1%. Conclusions:Levels of primary resistance in Buenos Aires are still low, despite a long history of ARV delivery and high coverage levels.

Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing

Single Molecule, Real-Time (SMRT®) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.

HLA Class-II Associated HIV Polymorphisms Predict Escape from CD4+ T Cell Responses

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.

Analysis of HIV Type 1 BF Recombinant Sequences from South America Dates the Origin of CRF12_BF to a Recombination Event in the 1970s

HIV-1 epidemics in South America are believed to have originated in part from the subtype B epidemic initiated in the Caribbean/North America region. However, circulation of BF recombinants in similar proportions was extensively reported. Information currently shows that many BF recombinants share a recombination structure similar to that found in the CRF12_BF. In the present study, analyzing a set of 405 HIV sequences, we identified the most likely origin of the BF epidemic in an early event of recombination. We found that the subtype B epidemics in South America analyzed in the present study were initiated by a founder event that occurred in the early 1970s, a few years after the introduction of these strains in the Americas. Regarding the F/BF recombinant epidemics, by analyzing a subtype F genomic segment within the viral gene gag present in the majority of the BF recombinants, we found evidence of a geographic divergence very soon after the introduction of subtype F strains in South America. Moreover, through analysis of a subtype B segment present in all the CRF12_BF-like recombination structure, we estimated the circulation of the subtype B strain that gave rise to that recombinant structure around the same time period estimated for the introduction of subtype F strains. The HIV epidemics in South America were initiated in part through a founder event driven by subtype B strains coming from the previously established epidemic in the north of the continent. A second introduction driven by subtype F strains is likely to have encountered the incipient subtype B epidemic that soon after their arrival recombined with them, originating the BF epidemic in the region. These results may explain why in South America the majority of F sequences are found as BF recombinants.

Increasing trends in primary NNRTI resistance among newly HIV-1-diagnosed individuals in Buenos Aires, Argentina

Our objective was to estimate primary resistance in an urban setting in a developing country characterized by high antiretroviral (ARV) coverage over the diagnosed population and also by an important proportion of undiagnosed individuals, in order to determine whether any change in primary resistance occurred in the past five years.

Divergent Strains of Human T-Lymphotropic Virus Type 1 (HTLV-1) within the Cosmopolitan Subtype in Argentina

HTLV-1 Cosmopolitan subtype Transcontinental subgroup A has been described among aboriginal communities from the northwest endemic area of Argentina. Moreover, Transcontinental subgroup A and the Japanese subgroup B were reported among blood donors from the nonendemic central region of the country. We carried out the first HTLV-1 phylogenetic study in individuals residing in Buenos Aires capital city. Phylogenetic analysis performed on the LTR region showed that all 44 new strains clustered within the Cosmopolitan subtype, with 42 (95.4%) belonging to Transcontinental subgroup A. Of them, 20 (45.5%) strains grouped in the large Latin American cluster and 4 (9.1%) in the small Latin American cluster. The majority of them belonged to individuals of nonblack origin, grouped with Amerindian strains. Three (6.8%) were closely related to South African references and two monophyletic clusters including only HIV/HTLV-1 coinfected individuals were observed. Interestingly, two (4.5%) new sequences (divergent strains) branched off from all five known Cosmopolitan subgroups in a well-supported clade. In summary, these findings show that HTLV-1 Cosmopolitan subtype Transcontinental subgroup A is infecting residents of Buenos Aires, a nonendemic area of Argentina, and confirm the introduction of divergent strains in the country.

A20 Deep sequencing reveals viral evolution in GAG within protective HLA Alleles B*57: 02, B*58: 01, and B*7 supertype individuals acutely infected with HIV-1 subtype C in Durban, South Africa

uals. Among the 25,251 polymorphic codon sites analysed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 co-infection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis dataset means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.

A12 Transmitted HLA pre-adapted polymorphisms in the GAG protein influences viral evolution in the new host

Daniela Celeste Monaco, Emory University Dario Alberto Dilernia, Emory University A Gartland, Fred Hutchinson Cancer Research Center K Qin, University of Alabama at Birmingham K Dennis, Emory University J Tang, University of Alabama at Birmingham J Gilmour, International AIDS Vaccine Initiative (IAVI) A Bansal, University of Alabama at Birmingham S Allen, Emory University P Goepfert, University of Alabama at Birmingham