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1.  Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial 
Journal of Virology  2013;87(13):7265-7281.
The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.
PMCID: PMC3700312  PMID: 23576510
2.  Origin of Human Immunodeficiency Virus Type 1 Quasispecies Emerging after Antiretroviral Treatment Interruption in Patients with Therapeutic Failure 
Journal of Virology  2002;76(14):7000-7009.
The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.
PMCID: PMC136319  PMID: 12072500
3.  Timing, Adherence, Resistance, and … Persistence? New Insight Into the Mechanisms of Failure of HIV Type 1 Postexposure Prophylaxis 
The Journal of Infectious Diseases  2013;208(10):1542-1544.
PMCID: PMC3805247  PMID: 24023256
HIV-1; postexposure prophylaxis; single-genome sequencing; viral sequestration
4.  Natural Killer Cell–Mediated Innate Sieve Effect on HIV-1: The Impact of KIR/HLA Polymorphism on HIV-1 Subtype-Specific Acquisition in East Africa 
The Journal of Infectious Diseases  2013;208(8):1250-1254.
Here we explore the association between killer cell immunoglobulin-like receptor (KIR)/HLA and human immunodeficiency virus type 1 (HIV-1) acquisition with different viral subtypes circulating in East Africa. In the prospective Cohort Development (CODE) cohort (Mbeya, Tanzania), carriers of KIR3DS1 and its putative ligand (HLA-A or HLA-B Bw4-80Ile alleles) showed increased HIV-1 acquisition risk (odds ratio [OR] = 3.46; 95% confidence interval [CI], 1.12–10.63; P = .04) and a trend for enrichment for subtype A and A-containing recombinants (78% vs 46%; OR = 4.05; 95% CI, .91–28.30; P = .09) at the expense of subtype C (11% vs 43%; OR = 0.17; 95% CI, .01–.97; P = .08). In vitro, only natural killer cells from KIR3DS1(+)/HLA-Bw4-80Ile(+) healthy donors showed a 2-fold increased capacity to inhibit replication of subtype C vs subtype A viruses (P = .01). These findings suggest the presence of an innate sieve effect and may inform HIV-1 vaccine development.
PMCID: PMC3778971  PMID: 23922366
HIV-1; innate immunity; KIR; HLA; sieve effect; subtypes; East Africa
5.  Nautilus: A Bioinformatics Package for the Analysis of HIV Type 1 Targeted Deep Sequencing Data 
AIDS Research and Human Retroviruses  2013;29(10):1361-1364.
The advent of next generation sequencing technologies is providing new insight into HIV-1 diversity and evolution, which has created the need for bioinformatics tools that could be applied to the characterization of viral quasispecies. Here we present Nautilus, a bioinformatics package for the analysis of HIV-1 targeted deep sequencing data. The DeepHaplo module determines the nucleotide base frequency and read depth at each position and computes the haplotype frequencies based on the linkage among polymorphisms in the same next generation sequence read. The Motifs module computes the frequency of the variants in the setting of their sequence context and mapping orientation, which allows for the validation of polymorphisms and haplotypes when strand bias is suspected. Both modules are accessed through a user-friendly GUI, which runs on Mac OS X (version 10.7.4 or later), and are based on Python, JAVA, and R scripts. Nautilus is available from
PMCID: PMC3785804  PMID: 23809062
6.  Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials 
A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein– Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
PMCID: PMC4152919  PMID: 24787274
Peripheral blood mononuclear cells; Cryopreservation; Repository
7.  Analysis of HLA A*02 Association with Vaccine Efficacy in the RV144 HIV-1 Vaccine Trial 
Journal of Virology  2014;88(15):8242-8255.
The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02+) participants than in A*02− participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02+ participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02+ participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.
IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at under registration no. NCT00223080.)
PMCID: PMC4135964  PMID: 24829343
9.  Short Communication: Investigation of Incident HIV Infections Among U.S. Army Soldiers Deployed to Afghanistan and Iraq, 2001–2007 
AIDS Research and Human Retroviruses  2012;28(10):1308-1312.
The U.S. Army initiated an investigation in response to observations of a possible increase in HIV incidence among soldiers deployed to combat. Human immunodeficiency virus (HIV)-infected U.S. Army soldiers are not eligible to deploy. Combat presents a health hazard to HIV-infected soldiers and they pose a threat to the safety of the battlefield blood supply and their contacts. All soldiers are routinely screened for HIV every 2 years and those who deploy are also screened both prior to and after deployment. Seroconversion rates were estimated for all soldiers who deployed to Afghanistan or Iraq in the period 2001–2007 and all active duty soldiers who did not. Seroconverters with an estimated date of infection, based on calculation of the midpoint between the last seronegative and first seropositive test date, that was either before or during deployment were eligible for inclusion. Confidential interviews and medical record reviews were conducted to determine the most likely time, geographic location, and mode of infection. Reposed predeployment samples were tested for HIV ribonucleic acid. The HIV seroconversion rate among all soldiers who deployed was less than the rate among those who did not deploy: 1.04 and 1.42 per 10,000 person-years, respectively. Among 48 cases, most were determined to have been infected in the United States or Germany and prior to deployment (n=20, 42%) or during rest and relaxation leave (n=13, 27%). Seven seronegative acute infections were identified in the predeployment period. Subtype was determined for 40 individuals; all were subtype B infections. All were acquired through sexual contact. These findings can inform development of preventive interventions and refinement of existing screening policy to further reduce HIV-infected deployed soldier person time.
PMCID: PMC3448093  PMID: 22280248
10.  RecDraw: A Software Package for the Representation of HIV-1 Recombinant Structures 
AIDS Research and Human Retroviruses  2010;26(12):1317-1321.
The crucial role of recombination in HIV-1 biology is being increasingly recognized. In vitro studies have shown that up to 30 strand-transfer events may occur per viral replication cycle. Thus, recombination may surpass mutation as a major mechanism driving HIV-1 evolution. Currently, recombinant strains comprise 37% of the full-genome HIV-1 sequence database, including sequences representing 47 Circulating Recombinant Forms (CRFs) and more than 250 different Unique Recombinant Forms (URFs). Mapping of recombination breakpoints helps establish relationships among strains that are related by descent, such as CRF07_BC and CRF08_BC in China, and sheds light on their origin and epidemic spread. Additionally, unrelated recombinants sharing common breakpoints may reflect recombination hotspots within the viral genome. Here we present a software tool, RecDraw, for the graphical representation and efficient comparison of recombinant HIV-1 structures and breakpoints. RecDraw is a platform-flexible, Java stand-alone application available through = 5&SubServiceID = 6.
PMCID: PMC3012000  PMID: 20961275
11.  Human Immunodeficiency Virus Type 1 Infection Is Associated with Increased NK Cell Polyfunctionality and Higher Levels of KIR3DL1+ NK Cells in Ugandans Carrying the HLA-B Bw4 Motif ▿  
Journal of Virology  2011;85(10):4802-4811.
Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1+ CD56dim NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1+ CD56dim NK cells were decreased, and levels of KIR2DL3+ CD56dim NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1+ NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4+ patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1+ and KIR2DL3+ NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1+ CD56dim NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1+ NK cells in Ugandans carrying the HLA-B Bw4 motif.
PMCID: PMC3126187  PMID: 21411516
12.  Cell Type-Specific Proteasomal Processing of HIV-1 Gag-p24 Results in an Altered Epitope Repertoire▿  
Journal of Virology  2010;85(4):1541-1553.
Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4+ T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4+ T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.
PMCID: PMC3028885  PMID: 21106750
13.  Viral and host factors associated with the HIV-1 viral load setpoint in adults from Mbeya Region, Tanzania 
The viral load setpoint (VLS) is an important predictor of HIV disease progression, but there is a lack of information regarding the VLS and its possible determinants in African populations.
Initially HIV negative adults from three distinct groups (female barworkers, females and males from the general population) were followed for up to four years. The VLS was calculated for 108 seroconverters and associations of the VLS with possible risk factors were analyzed using univariate and multivariate regression.
The median VLS for female barworkers, females and males from the general population were 69,850, 28,600 and 158,000 RNA copies/ml respectively. Significant associations with an elevated viral load were observed for male gender (Risk Ratio (RR)=1.83, 95% confidence interval (95%CI)=1.14–2.93), the expression of harmful HLA I alleles (RR=1.73, 95%CI=1.13–2.66) and multiple infection with different HIV-1 subtypes (RR=1.65, 95%CI =1.03–2.66). Barworkers were considerably more often infected with different HIV-1 subtypes than participants from the general population.
Our study confirms that gender and the expression of different HLA class I alleles are important determinants of the viremia at VLS and it also corroborates an earlier finding that multiple infection with different HIV-1 subtypes is associated with a higher VLS.
PMCID: PMC2958061  PMID: 20632457
HIV-1 infection; Acute infection; Viral load setpoint; Multiple infection; HLA class I alleles; Africa
14.  High Affinity Allele for the Gene of FCGR3A Is Risk Factor for HIV Infection and Progression 
PLoS ONE  2010;5(12):e15562.
We investigated the genetics of Fc receptors, which function as activating receptors on immune cells and help to control HIV through antibody-mediated cellular cytotoxicity. Thus, Fc receptors may be important for virus immunity but might also promote immune hyperactivation that would enhance infection.
Methodology/Principal Findings
We measured abundance of low and high activity alleles in two Fc receptor genes, FCGR2A and FCGR3A, for persons with HIV disease, natural virus suppressors (HIV+, without disease) and healthy controls to show whether genotypes were associated with infection and disease. Individuals homozygous for the high activity allele of FCGR3A (158VV) were predominantly found among HIV progressors and this group was also skewed toward higher allele frequencies for the V158 variant. Both of the HIV positive groups (progressors and natural virus suppressors) had significantly higher frequencies of the V158 allele compared with uninfected controls. There were no apparent associations among FCGR2A alleles and HIV status.
Our results indicate that high activity alleles of FCGR3A may be risk factors for HIV infection or progression and we need to understand how allelic variants affect the balance between virus control and immune activation.
PMCID: PMC3004964  PMID: 21187939
15.  High-Throughput High-Resolution Class I HLA Genotyping in East Africa 
PLoS ONE  2010;5(5):e10751.
HLA, the most genetically diverse loci in the human genome, play a crucial role in host-pathogen interaction by mediating innate and adaptive cellular immune responses. A vast number of infectious diseases affect East Africa, including HIV/AIDS, malaria, and tuberculosis, but the HLA genetic diversity in this region remains incompletely described. This is a major obstacle for the design and evaluation of preventive vaccines. Available HLA typing techniques, that provide the 4-digit level resolution needed to interpret immune responses, lack sufficient throughput for large immunoepidemiological studies. Here we present a novel HLA typing assay bridging the gap between high resolution and high throughput. The assay is based on real-time PCR using sequence-specific primers (SSP) and can genotype carriers of the 49 most common East African class I HLA-A, -B, and -C alleles, at the 4-digit level. Using a validation panel of 175 samples from Kampala, Uganda, previously defined by sequence-based typing, the new assay performed with 100% sensitivity and specificity. The assay was also implemented to define the HLA genetic complexity of a previously uncharacterized Tanzanian population, demonstrating its inclusion in the major East African genetic cluster. The availability of genotyping tools with this capacity will be extremely useful in the identification of correlates of immune protection and the evaluation of candidate vaccine efficacy.
PMCID: PMC2873994  PMID: 20505773

Results 1-15 (15)