Years Of HIV Infection Research Articles

Gag-Specific CD4 and CD8 T-Cell Proliferation in Adolescents and Young Adults with Perinatally Acquired HIV-1 Infection Is Associated with Ethnicity - The ANRS-EP38-IMMIP Study.

The ANRS-EP38-IMMIP study aimed to provide a detailed assessment of the immune status of perinatally infected youths living in France. We studied Gag-specific CD4 and CD8 T-cell proliferation and the association between the proliferation of these cells, demographic factors and HIV disease history. We included 93 youths aged between 15 and 24 years who had been perinatally infected with HIV. Sixty-nine had undergone valid CFSE-based T-cell proliferation assays. Gag-specific proliferation of CD4 and CD8 T cells was detected in 12 (16%) and 30 (38%) patients, respectively. The Gag-specific proliferation of CD4 and CD8 T cells was more frequently observed in black patients than in patients from other ethnic groups (CD4: 32% vs. 4%, P = 0.001; CD8: 55% vs. 26%, P = 0.02). Among aviremic patients, the duration of viral suppression was shorter in CD8 responders than in CD8 nonresponders (medians: 54 vs. 20 months, P = 0.04). Among viremic patients, CD8 responders had significantly lower plasma HIV RNA levels than CD8 nonresponders (2.7 vs. 3.7 log10 HIV-RNA copies/ml, P = 0.02). In multivariate analyses including sex and HIV-1 subtype as covariables, Gag-specific CD4 T-cell proliferation was associated only with ethnicity, whereas Gag-specific CD8 T-cell proliferation was associated with both ethnicity and the duration of viral suppression. Both CD4 and CD8 responders reached their nadir CD4 T-cell percentages at younger ages than their nonresponder counterparts (6 vs. 8 years, P = 0.04 for both CD4 and CD8 T-cell proliferation). However, these associations were not significant in multivariate analysis. In conclusion, after at least 15 years of HIV infection, Gag-specific T-cell proliferation was found to be more frequent in black youths than in patients of other ethnic groups, despite all the patients being born in the same country, with similar access to care.

Longitudinal changes in plasma Caspase-1 and Caspase-3 during the first 2 years of HIV-1 infection in CD4Low and CD4High patient groups.

Over 95% of CD4 cell death occurs by Caspase-1-mediated pyroptosis during HIV infection. Caspase-3-mediated apoptosis accounts for the death in a small proportion of infected CD4 cells. To date, there have been no reports on the dynamics of Caspase-1 and Caspase-3 and their relationship with disease progression in acute HIV-1 infection. In this study, two distinct HIV-1 patient groups were enrolled. The CD4High group maintained a CD4 level above450 cells/μl while CD4 levels in the CD4Low group dropped below 250 cells/μl within 2 years after infection. Blood samples were collected at 1, 2, 3, 4, 6, 12 and 24 months after HIV infection. Plasma Caspase-1 and Caspase-3 levels in the two patients groups were determined by a single-step ELISA using commercially available monoclonal antibodies. The results showed that Caspase-1 and Caspase-3 levels in the CD4High group increased rapidly and then decreased within a short time during early HIV-1 infection. In contrast, Caspase-1 and Caspase-3 levels in the CD4Low group were obviously increased after 1 year of HIV-1 infection.

Broad and persistent Gag-specific CD8+ T-cell responses are associated with viral control but rarely drive viral escape during primary HIV-1 infection.

We characterized protein-specific CD8 T-cell immunodominance patterns during the first year of HIV-1 infection, and their impact on viral evolution and immune control. We analyzed CD8 T-cell responses to the full HIV-1 proteome during the first year of infection in 18 antiretroviral-naïve individuals with acute HIV-1 subtype C infection, all identified prior to seroconversion. Ex-vivo and cultured interferon-γ ELISPOT assays were performed and viruses from plasma were sequenced within defined CTL Gag epitopes. Nef-specific CD8 T-cell responses were dominant during the first 4 weeks after infection and made up 40% of the total responses at this time; yet, by 1 year, responses against this region had declined and Gag responses made up to 47% of all T-cell responses measured. An inverse correlation between the breadth of Gag-specific responses and viral load set point was evident at 26 weeks after infection (P = 0.0081, r = -0.60) and beyond. An inverse correlation between the number of persistent responses targeting Gag and viral set point was also identified (P = 0.01, r = -0.58). Gag-specific responses detectable by the cultured ELISPOT assay correlated negatively with viral load set point (P = 0.0013, r = -0.91). Sequence evolution in targeted and nontargeted Gag epitopes in this cohort was infrequent. These data underscore the importance of HIV-specific CD8 T-cell responses, particularly to the Gag protein, in the maintenance of low viral load levels during primary infection, and show that these responses are initially poorly elicited by natural infection. These data have implications for vaccine design strategies.

Cost effectiveness of 'on demand' HIV pre-exposure prophylaxis for non-injection drug-using men who have sex with men in Canada.

Recent trials report the efficacy of continuous tenofovir-based pre-exposure prophylaxis (PrEP) for prevention of HIV infection. The cost effectiveness of 'on demand' PrEP for non-injection drug-using men who have sex with men at high risk of HIV acquisition has not been evaluated. To conduct an economic evaluation of the societal costs of HIV in Canada and evaluate the potential benefits of this PrEP strategy. Direct HIV costs comprised outpatient, inpatient and emergency department costs, psychosocial costs and antiretroviral costs. Resource consumption estimates were derived from the Centre Hospitalier de l'Université de Montréal HIV cohort. Estimates of indirect costs included employment rate and work absenteeism. Costs for 'on demand' PrEP were modelled after an ongoing clinical trial. Cost-effectiveness analysis compared costs of 'on demand' PrEP to prevent one infection with lifetime costs of one HIV infection. Benefits were presented in terms of life-years and quality-adjusted life-years. The average annual direct cost of one HIV infection was $16,109 in the least expensive antiretroviral regimen scenario and $24,056 in the most expensive scenario. The total indirect cost was $11,550 per year. Total costs for the first year of HIV infection ranged from $27,410 to $35,358. Undiscounted lifetime costs ranged from $1,439,984 ($662,295 discounted at 3% and $448,901 at 5%) to $1,482,502 ($690,075 at 3% and $485,806 at 5%). The annual cost of PrEP was $12,001 per participant, and $621,390 per infection prevented. The PrEP strategy was cost-saving in all scenarios for undiscounted and 3% discounting rates. At 5% discounting rates, the strategy is largely cost-effective: according to least and most expensive scenarios, incremental cost-effectiveness ratios ranged from $60,311 to $47,407 per quality-adjusted life-year. This 'on demand' PrEP strategy ranges from cost-saving to largely cost-effective. The authors believe it represents an important public health strategy for the prevention of HIV transmission.

Short communication: Longitudinal changes in peripheral blood NK cells during the first year of HIV-1 Infection in CD4Low and CD4High patient groups.

Natural killer (NK) cells may modulate the pathogenesis of primary HIV-1 infection. However, the relationship between the number and function of NK cells during an acute HIV-1 infection and HIV-1 disease progression remains to be elucidated. In this study, we enrolled two distinct patient groups. One group progressed to where their CD4 cell counts fell below 200 cells/μl within 2 years (CD4Low group), while the CD4 cell counts of the other group remained above 500 cells/μl for over 2 years (CD4High group). We compared the number and function of NK cells during the first year of HIV-1 infection between the two distinct groups. We found that the number of total NK cells and the number of cells in the CD56(dim)CD16(pos) subset rapidly decreased in both groups during early HIV-1 infection. The absolute number of total NK cells and CD56(dim)CD16(pos) NK cells was significantly higher in the CD4High group when compared to the CD4Low group during the first month of infection. No significant difference between the numbers of CD56(bright)CD16(neg) NK cells of the two groups was observed. However, more CD56(neg)CD16(pos) NK cells were found in the CD4Low group than in the CD4High group. We also found that NK cell function increased within the first 3 months of HIV-1 infection in the CD4High group and then exhibited a decreasing trend. However, in the CD4Low group, NK cell function did not increase significantly within the first 3 months of HIV-1 infection but then gradually increased. We concluded, therefore, that robust NK functioning cells that are present during an acute HIV-1 infection might be beneficial in controlling HIV-1 disease progression.

Longitudinal changes in total, 2-LTR circular, and integrated HIV-1 DNA during the first year of HIV-1 infection in CD4Low and CD4High patient groups with HIV-1 subtype AE.

The level of viral DNA in early HIV-1 infection is an important parameter in the prediction of disease progression. Few data have been published on the dynamics of HIV-1 DNA during the first year of HIV infection. In this study, two distinct HIV-1 patient groups were enrolled. Group 1 (CD4High group) maintained their CD4 above 450 cells/μL within 1 year, while Group 2 (CD4Low group) progressed to CD4 below 300 cells/μL. The amounts of total, 2-long terminal repeat (2-LTR) circular, and integrated HIV-1 DNA were determined in the peripheral blood mononuclear cells at 1, 3, 6, and 12 months after HIV infection. Reductions in the amount of total and integrated HIV-1 DNA were detected in the CD4High group during the first year of HIV infection but not in the CD4Low group. Disease progression may be related to the body's ability to control HIV-1 DNA during early HIV-1 infection.

Limited HIV infection of central memory and stem cell memory CD4+ T cells is associated with lack of progression in viremic individuals.

A rare subset of HIV-infected individuals, designated viremic non-progressors (VNP), remain asymptomatic and maintain normal levels of CD4+ T-cells despite persistently high viremia. To identify mechanisms potentially responsible for the VNP phenotype, we compared VNPs (average >9 years of HIV infection) to HIV-infected individuals who have similar CD4+ T-cell counts and viral load, but who are likely to progress if left untreated (“putative progressors”, PP), thus avoiding the confounding effect of differences related to substantial CD4+ T cell depletion. We found that VNPs, compared to PPs, had preserved levels of CD4+ stem cell memory cells (TSCM (p<0.0001), which was associated with decreased HIV infection of these cells in VNPs (r = −0.649, p = 0.019). In addition, VNPs had decreased HIV infection in CD4+ central memory (TCM) cells (p = 0.035), and the total number of TCM cells was associated with increased proliferation of memory CD4+ T cells (r = 0.733, p = 0.01). Our results suggest that, in HIV-infected VNPs, decreased infection of CD4+ TCM and TSCM, cells are involved in preservation of CD4+ T cell homeostasis and lack of disease progression despite high viremia.

Compare mDCs and pDCs between two distinct patients groups in acute HIV-1 infection.

The role of DCs in primary HIV-1 infection remains uncertain. In this study, we enrolled two different groups of subjects with acute HIV-1 infection. One group progressed to CD4 counts below 200 cells/μl within 2 years of HIV-1 infection (CD4 Low Group), while the other group maintained CD4 counts above 500 cells/μl (CD4 High Group). We did not find statistical difference in the pDC number between the two groups during acute HIV-1 infection. However, the mDC number was significantly lower in the CD4 Low Group than in the CD4 High Group.

Early suppressive antiretroviral therapy in HIV infection is associated with measurable changes in the corpus callosum.

The purpose of this study was to examine the impact of early suppressive antiretroviral therapy (ART) on brain structure and neurocognitive outcomes. We conducted an observational study of subjects within 1 year of HIV infection. Ten ART-naïve and 10 ART-suppressed individuals were matched for age and infection duration and age-matched to 10 HIV-seronegative controls. Quantitative brain imaging and neurocognitive data were analyzed. Subjects on suppressive ART had diminished corpus callosum structural integrity on macromolecular and microstructural imaging, higher cerebrospinal fluid percent, higher depression scores, and lower functional performance. Early suppressive ART may alter the trajectory of neurological progression of HIV infection, particularly in the corpus callosum.

HIV infection and Cushing's disease

Introduction: People with AIDS can have a dysfunction of the hypothalamic - pituitary-adrenal axis. With regard to HIV infection, most often mentioned is iatrogenic Cushing's syndrome or Pseudo-Cushing's Syndrome. So far there are described only two cases of Cushing disease in HIV -infected persons. Case report: A 48-year-old patient, after eleven years of HIV infection and a year since the introduction of HAART, was diagnosed with Cushing's disease based on cushingoid habitus, lack of suppression of cortisol in screening, elevated ACTH and pituitary tumor. She had transfenoidal surgery and histopathologic findings corresponded to basophilic adenoma. After the operation, short time on hydrocortisone substitution, she generally felt well with regular ART. Four years later, again easily bruising, facial redness, oily skin with acne, weight gain, uneven distribution of stomach adipose tissue, sweating, oligomenorrhea and high blood pressure. There was no rest/relapse of tumor on control pituitary MRI. Initially, elevated ACTH, valid cortisol in daily profiles, later the absence of the suppression of cortisol after 4 mg (LDST) and 8 mg (HDST) of dexamethasone along with maintenance of higher ACTH, indicate recurrence of clinical and laboratory relapse wherefore ketoconazole was introduced. Despite increasing doses of ketoconazole, she held slightly higher morning cortisol, ACTH and with persisting Cushing's syndrome. Conclusion: The coexistence of the two entities could lead to overlapping metabolic and phenotypic characteristics and the interaction between and/or synergism.

HIV-1 Transmission during Early Infection in Men Who Have Sex with Men: A Phylodynamic Analysis

Conventional epidemiological surveillance of infectious diseases is focused on characterization of incident infections and estimation of the number of prevalent infections. Advances in methods for the analysis of the population-level genetic variation of viruses can potentially provide information about donors, not just recipients, of infection. Genetic sequences from many viruses are increasingly abundant, especially HIV, which is routinely sequenced for surveillance of drug resistance mutations. We conducted a phylodynamic analysis of HIV genetic sequence data and surveillance data from a US population of men who have sex with men (MSM) and estimated incidence and transmission rates by stage of infection. We analyzed 662 HIV-1 subtype B sequences collected between October 14, 2004, and February 24, 2012, from MSM in the Detroit metropolitan area, Michigan. These sequences were cross-referenced with a database of 30,200 patients diagnosed with HIV infection in the state of Michigan, which includes clinical information that is informative about the recency of infection at the time of diagnosis. These data were analyzed using recently developed population genetic methods that have enabled the estimation of transmission rates from the population-level genetic diversity of the virus. We found that genetic data are highly informative about HIV donors in ways that standard surveillance data are not. Genetic data are especially informative about the stage of infection of donors at the point of transmission. We estimate that 44.7% (95% CI, 42.2%-46.4%) of transmissions occur during the first year of infection. In this study, almost half of transmissions occurred within the first year of HIV infection in MSM. Our conclusions may be sensitive to un-modeled intra-host evolutionary dynamics, un-modeled sexual risk behavior, and uncertainty in the stage of infected hosts at the time of sampling. The intensity of transmission during early infection may have significance for public health interventions based on early treatment of newly diagnosed individuals.

Dynamics of Viral Evolution and Neutralizing Antibody Response after HIV-1 Superinfection

Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.

Double Negative (DN) [CD3(+)CD4(−)CD8(−)] T cells correlate with disease progression during HIV infection

Although double negative T (DNT) cells (CD3+CD4-CD8-) share some characteristics with T regulatory cells, the relationship between DNT cells and disease progression in HIV infection is unclear. In this study, we analyzed the relationship between DNT cells and disease progression during the first 2 years of HIV infection. We found that DNT cell numbers tended to decrease with disease progression. There was a positive correlation between DNT cells and CD4 counts. The DNT cell numbers were significantly lower in the high viral load group compared with the low viral load group. Therefore, we conclude that DNT cells correlated with disease progression in HIV infection. These data provide valuable information for further understanding of the role of DNT cells during HIV infection.

High CCR5 Density on Central Memory CD4+ T Cells in Acute HIV-1 Infection Is Mostly Associated with Rapid Disease Progression

CD4+ central memory T cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, and the CCR5 density on the surface of CD4 T cells is an important factor in human immunodeficiency virus (HIV)-1 disease progression. We hypothesized that quantifying central memory cells and CCR5 expression in the early stages of HIV-infection could provide useful prognostic information. We enrolled two different groups of acute HIV-infected subjects. One group progressed to CD4 T cell numbers below 250 cells/µl within 2 years (CD4 Low group), while the other group maintained CD4 cell counts above 450 cells/µl over 2 years (CD4 High group). We compared the CCR5 levels and percentage of CD4 subsets between the two groups during the 1st year of HIV infection. We found no differences between the two groups regarding the percentage of naïve, central memory and effector memory subsets of CD4 cells during the 1st year of HIV-1 infection. CCR5 levels on CD4+ CM subset was higher in the CD4 Low group compared with the CD4 High group during the 1st year of HIV-1 infection. High CCR5 levels on CD4 central memory cells in acute HIV infection are mostly associated with rapid disease progression. Our data suggest that low CCR5 expression on CD4 central memory cells protects CD4 cells from direct virus infection and favors the preservation of CD4+ T cell homeostasis.

Impact of lamivudine monotherapy in failing patients with multidrug‐resistant HIV: final 48 weeks results (MONO‐AIFA FARM7PAZS3)

Purpose of the studyTo assess the impact of lamivudine (3TC) monotherapy in failing patients with multidrug‐resistant HIV and limited therapeutic options.MethodsProspective, open‐label, multicenter, randomised (1:1), pilot study. HIV‐1 failing pts with M184V mutation, HBsAg negative were assigned to 3TC 300mg QD for 24 weeks followed by a new regimen for 24 weeks (Arm A) or to a new regimen for 48 weeks (Arm B). The new HAART regimen was decided before randomisation in both groups, based on clinical history, genotype and viral tropism. Primary endpoint was the proportion of pts with HIV‐RNA&lt;50 copies/mL (VS) at week 48 (W48). ITT and OT analysis performed. Results described by median (IQR).Summary of results109 screened, 34 screening failures, 75 randomised, 72 initiated the assigned treatment [38 and 34 pts in Arm A and B, respectively]: 69% males; age: 47.4 (43.2–51.9) years; years of HIV infection: 17 (14–23); nadir CD4+: 150 (47–231) cells/μL. Similar baseline demographic and clinical characteristics were found in Arm A vs Arm B [CD4+: 413 (294–550) vs 377 227–520) cells/µL; HIV‐RNA: 3.94 (2.89–4.43) vs 3.66 (2.78–4.20) log10copies/mL; number of mutations: 9 (5–13) vs 9 (3–19); R5‐virus: 67% vs 50%; new regimen GSS: 2 (2–3) vs 2 (2–2); 60% vs 56% pts included in the new regimen at least two of the following drugs: DRV/r, MVC, RAL, ETR, T‐20]. At W48, pts with VS were 15/38 (39%) vs 17/34 (50%) in Arm A and B, respectively (ITT: P=0.477). In Arm A, 25/38 (66%) completed the 24‐weeks of monotherapy and 21 reached week 48 vs 27 pts in Arm B (P=0.045). Pts with VS were 15/21 (71%) vs 17/27 (63%) in Arm A and B, respectively (OT: P=0.758). VS according to nadir CD4+, screening HIVRNA and GSS of the new regimen shown in 1. SAEs occurred in 4 (11%) and 2 (6%) in Arm A and B, respectively; all but 1 in Arm A unrelated to the regimen; 4 CDC events: 3 oral candidiasis and 1 recurrence of CMV infection in Arm A; 1 oral candidiasis in Arm B. W48 CD4 change from baseline was: ITT:−4 (−108/+56) and 53 (−37/+110) cells/L (P=0.735); OT: 8 (−62/+58) and 68 (−24/+148) cells/L in arm A and B, respectively (P=0.500). Mutations associated with resistance at W48 were 4 (0–7) and 7 (2–13) in Arm A and B, respectively (P=0.031). R5 virus at W48: Arm A=55% vs Arm B=29% (P=0.124).ConclusionsUse of 3TC monotherapy was associated with greater discontinuation. It may be considered in patients without effective therapeutic options to favour virological efficacy of the subsequent regimen. Virological success according to Nadir CD4+, screening HIVRNA and GSS of the new regimen. ITT ANALYSIS Characteristic Strata Percent of patients with HIV‐RNA &lt; 50 copies/mL at week 48 in Arm A Percent of patients with HIV‐RNA &lt; 50 copies/mL at week 48 in Arm B P‐value Breslow‐Day Test P‐value Nadir Cd4+(cells/μL) &lt;200 10/24 42% 10/24 42% 0.999 0.173 ≥200 5/14 (36%) 7/10 (70%) 0.214 Screening HIV‐RNA 51–10000 15/26 (58%) 15/26 (58%) 0.999 0.092 (copies/mL) &gt;10000 0/12 2/8 (25%) 0.147 New Regimen GSS &lt;2 3/7 (43%) 3/8 (38%) 0.999 0.479 ≥2 12/31 (39%) 14/26 (54%) 0.294 OT Analysis Characteristic Strata Percent of patients with HIV‐RNA &lt; 50 copies/mL at week 48 in Arm A Percent of patients with HIV‐RNA &lt; 50 copies/mL at week 48 in Arm B P‐value Breslow‐Day Test P‐value Nadir Cd4+(cells/μL) &lt;200 10/12 (83%) 10/19 (53%) 0.128 0.027 ≥200 5/9 (56%) 7/8 (88%) 0.294 Screening HIV‐RNA 51–10000 15/17 (88%) 15/22 (68%) 0.251 0.052 (copies/mL) &gt;10000 0/4 (0%) 2/5 (40%) 0.444 New Regimen GSS &lt;2 3/4 (75%) 3/5 (60%) 0.999 0.816 ≥2 12/17 (71%) 14/22 (64%) 0.740

HIV-1 Subtypes and Recombinants in Northern Tanzania: Distribution of Viral Quasispecies

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28–50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies.

Evolutionary dynamics of HIV-1 subtype C accessory and regulatory genes in primary infection

The mean intra-patient diversity was found to be 0.11% (95%CI; 0.02 to 0.20) for vif, 0.23% (95%CI; 0.08 to 0.38) for vpr, 0.35% (95%CI; -0.05 to 0.75) for vpu, 0.18%(95% CI; 0.01 to 0.35 ) for tat exon 1 and 0.30% (95%CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95%CI; 0.28 to 0.64), vpr 0.44% (95%CI; 0.24 to 0.64), vpu 0.84% (95%CI; 0.55 to 1.13), tat exon 1 0.35% (95%CI; 0.14 to 0.56 ) and 0.42% (95%CI; 0.18 to 0.66) for rev exon 1 during the time period of 181 to 500 days p/s. Statistically significant increases in viral diversity were observed for vif (p=0.013) and vpu (p=0.002). Weak and sporadic associations between levels of viral diversity within the nonstructural genes and HIV-1 RNA load during primary infection were found. Positive and negative selection patterns over the first year post-seroconversion were assessed in each of these genes, providing insight into the selection pressures on these genes which are crucial for viral replication in-vivo. Conclusion

Viral Diversity and Diversification of Major Non-Structural Genes vif, vpr, vpu, tat exon 1 and rev exon 1 during Primary HIV-1 Subtype C Infection

To assess the level of intra-patient diversity and evolution of HIV-1C non-structural genes in primary infection, viral quasispecies obtained by single genome amplification (SGA) at multiple sampling timepoints up to 500 days post-seroconversion (p/s) were analyzed. The mean intra-patient diversity was 0.11% (95% CI; 0.02 to 0.20) for vif, 0.23% (95% CI; 0.08 to 0.38) for vpr, 0.35% (95% CI; −0.05 to 0.75) for vpu, 0.18% (95% CI; 0.01 to 0.35) for tat exon 1 and 0.30% (95% CI; 0.02 to 0.58) for rev exon 1 during the time period 0 to 90 days p/s. The intra-patient diversity increased gradually in all non-structural genes over the first year of HIV-1 infection, which was evident from the vif mean intra-patient diversity of 0.46% (95% CI; 0.28 to 0.64), vpr 0.44% (95% CI; 0.24 to 0.64), vpu 0.84% (95% CI; 0.55 to 1.13), tat exon 1 0.35% (95% CI; 0.14 to 0.56 ) and rev exon 1 0.42% (95% CI; 0.18 to 0.66) during the time period of 181 to 500 days p/s. There was a statistically significant increase in viral diversity for vif (p = 0.013) and vpu (p = 0.002). No associations between levels of viral diversity within the non-structural genes and HIV-1 RNA load during primary infection were found. The study details the dynamics of the non-structural viral genes during the early stages of HIV-1C infection.

Highly Sensitive and Specific Detection of Rare Variants in Mixed Viral Populations from Massively Parallel Sequence Data

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (<1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved >97% sensitivity and >97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the ∼10 kb genome, including 603 rare variants (<1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

Viral tropism by geno2pheno as a tool for predicting CD4 decrease in HIV-1-infected naive patients with high CD4 counts

To investigate the value of tropism (determined by genotypic testing) to predict CD4 depletion in HIV-infected antiretroviral-naive patients with high CD4 counts. Viral tropism was determined by geno2pheno (false positive rate = 10%) in 223 HIV-infected subjects naive to antiretrovirals with CD4 count ≥350 cells/μL and HIV-RNA >500 copies/mL enrolled in the ICONA Foundation Study for whom a stored plasma sample (baseline) was retrospectively tested. We monitored CD4 cell count and identified predictors of decline before antiretroviral therapy initiation, applying a mixed linear model with covariates (age, gender, tropism, HIV risk factor, calendar year of HIV infection, months from HIV diagnosis to baseline, hepatitis C virus status, CD4 and HIV-RNA at sample collection and duration of follow-up). Two hundred and twenty-three subjects met the eligibility criteria; 137 (61%) were male and the median age was 35 (31-40) years. Median follow-up was 16.4 (3.2-37.2) months. Median CD4 decrease during follow-up was -157 (-278 to -13) cells/μL. At baseline, 192 (86%) subjects were defined as harbouring R5 virus and 31 (14%) non-R5. Median CD4 count was 571 (458-729) cells/μL and median HIV-RNA was 4.08 (3.57-4.55) log(10) copies/mL. At multivariable analysis, a greater mean CD4 decrease was associated with non-R5 viral tropism (-159.9 ± 12.22, P = 0.0002) at baseline. Other significant covariates were female gender, older age, intravenous drug use, longer duration of follow-up, and higher CD4 cell count and higher HIV-RNA at sample collection. In patients with CD4 counts ≥350 cells/μL, non-R5 viral tropism by geno2pheno is predictive of CD4 decrease independent of their viral set point and CD4 counts.