Person:

Novitsky, Vladimir

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.

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.

To address whether sequences of viral gag and env quasispecies collected during the early post-acute period can be utilized to determine multiplicity of transmitted HIV's, recently developed approaches for analysis of viral evolution in acute HIV-1 infection [1], [2] were applied. Specifically, phylogenetic reconstruction, inter- and intra-patient distribution of maximum and mean genetic distances, analysis of Poisson fitness, shape of highlighter plots, recombination analysis, and estimation of time to the most recent common ancestor (tMRCA) were utilized for resolving multiplicity of HIV-1 transmission in a set of viral quasispecies collected within 50 days post-seroconversion (p/s) in 25 HIV-infected individuals with estimated time of seroconversion. The decision on multiplicity of HIV infection was made based on the model's fit with, or failure to explain, the observed extent of viral sequence heterogeneity. The initial analysis was based on phylogeny, inter-patient distribution of maximum and mean distances, and Poisson fitness, and was able to resolve multiplicity of HIV transmission in 20 of 25 (80%) cases. Additional analysis involved distribution of individual viral distances, highlighter plots, recombination analysis, and estimation of tMRCA, and resolved 4 of the 5 remaining cases. Overall, transmission of a single viral variant was identified in 16 of 25 (64%) cases, and transmission of multiple variants was evident in 8 of 25 (32%) cases. In one case multiplicity of HIV-1 transmission could not be determined. In primary HIV-1 subtype C infection, samples collected within 50 days p/s and analyzed by a single-genome amplification/sequencing technique can provide reliable identification of transmission multiplicity in 24 of 25 (96%) cases. Observed transmission frequency of a single viral variant and multiple viral variants were within the ranges of 64% to 68%, and 32% to 36%, respectively.

A systematic analysis of immune responses on a population level is critical for a human immunodeficiency virus type 1 (HIV-1) vaccine design. Our studies in Botswana on (i) molecular analysis of the HIV-1 subtype C (HIV-1C) epidemic, (ii) frequencies of major histocompatibility complex class I HLA types, and (iii) cytotoxic T-lymphocyte (CTL) responses in the course of natural infection allowed us to address HIV-1C-specific immune responses on a population level. We analyzed the magnitude and frequency of the gamma interferon ELISPOT-based CTL responses and translated them into normalized cumulative CTL responses. The introduction of population-based cumulative CTL responses reflected both (i) essentials of the predominant virus circulating locally in Botswana and (ii) specificities of the genetic background of the Botswana population, and it allowed the identification of immunodominant regions across the entire HIV-1C. The most robust and vigorous immune responses were found within the HIV-1C proteins Gag p24, Vpr, Tat, and Nef. In addition, moderately strong responses were scattered across Gag p24, Pol reverse transcriptase and integrase, Vif, Tat, Env gp120 and gp41, and Nef. Assuming that at least some of the immune responses are protective, these identified immunodominant regions could be utilized in designing an HIV vaccine candidate for the population of southern Africa. Targeting multiple immunodominant regions should improve the overall vaccine immunogenicity in the local population and minimize viral escape from immune recognition. Furthermore, the analysis of HIV-1C-specific immune responses on a population level represents a comprehensive systematic approach in HIV vaccine design and should be considered for other HIV-1 subtypes and/or different geographic areas.

Nucleoside analogue reverse transcriptase inhibitors are an integral component of combination antiretroviral treatment regimens. However, their ability to inhibit polymerase-γ has been associated with several mitochondrial toxicities, including potentially life-threatening lactic acidosis. A total of 650 antiretroviral-naive adults (69% female) initiated combination antiretroviral therapy (cART) and were intensively screened for toxicities including lactic acidosis as part of a 3-year clinical trial in Botswana. Patients were categorized as no lactic acidosis symptoms, minor symptoms but lactate <4.4 mmol/liter, and symptoms with lactate ≥ 4.4 mmol/liter [moderate to severe symptomatic hyperlactatemia (SH) or lactic acidosis (LA)]. Of 650 participants 111 (17.1%) developed symptoms and/or laboratory results suggestive of lactic acidosis and had a serum lactate drawn; 97 (87.4%) of these were female. There were 20 events, 13 having SH and 7 with LA; all 20 (100%) were female (p<0.001). Cox proportional hazard analysis limited to the 451 females revealed that having a higher baseline BMI was predictive for the development of SH/LA [aHR=1.17 per one-unit increase (1.08-1.25), p<0.0001]. Ordered logistic regression performed among all 650 patients revealed that having a lower baseline hemoglobin [aOR=1.28 per one-unit decrease (1.1-1.49), p=0.002] and being randomized to d4T/3TC-based cART [aOR=1.76 relative to ZDV/3TC (1.03-3.01), p=0.04] were predictive of the symptoms and/or the development of SH/LA. cART-treated women in sub-Saharan Africa, especially those having higher body mass indices, should receive additional monitoring for SH/LA. Women presently receiving d4T/3TC-based cART in such settings also warrant more intensive monitoring.

Cross-sectional prevalence studies based on immunoassays that discriminate between recent and long-term infections, such as the BED assay, have been widely used to estimate HIV incidence. However, individuals receiving highly active antiretroviral therapy tend to have lower BED levels and are associated with a higher risk for being mistakenly classified as recent infections. To assess the effect of short-term antenatal zidovudine (ZDV) and single-dose nevirapine (sdNVP) on the BED levels in HIV-1C infection, we measured longitudinal BED normalized optical density (OD-n) levels using stored plasma samples collected prenatally and postnatally from 159 pregnant HIV-infected women in Botswana who participated in the randomized clinical Mother-to-Child-Prevention study, the Mashi study. All women received ZDV from 34 weeks gestation through delivery and were randomized to receive either sdNVP or placebo during labor. Among 159 subjects, the OD-n levels decreased from baseline to delivery in 93 subjects (p=0.039), suggesting that short-course ZDV may decrease OD-n levels. sdNVP at delivery did not affect longitudinal BED OD-n levels postdelivery. However, sdNVP appeared to modify the association between CD4 count at delivery and OD-n levels postdelivery. When estimating HIV incidence with the BED assay, special care may be required regarding women who received short-term ZDV for prevention of mother-to-child transmission.

Laboratory cross-sectional assays are useful for the estimation of HIV incidence, but are known to misclassify individuals with long-standing infection as recently infected. The false recent rate (FRR) varies widely across geographic areas; therefore, accurate estimates of HIV incidence require a locally defined FRR. We determined FRR for Botswana, where HIV-1 subtype C infection is predominant, using the BED capture enzyme immunoassay (BED), a Bio-Rad Avidity Index (BAI) assay (a modification of the Bio-Rad HIV1/2+O EIA), and two multiassay algorithms (MAA) that included clinical data. To estimate FRR, stored blood samples from 512 antiretroviral (ARV)-naive HIV-1 subtype C-infected individuals from a prospective cohort in Botswana were tested at 18-24 months postenrollment. The following FRR mean (95% CI) values were obtained: BED 6.05% (4.15-8.48), BAI 5.57% (3.70-8.0), BED-BAI 2.25% (1.13-4.0), and a combination of BED-BAI with CD4 (>200) and viral load (>400) threshold 1.43% (0.58-2.93). The interassay agreement between BED and BAI was 92.8% (95% CI, 90.1-94.5) for recent/long-term classification. Misclassification was associated with viral suppression for BED [adjusted OR (aOR) 10.31; p=0.008], BAI [aOR 9.72; p=0.019], and MAA1 [aOR 16.6; p=0.006]. Employing MAA can reduce FRR to <2%. A local FRR can improve cross-sectional HIV incidence estimates.

BACKGROUND: Clinical laboratories in Botswana have relied entirely on the reference intervals for normal immunohaematological values provided by manufacturers' kits and textbooks. OBJECTIVES: The aim of this study was to determine the means, medians, 2.5th and 97.5th percentile reference intervals, for normal immunohaematological values in healthy adults in Botswana. METHOD: A total of 261 healthy participants comprising 126 men (48%) and 135 (52%) women were enrolled in the southern part of Botswana, and immunological and haematological laboratory parameters were measured. RESULTS: The mean age was 28.8 (95% Confidence Interval [CI] 27.7-29.8) years, with a median of 27 years and a range 18-66 years. The mean haemoglobin level was significantly lower for women (12.4 g/dL; 95% CI 12.1% - 12.7%) than men (15.1 g/dL; 95% CI 14.9% - 15.3%). The women's haemoglobin reference values (9.0 g/dL - 15.0 g/dL) levels were lower than observed in predominantly White populations (12.0 g/dL - 16.0 g/dL), but comparable with regional consensus reference intervals (9.5 g/dL - 15.8 g/dL) recently defined for East and Southern Africa. CONCLUSION: The established values provide an important tool for patient management and could influence decisions on inclusion of participants and adverse events in clinical trials conducted locally.