Olivier Manigart

Deciphering Human Immunodeficiency Virus Type 1 Transmission and Early Envelope Diversification by Single-Genome Amplification and Sequencing

ABSTRACT Accurate identification of the transmitted virus and sequences evolving from it could be instrumental in elucidating the transmission of human immunodeficiency virus type 1 (HIV-1) and in developing vaccines, drugs, or microbicides to prevent infection. Here we describe an experimental approach to analyze HIV-1 env genes as intact genetic units amplified from plasma virion RNA by single-genome amplification (SGA), followed by direct sequencing of uncloned DNA amplicons. We show that this strategy precludes in vitro artifacts caused by Taq-induced nucleotide substitutions and template switching, provides an accurate representation of the env quasispecies in vivo, and has an overall error rate (including nucleotide misincorporation, insertion, and deletion) of less than 8 × 10−5. Applying this method to the analysis of virus in plasma from 12 Zambian subjects from whom samples were obtained within 3 months of seroconversion, we show that transmitted or early founder viruses can be identified and that molecular pathways and rates of early env diversification can be defined. Specifically, we show that 8 of the 12 subjects were each infected by a single virus, while 4 others acquired more than one virus; that the rate of virus evolution in one subject during an 80-day period spanning seroconversion was 1.7 × 10−5 substitutions per site per day; and that evidence of strong immunologic selection can be seen in Env and overlapping Rev sequences based on nonrandom accumulation of nonsynonymous mutations. We also compared the results of the SGA approach with those of more-conventional bulk PCR amplification methods performed on the same patient samples and found that the latter is associated with excessive rates of Taq-induced recombination, nucleotide misincorporation, template resampling, and cloning bias. These findings indicate that HIV-1 env genes, other viral genes, and even full-length viral genomes responsible for productive clinical infection can be identified by SGA analysis of plasma virus sampled at intervals typical in large-scale vaccine trials and that pathways of viral diversification and immune escape can be determined accurately.

Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

ABSTRACT The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50 titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.

Inflammatory Genital Infections Mitigate a Severe Genetic Bottleneck in Heterosexual Transmission of Subtype A and C HIV-1

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier.

CLSI-derived hematology and biochemistry reference intervals for healthy adults in eastern and southern Africa.

Background Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial. Methods and Findings Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S.-derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials. Conclusions To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.

Maternal plasma viral load, zidovudine and mother-to-child transmission of HIV-1 in Africa: DITRAME ANRS 049a trial.

ObjectiveTo study the relationship between maternal plasma RNA levels and mother-to-child transmission (MTCT) of HIV-1 in African breastfed children. DesignNested case–control study within a randomized trial assessing the efficacy of a short maternal zidovudine (ZDV) regimen to reduce MTCT. MethodsEligible women received either 300 mg of ZDV twice a day until labour, 600 mg at the beginning of labour and 300 mg twice a day for 7 days post-partum or a placebo. The diagnosis of paediatric HIV-1 infection was based on PCR tests at days 1–8, 45, 90 and 180 then on serology performed at 3 monthl intervals. Plasma HIV-1 RNA was measured at inclusion and on day 8 after delivery for all women who did transmit HIV to their children (cases) using a Chiron branched DNA assay (sensitivity 50 copies/ml) and compared with women who did not transmit (two per case) matched for phase trial, treatment allocation and site. ResultsAt inclusion, mean log10 viral load was 4.6 among 55 transmitting mothers and 3.7 among 117 non transmitters (P = 0.0001). Among transmitters, the mean difference in log10 viral load between day 8 post-partum and inclusion was −0.13 in the ZDV group (n = 23) versus 0.27 in the placebo group (n = 32;P = 0.01); among non transmitters it was −0.35 for the ZDV group (n = 47) versus 0.27 in the placebo group (n = 70;P < 10−4). In multivariate logistic regression analysis, odds ratios for MTCT were 8.7 (95% confidence interval, 3.7–20.6) for 1 log10 increase of maternal RNA at inclusion and 4.2 (95% confidence interval, 1.7–10.3) for 1 log10 increase difference from inclusion to day 8 post-partum. ConclusionHigh maternal viral load at inclusion strongly predicts MTCT of HIV in Africa. A short ZDV treatment regimen decreases significantly maternal viral load from its pretreatment level.

The Diversity of Meningococcal Carriage Across the African Meningitis Belt and the Impact of Vaccination With a Group A Meningococcal Conjugate Vaccine

Background. Study of meningococcal carriage is essential to understanding the epidemiology of Neisseria meningitidis infection. Methods. Twenty cross-sectional carriage surveys were conducted in 7 countries in the African meningitis belt; 5 surveys were conducted after introduction of a new serogroup A meningococcal conjugate vaccine (MenAfriVac). Pharyngeal swab specimens were collected, and Neisseria species were identified by microbiological and molecular techniques. Results. A total of 1687 of 48 490 participants (3.4%; 95% confidence interval [CI], 3.2%–3.6%) carried meningococci. Carriage was more frequent in individuals aged 5–14 years, relative to those aged 15–29 years (adjusted odds ratio [OR], 1.41; 95% CI, 1.25–1.60); in males, relative to females (adjusted OR, 1.17; 95% CI, 1.10–1.24); in individuals in rural areas, relative to those in urban areas (adjusted OR, 1.44; 95% CI, 1.28–1.63); and in the dry season, relative to the rainy season (adjusted OR, 1.54; 95% CI, 1.37–1.75). Forty-eight percent of isolates had genes encoding disease-associated polysaccharide capsules; genogroup W predominated, and genogroup A was rare. Strain diversity was lower in countries in the center of the meningitis belt than in Senegal or Ethiopia. The prevalence of genogroup A fell from 0.7% to 0.02% in Chad following mass vaccination with MenAfriVac. Conclusions. The prevalence of meningococcal carriage in the African meningitis belt is lower than in industrialized countries and is very diverse and dynamic, even in the absence of vaccination.

Effect of Perinatal Zidovudine Prophylaxis on the Evolution of Cell-Free HIV-1 RNA in Breast Milk and on Postnatal Transmission

Perinatal zidovudine (ZDV) prophylaxis decreases rates of perinatal transmission of human immunodeficiency virus type 1 (HIV-1). Its relationship with levels of HIV-1 RNA in breast milk and postnatal transmission in breast-fed African children is unknown. At day 8 after delivery, levels of HIV-1 RNA in breast milk from 28 women who transmitted HIV-1 (Ts) postnatally and from 130 women who did not transmit HIV-1 (NTs) were lower for women receiving ZDV than for women receiving placebo. Levels of HIV-1 RNA in breast milk remained low over time in NTs but increased by 8-16-fold in Ts treated with ZDV from baseline to days 45/90 after delivery. Levels of HIV-1 RNA in breast milk at day 8 after delivery and the increase in levels of HIV-1 RNA in breast milk from day 8 to days 45/90 after delivery were independently associated with postnatal transmission. An increase in the levels of HIV-1 RNA in breast milk from day 8 to 45 after delivery was associated with maternal ZDV prophylaxis. The rebound in levels of HIV-1 RNA in breast milk after discontinuation of maternal antiretrovirals needs to be further explored--it may justify prolonging antiretroviral prophylaxis during the entire breast-feeding period.

Continuing Effectiveness of Serogroup A Meningococcal Conjugate Vaccine, Chad, 2013

In 2011, vaccination with a serogroup A meningococcal polysaccharide conjugate vaccine was implemented in 3 of 23 regions in Chad. Cases of meningitis declined dramatically in vaccinated areas, but an epidemic continued in the rest of Chad. In 2012, the remaining Chad population was vaccinated, and the epidemic was halted.

Timing and source of subtype-C HIV-1 superinfection in the newly infected partner of Zambian couples with disparate viruses

BackgroundHIV-1 superinfection occurs at varying frequencies in different at risk populations. Though seroincidence is decreased, in the negative partner of HIV-discordant couples after joint testing and counseling in the Zambia Emory HIV Research Project (ZEHRP) cohort, the annual infection rate remains relatively high at 7-8%. Based on sequencing within the gp41 region of each partners virus, 24% of new infections between 2004 and 2008 were the result of transmission from a non-spousal partner. Since these seroconvertors and their spouses have disparate epidemiologically-unlinked viruses, there is a risk of superinfection within the marriage. We have, therefore, investigated the incidence and viral origin of superinfection in these couples.ResultsSuperinfection was detected by heteroduplex mobility assay (HMA), degenerate base counting of the gp41 sequence, or by phylogenetic analysis of the longitudinal sequences. It was confirmed by full-length env single genome amplification and phylogenetic analysis. In 22 couples (44 individuals), followed for up to five years, three of the newly infected (initially HIV uninfected) partners became superinfected. In each case superinfection occurred during the first 12 months following initial infection of the negative partner, and in each case the superinfecting virus was derived from a non-spousal partner. In addition, one probable case of intra-couple HIV-1 superinfection was observed in a chronically infected partner at the time of his seroconverting spouses initial viremia. Extensive recombination within the env gene was observed following superinfection.ConclusionsIn this subtype-C discordant couple cohort, superinfection, during the first year after HIV-1 infection of the previously negative partner, occurred at a rate similar to primary infection (13.6% [95% CI 5.2-34.8] vs 7.8% [7.1-8.6]). While limited intra-couple superinfection may in part reflect continued condom usage within couples, this and our lack of detecting newly superinfected individuals after one year of primary infection raise the possibility that immunological resistance to intra-subtype superinfection may develop over time in subtype C infected individuals.

Baseline Morbidity in 2,990 Adult African Volunteers Recruited to Characterize Laboratory Reference Intervals for Future HIV Vaccine Clinical Trials

Background An understanding of the health of potential volunteers in Africa is essential for the safe and efficient conduct of clinical trials, particularly for trials of preventive technologies such as vaccines that enroll healthy individuals. Clinical safety laboratory values used for screening, enrolment and follow-up of African clinical trial volunteers have largely been based on values derived from industrialized countries in Europe and North America. This report describes baseline morbidity during recruitment for a multi-center, African laboratory reference intervals study. Methods Asymptomatic persons, aged 18–60 years, were invited to participate in a cross-sectional study at seven sites (Kigali, Rwanda; Masaka and Entebbe, Uganda; Kangemi, Kenyatta National Hospital and Kilifi, Kenya; and Lusaka, Zambia). Gender equivalency was by design. Individuals who were acutely ill, pregnant, menstruating, or had significant clinical findings were not enrolled. Each volunteer provided blood for hematology, immunology, and biochemistry parameters and urine for urinalysis. Enrolled volunteers were excluded if found to be positive for HIV, syphilis or Hepatitis B and C. Laboratory assays were conducted under Good Clinical Laboratory Practices (GCLP). Results and Conclusions Of the 2990 volunteers who were screened, 2387 (80%) were enrolled, and 2107 (71%) were included in the analysis (52% men, 48% women). Major reasons for screening out volunteers included abnormal findings on physical examination (228/603, 38%), significant medical history (76, 13%) and inability to complete the informed consent process (73, 13%). Once enrolled, principle reasons for exclusion from analysis included detection of Hepatitis B surface antigen (106/280, 38%) and antibodies against Hepatitis C (95, 34%). This is the first large scale, multi-site study conducted to the standards of GCLP to describe African laboratory reference intervals applicable to potential volunteers in clinical trials. Approximately one-third of all potential volunteers screened were not eligible for analysis; the majority were excluded for medical reasons.