Uterine epithelial cell regulation of DC-SIGN expression inhibits transmitted/founder HIV-1 trans infection by immature dendritic cells

Background

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.

Methodology/Principal Findings

Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.

Conclusions/Significance

Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1.     

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism

Objective

Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4⁺ T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms.

Study Design

ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions.

Results

DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein.

Conclusion

The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.     

Antigen-Presenting Cells Represent Targets for R5 HIV-1 Infection in the First Trimester Pregnancy Uterine Mucosa

Background

During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells) but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown.

Methodology/Principal Findings

To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14⁺ cells were the main targets of HIV-1 infection in the decidua. These infected CD14⁺ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization.

Conclusions/Significance

The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.     

HIV-1 disease progression is associated with bile-salt stimulated lipase (BSSL) gene polymorphism

Background

DC-SIGN expressed by dendritic cells captures HIV-1 resulting in trans-infection of CD4⁺ T-lymphocytes. However, BSSL (bile-salt stimulated lipase) binding to DC-SIGN interferes with HIV-1 capture. DC-SIGN binding properties of BSSL associate with the polymorphic repeated motif of BSSL exon 11. Furthermore, BSSL binds to HIV-1 co-receptor CXCR4. We hypothesized that BSSL modulates HIV-1 disease progression and emergence of CXCR4 using HIV-1 (X4) variants.

Results

The relation between BSSL genotype and HIV-1 disease progression and emergence of X4 variants was studied using Kaplan Meier and multivariate Cox proportional hazard analysis in a cohort of HIV-1 infected men having sex with men (n = 334, with n = 130 seroconverters). We analyzed the association of BSSL genotype with set-point viral load and CD4 cell count, both pre-infection and post-infection at viral set-point. The number of repeats in BSSL exon 11 were highly variable ranging from 10 to 18 in seropositive individuals and from 5–17 in HRSN with 16 repeats being dominant (>80% carry at least one allele with 16 repeats). We defined 16 to 18 repeats as high (H) and less than 16 repeats as low (L) repeat numbers. Homozygosity for the high (H) repeat number BSSL genotype (HH) correlated with high CD4 cell numbers prior to infection (p = 0.007). In HIV-1 patients, delayed disease progression was linked to the HH BSSL genotype (RH = 0.462 CI = 0.282–0.757, p = 0.002) as was delayed emergence of X4 variants (RH = 0.525, 95% CI = 0.290–0.953, p = 0.034). The LH BSSL genotype, previously found to be associated with enhanced DC-SIGN binding of human milk, was identified to correlate with accelerated disease progression in our cohort of HIV-1 infected MSM (RH = 0.517, 95% CI = 0.328–0.818, p = 0.005).

Conclusion

We identify BSSL as a marker for HIV-1 disease progression and emergence of X4 variants. Additionally, we identified a relation between BSSL genotype and CD4 cell counts prior to infection.     

Plasmodium falciparum Infection Significantly Impairs Placental Cytokine Profile in HIV Infected Cameroonian Women

Background

Placental cytokines play crucial roles in the establishment and maintenance of pregnancy as well as protecting the foetus from infections. Previous studies have suggested the implication of infections such as P. falciparum and HIV in the stimulation of placental cytokines. This study assessed the impact of P. falciparum on placental cytokine profiles between HIV-1 positive and negative women.

Materials and Methods

P. falciparum infection was checked in peripheral and placental blood of HIV-1 negative and positive women by the thick blood smear test. Cytokines proteins and messenger RNAs were quantified by ELISA and real time PCR, respectively. Non-parametric tests were used for statistical analyses.

Results

Placental and peripheral P. falciparum infections were not significantly associated with HIV-1 infection (OR: 1.4; 95% confidence interval (95%CI): 0.5–4.2; p = 0.50 and OR: 0.6; 95%CI: 0.3–1.4; p = 0.26, respectively). Conversely, placental P. falciparum parasitemia was significantly higher in the HIV-1 positive group (p = 0.04). We observed an increase of TNF-α mRNA median levels (p = 0.02) and a trend towards a decrease of IL-10 mRNA (p = 0.07) in placenta from HIV-1 positive women compared to the HIV negative ones leading to a median TNF-α/IL-10 mRNA ratio significantly higher among HIV-1 positive than among HIV-1 negative placenta (p = 0.004; 1.5 and 0.8, respectively). Significant decrease in median secreted cytokine levels were observed in placenta from HIV-1 positive women as compared to the HIV negative however these results are somewhat indicative since it appears that differences in cytokine levels (protein or mRNA) between HIV-1 positive and negative women depend greatly on P.falciparum infection. Within the HIV-1 positive group, TNF-α was the only cytokine significantly associated with clinical parameters linked with HIV-1 MTCT such as premature rupture of membranes, CD4 T-cell number, plasma viral load and delay of NVP intake before delivery.

Conclusions

These results show that P. falciparum infection profoundly modifies the placenta cytokine environment and acts as a confounding factor, masking the impact of HIV-1 in co-infected women. This interplay between the two infections might have implications in the in utero MTCT of HIV-1 in areas where HIV-1 and P. falciparum co-circulate.     

Evaluation of cervical mucosa in transmission bottleneck during acute HIV-1 infection using a cervical tissue-based organ culture

Background

Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this “transmission bottleneck” is as yet unknown.

Methodology/Principal Findings

A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution.

Conclusion

There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo.     

Estimating the Timing of Mother-to-Child Transmission of the Human Immunodeficiency Virus Type 1 Using a Viral Molecular Evolution Model

Background

Mother-to-child transmission (MTCT) is responsible for most pediatric HIV-1 infections worldwide. It can occur during pregnancy, labor, or breastfeeding. Numerous studies have used coalescent and molecular clock methods to understand the epidemic history of HIV-1, but the timing of vertical transmission has not been studied using these methods. Taking advantage of the constant accumulation of HIV genetic variation over time and using longitudinally sampled viral sequences, we used a coalescent approach to investigate the timing of MTCT.

Materials and Methods

Six-hundred and twenty-two clonal env sequences from the RNA and DNA viral population were longitudinally sampled from nine HIV-1 infected mother-and-child pairs [range: 277–1034 days]. For each transmission pair, timing of MTCT was determined using a coalescent-based model within a Bayesian statistical framework. Results were compared with available estimates of MTCT timing obtained with the classic biomedical approach based on serial HIV DNA detection by PCR assays.

Results

Four children were infected during pregnancy, whereas the remaining five children were infected at time of delivery. For eight out of nine pairs, results were consistent with the transmission periods assessed by standard PCR-based assay. The discordance in the remaining case was likely confused by co-infection, with simultaneous introduction of multiple maternal viral variants at the time of delivery.

Conclusions

The study provided the opportunity to validate the Bayesian coalescent approach that determines the timing of MTCT of HIV-1. It illustrates the power of population genetics approaches to reliably estimate the timing of transmission events and deepens our knowledge about the dynamics of viral evolution in HIV-infected children, accounting for the complexity of multiple transmission events.     

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children

Background

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5^broad viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.

Methodology/Principal Findings

Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5^narrow phenotype (n = 20), but R5^broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5^broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5^broad phenotype, however, the presence of the R5^broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5^broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5^broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn''s viral variant.

Conclusions/Significance

Our results show that R5^broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5^narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.     

Correlation between Vertical Transmission of Hepatitis B Virus and the Expression of HBsAg in Ovarian Follicles and Placenta

Background

The aim of this study was to investigate the correlation between the expression of hepatitis B surface antigen (HBsAg) in human ovary and placenta and the vertical transmission of hepatitis B virus (HBV).

Methodology/Principal Fidnings

Ovarian and placental tissue specimens of pregnant women infected with HBV were collected during cesarean section and immunostained for HBsAg. The sera of the corresponding newborns were tested for HBV markers and HBV DNA. HBsAg was detected in 15 out of 33 (45%) placental tissues and was further detected in capillary endothelial cells in 4 specimens (26%), of which 3 (75%) corresponding infants were infected with HBV in utero. Out of the 33 ovarian tissues, 7 (21%) were positive for HBsAg, of which 2 (28%) showed HBsAg in ovarian follicles and the 2 corresponding infants (100%) had intrauterine HBV infection.

Conclusions/Significance

HBsAg expression in cells of the ovarian follicle or placental capillary endothelium signal a higher risk for intrauterine HBV infection.     

Cell-Free (RNA) and Cell-Associated (DNA) HIV-1 and Postnatal Transmission through Breastfeeding

Introduction

Transmission through breastfeeding remains important for mother-to-child transmission (MTCT) in resource-limited settings. We quantify the relationship between cell-free (RNA) and cell-associated (DNA) shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum.

Materials and Methods

Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission.

Results

There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml) in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml) was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33–4.59) vs. aHR 1.52 (95% CI, 1.17–1.96), respectively]. At 6 months, cell-free and cell-associated levels (per ml) in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64–3.92) vs. 1.73 (95% CI 0.94–3.19), respectively].

Conclusions

The findings suggest that cell-associated virus level (per ml) is more important for early postpartum HIV-1 transmission (at 6 weeks) than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective drugs during lactation.     

Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission

Background

WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis.

Method

1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1–2, 4–6 and 12–16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%.

Results

50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39–64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus.

Conclusion

Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.     

Different regions of HIV-1 subtype C env are associated with placental localization and in utero mother-to-child transmission

HIV infections are initiated by a limited number of variants that diverge into a diverse quasispecies swarm. During in utero mother-to-child transmission (IU MTCT), transmitted viral variants must pass through multiple unique environments, and our previously published data suggest a nonstochastic model of transmission. As an alternative to a stochastic model of viral transmission, we hypothesize that viral selection in the placental environment influences the character of the viral quasispecies when HIV-1 is transmitted in utero. To test this hypothesis, we used single-template amplification to isolate HIV-1 envelope gene (env) sequences from both peripheral plasma and the placentas of eight nontransmitting (NT) and nine IU-transmitting participants. Statistically significant compartmentalization between peripheral and placental HIV-1 env was detected in one of the eight NT cases and six of the nine IU MTCT cases. In addition, viral sequences isolated from IU MTCT placental tissue showed variation in env V1 loop lengths compared to matched maternal sequences, while NT placental env sequences did not. Finally, comparison of env sequences from NT and IU MTCT participants indicated statistically significant differences in Kyte-Doolittle hydropathy in the signal peptide, C2, V3, and C3 regions. Our working hypothesis is that the hydropathy differences in Env associated with IU MTCT alter viral cellular tropism or affinity, allowing HIV-1 to efficiently infect placentally localized cells.     

Zidovudine Exposure in HIV-1 Infected Tanzanian Women Increases Mitochondrial DNA Levels in Placenta and Umbilical Cords

Background

Zidovudine (AZT) constitutes part of the recommended regimens for prevention and treatment of HIV-1 infection. At the same time, AZT as well as HIV-1 infection itself may induce mitochondrial damage. In this study, we analyzed the impact of prenatal AZT-exposure on mitochondrial alterations in HIV-infected women and their infants.

Methods

Mitochondrial DNA (mtDNA) levels in placentas of HIV-1 infected Tanzanian women with and without prenatal AZT exposure, and in the umbilical cords of their AZT-exposed/unexposed infants were quantified using real-time PCR. Furthermore, we checked for the most common mitochondrial deletion in humans, the 4977 base pair deletion (dmtDNA4977) as a marker for mitochondrial stress.

Results

83 women fulfilled the inclusion criteria. 30 women had been treated with AZT (median duration 56 days; IQR 43–70 days) while 53 women had not taken AZT during pregnancy. Baseline maternal characteristics in the two groups were similar. The median mtDNA levels in placentas and umbilical cords of women (311 copies/cell) and infants (190 copies/cell) exposed to AZT were significantly higher than in AZT-unexposed women (187 copies/cell; p = 0.021) and infants (127 copies/cell; p = 0.037). The dmtDNA4977 was found in placentas of one woman of each group and in 3 umbilical cords of AZT-unexposed infants but not in umbilical cords of AZT-exposed infants.

Conclusions

Antenatal AZT intake did not increase the risk for the common mitochondrial deletion dmtDNA4977. Our data suggests that AZT exposure elevates mtDNA levels in placentas and umbilical cords possibly by positively influencing the course of maternal HIV-1 infection.     

Restriction of HIV-1 Genotypes in Breast Milk Does Not Account for the Population Transmission Genetic Bottleneck That Occurs following Transmission

Background

Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established.

Methodology/Principal Findings

We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue.

Conclusions/Significance

Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.     

Restricted Genetic Diversity of HIV-1 Subtype C Envelope Glycoprotein from Perinatally Infected Zambian Infants

Background

Mother-to-child transmission of HIV-1 remains a significant problem in the resource-constrained settings where anti-retroviral therapy is still not widely available. Understanding the earliest events during HIV-1 transmission and characterizing the newly transmitted or founder virus is central to intervention efforts. In this study, we analyzed the viral env quasispecies of six mother-infant transmission pairs (MIPs) and characterized the genetic features of envelope glycoprotein that could influence HIV-1 subtype C perinatal transmission.

Methodology and Findings

The V1-V5 region of env was amplified from 6 MIPs baseline samples and 334 DNA sequences in total were analyzed. A comparison of the viral population derived from the mother and infant revealed a severe genetic bottleneck occurring during perinatal transmission, which was characterized by low sequence diversity in the infant. Phylogenetic analysis indicates that most likely in all our infant subjects a single founder virus was responsible for establishing infection. Furthermore, the newly transmitted viruses from the infant had significantly fewer potential N-linked glycosylation sites in Env V1-V5 region and showed a propensity to encode shorter variable loops compared to the nontransmitted viruses. In addition, a similar intensity of selection was seen between mothers and infants with a higher rate of synonymous (dS) compared to nonsynonymous (dN) substitutions evident (dN/dS<1).

Conclusions

Our results indicate that a strong genetic bottleneck occurs during perinatal transmission of HIV-1 subtype C. This is evident through population diversity and phylogenetic patterns where a single viral variant appears to be responsible for infection in the infants. As a result the newly transmitted viruses are less diverse and harbored significantly less glycosylated envelope. This suggests that viruses with the restricted glycosylation in envelope glycoprotein appeared to be preferentially transmitted during HIV-1 subtype C perinatal transmission. In addition, our findings also indicated that purifying selection appears to predominate in shaping the early intrahost evolution of HIV-1 subtype C envelope sequences.     

Infections in infants during the first 12 months of life: role of placental malaria and environmental factors

Background

The association between placental malaria (PM) and first peripheral parasitaemias in early infancy was assessed in Tori Bossito, a rural area of Benin with a careful attention on transmission factors at an individual level.

Methodology

Statistical analysis was performed on 550 infants followed weekly from birth to 12 months. Malaria transmission was assessed by anopheles human landing catches every 6 weeks in 36 sampling houses and season defined by rainfall. Each child was located by GPS and assigned to the closest anopheles sampling house. Data were analysed by survival Cox models, stratified on the possession of insecticide-treated mosquito nets (ITNs) at enrolment.

Principal Findings

Among infants sleeping in a house with an ITN, PM was found to be highly associated to first malaria infections, after adjusting on season, number of anopheles, antenatal care (ANC) visits and maternal severe anaemia. Infants born from a malaria infected placenta had a 2.13 fold increased risk to present a first malaria infection than those born from a non infected placenta ([1.24–3.67], p<0.01) when sleeping in a house with an ITN. The risk to present a first malaria infection was increased by 3.2 to 6.5, according to the level of anopheles exposure (moderate or high levels, compared to the absence of anopheles).

Conclusions

First malaria infections in early childhood can be attributed simultaneously to both PM and high levels of exposure to infected anopheles. Protective measures as Intermittent Preventive Treatment during pregnancy (IPTp) and ITNs, targeted on both mothers and infants should be reinforced, as well as the research on new drugs and insecticides. In parallel, investigations on placental malaria have to be strengthened to better understand the mechanisms involved, and thus to protect adequately the infants high risk group.     

Generation of a Recombinant Gag Virus-Like-Particle Panel for the Evaluation of p24 Antigen Detection by Diagnostic HIV Tests

Background

Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.

Methods

We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4^th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.

Results

Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4^th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.

Conclusions

The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.     

Phylogenetic Relatedness of Circulating HIV-1C Variants in Mochudi,Botswana

Background

Determining patterns of HIV transmission is increasingly important for the most efficient use of modern prevention interventions. HIV phylogeny can provide a better understanding of the mechanisms underlying HIV transmission networks in communities.

Methods

To reconstruct the structure and dynamics of a local HIV/AIDS epidemic, the phylogenetic relatedness of HIV-1 subtype C env sequences obtained from 785 HIV-infected community residents in the northeastern sector of Mochudi, Botswana, during 2010–2013 was estimated. The genotyping coverage was estimated at 44%. Clusters were defined based on relatedness of HIV-1C env sequences and bootstrap support of splits.

Results

The overall proportion of clustered HIV-1C env sequences was 19.1% (95% CI 17.5% to 20.8%). The proportion of clustered sequences from Mochudi was significantly higher than the proportion of non-Mochudi sequences that clustered, 27.0% vs. 14.7% (p = 5.8E-12; Fisher exact test). The majority of clustered Mochudi sequences (90.1%; 95% CI 85.1% to 93.6%) were found in the Mochudi-unique clusters. None of the sequences from Mochudi clustered with any of the 1,244 non-Botswana HIV-1C sequences. At least 83 distinct HIV-1C variants, or chains of HIV transmission, in Mochudi were enumerated, and their sequence signatures were reconstructed. Seven of 20 genotyped seroconverters were found in 7 distinct clusters.

Conclusions

The study provides essential characteristics of the HIV transmission network in a community in Botswana, suggests the importance of high sampling coverage, and highlights the need for broad HIV genotyping to determine the spread of community-unique and community-mixed viral variants circulating in local epidemics. The proposed methodology of cluster analysis enumerates circulating HIV variants and can work well for surveillance of HIV transmission networks. HIV genotyping at the community level can help to optimize and balance HIV prevention strategies in trials and combined intervention packages.