Irene N. Koulinska

Transmission of cell-free and cell-associated HIV-1 through breast-feeding.

Background:Transmission through breast-feeding is an important cause of infant HIV-1 infections in developing countries; however, its mechanism remains largely unknown. We have explored the association between cell-free virus (CFV) and cell-associated virus (CAV) levels in breast milk (BM), as reflected by viral RNA and proviral DNA, respectively, and the risk of infant HIV-1 infection after 6 weeks postpartum. Methods:Sixty-one HIV-positive mothers who transmitted HIV-1 by BM were matched to 61 HIV-positive nontransmitting mothers based on their infants age at sample collection. CFV and CAV were quantified in a single milk specimen per mother preceding the infants first HIV-positive result. Results:After adjusting for maternal CD4+ cell counts and disease stage, each 10-fold increase in CFV or CAV load was associated with an almost 3-fold increase in BM transmission. Whereas CAV load was predictive of transmission before and after 9 months postpartum, CFV was a significant predictor of transmission occurring only after 9 months. Phylogenetic analyses of the C2 to C5 env region showed that 85% of infants (11 of 13 infants) harboring viruses that clustered with CFV in their mothers milk were infected after 9 months postpartum. Conclusion:A reduction in milk CAV and CFV loads might significantly decrease HIV-1 transmission by breast-feeding.

A New Human Immunodeficiency Virus Type 1 Circulating Recombinant Form from Tanzania

It is becoming increasingly important to identify and to study human immunodeficiency virus type 1 (HIV-1) circulating recombinant forms (CRFs) with evidence of epidemic spread, since mosaic strains arise frequently, especially in populations where multiple subtypes cocirculate. We describe the almost complete nucleotide sequence of 3 subtype C and D recombinant viruses, selected from a pool of 13 D(gag)-D/C/D(env) perinatally infected infants from Dar es Salaam, Tanzania. All three genomes had cross-over points with approximately the same genomic localization. The subtype C-like sequences were located within pol, vif, vpr, vpu, the first exons of rev and tat, V3, and the U3-R regions of the LTR. Phylogenetic analyses of the full-length genomic sequences from these viruses showed the formation of a distinct subcluster on the HIV-1 subtype D branch. The pattern of recombination of genomes belonging to this new CRF, named CRF10_CD, might have resulted from independent recombination events occurring at high frequency or from a single source that originated earlier in this population. Future surveys will be needed to determine the potential of this CRF for epidemic spread.

Zinc supplementation to HIV-1-infected pregnant women: effects on maternal anthropometry, viral load, and early mother-to-child transmission.

Objective:To examine the effect of zinc supplementation to HIV-1-infected pregnant women on viral load, early mother-to-child transmission of HIV (MTCT), and wasting.Design:Double-blind placebo-controlled randomized clinical trial.Setting:Antenatal clinic in Dar es Salaam, Tanzania.Subjects:Four hundred HIV-1-infected pregnant women.Methods:Women 12–27 weeks of gestation were randomly assigned to receive a daily oral dose of 25 mg zinc or placebo from the day of the first prenatal visit until 6 weeks postdelivery. Weight and mid-upper arm circumference (MUAC) were measured monthly. HIV status of the babies was assessed at birth and at 6 weeks postpartum. Viral load was assessed in a random sample of 100 women at baseline and at the end of the study.Results:Zinc had no effects on maternal viral load or early MTCT. Supplementation was related to a significant threefold increase in the risk of wasting (reaching a MUAC value <22 cm) during an average 22 weeks of observation (RR=2.7, 95%CI=1.1, 6.4, P=0.03), and to a 4 mm decline in MUAC during the second trimester (P=0.02).Conclusions:Zinc supplementation to HIV-infected pregnant women offers no benefits on viral load or MTCT. The clinical relevance of an apparent decrease in MUAC associated with zinc supplementation is yet to be ascertained. These findings together with the lack of effect on fetal outcomes (reported previously) do not provide support for the addition of zinc supplements to the standard of prenatal care among HIV-infected women.Sponsorship:The US National Institute of Child Health and Human Development (NICHD R01 32257).

Subclinical Mastitis, Cell-Associated HIV-1 Shedding in Breast Milk, and Breast-Feeding Transmission of HIV-1

Background:Mastitis has been identified as a risk factor for mother-to-child transmission (MTCT) of HIV-1 through breast-feeding. It is unclear whether this association is mediated by increased cell-free virus (CFV) versus cell-associated virus (CAV) HIV shedding in breast milk. Methods:We examined the risk of MTCT associated with subclinical mastitis and the relation between mastitis and CFV or CAV shedding in breast milk. Fifty-nine women who transmitted HIV through breast-feeding (cases) were individually matched to 59 nontransmitting controls nested in a cohort from Tanzania. For each case, we selected a milk specimen obtained before the infants first positive test to quantify sodium (Na) and potassium (K) and measure CFV and CAV concentrations. Controls were matched on the childs age at the time of sample collection. Results:Women with a breast milk Na/K ratio suggestive of mastitis (>1.0) had an 11-fold greater odds of transmission (95% confidence interval [CI]: 1.2 to 98.1), compared to women with a Na/K ≤0.6, after adjusting for maternal CD4 cell count and vitamin A supplementation. Although mastitis was positively related to both CFV and CAV shedding in breast milk, only the association with the latter was strong and statistically significant. Conclusion:Increased cell-associated HIV-1 shedding in breast milk could mediate the association between mastitis and MTCT.

Hypermutation of HIV type 1 genomes isolated from infants soon after vertical infection.

Hypermutation involving excessive G-to-A substitutions in the dinucleotide context GA or GG is common among the lentiviruses and results in multiple stop codons across the genome. Hypermutated viruses have been associated with slower disease progression and might reflect an antiviral cell-defense mechanism. However, it is unclear how soon G-to-A substitutions are generated after infection and whether they occur randomly along the genome. In this report we describe for the first time hypermutated sequences detected at delivery and in the first weeks of life, which suggests that they could be either generated in utero and soon after birth and/or vertically transmitted. Hypermutated C2-C5 env clones were harbored in 13.2% of 243 infants and 18.6% of 199 mothers. A lower extent of hypermutation was found in infants than in mothers (Fishers exact p = 0.034), but there was no relationship between the percent hypermutated Gs and viral subtype or transmission status of the mother. Analyses of six hypermutated full-length HIV-1 clones showed that although all genes could be affected by G-to-A substitutions, there was a significant drop in the extent of hypermutation between the central polypurine tract and the beginning of env, indicating that hypermutation across the HIV-1 genome might occur in a specific pattern. The genomic regions most affected by hypermutation were pol and env while both polypurine tracts remained unaffected. A better understanding of the mechanism of hypermutation may reveal novel virus-host interactions that could be targeted in drug development.

Effect of vitamin supplements on HIV shedding in breast milk

BACKGROUND Supplementation in lactating HIV-1-infected women with preformed vitamin A and β-carotene (VA/BC) increases the risk of mother-to-child transmission of HIV through breastfeeding. Identifying a biological mechanism to explain this unexpected finding would lend support to a causal effect. OBJECTIVE The aim of the study was to evaluate the effect of VA/BC or multivitamin (B complex, vitamin C, and vitamin E) supplementation of HIV-infected women on HIV shedding in breast milk during the first 2 y postpartum. DESIGN We quantified viral (cell-free) and proviral (cell-associated) HIV loads in breast-milk samples collected ≤15 d after delivery and every 3 mo thereafter from 594 Tanzanian HIV-1-infected women who participated in a randomized trial. Women received 1 of the following 4 daily oral regimens in a 2 × 2 factorial fashion during pregnancy and throughout the first 2 y postpartum: multivitamin, VA/BC, multivitamin including VA/BC, or placebo. RESULTS The proportion of breast-milk samples with detectable viral load was significantly higher in women who received VA/BC (51.3%) than in women who were not assigned to VA/BC (44.8%; P = 0.02). The effect was apparent ≥6 mo postpartum (relative risk: 1.34; 95% CI: 1.04, 1.73). No associations with proviral load were observed. The multivitamin had no effects. In observational analyses, β-carotene but not retinol breast-milk concentrations were significantly associated with an increased viral load in milk. CONCLUSIONS VA/BC supplementation in lactating women increases the HIV load in breast milk. This finding contributes to explaining the adverse effect of VA/BC on mother-to-child transmission. β-Carotene appears to have an effect on breast-milk viral load, independent of preformed vitamin A. This trial was registered at clinicaltrials.gov as NCT00197756.

Common genetic arrangements among human immunodeficiency virus type 1 subtype A and D recombinant genomes vertically transmitted in Tanzania.

Human immunodeficiency virus type 1 (HIV-1) subtypes A, C, and D are cocirculating in Tanzania, and large numbers of recombinant genomes have been reported from this region. Here we describe full-length sequences of six unlinked HIV-1 subtype A and D recombinants. The samples came from newborns, indicating that the recombination patterns were vertically transmitted and were functionally competent. All six genomes had different recombination patterns with one to eight cross-over points frequently located at the beginning or end of functionally defined regions. In five of the six viruses most of gag, pol, tat, and rev and the intracytoplasmic domain of gp41 were classified as subtype D. In all but one genome, the external domain of gp41 and the majority of gp120 belonged to subtype A. A recombination site common to four of the six genomes was located at the transmembrane domain of gp41, at the end of the rev response element. The identification of subtype patterns among intersubtype recombinant genomes from recently infected individuals may reveal genetic determinants of improved viral fitness or advantage for transmission.

Association between Breast Milk Erythropoietin and Reduced Risk of Mother-to-Child Transmission of HIV

We examined the prospective associations between breast milk concentrations of erythropoietin, a factor with trophic effects on infant gut epithelia, and the risk of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) through breast-feeding in a study of 59 MTCT cases and 116 controls nested within a cohort of antiretroviral-naive HIV-infected Tanzanian women. Controls were matched to cases on the basis of the time from birth when the breast milk sample was collected. The risk of MTCT was inversely related to breast milk erythropoietin concentration (adjusted odds ratio for highest vs lowest erythropoietin concentration tertile, 0.34 [95% confidence interval, 0.14-0.82]; P = .02). These results suggest a protective effect of breast milk erythropoietin against MTCT.