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

Abstract

BackgroundBreast 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 FindingsWe 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/SignificanceDespite 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.

Origin and Evolution of HIV-1 in Breast Milk Determined by Single-Genome Amplification and Sequencing

“…Moreover, the milk virus env sequences from these groups of identical viruses produced functional pseudovirions, indicating that the groups of identical virus env sequences represent replication of functional virus variants. This clonal amplification of virus variants has been described for milk of chronically SIVinfected, lactating monkeys (38) and HIV-infected, lactating women (13,15). However, the presence of genetically identical viruses in HIV-infected, lactating women has not been confirmed previously by strict single-virus RNA genome amplification or assessed for functionality and persistence.…”

“…Each of the subjects with evidence of significant compartmentalization by both tests had a large number of identical or nearly identical sequences in milk (Table 3), which could contribute to an overestimation of potential compartmentalization (6,7,15). Therefore, the identical sequences within the same compartment were removed from the data set subjected to the SM and S nn analyses for all subjects.…”

“…Moreover, comparison of the levels of genetic diversity of the HIV env sequences of breast milk and plasma virus populations did not reveal consistently low genetic diversity among breast milk virus variants, which might be expected of a compartmentalized, small pool of virus that evolves distinctly from circulating virus. However, the women included in this study were selected for detectable virus loads in milk, and therefore, it is possible that the degree of compartmentalization of milk virus is substantial in women with Although early studies employing phylogenetic analysis of the variable region of HIV env after bulk cloning of breast milk and plasma viruses were contradictory as to whether these virus populations were compartmentalized (5,16), the application of single-genome amplification of fulllength HIV env and the consistency with data from nonhuman primate models (38) and other human cohorts (13,15) increase the strength of our finding that breast milk viruses are not compartmentalized phylogenetically from plasma viruses in the majority of subjects. Interestingly, two subjects displayed groups of identical and nearly identical viruses that included both blood and milk virus sequences (subjects 4403 and 5807).…”

“…However, recent studies of HIV-infected lactating women (13,15,16) and simian immunodeficiency virus (SIV)-infected lactating monkeys (38) have suggested that the virus present in milk is not genetically distinct from that in plasma. In addition, recent human and primate studies of the virus population in milk reported that groups of identical or nearly identical viruses were frequently detected in milk, suggesting that a proportion of viruses present in milk are produced by infected cells which are resident in the mammary gland (13,15,38). However, the origin, persistence, and functionality of these monotypic viruses are unknown.…”

The Breadth and Potency of Passively Acquired Human Immunodeficiency Virus Type 1-Specific Neutralizing Antibodies Do Not Correlate with the Risk of Infant Infection

“…Recent studies indicate that the viruses in breast milk and blood are intermingled and are not generally compartmentalized (15,17), suggesting that breast milk viruses are typical of circulating viruses. Moreover, the probability of infection per exposure during breastfeeding, measured either as per liter of breast milk ingested or by daily exposure, is generally similar to the probability of infection from one unprotected exposure during sexual transmission (38).…”