Monotypic Human Immunodeficiency Virus Type 1 Genotypes across the Uterine Cervix and in Blood Suggest Proliferation of Cells with Provirus

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HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P ‫؍‬ 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.

Section: Resultsmentioning

confidence: 99%

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

Salazar-Gonzalez

Salazar

Learn

et al. 2011

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“…First, populations of monotypic HIV-1 env sequences have been observed in plasma (15,19,29) and genital tract (33) specimens. Second, central memory CD4…”

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confidence: 99%

An Increasing Proportion of Monotypic HIV-1 DNA Sequences during Antiretroviral Treatment Suggests Proliferation of HIV-Infected Cells

Wagner¹,

McKernan²,

Tobin

et al. 2013

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Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656 env and pol sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%; P < 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4؉ T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.

“…The topology of each phylogenetic tree was examined, with particular attention to the identification of breast milk-or peripheral blood-specific clades. The compartmental structure of viral sequences in blood and milk specimens obtained from each subject was evaluated by the Slatkin and Maddison (SM) test, which evaluates variation from normalcy in the distribution of sequences over a predicted tree structure using MacClade (8,49). Each blood and milk specimen was included in SM analysis if at least five sequences were available from that specimen.…”

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confidence: 99%

“…No evidence for compartmentalization between right and left breast milk specimens was observed (data not shown). After monotypic sequences, which can inflate statistical estimates of population structure (7,8,21), were collapsed into a single sequence, 3 of 6 (50%) specimens no longer showed evidence of compartmentalization by SM testing (Table 3), reducing the overall rate to 3 of 17 (17.6%) specimens.…”

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confidence: 99%

Genetic Analyses of HIV-1 env Sequences Demonstrate Limited Compartmentalization in Breast Milk and Suggest Viral Replication within the Breast That Increases with Mastitis

Gantt

Carlsson

Heath

et al. 2010

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The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na ؉ ) concentration. The association between milk Na ؉ and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P ‫؍‬ 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P ‫؍‬ 0.621), but Na ؉ was correlated with greater genetic distance between milk and blood HIV-1 populations (P ‫؍‬ 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.