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

Abstract

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.

HIV-Specific Functional Antibody Responses in Breast Milk Mirror Those in Plasma and Are Primarily Mediated by IgG Antibodies

“…However, patient 3902 showed moderate to high neutralization potency at 9 months, with ID 50 s as high as 463. These results confirm that milk and plasma virus Envs have comparable neutralization sensitivities, as previously indicated by assessment of milk and plasma virus Env neutralization sensitivity using broadly neutralizing MAbs (55). The majority of the breast milk samples from uninfected lactating women displayed low levels of nonspecific HIV-neutralizing activity.…”

“…Thus, the role of neutralizing antibodies in vertical transmission of HIV is unclear. Viruses isolated from the breast milk and plasma of HIV-infected women have similar sensitivity to neutralization by broadly HIV-neutralizing monoclonal antibodies (MAbs) (55), but the ability of breast milk antibodies to neutralize autologous and heterologous HIVs has not been studied. Moreover, nonneutralizing antibodies, such as antibodies that mediate antibody-dependent cellular cytoxicity (ADCC) responses, may contribute to better clinical outcome and lower HIV/SIV load (2,17,41) and to the protection of vaccinated rhesus macaques against SHIV challenge (71).…”

Short Communication: HIV Type 1 Subtype C Variants Transmitted Through the Bottleneck of Breastfeeding Are Sensitive to New Generation Broadly Neutralizing Antibodies Directed Against Quaternary and CD4-Binding Site Epitopes

“…13,15 Others had similar findings for infants mostly infected intrapartum, though they only found differences in glycosylation sites and not variable loop lengths. 16 In this article, we present data on viral sequences from three mother-child pairs plus three additional infants where the infant became HIV DNA positive between 6 and 12 weeks postpartum through exposure to breast milk, with the goal of exploring whether known selective pressures influence this route of mother-to-child transmission.…”

“…12,13 Data are very limited for breastfeeding pairs, but one study of three breast milk transmission events found a similar bottleneck for HIV-1 subtype A.…”

Generation of Transmitted/Founder HIV-1 Infectious Molecular Clones and Characterization of Their Replication Capacity in CD4 T Lymphocytes and Monocyte-Derived Macrophages

“…All inferred genomes were intact and comprised of nine genes with open reading frames (LTR-gag-pol-vif-vpr-tat-rev-vpu-env-nef-LTR). Studies of these T/F virus sequences and progeny arising from them have given new fundamental insights into early virus evolution and the ontogeny of the antiviral immune response (1,26,31,34,46,56,66). We further hypothesized that the derivation with the Sequencher program (version 4.7; Gene Codes, Ann Arbor, MI) as previously described.…”