The statistically significantly larger proportion of genetically identical viruses in breast milk than in plasma for 6 of 12 subjects in this cohort, identified by a sequencing method that limits the possibility of virus resampling (20
), is striking. 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 SIV-infected, lactating monkeys (38
) and HIV-infected, lactating women (13
). 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. While we used only genetically identical env
genes for statistical analysis of monotypic viruses in milk and plasma in this study, the nearly identical viruses that cluster together on the phylogenetic trees certainly also represent clonally amplified variants, given the large amount of sequence diversity within a single chronically HIV-infected individual. Therefore, our report of the frequency of only the genetically identical viruses in milk likely underestimates the true frequency of clonally amplified viruses in milk. The high frequency of genetically identical and nearly identical clonally amplified virus variants in milk indicates that infected cells that have trafficked to or are resident in the mammary gland may produce at least a portion of the viruses present in the milk.
Despite evidence that breast milk virus may be produced by infected cells resident in the breast milk compartment, we found limited evidence of phylogenetic compartmentalization and distinct evolution of viruses in breast milk from that of circulating viruses by maximum likelihood tree analysis and phylogenetic and genetic distance-based tests of compartmentalization for the majority of subjects. Furthermore, longitudinal env
sequencing of milk and plasma virus variants revealed analogous virus evolution over time, indicating ongoing seeding of the breast milk virus population by blood variants. This lack of compartmentalization of virus in milk in the majority of subjects is in contrast to studies of virus populations in the male and female genital tracts, where compartmentalization of virus in genital secretions is frequently observed in the majority of subjects studied (1
). These contrasting results may indicate a difference in virus trafficking from the systemic circulation to the genital tract and the breast milk compartment (38
). 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 low milk virus loads. 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
), the application of single-genome amplification of full-length HIV env
and the consistency with data from nonhuman primate models (38
) and other human cohorts (13
) 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). Furthermore, both right and left breast milk virus variants populated the major cluster of genetically identical and nearly identical virus variants for subject 4403. Moreover, the genetic diversity of virus env
sequences in milk samples that displayed a large proportion of clonally amplified virus variants was not always more restricted than that in plasma (subjects 0601, 3305, and 3404). These findings, paired with the appearance of new clusters of clonally amplified virus variants that are closely related to concurrent blood virus variants in longitudinal milk virus sequencing, indicate ongoing bursts of local virus replication after seeding of the milk compartment by circulating virus variants. In fact, a recent comparison of breast milk HIV env
genetic diversity in antiretroviral-treated women revealed virus evolution under selective drug pressure in milk that was distinct from that in plasma, implicating local replication of breast milk virus (3
). This insight into the location of milk virus replication is important to the design of immunologic interventions to reduce virus content in the milk, as decreasing milk virus loads would likely lead to a reduction in the risk of transmission of HIV via breastfeeding.
Finally, full-length HIV env
sequencing of the milk virus population allowed us to assess whether a distinct virus env
sequence, predicted virus Env phenotype, or neutralization sensitivity was unique to breast milk virus env
genes. Analysis of milk virus env
mutations and deletions compared to those in plasma HIV env
sequences did not reveal a signature sequence or deletion unique to breast milk viruses. Furthermore, milk virus env
sequences were not consistently predicted to be glycosylated more or less than those of plasma viruses, did not consistently have a longer or shorter V region length than that of plasma viruses, and were not distinctly resistant to neutralization by plasmas of chronically HIV-infected subjects or broadly HIV-neutralizing antibodies. Therefore, while neutralization-resistant viruses have been associated with breast milk transmission of HIV (43
), a neutralization-resistant env
phenotype consisting of a small number of N-linked glycosylation sites and deletions in the variable loops (28
) was not more common in breast milk viruses than in plasma viruses. This lack of a predicted Env phenotype or neutralization phenotype unique to breast milk virus variants suggests that plasma and breast milk viruses are not compartmentalized from each other and evolve under similar humoral immune pressures.
Importantly, we did not find evidence of distinct breast milk virus evolution, a distinct degree of local virus replication, or a predicted virus Env phenotype in milk of transmitting subjects compared to nontransmitting subjects. This finding suggests that the degree of local breast milk virus replication or the virus Env phenotype may not contribute to the risk of virus transmission via breastfeeding. Subclinical mastitis, which is known to increase the risk of breast milk virus transmission (12
), was associated with long genetic distances between plasma and milk virus variants in a recent report (13
). In our study, subclinical mastitis was associated with a large proportion of clonally amplified viruses in milk for three of three subjects with mastitic milk (subjects 0404, 1209, and 3404), with evidence of compartmentalization between plasma and milk virus variants for two of three subjects with mastitic milk (subjects 0404 and 3404), and with higher genetic diversity of milk viruses than plasma viruses for one of three subjects with mastitic milk (subject 3404). These findings may suggest increased kinetics of virus replication in the breast milk compartment during subclinical mastitis and breast inflammation. Therefore, the possibility of increased local virus replication as a result of inflammatory processes in the mammary gland leading to a high risk of virus transmission via breast milk should be investigated further.
The absence of phylogenetic compartmentalization, distinct virus evolution, or unique virus env genetic or phenotypic features of milk viruses compared to those in plasma suggests that viruses in breast milk are replenished from the pool of circulating viruses. However, the high frequency of clonally amplified virus variants in milk suggests that a relatively small number of productively infected cells in mammary tissue transiently contribute a disproportionately large number of virions to the virus pool in milk. Therefore, there may be two mechanisms by which virus populates the breast milk compartment. The first is continual trafficking of cell-free or cell-associated virus from blood into the breast milk compartment. The second is transient local production of virus recently trafficked from the blood by HIV-infected cells in the mucosa of the mammary gland or in the milk. Therefore, immunologic and antiretroviral drug interventions to decrease breast milk virus load as a strategy to reduce transmission of HIV via breastfeeding may need to target both blood-derived virus trafficking into the mammary gland and local replication of HIV within the breast milk compartment.