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Local replication of simian immunodeficiency virus in the breast milk compartment of chronically-infected, lactating rhesus monkeys
© Permar et al; licensee BioMed Central Ltd. 2010
Received: 3 November 2009
Accepted: 1 February 2010
Published: 1 February 2010
Breast milk transmission remains a major mode of infant HIV acquisition, yet anatomic and immunologic forces shaping virus quasispecies in milk are not well characterized. In this study, phylogenic analysis of envelope sequences of milk SIV variants revealed groups of nearly identical viruses, indicating local virus production. However, comparison of the patterns and rates of CTL escape of blood and milk virus demonstrated only subtle differences between the compartments. These findings suggest that a substantial fraction of milk viruses are produced by locally-infected cells, but are shaped by cellular immune pressures similar to that in the blood.
While transmission via breastfeeding remains a significant mode of infant HIV acquisition, the mechanisms of this transmission are not well understood. The level of both cell-free and cell-associated virus in milk have been linked to the risk of HIV infection of breastfeeding infants [1–3]. Therefore, viral variants in the milk are a likely source of transmitted virus.
Recent studies suggest that mucosal compartments are distinct from systemic compartments in their HIV/SIV-specific immune responses and virus quasispecies [4–8]. The immune response in milk may shape compartment-specific virus, as seen in other anatomic compartments such as semen and cervicovaginal fluid [6, 9–11]. One study of the genetic diversity of milk virus suggested a difference in the dominant virus species in milk and peripheral blood . A second study subsequently reported that the predominant virus in blood and milk were genetically similar, suggesting an equilibrium of virus between these compartments . Thus, the production site of the virus transmitted via breastfeeding and the selection pressures that shape its genetic composition are not well understood.
It is well established that HIV/SIV-specific cellular immune responses are critical for control of systemic virus replication  and progression to AIDS . While HIV/SIV-specific cellular immune responses have been identified in milk [15, 16], the role of these responses in containing local virus replication is unknown. Immunodominant cytotoxic T lymphocyte (CTL) epitopes of SIV and HIV are under significant cellular immune pressure, leading to distinct mutations within these epitopes that facilitate virus escape from CTL recognition. Therefore, the rate at which these CTL escape mutations occur within a virus population may be indicative of the magnitude of the cellular immune pressure in that compartment. However, rates of CTL escape of mucosal and systemic virus quasispecies have not previously been compared. As the proportion of SIV-specific CD8+ T lymphocytes is two to three times higher in milk than in blood during acute SIV infection , a faster rate of CTL escape of the milk virus quasispecies than that in blood would indicate that this immune response in milk is acting on locally replicating virus.
The SIV-infected rhesus monkey model of HIV pathogenesis is an excellent model in which to study virus-specific immune responses and virus evolution in mucosal compartments, including breast milk . In the present study, we sought to evaluate genetic similarities between virus quasispecies in blood and milk of chronically SIVmac251-infected rhesus monkeys through phylogenetic comparison of SIV envelope (env) sequences. We then compared the rate of CTL escape of two Mamu-A*01-restricted immunodominant epitopes (Tat TL8 and Gag p11C) in blood and milk virus. Animals were maintained in accordance with the guidelines of the "Guide for the Care and Use of Laboratory Animals" (National Research Council, National Academic Press, Washington, D.C., 1996).
Groups of identical or nearly identical viruses in breast milk of chronically SIV-infected, lactating rhesus monkeys
Overall, the range of nucleotide sequence diversity of cell-free milk virus (monkey 206 = 1.03%, monkey 257 = 0.76%, monkey 403 = 0.75%) was similar to that in the cell-free blood virus (monkey 206 = 1.1%, monkey 257 = 0.85%, monkey 403 = 0.73%). Strikingly, groups of identical or nearly identical cell-free envs were identified in milk of all animals, while this was not observed for plasma-derived cell-free env sequences. These groups of envs were either identical or differed by a single nucleotide (0 - 0.04% nucleotide difference) and comprised one-third to three-quarters of the cell-free milk virus amplified in each animal (monkey 206 = 38.1%, monkey 257 = 76.5%, monkey 402 = 73.3%). Moreover, in animal 402, a single breast milk cell-associated virus variant had a sequence identical to that of a cluster of cell-free milk viruses, which suggests that the clonal amplification of cell-free milk virus variants is produced by a small number of productively-infected, resident cells. In fact, our findings are consistent with a recent report that actively replicating virus in breast milk evolves under drug pressure that is distinct from that in the plasma . Thus, locally-produced virus is likely the major source of virus present in breast milk.
However, we did not observe phylogenetic evidence of compartmentalization of blood and milk virus quasispecies, as the blood and milk viral variants were interspersed in the phylogenetic tree of sequences derived from each monkey. While the milk viruses did not cluster in the phylogenetic trees, all animals exhibited significant compartmentalization between blood and milk cell-free virus quasispecies as determined by the Slatkin-Maddison compartmentalization test of the minimum possible number of inter-compartment migration events compared to the distribution of migration events in 1000 randomized trees [22, 23] implemented by HYPHY software  (monkey 206: p = 0.001; monkey 402: p = 0.003; monkey 403: p = 0.001). When repeated identical and nearly identical sequences were removed from the data set subjected to the Slatkin-Maddison analysis, significant compartmentalization between blood and breast milk was only evident in animal 206 (monkey 206: p = 0.006; monkey 402: p = 0.795; monkey 403: p = 0.505). Therefore, whether breast milk and blood represent distinct anatomic compartments for SIV in the setting of chronic infection remains an open question.
Rate of CTL escape at immunodominant epitopes Tat TL8 and Gag p11C in breast milk virus
As a robust SIV-specific CD8+ T lymphocyte response occurs in breast milk during acute SIV infection, we investigated whether CTL escape of two Mamu-A*01-restricted immunodominant epitopes Tat TL8 and Gag p11C occurred at a different rate in milk and blood virus quasispecies. The Tat TL8 epitope was amplified by SGA with primers described above (6 to 31 amplicons per compartment). The Gag p11C epitope was analyzed by SGA with primers: outer forward (5' - GTCTGCGTCATCTGGTGCATTC - 3'), outer reverse (5' - TGTTTGTTCTGCTCTTAAGCTTTTGTAG - 3'), inner forward (5' - CAAAACAGATAGTGCAGAGACACCTAGTG - 3'), inner reverse (5' - GAAATGGCTCTTTTGGTCCTT - 3'). Virus quasispecies with at least one nonsynonymous mutation within an immunodominant epitope was defined as a CTL escape variant. CTL escape at the Tat TL8 epitope occurs rapidly in the blood so that all virus quasispecies have a mutated Tat TL8 epitope by day 28 of infection , whereas CTL escape at the Gag p11C epitope occurs much later due to structural constraints on the virus at that epitope [26, 27].
CTL escape of breast milk and blood virus quasispecies at the Tat TL8 epitope during acute SIV infection.
Days after SIV infection
We used the Spearman's rank correlation test to determine if the proportion of virus variants with CTL escape of Tat TL8 at day 18 after SIV infection was associated with the proportion of Tat TL8-specific CD8+ T lymphocytes or the magnitude of the milk virus load . The proportion of virus with evidence of CTL escape in the milk virus population most strongly correlated with the peak milk virus load (R2 = 0.986, p = 0.083). Therefore, the slightly slower rate of CTL escape mutation of the Tat TL8 epitope in milk is likely a result of a lower rate of virus replication in the breast milk compartment.
CTL escape at the Gag p11c epitope in breast milk and blood virus quasispecies during acute SIV infection.
Weeks after SIV infection
In summary, CTL escape of milk virus occurred with a pattern and rate similar to that seen in blood virus, suggesting that similar cellular immune pressures shape virus quasispecies evolution in these compartments. While SIV-specific CD8+ T lymphocyte responses during acute SIV infection appear in milk concurrently with the reduction of milk virus load from peak , we were not able to demonstrate that the high proportion of SIV-specific CD8+ T lymphocytes observed in milk drives earlier CTL escape of milk virus. However, the small number of evaluated monkeys and restricted sampling schedule make it difficult to demonstrate distinct rates of CTL escape in blood and milk virus. Although a limited number of animals were included in this study, clonal amplification of virus in milk and not in plasma of all animals studied suggests that replication of breast milk virus occurs locally.
This work was supported by the Center for HIV/AIDS Vaccine Immunology (SP and NL; AI067854), the Pediatric Infectious Disease Society/St. Jude Children's Hospital Basic Science Research Award (SP), and NIH K08AI087992 (SP).
We are grateful to Jesus Gonzalez-Salazar, Maria Salazar, Jeff Anderson, Aravind Basavapathruni, James Whitney, George Shaw, and Bette Korber for assistance with sequence analysis and data interpretation.
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