Target cell availability dictates mother-to-infant transmission of SIV Ann Chahroudi, MD, PhD Division of Pediatric Infectious Diseases and Yerkes National Primate Research Center, Emory University School of Medicine Atlanta, GA, USA Thank you to the conference organizers for giving me the chance to present our data today at AIDS 2012. I will be discussing some of our work in the nonhuman primate model that indicates that target cell availability dictates MTIT of SIV

Main features of SIV infection of natural vs. non-natural hosts I will first start by introducing the comparative AIDS model of SIV infection of natural and non-natural hosts.As many of you know, Natural hosts are west african nonhuman primate species that are naturally infected with SIV in the wild and include sooty mangabeys, african green monkeys, mandrills, and others. Humans and rhesus macaques are considered non-natural hosts because their infections are the result of either zoonotic transmission or experimental infection. The most striking features of SIV infection of natural hosts are their lack of progression to AIDS despite high viral loads. For the purpose of this talk, I would like to highlight another key feature and that is the differential rate of vertical transmission, with rare evidence of mother to infant transmission in natural hosts as compared to the more frequent transmission events seen in both humans and rhesus macaques. Chahroudi A et al, Science 2012

Low rates of mother-to-infant transmission in natural hosts THREE MECHANISTIC HYPOTHESES Low viral load in breast milk Innate inhibitory factors in milk (work in progress) Highly active immune responses in infants Restricted availability of CD4+CCR5+ target cells We propose 3 potential mechanisms to explain the low rates of mother to infant transmission of SIV in natural hosts. The first potential mechanism relates specifically to breastfeeding transmission and proposes that breast milk of natural hosts is less infectious due to either lower viral loads or innate inhibitory factors. Preliminary data we have generated but that I do not have time to show today is not supportive this hypothesis. The second potential mechanism is that infant natural hosts possess highly active immune responses against the virus. This seems unlikely however given the low level of Nab and ineffective T cell responses found in adult sooty mangabeys. The 3rd hypothesis is that infant natural hosts have limited CD4+CCR5+ target cells for SIV infection. We favor this mechanism as it has already been shown that adult sooty mangabeys restrict expression of CCR5 to an expendable subset of memory T cells, but the role of CCR5 in vertical SIV transmission in mangabeys had not been previously investigated. Chahroudi A et al, J Virol 2011

Experimental methods Multicolor flow cytometry to measure SIV target cells in bulk, naïve, and memory CD4+ T cells from infant sooty mangabey tissues at necropsy Direct comparison of the levels of SIV target cells found in the peripheral blood of infant sooty mangabeys and rhesus macaques To study the availability of SIV target cells we performed multicolor flow cytometry on cells isolated from infant sooty mangabey tissues at necropsy. We next performed a direct comparison of the levels of SIV target cells found in the peripheral blood of infant sooty mangabeys and rhesus macaques This table shows the age and cause of death of the sooty mangabey infants whose tissues we obtained at necropsy. All infants were born to SIV negative mothers and both parents were wild type for CCR5.

Infant sooty mangabeys have predominately naïve CD4+ T cells On this slide I am showing the level of naïve and memory CD4 T cells in the mangabey infants we studied. As might be expected based on age, the majority of CD4+ T cells have a naïve phenotype. Mean of values from 6 (GI tract) or 7 infants (lymphoid tissue)

Restricted expression of CCR5 on CD4+ T cells from infant mangabeys Ki67 = marker of T cell proliferation CCR5 = main HIV/SIV coreceptor on target cells We next measured the percentage of proliferating CD4+ T cells using the marker Ki67. We found robust levels of proliferation in total, naïve and memory cells, likely due to the developing immune system in these infants, And in the 3 graphs on the bottom half of the slide I think you can see that, despite these robust levels of proliferation, the frequency of CCR5 positivity among total, naïve, and memory CD4+ T cells is extremely low.

Spotlight on tissues related to breast milk transmission - 1 Milush JM, et al AIDS 2004 Oral inoculation of neonatal rhesus macaques with SIV Anatomical distribution of SIV DNA early after infection: Oral mucosa Tonsils Esophagus Lymphoid tissue So thus far I have shown you the low levels of CD4+CCR5+ T cells in classical gut and lymphoid tissues. We wanted to extend this finding to additional sites relevant to breastfeeding transmission. On this slide I am showing a cartoon from an article by Don Sodora’s where they orally administered SIV to neonatal rhesus macaques and found that early after infection, the virus is present in oral mucosa, tonsils, esophagus, and a number of different lymph nodes. We used this information to decide which additional tissues to obtain from the mangabey infants.

Spotlight on tissues related to breast milk transmission - 2 FYa1, euthanized DOL 10 FUa1, euthanized DOL 107 Oral mucosa Esophagus Here you can see representative flow plots of the oral mucosa, esophagus, and tonsils from 2 infants, 1 who died on DOL10 and another who died later, on DOL 107. It should be noted that this is the first time we have looked at these sites in mangabeys and while there were some technical challenges, I think you can clearly see that these tissues demonstrate a similar pattern to what we observed in the more classical tissues, with readily detectable proliferating cells but low levels of CCR5. Tonsil CD4 CD4 Ki67 CCR5 Ki67 CCR5

Infant macaques have ample SIV CD4+CCR5+ target cells This is the final piece of data I will show today. Here we directly compared the target cell availability in the peripheral blood of sooty mangabeys and rhesus macaques. We found that the non-natural host rhesus macaques have a greater percentage of CCR5+ CD4+ central and effector memory T cells in red as compared to sooty mangabeys in black. This difference was statistically significant. TN = CD28+CD95-CCR7+ TCM = CD28+CD95+CCR7+; TEM = CD28+/-CD95+CCR7-

Summary Mother-to-infant transmission of SIV is rare in natural hosts Robust proliferation of both naïve and memory T cells is present in infant sooty mangabeys Infant sooty mangabeys, unlike rhesus macaques, are characterized by limited availability of CD4+CCR5+ target cells for SIV Target cell restriction likely represents a key evolutionary adaptation to reduce the risk of mother-to-infant transmission in natural hosts In summary, We have previously Today I have shown you that And importantly I’d like to point out that Ivona Pandrea and Cris Apetrei have published data showing low levels of CCR5 in the blood of infant mandrills, another natural host. We believe that this key defining feature of natural hosts, that is, low expression of CCR5 on CD4 T cells, evolved as a mechanism to protect infants from vertical transmission of SIV.

Acknowledgements Silvestri Laboratory: Paul Carnathan Diane Carnathan Joana Yu Katie Sheehan Kathyrn Folkner Emily Cartwright Ron Trible Tayebeh Hashempoor Ankita Chowdhury Charlene Wang Alexandra Ortiz Tim Hayes Kiran Mir Steve Bosinger Thomas Vanderford Emory: Paul Spearman Eric Hunter Larry Anderson YNPRC: Benton Lawson Tracy Meeker Stephanie Ehnert Joyce Cohen, Maria Crane Cindy Courtney, Prachi Sharma, Anapatricia Garcia Barbara Cervasi Mirko Paiardini Francois Villinger Jim Else Duke: Sallie Permar Funding Sources: Emory CFAR03 Developmental Award NIH R37 AI66998 to G. Silvestri NIH P51 DO-00165 to YNPRC Thank you for your attention and I’d like to acknowledge my husband and collaborator, Guido Silvestri as well as members of his lab who contributed to this work, particularly Paul and Diane Carnathan. In addition, I would like to thank the veterinarians and animal resources staff at Yerkes. And finally thank you to the NIH and Emory CFAR for funding. Prelim data from Silvestri and Collman show that the pattern of alternative coreceptors follows CCR5 GPR15, CXCR6 – are utlilized in vitro and likely in vivo (as CCR5 knock out animals can get infected, albeit with slightly lower viral loads).

Gated on CD3+CD4+ T lymphocytes FYa1, euthanized DOL 10 FUa1, euthanized DOL 107 CD95 CD28 TN TM CD95 CD28 TN TM Oral mucosa Gated on CD3+CD4+ T lymphocytes