Dr. Anu Rebbapragada University of Toronto

1. Potential mucosal immune mechanisms for increased HIV susceptibility in women infected by Herpes simplex type 2 Dr. Anu Rebbapragada University of Toronto Anu Rebbapragada, Charles Wachihi, Chris Pettengell, Sherzana Sunderji, Sanja Huibner, Anthony Sheung, George Moussa, Anthony Mazzuli, Walter Jaoko, Blake Ball, Keith Fowke, Francis Plummer and Rupert Kaul

2. Interactions at the Mucosal Front HIV infects >40 million globally, mostly through sexual transmission, and most are now in women Mucosal vaccine and microbicide trials will need to monitor genital immunology and must consider that: • Globally, genital co-infections are “the norm” • CMV infects 50-90% of adults - more in Sub Saharan Africa (SSA) • HSV2 infects 20-60% of adults - more in SSA, more in women • Bacterial vaginosis, HPV, trichomoniasis: also very common

3. HIV and HSV2A Dangerous Liaison General population: • associated with a >3 fold increase in HIV acquisition by women, even in the absence of genital ulcers (Freeman E, 2006; Wald A, 2004) • In Sub-saharan African Women: HSV2 prevalence ~ 50% Core Transmitter Groups: • HSV2 has been associated with 6-fold increase in HIV acquisition in Nairobi female sex workers (FSWs; JAMA, 2004) • In FSWs: HSV2 prevalence group exceeds 75%

4. Wald A, Herpes. 2004. HSV2 may drive HIV transmission • In regions of high HSV2 seroprevalance: • Mathematical modeling predicts 50% of new HIV cases directly attributed to HSV2

5. Research Questions How does HSV2 infection increase HIV acquisition? • Is this simply due to epithelial micro-ulceration? • Or through negative effects on the female genital tract (FGT) immune milieu? What is the potential immune basis of enhanced HIV susceptibility?

6. HSV2 Study: Mucosal Sampling For all women: • HIV, CMV serology; HSV2 serology (Kalon) • HSV2 DNA from swab (Artus) • CMV DNA from CVL (Artus) • HIV-1 RNA from swab (Versant) • Endocervical cell populations by flow cytometry (scraper) • Mucosal Lymphocytes: CCR5+/CD69+ CD4+ T cells • Mucosal Dendritic cells: CD1a+ iDC, DC-SIGN+ iDC, TLR9+ iDC • Cytokines/chemokine levels in CVL by CBA (BD) • Immune Genes expression levels: QPCR (cytobrush)

7. 102 FSWs screened for possible study enrollment 11 FSWs taking ARV excluded • Cross-sectional survey during 2004-2005 • Female sex workers from Pumwani, Nairobi 91 FSWs enrolled HIV uninfected (N = 55) HIV infected (N = 36) ** LR = 14.5 P<0.001 HSV2 infected (42/55, 76%) HSV2 uninfected (13/55, 24%) HSV2 infected (36/36, 100%) ** LR = 16.5 P<0.001 0/42 (0%) Shedding HSV2 10/36 (26%) Shedding HSV2

8. Autofluorescence Granular Cells Side scatter Lymphocytes Forward scatter Endocervical Cell Populations DC-SIGN CD4 CD1a CD3

9. Impact of HSV2 status on FGT immune milieu Lymphocyte Populations

10. 20% Impact of HSV2 status on FGT immune milieu Dendritic Cell Populations

11. HSV2 reactivation & shedding HSV2 proteins transactivate HIV-LTR  HIV shedding •  DC-SIGN+ iDC • CCR5+ CD4 T cells  DC-SIGN+ iDC  TLR9+ iDC HIV infection One possible model for mucosal HIV-HSV2 synergy HSV2 infection

12. Conclusions • Powerful epidemiologic synergy between HIV and HSV2: • HIV infected FSWs more likely to be HSV2 infected and shed HSV2. • HIV target cells increased in FGT of HSV2 infectedFSWs: • 10 fold increase in DC-SIGN+ CD1a+ immature dendritic cells • 3 fold increase in CCR5+ CD4+ T cells 3. Interaction between HIV and HSV2 in the genital mucosa • Powerful immunological negative synergy 4. Novel Strategies to reduce HIV transmission: • Microbicides targeting DC-SIGN and/or CCR5

13. Dr.Rupert KaulKaul lab:Prameet Sheth, Chris Pettengel, Sanja Huibner, Bolette Bossen, Kamnoosh Shahabi, Sherzana Sunderji, Anthony SheungUniversity of Toronto:Mario Ostrowski, Sandy DerUniversity of Manitoba:Blake Ball, Keith Fowke, Frank PlummerUniversity of Nairobi:Joshua Kimani, Charles Wachihi, Elizabeth Ngugi, Walter Jaoko, Jane Kamene, Nyakio Chinga, Anne MiangiFunding:NIH, CIHR, CANVAC, Canada Research Chair