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

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 Interactions at the Mucosal Front

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% HIV and HSV2 A Dangerous Liaison 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%

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

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?

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

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

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

Impact of HSV2 status on FGT immune milieu Lymphocyte Populations

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

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

Conclusions 1. Powerful epidemiologic synergy between HIV and HSV2: HIV infected FSWs more likely to be HSV2 infected and shed HSV2. 4. Novel Strategies to reduce HIV transmission: Microbicides targeting DC-SIGN and/or CCR5 3. Interaction between HIV and HSV2 in the genital mucosa Powerful immunological negative synergy 2.HIV target cells increased in FGT of HSV2 infected FSWs: 10 fold increase in DC-SIGN+ CD1a+ immature dendritic cells 3 fold increase in CCR5+ CD4+ T cells

Dr.Rupert Kaul Kaul lab: Prameet Sheth, Chris Pettengel, Sanja Huibner, Bolette Bossen, Kamnoosh Shahabi, Sherzana Sunderji, Anthony Sheung University of Toronto: Mario Ostrowski, Sandy Der University of Manitoba: Blake Ball, Keith Fowke, Frank Plummer University of Nairobi: Joshua Kimani, Charles Wachihi, Elizabeth Ngugi, Walter Jaoko, Jane Kamene, Nyakio Chinga, Anne Miangi Funding: NIH, CIHR, CANVAC, Canada Research Chair