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Ken Rosenthal Afri-Can Forum Entebbe, Uganda Jan. 18, 2013

Anti-HIV-1 Activity of Elafin Depends on its Nuclear Localization and Altered Innate Immune Activation in Female Genital Epithelial Cells. Ken Rosenthal Afri-Can Forum Entebbe, Uganda Jan. 18, 2013. Trappin-2 and Elafin - biomarkers of resistance in HIV-R CSW.

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Ken Rosenthal Afri-Can Forum Entebbe, Uganda Jan. 18, 2013

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  1. Anti-HIV-1 Activity of Elafin Depends on its Nuclear Localization and Altered Innate Immune Activation in Female Genital Epithelial Cells Ken Rosenthal Afri-Can Forum Entebbe, Uganda Jan. 18, 2013

  2. Trappin-2 and Elafin - biomarkers of resistance in HIV-R CSW “Elevated elafin/trappin-2 in the female genital tract is associated with protection against HIV acquisition.” Iqbal SM et al. 2009; AIDS • SELDI-TOF MS – for E • ELISA – for both Tr/E • Both Trappin-2 (Tr) and Elafin (E) (Tr/E) were identified as biomarkers of resistance against HIV-1 in genital tract of HIV-resistant sex workers (Nairobi, Kenya) • Iqbal et al., 2009; AIDS

  3. Tr/E: structure Belong to Whey Acidic Protein (WAP family of proteins Two separate proteins, although often used interchangeably: - Trappin-2(Tr) - mature 9.9kDa (95aa) secreted protein - Elafin(E) - processed 6kDa (57aa) and secreted form of Tr Both Tr/E contain: - C terminus – with antiprotease inhibitory loop - N terminus – anchors Tr and E to extracellular matrix proteins (i.e., heparin, fibronectin, etc.) 40% homology between secretory protease inhibitor (SLPI), another WAP protein, and E • Moreau et al., 2008;Biochimie

  4. Horne et al., 2008; Reproduction

  5. Tr/E: functions • S. aureus • P.aeruginosa • C. albicans • K. Baranger, et al., 2008, FEBS • Antiprotease- due to antiprotease domain; • Antibacterial – due to cationic nature and disruption of bacterial cell wall or binding to DNA; • Immunomodulatory properties - • via inhibition of AP-1 and • NF-kB activation (in response • to LPS or polyI:C (Drannik et al., • in press) inflammatory stimuli); • Antiviral – due to potential direct effect on virus (was proposed for E against HIV, Ghosh et al., 2010, Immunology) or indirect effect on antiviral host immune responses (in lungs against Ad challenge, Roghanian et al., 2006, AJRCM)

  6. Rationale & hypothesis Genital tract epithelial cells (ECs) - first line of defense against pathogens ECs involved in innate antiviral responses - by recognizing pathogens with innate sensors(TLRs, NODs, RNA helicases, etc.) - by producing innateantimicrobial factors (defensins, secretory leukocyte proteinase inhibitor (SLPI), and trappin-2/elafin) Tr/E identified as biomarkers of HIV resistance, but their role in anti-HIV mucosal protection is still poorly understood We hypothesized that Tr/Eplay an important role in defense against HIV in the female genital mucosa through modulation of host/pathogen interaction by:decreasing viral infectivity and/or “quieting” host innate immune responses in response to HIV

  7. Elevated Tr/E in CVLs are associated with reduced HIV infection in vitro • Tr/E in CVLs • R5 • X4 • CVLs from HIV-R CSWs, with elevated Tr/E, are significantly more protective against R5 HIV, but not X4, infection of TZM-bl target cells • A. Drannik, et al., 2012, J Virology

  8. Tr/E contribute to natural anti-HIV activity of CVL in HIV-R CSW • Tr/E depletion from CVLs • R5 • X4 • Tr/E contribute up to 60% to anti-HIV activity of HIV-R CVLs • against R5 HIV infection of target cells, thus being among principal anti-HIV molecules of CVL • A. Drannik, et al., 2012, J Virology

  9. Anti-HIV activity of Tr/E • Trappin-2 • Elafin • Cells vs Virus • TZM-bl • HEC-1A • Each Tr/E has anti-HIV activity, and it is contextual • Tr/E act directly against HIV (in TZM-bl) or both HIV and cells (in HEC-1A), and likely via non-canonical receptors - Anti-viral effect(s) of Tr/E are mainly against R5 HIV, but not X4 (not shown) A. Drannik, et al., 2012, J Virology

  10. Anti-HIV activity of Tr/E depends on unrestricted N-terminus • NH2 • COOH • Only unblocked N-terminus Tr/E inhibited HIV attachment and transcytosis in HEC-1A cells. • Anti-HIV effect appeared primarily against R5 HIV, but not X4, except for E in transcytosis, and was independent of antiprotease activity. • A. Drannik, et al. PLoS ONE 2012

  11. Antiviral Tr/E are localized to the nucleus • Untagged exogenous Tr/E cannot be differentiated from endogenous proteins • Exogenous Tr/E with restricted C-terminus can enter the nucleus as well as inhibit HIV • Exogenous E with restricted N-terminus cannot enter the nucleus and does not inhibit HIV • A. Drannik, et al. PLoS ONE 2012

  12. Antiviral Tr/E are immuno-modulatory • Antiviral Tr/E also inhibited IL-8 secretion and mRNA expression of TLR3 and RIG-I in response to R5 HIV, which echoed with reduced mRNA of TLR2, TLR4 and RIG-I in ECs from HIV-R CSWs • Antiviral Tr/E attenuate HIV-induced NF-κB nuclear translocation in HEC-1A cells A. Drannik, et al. PloS ONE 2012

  13. Anti-HIV activity of E is more potent than Tr • Tr IC50 • Tr IC50 • E IC50 • E IC50 • Infection in TZM-bl cells • Transcytosis in HEC-1A cells • Determination of IC50 revealed that: • E is ~130x more potent than Tr in reducing infection of TZM-bl cells with • canonical HIV receptors • E is ~80x more potent than Tr in reducing HIV transcytosis in HEC-1A cells • with non-canonical receptors • E is ~ 1.5x more active against HIV than cells in HEC-1A cells (not shown) • A. Drannik, et al., 2012, J Virology

  14. Summary of Tr/E against HIV Tr/E are among principal anti-HIV molecules in CVL Anti-HIV activity of Tr/E is contextual and depends on viral strain, HIV receptors, as well as unrestricted N-terminus of E and its intranuclear localization, but not on antiprotease activity of Tr/E Tr/E reduce HIV-triggered NF-kB nuclear translocation, IL-8 secretion, and PRRs mRNA expression Both Tr/E are antiviral, with E significantly more potent against HIV than Tr Reduced mRNA expression of PRRs in ECs from HIV-R CSWs links Tr/E with immune modulation and HIV mucosal resistance

  15. Implications Tr/E may interfere with HIV-1 and HSV-2 pathogenesis through: Direct targeting of virus and limiting viral attachment/entry Moderating viral innate recognition and mounting inflammatory responses Altering viral acquisition and establishment of primary infection Targeting viral transcytosis/transmission and dissemination

  16. Acknowledgements McMaster University Dr. K. Rosenthal Anna G. Drannik Bethany Henrick Kakon Nag Xiao-Dan Yao Sumiti Jain Jennifer Newton Amy Patrick University of Manitoba T. Blake Ball Francis A. Plummer University of Nairobi Charles Wachihi Joshua Kimani Pumwani women participants! Supported by: CTC-VIMC & CAVD -

  17. Model of Antiviral Activity of Tr/E Virus (VSV-GFP, HSV-2, HIV) TLRs dsRNA Trappin-2 Elafin RLRs Direct antiviral effect Viral sensing RLRs, TLRs Attachment Viral binding receptors Antiviral state Transcytosis NF-kB IL-8, TNFa,IL-6

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