Tricks in HIV s bag to counter vaginal or rectal microbicides

Transcription

1 Tricks in HIV s bag to counter vaginal or rectal microbicides Florian Hladik, MD, PhD University of Washington Fred Hutchinson Cancer Research Center Seattle

2 Contribution of the various HIV invasion routes to HIV cases worldwide Female genital tract Male genital tract Intestinal tract Placenta Blood stream 12.6 million 10.2 million 7.3 million 0.5 million 2.6 million Hladik F. & McElrath J. Nature Reviews Immunology 2008, 8:

3 HIV vaccines April 23 rd, 1984 Margaret Heckler Secretary of Health and Human Services We expect that a preventive HIV vaccine will be ready for testing within two years

4 HIV microbicides

5 Transmission sites in the female genital tract Hladik F. & McElrath J. Nature Reviews Immunology 2008, 8:

6 Transmission sites in the male genital tract Hladik F. & McElrath J. Nature Reviews Immunology 2008, 8:

7 The mucosa a multi-opportunity site for HIV

8 HIV rapidly binds to intraepithelial vaginal T cells and productively infects them 2 hours Hladik F. et al. Immunity 2007, 26:

9 HIV s tricks to counter microbicides 1. Disseminate beyond the reach of microbicide action 2. Persist beyond the time of microbicide action 3. Induce changes in the mucosa that pave the way for future infection 4. Take advantage of co-existing STDs 5. Acquire drug resistance

10 1. HIV dissemination beyond the reach of microbicide action Dissemination of HIV beyond the mucosa Insufficient mucosal penetration of the microbicide

11 Ex vivo HIV infection model of the vaginal epithelium Epithelium Rete ridge Papilla Stroma

12 Ex vivo HIV infection model of the vaginal epithelium Challenge with virus Obtain mucosa from vaginal repair surgeries Isolate epithelial sheets by EDTA incubation overnight Harvest sheets and/or emigrated cells after 2 days Test for viral infection

13 HIV rapidly enters vaginal Langerhans cells 2 hours CD1a red, HIV-1 green

14 Intact virions are frequently found in perinuclear endocytic compartments of Langerhans cells

15 Newly bound HIV-1 may concentrate along the LC-T cell junction ( infectious synapse ) 2 hours CD1a red, HIV-1 green

16 2μ Vaginal Langerhans cells carry internalized virions into the submucosa

17 Vaginal Langerhans are not productively infected Total HIV DNA Integrated HIV DNA HIV-1 DNA copies / 10 5 cells HIV-1 DNA copies / 10 5 cells Tissue Donor Tissue Donor LC LC-TC Unpublished results

18 Nevertheless, vaginal Langerhans cells pass infectious HIV-1 to T cells HIV-1 Gag p24 (pg/ml) Langerhans / T cell conjugates Langerhans cells only Day 7 Day 16 Day 22 Day 28 Donor 1 Donor 2 Donor 3 Unpublished results

19 How does HIV bypass langerin-mediated degradation in vaginal Langerhans cells? Langerin CD1a Langerin & CD1a Vaginal side 50μ Langerin & CD1a 50μ 15μ 50μ Basal side Skin surface 15μ Basal side Langerin & CD1a Unpublished results

20 Dissemination of HIV beyond the mucosa Conclusion Prevention of productive infection of CD4 + T lymphocytes residing in the mucosa is the most important goal However, HIV clearly has alternatives to straight-forward infection of local T cells

21 Insufficient mucosal penetration of the microbicide An example: T-20 Fuzeon versus T-20 DAIDS 100 Relative viral integration (%) T-20 DAIDS (IC 50 : ) T-20 Roche (IC 50 : 0.23) Cellulose sulfate (IC 50 : 1.81) Concentration (µg/ml) McElrath M. J., Ballweber L. & Hladik F. Antimicrob. Agents Chemoth. 2010, 54:

22 2. Local HIV persistence beyond the time of microbicide action HIV in vaginal epithelial cells Keratin fibers 200nm Centriole Nucleus 200nm Nucleus Desmosomes Desmosomes 2μ Basal cell layer 200nm Unpublished results

23 3. Infection-independent effects of HIV on the mucosa that pave the way for future infection An example: HIV-1 triggers long-lasting expression of thymic stromal lymphopoetin (TSLP) in vaginal epithelium Fold mrna increase over uninfected controls Vaginal epithelial sheets Whole vaginal explants Vaginal stroma 0 4h 12h 24h 96h Unpublished results

24 HIV-1 triggers TSLP in vaginal epithelium Baseline 293T cell supernatant Day 1 1 ng HIV-1 p24 /ml 100 ng HIV-1 p24/ml Unpublished results

25 HIV-1 triggers TSLP in vaginal epithelium Baseline 293T cell supernatant Day 5 1 ng HIV-1 p24 /ml 100 ng HIV-1 p24/ml Unpublished results

26 4. Long-lasting effects of co-existing STDs Persistence of CD4 + T cells in genital herpes simplex lesions after healing CD4 + cells / mm 2 Lesion site Control site Weeks post heeling Zhu J., Hladik F. et al. Nature Medicine 2009, 15:

27 Contribution of the various HIV invasion routes to HIV cases worldwide Female genital tract Male genital tract Intestinal tract Placenta Blood stream 12.6 million 10.2 million 7.3 million 0.5 million 2.6 million Hladik F. & McElrath J. Nature Reviews Immunology 2008, 8:

28 CCR5 expression in the vaginal epithelium CD3 + T cells CCR CD3 CCR5 CD1a CD4

29 CCR5 expression in the rectal mucosa CCR5 green CD68 red Unpublished results

30 Computer-aided analysis of immunohistology Unpublished results

31 Stitching individual images together Unpublished results

32 CCR5 expression on rectal T cells and macrophages in situ CCR5 on CD3 + T cells CCR5 on CD68 + macrophages CCR5 fluorescence range 42.9% CCR5 fluorescence range 7.3% CCR5 mean fluorescence intensity CCR5 mean fluorescence intensity Unpublished results

33 Conclusion and thoughts Independent of the anatomical location, prevention of productive infection of CD4 + T lymphocytes residing in the mucosa is the most important goal However, alternative routes of HIV entry, indirect effects of exposure to HIV and the influence of pathogenic co-factors on HIV infection afford the virus a potential edge and may vary between anatomical sites

34 Acknowledgements Lamar Ballweber Allison Kreger Kimberly Smythe Barry Robinson Julie McElrath Jia Zhu Larry Corey Jagannadha Sastry Danielle Fontenot David Eschenbach Gretchen Lentz Michael Fialkow Funding Agencies: NIH NICHD & NIAID