The in vitro cell culture system has been widely used as a primary screening tool for evaluating anti-HIV-1 activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical tissue-derived organ culture model which mimics in vivo conditions and has been used to test microbicides for their ability to block HIV-1 transmission and measure inflammatory cytokines in response to microbicides and sexually transmitted infection-related bacteria. Our hypothesis is that a cervical tissue-based organ culture is an ideal system to test microbicides for toxicity in genital tissue and for its ability to block transmission of HIV-1 with varying phenotypic properties across the cervical epithelium in the presence of common environmental factors that are present in vagina, such as semen, vaginal fluid, and STI-related microorganisms.
Specific aims of the project are: 1) Evaluation of -nucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) from Project 1 and the entry inhibitor antimicrobial peptide retrocyclin RC101 from Project 2 alone or in combination and their formulations for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. 2) Assessment of cervical tissue inflammation and their changes in response to CSIC and RC101 from Projects 1 and 2, respectively, using the organ culture model. The expression of proinflammatory cytokines, such as 11-13, IL-6, IL-8 and TNF-a will be monitored by measuring their messages in the tissues by the real time PCR and secretion in the culture supernatant using the Luminex system;3) Evaluation of anti-HIV activity of CSIC and RC101 under physiologically relevant conditions. CSIC and RC101 alone or in combination and their formulations will be evaluated for their antiviral activities in the presence of semen and vaginal fluid. In addition the organ culture model will be expanded to measure STIrelated microorganisms, such as Neisseria gonorrhoeae and Prevotella melaninogenica by measuring proinflammatory cytokine response. Microbicides will then be evaluated for their antiviral activity in the aresence of the proinflammatory cytokines induced by these microorganisms.The proposed studies in this Droject will complement various other projects in this U19 grant application by providing a valuable in vitro cervical tissue-based assay which will bridge between the microbicide development (Projects 1 and 2), monkey model (Project 4) and Formulation Core (Core B).

Public Health Relevance

Since the microbicides will be topically applied to the vagina, it is important that they be tested for their antiviral activity and toxicity in a genital tissue (cervix) based in vitro assay before they are used in expensive and time-consuming clinical trials.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI082623-04
Application #
8380860
Study Section
Special Emphasis Panel (ZAI1-BP-A)
Project Start
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2012
Total Cost
$360,875
Indirect Cost
$66,992
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Eade, Colleen R; Diaz, Camila; Chen, Sixue et al. (2015) HIV-Enhancing Factors Are Secreted by Reproductive Epithelia upon Inoculation with Bacterial Vaginosis-Associated Bacteria. Protein Pept Lett 22:672-80
Wood, Matthew P; Cole, Amy L; Eade, Colleen R et al. (2014) The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2. Immunology 142:474-83
Wood, Matthew P; Cole, Amy L; Ruchala, Piotr et al. (2013) A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion. PLoS One 8:e55478
Eade, Colleen R; Cole, Amy L; Diaz, Camila et al. (2013) The anti-HIV microbicide candidate RC-101 inhibits pathogenic vaginal bacteria without harming endogenous flora or mucosa. Am J Reprod Immunol 69:150-8
Eade, Colleen R; Diaz, Camila; Wood, Matthew P et al. (2012) Identification and characterization of bacterial vaginosis-associated pathogens using a comprehensive cervical-vaginal epithelial coculture assay. PLoS One 7:e50106
Levinson, Pauline; Choi, Robert Y; Cole, Amy L et al. (2012) HIV-neutralizing activity of cationic polypeptides in cervicovaginal secretions of women in HIV-serodiscordant relationships. PLoS One 7:e31996
Eade, Colleen R; Wood, Matthew P; Cole, Alexander M (2012) Mechanisms and modifications of naturally occurring host defense peptides for anti-HIV microbicide development. Curr HIV Res 10:61-72
Penberthy, W Todd; Chari, Soumya; Cole, Amy L et al. (2011) Retrocyclins and their activity against HIV-1. Cell Mol Life Sci 68:2231-42
Rinehart, Matthew T; Drake, Tyler K; Robles, Francisco E et al. (2011) Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy. J Biomed Opt 16:120510
Micewicz, Ewa D; Cole, Amy L; Jung, Chun-Ling et al. (2010) Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin. PLoS One 5:e14360