Our laboratory and others have demonstrated the ability of CD8+ cells from HIV-infected individuals to suppress virus replication in CD4+ cells. We have determined that this non-lytic, non-MHG-restricted activity is mediated in part by a novel cytokine produced solely by CD8+ cells. This CD8+ cell antiviral factor (CAF) is made at highest levels by CD8+ cells from asymptomatic individuals and its production decreases with advancement to disease. Our studies indicate that CAF is heat and pH stable, and has a size of approximately 30 kD or less. It is resistant to trypsin and lyophilization and does not directly inactivate HIV. It is sensitive to staph V8 protease. The recent removal of CAF by cation columns suggests that this small protein is positively charged at pH 6.0. The major objective of the present proposal is to purify CAF so that its amino acid sequence can be determined and subsequent cloning and expression of the factor can be undertaken. The approaches used will include standard protein purification methods (i.e., column chromatography, isoelectric focusing and gel electrophoresis) and the development and use of a factor-specific monoclonal antibody. The monoclonal antibody would also be helpful in the final purification of the factor and also provide a means for derivation of a CAF-specific ELISA to rapidly assay for the factor, help identify cells producing CAF, and evaluate methods for enhancing its production. Other studies to assist in CAF purification include approaches to establish cell lines producing CAF, to enhance CAF production by CD8+ lymphocytes, and to develop more rapid assays for detection of CAF. The purification and characterization of this antiviral factor could have important therapeutic value in HIV infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030350-05
Application #
2065554
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1990-06-01
Project End
1997-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
5
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Mackewicz, Carl E; Wang, Baikun; Metkar, Sunil et al. (2003) Lack of the CD8+ cell anti-HIV factor in CD8+ cell granules. Blood 102:180-3
Mackewicz, Carl E; Craik, Charles S; Levy, Jay A (2003) The CD8+ cell noncytotoxic anti-HIV response can be blocked by protease inhibitors. Proc Natl Acad Sci U S A 100:3433-8
Mackewicz, Carl E; Yuan, Jun; Tran, Patti et al. (2003) alpha-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors. AIDS 17:F23-32
Levy, Jay A; Scott, Iain; Mackewicz, Carl (2003) Protection from HIV/AIDS: the importance of innate immunity. Clin Immunol 108:167-74
Levy, Jay A (2003) The search for the CD8+ cell anti-HIV factor (CAF). Trends Immunol 24:628-32
Mackewicz, C E; Lieberman, J; Froelich, C et al. (2000) HIV virions and HIV infection in vitro are unaffected by human granzymes A and B. AIDS Res Hum Retroviruses 16:367-72
Mackewicz, C E; Patterson, B K; Lee, S A et al. (2000) CD8(+) cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration. J Gen Virol 81:1261-4
Mackewicz, C E; Ridha, S; Levy, J A (2000) HIV virions and HIV replication are unaffected by granulysin. AIDS 14:328-30
Greco, G; Mackewicz, C; Levy, J A (1999) Sensitivity of human immunodeficiency virus infection to various alpha, beta and gamma chemokines. J Gen Virol 80 ( Pt 9):2369-73
Mackewicz, C E; Garovoy, M R; Levy, J A (1998) HLA compatibility requirements for CD8(+)-T-cell-mediated suppression of human immunodeficiency virus replication. J Virol 72:10165-70

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