A -- Immunogenetic Project

The Department of Health and Human Services/Centers for Disease Control and Prevention (CDC), Procurement and Grants Office (PGO), intends to award on a SOLE SOURCE BASIS a Purchase Order. The title of the acquisition is - The Immunogenetic Project.-(RFQ# 200-2006-Q-08577). A Purchase order will be awarded to Case Western Reserve University. The Contact for this acquisition is Marcus A. Powell, Contract Specialist, Telephone: (404) 639-8054, E-mail tzp2@cdc.gov, and the fax number is (404) 639-8095. Statement of Work: Title of Project: Prospective Evaluation of Immunologic and Immunogenetic Susceptibility to Mycobacterium tuberculosis infection and progression to disease C.1 Background and Need - In 1998, CDC initiated an immunologic contact study in collaboration with Case Western Reserve University (CWRU). Until 2002, CWRU's collaboration with CDC was at no cost to CDC. Beginning in 2002, CDC has funded CWRU through a sole source mechanism to continue to perform the immunologic assays required for this project. Developing and performing immunologic assays is a specialized skill. The sensitivity and specificity of immunologic assays for the same parameter vary from laboratory to laboratory. To maintain comparability of results, it is important that all samples be tested in a single laboratory. Since the immunologic testing for this study began at CWRU, it is essential to the scientific quality of the results that the remainder of the immunologic assays for the study also be performed in the same laboratory. CWRU has already tested 2,027 of the 3,000 samples which will be collected for this project. C.2 Project Objective - To identify cytokine surrogate markers for protective immunity to infection with Mycobacterium tuberculosis among contacts exposed to infectious tuberculosis patients. Results of this work serve as a critical step in the process of developing and testing new candidate vaccines for tuberculosis. C.3 Scope of Work - CWRU will perform cytokine assays for interferon-gamma, tumor necrosis factor-alpha, and interleukin 10 on 973 whole blood specimens (through specimen number 3000), as well as Elispot measurements for Interferon-gamma on a subset of 200 consecutive whole blood specimens. Results of these assays will be reported back to CDC on a quarterly basis. C.4 Technical Requirements - 1. Peripheral blood mononuclear cells will be separated from whole blood according to standard techniques, and cytokine assays will be set up the same day specimens are received. 2. DNA will be extracted from whole venous blood using the Puregene DNA isolation kit from Gentra Systems (Minneapolis, MN) according to standard techniques, and stored at -700C until immunogenetic testing is performed. 3. To assess MTB antigen-induced production of cytokines (IFN-gamma, TNF-alpha, TGF-beta, and IL-10) and beta chemokines (MIP1-alpha) previously shown to be involved in antituberculous immunity, a whole blood cell culture method will be used. This method has been applied previously in a field study evaluating immune responses in leprosy patients (Weir et al), and has been modified by investigators of TBRU to assess antituberculous immunity in individuals enrolled in a household contact study (designed to study immunological correlates of transmission of MTB infection) in Kampala, Uganda. In parallel, peripheral blood mononuclear cells (PBMC) will be separated from the remaining heparinized blood according to standard techniques and stored frozen in liquid nitrogen freezers for later use in assays evaluating frequencies of M. tuberculosis antigen-stimulated IFN-gamma-producing T cells (ELIspot technique). Elispot assays will be performed in batches and utilizing freeze-thawed PBMC. 4. Anti-coagulated whole blood will be diluted (10-fold) in RPMI 1640 tissue culture medium containing Hepes (15 mM), L-glutamine (2 mM) and Penicillin (50 U/ml) and streptomycin (5 micrograms/ml). r Eight 1ml aliquots of the diluted whole blood from each study subject are dispensed into 24-well tissue culture plates (Falcon), and plates containing diluted whole blood (2 wells each additive [stimulus]-free, with culture filtrate of MTB [5 mg/ml] are incubated at 370C, 5% CO2 for up to 5 days. Whole blood culture supernatants from the first four two wells are collected following 24hs of culture; the content of the remaining four wells is recovered following 5 days of incubation. Once collected, whole blood culture supernatants are stored frozen at - 700 C until assessment of cytokine and chemokine immunoreactivities (TNF-alpha in 24h supernatants; IFN-gamma and IL-10 immunoreactivities in 5 day samples). 5. For assessment of TNF-alpha and IFN-gamma immunoreactivities in whole blood culture supernatants commercially available "cytokine (ELISA) kits" are used. Levels of TNF-alpha, MIP1-alpha, and TGF-beta are measured with assay systems available from R&D Diagnostics with a lower detection limit of < 7 pg/ml of cytokine immunoreactivity (TNF-alpha, MIP1-alpha, and TGF-beta). PIERCE/ENDOGEN is the source of the ELISA kit evaluating IFN-gamma immunoreactivity in whole blood culture supernatants. In this assay levels of IFN-gamma > 2 pg/ml are significant. IL-10 immunoreactivity is assessed utilizing a kit from BioSource. This ELISA has a lower limit of detection of < 2 pg/ml of IL-10. 6. For ELIspot assays, viable cells will be cryopreserved. To freeze cells, freezing media (FCS containing 10% DMSO) will be prepared and chilled either by placing it on ice or placing it at - 200C for about 30 minutes. Prior to freezing, cells are washed in RPMI. The supernatant is decanted, the cell pellet is vortexed and freezing media then is added bit by bit while constantly swirling the tube. To assess frequencies of MTB antigen-stimulated, IFN-gamma-producing cells, the Elispot technique will be used. This assay (described in detail in Hirsch et al JID 2001) is in routine use in Dr. Hirsch's laboratories in Cleveland. Elispot microtiter plates (Athersys) are first coated by adding 100 ul/well of a monoclonal antibody to IFN-gamma (PIERCE/ENDOGEN, 4 ug/ml) diluted in PBS, and incubating the plates overnight at 40C. On the next morning PBMC are added and plates are incubated at 370C overnight. Following two additional incubations with capping antibody and streptavidin peroxidase on the next morning, plates are developed using AEC substrate. Numbers of spots in each well, which correspond to frequencies of IFN-gamma-producing cells, will be assessed using the ELIspot reader (CTL) in the CFAR Cytokine Core Facility at CWRU. Frequencies of IFN-gamma-producing cells will be correlated with cytokine content in whole blood supernatants and contact TST and disease status. C.5 Reporting Schedule - Cytokine assay and Elispot results will be entered into an excel spreadsheet and sent electronically to CDC on a quarterly basis. The preliminary results of all assays will be sent to the CDC no later than one year from the date of award. C.6 Special Considerations - None C.7 Government Furnished Property None C.8 References - See attached protocol Deliverables