AEGiS-15IAC: Validation of cytokine flow cytometry for measurement of immunogenicity in phase I/II clinical trials of HIV vaccines.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Validation of cytokine flow cytometry for measurement of immunogenicity in phase I/II clinical trials of HIV vaccines.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10075)

Gill D, Hayes P, Gilmour J
Imperial College, London, United Kingdom

BACKGROUND: Cytokine flow cytometry (CFC) is used to assess immunogenicity in HIV vaccine clinical trials. Assays are influenced by cell treatment, culture conditions and inter-operator variability. These factors should be characterised to increase assay reliability, for submission of data to regulatory authorities for vaccine approval.

METHODS: 15mer peptide pools covering HIV-1 Gag were used in CFC assay to measure IFNgamma positive T cells from unvaccinated HIV- subjects to determine assay noise. CMVpp65 peptides were used to generate data on peptide-specific responses. Comparisons were made using whole blood, cryopreserved PBMC and the use of CD28/49d co-stimulatory antibodies. Assay variation was assessed independently by 2 operators using identical samples.

RESULTS: Mean HIV peptide IFNgamma responses of unvaccinated HIV- subjects were 0.02% of CD3+CD4+ and 0.08% of CD3+CD4- T cells (assay noise). For CMV, CD3+CD4+ T cell responses were similar between whole blood and cryopreserved PBMC, but in CD3+CD4- T cells, a 75% reduction in background and 50% loss in the peptide specific response was seen in cryopreserved PBMC compared to whole blood. CD28/49d antibodies increased background by 100% yet CMV peptide-specific responses by 50%. Data from 2 operators were not significantly different in peptide responses though there was some variation in background.

CONCLUSIONS: Cut offs for true peptide specific responses were determined with 99% confidence to be 0.05% for CD3+CD4+ responses and 0.2% for CD3+CD4- T-cells. CD28/49d antibodies increase background more than peptide responses. While CD4 responses were unaffected, a loss of about 50% of CD8 CMV peptide responses were noted in cryopreserved PBMC. Inter-operator assay variation was minimal for peptide-specific responses. This study provides a detailed characterisation and validation of the CFC technique, allowing a more objective assessment of CFC assay performance.

Keywords: AEGIS, AIDS Vaccines, Flow Cytometry, Clinical Trials, Phase I, Cytokines, CD4-Positive T-Lymphocytes, HIV-1, T-Lymphocytes, Antigens, CD28, Antigens, CD4, Antigens, CD3, HIV, Genes, gag, HIV Infections, Clinical Trials, HIV Long-Term Survivors, Acquired Immunodeficiency Syndrome, HIV Core Protein p24, immunology, genetics

040711
A10075