Cellular immune responses induced by candidate HIV-1 vaccines are notoriously difficult to detect and quantitate. This is likely the result of both limited cellular immune responses and the highly qualitative nature of the in vitro assays used to measure them. Since it appears that the induction of strong, diverse, and long lasting cellular immune responses will be required for successful HIV-1 vaccination, development of a more sensitive and quantitative method to directly measure cellular responses in vivo will assist in comparing alternative vaccination strategies. In order to measure cellular immune responses in vivo, the investigators plan to quantitate CD4+ and CD8+ T-cell receptor (TCR) repertoire changes following canarypox vector and plasmid injection. Using a new assay capable of detecting TCR beta chain transcript expansion as small as 1 percent of a single V beta subfamily, the investigators will track the emergence of novel T-cell clones immediately following vaccination. The number, frequency, and half-life of CD4 and CD8 clonal expansions induced in vivo following vaccination therefore will be accurately quantified. This new technique for the analysis of cellular immune responses is based on the detection of TCR beta chain transcripts bearing novel CDR3 (VDJ) regions, and is capable of quantifying low frequency T-cell repertoire changes otherwise undetectable by the alternative in vivo methods presently available (V beta subfamily-specific semi-quantitative PCR, FACS using V beta-specific monoclonal antibodies, TCR mRNA spectratyping, or MHC tetramer analyses). The T-cell clones seen expanding in vivo following vaccination will be purified using a combination of cell sorting using V beta subfamily-specific monoclonal antibodies and TCR CDR3 sequence analysis, and their antigenic specificity will be determined by classical in vitro assays. The investigators therefore will use a new, high resolution, method of T-cell repertoire analysis to quantify cellular immune responses induced by different modes of vaccinations. The extent of T-cell repertoire changes induced by canarypox vectors and DNA injections in humans will be compared with that induced by the only effective lentiviral vaccination strategy to date, pre-infection with attenuated SIV. The development of this methodology may assist in the evaluation of cellular immune responses induced by future candidate vaccines.
| Gordon, C J; Delwart, E L (2000) Genetic diversity of primary HIV-1 isolates and their sensitivity to antibody-mediated neutralization. Virology 272:326-30 |
