RESUMO
Quantitative analysis of cell-mediated immune responses induced by candidate HIV vaccines requires robust procedures for collecting and processing human peripheral mononuclear blood cells (PBMCs). We evaluated several parameters in order to optimize a sample handling process that would be suitable for a multicenter clinical trial. Among the findings, systematic increases in the magnitude of IFN-gamma ELISpot responses were observed when the time from blood collection to PBMC freezing was reduced to <12 h. By implementing these improvements within an ongoing clinical trial, the estimated immunologic response rates to an adenovirus- based HIV vaccine increased by more than 20 percentage points to approximately 80% of the vaccine recipients against any of the vaccine antigens and the average levels of T cell response improved more than 3-fold. These studies establish the importance of optimal conditions for PBMC collection and handling to the success of a clinical development program.
Assuntos
Vacinas contra a AIDS/imunologia , Leucócitos Mononucleares/imunologia , Manejo de Espécimes , Vacinas contra a AIDS/uso terapêutico , Adenoviridae/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Ensaios Clínicos como Assunto , Criopreservação , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/prevenção & controle , Soronegatividade para HIV , Humanos , Imunidade Celular , Interferon gama/imunologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.
Assuntos
Vacinas contra a AIDS/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos HIV/análise , Soronegatividade para HIV , Interferon gama/metabolismo , Vacinas contra a AIDS/uso terapêutico , Ensaios Clínicos como Assunto , Reações Falso-Positivas , Antígenos HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1 , Humanos , Interferon gama/análise , Leucócitos Mononucleares/imunologia , Peptídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.