Enhanced Rates and Magnitude of Immune Responses Detected against an HIV Vaccine: Effect of Using an Optimized Process for Isolating PBMC

Abstract

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.

Loss of T cell responses following long-term cryopreservation

“…This is thought to be due to cells requiring a period of recovery following the harsh procedure of thawing to allow any cells which may be apoptotic or necrotic to die, leaving a healthy population of cells for functional studies. Indeed, as Kierstead et al (2007) report, we also measured a reduction in cell viability after resting, compared to immediately post thawing, suggesting that a population of cells still undergoes apoptosis or necrosis following thawing, even after an 18 h period of rest. However, our BSRI laboratory routinely uses PBMC directly after thawing without resting and has measured good T cell responses in short-term cryopreserved PBMC (Fig.…”

“…The loss of responses cannot be attributed to cryopreservation technique or to the shipping of samples, as suggested in previous studies (Disis et al, 2006;Bull et al, 2007;Kierstead et al, 2007), as similar losses were measured in samples processed and frozen at three independent laboratories. These changes occurred despite the observation that cell viability was generally maintained at acceptable levels of 81-95% (Table 2).…”

“…Lamoreaux et al (2006) found that resting PBMC can reduce background cytokine responses and therefore increase the specificity of the T cell response. Kierstead et al (2007) also report that resting cells overnight post thawing is essential for measurement of optimal T cell responses by ELISPOT or intracellular cytokine staining. This is thought to be due to cells requiring a period of recovery following the harsh procedure of thawing to allow any cells which may be apoptotic or necrotic to die, leaving a healthy population of cells for functional studies.…”

“…Some groups have suggested resting PBMC overnight after thawing before testing effector functions (Lamoreaux et al, 2006;Kierstead et al, 2007). It is possible that the resting process would remove cells destined for death and allow "stunned" cells to recover functional ability.…”

“…The reason for these inconsistent observations has not been well defined. Two recent studies show that the time elapsed between phlebotomy and cryopreservation can result in lower functional responses (Bull et al, 2007;Kierstead et al, 2007). Other factors may include the quality of the cryopreservation and storing procedures (which are generally not standardized), as well as the duration of cryopreservation.…”