ABSTRACTMycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growingMycobacterium smegmatiscan express bothMycobacterium tuberculosisantigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity ofM. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion (“high secretors”) and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8+T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV)gaggene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8+T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8+T cellsin vivo. The data also suggest that theseM. smegmatistransposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses.

Cryopreservation of peripheral blood mononuclear cells (PBMC) is a good alternative to testing fresh PBMC in clinical trials as functional immunologic assays may be batched to increase efficiency. Functional assays are essential for accurately evaluating the performance of vaccines, antiviral and anticancer agents. Optimal retention of PBMC functionality during cryopreservation depends on optimized and standardized cell isolation, freezing and storage conditions. Collection and analysis of PBMC is a significant technical challenge in multi-center clinical trials due to pre-analytical variability in processing, freezing and storage. It is vital that all centers participating in a multi-center collaboration operate with consistent protocols. To this end, Precision has developed a Laboratory Network (PLN) to ensure quality PBMC processing across multi-center trials. The current study was performed within the PLN to ensure samples processed at each laboratory achieved similar results. We report the recovery, viability and functionality of PBMC obtained from participating laboratories. Phenotypic characterization of the major cell subset was also analyzed and the percent obtained was comparable across laboratories. This data provides confidence that PBMC processing in a multi-center clinical trial operating with the same standard operating procedures (SOPs) produces comparable data, thus accelerating drug and vaccine development.

Cryopreserved peripheral blood mononuclear cells (PBMC) are a good alternative to fresh PBMC in clinical trials when samples are collected at multiple sites and processed later. Optimal retention of cryopreserved PBMC functionality depends on cell isolation, freezing and storage. SeraCare developed validated PBMC cryopreservation procedures to ensure functionality is maintained on cryopreservation. Two assays were performed to demonstrate the equivalence of cryopreserved vs. fresh PBMCs. Intracellular cytokine staining (ICS) quantitatively measures antigen specific T cell cytokine production at the single cell level. Using flow cytometry ICS analyzes multiple parameters on a single sample to obtain the precise cytokine producing T cell phenotype. Our data demonstrates production of IFN-γ, IL-2, TNF-α and IL-4 from cryopreserved PBMC suggesting full retention of PBMC functionality after cryopreservation. The antibody dependent cellular cytotoxicity (ADCC) assay measures natural killer cell mediated destruction of antibody targeted cells. ADCC is a powerful tool to measure immune responses in HIV and cancer immunotherapy. Most studies used fresh PBMC in ADCC assays, noting that cryopreserved PBMC result in lower activity. We show cryopreserved PBMCs have comparable ADCC activity to fresh PBMC. ADCC activity appeared donor specific and was not affected by cryopreservation. Our data suggest that cryopreservation has no adverse affect on PBMC functionality in immunological assays.