Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity.

Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2-3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold-12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100-1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs.

Using DNA Immunization to Elicit Monoclonal Antibodies in Mice, Rabbits, and Humans.

In recent vaccine studies, DNA immunization was found to effectively stimulate both innate and adaptive immunities to elicit high levels of antigen-specific antibody responses. The DNA molecule itself can activate multiple pathways of innate immunity. The in vivo production of antigens allows for presentation by major histocompatibility complexes, so that T-cell responses are generated to help in the development of antigen-specific B cells, leading to high-affinity antibody response. By using the same process, DNA immunization should also be effective at producing functionally potent monoclonal antibodies (mAbs). Furthermore, the in vivo expressed proteins can maximally maintain the native structures and go through appropriate post-transcriptional modifications. By combining such advantages, DNA immunization can be expected to play more important roles in the future to elicit mAbs against difficult targets from a wide range of host systems. The current report shares our group's experience in using DNA immunization to elicit mAbs in the mouse, rabbit, and human models.

RUNX1 induces DNA replication independent active DNA demethylation at SPI1 regulatory regions.

BACKGROUND: SPI1 is an essential transcription factor (TF) for the hematopoietic lineage, in which its expression is tightly controlled through a -17-kb upstream regulatory region and a promoter region. Both regulatory regions are demethylated during hematopoietic development, although how the change of DNA methylation status is performed is still unknown. RESULTS: We found that the ectopic overexpression of RUNX1 (another key TF in hematopoiesis) in HEK-293T cells induces almost complete DNA demethylation at the -17-kb upstream regulatory region and partial but significant DNA demethylation at the proximal promoter region. This DNA demethylation occurred in mitomycin-C-treated nonproliferating cells at both regulatory regions, suggesting active DNA demethylation. Furthermore, ectopic RUNX1 expression induced significant endogenous SPI1 expression, although its expression level was much lower than that of natively SPI1-expressing monocyte cells. CONCLUSIONS: These results suggest the novel role of RUNX1 as an inducer of DNA demethylation at the SPI1 regulatory regions, although the mechanism of RUNX1-induced DNA demethylation remains to be explored.