Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids

BACKGROUND: Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression. METHODOLOGY/PRINCIPAL FINDINGS: Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid. CONCLUSIONS/SIGNIFICANCE: Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival