Elucidating variations in the nucleotide sequence of Ebola virus associated with increasing pathogenicity

Background Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission. Results To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage. Conclusions Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV

r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors

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

Elucidating variations in the nucleotide sequence of Ebola virus associated with increasing pathogenicity

BACKGROUND: Ebolaviruses cause a severe and often fatal haemorrhagic fever in humans, with some species such as Ebola virus having case fatality rates approaching 90%. Currently, the worst Ebola virus outbreak since the disease was discovered is occurring in West Africa. Although thought to be a zoonotic infection, a concern is that with increasing numbers of humans being infected, Ebola virus variants could be selected which are better adapted for human-to-human transmission. RESULTS: To investigate whether genetic changes in Ebola virus become established in response to adaptation in a different host, a guinea pig model of infection was used. In this experimental system, guinea pigs were infected with Ebola virus (EBOV), which initially did not cause disease. To simulate transmission to uninfected individuals, the virus was serially passaged five times in naïve animals. As the virus was passaged, virulence increased and clinical effects were observed in the guinea pig. An RNAseq and consensus mapping approach was then used to evaluate potential nucleotide changes in the Ebola virus genome at each passage. CONCLUSIONS: Upon passage in the guinea pig model, EBOV become more virulent, RNA editing and also coding changes in key proteins become established. The data suggest that the initial evolutionary trajectory of EBOV in a new host can lead to a gain in virulence. Given the circumstances of the sustained transmission of EBOV in the current outbreak in West Africa, increases in virulence may be associated with prolonged and uncontrolled epidemics of EBOV