de Silva, A. D. | Search bioRxiv

bioRxiv2025

In vitro effects of selected angiogenesis-promoting drugs on human Ea.hy926 endothelial cells treated with critical-stage dengue hemorrhagic fever serum

Endothelial dysfunction is the major pathophysiological effect in severe dengue infection. The effects of three angiogenesis-promoting drugs, Simvastatin, Phenytoin, and Ascorbic acid on endothelial dysfunction was assessed using the simulation model using Ea.hy.926 cells treating with severe dengue hemorrhagic fever serum. Sera of patients with dengue fever (DF, n=5), dengue hemorrhagic fever (DHF, n=5), and healthy controls (HC, n=5) were treated on Ea.hy926 endothelial cells and distances were measured after 3-hour incubation. DHF-treated cells showed a significant increase in average cell-cell distances compared to HC and DF, simulating endothelial destabilization caused during DHF condition (p<0.05). Significant reductions in cell-cell distances were observed in the cells treated with 0.01 {micro}M and 1 {micro}M Simvastatin along with DHF serum, compared to the cells treated only with DHF serum (p<0.05). Among these two conditions tested, 0.01 {micro}M Simvastatin showed a significant reduction in the cell-cell distances (p<0.05) and a significant increase in cell sizes (p<0.05) in the presence of DHF serum. Ascorbic acid (40 {micro}M, 80 {micro}M) showed no significant effect towards the endothelial monolayer destabilization in the presence of DHF serum. Both concentrations of phenytoin (10 {micro}M, 30 {micro}M) showed a significant increase in the average cell-cell distances compared to cells treated with DHF (p<0.05). This preliminary study suggests that low-dose Simvastatin promotes endothelial membrane formation in the presence of DHF serum and the reduction in cell-to-cell distances may not due to cell proliferation but could mainly be due to the increase in the average cell size, a known effect of Simvastatin.

immunology↗

bioRxiv2020

T cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-reactivity with other flavivirus species.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. Here, we tested pools of epitopes derived from dengue (DENV), zika (ZIKV), Japanese Encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by Intracellular Cytokine Staining (ICS) using PBMCs of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccines. CD8 T cell responses recognized epitopes from multiple flaviviruses, however, the magnitude of cross-reactive responses was consistently several-fold lower than those to the autologous epitope pools, and associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing twenty-nine different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with more than 100-fold lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses, and in five out of eight cases more than 1000-fold lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and has implications for understanding immunity elicited by natural infection, and strategies to develop live attenuated vaccines against flaviviral species. ImportanceThe envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the non-structural (NS) and capsid (C) antigens which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses amongst different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses, and might thus impair vaccine performance.

immunology↗