Patra, T. | Search bioRxiv

bioRxiv2022

Modified E2 glycoprotein of hepatitis C virus enhances pro-inflammatory cytokines and protective immune response in mice

Hepatitis C virus (HCV) is characterized by a high number of chronic cases owing to an impairment of innate and adaptive immune responses. CD81 on the cell surface facilitates HCV entry by interacting with the E2 envelope glycoprotein. On the other hand, CD81/E2 binding on immune related cells may also influence host response outcome to HCV infection. Here, we performed site-specific amino acid substitution in the front layer of E2 sequence to reduce CD81 binding and evaluate HCV candidate vaccine potential. The altered sE2 protein (F442NYT), unlike sE2, displayed a significant reduction in CD81 binding, induced pro-inflammatory cytokines, and repressed anti-inflammatory response in primary monocyte-derived macrophages as antigen presenting cells. Further, sE2F442NYT stimulated CD4+T cell proliferation. Immunization of Balb/c mice with an E1/sE2F442NYT RNA-lipid nanoparticle (LNP) displayed improved IgG1 to IgG2a isotype switching, an increase in HCV pseudotype virus neutralizing antibodies, and resistance to challenge infection with a surrogate recombinant vaccinia virus expressing HCV E1-E2-NS2(aa134-966), unlike parental E1/sE2 immunization. Further investigation on modified E2 antigen for selection as antigen may provide helpful information for HCV vaccine development. One Sentence SummaryReduced HCV E2-CD81 binding and immune response

microbiology↗

bioRxiv2021

SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response

Increased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-{beta}-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.

immunology↗