The resulting RNA was electroporated into either C6/36 or Vero cells

The resulting RNA was electroporated into either C6/36 or Vero cells. and efficacy of DENV vaccines. Antibody-dependent enhancement is linked to antibodies targeting the fusion loop (FL) motif of the envelope protein, which is completely conserved in mosquito-borne flaviviruses and required for viral entry and fusion. In the current study, we utilized saturation mutagenesis and directed evolution to engineer a functional variant with a mutated FL (D2-FL), which is not neutralized by FL-targeting monoclonal antibodies. The FL mutations were combined with our previously evolved prM cleavage site to create a mature version of D2-FL (D2-FLM), which evades both prM- and FL-Abs but retains sensitivity to other type-specific and quaternary cross-reactive (CR) Abs. CR serum from heterotypic (DENV4)-infected non-human primates (NHP) showed lower neutralization titers against D2-FL and D2-FLM than isogenic wildtype DENV2 while similar neutralization titers were observed in serum from homotypic (DENV2)-infected NHP. We propose D2-FL and D2-FLM as valuable tools to delineate CR Ab subtypes in serum as well as an exciting platform for safer live-attenuated DENV vaccines suitable for na?ve individuals and children. Research organism: Viruses Introduction Dengue virus (DENV) is a member of the genus and is a major global 3-Hydroxyvaleric acid public health threat, with four major serotypes of DENV found worldwide. Dengue causes ~400 million infections each year, of which ~20% of cases present clinically, a subset of which may progress to severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Bhatt et al., 2013; Brady et al., 2012). DENV is transmitted through mosquito vectors, and globalization and global warming are increasing the endemic range of dengue 3-Hydroxyvaleric acid worldwide (Ebi and Nealon, 2016; Messina et al., 2019). The pathogenesis of dengue is complex as first-time infections are rarely severe and lead to serotype-specific immunity. However, reinfection with a different serotype increases the risk of developing DHF/DSS (Halstead, 1988). This is thought to be due to the phenomenon of antibody-dependent enhancement (ADE), in which poorly neutralizing cross-reactive (CR) antibodies (Abs) lead to enhanced viral uptake and infection of unique cell populations in an Fc-receptor-mediated manner (Katzelnick et al., 2017). ADE remains a major challenge for DENV vaccine development (Rey et al., 2018). The leading DENV vaccine platforms in clinical testing are tetravalent live-attenuated virus mixtures of all four serotypes. However, creating formulations that elicit a balanced response has proven challenging (Rabaa et al., 2017). Additionally, lab-grown strains differ from patient-derived DENVs in both maturation status and antigenicity (Raut et al., 2019). In particular, Abs 3-Hydroxyvaleric acid targeting the fusion loop (FL) have been reported to neutralize lab and patient strains with differing strengths and have been observed to facilitate Fc-receptor uptake in vitro and therefore ADE (Lai et al., 2013; Beltramello et al., 2010; Costin et al., Mouse monoclonal to CD81.COB81 reacts with the CD81, a target for anti-proliferative antigen (TAPA-1) with 26 kDa MW, which ia a member of the TM4SF tetraspanin family. CD81 is broadly expressed on hemapoietic cells and enothelial and epithelial cells, but absent from erythrocytes and platelets as well as neutrophils. CD81 play role as a member of CD19/CD21/Leu-13 signal transdiction complex. It also is reported that anti-TAPA-1 induce protein tyrosine phosphorylation that is prevented by increased intercellular thiol levels 2013). Currently, there is a single FDA-approved DENV vaccine, Dengvaxia. However, it is only approved for use in individuals aged 9C16 with previous DENV infection living in endemic areas and is contraindicated for use in na?ve individuals and younger children. In na?ve children, vaccination stimulated non-neutralizing CR Abs that increased the risk of severe disease after DENV infection (Wilder-Smith, 2019; Anonymous, 2019). Other DENV vaccines have been tested or are currently undergoing clinical trial, but 3-Hydroxyvaleric acid thus far none have been approved for use in the United States (Izmirly et al., 2020). The vaccine Qdenga has been approved in the European Union, Indonesia, and Brazil, although vaccine efficacy in adults, na?ve individuals, and with all serotypes has not yet been shown (Angelin et al., 2023). The.