Vidya Mangala Prasad

Flaviviruses, or orthoflaviviruses, are arthropod-borne RNA viruses that pose a significant global health threat and co-circulate in many regions across the world. Neutralizing antibodies against specific pathogenic flaviviruses, such as dengue virus, Zika virus, and West Nile virus, are well-known. However, vaccine and therapeutic development against many flaviviruses remains challenging due to their antigenic diversity and complex humoral responses. Recent studies have reported patient-derived neutralizing antibodies that are active against multiple flaviviruses. Structural characterization of these pan-flavivirus antibodies has identified quaternary epitopes that are conserved across multiple flaviviruses, emphasizing the importance of quaternary epitopes on native flaviviruses, which, in some cases, are also preserved across different virus morphologies. Understanding the basis of quaternary structural epitope recognition can aid in the design of immunogens that elicit broad protection, with a lower risk of infection enhancement. In this review, we discuss mechanistic insights of bnAbs targeting different quaternary epitopes found on pathogenic flaviviruses, and implications for next-generation pan-flavivirus vaccine strategies.

Dengue virus (DENV) is a global health threat, with four antigenically distinct serotypes impeding vaccine and therapeutic development. We describe here, structural features of patient-derived, heavy chain driven Edimer recognizing (HEDR) antibodies that achieve potent, pan-serotype activity by binding a conserved quaternary epitope on DENVs envelope (E) glycoprotein dimer. Single-particle cryoEM structures of three HEDR broadly neutralizing antibodies (bnAbs) complexed to DENV delineate epitope engagement via E-glycoprotein fusion loop region and conserved glycans. We also identify capsule-shaped DENV, with helical cryoEM reconstructions of Fab-bound tubular DENV from different strains revealing that the E-glycoprotein arrangement is determined by virus strain, not bnAb identity. Our findings elucidate key binding features of HEDR bnAbs and signify the need for intervention strategies capable of neutralizing diverse DENV morphologies.

Several trafficking pathways are operational at the plasma membrane, and both clathrin-dependent, and clathrin-independent endocytosis (CIE), can serve as virus entry portals. Our research has shown that the neurotropic flavivirus : Japanese encephalitis virus (JEV), infects neuronal cells via CIE. Here we have identified and characterized two essential host-factors for JEV trafficking in neuronal cells: Endophilin & Epidermal Growth Factor Receptor (EGFR). Through quantitative estimation of viral RNA copy number, we demonstrate that JEV entry in neuronal cells, was blocked by knock-down of Endophilin A isoforms, while activation of Endophilin-mediated endocytosis using a specific inhibitor of GSK-3β enhanced virus entry. Deletion mutants of Endophilin showed an essential role of SH ₃ , BAR, H ₀ domains for virus entry. High resolution fluorescence imaging of virions showed overlap with Endophilin A2 puncta. Virus entry led to rearrangements of the actin cytoskeleton, and was highly sensitive to any pharmacological actin perturbation. Virus endocytosis activated EGFR, and the specific kinase inhibitors Erlotinib, and Gefitinib, reduced virus entry and replication in cultured cells and mouse primary cortical neurons. Silencing of EGFR, competitive inhibition with receptor ligand EGF, and EGFR specific antibodies significantly impaired JEV binding and entry, indicating the crucial role of its ligand binding domain for virus attachment/receptor interaction. EGFR colocalized with virions at early time-points of infection, and the ED3 domain of the JEV-envelope protein showed specific interaction with EGFR through Bio-layer interferometry (BLI). Our study provides evidence for JEV entry in neuronal cells through an endocytic pathway involving Endophilin and EGFR.

Several trafficking pathways are operational at the plasma membrane, and both clathrin-dependent, and clathrin-independent endocytosis (CIE), can serve as virus entry portals. Our research has shown that the neurotropic flavivirus : Japanese encephalitis virus (JEV), infects neuronal cells via CIE. Here we have identified and characterized two essential host-factors for JEV trafficking in neuronal cells: Endophilin & Epidermal Growth Factor Receptor (EGFR). Through quantitative estimation of viral RNA copy number, we demonstrate that JEV entry in neuronal cells, was blocked by knock-down of Endophilin A isoforms, while activation of Endophilin-mediated endocytosis using a specific inhibitor of GSK-3β enhanced virus entry. Deletion mutants of Endophilin showed an essential role of SH ₃ , BAR, H ₀ domains for virus entry. High resolution fluorescence imaging of virions showed overlap with Endophilin A2 puncta. Virus entry led to rearrangements of the actin cytoskeleton, and was highly sensitive to any pharmacological actin perturbation. Virus endocytosis activated EGFR, and the specific kinase inhibitors Erlotinib, and Gefitinib, reduced virus entry and replication in cultured cells and mouse primary cortical neurons. Silencing of EGFR, competitive inhibition with receptor ligand EGF, and EGFR specific antibodies significantly impaired JEV binding and entry, indicating the crucial role of its ligand binding domain for virus attachment/receptor interaction. EGFR colocalized with virions at early time-points of infection, and the ED3 domain of the JEV-envelope protein showed specific interaction with EGFR through BLI. Our study provides evidence for JEV entry in neuronal cells through an endocytic pathway involving Endophilin and EGFR.

Pancreatic β‐cells secrete insulin stored in large dense core vesicles (LDCV) by fusion of vesicle and plasma membrane during a process called insulin exocytosis. Insulin secretion is biphasic with a fast first phase and a sustained second phase. Previous studies have pointed out that exocytosis of insulin can occur via (1) single LDCVs fusing with the plasma membrane to release their content or (2) multiple vesicles are involved during a process called compound exocytosis. Compound exocytosis represents a specialized form of secretion in which vesicles undergo homotypic fusion either before (multi‐vesicular exocytosis) or continuous fusion in a sequential manner from (sequential exocytosis) within the same site at the plasma membrane. We see that the number of multi‐vesicles is few and not localized in the vicinity of the plasma membrane. Studying the kinetics of this process and correlating it with biphasic insulin secretion is not possible since there are no specific probes to detect them. It is challenging to identify compound exocytosis with probes that exist for simple exocytosis. To advance our understanding, we need a fluorescent probe that could detect secretory vesicles undergoing compound exocytosis and allow us to distinguish it from other modes of exocytosis. Here, we used two cargo proteins (NPY and tPA) labeled with different fluorescent proteins (mCherry GFP and eGFP) and employed total internal reflection fluorescence microscopy (TIRF‐M) to capture distinct single‐granule and multi‐granular fusion events. We identified tPA‐GFP as a better probe for studying compound exocytosis, as it can detect both simple and sequential exocytosis reliably. Using these probes, we have studied the kinetics of compound exocytosis in human β‐cells. These observations, with additional experiments, may open a whole new field to study the impact of compound exocytosis on biphasic secretion of insulin. Identifying targets to increase the compound exocytosis process can help potentiate insulin secretion in diabetics.

Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate.

Enveloped RNA viruses have been causative agents of major pandemic outbreaks in the recent past. Glycans present on these virus surface proteins are critical for multiple processes during the viral infection cycle. Presence of glycans serves as a key determinant of immunogenicity, but intrinsic heterogeneity, dynamics, and evolutionary shifting of glycans in heavily glycosylated enveloped viruses confounds typical structure–function analysis. Glycosylation sites are also conserved across different viral families, which further emphasizes their functional significance. In this review, we summarize findings regarding structure–function correlation of glycans on enveloped RNA virus proteins.