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.

The existence of broadly cross-reactive antibodies that can neutralize diverse HIV-1 isolates (bnAbs) has been appreciated for more than a decade. Many high-resolution structures of bnAbs, typically with one or two well-characterized HIV-1 Env glycoprotein trimers, have been reported. However, an understanding of how such antibodies grapple with variability in their antigenic targets across diverse viral isolates has remained elusive. To achieve such an understanding requires first characterizing the extent of structural and antigenic variation embodied in Env, and then identifying how a bnAb overcomes that variation at a structural level. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and quantitative measurements of antibody binding kinetics, we show that variation in structural ordering in the V1/V2 apex of Env across a globally representative panel of HIV-1 isolates has a marked effect on antibody association rates and affinities. We also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit different degrees of structural dynamics throughout the trimer structures. Parallel HDX-MS experiments demonstrate that PGT145 bnAb has an exquisitely focused footprint at the trimer apex where binding did not yield allosteric changes throughout the rest of the structure. These results demonstrate that structural dynamics are a cryptic determinant of antigenicity, and mature antibodies that have achieved breadth and potency in some cases are able to achieve their broad cross-reactivity by “threading the needle” and binding in a highly focused fashion, thus evading and overcoming the variable properties found in Env from divergent isolates.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC₅₀). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

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.