Visualizing intermediate stages of viral membrane fusion by cryo-electron tomography.

Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion.

mRNA-based VP8* nanoparticle vaccines against rotavirus are highly immunogenic in rodents.

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.

An HIV-1 broadly neutralizing antibody overcomes structural and dynamic variation through highly focused epitope targeting.

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.