Batchelor, J. D. | Search bioRxiv

bioRxiv2022

A bivalent EBV vaccine induces neutralizing antibodies that block B and epithelial cell infection and confer immunity in humanized mice

Epstein Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers. Despite its prevalence and major impact on human health, there are currently no specific vaccines or treatments. Four viral glycoproteins, gp 350 and gH/gL/gp42 mediate entry into the major sites of viral replication, B cells and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and show that it elicits potent neutralizing antibodies that protect against infection in vivo. Based on structural analyses, we designed single chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticles. X-ray crystallographic analysis revealed that single chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild type proteins, and the protein spikes were observed by electron microscopy. Single chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and non-human primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123 or gH/gL+gp350D123 nanoparticles. While all control animals (6/6) were infected, only one mouse in each vaccine group that received immune IgG had transient low level viremia (1/6). Furthermore, no EBV lymphomas were detected in immune animals in contrast to non-immune controls. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans. One sentence summaryA bivalent gp350 and gH/gL/gp42 nanoparticle vaccine elicits neutralizing antibodies that protect against EBV infection and EBV lymphoma in vivo.

immunology↗

bioRxiv2020

The ΔF508 CFTR defect: molecular mechanism of suppressor mutation V510D and the contribution of transmembrane helix unraveling

Cystic fibrosis (CF) results from mutations within the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a transmembrane chloride channel found on the apical surface of epithelial cells. The most common CF-causing mutation results in a deletion of phenylalanine 508 ({Delta}F508-CFTR), a residue normally found within the NBD1 domain. Loss of F508 causes NBD1 to be less thermodynamically stable and prevents proper tertiary folding of CFTR. As a result, CFTR is not properly trafficked to the cell surface. Recently, progress has been made towards the development of small molecule "correctors" that can restore CFTR tertiary structure and stabilize the channel to overcome the instability inherent in {Delta}F508-CFTR. However, the resultant improvement in channel activity has been modest, and the need for potent correctors remains. To fully inform such efforts, a better understanding of the molecular pathology associated with {Delta}F508-CFTR is required. Here we present a comprehensive study of the impact of F508 deletion on both purified NBD1 and full-length CFTR. Through the use of homology modeling, molecular dynamics simulations, mutational analysis, biochemical, biophysical and functional characterization studies, we obtained insight into how the {Delta}F508 mutation may lead to helical unraveling of transmembrane domains 10 and 11 (TM10, TM11), and how the known suppressor mutations V510D and R1070W, as well as novel second site suppressor mutations (SSSMs) identified in this work, may act to rescue {Delta}F508-CFTR maturation and trafficking.

biochemistry↗