Biology subjects

Chumbe, A.

Publications and source records attributed to Chumbe, A..

4 recordsLinked to original sources

bioRxiv2024

Bispecific antibodies against the hepatitis C virus E1E2 envelope glycoprotein

Hepatitis C virus (HCV) currently causes about one million infections and 240,000 deaths worldwide each year. To reach the goal set by the World Health Organization (WHO) of global HCV elimination by 2030, it is critical to develop a prophylactic vaccine. Broadly neutralizing antibodies (bNAbs) target the E1E2 envelope glycoproteins on the viral surface, can neutralize a broad range of the highly diverse circulating HCV strains and are essential tools to inform vaccine design. However, bNAbs targeting a single E1E2 epitope might be limited in neutralization breadth, which can be enhanced by using combinations of bNAbs that target different envelope epitopes. We have generated 60 IgG-like bispecific antibodies (bsAbs) that can simultaneously target two distinct epitopes on E1E2. We combine non-overlapping E1E2 specificities into three types of bsAbs, each containing a different hinge length. The bsAbs show retained or increased potency and breadth against a diverse panel of HCV pseudoparticles (HCVpp) and HCV produced in cell culture (HCVcc) compared to monospecific and cocktail controls. Additionally, we demonstrate that changes in the hinge length of bsAbs can alter the binding stoichiometry to E1E2. These results provide insights into the binding modes and the role of avidity in bivalent targeting of diverse E1E2 epitopes, and suggest structural differences between HCVpp and HCVcc. This study illustrates how potential cooperative effects of HCV bNAbs can be utilized by strategically designing bispecific constructs. These new HCV bsAbs can guide vaccine development and unlock novel therapeutic and prophylactic strategies against HCV and other (flavi)viruses.

microbiology↗

bioRxiv2021

Structure of the hepatitis C virus E1E2 glycoprotein complex

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans, and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and infection, and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remains elusive. We present the cryo-electron microscopy (cryoEM) structure of the membrane-extracted full-length E1E2 heterodimer in complex with broadly neutralizing antibodies (bNAbs) AR4A, AT12009 and IGH505 at [~]3.5 [A] resolution. We resolve the long sought-after interface between the E1 and E2 ectodomains and reveal how it is stabilized by hydrophobic interactions and glycans. This structure deepens our understanding of the HCV fusion glycoprotein and delivers a blueprint for the rational design of novel vaccine immunogens and anti-viral drugs.

immunology↗

bioRxiv2020

Optimised cell systems for the investigation ofhepatitis C virus E1E2 glycoproteins

Great strides have been made in understanding and treating Hepatitis C virus (HCV) thanks in part to the development of the full-length cell-culture system, the pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterising neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones fail to produce infectious particles or produce particles that exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in HEK 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T and 293F exhibit important differences. We found that 293F-produced sE2 harbours mostly complex type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex type glycans and highmannose or hybrid type glycans. Moreover, sE2 produced in HEK 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

microbiology↗

bioRxiv2018

Genotype-matched Newcastle disease virus vaccine confers improved protection against genotype XII challenge: The importance of cytoplasmic tails in viral replication and vaccine design

Although typical Newcastle disease virus (NDV) vaccines can prevent mortality, they are not effective preventing viral shedding. To overcome this, genotype-matched vaccines have been proposed. To date, this approach has never been tested against genotype XII strains. In this study, we generated and assessed the protection against genotype XII challenge of two chimeric NDV vaccine strains (rLS1-XII-1 and rLS1-XII-2). The rLS1-XII-1 virus has the complete fusion protein (F) and the hemmaglutinin-neuraminidase (HN) open reading frames replaced with those from genotype XII strain NDV/peacock/Peru/2011 (PP2011) in a recombinant LaSota (rLS1) backbone. For rLS1-XII-2 cytoplasmic tails of F and HN proteins were restored to those of rLS1. In vitro studies showed that rLS1-XII-2 and the parental rLS1 strains replicate at higher efficiencies than rLS1-XII-1. In the first vaccine/challenge experiment, SPF chickens vaccinated with rLS1-XII-1 virus showed only 71.3% protection, whereas, rLS1 and rLS1-XII-2 vaccinated chickens were fully protected. In a second experiment, both rLS1-XII-2 and the commercial vaccine strain LaSota induced 100% protection. However, rLS1-XII-2 virus significantly reduced viral shedding, both in the number of shedding birds and in quantity of shed virus. In conclusion, we have developed a vaccine candidate capable of fully protecting chickens against genotype XII challenges. Furthermore, we have shown the importance of cytoplasmic tails in virus replication and vaccine competence.

microbiology↗