From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus

With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains.

Hemagglutinin stability determines influenza A virus susceptibility to a broad-spectrum fusion inhibitor Arbidol

Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral which binds to and prevents the fusion-associated conformational changes in the trimeric influenza hemagglutinin (HA). The rate-limiting step during HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion peptide stability on the pre-fusion HA: stabilized mutants were more sensitive, and destabilized ones resistant to Arbidol. Single-virion membrane fusion experiments for representative Wild Type and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not dependent on reduced Arbidol binding to HA at neutral pH. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a "tug" on the fusion peptide by the extension of the central coiled-coil on HA, and the key interactions stabilizing fusion peptide in the pre-fusion pocket. Arbidol increases the free-energy penalty for coiled-coil extension, but destabilizing mutations decrease the free-energy cost for fusion peptide release, accounting for the observed resistance. Our findings have broad implications for fusion-inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

Functional Analysis of Human and Feline Coronavirus Cross-Reactive Antibodies Directed Against the SARS-CoV-2 Fusion Peptide

To face the continuous emergence of SARS-CoV-2 variants, broadly protective therapeutic antibodies are highly needed. We here focused on the fusion peptide (FP) region of the viral spike antigen since it is highly conserved among alpha- and betacoronaviruses. First, we found that coronavirus cross-reactive antibodies are commonly formed during infection, being omnipresent in sera from COVID-19 patients, in ~50% of pre-pandemic human sera (rich in antibodies against endemic human coronaviruses), and even in feline coronavirus-infected cats. Pepscan analyses demonstrated that a confined N-terminal region of the FP is strongly immunogenic across diverse coronaviruses. Peptide-purified human antibodies targeting this conserved FP epitope exhibited broad binding of alpha- and betacoronaviruses, besides weak and transient SARS-CoV-2 neutralizing activity. Being frequently elicited by coronavirus infection, these FP-binding antibodies might potentially exhibit Fc-mediated effector functions and influence the kinetics or severity of coronavirus infection and disease.

A Novel Double Mosaic Virus-like Particle-Based Vaccine against SARS-CoV-2 Incorporates Both Receptor Binding Motif (RBM) and Fusion Domain

COVID-19 has emerged, and has rapidly become a major health problem worldwide, causing millions of mortalities. Vaccination against COVID-19 is the most efficient way to stop the pandemic. The goal of vaccines is to induce neutralizing antibodies against SARS-CoV-2 virus. Here, we present a novel double mosaic virus-like particle (VLP) displaying two independent neutralizing epitopes, namely the receptor binding motif (RBM) located in S1 and the fusion peptide (AA 817–855) located in S2. CuMVTT virus-like particles were used as VLP scaffold and both domains were genetically fused in the middle of CuMVTT subunits, which co-assembled into double mosaic particles (CuMVTT-DF). A single fusion mosaic particle (CuMVTT-FP) containing the fusion peptide only was used for comparison. The vaccines were produced in E. coli, and electron microscopy and dynamic light scattering confirmed their integrity and homogeneity. In addition, the CuMVTT-DF vaccine was well recognized by ACE2 receptor, indicating that the RBM was in native conformation. Both CuMVTT-FP and CuMVTT-DF vaccines induced high levels of high avidity IgG antibodies as well as IgA recognizing spike and RBD in the case of CuMVTT-DF. Both vaccine candidates induced virus-neutralizing antibodies indicating that the fusion peptide can independently induce virus-neutralizing antibodies. In contrast, CuMVTT-DF containing both RBM and fusion peptide induced a higher level of neutralizing antibodies suggesting that the new double mosaic vaccine candidate CuMVTT-DF consisting of two antigens in one VLP maybe an attractive candidate for scale-up in a bacterial fermentation process for clinical development.

Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein after infection and/or vaccination

ABSTRACTBackgroundControl of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal.MethodsThe epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential escape mutations by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue.ResultsIndividuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination.ConclusionsThe finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level.FundingThis work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

Novel neutralization mechanism of a human antibody with pan-coronavirus reactivity

The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.