Role of SARS-CoV-2 mutations in the evolution of the COVID-19 pandemic.

OBJECTIVES: The SARS-CoV-2 pandemic and large-scale genomic surveillance provided an exceptional opportunity to analyze mutations that appeared over three years in viral genomes. Here we studied mutations and their epidemic consequences for SARS-CoV-2 genomes from our center. METHODS: We analyzed 61,397 SARS-CoV-2 genomes we sequenced from respiratory samples for genomic surveillance. Mutations frequencies were calculated using Nextclade, Microsoft Excel, and an in-house Python script. RESULTS: A total of 22,225 nucleotide mutations were identified, 220 (1.0%) being each at the root of ≥836 genomes, classifying mutations as 'hyperfertile'. Two seeded the European pandemic: P323L in RNA polymerase, associated with an increased mutation rate, and D614G in spike that improved fitness. Most 'hyperfertile' mutations occurred in areas not predicted with increased virulence. Their mean number was 8±6 (0-22) per 1000 nucleotides per gene. They were 3.7-times more frequent in accessory than informational genes (13.8 versus 3.7/1000 nucleotides). Particularly, they were 4.1-times more frequent in ORF8 than in the RNA polymerase gene. Interestingly, stop codons were present in 97 positions, almost only in accessory genes, including ORF8 (21/100 codons). CONCLUSIONS: most 'hyperfertile' mutations did not predict emergence of a new epidemic, and some were stop codons indicating the existence of so-named 'non-virulence' genes.

What Is life? Rethinking Biology in Light of Fundamental Parameters.

Defining life is an arduous task that has puzzled philosophers and scientists for centuries. Yet biology suffers from a lack of clear definition, putting biologists in a paradoxical situation where one can describe at the atomic level complex objects that remain globally poorly defined. One could assume that such descriptions make it possible to perfectly characterize living systems. However, many cases of misinterpretation put this assumption into perspective. In this article, we focus on critical parameters such as time, water, entropy, space, quantum properties, and electrostatic potential to redefine the nature of living matter, with special emphasis on biological coding. Where does the DNA double helix come from, why cannot the reproduction of living organisms occur without mutations, what are the limitations of the genetic code, and why do not all proteins have a stable three-dimensional structure? There are so many questions that cannot be resolved without considering the aforementioned parameters. Indeed, (i) time and space constrain many biological mechanisms and impose drastic solutions on living beings (enzymes, transporters); (ii) water controls the fidelity of DNA replication and the structure/disorder balance of proteins; (iii) entropy is the driving force of many enzymatic reactions and molecular interactions; (iv) quantum mechanisms explain why a molecule as simple as hydrocyanic acid (HCN) foreshadows the helical structure of DNA, how DNA is stabilized, why mutations occur, and how the Earth magnetic field can influence the migration of birds; (v) electrostatic potential controls epigenetic mechanisms, lipid raft functions, and virus infections. We consider that raising awareness of these basic parameters is critical for better understanding what life is, and how it handles order and chaos through a combination of genetic and epigenetic mechanisms. Thus, we propose to incorporate these parameters into the definition of life.

Resistance-associated mutations to the anti-SARS-CoV-2 agent nirmatrelvir: Selection not induction.

Mutations associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance to antiprotease nirmatrelvir were reported. We aimed to detect them in SARS-CoV-2 genomes and quasispecies retrieved in our institute before drug availability in January 2022 and to analyze the impact of mutations on protease (3CLpro) structure. We sought for 38 3CLpro nirmatrelvir resistance mutations in a set of 62 673 SARS-CoV-2 genomes obtained in our institute from respiratory samples collected between 2020 and 2023 and for these mutations in SARS-CoV-2 quasispecies for 90 samples collected in 2020, using Python. SARS-CoV-2 protease with major mutation E166V was generated with Swiss Pdb Viewer and Molegro Molecular Viewer. We detected 22 (58%) of the resistance-associated mutations in 417 (0.67%) of the genomes analyzed; 325 (78%) of these genomes had been obtained from samples collected in 2020-2021. APOBEC signatures were found for 12/22 mutations. We also detected among viral quasispecies from 90 samples some minority reads harboring any of 15 nirmatrelvir resistance mutations, including E166V. Also, we predicted that E166V has a very limited effect on 3CLpro structure but may prevent drug attachment. Thus, we evidenced that mutations associated with nirmatrelvir resistance pre-existed in SARS-CoV-2 before drug availability. These findings further warrant SARS-CoV-2 genomic surveillance and SARS-CoV-2 quasispecies characterization.

From viral democratic genomes to viral wild bunch of quasispecies.

The tremendous majority of RNA genomes from pathogenic viruses analyzed and deposited in databases are consensus or "democratic" genomes. They represent the genomes most frequently found in the clinical samples of patients but do not account for the huge genetic diversity of coexisting genomes, which is better described as quasispecies. A viral quasispecies is defined as the dynamic distribution of nonidentical but closely related mutants, variants, recombinant, or reassortant viral genomes. Viral quasispecies have collective behavior and dynamics and are the subject of internal interactions that comprise interference, complementation, or cooperation. In the setting of SARS-CoV-2 infection, intrahost SARS-CoV-2 genetic diversity was recently notably reported for immunocompromised, chronically infected patients, for patients treated with monoclonal antibodies targeting the viral spike protein, and for different body compartments of a single patient. A question that deserves attention is whether such diversity is generated postinfection from a clonal genome in response to selection pressure or is already present at the time of infection as a quasispecies. In the present review, we summarize the data supporting that hosts are infected by a "wild bunch" of viruses rather than by multiple virions sharing the same genome. Each virion in the "wild bunch" may have different virulence and tissue tropisms. As the number of viruses replicated during host infections is huge, a viral quasispecies at any time of infection is wide and is also influenced by host-specific selection pressure after infection, which accounts for the difficulty in deciphering and predicting the appearance of more fit variants and the evolution of epidemics of novel RNA viruses.

The return of the "Mistigri" (virus adaptative gain by gene loss) through the SARS-CoV-2 XBB.1.5 chimera that predominated in 2023.

Severe acute respiratory syndrome coronavirus 2 XBB.1.5 is the first recombinant lineage to predominate at the country and global scales. Very interestingly, like the Marseille-4B subvariant (or B.1.160) and the pandemic variant B.1.1.7 (or Alpha) previously, it has its ORF8 gene inactivated by a stop codon. We aimed here to study the distribution of stop codons in ORF8 of XBB.1.5 and non-XBB.1.5 genomes. We identified that a stop codon was present at 89 (74%) ORF8 codons in ≥1 of 15 222 404 genomes available in GISAID. The mean proportion of genomes with a stop codon per codon was 0.11% (range, 0%-7.8%). In addition, a stop codon was detected at 15 (12%) codons in at least 1000 genomes. These 15 codons are notably located on seven stem-loop hairpin regions and in the signal peptide region for the case of the XBB.1.5 lineage (codon 8). Thus, it is very likely that stop codons in ORF8 gene contributed on at least three occasions and independently during the pandemic to the evolutionary success of a lineage that became transiently predominant. Such association of gene loss with evolutionary success, which suits the recently described Mistigri rule, is an important biological phenomenon very unknown in virology while largely described in cellular organisms.

Possible contribution of rare alleles of human ACE2 in the emergence of SARS-CoV-2 variants escaping the immune response.

Since the start of the SARS-CoV-2 pandemic, the rapid replacement of one lineage by another has been observed. Indeed, SARS-CoV-2 is evolving through a quasispecies mechanism leading to post-infection mutation selection under positive evolutionary pressure (host-driven viral evolution). These mutations may reduce the effectiveness of the specific neutralizing immune response against the virus. We provide here evidence that apart from the selection of SARS-CoV-2 variants by the immune system, selection by the cellular receptor can just as well select variants which escape neutralization.

Emergence of a second SARS-CoV-2 variant with a tremendous genetic leap from its ancestors.

The on-going emergence of the Omicron BA.2.86 variant is one of the major events in SARS-CoV-2 genetic evolution that remain enigmatic regarding the overall virus mutation rate, together with the emergence of the initial Omicron variant, BA.1. Indeed, the genomes of the Omicron BA.2.86 lineage, an offspring of the second major Omicron subvariant, BA.2, harbor 39 additional mutations in the spike compared to this ancestor. Here we comment on the phylogeny of BA.2.86, on the positions, and frequencies in other SARS-CoV-2, of mutations in its spike, and on the structural model of this protein that concentrates most of BA.2.86 additional mutations.

Structure of the Myelin Sheath Proteolipid Plasmolipin (PLLP) in a Ganglioside-Containing Lipid Raft.

BACKGROUND: Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as neurological disorders, type 2 diabetes, and cancer metastasis. To better understand how PLLP interacts with raft components (gangliosides and cholesterol), we undertook a global study combining in silico simulations and physicochemical measurements of molecular interactions in various PLLP-ganglioside systems. METHODS: In silico studies consisted of molecular dynamics simulations in reconstructed membrane environments. PLLP-ganglioside interaction measurements were performed by microtensiometry at the water-air interface on ganglioside monolayers. RESULTS: We have elucidated the mode of interaction of PLLP with ganglioside GM1 and characterized this interaction at the molecular level. We showed that GM1 induces the structuring of the extracellular loops of PLLP and that this interaction propagates a conformational signal through the plasma membrane, involving a cholesterol molecule located between transmembrane domains. This conformational wave is finally transmitted to the intracellular domain of the protein, consistent with the role of PLLP in signal transduction. CONCLUSIONS: This study is a typical example of the epigenetic dimension of protein structure, a concept developed by our team to describe the chaperone effect of gangliosides on disordered protein motifs which associate with lipid rafts. From a physiological point of view, these data shed light on the role of gangliosides in myelin formation. From a pathological point of view, this study will help to design innovative therapeutic strategies focused on ganglioside-PLLP interactions in various PLLP-associated diseases.

Host Membranes as Drivers of Virus Evolution.

The molecular mechanisms controlling the adaptation of viruses to host cells are generally poorly documented. An essential issue to resolve is whether host membranes, and especially lipid rafts, which are usually considered passive gateways for many enveloped viruses, also encode informational guidelines that could determine virus evolution. Due to their enrichment in gangliosides which confer an electronegative surface potential, lipid rafts impose a first control level favoring the selection of viruses with enhanced cationic areas, as illustrated by SARS-CoV-2 variants. Ganglioside clusters attract viral particles in a dynamic electrostatic funnel, the more cationic viruses of a viral population winning the race. However, electrostatic forces account for only a small part of the energy of raft-virus interaction, which depends mainly on the ability of viruses to form a network of hydrogen bonds with raft gangliosides. This fine tuning of virus-ganglioside interactions, which is essential to stabilize the virus on the host membrane, generates a second level of selection pressure driven by a typical induced-fit mechanism. Gangliosides play an active role in this process, wrapping around the virus spikes through a dynamic quicksand-like mechanism. Viruses are thus in an endless race for access to lipid rafts, and they are bound to evolve perpetually, combining speed (electrostatic potential) and precision (fine tuning of amino acids) under the selective pressure of the immune system. Deciphering the host membrane guidelines controlling virus evolution mechanisms may open new avenues for the design of innovative antivirals.

ACE2 receptor polymorphism in humans and animals increases the risk of the emergence of SARS-CoV-2 variants during repeated intra- and inter-species host-switching of the virus.

Like other coronaviruses, SARS-CoV-2 has ability to spread through human-to-human transmission and to circulate from humans to animals and from animals to humans. A high frequency of SARS-CoV-2 mutations has been observed in the viruses isolated from both humans and animals, suggesting a genetic fitness under positive selection in both ecological niches. The most documented positive selection force driving SARS-CoV-2 mutations is the host-specific immune response. However, after electrostatic interactions with lipid rafts, the first contact between the virus and host proteins is the viral spike-cellular receptor binding. Therefore, it is likely that the first level of selection pressure impacting viral fitness relates to the virus's affinity for its receptor, the angiotensin I converting enzyme 2 (ACE2). Although sufficiently conserved in a huge number of species to support binding of the viral spike with enough affinity to initiate fusion, ACE2 is highly polymorphic both among species and within a species. Here, we provide evidence suggesting that when the viral spike-ACE2 receptor interaction is not optimal, due to host-switching, mutations can be selected to improve the affinity of the spike for the ACE2 expressed by the new host. Notably, SARS-CoV-2 is mutation-prone in the spike receptor binding domain (RBD), allowing a better fit for ACE2 orthologs in animals. It is possibly that this may also be true for rare human alleles of ACE2 when the virus is spreading to billions of people. In this study, we present evidence that human subjects expressing the rare E329G allele of ACE2 with higher allele frequencies in European populations exhibit a improved affinity for the SARS-CoV-2 spike N501Y variant of the virus. This may suggest that this viral N501Y variant emerged in the human population after SARS-CoV-2 had infected a human carrying the rare E329G allele of ACE2. In addition, this viral evolution could impact viral replication as well as the ability of the adaptive humoral response to control infection with RBD-specific neutralizing antibodies. In a shifting landscape, this ACE2-driven genetic drift of SARS-CoV-2 which we have named the 'boomerang effect', could complicate the challenge of preventing COVID with a SARS-CoV-2 spike-derived vaccine.

Unravelling Antigenic Cross-Reactions toward the World of Coronaviruses: Extent of the Stability of Shared Epitopes and SARS-CoV-2 Anti-Spike Cross-Neutralizing Antibodies.

The human immune repertoire retains the molecular memory of a very great diversity of target antigens (epitopes) and can recall this upon a second encounter with epitopes against which it has previously been primed. Although genetically diverse, proteins of coronaviruses exhibit sufficient conservation to lead to antigenic cross-reactions. In this review, our goal is to question whether pre-existing immunity against seasonal human coronaviruses (HCoVs) or exposure to animal CoVs has influenced the susceptibility of human populations to SARS-CoV-2 and/or had an impact upon the physiopathological outcome of COVID-19. With the hindsight that we now have regarding COVID-19, we conclude that although antigenic cross-reactions between different coronaviruses exist, cross-reactive antibody levels (titers) do not necessarily reflect on memory B cell frequencies and are not always directed against epitopes which confer cross-protection against SARS-CoV-2. Moreover, the immunological memory of these infections is short-term and occurs in only a small percentage of the population. Thus, in contrast to what might be observed in terms of cross-protection at the level of a single individual recently exposed to circulating coronaviruses, a pre-existing immunity against HCoVs or other CoVs can only have a very minor impact on SARS-CoV-2 circulation at the level of human populations.

Lipid rafts and human diseases: why we need to target gangliosides.

Gangliosides are functional components of membrane lipid rafts that control critical functions in cell communication. Many pathologies involve raft gangliosides, which therefore represent an approach of choice for developing innovative therapeutic strategies. Beginning with a discussion of what a disease is (and is not), this review lists the major human pathologies that involve gangliosides, which includes cancer, diabetes, and infectious and neurodegenerative diseases. In most cases, the problem is due to a protein whose binding to gangliosides either creates a pathological condition or impairs a physiological function. Then, I draw up an inventory of the different molecular mechanisms of protein-ganglioside interactions. I propose to classify the ganglioside-binding domains of proteins into four categories, which I name GBD-1, GBD-2, GBD-3, and GBD-4. This structural and functional classification could help to rationalize the design of innovative molecules capable of disrupting the binding of selected proteins to gangliosides without generating undesirable effects. The biochemical specificities of gangliosides expressed in the human brain must also be taken into account to improve the reliability of animal models (or any animal-free alternative) of Alzheimer's and Parkinson's diseases.

Structural basis of botulinum neurotoxin serotype A1 binding to human SV2A or SV2C receptors.

Botulinum neurotoxin A1 (BoNT/A1) is the most potent natural poison in human. BoNT/A1 recognize the luminal domain of SV2A (LD-SV2A) and its glycosylation at position N573 (N573g) or the luminal domain of SV2C (LD-SV2C) and its glycosylation at position N559 (N559g) to bind neural membrane. Our computational data suggest that the N-glycan at position 480 (N480g) in the luminal domain of SV2C (LD-SV2C) indirectly enhanced the contacts of the neurotoxin surface with the second N-glycan at position 559 (N559g) by acting as a shield to prevent N559g to interact with residues of LD-SV2C. The absence of an N-glycan homologous to N480g in LD-SV2A leads to a decrease of the binding of N573g to the surface of BoNT/A1. Concerning the intermolecular interactions between BoNT/A and the protein part of LD-SV2A or LD-SV2C, we showed that the high affinity of the neurotoxin for binding LD-SV2C are mediated by a better compaction of its F557-F562 part provided by a π-π network mediated by residues F547, F552, F557 and F562 coupled with the presence of two aromatic residues at position 563 and 564 that optimize the binding of BoNT/A1 via cation-pi and CH-pi interaction. Finally, in addition to the well-known ganglioside binding site which accommodates a ganglioside on the surface of BoNT/A1, we identified a structure we coined the ganglioside binding loop defined by the sequence 1253-HQFNNIAK-1260 that is conserved across all subtypes of BoNT/A and is predicted to has a high affinity to interact with gangliosides. These data solved the puzzle generated by mutational studies that could be only partially understood with crystallographic data that lack both a biologically relevant membrane environment and a full glycosylation of SV2.

Convergent Evolution Dynamics of SARS-CoV-2 and HIV Surface Envelope Glycoproteins Driven by Host Cell Surface Receptors and Lipid Rafts: Lessons for the Future.

Although very different, in terms of their genomic organization, their enzymatic proteins, and their structural proteins, HIV and SARS-CoV-2 have an extraordinary evolutionary potential in common. Faced with various selection pressures that may be generated by treatments or immune responses, these RNA viruses demonstrate very high adaptive capacities, which result in the continuous emergence of variants and quasi-species. In this retrospective analysis of viral proteins, ensuring the adhesion of these viruses to the plasma membrane of host cells, we highlight many common points that suggest the convergent mechanisms of evolution. HIV and SARS-CoV-2 first recognize a lipid raft microdomain that acts as a landing strip for viral particles on the host cell surface. In the case of mucosal cells, which are the primary targets of both viruses, these microdomains are enriched in anionic glycolipids (gangliosides) forming a global electronegative field. Both viruses use lipid rafts to surf on the cell surface in search of a protein receptor able to trigger the fusion process. This implies that viral envelope proteins are both geometrically and electrically compatible to the biomolecules they select to invade host cells. In the present study, we identify the surface electrostatic potential as a critical parameter controlling the convergent evolution dynamics of HIV-1 and SARS-CoV-2 surface envelope proteins, and we discuss the impact of this parameter on the phenotypic properties of both viruses. The virological data accumulated since the emergence of HIV in the early 1980s should help us to face present and future virus pandemics.

Electrostatic Surface Potential as a Key Parameter in Virus Transmission and Evolution: How to Manage Future Virus Pandemics in the Post-COVID-19 Era.

Virus-cell interactions involve fundamental parameters that need to be considered in strategies implemented to control viral outbreaks. Among these, the surface electrostatic potential can give valuable information to deal with new epidemics. In this article, we describe the role of this key parameter in the hemagglutination of red blood cells and in the co-evolution of synaptic receptors and neurotransmitters. We then establish the functional link between lipid rafts and the electrostatic potential of viruses, with special emphasis on gangliosides, which are sialic-acid-containing, electronegatively charged plasma membrane components. We describe the common features of ganglioside binding domains, which include a wide variety of structures with little sequence homology but that possess key amino acids controlling ganglioside recognition. We analyze the role of the electrostatic potential in the transmission and intra-individual evolution of HIV-1 infections, including gatekeeper and co-receptor switch mechanisms. We show how to organize the epidemic surveillance of influenza viruses by focusing on mutations affecting the hemagglutinin surface potential. We demonstrate that the electrostatic surface potential, by modulating spike-ganglioside interactions, controls the hemagglutination properties of coronaviruses (SARS-CoV-1, MERS-CoV, and SARS-CoV-2) as well as the structural dynamics of SARS-CoV-2 evolution. We relate the broad-spectrum antiviral activity of repositioned molecules to their ability to disrupt virus-raft interactions, challenging the old concept that an antibiotic or anti-parasitic cannot also be an antiviral. We propose a new concept based on the analysis of the electrostatic surface potential to develop, in real time, therapeutic and vaccine strategies adapted to each new viral epidemic.

AmyP53 Prevents the Formation of Neurotoxic ß-Amyloid Oligomers through an Unprecedent Mechanism of Interaction with Gangliosides: Insights for Alzheimer's Disease Therapy.

A broad range of data identify Ca2+-permeable amyloid pores as the most neurotoxic species of Alzheimer's ß-amyloid peptide (Aß1-42). Following the failures of clinical trials targeting amyloid plaques by immunotherapy, a consensus is gradually emerging to change the paradigm, the strategy, and the target to cure Alzheimer's disease. In this context, the therapeutic peptide AmyP53 was designed to prevent amyloid pore formation driven by lipid raft microdomains of the plasma membrane. Here, we show that AmyP53 outcompetes Aß1-42 binding to lipid rafts through a unique mode of interaction with gangliosides. Using a combination of cellular, physicochemical, and in silico approaches, we unraveled the mechanism of action of AmyP53 at the atomic, molecular, and cellular levels. Molecular dynamics simulations (MDS) indicated that AmyP53 rapidly adapts its conformation to gangliosides for an optimal interaction at the periphery of a lipid raft, where amyloid pore formation occurs. Hence, we define it as an adaptive peptide. Our results describe for the first time the kinetics of AmyP53 interaction with lipid raft gangliosides at the atomic level. Physicochemical studies and in silico simulations indicated that Aß1-42 cannot interact with lipid rafts in presence of AmyP53. These data demonstrated that AmyP53 prevents amyloid pore formation and cellular Ca2+ entry by competitive inhibition of Aß1-42 binding to lipid raft gangliosides. The molecular details of AmyP53 action revealed an unprecedent mechanism of interaction with lipid rafts, offering innovative therapeutic opportunities for lipid raft and ganglioside-associated diseases, including Alzheimer's, Parkinson's, and related proteinopathies.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Mistigri rule.

The nature and dynamics of mutations associated with the emergence, spread, and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant ("Marseille-4") for 10 months since its onset in July 2020. Here, we analyzed and classified into subvariants and lineages 7453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1 ± 1.4 months, during which 4.1 ± 2.6 mutations accumulated. Growth rate was 0.079 ± 0.045, varying from 0.010 to 0.173. Most of the lineages exhibited a bell-shaped distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in SARS-CoV-2 of mink and in the Alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Structural Basis of Botulinum Toxin Type F Binding to Glycosylated Human SV2A: In Silico Studies at the Periphery of a Lipid Raft.

Botulinum neurotoxins are the deadliest microbial neurotoxins in humans, with a lethal dose of 1 ng/kg. Incidentally, these neurotoxins are also widely used for medical and cosmetic purposes. However, little is known about the molecular mechanisms that control binding of botulinum neurotoxin type F1 (BoNT/F1) to its membrane receptor, glycosylated human synaptic vesicle glycoprotein A (hSV2Ag). To elucidate these mechanisms, we performed a molecular dynamics simulation (MDS) study of initial binding kinetics of BoNT/F1 to SV2A. Since this toxin also interacts with gangliosides, the simulations were performed at the periphery of a lipid raft in the presence of both SV2A and gangliosides. Our study suggested that interaction of BoNT/F1 with SV2A is exclusively mediated by N-glycan moiety of SV2A, which interacts with aromatic residues Y898, Y910, F946, Y1059 and H1273 of this toxin. Thus, in contrast with botulinum neurotoxin A1 (BoNT/A1), BoNT/F1 does not interact with protein content of SV2A. We attributed this incapability to a barrage effect exerted by neurotoxin residues Y1132, Q1133 and K1134, which prevent formation of long-lasting intermolecular hydrogen bonds. We also provided structural elements that suggest that BoNT/F1 uses the strategy of BoNT/A1 combined with the strategy of botulinum neurotoxin type E to bind N-glycan of its glycoprotein receptor. Overall, our study opened a gate for design of a universal inhibitor aimed at disrupting N-glycan-toxin interactions and for bioengineering of a BoNT/F1 protein that may be able to bind protein content of synaptic vesicle glycoprotein for therapeutic purposes.

SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects.

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen.

A Vaccine Strategy Based on the Identification of an Annular Ganglioside Binding Motif in Monkeypox Virus Protein E8L.

The recent outbreak of Monkeypox virus requires the development of a vaccine specifically directed against this virus as quickly as possible. We propose here a new strategy based on a two-step analysis combining (i) the search for binding domains of viral proteins to gangliosides present in lipid rafts of host cells, and (ii) B epitope predictions. Based on previous studies of HIV and SARS-CoV-2 proteins, we show that the Monkeypox virus cell surface-binding protein E8L possesses a ganglioside-binding motif consisting of several subsites forming a ring structure. The binding of the E8L protein to a cluster of gangliosides GM1 mimicking a lipid raft domain is driven by both shape and electrostatic surface potential complementarities. An induced-fit mechanism unmasks selected amino acid side chains of the motif without significantly affecting the secondary structure of the protein. The ganglioside-binding motif overlaps three potential linear B epitopes that are well exposed on the unbound E8L surface that faces the host cell membrane. This situation is ideal for generating neutralizing antibodies. We thus suggest using these three sequences derived from the E8L protein as immunogens in a vaccine formulation (recombinant protein, synthetic peptides or genetically based) specific for Monkeypox virus. This lipid raft/ganglioside-based strategy could be used for developing therapeutic and vaccine responses to future virus outbreaks, in parallel to existing solutions.