Abstract: bioRxiv preprint doi: https://doi.org/10.1101/2022.11.24.517882; this version posted November 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects Celine Boschi1, David E. Scheim2*, Audrey Bancod1, Muriel Millitello1, Marion Le Bideau1, Philippe Colson1, Jacques Fantini3, Bernard La Scola1* 1 MEPHI, Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), Assistance Publique - Hôpitaux de Marseille (AP-HM), IHU Méditerranée Infection, 13005 Marseille, France 2 US Public Health Service, Commissioned Officer, Inactive Reserve, Blacksburg, VA 24060, USA 3 INSERM UMR S 1072, Aix-Marseille Université, 13015 Marseille, France *Correspondence: dscheim@alum.mit.edu, Tel.: +1 540 3208013; bernard.la-scola@univamu.fr, Tel.: +33 413732401 Keywords: SARS-CoV-2; COVID-19; spike protein; hemagglutination; sialic acid; CD147; electrostatic charge; glycophorin A bioRxiv preprint doi: https://doi.org/10.1101/2022.11.24.517882; this version posted November 28, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 21 ABSTRACT 22 Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses 23 indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells 24 (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID- 25 19. To provide further insight into these glycan attachments and their potential clinical relevance, 26 the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, 27 Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The 28 electrostatic potential of the central region of spike protein from these four lineages was studied 29 through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using 30 the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 31 spike protein glycan sites. The results of these experiments were, first, that spike protein from 32 these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower 33 threshold concentration of spike protein than for the three prior lineages and was much more 34 electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to 35 spike protein and reversed HA when added afterwards. These results validate and extend prior 36 findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore 37 suggest therapeutic options using competitive glycan-binding agents such as IVM and may help 38 elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines which 39 use spike protein as the generated antigen. bioRxiv preprint doi: https://doi.org/10.1101/2022.11.24.517882; this version posted November 28, 2022. The copyright holder for this..