Impact of mAb-induced A475V substitution on viral fitness and antibody neutralization of SARS-CoV-2 omicron variants in the presence of monoclonal antibodies and human convalescent sera

The emergence and rapid evolution of SARS-CoV-2 variants have posed a major challenge to the global efforts to control the COVID -19 pandemic. In this study, we investigated the potential of two SARS-CoV-2 variants, BA.2 and BA.5, to evade neutralization by a human monoclonal antibody targeting the virus’s spike RBD (mAb 1D1). By subjecting the viruses to serial propagation in the presence of the antibody, we found that BA.2 exhibited poor growth, whereas BA.5 regained robust growth with significantly higher kinetics than the parental virus. Genetic analysis identified a single mutation, A475V, in the spike protein of BA.5 that substantially reduced the neutralizing activities of monoclonal antibodies and convalescent sera. In addition, the A475V mutation alone in BA.2 moderately reduced the neutralizing activity but completely abolished the neutralizing effect of mAb 1D1 when F486V or L452R were also present. Our results shed light on the possible evolutionary development of SARS-CoV-2 variants under selection pressure by monoclonal antibodies and have implications for the development of effective antibody therapies and vaccines against the virus

Characterization of influenza A virus pseudotyped with the spikeprotein of porcine epidemic diarrhea virus

AbstractThe coronavirus spike protein and the influenza virus hemagglutinin are class I viral membrane fusion proteins. Whilethe two proteins display strong structural conservation and the mechanisms underlying membrane fusion are similar, theyshare no sequence similarity. Whether they are functionally interchangeable is currently unknown. In this study, we constructedscIAV-S, a single-cycle influenza A virus pseudotyped with the spike protein of porcine epidemic diarrhea virus(PEDV), and demonstrated that this virus could infect cultured cells and trigger massive syncytium formation. Treatmentwith endocytosis inhibitors did not affect syncytium formation by infected cells. Moreover, the infectivity of scIAV-S wasassociated with the degree of cell adaptation of PEDV-S. Intriguingly, scIAV-S lacking functional neuraminidase (NA)exhibited substantially higher infectivity, suggesting a pivotal role of the sialic acid in the binding/entry of PEDV. Together,scIAV-S offers a robust platform for the investigation of the entry mechanism of PEDV or, possibly, of other coronaviruses

Cross-Neutralization of SARS-CoV-2-Specific Antibodies in Convalescent and Immunized Human Sera against the Bat and Pangolin Coronaviruses

Coronaviruses isolated from bats and pangolins are closely related to SARS-CoV-2, the causative agent of COVID-19. These so-called sarbecoviruses are thought to pose an acute pandemic threat. As SARS-CoV-2 infection and vaccination have become more widespread, it is not known whether neutralizing antibodies to SARS-CoV-2 can cross-neutralize coronaviruses transmitted by bats or pangolins. In this study, we analyzed antibody-mediated neutralization with serum samples from COVID-19 patients (n = 31) and those immunized with inactivated SARS-CoV-2 vaccines (n = 20) against lentivirus-based pseudo-viruses carrying the spike derived from ancestral SARS-CoV-2, bat (RaTG13 or RshSTT182), or pangolin coronaviruses (PCoV-GD). While SARS-CoV-2, PCoV-GD, and RshSTT182 spikes could promote cell-cell fusion in VeroE6 cells, the RaTG13 spike did not. RaTG13, on the other hand, was able to induce cell-cell fusion in cells overexpressing ACE2. Dramatic differences in neutralization activity were observed, with the highest level observed for RaTG13, which was even significantly higher than SARS-CoV-2, PCoV-GD, and RshSTT182 pseudo-viruses. Interestingly, pseudo-viruses containing the chimeric protein in which the receptor-binding domain (RBD) of PCoV-GD spike was replaced by that of RaTG13 could be strongly neutralized, whereas those carrying RaTG13 with the RBD of PCoV-GD were significantly less neutralized. Because the high neutralizing activity against RaTG13 appears to correlate with its low affinity for binding to the human ACE2 receptor, our data presented here might shed light on how pre-existing immunity to SARS-CoV-2 might contribute to protection against related sarbecoviruses with potential spillover to the human host

A Single V672F Substitution in the Spike Protein of Field-Isolated PEDV Promotes Cell–Cell Fusion and Replication in VeroE6 Cells

While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells

SARS-CoV-2 Delta (B.1.617.2) variant replicates and induces syncytia formation in human induced pluripotent stem cell-derived macrophages

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity

In Vitro and In Vivo Attenuation of Vesicular Stomatitis Virus (VSV) by Phosphoprotein Deletion.

Vesicular stomatitis virus (VSV) is highly immunogenic and able to stimulate both innate and adaptive immune responses. However, its ability to induce adverse effects has held back the use of VSV as a potential vaccine vector. In this study we developed VSV-ΔP, a safe yet potent replication-defective recombinant VSV in which the phosphoprotein (P) gene was deleted. VSV-ΔP replicated only in supporting cells expressing P (BHK-P cells) and at levels more than 2 logs lower than VSV. In vivo studies indicated that the moderate replication of VSV-ΔP in vitro was associated with the attenuation of this virus in the mouse model, whereas mice intracranially injected with VSV succumbed to neurotoxicity. Furthermore, we constructed VSV and VSV-ΔP expressing a variety of antigens including hemagglutinin-neuraminidase (HN) from Newcastle disease virus (NDV), hemagglutinin (HA) from either a 2009 H1N1 pandemic influenza virus (pdm/09) or the avian H7N9. VSV and VSV-ΔP incorporated the foreign antigens on their surface resulting in induction of robust neutralizing antibody, serum IgG, and hemagglutination inhibition (HAI) titers against their corresponding viruses. These results indicated that VSV with P gene deletion was attenuated in vitro and in vivo, and possibly expressed the foreign antigen on its surface. Therefore, the P gene-deletion strategy may offer a potentially useful and safer approach for attenuating negative-sense RNA viruses which use phosphoprotein as a cofactor for viral replication

A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate