Significance of Conserved Regions in Coronavirus Spike Protein for Developing a Novel Vaccine against SARS-CoV-2 Infection.

Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2, which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants' proteins, especially in the spike protein (SP) used for viral entry. These mutations, especially in the SP, enable the virus to evade immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of a vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase its immunogenicity.

Protocol to evaluate the inflammatory response in human macrophages induced by SARS-CoV-2 spike-pseudotyped VLPs.

The excessive release of pro-inflammatory cytokines in COVID-19 patients is deleterious to organs. The contribution of SARS-CoV-2 spike protein (S) to the inflammatory response is essential to understand its pathogenesis and virulence. Here, we present a protocol to produce and characterize HIV- and SARS-CoV-2-based virus-like particles and then evaluate the inflammatory cytokines' protein and mRNA levels produced in human macrophages by S of SARS-CoV-2 original strain and Delta variant. This protocol is applicable in evaluating S from different emerging variants. For complete details on the use and execution of this protocol, please refer to Ao et al. (2022).1.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

SARS-CoV-2 Delta spike protein enhances the viral fusogenicity and inflammatory cytokine production.

The Delta variant had spread globally in 2021 and caused more serious disease than the original virus and Omicron variant. In this study, we investigated several virological features of Delta spike protein (SPDelta), including protein maturation, its impact on viral entry of pseudovirus and cell-cell fusion, and its induction of inflammatory cytokine production in human macrophages and dendritic cells. The results showed that SPΔCDelta exhibited enhanced S1/S2 cleavage in cells and pseudotyped virus-like particles (PVLPs). Further, SPΔCDelta elevated pseudovirus entry in human lung cell lines and significantly enhanced syncytia formation. Furthermore, we revealed that SPΔCDelta-PVLPs had stronger effects on stimulating NF-κB and AP-1 signaling in human monocytic THP1 cells and induced significantly higher levels of proinflammatory cytokine, such as TNF-α, IL-1ß, and IL-6, released from human macrophages and dendritic cells. Overall, these studies provide evidence to support the important role of SPΔCDelta during virus infection, transmission, and pathogenesis.

Identification and evaluation of the inhibitory effect of Prunella vulgaris extract on SARS-coronavirus 2 virus entry.

Until now, antiviral therapeutic agents are still urgently required for treatment or prevention of SARS-coronavirus 2 (SCoV-2) virus infection. In this study, we established a sensitive SCoV-2 Spike glycoprotein (SP), including an SP mutant D614G, pseudotyped HIV-1-based vector system and tested their ability to infect ACE2-expressing cells. Based on this system, we have demonstrated that an aqueous extract from the Natural herb Prunella vulgaris (NhPV) displayed potent inhibitory effects on SCoV-2 SP (including SPG614 mutant) pseudotyped virus (SCoV-2-SP-PVs) mediated infections. Moreover, we have compared NhPV with another compound, Suramin, for their anti-SARS-CoV-2 activities and the mode of their actions, and found that both NhPV and Suramin are able to directly interrupt SCoV-2-SP binding to its receptor ACE2 and block the viral entry step. Importantly, the inhibitory effects of NhPV and Suramin were confirmed by the wild type SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) virus infection in Vero cells. Furthermore, our results also demonstrated that the combination of NhPV/Suramin with an anti-SARS-CoV-2 neutralizing antibody mediated a more potent blocking effect against SCoV2-SP-PVs. Overall, by using SARS-CoV-2 SP-pseudotyped HIV-1-based entry system, we provide strong evidence that NhPV and Suramin have anti-SARS-CoV-2 activity and may be developed as a novel antiviral approach against SARS-CoV-2 infection.