A Vaccine of SARS-CoV-2 S Protein RBD Induces Protective Immunity.

The pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed great threat to the world in many aspects. There is an urgent requirement for an effective preventive vaccine. The receptor binding domain (RBD), located on the spike (S) gene, is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. The RBD protein is an effective and safe antigen candidate. The six-helix bundle (6HB) "molecular clamp" is a novel thermally-stable trimerization domain derived from a human immunodeficiency virus (HIV) gp41 protein segment. We selected the baculovirus system to fuse and express the RBD protein and 6HB for imitating the natural trimeric structure of RBD, named RBD-6HB. Recombinant RBD-6HB was successfully obtained from the cell culture supernatant and purified to high homogeneity. The purity of the final protein preparation was more than 97%. The results showed that the protein was identified as a homogeneous polymer. Further studies showed that the RBD-6HB protein combined with AL/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges. Our findings highlight the importance of trimerized SARS-CoV-2 S protein RBD in designing SARS-CoV-2 vaccines and provide a rationale for developing a protective vaccine through the induction of antibodies against the RBD domain.

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

A micro-sized vaccine based on recombinant Lactiplantibacillus plantarum fights against SARS-CoV-2 infection via intranasal immunization.

COVID-19 has globally spread to burden the medical system. Even with a massive vaccination, a mucosal vaccine offering more comprehensive and convenient protection is imminent. Here, a micro-sized vaccine based on recombinant Lactiplantibacillus plantarum (rLP) displaying spike or receptor-binding domain (RBD) was characterized as microparticles, and its safety and protective effects against SARS-CoV-2 were evaluated. We found a 66.7% mortality reduction and 100% protection with rLP against SARS-CoV-2 in a mouse model. The histological analysis showed decreased hemorrhage symptoms and increased leukocyte infiltration in the lung. Especially, rLP:RBD significantly decreased pulmonary viral loads. For the first time, our study provides a Lactiplantibacillus plantarum-vectored vaccine to prevent COVID-19 progress and transmission via intranasal vaccination.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Antiviral Effect of pIFNLs against PEDV and VSV Infection in Different Cells.

Type III and type I interferon have similar mechanisms of action, and their different receptors lead to different distributions in tissue. On mucosal surfaces, type III interferon exhibits strong antiviral activity. Porcine epidemic diarrhea virus (PEDV) is an economically important enteropathogenic coronavirus, which can cause a high incidence rate and mortality in piglets. Here, we demonstrate that porcine interferon lambda 1 (pIFNL1) and porcine interferon lambda 3 (pIFNL3) can inhibit the proliferation of vesicular stomatitis virus with an enhanced green fluorescent protein (VSV-EGFP) in different cells, and also show strong antiviral activity when PEDV infects Vero cells. Both forms of pIFNLs were shown to be better than porcine interferon alpha (pIFNα), the antiviral activity of pIFNL1 is lower than that of pIFNL3. Therefore, our results provide experimental evidence for the inhibition of PEDV infection by pIFNLs, which may provide a promising treatment for the prevention and treatment of Porcine epidemic diarrhea (PED) in piglets.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

The global succinylation of SARS-CoV-2-infected host cells reveals drug targets.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.

Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus.

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

RBD trimer mRNA vaccine elicits broad and protective immune responses against SARS-CoV-2 variants.

With the rapid emergence and spread of SARS-CoV-2 variants, development of vaccines with broad and potent protectivity has become a global priority. Here, we designed a lipid nanoparticle-encapsulated, nucleoside-unmodified mRNA (mRNA-LNP) vaccine encoding the trimerized receptor-binding domain (RBD trimer) and showed its robust capability in inducing broad and protective immune responses against wild-type and major variants of concern (VOCs) in the mouse model of SARS-CoV-2 infection. The protectivity was correlated with RBD-specific B cell responses especially the long-lived plasma B cells in bone marrow, strong ability in triggering BCR clustering, and downstream signaling. Monoclonal antibodies isolated from vaccinated animals demonstrated broad and potent neutralizing activity against VOCs tested. Structure analysis of one representative antibody identified a novel epitope with a high degree of conservation among different variants. Collectively, these results demonstrate that the RBD trimer mRNA vaccine serves as a promising vaccine candidate against SARS-CoV-2 variants and beyond.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates.

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.