M protein ectodomain-specific immunity restrains SARS-CoV-2 variants replication.

Introduction: The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear. Methods: We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay. Results: Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30. Discussion: The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections.

Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis.

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis.

Integration of single-cell RNA sequencing of endothelial cells and proteomics to unravel the role of ICAM1-PTGS2 communication in apical periodontitis: A laboratory investigation.

AIM: Endothelial cells (EDs) play a key role in angiogenesis and are associated with granulomatous lesions in patients with chronic apical periodontitis (CAP). This study aimed to investigate the diversity of EDs using single-cell ribonucleic acid sequencing (scRNA-seq) and to evaluate the regulation of intercellular adhesion molecule 1 (ICAM1) on the ferroptosis-related protein, prostaglandin-endoperoxide synthase 2 (PTGS2), in CAP. METHODOLOGY: EDs from the uploaded scRNA-seq data of five CAP samples (GSE181688 and GSE197680) were categorized using distinct marker genes. The interactions between vein EDs (veinEndo) and other cell types were analysed using CellPhoneDB. Differentially expressed proteins in the proteomics of human umbilical vein EDs (HUVECs) and THP-1-derived macrophages infected with Porphyromonas gingivalis were compared with the differentially expressed genes (DEGs) of VeinEndo in scRNA-seq of CAP versus healthy control periodontal tissues. The protein-protein interaction of ICAM1-PTGS2 in macrophages and HUVECs was validated by adding recombinant ICAM1, ICAM1 inhibitor and PTGS2 inhibitor using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence staining. RESULTS: EDs in patients with CAP were divided into eight subclusters: five vein ED, capillaries, arterials and EC (PLA). There were 29 mutually upregulated DEGs and two mutually downregulated DEGs in vein cells in the scRNA-seq data, as well as differentially expressed proteins in the proteomics of HUVECs. Real-time PCR and immunofluorescence staining showed that ICAM1 and PTGS2 were highly expressed in CAP, infected HUVECs, and macrophages. Recombinant protein ICAM1 may improve PTGS2 expression, reactive oxygen species (ROS), and Fe2+ levels and decrease glutathione peroxidase 4 (GPX4) and SLC7A11 protein levels. ICAM1 inhibitor may inverse the above changes. CONCLUSIONS: scRNA-seq revealed the diversity of EDs in CAP and identified the possible regulation of ICAM1 by the ferroptosis-related protein, PTGS2, in infected HUVECs and macrophages, thus providing a basis for therapeutic approaches that target the inflammatory microenvironment of CAP.

Integration of metalloproteome and immunoproteome reveals a tight link of iron-related proteins with COVID-19 pathogenesis and immunity.

Increasing clinical data show that the imbalance of host metallome is closely associated with different kinds of disease, however, the intrinsic mechanisms of action of metals in immunity and pathogenesis of disease remain largely undefined. There is lack of multiplexed profiling system to integrate the metalloproteome-immunoproteome information at systemic level for exploring the roles of metals in immunity and disease pathogenesis. In this study, we build up a metal-coding assisted multiplexed proteome assay platform for serum metalloproteomic and immunoproteomic profiling. By taking COVID-19 as a showcase, we unbiasedly uncovered the most evident modulation of iron-related proteins, i.e., Ft and Tf, in serum of severe COVID-19 patients, and the value of Ft/Tf could work as a robust biomarker for COVID-19 severity stratification, which overtakes the well-established clinical risk factors (cytokines). We further uncovered a tight association of transferrin with inflammation mediator IL-10 in COVID-19 patients, which was proved to be mainly governed by the monocyte/macrophage of liver, shedding light on new pathophysiological and immune regulatory mechanisms of COVID-19 disease. We finally validated the beneficial effects of iron chelators as anti-viral agents in SARS-CoV-2-infected K18-hACE2 mice through modulation of iron dyshomeostasis and alleviating inflammation response. Our findings highlight the critical role of liver-mediated iron dysregulation in COVID-19 disease severity, providing solid evidence on the involvement of iron-related proteins in COVID-19 pathophysiology and immunity.

Potent and broadly neutralizing antibodies against sarbecoviruses induced by sequential COVID-19 vaccination.

The current SARS-CoV-2 variants strikingly evade all authorized monoclonal antibodies and threaten the efficacy of serum-neutralizing activity elicited by vaccination or prior infection, urging the need to develop antivirals against SARS-CoV-2 and related sarbecoviruses. Here, we identified both potent and broadly neutralizing antibodies from a five-dose vaccinated donor who exhibited cross-reactive serum-neutralizing activity against diverse coronaviruses. Through single B-cell sorting and sequencing followed by a tailor-made computational pipeline, we successfully selected 86 antibodies with potential cross-neutralizing ability from 684 antibody sequences. Among them, PW5-570 potently neutralized all SARS-CoV-2 variants that arose prior to Omicron BA.5, and the other three could broadly neutralize all current SARS-CoV-2 variants of concern, SARS-CoV and their related sarbecoviruses (Pangolin-GD, RaTG13, WIV-1, and SHC014). Cryo-EM analysis demonstrates that these antibodies have diverse neutralization mechanisms, such as disassembling spike trimers, or binding to RBM or SD1 to affect ACE2 binding. In addition, prophylactic administration of these antibodies significantly protects nasal turbinate and lung infections against BA.1, XBB.1, and SARS-CoV viral challenge in golden Syrian hamsters, respectively. Importantly, post-exposure treatment with PW5-5 and PW5-535 also markedly protects against XBB.1 challenge in these models. This study reveals the potential utility of computational process to assist screening cross-reactive antibodies, as well as the potency of vaccine-induced broadly neutralizing antibodies against current SARS-CoV-2 variants and related sarbecoviruses, offering promising avenues for the development of broad therapeutic antibody drugs.

Single-cell RNA sequencing combined with proteomics of infected macrophages reveals prothymosin-α as a target for treatment of apical periodontitis.

INTRODUCTION: Chronic apical periodontitis (CAP) is a common infectious disease of the oral cavity. Immune responses and osteoclastogenesis of monocytes/macrophages play a crucial role in CAP progression, and this study want to clarify role of monocytes/macrophages in CAP, which will contribute to treatment of CAP. OBJECTIVES: We aim to explore the heterogeneity of monocyte populations in periapical lesion of CAP tissues and healthy control (HC) periodontal tissues by single-cell RNA sequencing (scRNA-seq), search novel targets for alleviating CAP, and further validate it by proteomics and in vitro and in vivo evaluations. METHODS: ScRNA-seq was used to analyze the heterogeneity of monocyte populations in CAP, and proteomics of THP-1-derived macrophages with porphyromonas gingivalis infection were intersected with the differentially expressed genes (DEGs) of macrophages between CAP and HC tissues. The upregulated PTMA (prothymosin-α) were validated by immunofluorescence staining and quantitative real time polymerase chain reaction. We evaluated the effect of thymosin α1 (an amino-terminal proteolytic cleavage product of PTMA protein) on inflammatory factors and osteoclast differentiation of macrophages infected by P. gingivalis. Furthermore, we constructed mouse and rat mandibular bone lesions caused by apical periodontitis, and estimated treatment of systemic and topical administration of PTMA for CAP. Statistical analyses were performed using GraphPad Prism software (v9.2) RESULTS: Monocytes were divided into seven sub-clusters comprising monocyte-macrophage-osteoclast (MMO) differentiation in CAP. 14 up-regulated and 21 down-regulated genes and proteins were intersected between the DEGs of scRNA-seq data and proteomics, including the high expression of PTMA. Thymosin α1 may decrease several inflammatory cytokine expressions and osteoclastogenesis of THP-1-derived macrophages. Both systemic administration in mice and topical administration in the pulp chamber of rats alleviated periapical lesions. CONCLUSIONS: PTMA upregulation in CAP moderates the inflammatory response and prevents the osteoclastogenesis of macrophages, which provides a basis for targeted therapeutic strategies for CAP.

High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2.

Mapping mutations and discovering cellular determinants that cause the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce infected cells to form syncytia would facilitate the development of strategies for blocking the formation of such cell-cell fusion. Here we describe high-throughput screening methods based on droplet microfluidics and the size-exclusion selection of syncytia, coupled with large-scale mutagenesis and genome-wide knockout screening via clustered regularly interspaced short palindromic repeats (CRISPR), for the large-scale identification of determinants of cell-cell fusion. We used the methods to perform deep mutational scans in spike-presenting cells to pinpoint mutable syncytium-enhancing substitutions in two regions of the spike protein (the fusion peptide proximal region and the furin-cleavage site). We also used a genome-wide CRISPR screen in cells expressing the receptor angiotensin-converting enzyme 2 to identify inhibitors of clathrin-mediated endocytosis that impede syncytium formation, which we validated in hamsters infected with SARS-CoV-2. Finding genetic and cellular determinants of the formation of syncytia may reveal insights into the physiological and pathological consequences of cell-cell fusion.

Metal-coding assisted serological multi-omics profiling deciphers the role of selenium in COVID-19 immunity.

Uncovering how host metal(loid)s mediate the immune response against invading pathogens is critical for better understanding the pathogenesis mechanism of infectious disease. Clinical data show that imbalance of host metal(loid)s is closely associated with the severity and mortality of COVID-19. However, it remains elusive how metal(loid)s, which are essential elements for all forms of life and closely associated with multiple diseases if dysregulated, are involved in COVID-19 pathophysiology and immunopathology. Herein, we built up a metal-coding assisted multiplexed serological metallome and immunoproteome profiling system to characterize the links of metallome with COVID-19 pathogenesis and immunity. We found distinct metallome features in COVID-19 patients compared with non-infected control subjects, which may serve as a biomarker for disease diagnosis. Moreover, we generated the first correlation network between the host metallome and immunity mediators, and unbiasedly uncovered a strong association of selenium with interleukin-10 (IL-10). Supplementation of selenium to immune cells resulted in enhanced IL-10 expression in B cells and reduced induction of proinflammatory cytokines in B and CD4+ T cells. The selenium-enhanced IL-10 production in B cells was confirmed to be attributable to the activation of ERK and Akt pathways. We further validated our cellular data in SARS-CoV-2-infected K18-hACE2 mice, and found that selenium supplementation alleviated SARS-CoV-2-induced lung damage characterized by decreased alveolar inflammatory infiltrates through restoration of virus-repressed selenoproteins to alleviate oxidative stress. Our approach can be readily extended to other diseases to understand how the host defends against invading pathogens through regulation of metallome.

Broad-spectrum humanized monoclonal neutralizing antibody against SARS-CoV-2 variants, including the Omicron variant.

Introduction: Therapeutic monoclonal antibodies (mAbs) against the SARS-CoV-2 spike protein have been shown to improve the outcome of severe COVID-19 patients in clinical trials. However, novel variants with spike protein mutations can render many currently available mAbs ineffective. Methods: We produced mAbs by using hybridoma cells that generated from mice immunized with spike protein trimer and receptor binding domain (RBD). The panel of mAbs were screened for binding and neutralizing activity against different SARS-CoV-2 variants. The in vivo effectiveness of WKS13 was evaluated in a hamster model. Results: Out of 960 clones, we identified 18 mAbs that could bind spike protein. Ten of the mAbs could attach to RBD, among which five had neutralizing activity against the ancestral strain and could block the binding between the spike protein and human ACE2. One of these mAbs, WKS13, had broad neutralizing activity against all Variants of Concern (VOCs), including the Omicron variant. Both murine or humanized versions of WKS13 could reduce the lung viral load in hamsters infected with the Delta variant. Conclusions: Our data showed that broad-spectrum high potency mAbs can be produced from immunized mice, which can be used in humans after humanization of the Fc region. Our method represents a versatile and rapid strategy for generating therapeutic mAbs for upcoming novel variants.

Neutralisation of SARS-CoV-2 by monoclonal antibody through dual targeting powder formulation.

Neutralising monoclonal antibody (mAb) is an important weapon in our arsenal for combating respiratory viral infections. However, the effectiveness of neutralising mAb has been impeded by the rapid emergence of mutant variants. Early administration of broad-spectrum mAb with improved delivery efficiency can potentially enhance efficacy and patient outcomes. WKS13 is a humanised mAb which was previously demonstrated to exhibit broad-spectrum activity against SARS-CoV-2 variants. In this study, a dual targeting formulation strategy was designed to deliver WKS13 to both the nasal cavity and lower airways, the two critical sites of infection caused by SARS-CoV-2. Dry powders of WKS13 were first prepared by spray drying, with cyclodextrin used as stabiliser excipient. Two-fluid nozzle (TFN) was used to produce particles below 5 µm for lung deposition (C-TFN formulation) and ultrasonic nozzle (USN) was used to produce particles above 10 µm for nasal deposition (C-USN formulation). Gel electrophoresis and size exclusion chromatography studies showed that the structural integrity of mAb was successfully preserved with no sign of aggregation after spray drying. To achieve dual targeting property, C-TFN and C-USN were mixed at various ratios. The aerosolisation property of the mixed formulations dispersed from a nasal powder device was examined using a Next Generation Impactor (NGI) coupled with a glass expansion chamber. When the ratio of C-TFN in the mixed formulation increased, the fraction of particles deposited in the lung increased proportionally while the fraction of particles deposited in the nasal cavity decreased correspondingly. A customisable aerosol deposition profile could therefore be achieved by manipulating the mixing ratio between C-TFN and C-USN. Dual administration of C-TFN and C-USN powders to the lung and nasal cavity of hamsters, respectively, was effective in offering prophylactic protection against SARS-CoV-2 Delta variant. Viral loads in both the lung tissues and nasal wash were significantly reduced, and the efficacy was comparable to systemic administration of unformulated WKS13. Overall, dual targeting powder formulation of neutralising mAb is a promising approach for prophylaxis of respiratory viral infections. The ease and non-invasive administration of dual targeting nasal powder may facilitate the widespread distribution of neutralising mAb during the early stage of unpredictable outbreaks.

Intranasal Boosting with Spike Fc-RBD of Wild-Type SARS-CoV-2 Induces Neutralizing Antibodies against Omicron Subvariants and Reduces Viral Load in the Nasal Turbinate of Mice.

The emergence of new immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and subvariants outpaces the development of vaccines specific against the dominant circulating strains. In terms of the only accepted immune correlate of protection, the inactivated whole-virion vaccine using wild-type SARS-CoV-2 spike induces a much lower serum neutralizing antibody titre against the Omicron subvariants. Since the inactivated vaccine given intramuscularly is one of the most commonly used coronavirus disease 2019 (COVID-19) vaccines in developing regions, we tested the hypothesis that intranasal boosting after intramuscular priming would provide a broader level of protection. Here, we showed that one or two intranasal boosts with the Fc-linked trimeric spike receptor-binding domain from wild-type SARS-CoV-2 can induce significantly higher serum neutralizing antibodies against wild-type SARS-CoV-2 and the Omicron subvariants, including BA.5.2 and XBB.1, with a lower titre in the bronchoalveolar lavage of vaccinated Balb/c mice than vaccination with four intramuscular doses of inactivated whole virion vaccine. The intranasally vaccinated K18-hACE2-transgenic mice also had a significantly lower nasal turbinate viral load, suggesting a better protection of the upper airway, which is the predilected site of infection by Omicron subvariants. This intramuscular priming and intranasal boosting approach that achieves broader cross-protection against Omicron variants and subvariants may lengthen the interval required for changing the vaccine immunogen from months to years.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo.

"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.

Vemurafenib Inhibits Enterovirus A71 Genome Replication and Virus Assembly.

Enterovirus A71 (EV-A71) infection is a major cause of hand, foot, and mouth disease (HFMD), which may be occasionally associated with severe neurological complications. There is currently a lack of treatment options for EV-A71 infection. The Raf-MEK-ERK signaling pathway, in addition to its critical importance in the regulation of cell growth, differentiation, and survival, has been shown to be essential for virus replication. In this study, we investigated the anti-EV-A71 activity of vemurafenib, a clinically approved B-Raf inhibitor used in the treatment of late-stage melanoma. Vemurafenib exhibits potent anti-EV-A71 effect in cytopathic effect inhibition and viral load reduction assays, with half maximal effective concentration (EC50) at nanomolar concentrations. Mechanistically, vemurafenib interrupts both EV-A71 genome replication and assembly. These findings expand the list of potential antiviral candidates of anti-EV-A71 therapeutics.

Virological features and pathogenicity of SARS-CoV-2 Omicron BA.2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 was a dominant circulating SARS-CoV-2 variant worldwide. Recent reports hint that BA.2 is similarly potent regarding antibody evasion but may be more transmissible than BA.1. The pathogenicity of BA.2 remains unclear and is of critical public health significance. Here we investigated the virological features and pathogenicity of BA.2 with in vitro and in vivo models. We show that BA.2 is less dependent on transmembrane protease serine 2 (TMPRSS2) for virus entry in comparison with BA.1 in vitro. In K18-hACE2 mice, BA.2 replicates more efficiently than BA.1 in the nasal turbinates and replicates marginally less efficiently in the lungs, leading to decreased body weight loss and improved survival. Our study indicates that BA.2 is similarly attenuated in lungs compared with BA.1 but is potentially more transmissible because of its better replication at the nasal turbinates.

The spike receptor-binding motif G496S substitution determines the replication fitness of SARS-CoV-2 Omicron sublineage.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Cross-variant protection against SARS-CoV-2 infection in hamsters immunized with monovalent and bivalent inactivated vaccines.

Rapid development and successful use of vaccines against SARS-CoV-2 might hold the key to curb the ongoing pandemic of COVID-19. Emergence of vaccine-evasive SARS-CoV-2 variants of concern (VOCs) has posed a new challenge to vaccine design and development. One urgent need is to determine what types of variant-specific and bivalent vaccines should be developed. Here, we compared homotypic and heterotypic protection against SARS-CoV-2 infection of hamsters with monovalent and bivalent whole-virion inactivated vaccines derived from representative VOCs. In addition to the ancestral SARS-CoV-2 Wuhan strain, Delta (B.1.617.2; δ) and Theta (P.3; θ) variants were used in vaccine preparation. Additional VOCs including Omicron (B.1.1.529) and Alpha (B.1.1.7) variants were employed in the challenge experiment. Consistent with previous findings, Omicron variant exhibited the highest degree of immune evasion, rendering all different forms of inactivated vaccines substantially less efficacious. Notably, monovalent and bivalent Delta variant-specific inactivated vaccines provided optimal protection against challenge with Delta variant. Yet, some cross-variant protection against Omicron and Alpha variants was seen with all monovalent and bivalent inactivated vaccines tested. Taken together, our findings support the notion that an optimal next-generation inactivated vaccine against SARS-CoV-2 should contain the predominant VOC in circulation. Further investigations are underway to test whether a bivalent vaccine for Delta and Omicron variants can serve this purpose.

Phosphatidic acid phosphatase 1 impairs SARS-CoV-2 replication by affecting the glycerophospholipid metabolism pathway.

Viruses exploit the host lipid metabolism machinery to achieve efficient replication. We herein characterize the lipids profile reprogramming in vitro and in vivo using liquid chromatography-mass spectrometry-based untargeted lipidomics. The lipidome of SARS-CoV-2-infected Caco-2 cells was markedly different from that of mock-infected samples, with most of the changes involving downregulation of ceramides. In COVID-19 patients' plasma samples, a total of 54 lipids belonging to 12 lipid classes that were significantly perturbed compared to non-infected control subjects' plasma samples were identified. Among these 12 lipid classes, ether-linked phosphatidylcholines, ether-linked phosphatidylethanolamines, phosphatidylcholines, and ceramides were the four most perturbed. Pathway analysis revealed that the glycerophospholipid, sphingolipid, and ether lipid metabolisms pathway were the most significantly perturbed host pathways. Phosphatidic acid phosphatases (PAP) were involved in all three pathways and PAP-1 deficiency significantly suppressed SARS-CoV-2 replication. siRNA knockdown of LPIN2 and LPIN3 resulted in significant reduction of SARS-CoV-2 load. In summary, these findings characterized the host lipidomic changes upon SARS-CoV-2 infection and identified PAP-1 as a potential target for intervention for COVID-19.