Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.</p

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10–15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection

An overview of vaccines against sars-cov-2 in the covid-19 pandemic era

The emergence of SARS-CoV-2 in late 2019 led to the COVID-19 pandemic all over the world. When the virus was first isolated and its genome was sequenced in the early months of 2020, the efforts to develop a vaccine began. Based on prior well-known knowledge about coronavirus, the SARS-CoV-2 spike (S) protein was selected as the main target. Currently, more than one hundred vaccines are being investigated and several of them are already authorized by medical agencies. This review summarizes and compares the current knowledge about main approaches for vaccine development, focusing on those authorized and specifically their immunogenicity, efficacy preventing severe disease, adverse side effects, protection, and ability to cope with emergent SARS-CoV-2 variants

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

<div><p>A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated <i>in vivo</i>. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.</p></div

Effect of SARS-CoV E protein PBM on host gene expression.

<p>(A) Comparison of gene expression in lungs of infected mice using microarrays: wt versus mock-infected, mutPBM versus mock-infected and mutPBM versus wt-infected mice. Red spots indicate upregulated gene transcripts (fold change, >2) and green spots indicate downregulated gene transcripts (fold change, <−2). Only genes with a FDR of <0.01 were considered as candidate genes. (B) Candidate genes that were downregulated in mutPBM infected mice compared wt infected ones, were grouped on Gene Ontology terms. Numbers on the <i>x</i> axis indicate DAVID FDR values. (C) Selection of differentially expressed genes found in at least one functional group using DAVID software. The numbers indicate the fold change for each gene in mutPBM versus wt-infected mice. (D) Expression of inflammatory cytokines evaluated by RT-qPCR. Three independent experiments were analyzed with similar results in all cases. Error bars represent standard deviations of the mean of results from three experiments.</p

Primers used for the generation of recombinant SARS-CoV-E-PBM mutants.

<p>Primers used for the generation of recombinant SARS-CoV-E-PBM mutants.</p

Effect of p38 MAPK inhibitor in rSARS-CoV-MA15-infected mice.

<p>16-week-old BALB/c mice were mock-infected or inoculated intranasally with 100,000 pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock pfu of wt virus. At 4 hpi and every 12 h from days 1 to 8, mock hpi and every 12 h from days 1 to 8, mock h from days 1 to 8, mock-infected and wt-infected mice were intraperitoneally injected with SB203580 (6 mg mg/kg of body weight/day). (A) Animals were monitored daily for mortality. Data represent three independent experiments with 5 mice per group. (B) The active phosphorylated (p-HSP27) and total HSP27 in lungs of three infected mice per condition were evaluated by Western blot analysis. (C) Phospho and total HSP27 amounts were quantified by densitometric analysis. The graph shows the phosphorylated HSP27/total HSP27 ratio at 2 dpi in lungs of mock dpi in lungs of mock-infected mice or mice infected with the parental virus, treated or not with SB203580. Error bars represent the means of three mice analyzed for each condition. Statistically significant data are indicated with one (<i>P</i><0.05) asterisk.</p

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine

<div><p>A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or <i>in vivo</i>, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness <i>in vitro</i> but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage <i>in vitro</i> and <i>in vivo</i>. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.</p></div