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1.
Int J Mol Sci ; 23(11)2022 May 27.
Article in English | MEDLINE | ID: covidwho-1869636

ABSTRACT

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype's differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Viral , Humans , Immunoglobulin G , Nucleocapsid , SARS-CoV-2
2.
Sci Rep ; 12(1): 8811, 2022 May 25.
Article in English | MEDLINE | ID: covidwho-1864764

ABSTRACT

In response to the COVID-19 pandemic, lateral flow assays (LFAs) for the detection of SARS-CoV-2 antigen have been proposed as a complementary option to the more costly and time consuming reverse-transcriptase polymerase chain reaction (RT-PCR). We assessed five commercially available SARS-CoV-2 antigen detecting LFAs (ASSUT EUROPE (Rome, Italy), Besthree (Taizhou, China), Encode (Zhuhai, China), Fortress (Antrim UK), and Hughes Medical (Buckinghamshire, UK), using samples collected from hospitalised individuals with COVID-19 and compared these results against established RT-PCR assays with the aim of estimating test performance characteristics. We performed a diagnostic accuracy study of the five LFAs on 110 inpatients with confirmed COVID-19 and 75 COVID-19 negative control participants. Assay evaluation was performed using a modified version of each manufacturer's protocol allowing for parallel testing of a single sample on multiple assays. Additional variables were studied including infection acquisition, oxygenation requirements at time of swabbing, and patient outcomes. The 110 patients were 48% (53) female, with mean age 67 years (range 26-100 years), and 77% (85) cases were community onset SARS-CoV-2. Across the five assays, sensitivity ranged from 64 (95% CI 53-73) to 76% (95% CI 65-85); Fortress performed best with sensitivity of 76% (95% CI 65-85). Specificity was high across all assays with 4/5 LFAs achieving 100%. LFA sensitivity was not dependant on RT-PCR cycle thresholds. SARS-CoV-2 antigen detecting LFAs may complement RT-PCR testing to facilitate early diagnosis and provide community testing strategies for identification of patients with COVID-19, however we find suboptimal test performance characteristics across a range of commercially available manufacturers, below WHO and MHRA pre-set sensitivity performance thresholds. With such variation in sensitivity between LFAs and PCR testing and between assay brands, we advise caution in the deployment of LFAs outside of environments with clinical oversight.


Subject(s)
COVID-19 , SARS-CoV-2 , Adult , Aged , Aged, 80 and over , COVID-19/diagnosis , Female , Humans , Immunologic Tests , Middle Aged , Nucleocapsid , Pandemics , SARS-CoV-2/genetics , Sensitivity and Specificity
3.
Microbiol Spectr ; 10(2): e0250721, 2022 Apr 27.
Article in English | MEDLINE | ID: covidwho-1779319

ABSTRACT

The multiplex capabilities of the new xMAP INTELLIFLEX DR-SE flow analyzer were explored by modifying a serological assay previously used to characterize the IgG antibody to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The goal was to examine the instrument's performance and to simultaneously measure IgM and IgG antibody responses against multiple SARS-CoV-2 antigens in a single assay. Specific antibodies against the SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N) proteins were investigated in 310 symptomatic case patients using a fluorescent microsphere immunoassay and simultaneous detection of IgM and IgG. Neutralization potential was studied using the addition of soluble angiotensin-converting enzyme 2 (ACE2) to block antibody binding. A profile extending to 180 days from symptom onset (DFSO) was described for antibodies specific to each viral antigen. Generally, IgM levels peaked and declined rapidly ∼3-4 weeks following infection, whereas S- and RBD-specific IgG plateaued at 80 DFSO. ACE2 more effectively prevented IgM and IgG binding in convalescent cases > 30 DFSO, suggesting those antibodies had greater neutralization potential. This work highlighted the multiplex and multi-analyte potential of the xMAP INTELLIFLEX DR-SE, and provided further evidence for antigen-specific IgM and IgG trajectories in acute and convalescent cases. IMPORTANCE The xMAP INTELLIFLEX DR-SE enabled simultaneous and semi-quantitative detection of both IgM and IgG to three different SARS-CoV-2 antigens in a single assay. The assay format is advantageous for rapid and medium-throughput profiling using a small volume of specimen. The xMAP INTELLIFLEX DR-SE technology demonstrated the potential to include numerous SARS-CoV-2 antigens; future work could incorporate multiple spike protein variants in a single assay. This could be an important feature for assessing the serological response to emerging variants of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/diagnosis , Humans , Immunoglobulin G , Immunoglobulin M , Nucleocapsid , Spike Glycoprotein, Coronavirus
4.
Biosens Bioelectron ; 209: 114222, 2022 Aug 01.
Article in English | MEDLINE | ID: covidwho-1778011

ABSTRACT

The 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel immunosensors targeting conserved protein sequences of the N protein of SARS-CoV-2 based on lab-produced and purified anti-SARS-CoV-2 nucleocapsid antibodies that are densely grafted onto various surfaces (diamond/gold/glassy carbon). Titration of antibodies shows very strong reactions up to 1:72 900 dilution. Next, we showed the mechanism of interactions of our immunoassay with nucleocapsid N protein revealing molecular recognition by impedimetric measurements supported by hybrid modeling results with both density functional theory and molecular dynamics methods. Biosensors allowed for a fast (in less than 10 min) detection of SARS-CoV-2 virus with a limit of detection from 0.227 ng/ml through 0.334 ng/ml to 0.362 ng/ml for glassy carbon, boron-doped diamond, and gold surfaces, respectively. For all tested surfaces, we obtained a wide linear range of concentrations from 4.4 ng/ml to 4.4 pg/ml. Furthermore, our sensor leads to a highly specific response to SARS-CoV-2 clinical samples versus other upper respiratory tract viruses such as influenza, respiratory syncytial virus, or Epstein-Barr virus. All clinical samples were tested simultaneously on biosensors and real-time polymerase chain reactions.


Subject(s)
Biosensing Techniques , COVID-19 , Epstein-Barr Virus Infections , Antibodies, Viral , Biosensing Techniques/methods , Boron , COVID-19/diagnosis , Carbon , Diamond , Gold , Herpesvirus 4, Human , Humans , Immunoassay/methods , Nucleocapsid , Nucleocapsid Proteins , SARS-CoV-2
5.
Tohoku J Exp Med ; 257(1): 57-64, 2022 May 27.
Article in English | MEDLINE | ID: covidwho-1770831

ABSTRACT

This study sought to evaluate the effects of two vaccine doses and the extent of SARS-CoV-2 infection among healthcare workers. We measured immunoglobulin G antibody titers against SARS-CoV-2 nucleocapsid and spike protein among healthcare workers at Gunma University Hospital. In March 2021, prior to BNT-162b2 vaccination, two of 771 participants were seropositive for nucleocapsid and spike protein, whereas 768 were seronegative. The remaining one participant was seropositive for nucleocapsid protein but seronegative for spike protein. A total of 769 participants were seropositive for spike protein after two vaccination doses. The two seropositive participants prior to vaccination showed the highest antibody titers after the second vaccination. They were probably infected with SARS-CoV-2 without clinical symptoms before March 2021. Four weeks after the second vaccination, a younger age was associated with higher antibody titers against SARS-CoV-2 spike protein. Thirty-two weeks after the second vaccination, blood samples were collected from 342 of 769 participants. Antibody titers at 32 weeks after the second vaccination significantly decreased compared with those at 4 weeks after the second vaccination among all age groups. The rate of decrease in antibody titers between 4 and 32 weeks after the second vaccination was greater in the female participants. No sex differences were observed in the antibody titers within each age group. BNT-162b2 vaccination thus induced seroconversion in an age-dependent manner. Serological screening could further establish the likelihood of subclinical SARS-CoV-2 infection.


Subject(s)
COVID-19 , Vaccines , Antibodies, Viral , COVID-19/prevention & control , Female , Health Personnel , Humans , Immunoglobulin G , Japan/epidemiology , Nucleocapsid , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
6.
Microbiol Spectr ; 10(1): e0127121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1752773

ABSTRACT

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.


Subject(s)
COVID-19/metabolism , Lipid Metabolism/physiology , Lipids/analysis , Lung/metabolism , Nucleocapsid/analysis , SARS-CoV-2 , Adolescent , Aged , Animals , COVID-19/pathology , Child, Preschool , Chlorocebus aethiops , Disease Outbreaks , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Lung/cytology , Lung/pathology , Lung/ultrastructure , Male , Microscopy, Immunoelectron , Middle Aged , Nucleocapsid/metabolism , Rabbits , SARS-CoV-2/ultrastructure , Vero Cells/virology
7.
Neurol Neuroimmunol Neuroinflamm ; 9(2)2022 03.
Article in English | MEDLINE | ID: covidwho-1745397

ABSTRACT

BACKGROUND AND OBJECTIVES: Information about humoral and cellular responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and antibody persistence in convalescent (COVID-19) patients with multiple sclerosis (PwMS) is scarce. The objectives of this study were to investigate factors influencing humoral and cellular responses to SARS-CoV-2 and its persistence in convalescent COVID-19 PwMS. METHODS: This is a retrospective study of confirmed COVID-19 convalescent PwMS identified between February 2020 and May 2021 by SARS-CoV-2 antibody testing. We examined relationships between demographics, MS characteristics, disease-modifying therapy (DMT), and humoral (immunoglobulin G against spike and nucleocapsid proteins) and cellular (interferon-gamma [IFN-γ]) responses to SARS-CoV-2. RESULTS: A total of 121 (83.45%) of 145 PwMS were seropositive, and 25/42 (59.5%) presented a cellular response up to 13.1 months after COVID-19. Anti-CD20-treated patients had lower antibody titers than those under other DMTs (p < 0.001), but severe COVID-19 and a longer time from last infusion increased the likelihood of producing a humoral response. IFN-γ levels did not differ among DMT. Five of 7 (71.4%) anti--CD20-treated seronegative patients had a cellular response. The humoral response persisted for more than 6 months in 41/56(81.13%) PwMS. In multivariate analysis, seropositivity decreased due to anti-CD20 therapy (OR 0.08 [95% CI 0.01-0.55]) and increased in males (OR 3.59 [1.02-12.68]), whereas the cellular response decreased in those with progressive disease (OR 0.04 [0.001-0.88]). No factors were associated with antibody persistence. DISCUSSION: Humoral and cellular responses to SARS-CoV-2 are present in COVID-19 convalescent PwMS up to 13.10 months after COVID-19. The humoral response decreases under anti-CD20 treatment, although the cellular response can be detected in anti-CD20-treated patients, even in the absence of antibodies.


Subject(s)
COVID-19/immunology , Immunity, Cellular , Immunity, Humoral , Multiple Sclerosis/immunology , Adult , Aged , Antibodies, Viral/analysis , Antigens, CD20/immunology , COVID-19/complications , Female , Humans , Immunoglobulin G/analysis , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Male , Middle Aged , Multiple Sclerosis/complications , Nucleocapsid/chemistry , Nucleocapsid/immunology , Retrospective Studies
8.
Clin Immunol ; 237: 108979, 2022 04.
Article in English | MEDLINE | ID: covidwho-1739616

ABSTRACT

We explored the performance of a whole blood interferon gamma release assay (IGRA) based on the stimulation of SARS-Cov2-specific T cells by purified recombinant proteins. Twenty volunteers vaccinated with BNT162b2 were selected first for T cell response evaluation using an in-house IGRA, a commercial IGRA, and ELISpot showing a S2 > S1 poly-epitopic response. Next, 64 vaccinated and 103 non-vaccinated individuals were tested for humoral and T cell response (IGRA-Spike/-nucleocapsid recombinant proteins). Following the second vaccine injection, humoral (100%) and IGRA-Spike T cell (95.3%) responses took place irrespective of sex, age, and vaccine type. The humoral response declined first, followed by IGRA-Spike T cell response after the second vaccine injection. Altogether, this study confirms the utility of the IGRA-Spike/-nucleocapsid assay to complement serology in COVID19 vaccinated individuals and those who have recovered from SARS-Cov2.


Subject(s)
COVID-19 , Interferon-gamma Release Tests , Antibodies, Viral , COVID-19/diagnosis , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Nucleocapsid , RNA, Viral , SARS-CoV-2 , T-Lymphocytes
9.
Genes (Basel) ; 13(3)2022 02 25.
Article in English | MEDLINE | ID: covidwho-1736872

ABSTRACT

The genus Betacoronavirus, consisting of four main subgenera (Embecovirus, Merbecovirus, Nobecovirus, and Sarbecovirus), encompasses all clinically significant coronaviruses (CoVs), including SARS, MERS, and the SARS-CoV-2 virus responsible for current COVID-19 pandemic. Very few molecular characteristics are known that are specific for the genus Betacoronavirus or its different subgenera. In this study, our analyses of the sequences of four essential proteins of CoVs, viz., spike, nucleocapsid, envelope, and RNA-dependent RNA polymerase (RdRp), identified ten novel molecular signatures consisting of conserved signature indels (CSIs) in these proteins which are specific for the genus Betacoronavirus or its subgenera. Of these CSIs, two 14-aa-conserved deletions found within the heptad repeat motifs 1 and 2 of the spike protein are specific for all betacoronaviruses, except for their shared presence in the highly infectious avian coronavirus. Six additional CSIs present in the nucleocapsid protein and one CSI in the RdRp protein are distinctive characteristics of either the Merbecovirus, Nobecovirus, or Sarbecovirus subgenera. In addition, a 4-aa insert is present in the spike protein, which is uniquely shared by all viruses from the subgenera Merbecovirus, Nobecovirus, and Sarbecovirus, but absent in Embecovirus and all other genera of CoVs. This molecular signature provides evidence that viruses from the three subgenera sharing this CSI are more closely related to each other, and they evolved after the divergence of embecoviruses and other CoVs. As all CSIs specific for different groups of CoVs are flanked by conserved regions, their sequences provide novel means for identifying the above groups of CoVs and for developing novel diagnostic tests. Furthermore, our analyses of the structures of the spike and nucleocapsid proteins show that all identified CSIs are localized in the surface-exposed loops of these protein. It is postulated that these surface loops, through their interactions with other cellular proteins/ligands, play important roles in the biology/pathology of these viruses.


Subject(s)
COVID-19 , Pandemics , Humans , Nucleocapsid/genetics , Phylogeny , SARS-CoV-2/genetics
10.
J Infect Dis ; 225(11): 1937-1947, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1722487

ABSTRACT

BACKGROUND: Within the ongoing AGEhIV Cohort Study in Amsterdam, we prospectively compared the incidence of and risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection between human immunodeficiency virus (HIV)-positive and HIV-negative participants. Moreover, we compared SARS-CoV-2 nucleocapsid antibody levels between participants with incident infection from both groups. METHODS: Starting in September 2020, consenting HIV-positive and HIV-negative participants were assessed every 6 months for incident SARS-CoV-2 infection, using combined immunoglobulin (Ig) A/IgM/IgG SARS-CoV-2 nucleocapsid antibody assay. Cumulative incidence of SARS-CoV-2 infection and associated risk factors were assessed from 27 February 2020 through 30 April 2021, using complementary log-log regression. In those with incident SARS-CoV-2 infection, nucleocapsid (N) antibody levels were compared between groups using linear regression. RESULTS: The study included 241 HIV-positive (99.2% virally suppressed) and 326 HIV-negative AGEhIV participants. The cumulative SARS-CoV-2 incidence by April 2021 was 13.4% and 11.6% in HIV-positive and HIV-negative participants, respectively (P = .61). Younger age and African origin were independently associated with incident infection. In those with incident infection, only self-reported fever, but not HIV status, was associated with higher N antibody levels. CONCLUSIONS: HIV-positive individuals with suppressed viremia and adequate CD4 cell counts had similar risk of SARS-CoV-2 acquisition and similar SARS-CoV-2 N antibody levels after infection compared with a comparable HIV-negative cohort. CLINICAL TRIAL REGISTRATION: NCT01466582.


Subject(s)
COVID-19 , HIV Infections , Antibodies, Viral , COVID-19/epidemiology , Cohort Studies , HIV , Humans , Immunoglobulin A , Immunoglobulin G , Nucleocapsid , SARS-CoV-2
11.
Nat Commun ; 13(1): 601, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1671558

ABSTRACT

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Genome, Viral , Mutation, Missense , SARS-CoV-2/genetics , COVID-19/enzymology , COVID-19/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Host-Pathogen Interactions , Humans , Nucleocapsid/genetics , Nucleocapsid/metabolism , Phosphorylation , Phylogeny , Protein Binding , SARS-CoV-2/classification , SARS-CoV-2/physiology , Saudi Arabia , Viral Load , Virus Replication
12.
Int J Infect Dis ; 117: 287-294, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1670581

ABSTRACT

OBJECTIVES: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. RESULTS: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. CONCLUSIONS: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.


Subject(s)
COVID-19 , Nucleic Acids , Adult , COVID-19/diagnosis , COVID-19 Testing , Humans , Nasopharynx , Nucleocapsid/genetics , Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Specimen Handling/methods , Tongue
13.
Cell ; 185(5): 896-915.e19, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1670278

ABSTRACT

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunity, Mucosal , Administration, Intranasal , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , COVID-19/virology , COVID-19 Vaccines/immunology , Cytokines/blood , Genetic Vectors/genetics , Genetic Vectors/immunology , Genetic Vectors/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutralization Tests , Nucleocapsid/genetics , Nucleocapsid/immunology , Nucleocapsid/metabolism , Pan troglodytes , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
14.
J Biol Chem ; 298(3): 101677, 2022 03.
Article in English | MEDLINE | ID: covidwho-1665144

ABSTRACT

In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein-an essential viral protein required for viral replication-and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs.


Subject(s)
COVID-19 , Nucleocapsid Proteins , SARS-CoV-2 , COVID-19/virology , Consensus , Humans , Nucleocapsid/genetics , Nucleocapsid/metabolism , Nucleocapsid Proteins/isolation & purification , Nucleocapsid Proteins/metabolism , RNA, Viral/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Viral Proteins/metabolism
15.
Am J Emerg Med ; 54: 81-86, 2022 04.
Article in English | MEDLINE | ID: covidwho-1664596

ABSTRACT

BACKGROUND: Emergency department (ED) workers have an increased seroprevalence of SARS-CoV-2 antibodies. However, breakthrough infections in ED workers have led to a reduced workforce within a strained healthcare system. By measuring levels of IgG antibodies to the SARS-CoV-2 nucleocapsid and spike antigens in ED workers, we determined the incidence of infection and described the course of antibody levels. We also measured the antibody response to vaccination and examined factors associated with immunogenicity. METHODS: We conducted a prospective cohort study of ED workers conducted at a single ED from September 2020-April 2021. IgG antibodies to the SARS-CoV-2 nucleocapsid antigen were measured at baseline, 3, and 6 months, and IgG antibodies to the SARS-CoV-2 spike antigen were measured at 6 months. RESULTS: At baseline, we found 5 out of 139 (3.6%) participants with prior infection. At 6 months, 4 of the 5 had antibody results below the test manufacturer's positivity threshold. We identified one incident case of SARS-COV-2 infection out of 130 seronegative participants (0.8%, 95% CI 0.02-4.2%). In 131 vaccinated participants (125 BNT162b2, 6 mRNA-1273), 131 tested positive for anti-spike antibodies. We identified predictors of anti-spike antibody levels: time since vaccination, prior COVID-19 infection, age, and vaccine type. Each additional week since vaccination was associated with an 11.1% decrease in anti-spike antibody levels. (95% CI 6.2-15.8%). CONCLUSION: ED workers experienced a low incidence of SARS-CoV-2 infection and developed antibodies in response to vaccines and prior infection. Antibody levels decreased markedly with time since infection or vaccination.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Emergency Service, Hospital , Health Personnel , Humans , Nucleocapsid , Prospective Studies , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus
16.
Int J Infect Dis ; 112: 103-110, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1654539

ABSTRACT

OBJECTIVES: Monitoring the antibody responses to SARS-CoV-2 infection and its correlation to clinical spectrum of disease is critical in understanding the disease progression and protection against re-infection. We assessed the nucleocapsid (N) and receptor-binding-domain of spike (SRBD) protein specific IgG and neutralizing antibody (NAb) responses in COVID-19 patients up to 8 months and its correlation with diverse disease spectrum. METHODS: During the first wave of the SARS-CoV-2 pandemic, from 284 COVID-19 patients, 608 samples were collected up to 8 months post infection. The patients were categorized as asymptomatic, symptomatic and severe. The N and SRBD IgG and NAb titers were evaluated and correlated with clinical data. RESULTS: A steep increase in antigen specific antibody titers was observed till 40 days post onset of the disease (POD), followed by a partial decline till 240 days. Severe disease was associated with a stronger SRBD IgG response and higher NAb titers. The persistence of antibody response was observed in 76% against N, 80% against SRBD and 80% for NAbs of cases up to 8 months POD. CONCLUSION: RBD and N protein specific IgG persisted till 240 days POD which correlated with NAb response, irrespective of individual`s symptomatic status indicating overall robust protection against re-infection.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Neutralizing , Antibodies, Viral , Humans , Nucleocapsid , SARS-CoV-2
18.
PLoS One ; 17(1): e0262169, 2022.
Article in English | MEDLINE | ID: covidwho-1633137

ABSTRACT

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10-23 and 2*10-13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys.


Subject(s)
COVID-19/pathology , Immunity, Humoral , Adult , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Asymptomatic Infections/epidemiology , COVID-19/immunology , COVID-19/virology , Female , Health Personnel , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Longitudinal Studies , Male , Middle Aged , Nucleocapsid/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology
20.
J Virol Methods ; 302: 114469, 2022 04.
Article in English | MEDLINE | ID: covidwho-1627760

ABSTRACT

SARS-CoV-2 RNA can be detected in respiratory samples for weeks after onset of COVID-19 disease. Therefore, one of the diagnostic challenges of PCR positive cases is differentiating between acute COVID-19 disease and convalescent phase. The presence of SARS-CoV-2 nucleocapsid antigen in serum and plasma samples of COVID-19 patients has been demonstrated previously. Our study aimed to characterize the analytical specificity and sensitivity of an enzyme-linked immunosorbent assay (Salocor SARS-CoV-2 Antigen Quantitative Assay Kit© (Salofa Ltd, Salo, Finland)) for the detection of SARS-CoV-2 nucleocapsid antigen in serum, and to characterize the kinetics of antigenemia. The evaluation material included a negative serum panel of 155 samples, and 126 serum samples from patients with PCR-confirmed COVID-19. The specificity of the Salocor SARS-CoV-2 serum nucleocapsid antigen test was 98.0 %. In comparison with simultaneous positive PCR from upper respiratory tract (URT) specimens, the test sensitivity was 91.7 %. In a serum panel in which the earliest serum sample was collected two days before the collection of positive URT specimen, and the latest 48 days after (median 1 day post URT sample collection), the serum N antigen test sensitivity was 95.6 % within 14 days post onset of symptoms. The antigenemia resolved approximately two weeks after the onset of disease and diagnostic PCR. The combination of simultaneous SARS-CoV-2 antigen and antibody testing appeared to provide useful information for timing of COVID-19. Our results suggest that SARS-CoV-2 N-antigenemia may be used as a diagnostic marker in acute COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Nucleocapsid , RNA, Viral , Sensitivity and Specificity
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