scholarly journals Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting

COVID ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 105-114
Author(s):  
Sahra Pajenda ◽  
Sebastian Kapps ◽  
Thomas Reiter ◽  
Raimundo Freire ◽  
Veronique A. J. Smits ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Posterior Part ◽  
Rapid Identification ◽  
Diagnostic Methods ◽  
Serum Samples ◽  
N Protein ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Global Pandemic ◽  
Human Coronaviruses

A novel beta coronavirus that emerged in late December 2019 triggered a global pandemic. Diagnostic methods for rapid identification of infected individuals were established in new biotechnological approaches. Vaccine production and application to individuals and measurement of SARS-CoV-2 antibodies also began. Serum samples from 240 health care workers were collected at three-month intervals over nine months. Indirect SARS-CoV-2 nucleocapsid IgG ELISA tests were used to identify humoral immune responses. All seropositive individuals and those with borderline ELISA values were tested with a specifically generated multipanel nucleocapsid fragment immunoblot. Of the 240 individuals, 24 showed seroconversion in ELISA after experiencing COVID-19. All of them showed a positive reaction against the full-length nucleocapsid protein in the immunoblot. The highest reactivity was seen either against fragment N(100–300) or in a minority against the posterior part N(200–419). In general, the staining pattern of COVID-19 patients showed four phenotypes. In contrast, three individuals classified as borderline by ELISA reacted exclusively with fragments N(1–220) and N(100–300) containing the octamer amino acid sequence FYYLGTGP, which is identical in human coronaviruses sharing this sequence with SARS-CoV-2. These represent a unique and thus fifth phenotype. This work suggests the existence of distinct phenotypic patterns of IgG production towards N-protein subdomains.

scholarly journals Antibody Responses to SARS-CoV-2 Antigens in Humans and Animals

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 684
Author(s):  
Hyunsuh Kim ◽  
Patrick Seiler ◽  
Jeremy C. Jones ◽  
Granger Ridout ◽  
Kristi P. Camp ◽  
...  
Keyword(s):  
Immune Responses ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Public Health Response ◽  
The Public ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Health Response ◽  
Seroepidemiologic Studies ◽  
Human Coronaviruses

To optimize the public health response to coronavirus disease 2019 (COVID-19), we must first understand the antibody response to individual proteins on the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the antibody’s cross reactivity to other coronaviruses. Using a panel of 37 convalescent COVID-19 human serum samples, we showed that the magnitude and specificity of responses varied across individuals, independent of their reactivity to seasonal human coronaviruses (HCoVs). These data suggest that COVID-19 vaccines will elicit primary humoral immune responses in naïve individuals and variable responses in those previously exposed to SARS-CoV-2. Unlike the limited cross-coronavirus reactivities in humans, serum samples from 96 dogs and 10 cats showed SARS-CoV-2 protein-specific responses focused on non–S1 proteins. The correlation of this response with those to other coronaviruses suggests that the antibodies are cross-reactive and generated to endemic viruses within these hosts, which must be considered in seroepidemiologic studies. We conclude that substantial variation in antibody generation against coronavirus proteins will influence interpretations of serologic data in the clinical and veterinary settings.

scholarly journals Immunological imprinting of the antibody response in COVID-19 patients

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Teresa Aydillo ◽  
Alexander Rombauts ◽  
Daniel Stadlbauer ◽  
Sadaf Aslam ◽  
Gabriela Abelenda-Alonso ◽  
...  
Keyword(s):  
Immune Response ◽  
Severe Acute Respiratory Syndrome ◽  
Antibody Response ◽  
Humoral Immune Response ◽  
Nucleocapsid Protein ◽  
Spike Protein ◽  
Ongoing Research ◽  
Variable Regions ◽  
Humoral Immune ◽  
Human Coronaviruses

AbstractIn addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection.

scholarly journals A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil

2013 ◽  
Vol 33 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Carla D. Marassi ◽  
Luciana Medeiros ◽  
Eduardo Figueiredo ◽  
Leila S. Fonseca ◽  
Rafael Duarte ◽  
...  
Keyword(s):  
Skin Test ◽  
Tuberculin Test ◽  
Diagnostic Methods ◽  
Test Analysis ◽  
Comprehensive Strategy ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Kappa Test ◽  
Nasal Swabs ◽  
Standard Tests

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.

scholarly journals Expression and novel alternative purification of the recombinant nucleocapsid (N) protein of SARS-CoV-2 in Escherichia coli for the serodiagnosis of COVID-19

2021 ◽  
Author(s):  
Jose David Rosales ◽  
William Quintero ◽  
Jhon Cruz ◽  
Balbino Perdomo ◽  
Militza Quintero ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein S ◽  
Exchange Chromatography ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Metal Exchange ◽  
Serological Tests ◽  
N Protein ◽  
Global Pandemic ◽  
Novel Strategy

The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach use SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDT's). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.

scholarly journals Diversity of Antigen Recognition by Serum Antibodies in Experimental Bovine Tuberculosis

1998 ◽  
Vol 66 (11) ◽  
pp. 5344-5349 ◽  
Author(s):  
Konstantin P. Lyashchenko ◽  
John M. Pollock ◽  
Roberto Colangeli ◽  
Maria Laura Gennaro
Keyword(s):  
Antibody Production ◽  
Bovine Tuberculosis ◽  
Tuberculin Test ◽  
Economic Problem ◽  
Antigen Recognition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Antibodies ◽  
Serum Samples ◽  
Humoral Immune ◽  
Humoral Immune Responses

ABSTRACT Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovisinvolved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.

scholarly journals Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses

2020 ◽  
Author(s):  
Noah Avery Schuster
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
N Gene ◽  
Nucleocapsid Proteins ◽  
Nucleocapsid Gene ◽  
N Protein ◽  
Global Pandemic ◽  
The Relationship

An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 16,500,000 cases and 650,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (S37 and A267) were found to exist only in its N protein, along with an extended β-hairpin that bends towards the nucleotide binding site. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, these data will enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.

Analysis of Humoral Immune Responses in Chikungunya Virus (CHIKV)-Infected Patients and Individuals Vaccinated With a Candidate CHIKV Vaccine

2019 ◽  
Vol 221 (10) ◽  
pp. 1713-1723
Author(s):  
Lisa Henss ◽  
Constanze Yue ◽  
Christine Von Rhein ◽  
Roland Tschismarov ◽  
Lia Laura Lewis-Ximenez ◽  
...  
Keyword(s):  
Immune Responses ◽  
Chikungunya Virus ◽  
Cross Reactivity ◽  
Similar Extent ◽  
Serum Samples ◽  
Neutralization Activity ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Cross Neutralization ◽  
Binding Avidity

Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu-like symptoms. The acute symptoms disappear after 1 week, but chronic arthralgia can persist for years. In this study, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors, and antibody epitope mapping was performed with a peptide array. Results The greatest CHIKV neutralization activity was observed 60–92 days after onset of symptoms. The amount of CHIKV-specific antibodies and their binding avidity and cross-reactivity with other alphaviruses increased over time. Chikungunya virus and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2- to 6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV, and the E2 ASR1 was the major antibody target. Conclusions These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

scholarly journals SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jun Wu ◽  
Boyun Liang ◽  
Cunrong Chen ◽  
Hua Wang ◽  
Yaohui Fang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Disease Onset ◽  
Immunoglobulin M ◽  
Late Time ◽  
N Protein ◽  
Contraction Phase ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Observation Period

AbstractLong-term antibody responses and neutralizing activities in response to SARS-CoV-2 infection are not yet clear. Here we quantify immunoglobulin M (IgM) and G (IgG) antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) of the spike (S) or the nucleocapsid (N) protein, and neutralizing antibodies during a period of 6 months from COVID-19 disease onset in 349 symptomatic COVID-19 patients who were among the first be infected world-wide. The positivity rate and magnitude of IgM-S and IgG-N responses increase rapidly. High levels of IgM-S/N and IgG-S/N at 2-3 weeks after disease onset are associated with virus control and IgG-S titers correlate closely with the capacity to neutralize SARS-CoV-2. Although specific IgM-S/N become undetectable 12 weeks after disease onset in most patients, IgG-S/N titers have an intermediate contraction phase, but stabilize at relatively high levels over the 6 month observation period. At late time points, the positivity rates for binding and neutralizing SARS-CoV-2-specific antibodies are still >70%. These data indicate sustained humoral immunity in recovered patients who had symptomatic COVID-19, suggesting prolonged immunity.

scholarly journals Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaori Saito ◽  
Tomohiko Ai ◽  
Akinori Kawai ◽  
Jun Matsui ◽  
Yoshiyuki Fukushima ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Serum Samples ◽  
N Protein ◽  
Detection Systems ◽  
Copy Numbers ◽  
Antigen Assay ◽  
Polymerase Chain ◽  
Human Coronaviruses ◽  
Automated Immunoassay

AbstractHere, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

scholarly journals Long-Term Humoral Immune Response against SARS-CoV-2 after Natural Infection and Subsequent Vaccination According to WHO International Binding Antibody Units (BAU/mL)

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2336
Author(s):  
Natalia Ruetalo ◽  
Bertram Flehmig ◽  
Michael Schindler ◽  
Lutz Pridzun ◽  
Angelika Haage ◽  
...  
Keyword(s):  
Natural Infection ◽  
Serum Samples ◽  
N Protein ◽  
Humoral Immune ◽  
Reference Preparation ◽  
Binding Antibody ◽  
One Year ◽  
Definition Of ◽  
The Individual

The new WHO reference standard allows for the definition of serum antibodies against various SARS-CoV-2 antigens in terms of binding antibody units (BAU/mL) and thus to compare the results of different ELISA systems. In this study, the concentration of antibodies (ABs) against both the S- and the N-protein of SARS-CoV-2 as well as serum neutralization activity were evaluated in three patients after a mild course of COVID-19. Serum samples were collected frequently during a period of over one year. Furthermore, in two individuals, the effects of an additional vaccination with a mRNA vaccine containing the S1-RBD sequence on these antibodies were examined. After natural infection, the antibodies (IgA, IgG) against the S1-protein remained elevated above the established cut-off to positivity (S-IgA 60 BAU/mL and S-IgG 50 BAU/mL, respectively) for over a year in all patients, while this was not the case for ABs against the N-protein (cut-off N-IgG 40 BAU/mL, N-IgA 256 BAU/mL). Sera from all patients retained the ability to neutralize SARS-CoV-2 for more than a year. Vaccination resulted in a rapid boost of antibodies to S1-protein but, as expected, not to the N-protein. Most likely, the wide use of the WHO reference preparation will be very useful in determining the individual immune status of patients after an infection with SARS-CoV-2 or after vaccination.

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