Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Also, three non-overlapping peptides belonging to the S2 domain of spike protein were assessed. Our results showed that while reactivity of patients’ sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (p<0.001), no significant differences were observed for reactivity of patients’ sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing aa 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and Western blotting. Altogether, our data suggest the involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of antigenic epitope in NP and RBD of SARS-CoV-2 could provide important advances for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

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Seeing beyond a Dilated Proventriculus: Diagnostic Tools for Proventricular Dilatation Disease in Psittacine Birds

Animals ◽

10.3390/ani11123558 ◽

2021 ◽

Vol 11 (12) ◽

pp. 3558

Author(s):

Jeann Leal de Araújo ◽

Raquel Rubia Rech

Keyword(s):

Molecular Techniques ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Postmortem Diagnosis ◽

Concise Review ◽

Life Threatening ◽

Polymerase Chain ◽

Proventricular Dilatation Disease ◽

Diagnostic Outcomes

Proventricular dilatation disease (PDD) is a life-threatening neurological disease caused by parrot bornaviruses (PaBVs) that affects several species worldwide. PDD can be clinically manifested as either a central nervous system condition or a gastrointestinal condition if the nerves and ganglia of the gastrointestinal tract are compromised. We intend to provide a concise review for veterinary clinicians and diagnosticians with focus on the main tools available for PDD diagnosis, including gross and histopathology, immunohistochemistry, molecular techniques and serology. We suggest that a combination of different strategies can increase the success of diagnostic outcomes, as tools such as reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) can be implemented for identification of bornaviral infections in live patients, and gross pathology, histopathology, immunohistochemistry and RT-PCR can provide reliable results for postmortem diagnosis of PDD.

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Development of an Indirect ELISA Based on Spike Protein to Detect Antibodies against Feline Coronavirus

Viruses ◽

10.3390/v13122496 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2496

Author(s):

Bo Dong ◽

Gaoqiang Zhang ◽

Xiaodong Zhang ◽

Xufei Chen ◽

Meiling Zhang ◽

...

Keyword(s):

Operating Characteristic ◽

Characteristic Curve ◽

Systemic Disease ◽

Analysis Data ◽

Spike Protein ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Pathogenic Virus ◽

Feline Coronavirus

Feline coronavirus (FCoV) is a pathogenic virus commonly found in cats that causes a benign enteric illness and fatal systemic disease, feline infectious peritonitis. The development of serological diagnostic tools for FCoV is helpful for clinical diagnosis and epidemiological investigation. Therefore, this study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) to detect antibodies against FCoV using histidine-tagged recombinant spike protein. FCoV S protein (1127–1400 aa) was expressed and used as an antigen to establish an ELISA. Mice and rabbits immunized with the protein produced antibodies that were recognized and bound to the protein. The intra-assay coefficient of variation (CV) was 1.15–5.04% and the inter-assay CV was 4.28–15.13%, suggesting an acceptable repeatability. iELISA did not cross-react with antisera against other feline viruses. The receiver operating characteristic curve analysis revealed an 86.7% sensitivity and 93.3% specificity for iELISA. Serum samples (n = 107) were tested for anti-FCoV antibodies, and 70.09% of samples were positive for antibodies against FCoV. The iELISA developed in our study can be used to measure serum FCoV antibodies due to its acceptable repeatability, sensitivity, and specificity. Additionally, field sample analysis data demonstrated that FCoV is highly prevalent in cat populations in Fujian province, China.

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Human Milk Antibodies Against SARS-CoV-2: A Longitudinal Follow-Up Study

Journal of Human Lactation ◽

10.1177/08903344211030171 ◽

2021 ◽

pp. 089033442110301

Author(s):

Hannah G. Juncker ◽

M. Romijn ◽

Veerle N. Loth ◽

Tom G. Caniels ◽

Christianne J.M. de Groot ◽

...

Keyword(s):

Human Milk ◽

Clinical Symptoms ◽

Immunoglobulin A ◽

Spike Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Follow Up Study ◽

Lactating Mothers ◽

Milk Antibodies ◽

Over Time

Background: Human milk contains antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) following Coronavirus Disease 2019 (COVID-19). These antibodies may serve as protection against COVID-19 in infants. However, the evolution of these human milk antibodies over time is unclear. Research Aim: To elucidate the evolution of immunoglobulin A (IgA) against SARS-CoV-2 in human milk after a SARS-CoV-2 infection. Methods: This longitudinal follow-up study included lactating mothers ( N = 24) who had participated in the COVID MILK study. To assess the evolution of SARS-CoV-2 antibodies, serum and human milk samples were collected 14–143 days after the onset of clinical symptoms related to COVID-19. Enzyme-Linked ImmunoSorbent Assay was used to detect antibodies against the ectodomain of the SARS-CoV-2 spike protein. Results: SARS-CoV-2 antibodies remain present up to 5 months (143 days) in human milk after onset of COVID-19 symptoms. Overall, SARS-CoV-2 IgA in human milk seems to gradually decrease over time. Conclusion: Human milk from SARS-CoV-2 convalescent lactating mothers contains specific IgA antibodies against SARS-CoV-2 spike protein up to at least 5 months post-infection. Passive viral immunity can be transferred via human milk and may serve as protection for infants against COVID-19. Dutch Trial Register on May 1st, 2020, number: NL 8575, URL: https://www.trialregister.nl/trial/8575 .

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Comparative Analysis of the Euroimmun CXCL13 Enzyme-Linked Immunosorbent Assay and the ReaScan Lateral Flow Immunoassay for Diagnosis of Lyme Neuroborreliosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00207-20 ◽

2020 ◽

Vol 58 (9) ◽

Author(s):

Katharina Ziegler ◽

Anca Rath ◽

Christoph Schoerner ◽

Renate Meyer ◽

Thomas Bertsch ◽

...

Keyword(s):

Immunosorbent Assay ◽

Roc Curve ◽

Point Of Care ◽

Lateral Flow ◽

Lyme Neuroborreliosis ◽

Diagnostic Tools ◽

Lateral Flow Immunoassay ◽

Enzyme Linked Immunosorbent Assay ◽

European Federation ◽

Point Of Care Test

ABSTRACT Diagnosis of Lyme neuroborreliosis (LNB) is challenging, as long as Borrelia-specific intrathecal antibodies are not yet detectable. The chemokine CXCL13 is elevated in the cerebrospinal fluid (CSF) of LNB patients. Here, we compared the performances of the Euroimmun CXCL13 enzyme-linked immunosorbent assay (CXCL13 ELISA) and the ReaScan CXCL13 lateral flow immunoassay (CXCL13 LFA), a rapid point-of-care test, to support the diagnosis of LNB. In a dual-center case-control study, CSF samples from 90 patients (34 with definite LNB, 10 with possible LNB, and 46 with other central nervous system [CNS] diseases [non-LNB group]) were analyzed with the CXCL13 ELISA and the CXCL13 LFA. Classification of patients followed the European Federation of Neurological Societies (EFNS) guidelines on LNB. The CXCL13 ELISA detected elevated CXCL13 levels in all patients with definite LNB (median, 1,409 pg/ml) compared to the non-LNB controls (median, 20.7 pg/ml; P < 0.0001), with a sensitivity of 100% and a specificity of 84.8% (cutoff value, 78.6 pg/ml; area under the receiver operating characteristic [ROC] curve, 0.93). Similarly, the CXCL13 LFA yielded elevated CXCL13 levels in 31 patients with definite LNB (median arbitrary value, 223.5) compared to the non-LNB control patients (median arbitrary value, 0; P < 0.0001) and had a sensitivity and specificity of 91.2% and 93.5%, respectively (cutoff arbitrary value, 22.5; area under the ROC curve, 0.94). The correlation between the CXCL13 levels obtained by ELISA and LFA was strong (Spearman correlation coefficient r = 0.89; P < 0.0001). The CXCL13 ELISA and the CXCL13 LFA are comparable diagnostic tools for the detection of CXCL13 in the CSF of patients with definite LNB. The advantage of the CXCL13 LFA is the shorter time to result.

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Role of Structural and Non-Structural Proteins and Therapeutic Targets of SARS-CoV-2 for COVID-19

Cells ◽

10.3390/cells10040821 ◽

2021 ◽

Vol 10 (4) ◽

pp. 821

Author(s):

Rohitash Yadav ◽

Jitendra Kumar Chaudhary ◽

Neeraj Jain ◽

Pankaj Kumar Chaudhary ◽

Supriya Khanra ◽

...

Keyword(s):

Host Cell ◽

Protein S ◽

Structural Similarity ◽

Cell Receptor ◽

Molecular Assembly ◽

The Body ◽

Spike Protein ◽

Structural Proteins ◽

Structural Protein ◽

Novel Coronavirus

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10–20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5′ end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.

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Comparative Study of Immune-Diagnostic Tools with Polymerase Chain Reaction in Sub-Clinical Leishmaniasis Isolates

Journal of Medicine ◽

10.3329/jom.v12i1.5422 ◽

2011 ◽

Vol 12 (1) ◽

pp. 34-39 ◽

Cited By ~ 2

Author(s):

Nazar M Abdalla

Keyword(s):

Polymerase Chain Reaction ◽

Skin Test ◽

Diagnostic Methods ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Clinical Form ◽

Chain Reaction ◽

Leishmanin Skin Test ◽

Wide Range ◽

Polymerase Chain

Objective: This study aimed to identify cases of leishmaniasis in the Nuba Mountain area, which is situated in a unique geographical site located in the centre of Sudanese leishmania belt. Wide range of investigations are available for detection of leishmania cases, but still the most reliable and easy test used as screening and epidemiological tool in field studies needs to be evaluated. The most commonly used conventional diagnostic methods direct microscopy and culture have some drawbacks in diagnosing subclinical cases of leishmaniasis. Materials and methods: In this study, comparative properties of various immune-diagnostic tools with Polymerase Chain Reaction used in sub-clinical leishmaniasis isolates were explored. The immune-diagnostic tools involved in this study include- Leishmanin Skin Test (LST), Enzyme Linked Immunosorbent Assay (ELISA) and Direct Agglutination Test (DAT). The study was conducted in the Green Valley village (Rashad Province, South Kordofan State) with a population of 332. Most of the villagers presented with sub-clinical form of leishmaniasis with minor symptoms and signs without the features found in clinical form of visceral leishmaniasis such as fever, diarrhoea, epistaxis, enlarged lymph nodes, spleen and liver. In this study we collected demographic, clinical and epidemiological data using special questionnaire. Leishmanin skin test (LST), ELISA, DAT and PCR for parasite DNA detection were used. Result: The final positive cases detected by PCR were 32 out of 332 belong to L. donovani species. The final positive cases detected by LST were 51.2% of the total population under study, while 11 out of the 37 tested samples were positive by ELISA. All of the 332 villagers showed negative readings by DAT with exception of three individuals who were positive with very high titers. Conclusion: DNA etxtraction and amplification with primers can be a good screening tool in subclinical leishmaniasis isolates. Keyword: Sub-clinical; Leishmaniasis; Leishmanin Skin Test; ELISA; DAT; PCR. DOI: 10.3329/jom.v12i1.5422J Medicine 2011; 12 : 34-39

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Localization of the gD-Binding Region of the Human Herpes Simplex Virus Receptor, HveA

Journal of Virology ◽

10.1128/jvi.75.1.171-180.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 171-180 ◽

Cited By ~ 41

Author(s):

J. Charles Whitbeck ◽

Sarah A. Connolly ◽

Sharon H. Willis ◽

Wangfang Hou ◽

Claude Krummenacher ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Binding Site ◽

Virus Entry ◽

Tumor Necrosis Factor Receptor ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Linear Epitopes ◽

Simplex Virus ◽

Tnfr Superfamily

ABSTRACT During virus entry, herpes simplex virus (HSV) glycoprotein D (gD) binds to one of several human cellular receptors. One of these, herpesvirus entry mediator A (HveA), is a member of the tumor necrosis factor receptor (TNFR) superfamily, and its ectodomain contains four characteristic cysteine-rich pseudorepeat (CRP) elements. We previously showed that gD binds the ectodomain of HveA expressed as a truncated, soluble protein [HveA(200t)]. To localize the gD-binding domain of HveA, we expressed three additional soluble forms of HveA consisting of the first CRP [HveA(76t)], the second CRP [HveA(77–120t)], or the first and second CRPs [HveA(120t)]. Biosensor and enzyme-linked immunosorbent assay studies showed that gD bound to HveA(120t) and HveA(200t) with the same affinity. However, gD did not bind to HveA(76t) or HveA(77–120t). Furthermore, HveA(200t) and HveA(120t), but not HveA(76t) or HveA(77–120t), blocked herpes simplex virus (HSV) entry into CHO cells expressing HveA. We also generated six monoclonal antibodies (MAbs) against HveA(200t). MAbs CW1, -2, and -4 bound linear epitopes within the second CRP, while CW7 and -8 bound linear epitopes within the third or fourth CRPs. None of these MAbs blocked the binding of gD to HveA. In contrast, MAb CW3 recognized a discontinuous epitope within the first CRP of HveA, blocked the binding of gD to HveA, and exhibited a limited ability to block virus entry into cells expressing HveA, suggesting that the first domain of HveA contains at least a portion of the gD binding site. The inability of gD to bind HveA(76t) suggests that additional amino acid residues of the gD binding site may reside within the second CRP.

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The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2, in vitro

10.1101/2021.03.19.435740 ◽

2021 ◽

Author(s):

Riho Tateyama-Makino ◽

Mari Abe-Yutori ◽

Taku Iwamoto ◽

Kota Tsutsumi ◽

Motonori Tsuji ◽

...

Keyword(s):

Serine Protease ◽

Protease Activity ◽

Oral Care ◽

Sodium Dodecyl ◽

Host Cells ◽

Spike Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Inhibitory Effects ◽

Docking Simulations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the mucosa of the tongue and gingiva, the oral cavity seems like it is an entry point for SARS-CoV-2. Daily oral care using mouthwash seems to play an important role in preventing SARS-CoV-2 infection. However, the relationship between daily oral care and the mechanisms of virus entry into host cells is unclear. In this study, we evaluated the inhibitory effects of ingredients that are generally contained in toothpaste and mouthwash on the interaction between the spike protein and ACE2 and on the serine protease activity of TMPRSS2 using an enzyme-linked immunosorbent assay and in vitro enzyme assay, respectively. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to the inhibitor-binding site of ACE2. In addition, tranexamic acid and 6-aminohexanoic acid, which act as serine protease inhibitors, exerted inhibitory effects on TMPRSS2 protease activity. Further experimental and clinical studies are needed to further elucidate these mechanisms. Our findings support the possibility that toothpaste and mouthwash contain ingredients that inhibit SARS-CoV-2 infection.

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Human Babesiosis in Europe

Pathogens ◽

10.3390/pathogens10091165 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1165

Author(s):

Anke Hildebrandt ◽

Annetta Zintl ◽

Estrella Montero ◽

Klaus-Peter Hunfeld ◽

Jeremy Gray

Keyword(s):

Clinical Management ◽

Diagnostic Tools ◽

Genetic Characteristics ◽

Susceptibility To Infection ◽

First Case ◽

The Usa ◽

Life Threatening ◽

History Of ◽

Etiological Agents ◽

Human Babesiosis

Babesiosis is attracting increasing attention as a worldwide emerging zoonosis. The first case of human babesiosis in Europe was described in the late 1950s and since then more than 60 cases have been reported in Europe. While the disease is relatively rare in Europe, it is significant because the majority of cases present as life-threatening fulminant infections, mainly in immunocompromised patients. Although appearing clinically similar to human babesiosis elsewhere, particularly in the USA, most European forms of the disease are distinct entities, especially concerning epidemiology, human susceptibility to infection and clinical management. This paper describes the history of the disease and reviews all published cases that have occurred in Europe with regard to the identity and genetic characteristics of the etiological agents, pathogenesis, aspects of epidemiology including the eco-epidemiology of the vectors, the clinical courses of infection, diagnostic tools and clinical management and treatment.

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Detection of cross-reactive IgA against SARS-CoV-2 spike 1 subunit in saliva

10.1101/2021.03.29.21253174 ◽

2021 ◽

Author(s):

Keiichi Tsukinoki ◽

Tatsuo Yamamoto ◽

Keisuke Handa ◽

Mariko Iwamiya ◽

Juri Saruta ◽

...

Keyword(s):

Breast Milk ◽

Recombinant Protein ◽

University Hospital ◽

Spike Protein ◽

Secretory Iga ◽

Enzyme Linked Immunosorbent Assay ◽

Receptor Binding Domain ◽

Blood Samples ◽

Milk Samples ◽

Mucosal Surfaces

AbstractAbundant secretory IgA (sIgA) in mucus, breast milk, and saliva provides immunity that prevents infection of mucosal surfaces. sIgA in pre-pandemic breast milk samples have been reported to cross-react with SARS-CoV-2, but whether it also occurs in saliva and, if so, whether it cross-reacts with SARS-CoV-2, has remained unknown. We aimed to clarify whether sIgA in saliva cross-reacts with SARS-CoV-2 spike 1 subunit in individuals who have not been infected with this virus. The study included 137 (male, n = 101; female, n = 36; mean age, 38.7 [24–65] years) of dentists and doctors in the Kanagawa Dental University Hospital. Saliva and blood samples were analyzed by PCR and immunochromatography for IgG and IgM, respectively. We then identified patients with saliva samples that were confirmed as PCR- and IgM-negative for COVID-19. Proportions of SARS-CoV-2 cross-reactive IgA-positive individuals were determined by enzyme-linked immunosorbent assay using a biotin-labeled spike S1-mFc recombinant protein covering the receptor-binding domain of SARS-CoV-2. The proportion of SARS-CoV-2 cross-reactive IgA-positive individuals was 46.7%, and this correlated negatively with age (r = −0.218, p = 0.01). The proportion of IgA-positive individuals ≥ 50 y was significantly lower than that of patients aged ≤ 49 y (p = 0.008). sIgA was purified from the saliva of all patients, and the salivary sIgA was found to suppress the binding of SARS-CoV-2 spike protein to the ACE-2 receptor. We found SARS-CoV-2 cross-reactive sIgA in the saliva of some participants who had never been infected with the virus, suggesting that sIgA helps prevent SARS-CoV-2 infection.

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