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1.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-305869

ABSTRACT

Background: Cell-mediated immunity to SARS-CoV-2 may define infection-risk in healthy and chronically immunosuppressed transplant patients.Methods: Blood samples from 64 unexposed healthy subjects including 21 with solid organ transplants, and 66 COVID-19 patients, including 26 with transplants were tested. Frequencies of SARS-CoV-2-reactive CD3+T-cells CD4+T-helper, CD8+T-cytotoxic and CD19+B-cells, which express CD154 were measured after stimulation with peptide mixtures representing the spike protein S antigen, its conserved C-terminal S2, and less conserved N-terminal S1 components.Findings: COVID-19 patients were sampled at median 11 days after diagnosis. S-reactive T- and B-cells were present in all samples and were significantly fewer in COVID-19 compared with healthy subjects. In logistic regression analysis using training and test sets, S-reactive CD3 and CD8 cells, age, race, and transplantation status distinguished COVID-19 patients from healthy subjects (test set AUC 0.9 [95%CI 0.83-0.97]). Among 66 COVID-19 patients S-reactive CD8 cells and age predicted respiratory failure (AUC 0.73 [95%CI 0.62-0.86]). S2-reactive T-cells also predicted COVID-19 infection (test set AUC 0.89 [95%CI 0.80-0.97]) and respiratory failure (AUC 0.73 [95%CI 0.60-0.86]). S1 antigen elicited minimal responses. COVID-19 subjects developed anti-spike-protein IgG antibody. Transplanted COVID-19 patients had significantly lower incidence of IgG to the receptor-binding-domain of the S1 antigen.Interpretations: In immunosuppressed and non-immunosuppressed individuals, SARS-CoV-2-spike-antigen-specific cellular immunity is directed toward conserved sequences. This immunity is decreased early after COVID-19 infection, more so with respiratory failure, and identifies individuals at-risk for COVID-19. Humoral immunity to less conserved viral sequences is impaired in chronically immunosuppressed patients early after infection.Funding: Intramural support from all participating institutions, NSF#2033307, NIH Grant number UL1 TR001450 (MUSC).Declaration of Interests: University of Pittsburgh Patent 9606019, author: RS, describes CMI testing for CMV, is licensed exclusively to Plexision, in which University and RS own equity. RS and CA developed Plexision’s patent-pending multi-variate CMI assay for SARS2. RS is Professor of Surgery at the University of Pittsburgh and Chief Scientific Officer of Plexision by permission of COI committee at the University. CA is a paid consultant to Plexision. All other authors declare no conflict of interest. Ethics Approval Statement: Human Subjects: COVID-19 patients were enrolled under IRB-approved protocols 2017-0365, Pro00101915, and 1551551 respectively, at the three centers in Washington, DC, Charleston, SC, and Edinburg, TX. De-identified residual cryopreserved PBL samples were tested under IRB-exempt protocol, and samples from healthy-NT subjects were tested under IRB approved protocol 6774 in the reference laboratory at Plexision.

2.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-295689

ABSTRACT

Over 1.2 million daily total tests are currently being performed for SARS-CoV-2, in the United States. The most common SARS-CoV-2 tests require RNA extraction and purification. Extraction of RNA is a time-consuming and costly step that requires a constant supply of reagents and accessories. With the current testing demand, the supply chain remains the bottleneck for RNA extraction. Here, we report Direct NP- a cost-effective extraction-free RT-qPCR based dualplex test for SARS-CoV-2 from NP swab specimens. Direct NP detects SARS-CoV-2 viral RNA from heat-denatured patient specimens using a dualplex RT-qPCR assay. Direct NP showed 92.5% positive percentage agreement (PPA) (95% Confidence Interval (CI) = 79.61% to 98.43%) and 97% negative percent agreement (NPA) (95% CI = 89.11 to 100%) with the CDC assay. Direct NP reduces the cost per test to $2, making it suitable for broad-scale testing while lowering the cost burden on the healthcare system.<br><br>Funding: This work was funded by the Enterprise Strategic Funding for Pandemic Response from the Medical University of South Carolina.<br><br>Declaration of Interests: The authors declare no competing interests.<br><br>Ethics Approval Statement: IRB review was waived by the Institutional Review Board for Human Research (IRB), a part of the Office of Research Integrity at the Medical University of South Carolina as this study is a process development and samples were deidentified and blinded.

3.
Sci Rep ; 11(1): 14232, 2021 07 09.
Article in English | MEDLINE | ID: covidwho-1303793

ABSTRACT

COVID-19 pandemic exerts a health care emergency around the world. The illness severity is heterogeneous. It is mostly unknown why some individuals who are positive for SARS-CoV-2 antibodies stay asymptomatic while others show moderate to severe disease symptoms. Reliable biomarkers for early detection of the disease are urgently needed to attenuate the virus's spread and help make early treatment decisions. Bioactive sphingolipids play a crucial role in the regulation of viral infections and pro-inflammatory responses involved in the severity of COVID-19. However, any roles of sphingolipids in COVID-19 development or detection remain unknown. In this study, lipidomics measurement of serum sphingolipids demonstrated that reduced sphingosine levels are highly associated with the development of symptomatic COVID-19 in the majority (99.24%) SARS-CoV-2-infected patients compared to asymptomatic counterparts. The majority of asymptomatic individuals (73%) exhibited increased acid ceramidase (AC) in their serum, measured by Western blotting, consistent with elevated sphingosine levels compared to SARS-CoV-2 antibody negative controls. AC protein was also reduced in almost all of the symptomatic patients' serum, linked to reduced sphingosine levels, measured in longitudinal acute or convalescent COVID-19 samples. Thus, reduced sphingosine levels provide a sensitive and selective serologic biomarker for the early identification of asymptomatic versus symptomatic COVID-19 patients.


Subject(s)
Acid Ceramidase/blood , COVID-19 , Carrier State , Lipid Metabolism , SARS-CoV-2/metabolism , Sphingolipids/blood , Sphingosine/blood , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers/blood , COVID-19/blood , COVID-19/diagnosis , Carrier State/blood , Carrier State/diagnosis , Female , Humans , Male , Middle Aged
4.
iScience ; 24(6): 102489, 2021 Jun 25.
Article in English | MEDLINE | ID: covidwho-1213295

ABSTRACT

The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral Spike protein, and approximately 3% were positive. Of these 3%, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them with those of asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Furthermore, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or post vaccination.

5.
Clin Chem ; 66(12): 1531-1537, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-745843

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody testing is an important tool in assessment of pandemic progress, contact tracing, and identification of recovered coronavirus disease 2019 (COVID-19) patients. We evaluated an orthogonal testing algorithm (OTA) to improve test specificity in these use cases. METHODS: A two-step OTA was applied where individuals who initially tested positive were tested with a second test. The first-line test, detecting IgG antibodies to the viral nucleocapsid protein, was validated in 130 samples and the second-line test, detecting IgG antibodies to the viral spike protein in 148 samples. The OTA was evaluated in 4333 clinical patient specimens. The seropositivity rates relative to the SARS-CoV-2 PCR positivity rates were evaluated from our entire patient population data (n = 5102). RESULTS: The first-line test resulted in a clinical sensitivity of 96.4% (95% CI; 82.3% to 99.4%), and specificity of 99.0% (95% CI; 94.7% to 99.8%), whereas the second-line test had a sensitivity of 100% (95% CI; 87.1% to 100%) and specificity of 98.4% (95% CI; 94.2% to 99.5%). Using the OTA, 78/98 (80%) of initially positive SARS-CoV-2 IgG results were confirmed with a second-line test, while 11/42 (26%) of previously diagnosed COVID-19 patients had no detectable antibodies as long as 94 days post PCR diagnosis. CONCLUSION: Our results show that an OTA can be used to identify patients who require further follow-up due to potential SARS CoV-2 IgG false positive results. In addition, serological testing may not be sufficiently sensitive to reliably detect prior COVID-19 infection.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/blood , SARS-CoV-2/chemistry , Adolescent , Adult , Aged , Aged, 80 and over , Algorithms , Antibodies, Viral/immunology , COVID-19/blood , Cohort Studies , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/immunology , Middle Aged , Phosphoproteins/immunology , Sensitivity and Specificity , South Carolina , Spike Glycoprotein, Coronavirus/immunology , Young Adult
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