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1.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-320935

ABSTRACT

We have previously shown that beta 2 -integrin conformational change is a very early activation marker that can be detected with fluorescent multimers of its ligand ICAM-1 for a rapid assessment of antigen-specific CD8 + T cells. Here, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4 + T cells using a monoclonal antibody (clone m24) specific for the open, high affinity conformation of the beta 2 -integrin. Kinetics of beta 2 -integrin activation were different on CD4 + and CD8 + T cells (several hours vs. few minutes, respectively), however, m24 antibody readily stained both cell types 4-6 hours after antigen stimulation. With this protocol, we were able to monitor CD4 + and CD8 + virus-specific T cells specific for CMV, EBV, HBV, and SARS-CoV-2 in whole blood or cryopreserved PBMCs of infected or vaccinated individuals. By costaining with m24 and CD154 antibodies, we assessed extremely low frequencies of polyfunctional CD4 + T cell responses to SARS-CoV-2 derived peptides. Our novel assay thus allows very sensitive and simultaneous screening of both CD4 + and CD8 + T cell reactivities with versatile applicability in clinical and vaccination studies, and for epitope discovery.

2.
Front Immunol ; 12: 798859, 2021.
Article in English | MEDLINE | ID: covidwho-1581315

ABSTRACT

SARS-CoV-2 antibodies in saliva serve as first line of defense against the virus. They are present in the mucosa, more precisely in saliva, after a recovered infection and also following vaccination. We report here the antibody persistence in plasma and in saliva up to 15 months after mild COVID-19. The IgG antibody response was measured every two months in 72 participants using an established and validated in-house ELISA assay. In addition, the virus inhibitory activity of plasma antibodies was assessed in a surrogate virus neutralization test before and after vaccination. SARS-CoV-2-specific antibody concentrations remained stable in plasma and saliva and the response was strongly boosted after one dose COVID-19 vaccination.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Immunoglobulin G/immunology , Saliva/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/immunology , Female , Humans , Male , Middle Aged , SARS-CoV-2
3.
Front Immunol ; 12: 753435, 2021.
Article in English | MEDLINE | ID: covidwho-1485058

ABSTRACT

Saliva is a body fluid with hitherto unused potential for the assessment of SARS-CoV-2 antibodies. Specific antibodies can indicate a past SARS-CoV-2 infection and allow to estimate the proportion of individuals with a potential protective immunity. First, we carefully characterized plasma samples obtained from adult control groups with and without prior SARS-CoV-2 infection using certified reference ELISAs. Simultaneously collected saliva samples of confirmed convalescent and negative individuals where then used to validate the herein newly developed ELISA for the detection of SARS-CoV-2 IgG antibodies in saliva. The saliva ELISA was applied to assess SARS-CoV-2 exposure in young children (N = 837) in the age between 1 and 10 years in Tübingen, Germany, towards the end of the first pandemic year 2020. Sensitivity and specificity of the new saliva ELISA was 87% and 100%, respectively. With 12% of all Tübingen children sampled via their respective educational institutions, estimates of SARS-CoV-2 antibody prevalence was 1.6%. Interestingly, only 0.4% preschool kids were positive compared to 3.0% of primary school children. Less than 20% of positive children self-reported symptoms within two months prior to saliva sampling that could be associated - but not exclusively - with a SARS-CoV-2 infection. The saliva ELISA is a valid and suitable protocol to enable population-based surveys for SARS-CoV-2 antibodies. Using non-invasive sampling and saliva ELISA testing, we found that prevalence of SARS-CoV-2 antibodies was significantly lower in young children than in primary school children.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing , COVID-19 , SARS-CoV-2/immunology , Saliva/immunology , Adult , COVID-19/diagnosis , COVID-19/immunology , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Germany , Humans , Infant , Male , Prospective Studies
4.
JMIR Res Protoc ; 10(10): e27739, 2021 Oct 08.
Article in English | MEDLINE | ID: covidwho-1480494

ABSTRACT

BACKGROUND: The world has been confronted with the COVID-19 pandemic for more than one year. Severe disease is more often found among elderly people, whereas most young children and adolescents show mild symptoms or even remain asymptomatic, so that infection might be undiagnosed. Therefore, only limited epidemiological data on SARS-CoV-2 infection in children and young adults are available. OBJECTIVE: This study aims to determine the prevalence of SARS-CoV-2 antibodies in children from the city of Tübingen, Germany, and to measure the incidence of new cases over 12 months. METHODS: SARS-CoV-2 antibodies will be measured in saliva as a surrogate for a previous SARS-CoV-2 infection. Children will be sampled at their preschools, primary schools, and secondary schools at three time points: July 2020, October to December 2020, and April to July 2021. An adult cohort will be sampled at the same time points (ie, adult comparator group). The saliva-based SARS-CoV-2-antibody enzyme-linked immunosorbent assay will be validated using blood and saliva samples from adults with confirmed previous SARS-CoV-2 infections (ie, adult validation group). RESULTS: The first study participant was enrolled in July 2020, and recruitment and enrollment continued until July 2021. We have recruited and enrolled 1850 children, 560 adults for the comparator group, and 83 adults for the validation group. We have collected samples from the children and the adults for the comparator group at the three time points. We followed up with participants in the adult validation group every 2 months and, as of the writing of this paper, we were at time point 7. We will conduct data analysis after the data collection period. CONCLUSIONS: Infection rates in children are commonly underreported due to a lack of polymerase chain reaction testing. This study will report on the prevalence of SARS-CoV-2 infections in infants, school children, and adolescents as well as the incidence change over 12 months in the city of Tübingen, Germany. The saliva sampling approach for SARS-CoV-2-antibody measurement allows for a unique, representative, population-based sample collection process. TRIAL REGISTRATION: ClinicalTrials.gov NCT04581889; https://clinicaltrials.gov/ct2/show/NCT04581889. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/27739.

5.
Wien Klin Wochenschr ; 133(17-18): 931-941, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1351300

ABSTRACT

BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV­2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV­2 S­protein and receptor binding domain (RBD), and SARS-CoV­2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2 µg, n = 47, 4 µg, n = 48, 6 µg, n = 46, 8 µg, n = 44, 12 µg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S­protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S­protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 µg group. Responses to 12 µg were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 µg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.


Subject(s)
COVID-19 , Nanoparticles , Vaccines , Adolescent , Adult , Antibodies, Viral , COVID-19/therapy , COVID-19 Vaccines , Double-Blind Method , Humans , Immunization, Passive , Immunogenicity, Vaccine , Lipids , Middle Aged , RNA, Messenger , SARS-CoV-2 , Young Adult
6.
Sci Rep ; 11(1): 11899, 2021 06 07.
Article in English | MEDLINE | ID: covidwho-1260951

ABSTRACT

The pandemic caused by SARS-CoV-2 resulted in increasing demands for diagnostic tests, leading to a shortage of recommended testing materials and reagents. This study reports on the performance of self-sampled alternative swabbing material (ordinary Q-tips tested against flocked swab and rayon swab), of reagents for classical RNA extraction (phenol/guanidine-based protocol against a commercial kit), and of intercalating dye-based one-step quantitative reverse transcription real-time PCRs (RT-qPCR) compared against the gold standard hydrolysis probe-based assays for SARS-CoV-2 detection. The study found sampling with Q-tips, RNA extraction with classical protocol and intercalating dye-based RT-qPCR as a reliable and comparably sensitive strategy for detection of SARS-CoV-2-particularly valuable in the current period with a resurgent and dramatic increase in SARS-CoV-2 infections and growing shortage of diagnostic materials especially for regions limited in resources.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/pathogenicity , Specimen Handling , COVID-19 Testing/methods , Humans , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription/physiology , Specimen Handling/methods , Time Factors
7.
Heliyon ; 7(6): e07147, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1252937

ABSTRACT

The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID-19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8+ T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8+ T cells suggested that convalescent patients have significantly diminished expression of both perforin and granzyme A. Using 1H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8+ T cytotoxic functions and changes the overall metabolic functions of immune cells.

8.
Nat Commun ; 12(1): 3109, 2021 05 25.
Article in English | MEDLINE | ID: covidwho-1243298

ABSTRACT

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody Formation , COVID-19/blood , Female , Gene Expression , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Mutation , Neutralization Tests , Protein Binding , Protein Domains/genetics , Receptors, Coronavirus/metabolism , Recombinant Proteins , SARS-CoV-2/genetics , Saliva/immunology , Saliva/virology
9.
Front Immunol ; 12: 626308, 2021.
Article in English | MEDLINE | ID: covidwho-1190310

ABSTRACT

We have previously shown that conformational change in the ß2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the ß2-integrin. The kinetics of ß2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining ß2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Host-Pathogen Interactions/immunology , Integrins/metabolism , Adult , Aged , Amino Acid Sequence , Binding Sites , COVID-19/genetics , COVID-19/immunology , COVID-19/metabolism , COVID-19/virology , Carrier Proteins/chemistry , Cytokines/metabolism , Cytomegalovirus/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Female , HLA Antigens/chemistry , HLA Antigens/immunology , Host-Pathogen Interactions/genetics , Humans , Immunohistochemistry , Immunophenotyping , Integrins/genetics , Intercellular Adhesion Molecule-1/chemistry , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Activation/immunology , Male , Middle Aged , Protein Binding , Protein Multimerization , SARS-CoV-2/immunology , T-Cell Antigen Receptor Specificity , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
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