Characterization of Adaptive Immune Responses to SARS-CoV-2 Updated Bivalent Booster

Bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and Omicron BA.4/5 spike proteins are successful in preventing infection from the original strain and Omicron variants, but the quality of adaptive immune responses is still not well documented. This study aims at characterizing adaptive immune responses to the bivalent booster vaccination in 46 healthy participants. Plasma and PBMC were collected prior and three weeks after bivalent booster. We measured anti-N, anti-S, and RBD IgM, IgA, IgG plasma titers against original, Omicron BA.1, and BA.5 variants (pending) as well as total anti-S IgG titers and surrogate Virus Neutralization capacity against the Alpha, Delta, and BA.1 variant. With spectral flow-cytometry we identified peripheral blood B-cells specific for the RBD of the S-protein of the original and BA.1 variants. T-cell-specific responses were assessed by cytokine release assay after stimulation with SARS-CoV-2 peptides from the original, BA.1, BA.4, and BA.5 variants (pending). Finally, we performed TRB and IGH repertoire studies on sorted CD4+, CD8+, CD19+ lymphocytes, to study breadth of SARS-CoV-2 specific clonotypes (pending). 27/46 participants were analyzed;9 had SARS-CoV-2 infection (COVID+), while 18 are infection naive (COVID-). In both groups, median time since last dose of SARS-CoV-2 vaccine (3rd or 4th) was 11 months. All subjects were positive for anti-S IgG prior to bivalent booster. The COVID + group displayed anti-S IgG pre-booster levels and neutralization against BA.1 higher than the COVID- group. Significant increase post-boost of total anti-S IgG and BA.1 neutralizing activity was detected in the COVID- but not in the COVID+ group;however, no difference in neutralization activity post-boost was detected between the two groups. Furthermore, the COVIDgroup showed significant increase in the frequency of CD19+ and CD27+ switched memory B-cells specific for BA.1 RBD in post-boost compared to pre-boost samples. However, post-boost frequencies of the same B-cells were higher in the COVID+ compared to the COVID- group. These preliminary findings confirm that among individual immunized with the original COVID-19 mRNAvaccine, prior COVID infection provides increased protection against SARS-CoV-2 variants. They also demonstrate that booster immunization with the bivalent vaccine induces robust adaptive immune responses against Omicron variant.[Formula presented][Formula presented]Copyright © 2023 Elsevier Inc.

Serotonin Release Assay: Functional Assay for Heparin- and Vaccine-Induced (Immune) Thrombotic Thrombocytopenia.

The serotonin release assay (SRA) has been the gold-standard assay for detection of heparin-dependent platelet-activating antibodies and integral for the diagnosis for heparin-induced thrombotic thrombocytopenia (HIT). In 2021, a thrombotic thrombocytopenic syndrome was reported after adenoviral vector COVID-19 vaccination. This vaccine-induced thrombotic thrombocytopenic syndrome (VITT) proved to be a severe immune platelet activation syndrome manifested by unusual thrombosis, thrombocytopenia, very elevated plasma D-dimer, and a high mortality even with aggressive therapy (anticoagulation and plasma exchange). While the platelet-activating antibodies in both HIT and VITT are directed toward platelet factor 4 (PF4), important differences have been found. These differences have required modifications to the SRA to improve detection of functional VITT antibodies. Functional platelet activation assays remain essential in the diagnostic workup of HIT and VITT. Here we detail the application of SRA for the assessment of HIT and VITT antibodies.

Interferon gamma release assay results and outcomes in COVID-19 patients

Introduction/Aim: The interferon gamma release assay (IGRA), used in diagnosing latent tuberculosis infections (LTBI), relies on the release of interferon gamma from T-cells exposed to M. tuberculosis specific peptides. An 'indeterminate' IGRA result is most commonly due an inadequate control (or 'mitogen') response, which may reflect underlying T-cell dysfunction, that is potentially associated with markers of severity in patients with COVID-19. The aim of this study was to determine associations and predictors of an indeterminate IGRA in hospitalised patients with COVID-19. Method(s): We performed a single centre, retrospective study on COVID-19 patients admitted to a tertiary referral hospital who had IGRA testing performed over a 5 months period. Demographics, markers of COVID-19 severity and other parameters were recorded, along with outcomes of COVID-19 infection. The primary outcomes included predictors of indeterminate IGRA results and associations with COVID-19 outcomes (severity, length of stay and mortality). Result(s): A total of 181 patients were included for analysis. Outcomes of IGRA testing included negative (n = 117) and indeterminate (n = 60) results. Patients with a positive IGRA (n = 4) were excluded from analysis. The odds of an indeterminate IGRA were increased with a higher severity grade of COVID-19 (OR 2.5;95% CI 1.3-4.9), immunosuppression at baseline (OR 2.3;95% CI 1.1-4.7) and when IGRA testing was done after immunosuppression for COVID-19 was commenced (OR 1.4;95% CI 1.1-1.8). A longer length of stay was more likely with an indeterminate IGRA compared to a negative result (OR 1.08;95% CI 1.03-1.14), No difference in mortality between the two IGRA subgroups was found. Conclusion(s): Our study demonstrates an indeterminate IGRA was associated with markers of disease severity and immunosuppression. In this cohort an indeterminate result was also associated with worse COVID-19 outcomes in hospitalised patients. This result could potentially be used as a prognostic marker for patients admitted with COVID-19.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

Humoral and Cellular Response and Associated Variables Nine Months following BNT162b2 Vaccination in Healthcare Workers.

In this study, we aimed to illustrate the trajectory of humoral and cellular immunity nine months after primary vaccination with the BNT162b2 mRNA vaccine among 189 healthcare workers (HCWs). Additionally, we endeavored to identify correlations between immunity parameters and a number of common variables and comorbidities. A total of 189 healthcare workers (HCWs), vaccinated against COVID-19, were finally included in the study. All of the subjects had received two doses of the BNT162b2 vaccine; had undergone antibody tests one, four and nine months post-vaccination; and had completed a medical questionnaire. Further samples taken at nine months were tested for cellular immunity. No participants had evidence of COVID-19 infection pre- or post-vaccination. An anti-S1 receptor binding domain (RBD) antibody assay was used to assess humoral response, and cellular immunity was estimated with an INF-γ release assay (IGRA). Statistical analysis was performed using STATA. We report a statistically significant antibody drop over time. Being above the age of 40 or a smoker reduces the rise of antibodies by 37% and 28%, respectively. More than half of the participants did not demonstrate T-cell activation at nine months. Female gender and antibody levels at four months predispose detection of cellular immunity at nine months post-immunization. This study furthers the qualitative, quantitative, and temporal understanding of the immune response to the BNT162b2 mRNA vaccine and the effect of correlated factors.

Longitudinal humoral and cell-mediated immune responses in a population-based cohort in Zurich, Switzerland between March and June 2022 - evidence for protection against Omicron SARS-CoV-2 infection by neutralizing antibodies and spike-specific T-cell responses.

OBJECTIVES: The correlate(s) of protection against SARS-CoV-2 remain incompletely defined. Additional information regarding the combinations of antibody and T cell-mediated immunity which can protect against (re)infection is needed. METHODS: We conducted a population-based, longitudinal cohort study including 1044 individuals of varying SARS-CoV-2 vaccination and infection statuses. We assessed spike (S)- and nucleocapsid (N)-immunoglobulin(Ig)G and wildtype, Delta, and Omicron-neutralizing antibody (N-Ab) activity. In a subset of 328 individuals, we evaluated S, membrane (M), and N-specific T cells. Three months later, we reassessed Ab (n = 964) and T cell (n = 141) responses and evaluated factors associated with protection from (re)infection. RESULTS: At the study start, >98% of participants were S-IgG seropositive. N-IgG and M/N-T-cell responses increased over time, indicating viral (re)exposure, despite existing S-IgG. Compared to N-IgG, M/N-T cells were a more sensitive measure of viral exposure. High N-IgG titers, Omicron-N-Ab activity, and S-specific-T-cell responses were all associated with a reduced likelihood of (re)infection over time. CONCLUSION: Population-level SARS-CoV-2 immunity is S-IgG-dominated, but heterogeneous. M/N-T-cell responses can distinguish previous infection from vaccination, and monitoring a combination of N-IgG, Omicron-N-Ab, and S-T-cell responses may help estimate protection against SARS-CoV-2 (re)infection.

Erythema nodosum -Expanding diagnostic workout in COVID-19 pandemic

Case report Erythema nodosum (EN) is considered a delayed type IV hypersensitivity reaction, triggered by exposure to an antigen, which diagnostic workout is usually challenging. Several conditions have been described as possible causes for EN, including infections, sarcoidosis, pregnancy, neoplasic and inflammatory diseases. Rarely, vaccines such as tetanus, diphtheria, BCG, hepatitis B, human papillomavirus, malaria, rabies, smallpox, typhoid, and cholera have been associated with subsequent EN. We present a 31-year- old leucodermic female with suppurative adenitis, who developed painful erythematous nodules on the pretibial area of the lower limbs. Ten days prior to presentation she had received the first dose of the COVID-19 mRNA-1273 vaccine. Fever, lymphadenopathy, fatigue, weight loss, arthritis, cough, diarrhoea, other organ-specifc symptoms and close contact with tuberculosis were excluded. She was under oral contraception for several years, that was not discontinued. Pregnancy was excluded. No positive signs were detected on physical examination besides the referred nodules. Laboratory tests revealed a normal complete blood count, erythrocyte sedimentation rate, C-reactive protein, antistreptolysin O titer, renal and hepatic tests. Interferon-gamma release assay was negative. Circulating rheumatoid factor was normal, anti-nuclear, anti-double stranded DNA and anti-neutrophil cytoplasmatic antibodies were negative. Angiotensin converting enzyme and protein electrophoresis were normal. Hepatitis B and C, HIV 1/2 and syphilis serologic profiles were negative. Urinalysis and fecal calprotectin were unremarkable. The patient was treated with naproxen and topic betamethasone dipropionate. Violaceous involution was reported, with complete resolution of the EN lesions over the following month. In the literature, there are rare reports of EN following SARS-COV2 infection and also after COVID-19 vaccination. To our knowledge this is the second report of EN after the COVID-19 mRNA-1273 vaccine. This case highlights the importance of clinical awareness for the possible association of COVID-19 vaccination and EN, adding to the already extensive list of causes included in the etiological investigation of these patients.

Rapid cytokine release assay in whole blood determines longitudinal T-cell responses against ancestral, Delta, and Omicron SARS CoV-2 variants

Background: A simple, accurate and rapid whole blood-based T-cell test was previously developed to determine SARS-CoV- 2- specific T-cell immunity. Herein, the test was utilized to measure the magnitude of T-cell responses up to 6 months post-vaccination, assess the effects of vaccine boosters, and to elucidate any effect that Delta and Omicron variants may have on T-cell immunity. Method(s): Immunocompetent individuals (n = 44) were recruited to donate a blood sample between one-and six-months post-vaccination. Whole blood was stimulated overnight with peptides spanning immunodominant regions specific for ancestral SARS-CoV- 2. Blood plasma samples were analysed for IL-2 production via Luminex xMAP cytokine array, as this was previously demonstrated to be the most accurate biomarker for the test. Following booster vaccinations, 58 individuals donated a blood sample between one-and four-months post-booster and T-cell responses after overnight stimulations were assessed. Additionally, 30 samples were stimulated with peptides from the immunodominant regions of the Delta and Omicron SARS-CoV- 2 variants and IL-2 levels were compared. Result(s): A comparison of T-cell responses from samples donated up to one-month and six-months post-vaccination revealed no significant differences in the magnitude of IL-2 production (p = 0.9252), with near equivalent means. Booster vaccinations increased the magnitude of the T-cell response in 69% of individuals analysed, with the mean production of IL-2 rising from 77pg/ml six-months pre-booster to 141pg/ml 3-weeks post-booster. Analysis of the longevity of post-booster T-cell response demonstrated no significant differences in the magnitude of IL-2 (p = 0.8141) production, with near equivalent means at one-month and 4-months post-booster (119pg/ml and 111pg/ml, respectively). Finally, no significant differences in the magnitude of IL-2 were observed between samples stimulated with either ancestral, Delta or Omicron peptides, with the means of each group being highly comparable. Conclusion(s): Results from this rapid whole blood-based T-cell test indicate that T-cell immunity to multiple variants of SARS-CoV- 2 within immunocompetent cohorts does not wane significantly over time. However, booster vaccinations may be important for individuals who have lower levels of immunity following their first complete vaccination doses. This test could be a valuable tool in the assessment of SARS-CoV- 2 immunity in multiple cohorts of clinical vulnerable individuals.

Glucocorticoids selectively affect the memory T cell response to SARS-Cov2 spike in vaccinated and post-infected patients with systemic lupus erythematosus.

Immune response to vaccines and pathogens remains unclear in patients with systemic lupus erythematosus (SLE). To investigate this, a single-center retrospective study was conducted with 47 SLE patients vaccinated against COVID-19, including 13 who subsequently developed an asymptomatic/mild disease. As compared to controls, post-vaccine response against Spike was reduced in SLE patients when considering both memory T-cells in a whole blood interferon gamma release assay (IGRA-S) and IgG anti-Spike antibody (Ab) responses. The SLE-associated defective IGRA-S response was associated with a serum albumin level below 40 g/L and with the use of glucocorticoids, while a defective IgG anti-Spike Ab response was associated with lower levels of anti-dsDNA and anti-SSA/Ro 52 kDa Abs. IGRA-S and IgG anti-Spike responses were independent from SLE activity and clinical phenotype, low complement, hypergammaglobulinemia, and lymphopenia. As compared to controls, SLE patients showed a rapid decay of anti-Spike T-cell memory and stable IgG anti-Spike Ab responses. In conclusion, both T cell and humoral anti-Spike responses were independently affected in our SLE patients cohort, which supports the exploration of both responses in the follow-up of SLE patients and especially in those receiving glucocorticoids.

Evaluation of T cell responses with the QuantiFERON SARS-CoV-2 assay in individuals with 3 doses of BNT162b2 vaccine, SARS-CoV-2 infection, or hybrid immunity.

Cellular immunity after SARS-CoV-2 infection or immunization may be important for long-lasting protection against severe COVID-19 disease. We investigated cellular immune responses after SARS-CoV-2 infection and/or vaccination with an interferon-γ release assay (QuantiFERON, QFN), in parallel, with humoral immunity assessment. We recruited 41 participants: unvaccinated convalescent children and adults and vaccinated uninfected or vaccinated convalescent adults. All vaccinated adults had received three doses of the BNT162b2 COVID-19 vaccine at 6.2 to 10.9 months prior to their inclusion to the study. All the unvaccinated participants were tested negative with QFN. Regarding the vaccinated population, 50% (8/16) of the vaccinated uninfected adults and 57.1% (8/14) of the vaccinated convalescent adults were tested positive. QFN did not detect T cell responses in unvaccinated individuals and in a significant number of vaccinated individuals. Further comparative studies with different immunoassays are required to elucidate whether this is the result of waning immunity or low sensitivity of the assay.

Comparative analysis between STANDARD-E Covi-FERON ELISA with pre-existing IFN-γ release assays and determination of the optimum cutoff value for assessment of T-Cell response to SARS-CoV-2.

BACKGROUND: Interferon-gamma (IFN-γ) release assays (IGRAs) are useful for the assessment of the T-cell response to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). We aimed to assess the performance of the newly developed IGRA ELISA test compared to the pre-existing assays and to validate the cutoff value in real-world conditions. METHODS: We enrolled 219 participants and assessed agreement between STANDARD-E Covi-FERON ELISA with Quanti-FERON SARS-CoV-2 (QFN SARS-CoV-2), as well as with T SPOT Discovery SARS-CoV-2 based on Cohen's kappa-index. We further determined the optimal cutoff value for the Covi-FERON ELISA according to the immune response to vaccinations or infections. RESULTS: We found a moderate agreement between Covi-FERON ELISA and QFN SARS-CoV-2 before vaccination (kappa-index = 0.71), whereas a weak agreement after the first (kappa-index = 0.40) and second vaccinations (kappa-index = 0.46). However, the analysis between Covi-FERON ELISA and T SPOT assay demonstrated a strong agreement (kappa-index >0.7). The cut-off value of the OS (original spike) marker was 0.759 IU/mL with a sensitivity of 96.3% and specificity of 78.7%, and that of the variant spike (VS) marker was 0.663 IU/mL with a sensitivity and specificity of 77.8% and 80.6%, respectively. CONCLUSION: The newly determined cut-off value may provide an optimum value to minimize and prevent the occurrence of false-negative or false-positive during the assessment of T-cell immune response using Covi-FERON ELISA under real-world conditions.

Covid-19 Vaccination and Covid 19 Infection in Atypical Hemolytic Uremic Syndrome: One Single Center Study in Taiwan

Introduction: Acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia with multiple organ thrombotic microangiopathy (TMA) are typical characteristic presentation of Atypical hemolytic uremic syndrome(aHUS). Infection, pregnancy, operation, and some medication can be a trigger factor to induce the complement system over activation and induce atypical hemolytic uremic syndrome unstable to a life-threatening condition. Both SARS-CoV-2(Severe Acute Respiratory Syndrome Coronavirus 2) infection and COVID 19 vaccination are reported to be the trigger factors for aHUS. There are no clinical trial enrolled aHUS cases to COVID 19 vaccine or anti SARS-CoV2 agent. Therefore, aHUS became a tough medical issue in this pandemic status. In this study, we evaluate the efficacy and disease activity of aHUS after COVID 19 vaccination. Meanwhile, we analysis the severity of COVID 19 infection in our 21 aHUS cases. Method(s): There are 21 aHUS cases enrolled this study from April 2022 to September 2022. Each cases with regular blood sampling which include hemolysis markers (Hemoglobin, Platelet count, LDH, CH50, haptoglobin, Blood smear), renal function and urine analysis every months. While them had COVID 19 vaccination or COVID 19 infection, the above blood sampling and urine analysis should be followed up two weeks later. Once the aHUS cases became severe condition and need hospitalization, our medical team must visit these cases closely and monitor if any new critical issue happen. We confirmed the serum SARS-CoV-2 Spike IgG and Interferon-gamma (IFNgamma) release assay testing for the vaccination efficacy analysis. Result(s): 21 aHUS cases all had COVID 19 vaccination, 2 cases received 1 dose vaccine, 6 cases received 2 doses vaccine and 13 cases received 3 doses vaccine. Only one case with aHUS unstable after Moderna vaccine injection which is self-limited gradually and didn't need extra dose of anti-complement therapy. Interestingly, this case with stable aHUS disease activity while he switches to Pfizer-BioNTech vaccine as his 2nd dose. The SARS CoV-2 Spike IgG level and IFNgamma level are corelated to the dosage of COVID 19 vaccination, the higher doses with the higher level. The SARS-CoV2 spike IgG and IFNgamma level without lower response to the group with regular anti-C5 treatment. For those complete three dose vaccination cases, mix type of COVID-19 vaccination (AZ/mRNA) with better efficacy trend to fix type of mRNA. During this study period, there are 4 cases with COVID 19 infection. One case (already had 2 doses COVID 19 vaccination) needed hospitalization and improved after remdesivir and dexamethasone treatment who with mild aHUS disease activity progression. Two cases (complete three doses COVID 19 vaccination) with stable aHUS disease activity after Molnupiravir treatment. One case (complete three doses COVID 19 vaccination) refused Molnupiravir treatment and had mild aHUS disease activity progression. Conclusion(s): According to our study, we recommend the aHUS patient to have COVID 19 vaccination and multiple doses are more protective for them. aHUS disease activity should be close monitor especially after COVID 19 vaccination, during COVID 19 infection and after COVID 19 infection. Remdesivir and Molmupiravir are relative safe to use for aHUS cases. No conflict of interestCopyright © 2023

COVID-19 and Tuberculosis: Two Knives in a Sheath

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) has caused a global human outbreak, making it a more serious threat to human health than any other infectious disease. Coronavirus infectious disease 2019 (COVID-19) has severely affected the lifestyles of people around the world and caused high mortality throughout the world. In both pandemic and seasonal influenza, co-infection of COVID-19 with other diseases has been linked to worse outcomes. The literature revealed that it is char-acteristically associated with comorbidities such as hypertension, blood pressure, obesity, cardiovascular diseases, and other microbial infections. Furthermore, microbial coinfections worsen respiratory viral infections and are a common cause of death in influenza pandemics. Deplorably, Tuberculosis (TB) is also a dreadful lung infection and attains cytokine equilibrium with host cells to maintain the latent stage. Studies showed that human coronaviruses (hCoV) activate latent TB to an active state due to unregulated cytokine production, called a cytokine storm. The present review concisely discusses the reason and status of co-infection of COVID-19 with TB based on previous case reports, cohorts, and scientific studies. COVID-19 patients are prone to be infected with TB and vice-versa in TB-prone areas. The therapeutic opportunities for overcoming the COVID-19 induced cytokine storm have also been emphasized by the present clinical trial candidates. In conclusion, we recommend categorizing the patients based on their medical history and cured or latent TB patients should be particularly closely monitored. They should be tested for Interferon Gamma Release Assay (IGRA) regularly on and after COVID-19 infection.Copyright © 2022 Bentham Science Publishers.

Investigation of cellular and humoral immune responses following COVID-19 vaccination in respiratory healthcare professionals

Background: SARS-CoV-2 vaccines are expected to induce both cellular and humoral immune responses, however, the dynamics and correlation between the two types of immunity are not precisely understood. Aim(s): Assessing IgG levels and T-cell response induced by SARS-CoV-2 vaccines and investigating the correlation between cellular and humoral immune responses. Method(s): Blood samples were taken from 166 respiratory healthcare professionals at four time-points: first before administering the booster vaccine, then on day 28, 56 and 120 post-vaccination. For the assessment of humoral immune response anti-Spike protein IgG ELISA was used, while T-cell response was tested by interferon-gammarelease-assay. Result(s): Out of 166 patients, 120 individuals presented positive result for interferon-gamma, while 100 had positive results for IgG. The positivity rate of cellular immune response was found to be significantly higher than humoral between the second and third doses of anti-COVID vaccines (P<0,05). Participants, who have had SARS-CoV-2 infection before the first two shots, the immune response was detectable at a statistically higher rates than in the COVID naive group. The third dose triggered different dynamics regarding the IgG titers and T-cell response. Four months after the administration of the booster shot both humoral and cellular immune response were detectable simultaneously. Conclusion(s): After the first two doses, the cellular immune response was found to last longer than humoral, especially in previously infected individuals. Measuring the T-cell responses to SARS-CoV-2 vaccines may complement the antibody tests currently used in clinical practice.

SARS-CoV-2 specific T-cell humoral response assessment after COVID-19 vaccination using a rapid direct real-time PCR amplification.

OBJECTIVES: The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10, and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release. METHODS: Whole blood (Li-He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li-He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China). RESULTS: Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and the time between the last vaccination or positivity. CONCLUSIONS: Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific T cells, especially when rapid analyses are required, such as for organ transplantation.

Performance of an interferon-γ release assay-based test for cell-mediated immunity to SARS-CoV-2.

In search for immunological correlates of protection against acute coronavirus disease 2019 (COVID-19) there is a need for high through-put assays for cell-mediated immunity (CMI) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We established an interferon-γ release assay -based test for detection of CMI against SARS-CoV-2 spike (S) or nucleocapsid (NC) peptides. Blood samples obtained from 549 healthy or convalescent individuals were measured for interferon-γ (IFN-γ) production after peptide stimulation using a certified chemiluminescence immunoassay. Test performance was calculated applying cutoff values with the highest Youden indices in receiver-operating-characteristics curve analysis and compared to a commercially available serologic test. Potential confounders and clinical correlates were assessed for all test systems. 522 samples obtained from 378 convalescent in median 298 days after PCR-confirmed SARS-CoV-2 infection and 144 healthy control individuals were included in the final analysis. CMI testing had a sensitivity and specificity of up to 89% and 74% for S peptides and 89% and 91% for NC peptides, respectively. High white blood cell counts correlated negatively with IFN-γ responses but there was no CMI decay in samples obtained up to one year after recovery. Severe clinical symptoms at time of acute infection were associated with higher measures of adaptive immunity and reported hair loss at time of examination. This laboratory-developed test for CMI to SARS-CoV-2 NC peptides exhibits excellent test performance, is suitable for high through-put routine diagnostics, and should be evaluated for clinical outcome prediction in prospective pathogen re-exposure.

Comparison of Two Commercially Available Interferon-γ Release Assays for T-Cell-Mediated Immunity and Evaluation of Humoral Immunity against SARS-CoV-2 in Healthcare Workers.

Cellular immunity against SARS-CoV-2 is an important component of the immune response to the virus. At present, two such tests based on interferon-gamma release (interferon-γ release assays, IGRAs) are available-Quan-T-Cell SARS-CoV-2 by EUROIMMUN and T-SPOT.COVID by Oxford Immunotec. In this paper, we compared the results of these two tests in 90 subjects employed at the Public Health Institute Ostrava who had previously undergone COVID-19 infection or were vaccinated against that disease. To the best of our knowledge, this is the first head-to-head comparison of these two tests evaluating T-cell-mediated immunity against SARS-CoV-2. In addition, we also evaluated humoral immunity in the same individuals using the in-house virus neutralization test and IgG ELISA assay. The evaluation yielded similar results for both IGRAs, with Quan-T-Cell appearing to be insignificantly (p = 0.08) more sensitive (all 90 individuals were at least borderline positive) than T-SPOT.COVID (negative results found in five patients). The overall qualitative (presence/absence of immune response) agreement of both tests with virus neutralization test and anti-S IgG was also excellent (close or equal to 100% in all subgroups, with the exception of unvaccinated Omicron convalescents, a large proportion of whom, i.e., four out of six subjects, were IgG negative while at least borderline positive for T-cell-mediated immunity measured by Quan-T). This implies that the evaluation of T-cell-mediated immunity is a more sensitive indicator of immune response than the evaluation of IgG seropositivity. This is true at least for unvaccinated patients with a history of being infected only by the Omicron variant, but also likely for other groups of patients.

Assessment of humoral and cellular immunity after bivalent BNT162b2 vaccination and potential association with reactogenicity.

OBJECTIVES: This study investigated the feasibility and clinical value of using a novel, automated and high-throughput SARS-CoV-2 Interferon Gamma Release Assay (IGRA), combined with total anti-SARS-CoV-2 antibodies assessment, for evaluating the immune response after bivalent BNT162b2 vaccination. METHODS: A cohort of healthcare workers, who already underwent primary vaccination and boosting with monovalent BNT162b2 vaccine, received a booster dose of the new BNT162b2 bivalent formulation. Blood samples were taken immediately before vaccination (T0) and 1 month afterwards (T1). Humoral and cellular immunity were assayed with Roche Elecsys Anti-SARS-CoV-2 and Roche Elecsys IGRA SARS-CoV-2, respectively. RESULTS: The study population consisted of 51 subjects (median age: 43 years; 51% females). Total anti-SARS-CoV-2 antibodies and IGRA SARS-CoV-2 values increased at T1 from 9,050 to 25,000 BAU/mL (p<0.001), and from 0.44 to 0.78 IU/mL (p=0.385), accounting for median increase of 2.0 and 1.6 folds, respectively. Increased T1 values of total anti-SARS-CoV-2 antibodies and IGRA SARS-CoV-2 were recorded in 100% and 68.6% subjects, respectively. In those with baseline values below the median, post-vaccine levels displayed larger increases of 3.3 and 5.1 folds for anti-SARS-CoV-2 total antibodies and IGRA SARS-CoV-2, respectively. The variation of total anti-SARS-CoV-2 antibodies was inversely associated with their T0 values (r=-0.97; p<0.001), whilst that of IGRA SARS-CoV-2 was inversely associated with its T0 value (r=-0.58; p<0.001). No other signifcant associations were found with demographical or clinical variables, including side effects. CONCLUSIONS: The bivalent BNT162b2 vaccine booster enhances humoral and cellular immunity against SARS-CoV-2, especially in recipients with lower baseline biological protection.

A pilot study on COVID-19 T-Cell in vaccinated healthcare workers, using Interferon-gamma Release Assay

As is known T-cells play a central role in the immunological response [1]. Nowadays new assays are being developed for the indirect quantification of T-cell memory activity [2]. The aim of this work was to demonstrate Interferongamma Release Assay (IGRA) test could be useful for vaccination monitoring. 23 vaccinated healthcare workers were enrolled in the study after 8 months of the Pfizer BioNTech vaccination. The antibody levels were assessed through Chemiluminescence immunoassay. T cells were indirectly analyzed by an ELISA against INFgamma. Lymphocyte subtyping was evaluated. Statistical analyses were processed. The patients were divided into 3 different groups based on S-RBD and ACE-2 antibody levels: the S-RBD and ACE-2 antibodies were significantly lower in Group 1 than in Group 2 (p<0.001). However, T cells revealed no significant difference between Group 1 and Group 2. Group 3 was the negative control. The results supported the actual role of SARS-CoV-2 T cell, expressed after the vaccine administration and persisting at high concentration over time, despite the antibody levels [3] [4]. Consequently, the new IGRA test was revealed to be an immunological screening that offers information on the protection from SARS-CoV-2 and suggests new strategies for doses administration.