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.

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.

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 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.

Evaluation of ichroma™ COVID-19 interferon gamma release assay for detection of vaccine-induced immunity in healthcare workers.

OBJECTIVES: We compared the performance of a new interferon gamma release assay (IGRA) format assay, the ichroma™ COVID-19 IGRA (IGRA-SARS), with that of the widely used QuantiFERON SARS-CoV-2 ELISA kit (QFN-SARS) in vaccinated healthcare workers (HCWs). Additionally, we analyzed the long-term changes in IGRA results after the final vaccine dose. METHODS: A total of 383 specimens from 281 HCWs were enrolled in this study, and the results of SARS-IGRA and QFN-SARS assays were compared. In addition, we performed the receive operator curve analysis to estimate the optimal cut-off value for IGRA-SARS. RESULTS: For all specimens, IGRA-SARS and QFN-SARS showed 75.7% and 64.2% of the positive results, respectively. The absolute agreement between IGRA-SARS and QFN-SARS was 80.0%, and the Fleiss' κ value was 0.525, indicating moderate agreement. ROC curve analysis of the IGRA-SARS results showed a cut-off value of >0.254 IU/mL, which was consistent with the manufacturer's specifications. The positive rates of both IGRA assays decreased significantly after a postvaccination period of 6 months. CONCLUSIONS: IGRA-SARS showed acceptable performance in the detection of vaccine-induced immunity against COVID-19; however, harmonization of IGRA assays has not yet been achieved. Additionally, the significant decline of positive rates of IGRA after the last vaccination would support the necessity of booster vaccination after a postvaccination period of 6 months.

Immunity following SARS-CoV-2 vaccination in autoimmune neurological disorders treated with rituximab or ocrelizumab

Introduction: B cell-depleting therapy targeting CD20 molecule with rituximab (RTX) or ocrelizumab (OCR) affects humoral immune response after vaccination. It remains unclear whether these therapies influence T-cell-mediated immune response against SARS-CoV-2 after immunization. Aim(s): We aimed to evaluate the humoral and cellular immune response to the COVID-19 vaccine in a cohort of patients with multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), and myasthenia gravis (MG). Method(s): Patients with MS (83), NMOSD (19), MG (7) under RTX (n=47) or OCR treatment (n=62) were vaccinated twice with mRNA BNT162b2 vaccine. Antibodies were quantified using the SARS-CoV-2- IgG chemiluminescence immunoassay targeting the spike protein. SARS-CoV-2-specific T-cell responses were quantified by interferon gamma release assays (IGRA). Immunocompetent vaccinated individuals (n=41) were included as controls. Result(s): Almost all immunocompetent controls developed antibodies against the SARS-CoV-2 trimeric spike protein, but only 42.05% of the patients under anti-CD20 (RTX or OCR) treatment seroconverted. There was no correlation between circulating B cells and the levels of antibodies. Even patients with a low proportion of circulating CD19+ B cells (<1%, 71 patients) had detectable SARS-CoV-2 specific antibody responses. This response was even higher in patients with longer than 3 weeks intervals of vaccination. SARS-CoV-2 specific T cell response measured by released interferon gamma was detected in 94.39% of the patients, independently of a humoral immune response. Conclusion(s): The majority of MS and NMOSD patients developed SARS-CoV-2-specific T cell response. The data suggest that vaccination can induce SARS-CoV-2-specific antibodies in a part of anti-CD20 treated patients. The response represented by levels of antibodies was better in individuals, who completed vaccination within more than 3 weeks.

Different type and timing of S1P receptor modulator therapy impacts T and B cell response after SARS-CoV2 vaccination

Introduction: The generation of humoral and cellular responses by SARS-CoV2 vaccination in patients with multiple sclerosis (pwMS) especially onsphingosin-1-phosphat receptor (S1PR) modulator treatment is not yet understood. In this study we aim to differentiate the immunological response profiles after 2 mRNA SARS-CoV2 vaccinations depending on timing and unselective vs. selective S1PR modulators in pwMS. Method(s): We conducted a cross-sectional study among pwMS on ozanimod (OZA), fingolimod (FTY) or without disease-modifying treatment. Two courses of mRNA SARS-CoV-2 vaccinations were performed on treatment resp. before treatment start. Demographic data on age, sex and disease progression did no significantly differ in selected groups. Blood was analyzed for SARS-CoV-2 spike protein-specific antibodies and for CD4 and CD8 T cell response by interferon-gamma release assay upon stimulation. Result(s): All untreated pwMS (n=31) developed positive antibodies (930.9 +/-803.7 BAU/mL), but only in 50% positive T cell responses were seen (S1 0.39 +/-0.68;S2 0.43 +/-0.67 IU/mL). PwMS on longterm FTY treatment (n=86) showed B cell responses (45.0 +/-117.9 BAU/mL) only in 27,9% and T cell responses (S1 0.11 +/-0.86;S2 0.01 +/-0.05 IU/mL) only in 5,9% both with significant lower titers compared to untreated pwMS. In contrast, pwMS on OZA (n= 22) developed B cell responses in 84.2% of patients (703.8 +/-837.5 BAU/mL) with lower titers than untreated pwMS. T cell responses were present in only 4,5% of patients on OZA (S1 0.02 +/-0.05;S2 0.03 +/-0.09 IU/mL). When patients were vaccinated before OZA start (n=25) and tested later on OZA treatment, all patients presented with positive antibody titers comparable to untreated patients (1466.2 +/-838.8 BAU/mL). However, T cell responses could be detected only in 19,0% of these OZA patients (S1 0.05 +/-0.11;S2 0.06 +/-0.14 IU/mL). In summary, only 15.8% OZA, but 69,8% FTY patients did neither develop B nor T cell response. In contrast untreated patients as well as vaccinated patients before OZA treatment start present with at least one B or T cell response. Conclusion(s): In this study, we present that B and T cell responses to SARS-CoV2 mRNA vaccines are selectively affected by different S1PR modulators. Vaccination before start of S1PR modulation induced a stable B cell response which could be demonstrated on treatment. These findings should be considered for vaccination strategies during S1PR modulatory therapy.

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine.

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.

ANTIBODY AND T-CELL RESPONSE SIX MONTHS AFTER INITIATION OF BNT162B2 VACCINATION IN PATIENTS WITH MULTIPLE MYELOMA OR CHRONIC LYMPHATIC B-CELL LEUKEMIA COMPARED TO HEALTHY CONTROLS

Background: The ongoing COVID-19 pandemic has resulted in more than 419 million cases and more than 5.9 million deaths. Preious studies hae indicated inferior responses to SARS-CoV-2 accination across different hematological diseases. Through this prospectie cohort study, we examined the deelopment and durability of anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients with either multiple myeloma (MM) or Chronic Lymphatic B-cell Leukemia (B-CLL) six months after accination and compared to immunocompetent controls. Aims: We aimed to inestigate the durability of immune responses to COVID-19 accination in patients with MM or B-CLL compared to healthy controls, and to identify risk factors for humoral non-response, including type of diagnosis. Methods: We measured anti-receptor binding domain (RBD) IgG after two doses of BNT162b2 in 179 patients (MM: n=78, B-CLL: n=101) and 179 age and sex matched healthy controls up to six months after first accination. Anti- RBD IgG leels and neutralizing capacity of antibodies were measured at first and second dose of BNT162b2 and two and six months after first dose. Humoral response was defined as anti-RBD IgG > 225 AU/mL with a neutralizing index ≥ 25%. Humoral non-response was defined as the absence of a humoral response. T-cell responses were assessed six months after the first dose using an ELISA-based interferon-gamma release assay. A positie T-cell response was defined as IFN-γ release > 200 mIU/mL. Data on diagnoses were obtained through medical records, and data on accination status were obtained from the Danish Vaccination Register. Results: In patients with MM or B-CLL, the geometric mean concentration (GMC) of anti-RBD IgG increased from baseline 1.49 AU/mL (95% CI: 1.21-1.84) to three weeks after the first accine dose 15.10 AU/mL (95% CI: 9.39- 24.29) and after receiing the second dose 1179.60 AU/mL (95% CI: 727.78-1919.85). From two to six months after first accine there was a significant decline in the GMC of anti-RBD IgG to 252.75 AU/mL (95% CI: 159.17-403.43). The mean neutralizing capacity in patients with MM or B-CLL was lower than in controls at all time points after the first accine dose. Six months after first accine dose, 79 of 179 (44.1%) patients with MM or B-CLL had a positie humoral response, while this was the case for 170 of 179 controls (95.0%), p<0.001. Haing MM or B-CLL was significantly associated with risk of humoral non-response. This was most pronounced in B-CLL patients who had an age and sex adjusted risk ratio (RR) of 12.25 (95% CI: 6.42-23.38, p< 0.001) of humoral non-response compared to healthy controls. For MM patients the RR was 4.65 (95% CI: 2.21-9.80, p< 0.001). T-cell response was assessed in a subset of 48 patients with MM (n=28) or B-CLL (n=20) and 26 controls, six months after first accine dose. A total of 21 (43.8%) patients with MM (12/28) or B-CLL (9/20) and 14 (53.8%) controls had a positie T-cell response (p =0.56). Seen of 20 (35.0%) patients with MM or B-CLL who did not deelop a humoral response, deeloped a T-cell response (MM: 3/8, B-CLL: 4/12), while 14 of 28 (50.0%) patients with MM or B-CLL who deeloped a humoral response deeloped a T-cell response (p =0.46, MM: 9/11, B-CLL: 5/8). In healthy controls 14 of 25 (56.0%) people who deeloped a humoral response also deeloped a T-cell response. Summary/Conclusion: Humoral response to BNT162b2 was impaired in patients with MM or B-CLL compared to healthy controls. Both patients with MM and B-CLL were at higher risk of humoral non-response compared to healthy controls.

CELLULAR IMMUNE RESPONSE TO THE BNT162B2 VACCINE IN LYMPHOMA PATIENTS

Background: In most individuals, protective humoral and cellular immunity develops after two doses of the BNT162b2 Pfizer vaccine. In patients with lymphoma, humoral response is weaker and almost universally abrogated in patients who received anti-CD20 monoclonal antibodies. Whether cellular immune response is also abrogated is unknown. Aims: To determine whether patients with lymphoma develop specific T-cell mediated cellular response to BNT162b2 Pfizer vaccine. Methods: We included patients with lymphoma above the age of 18 years who received two doses of the BNT162b2 Pfizer vaccine and collected clinical and demographics data. T-cell immune response to the vaccine was analysed in patients' blood samples stimulated by spike antigen and quantified by two methods: (1) Interferon-gamma (IFNg)- release assay (IGRA, EuroImmun, Germany)- IFNg was quantified by ELISA (DuoSet, R and D Systems, Minneapolis, Minnesota, USA) and response above 50 pg/ml was considered positive. (2) Flow cytometry- Quantification of the T cell activation markers, CD134+ CD25+CD4+ T-cells was performed (Act-T4 CellTM kit, Cytognos, Spain), and any response above 0 was considered positive. Humoral response was measured by SARS-CoV-2 IgG II Quant (Abbott©) assay. The positive cut-off was set at 50AU/ml. Blood samples were drawn approximately 4 months after the second vaccination. Results: Sixty-nine lymphoma patients, treated with two vaccine doses, were included in this study. Median age was 66 (range: 30-84) and 39 (57%) were males. Sixty-two patients (90%) had non-Hodgkin lymphoma (NHL) including 18 with DLBCL, 26 with follicular lymphoma and 14 with marginal zone lymphoma. Seven (10%) patients had Hodgkin lymphoma. In this cohort, 70% (n=49) of the patients received anti CD20 MoAb, and 35% of them (n=27) were still on anti CD20 treatment. Thirteen patients received bendamustine-based immunochemotherapy. At the time of assessment (median 4.8 months after the 2nd vaccine) anti-spike antibodies were detected in only 42% (N = 29) of patients. In comparison, there was an increase in specific T cell response by any assay (IGRA and Flow) in 49% of patients (n = 34). The correlation between the IGRA and flow data was 0.7 (pearson correlation, P = 0.01). However, no correlation between humoral (qualitative and quantitative) and T cell response was shown, regardless of the assay applied. Cellular response was not corelated with the time elapsing from last immunochemotherapy. In the anti-CD20 MoAb treated cohort, of which 27 patients were still on active treatment at the time of vaccination, only 2 patients (7%) developed a humoral immune response, while cellular immunity was elicited in 52% (N = 15) patients (ELISA assay). In the Bendamustine treated cohort, with a median time from end of treatment to vaccination of 23 months (1-106 months), humoral but not cellular response correlated positively with the time from treatment completion to vaccination (p=0.04). Summary/Conclusion: The rate of cellular and humoral response to two doses of the BNT162b2 Pfizer vaccine in lymphoma patients was found to be significantly abrogated. In this small cohort, 49% of patients developed a cellular response despite a severely abrogated humoral immunity. These findings suggest that vaccine administration should be considered even early after anti CD20 therapy despite the reduced humoral immunity. These findings should be validated in studies with a higher number of patients.