Humoral and cellular immune response to SARS-CoV-2 mRNA BNT162b2 vaccine in pediatric kidney transplant recipients compared with dialysis patients and healthy children.

BACKGROUND: Compared with the general population, the immune response to COVID-19 mRNA vaccines is lower in adult kidney transplant recipients (KTRs). However, data is limited for pediatric KTRs. In this study, we aimed to assess humoral and cellular immune responses to the COVID-19 mRNA vaccine in pediatric KTRs. METHODS: This multicenter, prospective, case-control study included 63 KTRs (37 male, aged 12-21 years), 19 dialysis patients, and 19 controls. Humoral (anti-SARS-CoV2 IgG, neutralizing Ab (nAb)) and cellular (interferon-gamma release assay (IGRA)) immune responses were assessed at least one month after two doses of BNT162b2 mRNA vaccine. RESULTS: Among COVID-19 naïve KTRs (n = 46), 76.1% tested positive for anti-SARS-CoV-2 IgG, 54.3% for nAb, and 63% for IGRA. Serum levels of anti-SARS-CoV-2 IgG and nAb activity were significantly lower in KTRs compared to dialysis and control groups (p < 0.05 for all). Seropositivity in KTRs was independently associated with shorter transplant duration (p = 0.005), and higher eGFR (p = 0.007). IGRA titer was significantly lower than dialysis patients (p = 0.009). Twenty (43.4%) KTRs were positive for all immune parameters. Only four of 11 seronegative KTRs were IGRA-positive. COVID-19 recovered KTRs had significantly higher anti-SARS-CoV-2 IgG and nAb activity levels than COVID-19 naïve KTRs (p = 0.018 and p = 0.007, respectively). CONCLUSIONS: The humoral and cellular immune responses to SARS-CoV-2 mRNA BNT162b2 vaccine are lower in pediatric KTRs compared to dialysis patients. Further prospective studies are required to demonstrate the clinical efficacy of the mRNA vaccine in KTRs. This prospective study was registered in ClinicalTrials.gov (NCT05465863, registered retrospectively at 20.07.2022). A higher resolution version of the Graphical abstract is available as Supplementary information.

T Lymphocyte Subset Counts and Interferon-Gamma Production in Adults and Children with COVID-19: A Narrative Review.

Adults and children exhibit a broad range of clinical outcomes from SARS-CoV-2 infection, with minimal to mild symptoms, especially in the pediatric age. However, some children present with a severe hyperinflammatory post-infectious complication named multisystem inflammatory syndrome in children (MIS-C), mainly affecting previously healthy subjects. Understanding these differences is still an ongoing challenge, that can lead to new therapeutic strategies and avoid unfavorable outcomes. In this review, we discuss the different roles of T lymphocyte subsets and interferon-γ (IFN-γ) in the immune responses of adults and children. Lymphopenia can influence these responses and represent a good predictor for the outcome, as reported by most authors. The increased IFN-γ response exhibited by children could be the starting point for the activation of a broad response that leads to MIS-C, with a significantly higher risk than in adults, although a single IFN signature has not been identified. Multicenter studies with large cohorts in both age groups are still needed to study SARS-CoV-2 pathogenesis with new tools and to understand how is possible to better modulate immune responses.

Cellular and humoral immunogenicity of the COVID-19 vaccine and COVID-19 disease severity in individuals with immunodeficiency.

Background: A well-coordinated adaptive immune response is crucial for limiting COVID-19 disease. Some individuals with immunodeficiency are at a high risk of developing severe COVID-19. Therefore, the development of standardized methods for measuring different arms of the vaccine response in the setting of immunodeficiency is of particular interest. In this study, we compared the vaccine response of individuals living with immunodeficiency with healthy controls in terms of interferon gamma (IFN-γ) production and spike protein-specific antibody level post primary COVID-19 vaccination and booster vaccines. Additionally, the disease severity of those individuals who contracted COVID-19 was assessed. Methods: Whole blood was stimulated overnight from 71 participants and 99 healthy controls. Commercially available PepTivator® peptide pool and trimeric spike protein stimulation were used. ELISA was used to analyze IFN-γ levels. The total SARS-CoV-2 spike protein antibody titre was measured using a Roche Elecsys® S total antibody assay. Patient characteristics, COVID-19 infection status and IDDA 2.1 'Kaleidoscope' scores were recorded. Vaccine responses were scored from zero to three. Results: 99% of healthy controls, 89% of individuals with IEI and 76% with secondary immunodeficiency (SID) had an IFN-γ level above the validated reference range after peptide mix stimulation following primary vaccination. There was an increase in IFN-γ levels in patients with inborn errors of immunity (IEI) following the booster vaccine (p = 0.0156). 100% of healthy controls, 70% of individuals living with IEI and 64% of individuals living with SID had detectable spike protein-specific antibody levels following the primary vaccination. 55% of immunodeficiency patients who had mild COVID-19 and 10% with moderate/severe COVID-19 had detectable antibody and IFN-γ levels post vaccine. The mean pre-infection IDDA 2.1 scores were higher in individuals who developed moderate/severe COVID-19 (25.2 compared to 9.41). Conclusions: Covid whole-blood IGRA is a highly accurate, straightforward and robust assay and can be easily adapted to measure cellular response to COVID-19. A complete evaluation of the vaccine response may be particularly important for individuals living with immunodeficiency. A clinical immunodeficiency score and a validated vaccine response score may be valuable tools in estimating COVID-19 disease risk and identifying individuals living with immunodeficiency who may benefit from enhanced vaccination schedules.

Humoral and cellular response after BNT162b2 vaccine booster in hemodialysis patients and kidney transplant recipients.

BACKGROUND: Vulnerable populations, such as hemodialysis (HD) patients and kidney transplant (RTx) recipients, have priority for anti-COVID-19 vaccination, because of their impaired immune status. Here, we investigated the immune response after vaccination with BNT162b2 (two doses plus booster) in HD and RTx patients. METHODS: A prospective, observational study was started in two homogeneous groups of 55 HD and 51 RTx patients previously matched from a cohort of 336 patients. Anti-RBD IgG levels, assayed after the second dose with BNT162b2 mRNA, were used to stratify subjects into quintiles. After the second dose and after booster, anti-RBD and IGRA test were evaluated in RTx and HD, belonging to the first and fifth quintiles. RESULTS: After the second dose of vaccine, the median circulating levels of anti-RBD IgG were significantly higher in HD (1456 AU/mL) compared to RTx (27.30 AU/mL). IGRA test showed significantly higher values in the HD (382 mIU/mL) compared with the RTx (73 mIU/mL). After the booster, humoral response increased significantly in both HD (p = 0.0002) and RTx groups (p = 0.009), whereas the T-cellular immunity remained essentially stable in most patients. In RTx patients with a low humoral response after the second dose, the third dose did not significantly strengthen either humoral or cellular immunity. CONCLUSIONS: For HD and RTx, there is great variability in the humoral response to anti-COVID-19 vaccination, with a stronger response in the HD group. The booster dose was ineffective at reinforcing the humoral and cellular immune response in most RTx patients hyporesponsive to the second dose.

Monitoring of Both Humoral and Cellular Immunities Could Early Predict COVID-19 Vaccine Efficacy Against the Different SARS-CoV2 Variants.

Reliable immunoassays are essential to early predict and monitor vaccine efficacy against SARS-CoV-2. The performance of an Interferon Gamma Release Assay (IGRA, QuantiFERON® SARS-CoV-2), and a current anti-spike serological test, compared to a plaque reduction neutralization test (PRNT) taken as gold standard were compared. Eighty vaccinated individuals, whose 16% had a previous history of COVID-19, were included in a longitudinal prospective study and sampled before and two to four weeks after each dose of vaccine. In non-infected patients, 2 doses were required for obtaining both positive IGRA and PRNT assays, while serology was positive after one dose. Each dose of vaccine significantly increased the humoral and cellular response. By contrast, convalescent subjects needed a single dose of vaccine to be positive on all 3 tests. Both IGRA and current serology assay were found predictive of a positive titer of neutralizing antibodies that is correlated with vaccine protection. Patients over 65 or 80 years old had a significantly reduced response. The response tended to be better with the heterologous scheme (vs. homologous) and with the mRNA-1273 vaccine (vs. BNT162b2) in the homologous group, in patients under 55 and under 65 years old, respectively. Finally, decrease intensity or absence of IGRA response and to a less extent of anti-spike serology were also correlated to reinfection which has occurred during the follow up. In conclusion, both IGRA and current anti-spike serology assays could be used at defined thresholds to monitor the vaccine response against SARS-CoV-2 and to simply identify non-responding individuals after a complete vaccination scheme. Two available specific tests (IGRA and anti-spike antibodies) could early assess the vaccine-induced immunity against SARS-CoV-2 at the individual scale, to potentially adapt the vaccination scheme in non-responder patients.

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.

Evaluation of SARS-CoV-2-Specific T-Cell Activation with a Rapid On-Chip IGRA.

Interferon-gamma release assays (IGRAs) that measure pathogen-specific T-cell response rates can provide a more reliable estimate of protection than specific antibody levels but have limited potential for widespread use due to their workflow, personnel, and instrumentation demands. The major vaccines for SARS-CoV-2 have demonstrated substantial efficacy against all of its current variants, but approaches are needed to determine how these vaccines will perform against future variants, as they arise, to inform vaccine and public health policies. Here we describe a rapid, sensitive, nanolayer polylysine-integrated microfluidic chip IGRA read by a fluorescent microscope that has a 5 h sample-to-answer time and uses ∼25 µL of a fingerstick whole blood sample. Results from this assay correlated with those of a comparable clinical IGRA when used to evaluate the T-cell response to SARS-CoV-2 peptides in a population of vaccinated and/or infected individuals. Notably, this streamlined and inexpensive assay is suitable for high-throughput analyses in resource-limited settings for other infectious diseases.

Long-Term Accuracy of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Interferon-γ Release Assay and Its Application in Household Investigation.

BACKGROUND: An immunodiagnostic assay that sensitively detects a cell-mediated immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed for epidemiological investigation and for clinical assessment of T- cell-mediated immune response to vaccines, particularly in the context of emerging variants that might escape antibody responses. METHODS: The performance of a whole blood interferon-gamma (IFN-γ) release assay (IGRA) for the detection of SARS-CoV-2 antigen-specific T cells was evaluated in coronavirus disease 2019 (COVID-19) convalescents tested serially up to 10 months post-infection and in healthy blood donors. SARS-CoV-2 IGRA was applied in contacts of households with index cases. Freshly collected blood in the lithium heparin tube was left unstimulated, stimulated with a SARS-CoV-2 peptide pool, and stimulated with mitogen. RESULTS: The overall sensitivity and specificity of IGRA were 84.5% (153/181; 95% confidence interval [CI]: 79.0-89.0) and 86.6% (123/142; 95% CI: 80.0-91.2), respectively. The sensitivity declined from 100% (16/16; 95% CI: 80.6-100) at 0.5-month post-infection to 79.5% (31/39; 95% CI: 64.4-89.2) at 10 months post-infection (P < .01). The IFN-γ response remained relatively robust at 10 months post-infection (3.8 vs 1.3 IU/mL, respectively). In 14 households, IGRA showed a positivity rate of 100% (12/12) and 65.2% (15/23), and IgG of 50.0% (6/12) and 43.5% (10/23) in index cases and contacts, respectively, exhibiting a difference of + 50% (95% CI: +25.4 to +74.6) and +21.7% (95% CI: +9.23 to +42.3), respectively. Either IGRA or IgG was positive in 100% (12/12) of index cases and 73.9% (17/23) of contacts. CONCLUSIONS: The SARS-CoV-2 IGRA is a useful clinical diagnostic tool for assessing cell-mediated immune response to SARS-CoV-2.

Clinical Performance of a Standardized Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Interferon-γ Release Assay for Simple Detection of T-Cell Responses After Infection or Vaccination.

BACKGROUND: We evaluated a standardized interferon-γ (IFN-γ) release assay (IGRA) for detection of T-cell immune response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. METHODS: This prospective study included patients with coronavirus disease 2019 (COVID-19) with different severity of illness and follow-up (FU), vaccinated subjects, and healthy unvaccinated persons. SARS-CoV-2 T-cell response was measured using a specific quantitative IGRA in whole blood (Euroimmun, Germany) and TrimericS-IgG and neutralizing antibodies with validated serological platforms. Positivity of reverse transcription-polymerase chain reaction or vaccination was considered as the reference standard. RESULTS: A total of 239 individuals were included (152 convalescent, 54 vaccinated, and 33 uninfected unvaccinated). Overall sensitivity, specificity, and positive- and negative-predictive values (95% confidence interval) of the IGRA were 81.1% (74.9-86%), 90.9% (74.5-97.6%), 98.2% (94.5-99.5%), and 43.5% (31.8-55.9%), respectively. All vaccinated SARS-CoV-2-naive subjects had positive IGRA at 3 months. In convalescent subjects the magnitude of IFN-γ responses and IGRA accuracy varied according to disease severity and duration of FU, with the best performance in patients with severe COVID-19 at 3 months and the worst in those with mild disease at 12 months. The greatest contribution of IGRA to serological tests was observed in patients with mild disease and long-term FU (incremental difference, 30.4%). CONCLUSIONS: The IGRA was a reliable method of quantifying T-cell response after SARS-COV-2 infection or vaccination. In convalescent patients, the sensitivity is largely dependent on disease severity and time since primary infection. The assay is more likely to add clinical value to serology in patients with mild infections.

Coordinated innate and T-cell immune responses in mild COVID-19 patients from household contacts of COVID-19 cases during the first pandemic wave.

Objective: To better define the immunopathogenesis of COVID-19, the present study aims to characterize the early immune responses to SARS-CoV-2 infection in household contacts of COVID-19 cases. In particular, innate, T- and B-cell specific responses were evaluated over time. Methods: Household contacts of COVID-19 cases screened for SARS-CoV-2 infection by nasopharyngeal swab for surveillance purposes were enrolled (T0, n=42). Of these, 28 subjects returned for a follow-up test (T1). The innate response was assessed by detecting a panel of soluble factors by multiplex-technology in plasma samples. Cell-mediated response was evaluated by measuring interferon (IFN)-γ levels by ELISA in plasma harvested from whole-blood stimulated with SARS-CoV-2 peptide pools, including spike (S), nucleocapsid (N) and membrane (M) proteins. The serological response was assessed by quantifying anti-Receptor-Binding-Domain (RBD), anti-Nucleocapsid (N), whole virus indirect immunofluorescence, and neutralizing antibodies. Results: At T0, higher levels of plasmatic IFN-α, IL-1ra, MCP-1 and IP-10, and lower levels of IL-1ß, IL-9, MIP-1ß and RANTES were observed in subjects with positive swab compared to individuals with a negative one (p<0.05). Plasmatic IFN-α was the only cytokine detectable in subjects with positive SARS-CoV-2 swabs with high accuracy for swab score positivity (0.93, p<0.0001). Among subjects with positive swabs, significant negative correlations were found among the RT-PCR cycle threshold values reported for genes S and N and IFN-α or IP-10 levels. At T0, the IFN-γ T-cell specific response was detected in 50% (5/10) of subjects with positive swab, while anti-RBD/anti-N antibodies showed a positivity rate of 10% (1/10). At T1, the IFN-γ T-cell specific response was detected in most of the confirmed-infection subjects (77.8%, 7/9), whereas the serological response was still observed in a minority of them (44.4%, 4/9). Overall, the swab test showed a moderate concordance with the T-cell response (78.6%, k=0.467), and a scarce concordance with the serological one (72.9%, k=0.194). Conclusions: Plasmatic IFN-α and the IFN-γ T-cell specific response appear early even in the absence of seroconversion, and show a greater positivity rate than the serological response in household contacts with positive swab.

B- and T-Cell Responses After SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis Receiving Disease Modifying Therapies: Immunological Patterns and Clinical Implications.

Background: Vaccination campaign to contrast the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised the issue of vaccine immunogenicity in special populations such as people with multiple sclerosis (PwMS) on highly effective disease modifying treatments (DMTs). While humoral responses to SARS-CoV-2 mRNA vaccines have been well characterized in the general population and in PwMS, very little is known about cell-mediated responses in conferring protection from SARS-CoV-2 infection and severe coronavirus disease-2019 (COVID-19). Methods: PwMS on ocrelizumab, fingolimod or natalizumab, vaccinated with two doses of mRNABNT162b2 (Comirnaty®) vaccine were enrolled. Anti-Spike (S) and anti-Nucleoprotein (N) antibody titers, IFN-gamma production upon S and N peptide libraries stimulation, peripheral blood lymphocyte absolute counts were assessed after at least 1 month and within 4 months from vaccine second dose administration. A group of age and sex matched healthy donors (HD) were included as reference group. Statistical analysis was performed using GraphPad Prism 8.2.1. Results: Thirty PwMS and 9 HDs were enrolled. All the patients were negative for anti-N antibody detection, nor reported previous symptoms of COVID-19. Peripheral blood lymphocyte counts were assessed in PwMS showing: (i) reduction of circulating B-lymphocytes in PwMS on ocrelizumab; (ii) reduction of peripheral blood B- and T-lymphocyte absolute counts in PwMS on fingolimod and (iii) normal B- and T-lymphocyte absolute counts with an increase in circulating CD16+CD56+ NK-cells in PwMS on natalizumab. Three patterns of immunological responses were identified in PwMS. In patients on ocrelizumab, anti-S antibody were lacking or reduced, while T-cell responses were normal. In patients on fingolimod both anti-S titers and T-cell mediated responses were impaired. In patients on natalizumab both anti-S titers and T-cell responses were present and comparable to those observed in HD. Conclusions: The evaluation of T-cell responses, anti-S titers and peripheral blood lymphocyte absolute count in PwMS on DMTs can help to better characterize the immunological response after SARS-CoV-2 vaccination. The evaluation of T-cell responses in longitudinal cohorts of PwMS will help to clarify their protective role in preventing SARS-CoV-2 infection and severe COVID-19. The correlation between DMT treatment and immunological responses to SARS-CoV-2 vaccines could help to better evaluate vaccination strategies in PwMS.

Cellular and Humoral SARS-CoV-2 Vaccination Responses in 192 Adult Recipients of Allogeneic Hematopoietic Cell Transplantation.

To determine factors influencing the vaccination response against SARS-CoV-2 is of importance in recipients of allogeneic hematopoietic cell transplantation (allo-HCT) as they display an increased mortality after SARS-CoV-2 infection, an increased risk of extended viral persistence and reduced vaccination response. Real-life data on anti-SARS-CoV-2-S1-IgG titers (n = 192) and IFN-γ release (n = 110) of allo-HCT recipients were obtained using commercially available, validated assays after vaccination with either mRNA (Comirnaty™, Pfizer-BioNTech™, NY, US and Mainz, Germany or Spikevax™, Moderna™, Cambridge, Massachusetts, US) or vector-based vaccines (Vaxzevria™,AstraZeneca™, Cambridge, UK or Janssen COVID-19 vaccine™Johnson/Johnson, New Brunswick, New Jersey, US), or after a heterologous protocol (vector/mRNA). Humoral response (78% response rate) was influenced by age, time after transplantation, the usage of antithymocyte globulin (ATG) and ongoing immunosuppression, specifically corticosteroids. High counts of B cells during the vaccination period correlated with a humoral response. Only half (55%) of participants showed a cellular vaccination response. It depended on age, time after transplantation, ongoing immunosuppression with ciclosporin A, chronic graft-versus-host disease (cGvHD) and vaccination type, with vector-based protocols favoring a response. Cellular response failure correlated with a higher CD8+ count and activated/HLA-DR+ T cells one year after transplantation. Our data provide the basis to assess both humoral and cellular responses after SARS-CoV2 vaccination in daily practice, thereby opening up the possibility to identify patients at risk.

Glucocorticoid use as a cause of non-cellular immune response to SARS-Cov2 Spike in patients with immune system diseases.

Disease modifying therapies compromise immune response to SARS-Cov2 or its vaccine in patients with immune system diseases (ISD). Therefore, analysis of the humoral and cellular responses against Spike is of utmost importance to manage ISD patients. A single-center retrospective study was conducted to evaluate the impact of COVID-19 immunization in 87 ISD patients and 81 healthy controls. We performed a whole blood interferon gamma release assay using SARS-Cov2 Spike and Nucleocapsid recombinant proteins in order to evaluate T-cell memory response, and an IgG anti-Spike ELISA to evaluate humoral response. Cellular (26.4%) and humoral (44.8%) responses were negative against Spike in ISD patients following COVID-19 immunization. In univariate analysis, an anti-Spike T cell defective response was associated with the use of glucocorticoids (Odds ratio [OR] = 10.0; p < 10-4), serum albumin level ≤40 g/L (OR = 18.9; p < 10-4), age over 55 years old (OR = 3.9, p = 0.009) and ≤2 vaccine injections (OR = 4.9; p = 0.001). The impact of glucocorticoids persisted after adjustment for age and number of vaccine injections (OR = 8.38, p < 0.001). In contrast, the humoral response was impacted by the use of anti-CD20 mAb (OR = 24.8, p < 10-4), and an extended time since immunization (≥75 days; OR = 4.3, p = 0.002). Double defective cellular/humoral responses (6.9%) were typically encountered in glucocorticoids and/or anti-CD20 mAb treated ISD with a serum albumin level ≤40 g/L (OR = 17.5; p = 0.002). Glucocorticoid usage, B cell depleting therapies, and a low serum albumin level were the main factors associated with a non-response to COVID-19 immunization in ISD patients. These results need further confirmation in larger studies.

Commercialized kits to assess T-cell responses against SARS-CoV-2 S peptides. A pilot study in health care workers.

Background: It is crucial to assess the levels of protection generated by natural infection or SARS-CoV-2 vaccines, mainly in individuals professionally exposed and in vulnerable groups. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection. Our aim was to assess the feasibility of a validated assay of T-cell responses. Methods: Twenty health-care-workers (HCW) were included. Antibody test to SARS-CoV-2 N and S-proteins in parallel with a commercially available whole-blood-interferon-gamma-release-assay (IGRA) to S-peptides and two detection methods, CLIA and ELISA were determined. Results: IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. The correlation by the two detection methods was very high (R > 0.8) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between specific antibody levels and the IGRA assay in the ability to detect immune response to SARS-CoV-2, there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one, with Ig(S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA-test. Conclusion: Whole-blood-IGRA-tests amenable to automation and constitutes a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become a valuable correlate of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health-care-workers.

Introducción: Es fundamental evaluar los niveles de protección inmune en infectados o tras la vacunación frente a SARS-CoV-2. La cuantificación de la respuesta inmune celular T puede complementar la determinación de anticuerpos. Evaluamos la viabilidad de un ensayo comercial validado de respuesta celular T específica frente a SARS-CoV-2. Métodos: Se incluyeron veinte trabajadores sanitarios (TS). Medimos anticuerpos contra las proteínas N y S de SARS-CoV-2 y realizamos el ensayo de liberación de interferón-gamma (IFNγ) en sangre completa (IGRA) frente a péptidos de la proteína S. IFNγ se determinó mediante dos métodos de detección: CLIA y ELISA. Resultados: IGRA detectó respuesta celular T en TS tanto infectados como vacunados. La correlación de los dos métodos de detección de IFNγ fue muy alta (R >0,8) y la sensibilidad y la especificidad variaron entre 100 y 86% y 100-73% respectivamente. Hubo una concordancia muy alta entre los niveles de anticuerpos específicos y el ensayo IGRA aunque la correlación cuantitativa fue relativamente baja. En el grupo de infectados, una dosis de vacuna fue suficiente para alcanzar el «plateau¼ de respuesta inmune. IGRA fue claramente positivo en un profesional vacunado inmunosuprimido que presentaba anticuerpos contra la proteína S negativos. Conclusiones: IGRA frente a péptidos de la proteína-S es susceptible de automatización y constituye una herramienta prometedora para medir la respuesta inmune celular frente a SARS-CoV-2; es aplicable a un gran número de muestras y puede servir para valorar la protección, particularmente en los grupos vulnerables en riesgo de volver a exponerse a la infección, como los TS.

Accuracy of QuantiFERON SARS-CoV-2 research use only assay and characterization of the CD4+ and CD8+ T cell-SARS-CoV-2 response: comparison with a homemade interferon-γ release assay.

OBJECTIVES: In this study, we aimed to characterize the SARS-CoV-2-specific T cell response detected by the QuantiFERON SARS-CoV-2 research use only assay in terms of accuracy and T cell subsets involved compared with a homemade interferon (IFN)-γ release assay (IGRA). METHODS: We evaluated T cell response by the standardized QuantiFERON SARS-CoV-2 tubes (antigen [Ag]1 and Ag2) and a homemade IGRA quantifying IFN-γ response to SARS-CoV-2 spike peptides (homemade-IGRA-SPIKE test). We evaluated the T cell subsets mediating the specific response using flow cytometry. RESULTS: We prospectively enrolled 66 individuals: COVID-19 or post-COVID-19 subjects and NO-COVID-19-vaccinated subjects, including healthy donors and immunocompromised subjects. The standardized kit detected 62.1% (41/66) of T cell responders. Ag2 tube showed a higher IFN-γ quantitative and qualitative response. Ag1 tube response was mainly mediated by CD4+ T cells; Ag2 tube response was mediated by CD4+ and CD8+ T cells. The homemade-IGRA-SPIKE test detected a higher number of responders (52/66, 78.8%) than the QuantiFERON SARS-CoV-2 assay (P = 0.056). The response was found in both T cell subsets, although a higher magnitude and response rate was observed in the CD4+ T cell subset. CONCLUSION: The QuantiFERON SARS-CoV-2 response is mediated by CD4+ and CD8+ T cells. A lower number of responders is found compared with the homemade-IGRA-SPIKE test, likely because of the different peptide composition.

T-Cell Assay after COVID-19 Vaccination Could Be a Useful Tool? A Pilot Study on Interferon-Gamma Release Assay in Healthcare Workers.

Background: SARS-CoV-2 T-cells are crucial for long-term protection against reinfection. The aim was to demonstrate the Interferon-gamma Release Assay (IGRA) test could be useful for vaccination monitoring. Methods: In a prospective cohort of 98 vaccinated healthcare workers for SARS-CoV-2, we selected 23 people in low-antibodies (Group 1, N = 8), high-antibodies (Group 2, N = 9), and negative control groups (Group 3, N = 6). SARS-CoV-2-specific humoral and cellular responses were analyzed at 8 months after two doses of Pfizer BioNTech, evaluating anti-RBD (Receptor Binding Domain) and RBD-ACE2 (Angiotensin Converting Enzyme-2) blocking antibodies in sera through a Chemiluminescence Immunoassay (CLIA) and T-cells through the IGRA test in heparinized plasma. Moreover, lymphocyte subtyping was executed by a flow cytometer. Statistical analysis was performed. Results: The data confirmed that RBD and RBD-ACE2 blocking ACE2 antibody levels of Group 1 were significantly lower than Group 2; p < 0.001. However, T-cells showed no significant difference between Group 1 and Group 2. Conclusions: This work suggests the need for new strategies for booster doses administration.

Performance of Whole Blood Stimulation Assays for the Quantification of SARS-CoV-2 Specific T-Cell Response: A Cross-Sectional Study.

Since the identification of the new severe acute respiratory syndrome virus 2 (SARS-CoV-2), a huge effort in terms of diagnostic strategies has been deployed. To date, serological assays represent a valuable tool for the identification of recovered COVID-19 patients and for the monitoring of immune response elicited by vaccination. However, the role of T-cell response should be better clarified and simple and easy to perform assays should be routinely introduced. The main aim of this study was to compare a home-made assay for whole blood stimulation with a standardized ELISpot assay design in our laboratory for the assessment of spike-specific T-cell response in vaccinated subjects. Even if a good correlation between the assays was reported, a higher percentage of responder subjects was reported for immunocompromised subjects with ELISpot assay (56%) than home-made whole blood stimulation assay (33%). Additionally, three commercial assays were compared with our home-made assay, reporting a good agreement in terms of both positive and negative results.

The Pitfalls of Mining for QuantiFERON Gold in Severely Ill Patients With COVID-19.

Objective: To assess the proportion of indeterminate QuantiFERON-TB Gold Plus (QFT-Plus) results in patients admitted for severe coronavirus disease 2019 (COVID-19) pneumonia and evaluate the factors associated with indeterminate QFT-Plus results. Patients and Methods: Data on COVID-19 admissions at Mayo Clinic in Florida were extracted between October 13, 2020, and September 20, 2021, and data from a prepandemic cohort were extracted between October 13, 2018, and September 20, 2019. A secondary analysis of the COVID-19 cohort was performed using gradient boosting modeling to generate variable importance and SHapley Additive exPlanations plots. Results: Our findings demonstrated more indeterminate QFT-Plus test results in patients hospitalized for severe COVID-19 infection than in patients without COVID-19 (139 of 495, 28.1%). The factors associated with indeterminate QFT-Plus test results included elevated levels of C-reactive protein, ferritin, lactate dehydrogenase and interleukin-6 and included lower levels of leukocyte, lymphocyte, and platelet counts. Conclusion: The patients with severe COVID-19 had a higher likelihood of indeterminate QFT-Plus results, which were associated with elevated levels of inflammatory markers consistent with severe infection. Interferon-gamma release assay screening tests are likely confounded by COVID-19 infection itself, limiting the screening ability for latent tuberculosis infection reactivation. Indeterminate QFT-Plus results may also require follow-up QFT-Plus testing after patient recovery from COVID-19, increasing the cost and complexity of medical decision making and management. Additional risk assessments may be needed in this patient population for screening for latent tuberculosis infection in patients with severe COVID-19.

Cellular and humoral immune response to SARS-CoV-2 vaccination and booster dose in immunosuppressed patients: An observational cohort study.

BACKGROUND: Humoral and cellular immune responses to SARS-CoV-2 vaccination among immunosuppressed patients remain poorly defined, as well as variables associated with poor response. METHODS: We performed a retrospective observational cohort study at a large Northern California healthcare system of infection-naïve individuals fully vaccinated against SARS-CoV-2 (mRNA-1273, BNT162b2, or Ad26.COV2.S) with clinical SARS-CoV-2 interferon gamma release assay (IGRA) ordered between January through November 2021. Humoral and cellular immune responses were measured by anti-SARS-CoV-2 S1 IgG ELISA (anti-S1 IgG) and IGRA, respectively, following primary and/or booster vaccination. RESULTS: 496 immunosuppressed patients (54% female; median age 50 years) were included. 62% (261/419) of patients had positive anti-S1 IgG and 71% (277/389) had positive IGRA after primary vaccination, with 20% of patients having a positive IGRA only. Following booster, 69% (81/118) had positive anti-S1 IgG and 73% (91/124) had positive IGRA. Factors associated with low humoral response rates after primary vaccination included anti-CD20 monoclonal antibodies (P < 0.001), sphingosine 1-phsophate (S1P) receptor modulators (P < 0.001), mycophenolate (P = 0.002), and B cell lymphoma (P = 0.004); those associated with low cellular response rates included S1P receptor modulators (P < 0.001) and mycophenolate (P < 0.001). Of patients who had poor humoral response to primary vaccination, 35% (18/52) developed a significantly higher response after the booster. Only 5% (2/42) of patients developed a significantly higher cellular response to the booster dose compared to primary vaccination. CONCLUSIONS: Humoral and cellular response rates to primary and booster SARS-CoV-2 vaccination differ among immunosuppressed patient groups. Clinical testing of cellular immunity is important in monitoring vaccine response in vulnerable populations.