ABSTRACT
Background: Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19 (1). Furthermore, vaccination-induced CD4 and CD8 T-cell responses have been suggested to have a protective role in COVID-19 (2). If T-cell responses are diminished after vaccination in immuno-compromised individuals is not known to date. Objectives: To investigate cellular immunity following mRNA vaccination against COVID-19 in healthy individuals and patients undergoing B-cell depletion therapy. Methods: In this interim analysis of the CoVVac study (NCT04858607), we analyzed T-cell responses in autoimmune patients treated with B-cell depleting therapy (BD, n=41) and age-matched healthy controls (HCs, n=50) 3-4 weeks after the second dose of mRNA vaccination against COVID-19. Therefore, we isolated PBMCs and stimulated them with a peptide pool covering the spike protein in vitro. Reactive CD4 and CD8 T-cells were determined by staining for IFNg, TNFa, IL-2 and GzmB by fow cytometry. Anti-SARS-CoV-2 antibody assays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed to elucidate concomitant B-cell responses. Results: We observed signifcant alterations in anti-SARS-CoV-2 antibody responses in our cohort, the frequency of IFNg+ and IL-2+ CD4 and CD8 T-cells was similar in BD patients and controls. On the other hand, TNFa+ CD4 T-cells were signifcantly enriched in healthy controls versus BD patients (p=0.017) and correlated signifcantly with antibody titres (p=0.003). Similarly, GzmB+ CD8 T-cells were signifcantly diminished in our patient cohort (p<0.001) and also showed a signifcant correlation with antibody titres (p<0.001). Overall, the frequency of GzmB+ CD8 T-cells correlated very well with reactivity of T-cell subsets for other cytokines. This effect, however, is lost in the BD cohort. No difference was observed in the frequency of TNFa+ CD8 T-cells between the groups. Only 21 (42%) healthy individuals and 14 (34%) patients showed reactive T-cells for all the cytokines tested. This observation is mainly explained by a lack of cytokine production of CD8 T-cells in 26 (52%) HCs and 27 (66%) BD patients. In turn, 22 (44%) HCs and 17 (42%) patients didn't show any IL-2 producing CD8 cells. Of note, only 2 (4%) of HCs showed no GzmB+ CD8 T-cells whereas the number increased to 15 (37%) of BD individuals (p<0.001). In contrast, 42 (84%) HCs as well as 32 (78%) of patients showed production of all IFNg, TNFa and IL-2 in CD4 T-cells. Conclusion: Our data suggest that most patients with B-cell depleting therapy are able to mount T-cell responses similar to those of healthy individuals while a minority of these patients did not show complete immunity against SARS CoV-2. Further analyses are needed to better understand a possible link of B-cell depletion therapy and CD8 T-cell responses.
ABSTRACT
Tocilizumab (TCZ), an interleukin-6 (IL-6) receptor-blocking monoclonal antibody, is used to treat variousrheumatologic conditions and cytokine release syndrome in CAR-T cell therapy and has been repurposed to treatCOVID-19-related hyperinflammation. There are limited data available reporting how TCZ affects the immuneresponse in the context of COVID-19. To investigate this question, we recruited patients treated with TCZ as part ofa COVID-19 biobanking protocol (A-28063295) to study immune parameters that might be affected. We enrolled 19patients who were treated with a range of 40-200mg TCZ as part of a low-dose TCZ trial (COVIDOSE, reportedseparately as abstract A-94803796), and 11 patients who received 400mg TCZ on a standard-of-care expanded-access basis. As IL-6 acts as a stimulant of B-cell proliferation, plasma cell maturation, and antibody responses, weevaluated whether blocking the IL-6 receptor with TCZ therapy impairs antibody generation to SARS-CoV-2. Toevaluate antibody levels in these patients, we performed ELISAs against the SARS-CoV-2 spike glycoprotein and itsreceptor-binding domain (RBD). The spike glycoprotein, a structural protein of SARS-CoV-2, is a crucial componentin the recognition, attachment, and entry of the virus into host cells. Specifically, the RBD is responsible for bindingthe ACE2 receptor on human cells, and likely serves as a major target for neutralizing antibodies. To establish if theformation and persistence of antibodies was affected by TCZ treatment, we analyzed serum and plasma sampleslongitudinally from 29 patients treated with TCZ and 26 control patients. To account for potential variability betweenplates, the measured optical density (OD) values were normalized to the OD for COVID-19-negative control serumat 1:50 dilution, and the same negative control was tested on each plate. Titers were calculated as the linearinterpolation of the inverse dilution at which the normalized OD value crossed a threshold of 1, representing themaximum OD measured for the negative control. Anti-spike and anti-RBD antibodies increased significantly overtime in both TCZ-treated patients and controls (p < 0.005 for both). Increasing antibody titers throughout the diseasecourse followed a similar trajectory in TCZ-treated patients compared to control patients, suggesting that TCZtreatment does not impede the generation of antibodies to SARS-CoV-2. Additionally, TCZ-treated patients achievedcomparable maximal observed antibody titers to control patients (average maximal log10 (titer) of 5.42 and 4.96 forspike and of 4.39 and 4.44 for RBD, respectively). These data suggest that TCZ does not impair the induction ofanti-SARS-CoV-2 antibodies.
ABSTRACT
The clinical spectrum of SARS-CoV-2 (COVID-19) infection ranges from asymptomatic infection to fatal pneumonia, but the determinants of outcome are not well understood. To characterize the immune response to COVID-19, weestablished a protocol to collect biologic specimens from patients with confirmed or suspected COVID-19. BetweenApril 9th and June 8th, 2020, we enrolled 146 inpatients and 169 outpatients at the University of Chicago. Wehypothesized that the complex interplay of viral, environmental, and host genetic factors may influence diseaseseverity in patients with COVID-19. To probe for genetic predispositions that may influence outcomes, we collectedgermline DNA from 140 patients spanning the breadth of clinical severity, which will be sequenced for SNPs ingenes previously implicated in immune responsiveness and ARDS. To determine whether a pattern of commensalbacteria correlates with disease severity, we will analyze the composition of airway microbiota from 226nasopharyngeal swabs, using viral quantification and 16S sequencing. Longitudinal serum samples from 156patients were obtained to probe for the presence of antibodies using an ELISA against the spike protein of SARS-CoV-2. In tandem, 36-color flow cytometry on PBMCs, from the same patients, will characterize immune cellphenotypes influenced by infection. We also hypothesized that by characterizing mechanisms of immune-hyperresponsiveness, we may elucidate key biologic pathways that inform the development of novel therapeutics.To determine if severity of disease and response to therapy correlates with soluble factors, we are performing 44-plex cytokine Luminex assays on serum samples. We will probe the adaptive immune response using an ELISAagainst the SARS-CoV-2 RBD domain, and by performing IFN-g ELISPOT analysis against peptide pools fromSARS-CoV-2 proteins. We developed a bioinformatic pipeline to integrate clinical data with the results from thediverse data types and will adopt a machine learning approach to identify parameters contributing to diseaseseverity, response to therapies, and outcomes. In establishing this protocol, there were significant biosafetyconsiderations. To limit potential exposure and virus transmission, research coordinators contacted inpatients byphone for an informed consent discussion, and patients completed the consent form electronically using REDCap(n=61). Inpatients who were unable to navigate the electronic consent were visited with a paper consent (n= 85).Samples were processed in a BSL2 laboratory with enhanced biosafety precautions. Where feasible, samples werecollected into reagents such as Zymo DNA/RNA shield to immediately inactivate the virus. Other safety measuresincluded heat inactivation of some samples and use of a laminar flow washer to minimize aerosolization duringFACS staining. In summary, we have established a biorepository of specimens from patients with COVID-19, including a subset with active cancer or a history of the disease (n=22).