key: cord-0743175-k2jzbijb authors: Ram, Ron; Freund, Tal; Halperin, Tami; Ben-Ami, Ronen; Amit, Odelia; Bar-On, Yael; Beyar-Katz, Ofrat; Eilaty, Nili; Gold, Ronit; Kay, Sigi; Glait-Santar, Chen; Hagin, David title: Immunogenicity of a third dose of the BNT162b2 mRNA Covid-19 Vaccine in Patients with impaired B cell reconstitution after cellular therapy – a Single Center Prospective Cohort Study date: 2022-02-16 journal: Transplant Cell Ther DOI: 10.1016/j.jtct.2022.02.012 sha: 60a32916ec16cbcd1a8320842186290285872c88 doc_id: 743175 cord_uid: k2jzbijb Patients with delayed B-cell reconstitution / B-cell aplasia after cellular therapy show decreased immunogenicity to the BNT162b2 mRNA COVID-19 vaccine. We prospectively evaluated both humoral and cellular immune response to a third vaccine dose in patients after allogeneic HCT (n=10) or CD19-based CAR-T therapy (n=6) with low absolute B cell numbers and who failed to mount a humeral response after 2 vaccine doses. Humoral response was documented in 40% and 17% after allogeneic HCT and CAR-T therapy, respectively. None of the patients with complete B cell aplasia developed anti-vaccine antibodies. Cellular response was documented in all patients after allogeneic HCT and in 83% of the patients after CAR-T. T-cell subclasses levels were not predictive for response, while a longer duration from infusion of cells was associated with a better cellular response. We conclude that cellular response develops with repeated vaccine doses even in patients with B-cell aplasia or delayed B-cell reconstitution, and these patients should therefore be vaccinated. These results should be considered in future studies analyzing immunogenicity in this population. Larger and longer follow-up studies are required to confirm whether cellular immunogenicity translates into vaccine efficacy. The BNT162b2 mRNA COVID-19 (Pfizer/BioNTech) vaccine was approved by both the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the prevention of COVID-19, based on a phase 3 study that showed 94.6% efficacy 1 . Patients after allogeneic hematopoietic cell transplantation (HCT) and chimeric antigen receptor T-cell therapy (CAR-T) targeting CD19 + cells, often display a delayed B-cell reconstitution pattern [2] [3] [4] . The concern for delayed reconstitution has resulted in a general recommendation to start vaccinating against COVID-19 at least 3-6 months after HCT or CAR-T infusion 5, 6 . We have previously shown that while post-vaccination positive serology was documented in 75% of patients after allogeneic HCT, only 36% of patients after CAR-T infusion had documented serum antibodies 7 . Based on studies showing efficacy of a third vaccine dose in solid organ transplanted patients, we hypothesized that similarly, patients with poor B-cell reconstitution and no evidence for immunogenicity, may benefit from a third dose of the anti-SARS-CoV2 vaccine 8 . This prospective study was performed at the BMT Long-Term Follow-up (LTFU) clinic at the Tel Aviv Sourasky Medical Center. Patients were eligible for this study if they had previously received 2 doses of the BNT162b2 mRNA COVID-19 (Pfizer/BioNTech) vaccine, were tested negative for the presence of anti-spike antibodies after 2 vaccine doses, and had evidence of Bcell dysfunction (either complete B-cell aplasia or B-cell lymphopenia with an absolute CD19 count of <150 cell/microL). All patients received the BNT162b2 third dose 5 months after the second vaccine dose. The study was approved by the hospital ethics committee (#1067-20 amended) and was registered in ClinicalTrials.gov (NCT04724642). All patients signed informed consent prior to enrollment. Patients were vaccinated through the national Israeli vaccination program in mid-July 2021. All patients had a baseline quantification of absolute numbers of peripheral blood CD19 + , CD4 + , and CD8 + cells (cells/microL). All patients were reassessed for suitability to vaccination prior to the third dose of BNT162b2 mRNA COVID-19 vaccine, including physical examination, assessment of GVHD status, complete blood count and liver function tests. Patients were interviewed for post-vaccination adverse events one week after the administration of the third vaccine dose and underwent physical evaluation and repeated laboratory tests. Patients were then reassessed 21 days after the booster vaccine dose and had blood samples taken for SARS-CoV-2 serology and cellular response assays. The primary endpoint was evaluation of the humoral and cell-mediated response to the vaccine (measured by anti-spike IgG titers and intracellular cytokine staining, respectively). Adverse events were graded according to CTCAE v5.0 and chronic GVHD according to the NIH 2014 grading and response criteria 9 . Measurement of anti-spike antibody titers and evaluation of cellular response are described in the supplementary methods appendix. In brief, antibody titers were measured using a commercial automated SARS-CoV-2 IgG assay designed to detect IgG antibodies against the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 wild-type spike protein (anti-spike) (Abbott, Ireland). Cellular response was assessed by stimulating PBMCs with a spike-protein peptide mix, followed by flow-cytometry based evaluation of stimulation induced CD154 upregulation, IFN production or TNFproduction (gating strategy is shown in Supp Figure 1 ). Complete cellular response was defined as production of both cytokines and CD154 upregulation, while partial cellular response was defined as either CD154 upregulation, IFN production, TNF production or a combination of any two. Continuous variables were described as the mean, median, standard deviation and range of values, as applicable. Categorical data were described with contingency tables including frequency and percent. Antibody titers were compared between patient groups using either Pearson Chi-Square or t test, as appropriate. A two-sided P value of <.05 considered to be statistically significant. We compared both the humoral and the cellular responses of patients to healthy volunteers (n=4). IBM SPSS Statistics, version 27 was used to perform all analyses. Twenty-five patients (32%), out of 77 patients who were included in the original vaccination program, did not have humoral response after two vaccine doses. Of these 25 patients, 16 patients had either complete B-cell aplasia (n=7) or a low absolute CD19 count of (<150 cell/microL) (n=9) and were eligible for enrollment according to this study protocol. Ten patients were post allogeneic HCT and 6 were post CAR-T infusion. All 16 patients received the third dose of the vaccine in mid-July 2021, after a median time of 5.2 (range, 5.1-5.6) months from the second vaccine dose. Table 1 depicts the characteristic of all on-protocol patients. Non-hematologic vaccine-related adverse events were observed in 2 patients (13%) and included muscle crumps and generalized pain (both graded as grade 2). There were no grade 3-4 nonhematologic adverse events. One patient with moderate chronic GVHD developed a transient There was no statistically significant difference in absolute CD4, CD8 or CD4/CD8 ratio values between patients with partial cellular response compared to patients with complete cellular response (p=.3, p=.4, and p=.7, respectively). Mean duration from infusion of cells was longer in patients with complete cellular response, compared to those with partial cellular response (34.1±23.9 months vs. 16.6±7.4 months, p=.085), however, overall cellular response did not correlate with time from infusion (R 2 =.06 for CD154 and R 2 =.07 for both IFN and TNFFigure 2. In contrast, serology was modestly correlated with time from infusion (R 2 =.15)Figure 2. Mean B-cell blood levels was higher in patients who had complete cellular response compared to patients who had partial cellular response; however this was not statistically significant (47±S.D.67 vs. 99±S.D. 60 cell/microL, respectively, p=.12). Out of the 10 patients who did not have evidence for humoral response, cellular response was documented in 90%. The only patient who did not have either cellular nor humoral response, was a patient with acute lymphoblastic leukemia in complete disease remission. This patient is currently 17 months after anti-CD19 CAR-T therapy with complete B-cell aplasia. In this study we evaluated the response to a third dose of the BNT162b2 mRNA COVID-19 vaccine in patients with a low B-cell count or complete B-cell aplasia, who did not develop humoral response after 2 vaccine doses. To our knowledge, this is the first study that focused on this subgroup of patients who are post cellular therapy. We show that in this specific population, the vaccine was relatively safe, and while humoral response was documented in minority of the patients (17% after CAR-T and 40% after allogeneic HCT), cellular response (whether documented in all 3 domains or only in 1-2) was more prevalent. While in patients after solid organ transplantation, a third vaccine dose resulted in reappearance of humoral immunogenicity, patients after allogeneic HCT may have both quantitative and qualitative defects of the B-cell compartment, and thus, revaccination may not necessarily result in augmentation of antibody production 8, 10 . In addition, B-cell aplasia after additional cellular therapies, is further associated with decreased response to vaccination. Conversely, both patients with B-cell aplasia and with B-cell dysfunction, exhibited a substantial cellular response, which may be sufficient for protective immunogenicity. Similar to other publications, we found that humoral and a more robust cellular immunity were positively associated with a longer time since HCT 7, 11, 12 . Interestingly, our findings that a greater cellular response in patients after CAR-T infusion also depends on the time elapsed from the CAR-T infusion, suggests that recovery of the B-cell number and function also impacts T-cell responsiveness. In addition, it is possible that longer duration of disease control allows better reconstitution of the T-cell compartment. Our study is limited by the small number of patients. Despite this, it is reassuring to see that patients in this study developed significant cellular reactivity even in the absence of humoral immunity. The significance of this reactivity and its relevance to vaccine efficacy is unknown. Evidence for persistence of post-vaccine anti-SARS-CoV-2 specific T-cell clones is scarce. In fact, there are only limited data that show effective cellular immunogenicity in the absence of humoral function 13 . In a broader view, no clinical or immunologic variables, as well as tests to monitor immune function have been established to predict vaccine responsiveness in patients after cellular therapy. In the absence of such data, we recommend all patients to continue following general preventive precautions. Longitudinal studies in this vulnerable population are essential to gain more experience and to confirm whether cellular immunogenicity translates into vaccine efficacy. In addition, exploring novel ways of vaccine combinations and prophylactic, or early administration of monoclonal antibodies or the use of novel anti-viral drugs, are warranted. The presence of anti-SARS-CoV-2 IgG antibodies was evaluated by using a commercial PBMCs were plated in a 96-well plate at a concentration of 0.5-1X10 6 PBMCs/100L and incubated at 37 °C and 5% CO 2 with 2L of either complete pooled S-peptide mix, CytoStim TM for positive control or 10% DMSO in sterile water for negative control. After two hours, Brefeldin A was added to each well and cells were incubated for additional 4 hours. Cells were then stained with viability dye, followed by fixation, permeabilization and staining for surface markers (CD3, CD20, CD14, CD4, CD8, CD154) and intracellular cytokines (TNF and IFN). Following staining, samples were acquired using BD FACSCanto II, and 20,000 CD4 + events were collected for each sample. Analysis was performed on gated CD4 + T cells and the absolute number of activated CD154 + , INF + or TNF + cells was recorded and normalized for 1X10 6 CD4 + T-cells. In order to calculate the actual response rate, the absolute number of positive events in the unstimulated negative control was deducted from the absolute number of events in the S-stimulated samples, as shown in the following formula: Safety and Efficacy of the BNT162b2 mRNA Covid-19 The New England journal of medicine Humoral Immune Reconstitution Kinetics after Allogeneic Hematopoietic Stem Cell Transplantation in Children: A Maturation Block of IgM Memory B Cells May Lead to Impaired Antibody Immune Reconstitution. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation Allogeneic hematopoietic stem cell transplantation recipients have defects of both switched and igm memory B cells. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation Immune reconstitution and infectious complications following axicabtagene ciloleucel therapy for large B-cell lymphoma B Cell Reconstitution and Influencing Factors After Hematopoietic Stem Cell Transplantation in Children Safety and Immunogenicity of the BNT162b2 mRNA COVID-19 Vaccine in Patients after Allogeneic HCT or CD19-based CART therapy-A Single-Center Prospective Cohort Study. Transplantation and cellular therapy Three Doses of an mRNA Covid Vaccine in Solid-Organ Transplant Recipients. The New England journal of medicine Measuring therapeutic response in chronic graft-versus-host disease. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: IV. The 2014 Response Criteria Working Group report. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation Safety and Immunogenicity of a Third Dose of SARS-CoV-2 Vaccine in Solid Organ Transplant Recipients: A Case Series Antibody response after third BNT162b2 dose in recipients of allogeneic HSCT Vaccine Was Well Tolerated and Highly Immunogenic in Young Adults in Long Follow-Up after Haematopoietic Stem Cell Transplantation Impaired Humoral but Substantial Cellular Immune Response to Variants of Concern B1.1.7 and B.1.351 in Hemodialysis Patients after Vaccination with transplantation or CAR-T infusion. Filled dots represent samples from patients post HCT and target-shape dots Mizrahi for the substantial help with the logistic support. Competing interests -All authors declare no conflicts of interests to disclose.