Humoral and cellular immune response in patients with hematological disorders after two doses of BNT162b2 mRNA COVID‐19 vaccine: A single‐center prospective observational study (NCT05074706)

Abstract Hematological patients at higher risk of severe COVID‐19 were excluded from the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccine trials. In this single‐center observational prospective study (NCT05074706), we evaluate immune response in the hematological patients followed at the Hematological Division of San Gerardo Hospital, Monza (Italy) deemed to be severely immunosuppressed after vaccination with two doses of the BNT162b2 vaccine. Anti‐SARS‐CoV‐2 immunoglobulin G titers above the cutoff value of 33.8 BAU/ml were detected in 303 (80.2%) out of the 378 patients enrolled. Patients with lymphoproliferative disorders had a significant lower probability of immunization (43.2% vs. 88.4%, p < 0.001). Patients treated with anti‐CD20 showed a significantly lower probability of immunization compared to all other treatments (21.4%, p < 0.0001). Among 69 patients who failed seroconversion, 15 patients (22.7%) showed a positive T‐cell response. Patients previously treated with anti‐CD20 were 2.4 times more likely to test positive for T‐cell responses (p = 0.014). Within a follow‐up of 9 months from the second COVID‐19 vaccination, symptomatic SARS‐CoV‐2 infections were reported by 20 patients (5.3%) and four of them required hospitalization. Successful serological or T‐cell‐mediated immunization conferred protection from symptomatic COVID‐19. Patients treated with anti‐CD20 who were not seroconverted after vaccination might still be protected from COVID‐19 due to the T‐cell immune response.


INTRODUCTION
COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared pandemic in March 2020. Patients with hematological malignancies have been excluded from the SARS-CoV-2 vaccine trials, despite being at higher risk of severe COVID-19 with a high mortality rate of 30%-37% [1][2][3]. However, most health authorities worldwide have designated these patients as a priority for COVID-19 vaccination even in the absence of efficacy data given their status of highly immunosuppressed patients [4]. Follow-up studies on seroconversion in cancer patients with COVID-19 demonstrated that while most will develop antibody response similar to the general population, subgroups of cancer patients with hematologic malignancies, receiving anti-CD20 antibody therapies and stem-cell transplantation, exhibit lower rates of seroconversion (70%) in comparison with all other hematologic patients (85%) [5]. However, T-cell response is induced by natural SARS-CoV-2 infection or vaccination and plays a central protective role as it usually does in viral infections. It has been reported that SARS-CoV-2-specific T-cell responses were inhibited in cancer patients and there is limited information about the T-cell-mediated vaccine responses after anti-CD20 treatment [6].
To gain more insights in the immunogenicity of mRNA vaccines in patients with hematologic malignancies, we analyzed the antibody as well as the T-cell response after the second dose of the BNT162b2 vaccination in hematological patients with important degree of immunosuppression.

Study participants and data collection
All the hematological patients followed at the Hematological Division

Test for T-cell response
To test cellular immune response, cells from lithium heparinized whole blood were stimulated with SARS-CoV-2-specific peptides, covering domains of the spike and nucleocapsid proteins. SD Biosensor Covi-FERON Tubes 500 (Table S2)

Statistical analysis
Descriptive analysis on the characteristics of enrolled patients was

B-cell response following SARS-CoV-2 vaccination
Anti-SARS-CoV-2 IgG titers above the cutoff value of 33. 8  treated with anti-CD20 or BTKI was even clearer when analyzing the subgroup of patients affected by lymphoproliferative disorders, in whom a recent treatment with anti-CD20 or BTKI reduced the likelihood of seroconversion by 74% and 65%, respectively (Table S6).  Table S7. Patients with lymphoprolif-erative disorders had a higher percentage of patients with a positive Covi-FERON test (30%, 95% CI 18.1-45.4), although the difference was not statistically significant (p = 0.402) ( Table 4). Patients previously treated with anti-CD20 were 2.4 times more likely to be tested positive for Covi-FERON than patients treated with other regimens and this difference was statistically significant (p = 0.014) ( Table 5).

Efficacy
Within a follow-up of 9 months from the second COVID-19 vac- Abbreviations: ALL, acute lymphoblastic leukemia; Allo-HSCT, allogenic hematopoietic stem-cell transplantation; AML, acute myeloid leukemia; CI, confidence interval; CLL, chronic lymphocytic leukemia; CMPD Ph negative, chronic myeloproliferative disorders Philadelphia negative; HL, Hodgkin lymphoma; MDS, myelodysplastic syndrome; MM, multiple myeloma; NHL, non-Hodgkin lymphoma. a Compared to all patients considered in the denominator of the relative risk with an absolute risk equal to 5.4% with 95% CI = (3.3%; 8.2%). b On the null hypothesis of the relative risk equal to 1. c Immune thrombocytopenic purpura, hemolytic anemia, thrombotic thrombocytopenic purpura, and medullary aplasia.   (Table 7). However, patients treated with anti-CD20 or BTKI had no higher risk of developing a severe COVID-19 compared to all other treatment regimens (p = 0.78 and 0.886, respectively).

DISCUSSION
In our analysis, the possibility of an antibody response to two-dose  [7,9]. An important treatment-related factor identified in this analysis is active anti-CD20 therapy. Recent studies on small cohorts of rituximab-treated patients with immune-mediated inflammatory disease provided some initial evidence that T-cell-mediated immune response is maintained even in the absence of a humoral anti-SARS-CoV-2 response [10,11].
Even in our study, performed on 378 hematological patients, active treatment with anti-CD20 monoclonal antibodies exhibited low seroconversion rates (21.4%, 95% CI 4.7-50.8) but relatively high rates of positive Covi-FERON tests, indicating successful immunization. These serological results are in line with previous findings of reduced response to anti-SARS-CoV-2 vaccines in patients with hematological malignancies after exposure to B-cell-depleting agents [9,[12][13][14].
However, it is interesting that this subgroup of patients without seroconversion, probably due to active anti-CD20 therapy, showed the presence of a T-cell immune response in over half of them. Other studies demonstrated the presence of T-cell response in hematological patients without antibody response [9,15].  [20]. Moreover, we did not analyze other patient-specific factors that may have influenced results, such as age, comorbidities, and concomitant treatments. Analysis of immunogenicity of added booster vaccination dose in hematological patients was out of the scope of the present work and will need further evaluation.

CONCLUSIONS
Overall, these results indicate that 85%-90% of hematological patients considered to be immunosuppressed are able to mount a positive immune response, either serological or cellular based, to SARS-CoV-2 vaccination. In addition, patients with recent or ongoing anti-CD20 treatment who suffer from insufficient humoral immune response after two COVID-19 vaccinations might still benefit from vaccination due to the cellular immune response, sufficient to ensure a mild course of disease. This observation should be taken into account while planning consistently effective strategy for hematological patients who remain seronegative after two-dose vaccination. It is possible that patients with hematological malignancies who exhibited a poor humoral immune response may still be protected by a good cellular immune response.

PATIENT CONSENT STATEMENT
Prior to this study, the principal investigator or investigators obtained written informed consent based on patient free will.

DATA AVAILABILITY STATEMENT
All data generated during this study are available upon justified request to the corresponding author. The authors affirm that this manuscript is an honest, accurate, and transparent account of the study being reported and that no important aspects of the study have been omitted.