Additional booster doses in patients with chronic lymphocytic leukemia induce humoral and cellular immune responses to SARS‐CoV‐2 similar to natural infection regardless ongoing treatments: A study by ERIC, the European Research Initiative on CLL
Authors: A. Campanella, A. Capasso, S. Heltai, C. Taccetti, E. Albi, Y. Herishanu, S. Haggenburg, T. Chatzikonstantinou, M. Doubek, M. Kättström, K. Giannopoulos, M. Simkovic, C. Moreno, M. Massaia, H. Bumbea, S. Alshemmari, P. Ranghetti, E. Perotta, F. Martini, E. Sant'Antonio, M. Colia, C. Combi, S. Levi, A. P. Kater, M. Hazenberg, I. S. Nijhof, Q. Hofsink, C. Demosthenous, J. Kotaskova, J. Zaleska, F. Vrbacky, A. Mora Raya, D. Bisogno, I. E. Tripoli, V. M. Popov, V. Roman, N. Stavroyianni, M. Karypidou, E. Scarano, M. Locatelli, M. Frenquelli, L. Scarfò, K. Stamatopoulos, P. Ghia
Journal: American Journal of Hematology (2024)

Profound immune dysregulation and impaired response to the SARS-CoV-2 vaccine put patients with chronic lymphocytic leukemia (CLL) at risk of severe COVID-19. We compared humoral memory and T-cell responses after booster dose vaccination or breakthrough infection. (Green) Quantitative determination of anti-Spike specific antibodies. Booster doses increased seroconversion rate and antibody titers in all patient categories, ultimately generating humoral responses similar to those observed in the postinfection cohort. In detail, humoral response with overscale median antibody titers arose in >80% of patients in watch and wait, off-therapy in remission, or under treatment with venetoclax single-agent. Anti-CD20 antibodies and active treatment with BTK inhibitors (BTKi) represent limiting factors of humoral response, still memory mounted in ~40% of cases following booster doses or infection. (Blue) Evaluation of SARS-CoV-2-specific T-cell responses. Number of T-cell functional activation markers documented in each patient. The vast majority of patients, including those seronegative, developed T-cell responses, qualitatively similar between treatment groups or between vaccination alone and infection cases. These data highlight the efficacy of booster doses in eliciting T-cell immunity independently of treatment status and support the use of additional vaccination boosters to stimulate humoral immunity in patients on active CLL-directed treatments. In the past 3 years, SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) leading to coronavirus disease 2019 (COVID-19) has threatened the life of patients with chronic lymphocytic leukemia (CLL). Though declining over time thanks to effective interventions, the case fatality rate and the percentage of patients with CLL experiencing severe infections remained higher than those of the general population.1 This vulnerability is unsurprising, considering that CLL per se is associated with a characteristic immune dysfunction,2 and that CLL-directed therapies may further worsen immune suppression. Factors leading to a higher infectious risk have been also associated with an impaired response to COVID-19 vaccines.3, 4 To some extent, booster doses increased vaccine immunogenicity in patients with CLL,5, 6 though the dynamics of cellular responses remain controversial. Indeed, some studies suggested that specific T cells may be generated and be functional independently of seroconversion and anti-CLL therapies,7-9 while others indicated a certain degree of impaired T-cell immunity,10 mostly seen in patients on active CLL-directed treatment.11 Besides, the potential differences from the response to natural infection by SARS-CoV-2 remain to be thoroughly assessed. In this work, thanks to a large multicenter study coordinated by ERIC (Table S1), we prospectively collected data concerning humoral and cellular responses to SARS-CoV-2 vaccination or breakthrough infection in a cohort of 596 patients diagnosed with CLL, well-annotated for disease status and type of anti-leukemic therapy (Table S2). Within this cohort, we investigated the humoral response generated by the primary 2-dose vaccination cycle in 533 cases (Figure S1). We confirmed the suboptimal humoral response of patients with CLL. In more detail, seroconversion rates were statistically higher in both patients in remission after therapy (79%; 48/61) and treatment naïve (watch and wait) patients (65%; 123/189) in comparison to patients on active treatment with single-agent Bruton tyrosine kinase inhibitors (BTKi) (26%; 50/194) or venetoclax in combination with BTKi (8%; 1/12) or patients exposed to anti-CD20 antibodies <12 months before vaccination (12%; 5/41) (Table 1A). Notably, among actively treated patients, those receiving venetoclax as single-agent achieved significantly higher rate of seroconversion (55%; 10/18) compared to the others (Table 1A). Antibody concentrations showed a similar trend; patients in remission presented the highest median antibody titer (585 U/mL), while those on therapy with single-agent BTKi the lowest (22.6 U/mL, p < .01) (Table 1A). Among numerically major subgroups, we identified by univariate analysis patient characteristics associated with seroconversion rate. In patients in watch and wait, unmutated immunoglobulin heavy variable genes (u-IGHV) associated with a lower, while deletion(13q) with a higher seroconversion rate. Deletion(17q) had a negative impact on seroconversion in patients on single-agent BTKi. In both subgroups, serum immunoglobulin deficiency (IgG≤6g/dL) paralleled with low seroconversion rate (Table S3). Then, we analyzed humoral responses in 268 patients after the first booster dose of vaccine (Figure S1 and Table S2). Although the rate of patients showing humoral memory increased compared to the post-2 dose assessment, only 47% (52/112) of single-agent BTKi, 27% (3/11) of BTKi plus venetoclax treated patients, and 32% (6/19) of patients exposed to anti-CD20 exhibited antibody responses, mostly with a low median antibody titer (Table 1A). At variance, patients in remission or in watch and wait were positive in 100% (22/22) and 85% (73/86) of cases, respectively, with a median antibody titer above the upper limit of quantification (>2500 U/mL) (Table 1A). The presence of u-IGHV genes negatively impacted seroconversion in watch and wait patients, whereas trisomy of chromosome 12 and mutation of the TP53 gene did so in cases under single-agent BTKi (Table S3). Focusing on patients who failed to achieve a humoral response after two doses with available data after the booster (N = 118) (Figure S1), the majority (N = 95) were actively treated. Of these patients, only 28% (20/71) of those under single-agent BTKi, 33% (3/9) of those under BTKi plus venetoclax, and 14% (2/14) of those exposed to anti-CD20 seroconverted following the booster dose (Table 1A). Last, we evaluated, in 76 patients, the effect of the mRNA second booster dose (Figure S1). Only 40% (14/35) of those on active single-agent BTKi were seropositive, still with a low median antibody titer (33 U/mL) (Table 1A). Both the seroconversion rate (80% 4/5;) and the median antibody titers (2500 U/mL) of patients treated with venetoclax were more similar to those of patients in watch and wait (86%; 24/28) and in remission (100%; 6/6) (Table 1A), although small numbers preclude drawing definitive conclusions. In the cohort of patients persistently seronegative after the third dose and followed longitudinally, the fourth vaccine dose elicited an antibody response in only 21% (4/19) of those on active BTKi treatment with very low antibody titers (median 1.34 U/mL) (Table 1A). We also analyzed the humoral responses in 66 patients with SARS-CoV-2 infection 4–16 weeks before the time of sampling (Figure S1); all but four patients had also received at least the primary cycle of vaccination before the infection. Infection induced the highest seroconversion rates in patients in watch and wait (85%; 11/13) and in clinical remission after therapy (80%; 4/5) (Table 1A). Among treated patients, none (0/3) of those infused with anti-CD20, 66% (22/33) of those on single-agent BTKi, and 50% (3/6) of patients on BTKi plus venetoclax showed detectable antibodies, while all (6/6) patients treated with single-agent venetoclax seroconverted (Table 1A). Median antibody titers paralleled the observed seroconversion rates, being highest for watch and wait and off-therapy in remission patient subgroups (>2500 U/mL) and lowest for patients on BTKi (105 U/mL; p < .01) (Table 1A). In 59 postinfection patients previously analyzed for seroconversion we additionally evaluated SARS-CoV-2-specific T-cell responses (Figure S1) through an established assay that monitors the expression of multiple T-cell functional activation markers (n = 7), including surface activation induced markers and IL-2, IFN-γ, and TNF-α secretion in response to spike peptides12 (Table S4 and Figure S2). All patients exhibited some degree of spike-specific T-cell activation. In particular, 92% (54/59) and 68% (40/59) of cases showed at least one marker of spike-specific CD4+ or CD8+ T cells, respectively (Table 1B). In parallel, we considered the co-expression of functional markers to further dissect the quality of the response. BTKi treated patients (n = 30 in single-agent and n = 5 plus venetoclax) showed similar frequencies of spike specific T cells as compared to untreated cases (n = 10 watch and wait and n = 5 off-therapy in remission) (Table 1B and Figure S3 and Graphical Abstract). No evaluated patient characteristic influenced T-cell responses (Table S5). We also analyzed T-cell responses in 27 post-only-vaccination patients previously analyzed for seroconversion (Figure S1). We observed T-cell responses with at least one marker of spike-specific T cell activation in the vast majority of cases (85%; 23/27) (Table 1B). Moreover, vaccinated patients exhibited a pattern of expression of functional markers similar to postinfection patients. Also in this category, treatment status did not impact the quality of T-cell responses. We observed no statistically significant differences when we compared vaccinated groups stratified by treatment status with the respective postinfection counterparts, thus excluding potential biases deriving from the few untreated cases present among the vaccinated patients (Table 1B). Importantly, 92% (12/13) of seronegative vaccinated cases exhibited T-cell responses in terms of marker frequencies comparable to the whole vaccinated group (Table 1B and Figure S3 and Graphical Abstract). In summary, we report that after additional booster doses the vast majority of both treatment-naïve and in remission patients mounted a humoral response with a median antibody titer above the measurable scale, with increasing rates up to the fourth dose, as previously indicated.6 In line with previous studies showing that anti-S1-immunoglobulin concentration correlates to the neutralizing capacity of these antibodies also in cases with hematologic malignancies,13 our data support that effective humoral memory can develop in these subgroups of patients with CLL. Whether this immunization might be functional against emerging novel SARS-CoV-2 variants remains to be tested. Importantly, we were able to delve deeper into the impact of treatment and show that ongoing BTKi and anti-CD20 monoclonal antibody treatments remain limiting factors also beyond the primary vaccination cycle. Having said that, booster doses did increase the seroconversion rate and the antibody titers, also in our cohort of BTKi-treated patients, though at a lower extent thus suggesting the appropriateness of administering additional booster doses even in seronegative patients. Notably, the majority of vaccinated patients successfully developed some degree of immune cellular response. In particular, it is of great clinical interest that no evident detrimental association emerged between cellular memory and the anti-leukemic treatments, at variance with the humoral response (Figure S3 and Graphical Abstract). Though this is potentially reflective of the efficacy of the vaccination in terms of eliciting a universal T-cell response, it remains to be established if and how this may indeed correlate with actual clinical protection against COVID-19. To this end, we were able to compare the rate of seroconversion and the quality of T cell responses after vaccination only and after a natural SARS-CoV-2 infection. The data collected in the postinfection group of patients with CLL show that seroconversion occurs at a rate and magnitude similar to that observed in post-booster cases, confirming that treatment with BTKi or anti-CD20 antibodies is a limiting factor for developing a humoral response also in post-infected individuals. That notwithstanding, it is remarkable that all patients analyzed postinfection showed cellular immune memory responses, qualitatively independent of treatment status and at a degree comparable to that of vaccinated patients (Table 1B and Figure S3 and Graphical Abstract). Future research is needed to establish the level of humoral and cellular memory sufficient to protect against the severe and deadly consequences of SARS-CoV-2 infection that remain high in patients with CLL. In this context, our data support the prompt use of additional vaccination boosters in patients with CLL, especially in those persistently seronegative regardless of their treatment status. That said, it is advisable to optimize the timing of the start of anti-leukemic therapies in order to shorten the time (and number of boosters) needed to achieve effective immune memory: in particular, during follow-up, either before treatment or after effective therapy, remains the best setting where to promptly administer vaccines. ACam, ACap, PG, KS, and LS designed the study, collected and analyzed the data, and wrote the manuscript. SH, CT, MF, PR, EP, MK, and ML performed the analyses and analyzed the data. EA, FM, ES, MC, CC, YH, SH, TC, MD, MK, KG, MS, CM, MM, HB, SA, SL, APK, MH, ISN, QH, CD, JK, JZ, FV, AMR, DB, IET, VMP, VR, NS, ES collected samples and provided clinical data. The project was supported in part by Fondazione Veronesi (ID 1852164), Janssen (IBR-I-20-EMEA-014-V01/54179060CLL4024; NOPRODCLL4001), and MH CZ—DRO (FNBr, 65269705) and AIRC 5 per mille program 2018 (ID 21198). [Correction added on February 21, 2024, after first online publication: The preceding sentence, the seroconversion rate and statistical significance of BTK inhibitors (single-agent) in Table 1 and paragraph 6 were corrected.] YH: honoraria from Janssen, AbbVie, AstraZeneca, Meidson, Lilly, Roche; research grant from Janssen. TC: honoraria from AbbVie. HB: honoraria from Abbvie, Astra Zeneca, Alvogen, Astellas, Amgen, Bristol Meyers Squibb, Janssen, Novartis, Pfizer, Roche, Sobi, Sandoz, Swixx, Takeda. MD: honoraria and advisory boards from AOP Orphan, AbbVie, AstraZeneca, Janssen, Glaxo-Smith-Kline. MS: travel grants and honoraria from Janssen, AbbVie, AstraZeneca. NS: honoraria from AbbVie, Janssen, and AstraZeneca. LS: honoraria from AbbVie, AstraZeneca, BeiGene, Janssen, Lilly, speakers bureau Octapharma. KS: honoraria from from AbbVie, AstraZeneca, Janssen; research support from AbbVie, AstraZeneca, Gilead Sciences, Janssen, and Roche. PG: honoraria from AbbVie, Arqule/MSD, AstraZeneca, Celgene/Juno/BMS, Janssen, Loxo/Lilly, Roche; research support from AbbVie, AstraZeneca, Janssen, Gilead, Sunesis. The other authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.