key: cord-0684966-hgc4zvsj
authors: Herishanu, Yair; Rahav, Galia; Levi, Shai; Braester, Andrei; Itchaki, Gilad; Bairey, Osnat; Dally, Najib; Shvidel, Lev; Ziv-Baran, Tomer; Polliack, Aaron; Tadmor, Tamar; Benjamini, Ohad
title: Efficacy of a Third BNT162b2 mRNA COVID-19 Vaccine Dose in Patients with CLL who Failed Standard Two-dose Vaccination
date: 2021-12-06
journal: Blood
DOI: 10.1182/blood.2021014085
sha: aeb7bc5201aa5c51899df872586069fe6f589fc1
doc_id: 684966
cord_uid: hgc4zvsj

Patients with chronic lymphocytic leukemia (CLL) have an impaired antibody response to COVID-19 vaccination. Here, we evaluated the antibody response to a third BNT162b2 mRNA vaccine in patients with CLL/small lymphocytic lymphoma (SLL) who failed to achieve a humoral response after standard two-dose vaccination regimen. Anti-SARS-CoV-2S and neutralizing antibodies were measured 3 weeks after administration of the third dose. In 172 patients with CLL the antibody response rate was 23.8%. Response rate among actively treated patients (12.0%, n=12/100) was lower compared to treatment-naïve patients (40.0%, n=16/40; OR=4.9, 95% CI 1.9-12.9; p<0.001) and patients off-therapy (40.6%, n=13/32; OR=5.0, 95% CI 1.8-14.1; p<0.001), (p<0.001). In those actively treated with BTK inhibitors or venetoclax ± anti-CD20 antibody, response rates were extremely low (15.3%, n=9/59 and 7.7%, n=3/39, respectively). Only one of the 28 patients (3.6%) treated with anti-CD20 antibodies <12 months prior to vaccination responded. The anti-SARS-CoV-2S antibody levels correlated linearly with neutralizing antibody titers (r=0.732, p<0.001). In a multivariate analysis, the independent variables that were associated with response included lack of active therapy (OR=5.6, 95% CI 2.3-13.8; p<0.001) and serum IgA levels ≥80 mg/dL (OR=5.8, 95% CI 2.1-15.9; p<0.001) In conclusion, in patients with CLL/SLL who failed to achieve a humoral response after standard two-dose BNT162b2 mRNA vaccination regimen, close to a quarter responded to the third dose of vaccine. The antibody response rates were lower during active treatment and in patients with a recent exposure (<12 months prior to vaccination) to anti-CD20 therapy. Clinical Trial # NCT04862806

1. In patients with CLL/SLL who failed to respond to two BNT162b2 doses, close to a quarter responded to the third dose of vaccine.

2. Antibody mediated responses were lower during active treatment and following exposure to anti-CD20 therapy.

Patients with chronic lymphocytic leukemia (CLL) have an impaired antibody response to COVID-19 vaccination. Here, we evaluated the antibody response to a third BNT162b2 mRNA vaccine in patients with CLL/small lymphocytic lymphoma (SLL) who failed to achieve a humoral response after standard two-dose vaccination regimen. Anti-SARS-CoV-2S and neutralizing antibodies were measured 3 weeks after administration of the third dose.

In 172 patients with CLL the antibody response rate was 23.8%. Response rate among actively treated patients (12.0%, n=12/100) was lower compared to treatment-naïve patients (40.0%, n=16/40; OR=4.9, 95% CI 1.9-12.9; p<0.001) and patients off-therapy (40.6%, n=13/32; OR=5.0, 95% CI 1.8-14.1; p<0.001), (p<0.001). In those actively treated with BTK inhibitors or venetoclax ± anti-CD20 antibody, response rates were extremely low (15.3%, n=9/59 and 7.7%, n=3/39, respectively). Only one of the 28 patients (3.6%) treated with anti-CD20 antibodies <12 months prior to vaccination responded. The anti-SARS-CoV-2S antibody levels correlated linearly with neutralizing antibody titers (r=0.732, p<0.001). In a multivariate analysis, the independent variables that were associated with response included lack of active therapy (OR=5.6, 95% CI 2.3-13.8; p<0.001) and serum IgA levels ≥80 mg/dL (OR=5.8, 95% CI 2.1-15.9; p<0.001)

In conclusion, in patients with CLL/SLL who failed to achieve a humoral response after

The coronavirus disease 2019 is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations ranged from asymptomatic infection to life-threatening disease 1, 2 . Severe illness is more likely to occur in elderly patients and in those who have significant underlying medical conditions 1,2 . Since 2021, variants of the virus have emerged and become dominant in many countries, with the Delta variant being currently one of the most virulent 3 .

Patients with chronic lymphocytic leukemia (CLL) have an increased risk for severe COVID-19 and subsequent mortality 4, 5 . Recent reports have suggested that mortality from COVID-19 in patients with CLL has decreased over time, possibility due to better patient management 6 . Seroconversion after the acute phase of SARS-CoV-2 infection is seen in 60-82% of patients with CLL [6] [7] [8] . Patients with CLL may develop persistent COVID-19 infection, as result of their inability to effectively eliminate the virus. In such cases, prolonged shedding of infectious SARS-CoV-2 virus and additional in-host genomic evolution may eventually lead to development of new virus variants 6 . Furthermore, the immune response to COVID-19 vaccination is reduced in patients with CLL/small lymphocytic lymphoma (SLL) 9-11 and depends on continuing disease activity and treatment and is particularly low during therapy at the time of vaccination 9,11 .

Administration of a third dose of mRNA-1273 COVID-19 vaccine to organ-transplant recipients two months after the second dose, has been shown to be safe and to increase antibody titers compared to placebo. Moreover, 44% of solid-organ transplants who had been seronegative after two doses of BNT162b2 became seropositive after a third vaccine dose 12 .

In the light of the above we decided to evaluate the serologic response to a third BNT162b2 mRNA COVID-19 vaccine in patients with CLL/SLL who failed to achieve a humoral response after the standard two-dose vaccination regimen.

This prospective study, conducted in the framework of the Israeli CLL study group, investigated the efficacy of a third BNT162b2 mRNA COVID-19 vaccine dose in seronegative patients with CLL/SLL who were followed at 7 medical centers in Israel. The study was approved by the institutional review board of each participating center and is registered in ClinicalTrials.gov, number NCT04862806. All subjects provided informed consent and were vaccinated through a national Israeli vaccination program of administering a third vaccine dose to immunocompromised subjects. Eligibility criteria for the study included: diagnosis of CLL/SLL according to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria 13 , age 18 years or older, no known history of SARS-CoV-2 infection and failure to obtain a serologic response 2-3 weeks after the second BNT162b2 vaccine dose.

Serum from the peripheral blood of patients with CLL/SLL were collected before and 3 weeks after administration of the third vaccine. The primary endpoint was to determine the proportion of subjects acquiring anti-SARS-CoV-2S antibodies. Serum samples were analyzed using the Architect AdviseDx SARS-CoV-2 IgG II (Abbot, Lake Forest, Illinois, USA), which detects immunoglobulin class G (IgG) antibodies to the receptor binding domain (RBD) of the S1 subunit of the spike (S) protein, with a positive cutoff of >50 AU/mL.

A surrogate viral assay was used to test antiviral humoral response based on a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S.

The neutralizing activities was assessed in a high-throughput fluorescent reporter assay as previously published 14 (the assay was kindly provided by Gert Zimmer, University of Bern, Switzerland).

All subjects were questioned about local or systemic adverse events (AEs) that had occurred within 7 days after each vaccine dose. In patients who developed COVID-19 after vaccination, disease severity was defined according to the criteria of the World Health Organization 15 . Relevant data was also extracted from the medical records and included: demographics, complete blood count, Binet stage, serum immunoglobulin (Ig) levels, mutational status of the immunoglobulin heavy chain variable (IGHV) gene (using a cut off of 98% identity to the germ-line sequence) and analyses of genomic aberrations by fluorescent in situ hybridization (FISH) 16 variables. Variables which were associated with a serologic response at a significant level of p<0.1 in the univariate analysis were then included in the multivariate analysis performed by binary logistic regression. Log10 transformation of quantitative variables into approximately normally distributed variables. Pearson's correlation coefficient between normally distributed quantitative variables was calculated. WINPEPI version 11.65 was used to calculate odds ratio (OR) and its 95% confidence interval (CI), and the mid-p exact CI when at least one cell had a limited number of subjects. Statistical significance was determined as p<0.05, and all statistical tests were 2-sided.

From July 2020 through August 2021, a total of 172 patients with CLL/SLL were included in the study (patient baseline demographic and disease characteristics are summarized in Table  1 ). All patients failed to produce anti-SARS-CoV-2 S antibodies after the standard two-dose BNT162b2 mRNA vaccination regimen and were seronegative before the third vaccine dose.

The median age was 72.1 years (IQR, 68.1-77.7) and 121 (69.9%) were males. Forty patients (23.3%) were treatment-naïve, 100 (58.1%) on-active therapy, 32 (18.6%) previously treated (off-therapy). Among the off-therapy patients, 24 (75%) were in remission (CR: n=18; PR: n=6) and 8 (25%) in relapse. The median time from CLL diagnosis to the third vaccination was 63.5 months (IQR, 61-162), the median time from second to the third vaccine was 179 days (IQR, 175-187) and the median time from the third vaccine dose to serology testing was 21 days (IQR, [21] [22] . Furthermore, in patients on-therapy, the median time from initial treatment to third vaccination was 17.7 months (IQR, 8.3-36.4).

Antibody response to the third BNT162b2 mRNA dose was seen in 41 of 172 (23.8%, Table   2 ) patients with a median antibody level of 2 AU/mL (IQR, 0-40.8, Supplementary Table 1, Figure 1A -B). In a univariate analysis, the variables that were significantly associated with response were: lack of active therapy (including previously untreated patients and those off therapy; OR=5.0, 95% CI 2.2-11.5, p<0.001), ≥12 months from the last anti-CD20 therapy to the third vaccination ( Figure   1A ] and higher antibody levels [median=8, (IQR, 0.3-509.8), median=6 (IQR, 0.0-764.5); respectively; Figure 1B and Supplementary Table 1) compared to actively treated patients (12.0%, n=12 of 100; OR=4.9, 95%CI 1.9-12.9, p<0.001, and OR=5.0, 95% CI 1.8-14.1, p<0.001, respectively, Table 2 ; and median=0.0, IQR, 0-10.3, Figure 1A -B and Supplementary Table 1 ; p<0.001). Among patients off-therapy, the serologic response rates among patients in CR, PR and relapse were 38.9%, 50% and 37.5%, respectively (p=0.894).

The median time from last treatment to the third vaccine dose, in these patients, was 11.5 months (IQR 7.3-51.6) in the responders compared to 10.2 months (IQR 4.0-20.9) in the nonresponders (p=0.465).

Among the 100 patients on treatment at the time of vaccination, 99 (99%) were treated with novel agents, including BTK inhibitors (ibrutinib or acalabrutinib, n=59), venetoclax ± anti-CD20 antibody (rituximab or obinutuzumab, n=39) and one patient treated with idelalisib.

Antibody response rate in patients receiving BTKi was 15.3% (n=9 of 59) compared to 7.7% (n=3 of 39) in patients treated with venetoclax ± anti-CD20 antibodies (OR=2.2, 95% CI 0.6-10.4, p=0.353, Figure 1C and Table 2 ). Five patients were treated with venetoclax alone, of A total of 94 patients with CLL had been previously exposed to anti-CD20 therapy; 28 within the last 12 months prior to vaccination (21 patients <6 months and 7 patients between 6-12 months; median=3.9 months, IQR, 1.2-6.6) and 66 patients, ≥12 months before vaccination (median=49.2 months, IQR, 19.1-70.7). Most patients (n=24, 85.7%) exposed to anti-CD20 antibodies <12 months prior to vaccination, received it in combination with venetoclax. Only one of the 28 patients (3.6%), treated with anti-CD20 antibodies within the last 12 months, has responded vs. 22 .7% (n=15 of 68) of those exposed to anti-CD20 therapy ≥12 months prior to vaccination (p=0.033, Table 1 ). In patients who received anti-CD20 antibody ≥12 months, there was no statistically significant difference in median of time since last anti-CD20 treatment between responsive (64.5 months, IQR, 18.3-83.6) compared to non-responsive patients (44.3 months, IQR, 20.5-66.5) (p=0.288). However, after controlling for other active treatments, each month that elapsed from the end of administration of the anti-CD20 therapy increased the odds for response by 1.03 times (OR=1.03, 95% CI 1.01-1.05; p=0.020). In a multivariate analysis, the independent variables that were associated with response included lack of active therapy (OR=5.6, 95% CI 2.3-13.8; p<0.001) and serum IgA levels ≥80 mg/dL (OR=5.8, 95% CI 2.1-15.9; p<0.001).

Samples taken from 54 patients were also evaluated for the production of neutralizing antibodies. As shown in Figure 1D , the anti-SARS-CoV-2 RBD antibody levels linearly correlated with neutralizing antibodies titers (log transformed, r=0.732 and p<0.001). Table 2 ). Two patients were actively treated with ibrutinib, one was treatment-naïve and one had been previously treated with obinutuzumab monotherapy.

Overall, one patient died because of COVID-19 while all the others recovered. Among these patients, three were seronegative after a third vaccine dose and another seropositive with a low antibody titer (138 AU/mL). 

This study evaluated the serologic response to a third BNT162b2 mRNA vaccine dose in patients with CLL/SLL who had failed to respond to the standard two-dose vaccination program. The overall response rate was 23.8%, while in univariate analysis lack of active treatment, ≥12 months from the last anti-CD20 therapy and higher serum IgG and IgA levels, were associated with a better response rate. In multivariate analysis, lack of active therapy and serum IgA levels ≥80 mg/dL were the only independent variables associated with serologic response. In our cohort the response rate was lower than the 44% reported for organ transplant recipients, who had been seronegative after two doses of BNT162b2, and responded to the third vaccine dose 12 In treatment-naïve and previously treated patients, the response rate after vaccination approached 40% compared to only 12% in those currently on active therapy. Most of the latter were treated with BTKis or venetoclax plus anti-CD20 antibody. Treatment with ibrutinib has been reported to result in partial reconstitution of humoral immunity and normal B-cell subpopulations in patients with CLL, but at the same time, also decreased serum IgG levels starting at 6 months of therapy and becoming more profound by 24 months. The poorer response to the vaccine given after exposure to rituximab or obinutuzumab within the last 12 months prior to vaccination and the improved incremental response after 12 months, are consistent with the kinetics of rapid peripheral blood B cell depletion after anti-CD20 therapy, followed by a recovery starting between 6-9 months after treatment with a return to normal counts at 12 months 19 . Even after taking into consideration the relatively low number of patients treated with venetoclax monotherapy. The response rate still appeared to be higher compared to that seen after BTKis or with venetoclax plus anti-CD20 antibody. This also seemed to be the case after administration of the second dose of vaccine 20, 21 .

In CLL, hypogammaglobinemia is consistently associated with lower humoral responses to COVID-19 vaccination 9,20,22 even after the third vaccination. Low immunoglobulin levels are commonly encountered in patients with CLL and decrease even more with the duration and progression of disease 23, 24 . Hypogammaglobinemia is a major risk factor for infections in patients with CLL 24 and it is known that immunization with other vaccines are also more efficient if immunoglobulin levels are better preserved 25 .

Overall, the response after the third vaccine had a pattern similar to that observed after the first two doses given in CLL and this correlated with the degree of immunosuppression accompanying the disease and therapy. While, in our study patients received a homologous mRNA vaccine regimen, a recent report suggested that heterologous prime-boost vaccination may indeed facilitate a stronger response 26 . Given the low response rate evident in our cohort, the latter approach in CLL deserves further investigation.

In addition, higher anti-SARS-Cov-2 S-RBD IgG titers were associated with younger age (≤65 years), lack of active treatment and higher serum IgG and IgA levels. Similarly, we and others have already reported a better response rate and higher antibody titers after two BNT162b2 vaccine doses in younger patients with CLL 9,27 . The anti-SARS-Cov-2 S-RBD IgG titers also linearly correlated with neutralizing antibodies titers, similar to our findings recorded after the second BNT162b2 dose in patients with CLL 20 . Recently, anti-SARS-Cov-2 levels have been shown to be clinically meaningful in terms of protection against COVID-19 28 .

Among fully vaccinated health care workers, the occurrence of breakthrough infections with SARS-CoV-2 correlated with the levels of neutralizing antibodies and SARS-CoV-2s antibodies, measured both within the first month after the second vaccine dose and periinfection 28 .

CD8 T cell immunity has been shown to be a critical parameter for survival in patients with hematologic malignancy with COVID-19 29 and vaccination with two doses of BNT162b2 has been reported to induce cellular response in only about half of patients with hematologic malignancies 27 . The T-cell response generally correlated with the anti-S IgG levels, but some patients who had no humoral response also achieved T-cell responses 27 . Nevertheless, the effects of a third COVID-19 vaccine dose on cellular immunity still needs to be studied further in CLL, in order to better understand its protective role in patients who failed to achieve a humoral response.

In immunocompetent individuals the immunity after BNT162b2 vaccination has been reported to wane after six months 30 . Neutralization levels are highly predictive of immune protection 31 and a BNT162b2 booster dose can increase the antibody neutralization level by an average of 5-7 times, compared to that obtained after a second dose 32 . In subjects aged ≥ 60 years, a booster dose of the BNT162b2 vaccine has been shown to substantially lower the number of confirmed cases of COVID-19 and decrease severity of the disease 33 . The potential risk for severe COVID-19 disease and its complications in patients with CLL, outweigh the lack of vaccine response. Although, our study focused primarily on the humoral reactivity in patients who failed to respond to the initial vaccination, we suggest that a booster should be considered for all patients with CLL who had been vaccinated with mRNA vaccines including those receiving the booster while still on active anti-CLL therapy. On a case-by-case basis it may be appropriate to delay the start of therapy in order to allow for COVID-19

vaccination. Patients who have not responded to the booster and have been treated with fixed duration treatment (e.g. including with anti-CD20 antibody) may perhaps still respond to an additional booster dose after a period of time that would l allow for immune reconstitution; however the vaccine type to be used (e.g. heterologous prime-boost vaccination) and optimal timing still need to be studied more. Until now there has been no recommendation for routine serology testing in patients with CLL after COVID-19 vaccination. However, knowing the degree of response obtained would probably help for improved risk stratification and future management. In this respect, regardless of the immune response to vaccination, it is important that patients with CLL continue to take all necessary precautions.

In conclusion it is noteworthy that almost a quarter of the patients with CLL/SLL who failed to achieve a humoral response after standard two-dose BNT162b2 mRNA vaccination regimen, responded to the third dose of vaccine. The antibody mediated response continued to be markedly impaired during active treatment and after recent exposure (<12 months prior to vaccination) to anti-CD20 therapy. These findings are of practical importance as they show that a third dose can still achieve seroconversion even in the more immunosuppressed subgroup of patients with CLL. 

White blood cells, median (IQR), (10 9 shown for the all cohort and according to the disease status; All CLL patients (n=172), Treatment naïve (n=40), On-therapy (n=100) and Off-therapy (n=32). (C) Response rate in patients with CLL treated with Bruton's kinase inhibitor (BTKi, n=59) and Venetoclax (Ven) ± anti-CD20 antibody (n=39). (D) Correlation between serological titers and neutralizing antibody levels following log transformation (n=24), (Pearson's correlation coefficient r=0.732, p<0.001; r 2 =0.536). In additional 30 patients the anti-SARS-CoV-2 and neutralizing antibodies levels were negative and therefore were invalid for analysis. 

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The authors would like to thank the clinical study coordinators and nurses at the hematology department in the Tel-Aviv Sourasky, Bnai Zion, Rabin and Chaim Sheba medical centers.

YH-Initiated the trial, designed the study, collected and analyzed data and wrote the paper.OB and TT-Initiated the trial, designed the study and collected the data. SL and TVB-Analyzed the data. OsB, GR, AB, GI, ND, LS-collected the data. AP-critical reading of the manuscript

The work was supported by a grant by Janssen pharmaceutical number EV00261620 YH reports honoraria from AbbVie, Janssen, Astra-Zeneca, Medison and Roche outside the submitted work. TT reports honoraria from AbbVie, Janssen, Astra-Zeneca, Novartis, Pfizer, and Roche outside the submitted work. OB reports honoraria from AbbVie, Janssen and Astra-Zeneca outside the submitted work.The remaining authors declare no competing financial interests.