Functional immune responses against SARS-CoV-2 variants of concern after fourth COVID-19 vaccine dose or infection in patients with blood cancer

Summary Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.


In brief
Fendler et al. evaluate neutralizing antibody (Nab) and cellular responses to a fourth COVID-19 vaccination in patients with blood cancer. The proportion of patients with detectable NAb and T cell responses increased after the fourth vaccine dose; however, some had suboptimal NAb responses, in particular, those receiving B cell-depleting therapies.

INTRODUCTION
A third COVID-19 vaccine dose induces functional immune responses in most patients with cancer, including neutralizing antibodies (NAbs) against variants of concern (VOCs) and T cell responses. However, some patients with blood cancer, especially those receiving B cell-depleting therapies, have inadequate immune responses even after a third dose 1,2 and may, in turn, have a higher risk of breakthrough infection. Regarding the Omicron BA.1 variant, NAb response increased after three doses in patients with solid cancer, but a substantial proportion of patients with blood cancer still lacked NAb responses against Omicron BA.1. 3,4 Additional sublineages with immune-evasive properties, such as BA.4. and BA.5, are now prevalent in many countries, including the UK. 5 Amid widespread lifting of COVID-19 public health measures and high rates of community transmission of Omicron, a significant proportion of COVID-19 deaths still occur in patients with blood cancer. 6 In the UK, a fourth vaccine dose was recommended in December 2021 for patient groups, including patients with blood cancer. It remains unknown whether this has an impact on those with suboptimal responses following three doses.
Here, we report the follow-up findings from CAPTURE, a prospective longitudinal cohort study assessing the functional immune responses to COVID vaccinations in patients with cancer. We report immune responses in patients with blood cancer who received a fourth vaccine dose in December 2021 to February 2022. Longitudinal sampling within CAPTURE was used to identify patients with breakthrough infections (BTIs) and to describe their NAb responses before and after infection.

RESULTS
We evaluated 80 patients with blood cancer who received a third and fourth dose of BNT162b2 after two doses of ChAdOx1 (n = 45, 56%) or BNT162b2 (n = 35, 44%) (Table 1). Furthermore, we evaluated 51 patients (n = 40 solid cancer, n = 11 blood cancer) with BTI at least 7 days following the second or third dose of the COVID-19 vaccine ( Table 2).
Blood was collected at a median of 28 days (range 8-60 days) after the third dose and 18 days (range 6-67 days) after the fourth dose. NAbs were measured using an established microneutralization assay, [7][8][9] and IC 50 titers (NAbT) of <40 (below the quantitative range) were considered undetectable.
Multivariable logistic regression analysis (MVA; CD4 + and CD8 + T cell responses were analyzed using an activation-induced marker (AIM) assay (using CD137 and OX40 as markers for CD4 + T cell activation and CD137 and CD69 as markers for CD8 + T cell activation) after stimulation with a peptide pool against full-length WT spike or Omicron spike in 39/80 patients with blood cancer (Table 1). T cell responses were considered positive if a 2-fold increase in AIM-positive T cells was detected after peptide stimulation vs. unstimulated control. 11 Thirty-two of thirty-nine (82%) patients were evaluable and had matched samples after the third and fourth doses.
Considering T cell responses against Omicron spike, 31% (10/32) of patients with blood cancer had CD4 + T cell responses after three doses compared with 59% (19/32) after four doses (McNemar test, p = 0.0077) ( Figure 1C), while 34% (11/32) had CD8 + T cell responses after three and 44% (14/32) after four doses (McNemar test, p = 0.51) ( Figure 1D). Considering T cell responses to the WT spike, 59% (19/32)  Taken together, these data indicate that patients with blood cancer benefit from a fourth vaccine dose, indicated by increases in the proportion of patients with NAb and T cell responses against VOCs.
Within CAPTURE, we identified 57 participants (n = 41 solid cancer, n = 16 blood cancer) with BTI, defined here as a positive SARS-CoV-2 RT-PCR and/or lateral flow antigen test at least 7 days following the second COVID-19 vaccine. All infections were detected during routine clinical care following two vaccine  Report ll OPEN ACCESS doses (36 patients, n = 33 solid cancer, n = 3 blood cancer) or three or four vaccine doses (21 patients, n = 8 solid cancer, n = 13 blood cancer) ( Table 2). The median time from the most recent vaccine dose to infection was 79 days (IQR 66-139). Most patients had mild COVID-19 (n = 42/57; WHO score 2-3). 12 The most common symptoms were cough (n = 29), fever (n = 22), or coryza (n = 20); and nine patients were asymptomatic (WHO score 1). Six patients had moderate (n = 2, WHO score 4-6) or severe COVID-19 (n = 4, WHO score 7-10) requiring hospitalization and treatment with oxygen therapy (n = 5), corticosteroids (n = 5), and IL-6 monoclonal antibodies (n = 3). Four patients died within 28 days of a positive SARS-CoV-2 test. Eight patients with blood cancer received antiviral therapies or monoclonal antibodies (remdesivir, n = 4; molnupiravir, n = 2; sotrovimab, n = 2) for treatment of acute SARS-CoV-2 infection. Patients with BTI following the second dose were considered as being infected with the Delta variant given the high prevalence of this variant at the time. In contrast, 19/21 patients infected following the third vaccine dose were infected from December 2021 onward at the peak of the Omicron wave, and these infections were subsequently considered as Omicron infections.
Convalescent blood samples were available for 51/57 patients (n = 36 infected after the second dose, and n = 15 infected after the third dose). During convalescence, 32/36 patients with BTI after two doses had detectable NAbT against Delta ( Figures 1E  and 1F). Following the third dose, 15/15 patients had detectable NAbT against Omicron after infection ( Figures 1E and 1G). In addition, blood samples between the most recent vaccine and the infection were available for 25 patients (n = 12 infected after second dose, n = 13 infected after third dose) ( Figure 1E). Eight of twelve patients infected after two vaccine doses had undetectable NAbT against Delta or their NAbT declined before infection ( Figure 1F), and 8/13 patients infected after three doses had undetectable NAbT against Omicron before infection ( Figure 1G). NabTs against WT were detected in all but one patient (after two vaccine doses) before infection.
Notably, patients with Delta and Omicron BTI had evidence of a degree of boosting cross-reactive neutralizing responses against the other variants, consistent with previous reports that cross-reactivity is observed in previously vaccinated patients. 13 Of the three patients with no detectable convalescent NAb, two were blood cancer patients with severe COVID-19 who later died, and one patient had a solid cancer with mild COVID-19. In summary, our data are consistent with published data in healthy individuals 14 in that low variant-specific NAb responses may contribute to infection risk.

DISCUSSION
We demonstrate that patients with blood cancer can benefit from a fourth vaccine dose, even if they had an undetectable response after three doses, especially when considering immune responses to Omicron BA.1 or BA.2. In a cohort of health care workers, a fourth dose of BNT162b2 after three doses of the same vaccine elicited an increase in spike and neutralizing titers, Targeted therapy, <28 days 17 (30) 10 (28)  7 (33) Anti-PD-L1 ± anti-CTLA-4, <6 months 7 (12) 7 (19) 0 (0) Anti-CD20 mAb, <12 months 3 (5) 1 (3)  2 (10) HSCT, ever 4 (7) 0 (0) 4 (19) Other medication   Report ll surpassing titers immediately after the third dose. 15 Our data show a nuanced picture in patients with blood cancer, where especially NAbTs against Omicron (which were undetectable in a substantial proportion after the third dose) were increased by the fourth dose. These findings highlight the need to consider variant-specific responses in determining which patients may benefit from additional vaccine doses or therapies using antiviral prophylaxis or monoclonal antibodies. Recent reports confirmed a higher risk of Omicron BTIs compared with Delta BTIs in individuals both with and without cancer, likely resulting from Omicron escaping vaccine-induced immunity. 16,17 In keeping with these findings, we observed both Delta and Omicron breakthroughs in our cohort, which were associated with low NAbTs against the respective variant. Our study has several limitations. First, the heterogeneity and size of the cohort limits subgroup analyses, and specific studies in each cancer type are needed to define risk factors for low NAb responses beyond B cell-depleting therapies and to define the determinants of T cell responses. Second, the precise correlate of protection from BTI remains undefined, and prospective studies are needed to accurately estimate infection risk after three and four vaccine doses in patients with blood cancer. Reports after three vaccine doses confirm the high clinical efficacy of COVID-19 vaccines in the general population and elderly individuals [18][19][20][21] and an additional benefit in older and at-risk individuals who had received a fourth dose. 22,23 Comparable data are currently lacking in patients with cancer, but our observations in patients with BTIs agree with models and data in healthy populations suggesting a direct association of NAbT with infection risk, 14,24 although our study was not designed to definitively address a direct association.
Third, we have not generated data on NAbTs against the Omicron subvariants BA.4 and BA.5, which have become the prevalent circulating variants in a number of countries, including the UK. 5 Reports indicate that, while booster vaccination increases responses to all Omicron sublineages, BA.4 and BA.5 show greater immune escape; therefore, patients with blood cancer will likely have less protection against these variants. [25][26][27] Overall, our data highlight the benefit of a fourth vaccine dose in patients with blood cancer and confirm that patients with B cell-depleting therapies are at the highest risk of having impaired NAb responses.

Limitations of the study
We acknowledge limitations of our study. First, cohort size and heterogeneity limited subgroup analyses, and larger and/or subtype-or treatment-specific cohorts are needed to evaluate immune responses in particular groups. Second, we were unable to directly assess immune responses to the Omicron sublineages BA.4 and BA.5; these are circulating in many countries and have been reported to show greater degree of immune escape that BA.1 and BA.2 sublineages. Finally, our study did  While the aim of our study was not to define precise correlates of immune protection, a means to identify patients with suboptimal protection should be a priority for the community. Prospective, adequately powered studies to address this are especially important in view of updated vaccine design and in the context of emergent variants.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
The authors have contributed this article on behalf of the CAPTURE Consortium. We thank the clinical leads and subunit teams for recruiting patients to the study, including F. We thank Prof. Wendy Barclay of Imperial College and the wider Genotype to Phenotype Consortium, for the Alpha and Delta strains used in this study, and Max Whiteley and Thushan I. de Silva at The University of Sheffield and Sheffield Teaching Hospitals NHS Foundation Trust for providing source material. We thank Prof. Gavin Screaton of the University of Oxford for the Omicron strain used in this study. We also thank volunteer staff at the Francis Crick Institute and the Crick COVID-19 Consortium.
Due to the pace at which the field is evolving, we acknowledge researchers of COVID-19, particularly those furthering our understanding of the COVID-19 vaccine-induced immune response. We apologize for the work that was not cited. This

Materials availability
All requests for resources and reagents should be directed to the lead contact author. All reagents will be made available on request after completion of a Materials Transfer Agreement.
Data and code availability d Data: All data reported in this paper will be shared by the lead contact upon request. d Code: This paper does not report original code. d Additional information: Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Study design CAPTURE (NCT03226886) is a prospective, longitudinal cohort study that commenced recruitment in May 2020 at the Royal Marsden NHS Foundation Trust. Adult patients with a current diagnosis or history of invasive cancer are eligible for enrolment. 29 Inclusion criteria are intentionally broad, and patients were recruited irrespective of cancer type, stage, or treatment. The primary endpoint of the CAPTURE study was the seroconversion rate in cancer patients at 14-28 days following the second dose of vaccine. 30 Exploratory endpoints include evaluation of neutralising responses to SARS-CoV-2 variants of concern (VOC). CAPTURE received ethical approval as a substudy of the TRACERx Renal Study (NCT03226886). TRACERx Renal was initially approved by the NRES Committee London, Fulham, on January 17, 2012 (11/LO/1996). The CAPTURE protocol was part of Substantial Amendment 9 and received approval by the Health Research Authority on April 30, 2020, and the NRES Committee London, Fulham on May 1, 2020. CAPTURE is conducted in accordance with the ethical principles of the Declaration of Helsinki, Good Clinical Practice and applicable regulatory requirements. All patients provided written, informed consent to participate. The Chief Investigator, Samra Turajlic is responsible for the oversight of all aspects of study conduct and governance.

Study schedule and follow-up
Detailed sampling schedule and methodology were described previously. 29 Patients eligible for a third and fourth vaccine dose were invited to receive the vaccine in our institution. Samples were collected following the third vaccine dose (Post-V3; 14-28 days post third vaccination) and following fourth vaccine dose (Post-V4; 7-28 days post fourth vaccine dose).
The study protocol did not mandate screening for breakthrough SARS-CoV-2 infections and all breakthrough infections were detected during the course of routine clinical care. Where breakthrough infections were reported, an additional post-infection blood sample was sought at least 14 days following the positive SARS-CoV-2 test.

Patient data
Demographic, epidemiological and clinical data (e.g. cancer type, cancer stage, treatment history, history of SARS-CoV-2 infection) were collected from the internal electronic patient record and prospectively from patients. Pseudonymised data was entered into a cloud-based electronic database (Ninox Software, Berlin, Germany). Chemotherapy, targeted therapy (small molecule inhibitors or monoclonal antibodies) or endocrine therapy was deemed to be current if given within 28 days of vaccination. Treatment with immune checkpoint inhibitors (CPI) within six months was considered significant given the prolonged receptor occupancy reported with these agents. 31 Treatment with ant-CD20 monoclonal antibodies within 12 months was considered. Concomitant medications were recorded for: corticosteroids (considered significant if >10 mg prednisolone equivalent given for at least seven days); GCSF when delivered within 48 h of vaccination or five days in the case of pegylated preparation; and other immunosuppressive drugs taken within 48 h of vaccination.

Definition of breakthrough SARS-CoV-2 infection
We considered patients to have had a breakthrough SARS-CoV-2 infection if they had SARS-CoV-2 positive RT-PCR (tests conducted as part of routine clinical care) at least seven days following the second COVID-19 vaccine dose. Breakthrough infections after the second vaccine dose were considered delta infections while breakthrough infections after the third vaccine dose were considered omicron infections based on the high prevalence of the respective variants at the time.