key: cord-0704925-9ugklhly
authors: Lasagna, A.; Lilleri, D.; Agustoni, F.; Percivalle, E.; Borgetto, S.; Alessio, N.; Comolli, G.; Sarasini, A.; Bergami, F.; Sammartino, J.C.; Ferrari, A.; Zavaglio, F.; Arena, F.; Secondino, S.; Falzoni, M.; Schiavo, R.; Lo Cascio, G.; Cavanna, L.; Baldanti, F.; Pedrazzoli, P.; Cassaniti, I.
title: Analysis of the humoral and cellular immune response after of a full course of BNT162b2 anti-SARS-CoV-2 vaccine in cancer patients treated with PD-1/PD-L1 inhibitors with or without chemotherapy: an update after six months of follow up
date: 2021-12-11
journal: ESMO Open
DOI: 10.1016/j.esmoop.2021.100359
sha: 2895dca8863643a597bdce4f8624108c623a09de
doc_id: 704925
cord_uid: 9ugklhly

BACKGROUND: The durability of immunogenicity of SARS-CoV-2 vaccination in cancer patients remains to be elucidated. We prospectively evaluated the immunogenicity of the vaccine in triggering both the humoral and the cell-mediated immune response in cancer patients treated with anti PD-1/PD-L1 with or without chemotherapy six months after BNT162b2 vaccine. PATIENTS AND METHODS: In the previous study, 88 patients were enrolled, while the analyses below refer to the 60 patients still on immunotherapy at the time of the follow up. According to previous SARS-CoV-2 exposure, patients were classified in SARS-CoV-2 naïve (without previous SARS-CoV-2 exposure) and SARS-CoV-2 experienced (with previous SARS-CoV-2 infection). Neutralizing antibody (NT Abs) titer against B.1.1 strain and total anti-Spike IgG concentration were quantified in serum samples. ELISpot assay was used for quantification of anti-Spike interferon gamma (IFNγ) producing cells/10(6) peripheral blood mononuclear cells (PBMC). Fifty patients (83.0%) were on immunotherapy alone, while ten patients (7%) were on chemo-immunotherapy. We analyzed separately patients on immunotherapy and patients on chemo-immunotherapy. RESULTS: Median T-cell response at six months was significantly lower than that measured at three weeks after vaccination (50 interquartile range (IQR) 20-118.8 vs 175IQR 67.5-371.3 IFNγ producing cells/10(6) PBMC;p<0.0001). The median reduction of IgG concentration was 88% in SARS-CoV-2 naïve subject, 2.1% in SARS-CoV-2 experienced subjects. SARS-CoV-2 NT Abs titer was stable maintained in SARS-CoV-2 experienced subjects while a significant decrease was observed in SARS-CoV-2 naïve subjects (from median 1:160 IQR 1:40-1:640 to median 1:20 IQR 1:10-1:40;p<0.0001). A weak correlation was observed between SARS-CoV-2 NT Abs and Spike-specific IFNγ producing cells at both six months and three weeks after vaccination (r=0.467;p=0.0002 and r=0.428;p=0.0006,respectively). CONCLUSIONS: Our work highlights a reduction in the immune response in cancer patients, particularly in SARS-CoV-2 naïve subjects. Our data support administering third dose of COVID-19 vaccine to cancer patients treated with PD1/PD-L1 inhibitors.

immunotherapy alone, while ten patients (7%) were on chemo-immunotherapy. We analyzed separately patients on immunotherapy and patients on chemo-immunotherapy.

Results: Median T-cell response at six months was significantly lower than that measured at three weeks after vaccination (50 interquartile range (IQR) 20-118.8 vs 175IQR 67.5-371. 3 IFNγ producing cells/10 6 PBMC;p<0.0001). The median reduction of IgG concentration was 88% in SARS-CoV-2 naïve subject, 2.1% in SARS-CoV-2 experienced subjects. SARS-CoV-2 NT Abs titer was stable maintained in SARS-CoV-2 experienced subjects while a significant decrease was observed in SARS-CoV-2 naïve subjects (from median 1:160 IQR 1:40-1:640 to median 1:20 IQR 1:10-1:40;p<0.0001). A weak correlation was observed between SARS-CoV-2 NT Abs and Spike-specific IFNγ producing cells at both six months and three weeks after vaccination (r=0.467;p=0.0002 and r=0.428;p=0.0006,respectively).

Conclusions: Our work highlights a reduction in the immune response in cancer patients, particularly in SARS-CoV-2 naïve subjects. Our data support administering third dose of COVID-19 vaccine to cancer patients treated with PD1/PD-L1 inhibitors.

Keywords: BNT162b2 anti-SARS-CoV-2 vaccine, cancer, PD-1/PD-L1 inhibitors, neutralizing antibody, Spike-specific T cell response, third dose

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have demonstrated high efficacy in reducing symptomatic infections and days of hospitalization [1] .

The first comprehensive meta-analysis about the immunogenicity and safety of anti-SARS-CoV-2 vaccines for patients with cancer has showed that there is no reduced rate of seroconversion compared with the control (RR 0.95, 95% IC 0.90 to 1.01, p = 0.09, I2 = 73.5%, p = 0.01) in solid cancer patients, even if a significant decrease was observed in hematological patients (RR 0.62, 95% IC 0.41 to 0.92, p = 0.02, I2 = 96.2%, p < 0.001) [2] . Additionally, patients with solid tumors vaccinated during chemotherapy courses have showed both reduced anti-RBD antibody concentration and neutralizing antibody response at 28 days after booster administration [3] .

To date, only one study reported a six-months follow-up of SARS-CoV-2 vaccine immunogenicity, efficacy and safety in cancer patients than respect to control group, revealing no differences between the two cohorts. Additionally, the decline of antibody concentration was similar six months after the second dose in the two groups, though the majority of patients was still seropositive [4] .

In our previous paper [5] , we highlighted the immunogenicity of the vaccine in triggering both the humoral and the cell-mediated immune response in cancer patients treated with anti PD-1/PD-L1 with or without chemotherapy after a full course of COVID-19 vaccine.

This study prospectively evaluated these outcomes six months after BNT162b2 anti-SARS-CoV-2 vaccine.

Patients with cancer receiving a full course of vaccine during an anti-PD-1/anti-PD-L1 therapy with or without chemotherapy were enrolled. As detailed in our previous report, the Inclusion Criteria were: i) patients aged 18 and older; ii) life-expectancy ≥ 6 months; iii) confirmed histological diagnosis of solid tumors; iv) vaccination with BNT162b2 mRNA vaccine; v) signing of informed consent. A previous infection with SARS-CoV-2 was not an exclusion criterion.

Patients were defined as "SARS-CoV-2 experienced" if they had a documented past positive SARS-CoV-2 RNA in nasopharyngeal swab and/or positive anti-Spike IgG at time of enrollment (before vaccination). Otherwise, they were classified as "SARS-CoV-2 naïve".

Patients were enrolled in two Oncology Units of Northern Italy (Fondazione IRCCS Policlinico San Matteo, Pavia and AUSL Ospedale Guglielmo Da Saliceto, Piacenza).

The study (Co-Vax) was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement for reporting observational studies [6] and was approved by the local Ethics Committee (Comitato Etico Area Pavia) and Institutional Review Board (P-20210023530). All the subjects signed an informed written consent before the enrollment. This is a prospective follow-up report of the primary study. 

The patients were monitored 26-27 weeks after the second dose with blood samples for humoral and cell-mediated immune response evaluation. Throughout the study, all patients underwent a nasopharyngeal swab before each cycle of immunotherapy.

In the first publication of this study [5] , the primary endpoint was percentage of patients with a significant increase in Spike-specific IFNγ-producing T cells between baseline and 3 weeks after second vaccination dose. In the present study we provided an update on the duration of immune response after BNT162b2 mRNA vaccination at 26-27 weeks (six months), analyzing both Spikespecific IFNγ-producing T cells and humoral response (total IgG concentration and SARS-CoV-2 NT Abs titer). Subjects were defined as "full responders" if there were positive anti-S IgG concentration, SARS-CoV-2 NT Abs titer and Spike-specific IFNγ-producing T cells. Additionally, we evaluated the incidence of virologically confirmed COVID-19 cases during the entire period of the study.

Peripheral blood mononuclear cells (PBMC) were isolated from heparin-treated blood by standard density gradient centrifugation. Briefly, PBMC (2x10 5 /100μl culture medium per well) were stimulated in duplicate for 24 h in 96-well plates (coated with anti-IFN-γ monoclonal capture antibody) with peptide pools (15mers, overlapping by 10 aminoacids, Pepscan, Lelystad The Netherlands) representative of the spike protein (S) at the final concentration of 0.25 µg/ml. Phytoheamagglutinin (PHA; 5 µg/mL) was used as positive control, and medium alone as negative control. Enzyme linked immunospot assay was performed according to our previous protocol [5] .

Responses ≥10 Spike-specific IFNγ-producing T cells/million PBMC were considered positive based on background results obtained with negative control (mean spot forming units+2SD).

In a subset of patients, phenotypical characterization of Spike-specific IFNγ-producing T cell response was performed, according to our previous protocol [5] . 

The quantitative characterization of Spike-specific IgG antibodies was performed by Trimeric assay (Liaison, Diasorin, Saluggia, Italy) and results were given as BAU/mL (positive results > 33.8 BAU/mL). Additionally, neutralizing antibody (NT Abs titer) titer against B.1.1 strain were measured as previously reported [5] and results were given as positive when NT Abs was higher or equal than 1:10 [7, 8] .

GraphPad Prism 8.3.0 GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. A two-sided P value <0.05 is considered statistically significant. Data were described with the median and interquartile range if continuous and as counts and percentage if categorical. Comparison between two groups was performed using the Mann-Whitney (unpaired samples) or Wilcoxon (paired samples) test while Spearman's test was used for the correlation analysis. Fisher exact test was used for comparison of categorical variables.

The original study cohort [5] In the first paper, complete analyses were performed in 73/78 subjects who received complete vaccination schedule. In the follow-up paper, we were able to collect samples at six months in only 60 out of 73. In particular, we have excluded thirteen patients: one patient refused the blood sample, six patients died and six patients were no longer on immunotherapy.

Fifty patients (50/60, 83.0%) were on immunotherapy alone, while ten patients (10/60, 7%) were on chemo-immunotherapy (Supplementary file 1).

Spike-specific IFNγ producing cells/10 6 PBMC measured after six months post-vaccination was 

Immune response at six months after vaccination was compared in 9 subjects treated with chemoimmunotherapy and 43 subjects treated with only immunotherapy. In order to avoid confounding factors, only SARS-CoV-2 naïve subjects vaccinated with two BNT162b2 doses were analyzed.

Overall, the rate of "full responders" was 0.78 (IC95 0.45-0.96) and 0.98 (IC95 0.88-0.99) in the two groups, respectively, even if the difference was not statistically significant (p=0.0738). Figure 4A , the median number of Spike-specific IFNγ-producing cells/10 6 PBMC in patients treated with chemo-immunotherapy was slightly lower (median 20 IQR 5-60 IFNγ producing cells/10 6 PBMC) compared to that observed in only immunotherapy patients (median 45 IQR 20-120

IFNγ producing cells/106 PBMC; p=0.0569).

Similarly, even if the differences were not statistically significant, humoral response against Spike antigen at six months was lower in patients receiving chemo-immunotherapy group in terms of both Figure 4C ).

In our cohort, age is not correlated with humoral response at six months even if a negative weak correlation was observed between age and Spike-specific IFNγ-producing cells/10 6 PBMC recorded at six months after vaccination (r=-0.3593; p=0.0065) but not at three weeks after vaccine (r=-0.1541; p=0.2567). No differences were reported when gender, tumor stage (III vs IV) and type of tumor (lung vs other) were compared.

A full course of mRNA anti-SARS-CoV-2 vaccines is highly effective in preventing a symptomatic SARS-CoV-2 infection [9] , but the durability of this protection is not known yet. The first real-world data on the duration of the humoral response six months after the full course of BNT162b2 COVID-19 vaccine indicate a considerable decrease of both IgG concentration and NT Abs titer, especially among men, people 65 years of age or older and among immunocompromised subjects [10] .

Our current study is a longitudinal follow-up of patients with cancer who had been on immunotherapy at the time of vaccination and had remained on treatment throughout the six-month study period.

Our data confirm the reduction of both humoral and cell-mediated responses after six months of full course of COVID-19 vaccine. In particular, the median number of Spike-specific IFNγ-producing cells was significantly lower than that measured at three weeks after vaccination in SARS-CoV-2 naïve patients, while in SARS-CoV-2 experienced subjects the reduction of median number of Spikespecific IFNγ-producing cells reduction was less marked, thus suggesting that a triple exposure to SARS-CoV-2 antigens may be able to induce a higher and more persistent cell-mediated immune response. This difference was also confirmed by the analysis of humoral response kinetics since positive SARS-CoV-2 NT Abs titer and IgG concentration were stable maintained in SARS-CoV-2 experienced subjects, but not in SARS-CoV-2 naïve patients.

About the analyzed clinical variables (age, sex, PD-L1 status, TNM staging, type of treatment, with or without chemotherapy), the chemo-immunotherapy seems to determine a reduction in humoral and Spike-specific IFNγ-producing cells, even if the difference was not statistically significant. The lack of significance might be due to the small sample size; thus, larger prospective studies are mandatory.

In our cohort, age is not correlated with humoral response at six months but while a negative weak correlation was observed between age and Spike-specific IFNγ-producing cells/10 6 PBMC recorded at six months after vaccination.

In our cohort of cancer patients, the administration of a full course of the mRNA vaccine provides a durable immune response in SARS-CoV-2 experienced subjects, while in SARS-CoV-2 naïve patients the durability seems less marked. Prior natural COVID-19 history leads to the formation of a strong immune response and our data highlight that the vaccine could act as a booster for SARS-CoV-2 experienced subjects.

In this setting, a "real life" study reported a vaccine protection against acquisition of SARS-CoV-2 infection reported of 83% in the overall population and of 93% SARS-CoV-2-experienced subjects [11] . Importantly, in our cohort, including seronegative patients, no cases of COVID-19 were documented. Since no control group has been included in this study, real data on vaccine efficacy in our cohort are difficult to be extrapolated. So far, the absence of SARS-CoV-2 positive cases after vaccination might be related to vaccination itself but also to the strict adherence of the patients to social distancing, facial mask use and hand sanification.

To date, the third dose administration programs are ongoing worldwide, but the data about the need of its administration are scarce, above all in frail and immune-compromised patients. Preliminary data confirmed that a third BNT162b2 dose leads to an increase NT Abs titer in cancer patient [12] .

Our paper confirms the immunogenicity of the BNT162b2 anti-SARS-CoV-2 vaccine even at six months, but highlights a reduction in the immune response in cancer patients, particularly in the SARS-CoV-2 naïve subjects. These data strongly suggest the priority to administer the third dose of COVID-19 vaccine and add a significant contribution in the management of patients with cancer.

The strength of our paper is the simultaneous tracking of humoral and cellular immune response with the detection of both anti-Spike IgG concentration and NT Abs titers and ELISpot assay in a welldefined and homogeneous population over time. The availability of baseline data for each patient of both cellular and immunological status allows us to demonstrate that the response depends only on the vaccine eliminating other confounding factors. On the other hand, the use of ex-vivo ELISpot assay limited the analysis of T-cell response to only IFN-γ producing cells in response to Spike antigen. Thus, other cells producing different cytokines have not been studied. Furthermore, the absence of COVID-19 cases in our cohort, limits the speculations in terms of protective T-cell response. Of note, we carried out the assessment at the six-month follow-up before the patients received their third dose of vaccine; in this way, we were able to eliminate an important confounding factor. The limitations of our paper are the lack of a control group and the small sample size that enable us to extend our conclusions in a definitive way.

J o u r n a l P r e -p r o o f

Our data confirm the durable of the vaccine both in the humoral and in the cell-mediated immune response in patients with cancer treated with anti-PD-1/PD-L1, but demonstrate also a significant reduction, particularly in SARS-CoV-2 naïve patients. Our data support administering a third dose of COVID-19 vaccine to patients with cancer treated with PD1/PD-L1 inhibitors.

All authors contributed to the article and approved the submitted version. 

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

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