key: cord-0794072-tczy4k9n
authors: Lasagna, A.; Bergami, F.; Lilleri, D.; Percivalle, E.; Quaccini, M.; Alessio, N.; Comolli, G.; Sarasini, A.; Sammartino, J.C.; Ferrari, A.; Arena, F.; Secondino, S.; Cicognini, D.; Schiavo, R.; Lo Cascio, G.; Cavanna, L.; Baldanti, F.; Pedrazzoli, P.; Cassaniti, I.
title: Immunogenicity and safety after the third dose of BNT162b2 anti-SARS-CoV-2 vaccine in patients with solid tumors on active treatment: a prospective cohort study
date: 2022-03-11
journal: ESMO Open
DOI: 10.1016/j.esmoop.2022.100458
sha: 3d5dbb95b5eb83a2449882459eb5d615f49f46a3
doc_id: 794072
cord_uid: tczy4k9n

BACKGROUND: Although a full course of COVID-19 vaccine is effective in cancer patients, the duration of the protection and the efficacy of a booster dose against the new variants remain unknown. We prospectively evaluated the immunogenicity of the third dose of the SARS-CoV-2 BNT162b2 mRNA vaccine in cancer patients undergoing active treatment. PATIENTS AND METHODS: Patients with solid cancer, vaccinated with a booster dose during active treatment, were enrolled in this study. Patients were classified in SARS-CoV-2 naïve (without previous COVID-19 infection) and SARS-CoV-2 experienced (with previous COVID-19 infection). Neutralizing antibody (NT Abs) titer and total anti-Spike IgG concentration were quantified in serum. Heparinized whole blood samples were used for SARS-CoV-2 Interferon Gamma Release Assay (IGRA). The primary endpoint was to assess the increase of IgG antibody level between baseline and 3 weeks after the booster. RESULTS: 142 consecutive patients were recruited. In SARS-CoV-2 naïve subjects, median level of IgG was 157 BAU/mL (interquartile range (IQR) 62-423) at T0 and reached median of 2080 (IQR 2080-2080) at three weeks after booster administration (T1; p<0.0001). A median 16-fold increase of SARS-CoV-2 NT Abs titre (IQR 4-32) was observed in naïve subjects (from median 20 IQR 10-40 to median 640 IQR 160-640; p<0.0001). Median IFN-γ level at T1 was significantly higher than that measured at T0 in SARS-CoV-2 naïve subjects (p=0.0049) but not in SARS-CoV-2 experienced patients. The median level of SARS-CoV-2 NT Abs was 32-fold lower against Omicron compared to wild type strain (p=0.0004) and 12-fold lower compared to Delta strain (p=0.0110) CONCLUSIONS: The third dose is able to trigger both the humoral and the cell-mediated immune response in cancer patients on active treatment. Our preliminary data about the neutralization of the SARS-CoV-2 vaccine against variants of concern (VOCs) seem to confirm the vaccine lower activity.

The SARS-CoV-2 pandemic caused over 359 million cases and more than 5 million deaths worldwide, including 10,212,621 cases and 144,343 deaths in Italy on January 26th, 2022 [1] . Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, a strong effort has been made for the development of protective vaccines. The BNT162b2 vaccine was the first authorized for active immunization with a 95% protection against SARS-CoV-2 infection in a phase 2/3 trial J o u r n a l P r e -p r o o f [2] . The efficacy of this vaccine was demonstrated as early as 11 days after the first dose in 91.7% of cases and it remained 91.3% (95% confidence interval [CI] , 89.0 to 93.2) with a six-month follow-up [3] .

Patients with cancer have an increased risk of infection and COVID-19-related morbidity and mortality compared with the non-cancer population [4] . A recent meta-analysis has demonstrated that the patients with cancer had a suboptimal seroconversion rate after COVID-19 vaccination when compared with the non-cancer patients but a notable increase in humoural response in the group with complete COVID-19 immunization schedule was already observed [5] . These findings have been confirmed also by our previously published data. The rate of patients with cancer in immunotherapy with or without chemotherapy, developing positive antibody level measured by S1/S2 assay was high following two vaccine doses (95%), but only one third of our patients developed a positive antibody response after the first dose. Furthermore, over 90% of patients developed a sustained Spike-specific T-cell response after the full course of BNT162b2 anti-SARS-CoV-2 vaccine [6] .

The ongoing discovery of new variants of SARS-CoV-2 has brought major challenges to the effectiveness of vaccines. Studies have shown that the serum neutralizing antibody titers against the Delta variant after the inoculation with the BNT162b2 SARS-CoV-2 vaccine are 1.41-11.30 times lower than the original strain WT/D614G [7] . The SARS-CoV-2 Omicron variant (BA.1/B.1.1.529) has 34 mutations in the spike protein, which is the main target of neutralizing antibodies [8] with the consequent potential ability to escape vaccine-induced immunity.

Based on available data, the Centers for Disease Control (CDC) authorized a third dose of COVID-19 vaccine (''booster'' shots) for immunosuppressed patients [9] . On July 30th, 2021, the Israeli Ministry of Health approved the use of the booster with BNT162b2 anti-SARS-CoV-2 vaccine and Arbel and colleagues have demonstrated that the subjects who had received a booster had 90% lower mortality due to COVID-19 than those who had not received the third dose (95% confidence interval [CI], 0.07 to 0.14; P<0.001) [10] .

J o u r n a l P r e -p r o o f All the above data have many practical implications also in the patients with cancer on active treatments. While the full course of COVID-19 vaccine is proved to be effective in the patients with cancer, the question remains how long the protection lasts, whether it is necessary to give everyone a third dose, how to monitor this population over time and if a booster dose may enhance the breadth of protection against the new variants.

The aim of the present study is to evaluate the immunogenicity and safety of the third dose of the SARS-CoV-2 BNT162b2 mRNA vaccine in the patients with cancer undergoing active treatment.

We conducted a prospective, observational cohort study in order to assess the humoral and cell- According to our previous paper [11] , we have defined "SARS-CoV-2 experienced" those patients with a documented past positive SARS-CoV-2 RNA in nasopharyngeal swab and/or positive anti-Spike IgG before the first dose of vaccination, otherwise, they were classified as "SARS-CoV-2 naïve".

The primary endpoint was to assess the increase of IgG antibody level between the baseline and 3 weeks after the third dose of BNT162b2 SARS-CoV-2 vaccine.

The secondary end-points were: iii) the evaluation of the incidence of adverse reactions to the COVID-19 vaccine, local and systemic, solicited and unsolicited, within the period of three weeks after the vaccination.

This study was conducted according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement for reporting observational studies [12] . It was approved by the local Ethics Committee (Comitato Etico Area Pavia) and Institutional Review Board (P-0103665/21). All the subjects signed an informed written consent before the enrollment.

The subjects were monitored before BNT162b2 booster (T0) and after 21 days (T1). Blood samples for humoral and cell-mediated immune response evaluation were obtained at each time point.

Chemiluminescent assay (Liaison SARS-CoV-2 trimeric, Diasorin) was used for Spike IgG quantification. The results higher than 33.8 BAU/mL were given as positive. SARS-CoV-2

Neutralizing antibody serum (NT Abs) titre was determined as previously reported [6] . Briefly, 50 µl of serum in serial fourfold dilution were added in two wells of a flat bottom tissue culture microtiter plate (COSTAR, Corning Incorporated, NY 14831, USA). The same volume of 100 TCID50 of SARS-CoV-2 strain was added and plates were incubated at 33°C in 5% CO2, according to our local protocol [11] . After 1 hour incubation at 33°C 5% CO2, VERO E6 cells were added to each well. After 72 h of incubation at 33°C 5% CO2 the same conditions, plates were antibodies. Neutralizing titer was the maximum dilution with the reduction of 90% of CPE. All the experiments were performed in BSL3 facility. The results higher or equal to 1:10 serum titer were considered positive, according to our protocol [13] .

Heparinized whole blood samples were used for SARS-CoV-2 IGRA, according to the manufacturer's instructions (Euroimmun, Lübeck, Germany). Briefly, 500 µl of sample was added to stimulator tube coated with Spike antigen and to negative and positive control tubes. All the tubes were incubated overnight at 37°C, 5% CO2 and, then, the samples were centrifuged and the plasma supernatant was then collected and stored at -80°C until testing. Interferon gamma (IFNγ) was detected automatically in the supernatants by an enzyme-linked immuno-sorbent assay (ELISA, Euroimmun, Lübeck, Germany) using the Euroimmun Analyzer I according to the manufacturer's instructions. IFN-γ response was defined as Spike-stimulated minus unstimulated. Results higher than 200 IU/mL were given as positive, while results ranging from 100 to 200 IU/mL were defined as borderline. In case of inadequate response to the positive control, the result was given as "indeterminate".

Adverse events (AEs) were obtained with a questionnaire with specific yes/no questions regarding local reactions (i.e. pain at the injection site) and systemic reactions (i.e. fever >38 °C).

Data were described with the median and interquartile range (IQR) 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). When the comparison between three or more groups was performed, Kruskav-Wallis test with post-hoc Dunn's comparisons was used.

Spearman's test was used for the correlation analysis. Fisher exact test was used for comparison of categorical variables. 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.

From September 17th to November 17th, 2021, 142 consecutive patients with solid cancer, vaccinated with a booster dose during active treatment, were recruited in this study (56 females and 86 males; median age 66 years; range 26-88); among them, 11 (7.7%) reported a SARS-CoV-2 infection before the first dose of vaccination (SARS-CoV-2 experienced).

The detailed demographic and clinical characteristics are reported in Table 1 . Figure 1B) . Age inversely correlated with total IgG level at baseline (T0) but not at the time of follow-up (T1) (Figure 2) . No difference was observed in terms of median response between patients treated with immunotherapy and chemotherapy (p>0.05). A stronger correlation between SARS-CoV-2 NT Abs and total IgG level was observed at T0 (r= 0.76; p<0.0001) compared to T1 (r=0.27, p=0.0081). No correlation as regards the number of days was observed from the first to the third vaccination and SARS-CoV-2 NT Abs/total IgG. 

In a small cohort of patients (N = 10), SARS-CoV-2 NT Abs responses against the circulating Delta Omicron compared to the wild type strain (p=0.0004) and 12-fold lower compared to Delta strain (p=0.0110) (Figure 4 ).

The most common side effects observed after the vaccine were pain at the injection site (64.8%, 92/142) and fever (24.6%, 35/142). No hypersensitivity adverse events, thrombosis, or vaccinerelated anaphylaxis were reported. Side-effects were typically resolved within the first 24 h after vaccination ( Table 2 ).

In this paper we have presented the results of a prospective observational study investigating the immunogenicity of the booster with BNT162b2 anti-SARS-CoV-2 vaccine in the patients with cancer on active treatment.

Two doses of mRNA anti-SARS-CoV-2 vaccines are effective in preventing a symptomatic SARS-CoV-2 infection [2] , but a decrease of both IgG concentration and NT Abs titer has been demonstrated six months after the vaccination, especially among the immunocompromised subjects [13] . Moreover, the emergence of the Delta variant in June 2021 has highlighted the immune evasion with the waning of vaccine-elicited immunity [14] . Caniels et al have observed a substantial reduction in binding and neutralization potency of antibodies against three SARS-CoV-2 variants of concern (VOCs) (B.1.1.7, B.1.351 and P.1) in convalescent or immunized sera after a full course of BNT162b2 SARS-CoV-2 vaccine [15] . The emergence of SARS-CoV-2 variants and the reduction of NT Abs titer in vaccinated or previously infected subjects has justified the approval by CDC of the third dose of COVID-19 vaccine [9] .

To date, few studies have reported the immunogenicity of the third dose in the patients with cancer.

Rottenberg and colleagues have demonstrated higher antibody levels after the booster in a cohort of J o u r n a l P r e -p r o o f reinforcement of anti-COVID-19 immunity among the total 88 patients with cancer enrolled, with a statistically significant seroconverting anti-S IgG levels after booster vaccination (p = 0.000062) [18] . Debie and colleagues have reported that the booster induced significantly higher anti-receptorbinding domain (RBD) IgG levels 28 days post-third dose than 28 days post-second dose in 141 cancer patients [19] . Finally, the data from a large cohort of patients with hemato-oncological tumors have confirmed the increased humoral response after the third dose of BNT162b2 SARS-CoV-2 vaccine [20] .

According to these studies, our data have demonstrated the seroconversion in all patients except one. The patient who failed to successfully seroconvert at T1 was under daily corticosteroid treatment during the vaccination schedule for concomitant rheumatoid arthritis. This negative association between steroid treatment and IgG levels has been described in many papers [21] [22] [23] .

We have also evaluated the NT Abs titer. In our cohort, the neutralizing activity was significantly higher in seroconverted patients, with a positive correlation between IgG levels and NT Abs titre. This correlation has also been reported in other papers: Di Noia and colleagues have found a strong correlation between the levels of anti-S IgG and the titre of NT Abs [23] . Also Levin et at have reported a strong correlation between IgG and NT Abs for the whole six months after the full course of vaccine (Spearman's rank correlation between 0.68 and 0.75), with a time-dependent regression relationship [14] .

No differences were reported when the type of anticancer treatment (chemotherapy or immunotherapy) was correlated with the SARS-CoV-2 humoral response, while the age inversely correlated with total IgG level and NT Abs titre at baseline (T0) but not after the booster (T1). This finding confirms that the age is an unfavourable prognostic factor for long lasting humoral response after the two-doses of SARS-CoV-2 vaccine, but the booster is immunogenic also in older subjects, as highlighted by Mwimanzi and colleagues too [24] .

Furthermore, we have also investigated the development of Spike-specific cell-mediated immune J o u r n a l P r e -p r o o f response using the SARS-CoV-2 Interferon Gamma Release Assay (IGRA) in a sample of subjects.

In SARS-CoV-2 naïve subjects after the booster, the median IFN-γ level three weeks after the booster was significantly higher than that measured before, while this difference was not reported in SARS-CoV-2 experienced subjects. This evidence confirms the hypothesis that the triple exposure to SARS-CoV-2 antigens is able to induce a higher cell-mediated immune response.

Moreover the third SARS-CoV-2 vaccine dose seems to increase the activity of neutralization againts VOCs also in the immunocompromised patients [25] [26] We have analyzed a small sample (N = 10) with the evidence of the median level of SARS-CoV-2 NT Abs 32-fold lower against Omicron compared to the wild type strain (p=0.0004) and 12-fold lower compared to Delta strain (p=0.0110).

No severe adverse events were reported.

The strength of our data consists in the simultaneous detection of both humoral and cellular immune response. One limit is the low numerosity (N = 142), even though this cohort of the patients with cancer is one of the largest available at the moment [17, 18, 19, 20, 27] . Another limit is the lack of a control group that enables us to extend our conclusions in a definitive way. Finally, at the time of writing, the post-third dose data about the activity of neutralization againts VOCs are available for only a small subset of participants.

Our data support the immunogenicity of the booster dose in the patients with cancer. The third dose J o u r n a l P r e -p r o o f is able to trigger both the humoral and the cell-mediated immune response in the patients with cancer on active treatment, independently from the type of therapy. Our preliminary data about the neutralization of the SARS-CoV-2 vaccine against VOCs seem to confirm the vaccine lower activity, but further studies would be designed to address this issue.

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. Correlation between IgG level measured at T0 or T1 and age was measured. P value and r were calculated using Spearman's' test and given in the graph. 

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Immunogenicity and risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection after Coronavirus Disease 2019 (COVID-19) vaccination in patients with cancer: a systematic review and meta-analysis

A snapshot of the immunogenity, efficacy and safety of a full course of BNT162b2 anti-SARS-CoV-2 vaccine in J o u r n a l P r e -p r o o f cancer patients treated with PD-1/PD-L1 inhibitors: a longitudinal cohort study

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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

The STROBE guidelines

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Assessment of response to a third dose of the SARS-CoV-2 BNT162b2 mRNA vaccine in patients with solid tumors undergoing active treatment

Antibody titres before and after a third dose of the SARS-CoV-2 BNT162b2 vaccine in patients with cancer

Third dose of SARS-CoV-2 vaccination in hemato-oncological patients and health care workers: immune response and adverse events _ a retrospective cohort study

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Table 2: Side effects after the third dose of BNT162b2 anti-SARS-CoV-2 vaccine. Side effects Num patients / percentage

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