key: cord-0321104-j8ojkbhb authors: Azzolini, E.; Pozzi, C.; Germagnoli, L.; Oresta, B.; Carriglio, N.; Calleri, M.; Selmi, C.; De Santis, M.; Finazzi, S.; Carlo-Stella, C.; Bertuzzi, A.; Motta, F.; Ceribelli, A.; Mantovani, A.; Bonelli, F.; Rescigno, M. title: A booster dose of mRNA-based COVID-19 vaccines fosters the development of an immune response in immunosuppressed fragile patients. date: 2022-01-21 journal: nan DOI: 10.1101/2022.01.21.22269633 sha: 8e23327232f27aa9be04ee905075fee480943651 doc_id: 321104 cord_uid: j8ojkbhb SARS-CoV-2 vaccination has proven effective in inducing an immune response towards the Spike protein in healthy individuals and is progressively allowing to overcome the pandemic. Recent evidence has shown that response to vaccination in some vulnerable patients may be diminished, and it has been proposed a booster dose. We tested the kinetic of development of serum antibodies to the trimeric form of the SARS-CoV-2 Spike protein, their neutralizing capacity, the CD4 and CD8 IFN-{gamma} T cell response in 328 subjects, including 131 fragile individuals (cancer, rheumatologic, and hemodialysis patients), 160 healthcare workers (HCW) and 37 subjects older than 75 yo, after vaccination with two or three doses of mRNA vaccines. We stratified the patients according to the type of treatment. We found that fragile patients, depending on the type of treatment, poorly respond to SARS-CoV-2 mRNA vaccines. However, an additional booster dose of vaccine induced a good immune response in almost all of the patients except those receiving anti-CD20 antibody. Similarly to HCW, previously infected and vaccinated fragile individuals demonstrate a stronger SARS-CoV-2 specific immune response than those who are vaccinated without prior infection. From December 2020 several anti-SARS-CoV-2 vaccines have been approved by the drug authority agencies for emergency use for the prevention and management of COVID-19. SARS-CoV-2 vaccination has proven to be effective in protecting against hospitalization and death in Israel (Haas et al., 2021) , and, as shown by the COVID-19 vaccine breakthrough infection surveillance, also in the US even towards the Delta variant (Scobie et al., 2021) . This indicates that vaccines can help control COVID-19 severity and the pandemic itself. Indeed, all of the vaccines approved so far have proven great efficacy in activating an immune response in healthy individuals (Abu Jabal et al., 2021; Arunachalam et al., 2021a; Arunachalam et al., 2021b; Dagan et al., 2021; Haas et al., 2021; Polack et al., 2020; Voysey et al., 2021; Walsh et al., 2020 ) and we and others have shown that one dose is sufficient in boosting the immune response in SARS-CoV-2 previously exposed subjects (Krammer et al., 2021a; Krammer et al., 2021b; Levi et al., 2021; Saadat et al., 2021; Sadoff et al., 2021; Samanovic et al., 2021) . However, the ability of mRNAbased SARS-CoV-2 vaccines to immunize primary or treatment-induced immune compromised individuals has recently been questioned (Collier et al., 2021) . In particular, patients with inflammatory bowel disease under infliximab treatment (Kennedy et al., 2021) , patients who have received an allogeneic stem cell transplantation (Lafarge et al., 2021) , cancer patients (Zeng et al., 2021) , methotrexate treatment (Mahil et al., 2021) , kidney transplant or hemodialysis (Danthu et al., 2021) (Bachelet et al., 2021) or multiple sclerosis (Apostolidis et al., 2021) have all demonstrated a reduced ability to mount an immune response, potentially adversely affecting protection offered by vaccines. However, studies in which a comprehensive comparative analysis of both humoral and cellular immune responses after a third dose of vaccine is lacking. Indeed, the type of immunomodulatory treatment may have a differential effect according to the immune cell which is targeted. For instance, B-cell-directed therapies for hematological malignancies have been shown to affect the production of antibodies in response to SARS-CoV-2 vaccination due to B-cell depletion and/or disruption of the B-cell receptor signaling pathway while leaving unaltered the T cell response (Apostolidis et al., 2021) . This T cell response may compensate for the B cell response and may explain why anti-CD20 treated patients are still protected from COVID-19 (Huang et al., 2021) . By contrast, a general immune suppression due to drug treatments or the disease itself may affect both All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January https://doi.org/10.1101 https://doi.org/10. /2022 4 humoral and cellular responses. Hence, it is very important to evaluate the immunization status and the duration of response in fragile patients undergoing SARS-CoV-2 vaccination and relate it to the type of treatment. Here, we compared the antibody production, CD4 and CD8 T cell response to the vaccine spike protein, as well as the neutralization potential of the antibody response in response to 2 or 3 doses of SARS-CoV-2 vaccine in 328 subjects including healthcare workers (HCW), elderly subjects (>75 yo) and immune compromised patients with different pathologies either in hemodialysis, with cancer or rheumatological diseases in relation to their treatments. We show that one of the major determinants of a successful immune response was the immune status, exposure to SARS-CoV-2 infection and type of treatment at the time of vaccination and that three doses of vaccine allowed achieve immunization even in immunocompromised individuals. However, as expected, anti-CD20 treatment impaired the development of an antibody response even after the third dose, suggesting that patients under this treatment should wait to receive the shots after interrupting the therapy. Patients under mycophenolate also respond poorly to vaccination, but interruption of therapy for just one week allows activation of the immune response. We also show that SARS-CoV-2 recovered frail individuals, similarly to healthy subjects (Krammer et al., 2021a; Krammer et al., 2021b; Levi et al., 2021; Saadat et al., 2021; Sadoff et al., 2021; Samanovic et al., 2021) , achieved a strong immune response, quicker than naïve subjects. Overall, this study highlights a need in a booster dose of vaccine in immunosuppressed fragile individuals, which should however consider their immune status and treatment. SARS-CoV-2 recovered patients instead, should be considered for the booster dose on an individual basis. In this observational study we analyzed the antibody production, the CD4 and CD8 T cell and the neutralizing antibody response to SARS-CoV-2 Spike protein in 328 subjects (Table 1) Comirnaty). For fragile patients we investigated the humoral and cellular immune response also at 2 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January https://doi.org/10.1101 https://doi.org/10. /2022 5 weeks after the third (booster) dose (T4). In particular, 13 (44%) cancer patients, 31 (65%) patients with rheumatic disease and 44 (83%) patients in hemodialysis received the third dose. Moreover, for HCW and cancer patients we tested the kinetics of B and T cell development before vaccination (T0) at 21-28 days after the first dose (T1), 10-26 days after dose 2 (T2) and 2-4 months (T3) after the second dose (Fig. 1) . For cancer patients we analyzed also the immune response 2 weeks after the booster dose (T4). 62 individuals had been previously exposed to SARS-CoV-2 (Table 1) and among these, only 6 out of 18 (33%) cancer patients, 1 out of 5 (20%) hemodialysis patients and 1 (100%) rheumatic disease patient received the third dose. The immune response was correlated with the type of pathology, the immune status and the treatment ( Table 2) . SARS-CoV-2 particle internalization is mediated by the binding of the trimeric form of the Spike protein with the ACE-2 receptor on host cells (Hoffmann et al., 2020) . We chose to test the level of IgG antibodies directed to the trimeric form of Spike protein (LIAISON ® SARS-CoV-2 TrimericS IgG, DiaSorin) to have a better correlation with neutralizing antibodies. Nevertheless, we also tested the neutralization ability of the ensued antibodies via a surrogate test of Spike neutralization (cPass™, GenScript). As shown in Suppl. figure 1A, while the antibody response was induced in healthcare workers already after the first vaccine dose (T1) and reached a climax 10 days after the second dose (T2), it was either undetectable in cancer patients receiving anti-CD20 treatment (blue triangles, category 2) or reduced in patients receiving other drugs with low/medium impact to the immune system (orange and green/yellow triangles, categories 0 or 1, respectively) at any time point between T0 and T3 ( Fig. S1 A) . In those patients that experienced an antibody response, the titers were much lower than those of the HCW suggesting that the amplitude of the antibody response was compromised. However, a booster dose of vaccine increased the antibody titers at levels similar to those of HCW, except for anti-CD20 treated cancer patients which remained undetectable ( Fig. S1 A, T4). As the latter patients were discouraged to take a booster dose, we could test only three out of eight patients who insisted to receive it. Wherever detectable, the antibodies were neutralizing and were preserved at least four months after vaccination (T3), but only in those patients that were not in active treatment at the time of vaccination (orange All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint 6 triangles, Fig. S1 A and Table 2 ). By contrast, the antibodies raised in HCW were all neutralizing (Fig. S1 A). Regarding SARS-CoV-2 previously exposed individuals, while nearly all HCW required one single dose to reach a very strong neutralizing antibody response, as we and other previously described (Krammer et al., 2021a; Krammer et al., 2021b; Levi et al., 2021; Saadat et al., 2021; Sadoff et al., 2021; Samanovic et al., 2021) , SARS-CoV-2 naturally infected cancer patients required two doses to reach comparable neutralizing antibodies (Fig. S1 B) , but almost all of them (16 out of 18) developed IgG antibody response, even if cancer patients were under active treatment at the time of vaccination (12 out of 18, Fig. S1 B and Table 2 ). In particular, ten were treated with drugs belonging to category 0, one with drug of category 1 (green triangles, Doxorubicin+Cisplatin) and one with anti-CD20 (blue triangles, category 2) (Fig. S1 B and Table 2 ). A booster dose increased the amount of serum antibodies, particularly the neutralizing antibodies ( Fig. S1 B, T4). The only naturally infected cancer patient under active anti-CD20 treatment did not increase antibodies even after the second dose (T2) (blue triangles, Fig. S1 B) , and was advised to take a third dose after stopping the anti-CD20 treatment. A higher number of patients under this treatment is required to reach any conclusions. The induction of a CD4 or CD8 T cell response is an additional arm of an effective vaccination. We thus evaluated the kinetic of anti-Spike T cell response activation in the two groups, by using specific CD4 (Ag1) and CD4 plus CD8 (Ag2) T cell epitopes of the Spike protein. As shown in Suppl. figures 2A, we found that the T cell response (both to Ag1 and Ag2) was low in general in cancer patients and was observed only in three out of seven patients under anti-CD20 treatment at T2. Interestingly, the peripheral blood T cell response dropped three months after vaccination in a good proportion of subjects, including HCW, and in 9 out of 23 (Ag1) and in 5 out of 23 (Ag2) was below the threshold of positivity selected for this study. The booster dose to cancer patients re-elevated the T cell response to levels similar to those after the second dose but we did not observe further enhancement like that of the antibody response. Anti-CD20 treated patients that did not show a T cell response after the second dose, did not benefit from the booster dose (Fig. S2 A) . As shown in Fig. S2 B, the T cell response was boosted in all of naturally All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint infected subjects at T2, regardless of being HCW or cancer patients with or without treatment (even anti-CD20) and it was high at 3-4 months after vaccination (T3) or at two weeks after the booster dose (T4). The immune response is compromised in a substantial proportion of patients in hemodialysis and in some rheumatologic patients but can be boosted by a third vaccine dose. Prompted by the intriguing results on cancer patients and the dependence of the immune response on the pharmacologic treatment, we evaluated whether other categories of fragile patients displayed a compromised immune response to the vaccine and the outcome after a booster dose. Thus, we tested the trimeric antibody levels, their neutralization ability and T cell responses at 2-3 months from the second dose and at two weeks after the booster dose in patients with rheumatic diseases or in patients in hemodialysis. As patients in hemodialysis were older, we also included a group of elderly people (≥75 yo) receiving the vaccine. As shown in Fig. 2 A, patients in hemodialysis had a significant reduction in trimeric antibody response at 3 months after the second dose of vaccine (T3) compared to health care workers (P<0.0001) and a drastic but not significative reduction versus older subjects. This response reflected also a significant reduction (P<0.0001) in the neutralizing ability of the antibodies (Fig. 2 B ) also in older subjects (P=0.0026). Rheumatic disease patients instead, as a group, had a reduction in IgG trimeric antibody response, which was not statistically significant, however, the neutralization potential was significantly reduced (P=0.0499) as compared to that of HCW individuals (Fig. 2, A and B) . Notably, 4 patients had no neutralizing antibodies, although 2 of them had a positive antibody test. As shown in figure 2C and 2D, the T cell response (both Ag1 and Ag2) was significantly lower as compared to HCW in hemodialysis patients, but not in the other patients. When we analyzed the response at two weeks after the third dose (T4) we observed that all rheumatic patients and dialysis patients (except for one patient of each class) had increased the serum levels of antibodies which were also neutralizing except for one patient having detectable trimeric antibodies (134 BAU/ml) which were not neutralizing (Fig. 2 However, although the T cell response was boosted, with a statistically significant increase only in dialysis patients, it remained below the limit of positivity set in this study for many patients (Fig. 2 , C and D). As observed also for cancer patients, previously exposed to SARS-CoV-2 patients displayed the highest levels of neutralizing antibodies which remained high also after the booster dose (Fig. S3 , A and B). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10. 1101 Moreover, the T cell responses remained higher in SARS-CoV-2 experienced patients than naïve HCW (Fig. S3, C and D) . The immune response depends on the type of treatment or immune status of the patients. Having observed a clear reduction in antibody levels in cancer or hemodialysis patients and in some rheumatologic disease patients, we analyzed whether the observed differences were linked to an immune depressed state induced by the treatment or by their disease. As described in the methods section, we classified the patients according to the type of treatment (cancer and rheumatic disease patients) or an immunoscore related to the disease for which the patients are in dialysis and their comorbidities. As shown in Fig. 3 A, the type of treatment (no treatment or low (0), medium (1) or high (2) interference with the immune system) or the worsening of the immunoscore in hemodialysis patients (low (0), medium (1) or high (2) immune compromised) impacted on the profile of the immune response with a progressive reduction of both antibody levels and neutralization potential. Interestingly, patients distributed quite homogenously in the three categories suggesting that their immune status, rather than the disease itself, was responsible for the impaired immune response. Particularly affected were patients belonging to category 2: patients in hemodialysis with an high immune compromised immunescore, rheumatic disease patients treated with mycophenolate or methotrexate and cancer patients treated with anti-CD20 ( Fig. 3 A, blue crosses, yellow or pink circles and blue triangles, respectively). It should be noted that patients under methotrexate stopped treatment one week after getting vaccinated and indeed they all developed neutralizing antibodies (Fig. 3, A and B , pink circles). Interestingly, the third dose (T4) allowed patients in category 2 to achieve levels of antibodies similar to those in category 1 at 2 / 4 months after the second dose (T3) except for patients treated with anti-CD20 antibody (Fig. 3 A, blue triangles) and one patient with mycophenolate ( Fig. 3 A, yellow circle) . Interestingly, the latter patient was advised to stop treatment for one week after vaccination, but did not follow the advice. The booster dose increased significantly the neutralization ability of IgG in all the categories (Fig. 3 B) . The T cell response also was affected particularly by the category of drugs with high interference with the immune system or by an immune compromised status (category 2), but differences with patients belonging to category 0 or 1 were not All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint striking at T3. Interestingly, T cell response (both to Ag1 and Ag2) was statistically significantly boosted with a third dose only in category 2 patients (Fig. 3, C and D) . Interestingly, when analyzing the correlation between antibody levels and neutralization potential, we found that in the group of patients in the category 2 (treated with drugs with high interference with the immune system or immune compromised patients) levels of trimeric antibody above 100 BAU/ml after the second dose are most likely to correspond to a positive neutralization test (>30%) (Fig. 4 A) . The booster dose allowed most of the patients achieve a neutralizing antibody response, and it was confirmed that a level of antibodies above 100 BAU/ml correlated with a positive neutralization test (Fig. 4 B) . Here we show that, upon vaccination, elderly subjects and patients under treatments that have little or no interference with the immune system develop an immune response which is slightly reduced but comparable to that of healthy individuals, while those immunosuppressed (with an immunoscore equal to 2) or under immunosuppressive treatments are strongly impaired in the ability to activate an antibody response (i.e., cancer patients treated with anti-CD20 therapy or rheumatic disease patients under active treatment of mycophenolate). In some cases, the immune response is not initiated at all. However, a third booster dose allows to achieve levels of neutralizing antibodies similar to those of HCW after the second vaccine dose (T2) except for anti-CD20 treated cancer patients. By stratifying patients according to treatment, we show that anti-CD20 and mycophenolate are the drugs with the highest impact on the development of a correct immune response. By contrast, methotrexate which is associated with specific immune inhibitory drugs did not have a major impact on the immune response, but it has to be considered that methotrexate therapy was stopped for one week after every dose of vaccine, whereas mycophenolate was not interrupted at the time of the first and second dose vaccination. This suggests that, wherever possible, treatment having an impact on the immune system should be interrupted or delayed in order to favor the development of an immune response. Indeed, at the administration of the third dose, mycophenolate was interrupted and this resulted in a proficient activation of the immune response. The patient who did not follow the advise of interrupting mycophenolate resulted in an undetectable antibody and T cell response even after the third booster dose, confirming that treatment should be stopped to favor All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint the development of an immune response. Interestingly, as expected the antibody response to the trimeric form of Spike was undetectable in individuals under anti-CD20 treatment, and the situation did not change after the third dose. Interestingly also a patient that had interrupted anti-CD20 five months earlier still did not display antibodies to the Spike trimeric protein. This is in line with a recent report showing that patients with B-cell lymphoma (BCL) receiving B-cell-directed therapies should be vaccinated at least nine months from the last treatment to improve antibody titers (Ghione et al., 2021) . By contrast the T cell response to AG1 and AG2 spike peptides was observed in three out of seven patients under anti-CD20 treatment at T2. This to us was unexpected as it has been shown that anti-CD20 treated multiple sclerosis patients had a similar ability to induce T cells to the spike protein as healthy subjects (Apostolidis et al., 2021) . This suggests that cancer patients may have an additional impairment in inducing the T cell response which is probably unrelated to the active treatment. This makes cancer patients a very vulnerable category that needs further attention. It would be important to correlate the vaccine immune response to the stage of disease as the immune system may be depressed as a consequence of the immunosuppressive status generated by the cancer itself. Indeed, it has been shown that COVID-19 mortality was statistically significantly higher in cancer patients with an active disease (Pinato et al., 2020) . Also, the immune status of the patients is strongly correlated with the ensued immune response as indicated by the impact of disease and immunoscore of patients in hemodialysis. In conclusion, fragile patients should be tested periodically to assess the development and status of an immune response and should be considered individually and on the basis of their active treatments with regards to a potential booster dose. Those that are not immunized should be prioritized to receive a booster dose of vaccine and be re-evaluated afterwards for effective immunization. However, the therapeutic schedule should be modulated (interrupted or delayed) to favor an immune response to the vaccine. Particular attention should be given to patients with antibody levels below 100 BAU/ml because these antibodies are unlikely to exert a neutralizing activity. A different scenario is observed in patients previously exposed to SARS-CoV-2. These patients reach maximal response after two doses of vaccine, still one subject under anti-CD20 treatment failed to activate an antibody response but developed a T cell response. More SARS-CoV-2 exposed patients should be tested with immunosuppressive treatments to draw conclusions. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint We tested the IgG antibody response, the CD4 and CD8 T cell activation and the neutralizing antibody response to SARS-CoV-2 spike protein developed after mRNA SARS-CoV-2 vaccination (Spikevax or Moderna mRNA-1273 -Comirnaty or BNT162b2 Pfizer-BioNTech) as a part of two observational studies approved by the Ethical Committee of Istituto Clinico Humanitas, in compliance with the Declaration of Helsinki principles. The studies were conducted at Istituto Clinico Humanitas and comprised a longitudinal sample collection, including healthcare workers (n=160) and cancer patients (n=30) and a cross-sectional sample collection, including elderly subjects (n=37), patients with rheumatic diseases (n=48) and patients in hemodialysis (n=53). Fragile patients received also a third dose (booster) ± 5 months after the second dose. Analyzed time points were: the day of the first dose (T0), 21-28 days after the first dose (T1), 10-26 days after the second dose (T2), 2-4 months after the second dose (T3) and 2 weeks after the third dose (T4). At each scheduled time point, as shown in Figure 1 , serum and lithium-heparin whole blood samples were collected from enrolled individuals. Study inclusion criteria included a vaccination with an authorized COVID-19 vaccine (according to Italian regulation and guidelines), age of 18 years or greater, and willingness and ability to provide informed consent. Study exclusion criteria included lack of willingness and ability to provide informed consent, or a lack of properly collected and stored samples. Demographic and clinical information for healthy subjects (healthcare workers and elderly) and patients can be found in Tables 1 and 2 . Experiments were conducted in a blinded fashion with designated members of the clinical team, who did not run the assays, having access to the sample key until data were collected, at which point researchers of the team were unblinded. All individuals enrolled in the studies provided an informed consent as part of the protocols (CLI-PR-2102 and CLI-PR-2108;). These studies began in February 2021 (CLI-PR-2102) and June 2021 (CLI-PR-2108) and are continuing with participant's follow-up. Enrolled individuals did not receive compensation for their participation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint 12 Cancer and rheumatic disease patients were classified according to the type of treatment: no active treatment or low (category 0), medium (category 1) or high (category 2) interference with the immune system ( Table 2 ). In particular, drugs with low interference with the immune system (category 0) included: Tyrosine Kinase Inhibitor, TKI (Imatinib), EGFR TKI (Osimertinib), chemotherapy (Lenalidomide, Docetaxel, Gemcitabine, Nab-paclitaxel), hormone therapy, anti-HER2 agents (Pertuzumab, Trastuzumab), chemotherapy + anti-PDL1 (Carboplatin+etoposide+Atezolizumab) (for cancer patients) and ursodeoxycholic acid for rheumatic diseases patients; drugs with medium interference with the immune system (category 1) were: Doxorubicin (with Cisplatin or with Ifosfamide or present in ABVD) (for cancer patients) and anti-TNF Ab -Infliximab, Certolizumab, Adalimumab, Golimumab; TNF blocker -Etanercept; JAK1/2 inhibitor -Baricitinib; CD80/CD86 blocker -Abatacept (for rheumatic diseases patients); immunosuppressive drugs (category 2) were rituximab, Obinutuzumab (for cancer patients), mycophenolate and methotrexate in combination with immune inhibitory drugs (for rheumatic disease patients). Treatment with methotrexate or Baricitinib (JAK1/2 inhibitor) was stopped one week after every dose of vaccine, whereas treatment with mycophenolate was stopped one week only after the third dose of vaccine. Patients in hemodialysis were classified with an immunoscore related to the disease for which the patients are in dialysis and their comorbidities: low (category 0), medium (category 1) or high (category 2) immune compromised ( Table 2) . Serum samples were tested using LIAISON ® SARS-CoV-2 TrimericS IgG (DiaSorin, Italy), a quantitative CE-marked assay for the detection of IgG antibodies recognizing the native trimeric Spike glycoprotein of SARS-CoV-2 (Bonelli et al., 2021) . According to manufacturer's instruction for use, the presence of an immune response in vaccine recipients was 100.0% (95% CI 96.3-100.0%) in 102 samples collected after Immunoglobulin (NIBSC 20:136), the readout was updated and the assay currently calculates the levels of SARS-CoV-2 IgG antibodies in binding antibody units (BAU/mL) (Perkmann et al., 2021) . Samples ≥ 33.8 BAU/mL were considered positive. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January https://doi.org/10.1101 https://doi.org/10. /2022 Neutralization was assessed by ELISA with cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript, China), a qualitative CE-marked assay for the detection of circulating neutralizing antibodies that block the interaction between the receptor binding domain (RBD) of the viral spike glycoprotein with the ACE2 cell surface receptor (Tan et al., 2020) . Samples were analyzed following manufacturer's instruction for use. Samples ≥ 30% signal inhibition were considered positive. T cell-mediated responses were analyzed using QuantiFERON SARS-CoV-2 Research Use Only assay (QIAGEN, Germany), following manufacturer's instruction for use. Briefly, fresh lithium-heparin whole blood samples were incubated in tubes containing different cocktails of SARS-CoV-2-specific antigens (Ag1 and Ag2) at 37°C for 16 to 24 h within 16 h of sampling, followed by centrifugation at 2,700 × g at room temperature for 15 min for plasma retrieval. Plasma from stimulated samples was analyzed using LIAISON XL instrument (DiaSorin, Italy) for detection of IFN-γ, according to the standard procedures recommended by the manufacturer. For this study, positive results were defined as ≥ 0.25 IU/mL, after background tube (nil) IU/mL was subtracted from Ag1 and Ag2 values. We defined this tentative cut-off threshold based on previous experience with the QuantiFERON test but this is arbitrary as other studies have defined a lower cut off between 0.15 and 0. 2(Van Praet et al., 2021) . Data were analyzed for normal distribution (Shapiro-Wilk test) before any statistical analyses. Individual values are presented as spaghetti plots or as box plots showing the interquartile range, median, and minimum-to-maximum whiskers. The differences between matched time points were analyzed using the non-parametric Friedman test with Dunn's multiple comparisons test. The comparison of multiple groups was carried out using the non-parametric Kruskal-Wallis test followed by Dunn's multiple comparisons test. In order to gauge the correlation between IgG values in plasma (x variable) and the % of neutralization (y variable), a non-parametric Spearman's rank correlation test was performed. A probability value of P < 0.05 was considered significant. All statistics and reproducibility information are reported in the figure legends. Data analyses were carried out using GraphPad Prism version 8. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10. 1101 Online supplemental material Fig. S1 shows the IgG antibody response and its neutralizing activity in vaccinated naïve and SARS-CoV-2 naturally infected health care workers (HCW) and cancer patients at different time points. All authors contributed to revise the manuscript for important intellectual content, were responsible for the decision to submit for publication, and approved the final submitted version of the manuscript. E.A and C.P conceived the study, analyzed the data and wrote the manuscript. Individual participant data that underlie the results reported in this article (tables, figures), after coding, will be available on the data repository Zenodo at the link (ongoing) with restricted license however available upon request. Patient informed consent does not allow for deposition of clinical data in public access repositories. Interested researchers should contact biblioteca@humanitas.it to inquire about access; requests for noncommercial academic use will be considered and require ethics review. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January https://doi.org/10.1101 https://doi.org/10. /2022 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. Glomerulopathy after liver transplantation 1 0 0 0 1 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (0), medium (1) or high (2) interference with the immune system. Patients in hemodialysis were classified with an immunoscore related to the disease for which the patients are in dialysis and their comorbidities: low (0), medium (1) or high (2) immune compromised. IgG antibody response (A), its neutralizing activity (B) and anti-spike T cell response activation, by using specific CD4 (Ag1, C) and CD4 plus CD8 (Ag2, D) T cell epitopes of the spike protein were measured in All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint 24 serum and plasma of vaccinated naïve health care workers (HCW, n=104), elderly people ≥ 75 yo (n=35), cancer patients (n=9), patients with rheumatic diseases (n=47) or patients in hemodialysis (n=48) at 2-4 months after second dose (black, T3) and in serum and plasma of cancer patients (n=7), patients with rheumatic diseases (n=30) or patients in hemodialysis (n=43) 2 weeks after the booster dose (red, T4). As a control, we indicated values of IgGs, their neutralizing activity and anti-spike T cell response activation of vaccinated naïve health care workers (HCW, n=119) at 10 days after the second dose (T2). The box plots show the interquartile range, the horizontal lines show the median values, and the whiskers indicate the minimum-to-maximum range. Each dot corresponds to an individual subject. P values were determined IgG antibody response (A), its neutralizing activity (B) and anti-spike T cell response activation, by using specific CD4 (Ag1, C) and CD4 plus CD8 (Ag2, D) T cell epitopes of the spike protein were measured in serum and plasma of vaccinated naïve patients with cancer (n=9), rheumatic diseases (n=47) or patients in hemodialysis (n=48) at 2-4 months after second dose (T3) and 2 weeks after the booster dose (T4). Cancer and rheumatic disease patients were classified according to the type of treatment: no active treatment or low (category 0), medium (category 1) or high (category 2) interference with the immune system, whereas patients in hemodialysis were classified with an immunoscore related to the disease for which the patients are in dialysis and their comorbidities: low (category 0), medium (category 1) or high (category 2) immune compromised. The distribution of patients in each category and the type of treatment are indicated in the legend. Samples (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 21, 2022. ; https://doi.org/10.1101/2022.01.21.22269633 doi: medRxiv preprint 25 minimum-to-maximum range. Each dot corresponds to an individual subject. P values were determined using 2-tailed Kruskal-Wallis test with Dunn's multiple comparisons post test. P values are reported. values in serum (x variable) and the % of neutralization (y variable) was performed in each category of fragile patients at T3 (0, n=23; 1, n=43; 2, n=38) (A) and at T4 (0, n=18; 1, n=30; 2, n=32) (B). A nonparametric Spearman's rank correlation test was performed. Samples Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy Systems vaccinology of the BNT162b2 mRNA vaccine in humans Humoral response after SARS-CoV-2 mRNA vaccines in dialysis patients: Integrating anti-SARS CoV-2 Spike-Protein-RBD antibody monitoring to manage dialysis centers in pandemic times Evaluation of the automated LIAISON((R)) SARS-CoV-2 TrimericS IgG assay for the detection of circulating antibodies COVID-19 mRNA Vaccine Immunogenicity in Immunosuppressed Individuals BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting Humoral Response after SARS-CoV-2 mRNA Vaccination in a Cohort of Hemodialysis Patients and Kidney Transplant Recipients Impaired humoral responses to COVID-19 vaccination in patients with lymphoma receiving B-cell-directed therapies Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell CD8 T cells compensate for impaired humoral immunity in COVID-19 patients with hematologic cancer D.s. Contributors to the. 2021. Infliximab is associated with attenuated immunogenicity to BNT162b2 and ChAdOx1 nCoV-19 SARS-CoV-2 vaccines in patients with IBD Antibody Responses in Seropositive Persons after a Single Dose of SARS-CoV-2 mRNA Vaccine No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Robust spike antibody responses and increased reactogenicity in seropositive individuals after a single dose of SARS-CoV-2 mRNA vaccine Coronavirus disease 2019 in immunocompromised patients: a comprehensive review of coronavirus disease 2019 in hematopoietic stem cell recipients The antibody response to SARS-CoV-2 increases over 5 months in patients with anosmia/dysgeusia. medRxiv 2021 The effect of methotrexate and targeted immunosuppression on humoral and cellular immune responses to the COVID-19 vaccine BNT162b2: a cohort study Anti-Spike Protein Assays to Determine SARS-CoV Antibody Levels: a Head-to-Head Comparison of Five Quantitative Assays Clinical portrait of the SARS-CoV-2 Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine Binding and Neutralization Antibody Titers After a Single Vaccine Dose in Health Care Workers Previously Infected With SARS-CoV-2 Poor antigen-specific responses to the second BNT162b2 mRNA vaccine dose in SARS-CoV-2-experienced individuals Monitoring Incidence of COVID-19 Cases, Hospitalizations, and Deaths, by Vaccination Status -13 U A SARS-CoV-2 surrogate virus neutralization test based on antibody Humoral and cellular immunogenicity of the BNT162b2 mRNA Covid-19 Vaccine in nursing home residents Safety and All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted The reagents in this study were supplied by DiaSorin (Italy) and QuantiFERON SARS-CoV-2 tubes by QIAGEN (Germany). We acknowledge the full financial support of DiaSorin S.p.A. for CLI-PR-2102 and CLI-PR-2108 clinical trials. In DiaSorin we would like to thank Elisa Ghezzi, Clara Rossini, and Chiara Mauro for neutralization testing and Alice Bianchi for T cell response testing. We thank Jenny Howard, Francis Stieber, and Vladyslav Nikolayevskyy from QIAGEN for their scientific support during the study.We would like to thank all the employees and the patients that volunteered to participate to this study, all the vaccinating doctors, the nurses and personnel that collected the samples, the laboratory technicians that run the serological tests and Humanitas Operations Management and Customer Care that coordinated vaccinations and blood draws. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted January https://doi.org/10.1101 https://doi.org/10. /2022