key: cord-0855669-oltfopmh authors: Grupper, Ayelet; Rabinowich, Liane; Ben-Yehoyada, Merav; Katchman, Eugene; Baruch, Roni; Freund, Tal; Hagin, David; Shlomo, Shani Ben; Schwartz, Doron; Schwartz, Idit F.; Shashar, Moshe; Bassat, Orit Kliuk-Ben; Halperin, Tami; Turner, Dan; Saiag, Esther; Goykhman, Yaacov; Shibolet, Oren; Levy, Sharon; Houri, Inbal; Katchman, Helena title: Humoral response to the third dose of SARS-CoV-2 vaccine in kidney transplant recipients. date: 2022-03-01 journal: Transplant Proc DOI: 10.1016/j.transproceed.2022.02.011 sha: 0938d201fd9d369dd4c516457b9191635117b587 doc_id: 855669 cord_uid: oltfopmh Background: The majority of solid organ transplant recipients did not develop an appreciable serologic response following two doses of mRNA SARS-CoV-2 vaccine. Methods: We analyzed the humoral response following third dose of BNT162b2 vaccine in 130 kidney transplant recipients, compare to 48 healthcare workers, and associated factors, including pre-vaccine cellular immune response, by evaluating intracellular cytokine production following stimulation of donor's peripheral blood mononuclear cells. Results: Following two doses, most of controls (47/48, 98%) and only 40% (52 of 130) kidney recipients were seropositive (p<0.001). Most seronegative recipients developed a serologic response after the booster (47 out 78, 60%), thus bringing the total number of seropositive recipients to 99/130 (76%). Following the third dose, there was a significant increase in antibodies titers in both groups. Decreased humoral response was significantly associated with an older age, lower lymphocyte count, and a lower level of antibodies before booster administration. CD4+TNFα+ and CD4+INFγ+ were correlated with mean increase in antibody titers. Conclusions: A third dose of BNT162b2 mRNA vaccine in kidney recipients is safe, and effectively results in increased IgG anti-S levels, including in individuals who were seronegative after two doses. Long-term studies of the length of the immune response and protection are required. recipients developed a serologic response after the booster (47 out 78, 60%), thus bringing the total number of seropositive recipients to 99/130 (76%). Following the third dose, there was a significant increase in antibodies titers in both groups. Decreased humoral response was significantly associated with an older age, lower lymphocyte count, and a lower level of antibodies before booster administration. CD4 + TNF + and CD4 + INF + were correlated with mean increase in antibody titers. Conclusions: A third dose of BNT162b2 mRNA vaccine in kidney recipients is safe, and effectively results in increased IgG anti-S levels, including in individuals who were seronegative after two doses. Long-term studies of the length of the immune response and protection are required. An infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting disease, coronavirus disease 2019 , have affected millions of people worldwide. Solid organ transplant (SOT) recipients are in increased risk of morbidity and mortality from COVID-19 due to their comorbidities and chronic immunosuppression state [1, 2] . Vaccines to prevent SARS-CoV-2 infection are considered the most promising approach for controlling the pandemic and are being vigorously pursued. Recently, studies demonstrated that in contrast to immunocompetent individuals, the majority of SOT recipients did not mount an appreciable serologic response [3] [4] [5] , and showed decreased cellular immunity [6] , following two doses of mRNA SARS-CoV-2 vaccine. Those observations, together with a suggested correlation between breakthrough infection with lower antibody levels after two doses of vaccine in the general population [7] as well as SOT recipients [8] , has led experts to recommend administration of a booster (third) dose to certain immunocompromised individuals, including SOT recipients [9] [10] [11] . First reports on administration of a third dose of the mRNA vaccine to SOT recipients has been shown to improve the immune response without causing any short-term, serious adverse events [12, 13] . However, the timing of the booster dose was less than 3 months after the second dose. In the present study, we aimed to quantify the humoral response following the third (booster) dose of the BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 mRNA vaccine, that was done 6 months after the second dose, among kidney transplant recipients, and associated factors, including magnitude of cellular immune response before the booster dose. The results were compare to a cohort of immunocompetent healthcare workers. Included were only participants with negative serology to SARS-CoV-2 nucleocapsid (N) protein, in order to exclude participants with prior exposure to the virus and evaluate the immune response to the vaccine itself, The study group included 132 adult kidney transplant recipients, who received 2 doses (21 days apart) and a third dose, at least 5 months after the second dose, of BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 mRNA vaccine,. The control group, composed of 48 immunocompetent healthcare workers, vaccinated according to the same protocol as the study group. Blood samples were collected prior to receiving the booster dose (same day or 1 day before), and 10-25 days following it. Freshly collected blood in clot activator gel tube was centrifuged at 3500 rpm for 4 minutes. The sera were separated and stored at 4 O C for analysis. The study was approved by the Tel Aviv Medical Center institutional ethical review board, and all participants provided written informed consent. Humoral Immune Response: Levels of antibodies targeting SARS-CoV-2 spike protein (IgG S1) were measured twice using the AdviseDx SARS-CoV-2 IgG II Quant assay (Abbott, Abbott Park, IL) on an Architect i200SR analyzer (Abbott). A cutoff value ≥50AU/ml was considered a significant antibody response, as previously suggested [14] . The results of this assay have been shown to correlate with in vitro neutralization of SARS-CoV-2 [15] . Included in the study participants who were never been positive to polymerase chain reaction (PCR) to SARS-CoV-2. In addition, every participant was tested to IgG antibodies against the SARS-CoV-2 nucleocapsid protein. This test was performed with an Architect i2000SR analyzer (Abbot Diagnostics, IL, USA) and Abbott chemistry according to the manufacture instructions. A cutoff of 1.4 index (S/C) was used [15] . This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Two transplant recipients were excluded from the study due to positive test of IgG antibodies to SARS-CoV-2 nucleocapsid protein as a marker of past exposure to the natural virus. Both patients had positive levels of anti-spike antibodies as well. The study group composed of 130 kidney transplant recipients, transplanted between January 1, 1996 and May 1, 2021. Four of them had liver and kidney transplant, and 8 had simultaneous kidney and pancreas transplantation (SPK). Sixteen recipients had the first and second doses of SARS-CoV2 vaccine before transplantation (and the third dose was given after transplantation). The mean time from the transplantation was 66 months, 25 (19.2%) patients in this group were transplanted during the year preceding the vaccination, but none of them in the 3 months before vaccination. The control group included 48 immunocompetent healthy individuals. As shown in Table 1 , participants in the control group were younger and had a higher prevalence of females. The time intervals between the first and the third dose of the vaccine, and between the third vaccine dose and the postvaccine sample collection were similar between the two groups. Maintenance immunosuppression of most recipients included low dose prednisone, tacrolimus and MMF. Clinical outcomes and adverse reactions after the third dose administration: After receiving the third vaccine dose, three participants developed COVID- 19 : one kidney recipient was tested seronegative 21 days after the third dose and is currently admitted with severe COVID19. Two participants, one kidney recipients and one healthcare worker, were tested positive in the post-vaccine sample and suffered only mild disease, 24-and 14-days post vaccination. In both recipients and conrtol groups, the booster dose of vaccination was safe, with no biopsy-proven acute rejections (in the study group), severe allergic reactions, or new neurological diagnoses (Guillain-Barre syndrome, Bell's palsy, zoster or other neuropathies) during a mean follow up period of 46 (±11) days after administration of the booster. Following two doses and just before the third dose, most of controls had detectable levels of IgG anti spike antibodies (47/48, 99%) while only 40% (52 of 130) recipients were seropositive (p<0.001). Similar to our previous work (4), before the booster the levels of anti-spike IgG in the recipients group (median=35 AU/mL (IQR 0-135)) was significantly lower than in the controls (median=687 AU/mL (IQR 308-1139)), p=0.003 ( Figure 2 ). However, when considering only seropositive participants, mean antibody levels were similar in both groups (median=189 (IQR 93-682) vs 709 (336-1142) AU/mL, study vs controls respectively, p=0.43). Unsurprisingly, all seropositive patients in the control group and in the study group remained seropositive after the third dose of the vaccine. In the control group, the only seronegative individual converted after receiving the third vaccine dose. Among the study group, most of the 78 kidney recipients who were seronegative before the booster, developed a serologic response of significant levels of IgG anti S after the booster (47 out 78, 60%) ( Figure 1) thus bringing the total number of seropositive recipients to 99 out of 130 (76%, as compared to just 40% after 2 vaccine doses). Following the third dose, there was a significant increase in antibodies titers in both groups. The mean increase in antibody titers was significantly higher in the controls (median=1278 (IQR= 68-7075) vs 28358 (15951-36766) AU/mL for study vs controls, respectively, p<0.001), Figure 1 . Univariate and multivariate analysis of variables associated with the risk of negative humoral response following the third vaccine dose are demonstrated in Table 2 . Age was inversely correlated to mean increase in antibody titers in both groups (correlation coefficient=-0.359; p<0.001 and -0.213; p=0.04 for study and controls, respectively). In addition, levels of antibody before the third dose were significantly correlated with increased titer following it (correlation coefficient=0.515 and 0.64, for study and control groups, respectively, p<0.001 for both). Decreased humoral response was significantly correlated with a lower lymphocyte count, and a lower level of antibodies before the vaccine booster administration. In addition, every year of age increased the risk of having a negative serology by 5%. The differences in the magnitude of the humoral immune response to the booster in both groups according to patients age are shown in Figure 3 . Comparison of the participants (controls and kidney transplant recipients) in the different age groups <50, 50-59 and >60, has revealed significant benefit for the younger groups in the magnitude of antibody level after the booster administration and in the delta of antibody levels before and after the booster. The difference in mean antibody levels before booster administration between participants of the control group aged <50 and 50-59 years did not reached a statistical difference (p=0.06), older individuals (>60 years old) in the control group had a significantly lower antibody level. For all age groups, serologic response was lower in study vs control group both before and after receiving the booster. However, booster vaccination lead to significant increase of antibody levels in both groups and all age groups (p<0.05 for all comparisons). Intracellular cytokine staining stimulated PBMCs was performed before the third vaccine dose in 14 healthy controls and 14 kidney transplant recipients. For this purpose, donors' cells were stimulated with a pooled S-peptide mix or controls, and intracellular cytokine production was evaluated by flow cytometry, gating on the CD4 + population. Mean percentage of CD4 + TNF + and CD4 + INF + cells was significantly higher in controls compare to kidney recipients ( Figure 4) . Interestingly, in a correlation analysis, CD4 + TNF + significantly correlated with the mean increase in antibody titers after the vaccine boost (correlation coefficient=0.58; p=0.029), correlation of CD4 + INF + with mean antibody increase showed a trend toward statistical significance (correlation coefficient=0.49; p=0.07). Healthy controls and transplanted patients did not show appreciable CD8 response (data not shown). Data evaluating the response to SARS-CoV-2 vaccines have been exponentially accumulating over the last several months. There is a consistent evidence that both humoral and cellular immune responses to SARS-CoV-2 vaccines are significantly reduced in kidney transplant recipients [18, 19] . Observation of the waning immunity demonstrated by decreased antibody levels in vaccinated subjects as well as correlation between breakthrough infections and the time that has passed since the second vaccine dose, has led experts including the FDA to recommend the administration of a booster vaccine dose to individuals over the age of 65, as well as immunocompromised patients and healthcare workers of any age, in whom the time interval since their second vaccine dose is over 5 months [20, 21] . This study describes the Anti-S1 IgG antibody response of kidney transplant recipients following a booster dose of the BNT162b2 (Pfizer-BioNTech) vaccine, and highlights parameters associated with it. We also studied the cellular response after the second dose of vaccine and showed it could serve as prediction for post-booster humoral response. Our pivotal finding was that most of kidney recipients (60%) remained seronegative following the second dose of the vaccine, as compared to ~3% in the healthy controls. However, the majority of kidney recipients who did not develop response after the second dose, showed a seroconversion after the booster vaccine administration, raising the level of seropositivity in the of recipients to 76%. The clinical importance of this laboratory finding is supported by growing evidences, suggesting that elapsed time has a major role in breakthrough infections due to wanning immunity and declining of antibody titers [0]. Bergwerk et al [22] described breakthrough infections with SARS-CoV-2 in a cohort of healthcare workers and a correlation of these infections with the declining of neutralizing antibody titers in the peri-infection period. According to the data of recent studies, including data from Israel, the booster is effective in reducing infections, as well as the rates of severe infections and hospitalizations due to COVID-19 [23, 24] . The data on the cellular immune response performed in our study demonstrated, unsurprisingly, significantly reduced cellular response in kidney transplant recipients in comparison to healthy controls. Somehow encouraging is our finding, showing that the presence of measurable levels of cellular immunity was associated with a better response following the third vaccine dose, despite lower absolute antibody levels. The predictors of blunt humoral response among transplant recipients after third dose of vaccine were decreased antibody response after the second dose, advanced age, and low lymphocyte count at the time of booster administration. Advanced age was consistently related to reduced antibody response in immunocompetent patients after COVID-19 [25] , as well as after SARS-CoV-2 vaccination [26, 27] , and in kidney recipients as well [28] . In concordance with previous studies, our study shows inferior serologic response after the third dose of vaccine, similar to the 2-dosevaccination schedule, in addition to lower surge in net antibody titers following the booster in the elderly patients. Recent studies, demonstrated that the serological response to vaccination agianst SARS-CoV-2 was affected by the net burden of patients' immunosuppression [4, 5] . After adjusting to other variables, we were not able to show this correlation in the present study. While it's tempting to speculate that booster vaccination overcome the barrier of high burden of immunosuppression, more realistic explanation might be that the lower titer of antibody before the booster has a strong statistical significancy which overcome other clinical parameters related to it. Strengths of this study include its novelty. It is the first published data on booster administration 6 months after the second dose of the vaccine, and correlation to cellular response before. Validation of the findings were done by a comparison to immunocompetent individuals, vaccinated on similar schedule. Exclusion of participants with IgG antibodies to nucleocapsid protein eliminates the possibility of response to the virus itself and therefore contributing to validation of our results. Limitations of the study include a short follow-up period and absence of assessing the cellular immune response following the third dose, those preclude us to address full spectrum of its immunogenicity as well as the clinical implications. Despite that, the accumulating data of significantly reduced immune response *P value=0.06 for controls age <50 and 50-59 before booster administration. For all other comparisons of study vs control, before vs after booster: p<0.05. in α+ (activation ratio) TNF + and CD4 γ+ INF + Mean and SD of CD4 : Figure 4 participants of both study group. P=0.048 and 0.046 respectively An initial report from the French SOT COVID Registry suggests high mortality due to COVID-19 in recipients of kidney transplants Is COVID-19 infection more severe in kidney transplant recipients? 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