BACKGROUND:Antibody-mediated rejection (AMR) is a major cause of kidney allograft failure. A substantial proportion of AMR occurs without detectable donor-specific antibodies (DSA), and the clinical relevance of DSA-negative AMR remains incompletely defined, particularly in regions with limited access to advanced immunological testing. METHODS:We conducted a retrospective analysis of kidney transplant recipients at a single center in Uruguay between 2018 and 2024. Patients with for-cause biopsies fulfilling contemporary Banff criteria for active AMR were included; chronic active AMR and non-immunologic lesions were excluded. Donor-specific antibodies were assessed at the time of biopsy using class I and II screening assays. Clinical, histological, and outcome data were compared between DSA-positive and DSA-negative AMR. Quantitative histological analyses, including an AMR/MVI index and an activity score, were used to assess microvascular injury and inflammatory burden. RESULTS:Among 458 kidney transplant recipients, 93 (20%) developed AMR, of whom only 24% had detectable DSA. DSA-positive patients showed distinct clinical and histological features, including more severe microvascular inflammation and a higher frequency of concomitant T cell-mediated rejection. A high proportion of cases were classified within the severe AMR/MVI category, and activity scores were significantly higher in DSA-positive patients compared with international cohorts. Graft survival was significantly reduced in patients with AMR, with no significant differences between DSA-positive and DSA-negative cases. CONCLUSIONS:In this single-center cohort, most AMR episodes occurred without detectable DSA. The predominance of high AMR/MVI scores suggests that patients present with later-stage histological injury at diagnosis. The use of AMR/MVI scores enabled comparison with international cohorts. These findings highlight the need for earlier diagnosis of AMR and regional limitations in comprehensive DSA assessment in Latin America.

BACKGROUND:Targeting emerging immunoregulatory molecules may open new therapeutic perspectives to control alloresponses and alleviate the burden of immunosuppressors. Transmembrane protein 176B (TMEM176B) is an intracellular cation channel highly expressed by myeloid cells. We have shown that TMEM176B is associated with allograft tolerance. Moreover, it controls the tolerogenic function of dendritic cells and inhibits NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome. Here, we speculated that pharmacological activation of TMEM176B by the nitroalkene derivative of 5-(2-nitroethenyl) salicylic acid (SANA) may prolong allograft survival through active immunoregulatory mechanisms. METHODS:SANA impact on TMEM176B activity was studied in vitro and in vivo. We assessed the potential efficacy of SANA treatment in prolonging graft survival in 2 allograft models: a mouse skin model with minor mismatches and a rat heart model with fully mismatches. Wild type and Tmem176b-/- recipient mice were used. Graft survival and innate and adaptive immune response were analyzed at the skin graft and draining lymph nodes through flow cytometry studies. RESULTS:SANA was identified as an activator of TMEM176B-dependent ion transport. SANA prolongs allograft survival in a Tmem176b -dependent manner. SANA triggered the expression of the immunoregulatory enzyme heme oxygenase-1 in wild type but not in Tmem176b-/- MHCII + CD11c high CD11b + conventional dendritic cells within the graft. SANA therapy was associated with increased CD4 + Forkhead box P3 + regulatory T (Treg) in the graft. The heme oxygenase-1 inhibitor tin protoporphyrin IX completely blocked the effect of SANA on graft survival and Treg in vivo. Furthermore, Treg modulation by anti-CD3 antibodies improves the graft-protecting effect of SANA. CONCLUSIONS:SANA-mediated activation of TMEM176B triggers an immunoregulatory pathway that prolongs skin and heart graft survival.

IntroductionThe humoral response after SARS-CoV-2 vaccination and boosters in kidney transplant recipients (KTRs) is heterogeneous and depends on immunosuppression status. There is no validated immune measurement associated with serological response in clinical practice. Multicolor flow cytometric immunophenotyping could be useful for measuring immune response. This study aimed to study B- and T-cell compartments through Standardized EuroFlow PID Orientation after SARS-CoV-2 vaccination and their association with IgG SARS-CoV-2 seropositivity status after two doses or boosters.MethodsWe conducted a multicenter prospective study to evaluate humoral response after SARS-CoV-2 vaccination in KTRs. Heterologous regimen: two doses of inactivated SARS-CoV-2 and two boosters of BNT162b2 mRNA (n=75). Homologous vaccination: two doses of BNT162b2 mRNA and one BNT162b2 mRNA booster (n=13). Booster doses were administrated to KTRs without taking into account their IgG SARS-CoV-2 seropositivity status. Peripheral blood samples were collected 30 days after the second dose and after the last heterologous or homologous booster. A standardized EuroFlow PID Orientation Tube (PIDOT) and a supervised automated analysis were used for immune monitoring cellular subsets after boosters.ResultsA total of 88 KTRs were included and divided into three groups according to the time of the first detected IgG SARS-CoV-2 seropositivity: non-responders (NRs, n=23), booster responders (BRs, n=41), and two-dose responders (2DRs, n=24). The NR group was more frequent on mycophenolate than the responder groups (NRs, 96%; BRs, 80%; 2DRs, 42%; p=0.000). Switched memory B cells in the 2DR group were higher than those in the BR and NR groups (medians of 30, 17, and 10 cells/ul, respectively; p=0.017). Additionally, the absolute count of central memory/terminal memory CD8 T cells was higher in the 2DR group than in the BR and NR groups. (166, 98, and 93 cells/ul, respectively; p=0.041). The rest of the T-cell populations studied did not show a statistical difference.Conclusionswitched memory B cells and memory CD8 T-cell populations in peripheral blood were associated with the magnitude of the humoral response after SARS-CoV-2 vaccination. Boosters increased IgG anti-SARS-CoV-2 levels, CM/TM CD8 T cells, and switched MBCs in patients with seropositivity after two doses. Interestingly, no seropositivity after boosters was associated with the use of mycophenolate and a lower number of switched MBCs and CM/TM CD8 T cells in peripheral blood.

Vaccination is the cornerstone in the fight against the ongoing COVID-19 pandemic.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar, 2Benotmane I. Gautier-Vargas G. Cognard N. et al.Low immunization rates among kidney transplant recipients who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine.Kidney Int. 2021; 99: 1498-1500https://doi.org/10.1016/j.kint.2021.04.005Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar, 4Benotmane I. Gautier G. Perrin P. et al.Antibody response after a third dose of the mRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients with minimal serologic response to 2 doses.JAMA. 2021; 326: 1063https://doi.org/10.1001/jama.2021.12339Crossref Scopus (163) Google Scholar, 5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Kidney transplant recipients (KTRs) had a weak immunologic response after 2 doses of either vaccine scheme.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar, 2Benotmane I. Gautier-Vargas G. Cognard N. et al.Low immunization rates among kidney transplant recipients who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine.Kidney Int. 2021; 99: 1498-1500https://doi.org/10.1016/j.kint.2021.04.005Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar,5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,S1–S9 Severe COVID-19 breakthrough infection has been reported in KTR with 2 doses of mRNA vaccine.3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar,S10,S11 On the basis of these data and evidence that boosters improve immunogenicity, international societies recommended a third dose in KTR and recently a fourth dose in a case-by-case basis.4Benotmane I. Gautier G. Perrin P. et al.Antibody response after a third dose of the mRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients with minimal serologic response to 2 doses.JAMA. 2021; 326: 1063https://doi.org/10.1001/jama.2021.12339Crossref Scopus (163) Google Scholar, 5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 6Reynolds C.J. Gibbons J.M. Pade C. et al.Heterologous infection and vaccination shapes immunity against SARS-CoV-2 variants.Science. 2022; 375eabm0811https://doi.org/10.1126/science.abm0811Crossref Scopus (21) Google Scholar, 7Caillard S. Thaunat O. Benotmane I. et al.Antibody response to a fourth messenger RNA COVID-19 vaccine dose in kidney transplant recipients: a case series.Ann Intern Med. 2022; 175: 455-456https://doi.org/10.7326/L21-0598Crossref PubMed Scopus (38) Google Scholar, 8Prendecki M, Thomson T, Candice L, et al. Comparison of humoral and cellular responses in kidney transplant recipients receiving BNT162b2 and ChAdOx1 SARS-CoV-2 vaccines. medRxiv. Published July 14, 2021. https://doi.org/10.1101/2021.07.09.21260192Google Scholar,S12–S14Different methods to assess immune response after vaccines, such as humoral and T cell function, have been used. However, many of these studies cannot be compared because of the lack of standardized assays.9Ikizler T.A. Coates P.T. Rovin B.H. Ronco P. Immune response to SARS-CoV-2 infection and vaccination in patients receiving kidney replacement therapy.Kidney Int. 2021; 99: 1275-1279https://doi.org/10.1016/j.kint.2021.04.007Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The World Health Organization International recommended standardization of antibody measurement by using binding antibody units per milliliter (BAU/ml) for binding serologic tests.S9 A cutoff value of 264 BAU/ml anti-receptor binding domain (RBD) IgG had been correlated with protection of symptomatic disease in the healthy population,S15 whereas Barrière et al.S16 proposed a 3-zone classification of anti–SARS-CoV-2 antibody levels for patients with cancer: a group of nonresponders with specific antibody levels <40 BAU/ml, a group that responds to vaccines with levels >260 BAU/ml, and a third area of uncertainty between these 2 values. Although they may be approximate values, the idea of this kind of classification can also be useful for its application in KTR patients.We are carrying on a multicenter, prospective, observational study for monitoring anti–SARS-CoV-2 specific IgG in KTR.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar Here, we present a second report on antibody levels after 4 doses of heterologous vaccination and 3 doses of homologous scheme stratified by the number of doses required to achieve seroconversion compared with healthy control.In Uruguay, 90% of KTRs received inactivated SARS-CoV-2 (CoronaVac, Sinovac Biotech Ltd., Beijing, People’s Republic of China) and 10% BNT162b2 mRNA (Pfizer, Manhattan, NY/BioNTech, Mainz, Germany), the most used vaccines worldwide, according to definitions of the Ministry of Public Health (details in the Supplementary Material). After the demonstration that only 29% of KTR seroconverted,1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar the Ministry of Public Health approved heterologous and homologous booster/s in all KTRs, irrespectively of seroconversion status. The heterologous vaccination group received 2 doses of inactivated SARS-CoV-2 (CoronaVac) and 2 BNT162b2 mRNA boosters 30 days apart, and the homologous vaccination group received 3 doses of the BNT162b2 mRNA vaccine (Supplementary Figure S1).Among all 1400 KTRs in follow-up in Uruguay (90% received 2 doses of CoronaVac and 10% 2 doses of mRNA-based vaccines), all were invited to participate in the study. Of these, 289 patients accepted to participate and were enrolled to study immunologic response to SARS-CoV-2 after 2 doses of vaccines.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar From this initial cohort, we only included for the present analysis patients who had blood samples available after 30 days of 2 initial doses and boosters according to the vaccination scheme (heterologous n = 92; homologous n = 17). A total of 180 patients who did not have all the samples available were excluded. Patients included in the study had no prior confirmed COVID-19 diagnosis until homologous or heterologous vaccination scheme was completed.All individuals declared not to have had any sign or symptom compatible with COVID-19 before vaccination. All patients underwent serologic testing for anti-RBD specific IgG 30 days after 2 initial doses and posteach booster. Patients were stratified into the following 4 groups according to the number of doses required to achieve seroconversion: seroconversion achieved after 2 doses (seroconversion-2D), 3 doses (seroconversion-3D), 4 doses (seroconversion-4D), and no seroconversion. In addition, a subanalysis between homologous or heterologous vaccine schemes was performed. Control group was constituted of 82 healthy individuals, fully vaccinated, with 2 doses of CoronaVac or BNT162b2 between April 1 and May 31, according to the recommendations of the Uruguayan National Health Authority considering age and comorbidities. No specific anti-RBD IgG antibodies were found before vaccination in the serum samples of the healthy control group (baseline seronegative). We do not had information on this regard in the KTR group. Seroconversion was defined as anti–RBD-SARS-CoV-2 specific IgG >10 BAU/ml.Baseline patients’ characteristics according to seroconversion groups are detailed in Table 1 and according to vaccine scheme in Supplementary Tables S1 and S2. Seroconversion after 2 doses was 26% globally, and it improved an additional 31.2% with the third dose (30.4% heterologous and 34.7% homologous vaccination, respectively) (Figure 1a). In the heterologous regimen, 21% of the patients seroconverted after the fourth dose of BNT162b2 mRNA (Figure 1a). At the end of heterologous and homologous vaccination, 71.7% and 70.6% of the patients seroconverted, respectively. In the seronegative group, a greater proportion of patients were treated with mycophenolate (96.7% vs. 45%, 85% and 87% in Seroconversion-2D, -3D, and -4D groups: respectively, P = 0.000) (Table 1); they also had less time since transplantation (median of 45 months vs. 113, 84, and 68 months in Seroconversion-2D, -3D, and -4D, respectively, P = 0.415) and lower lymphocyte count (median 1900/μl vs. 2400, 2010, and 2600/μl inSeroconversion-2D, -3D, and -4D, P = 0.415), statistically nonsignificant.Table 1Clinical characteristics of patients according to the number of doses to achieve seroconversion after 3D homologous and 4D heterologous SARS-CoV-2 vaccinationsVariablesSeroconversion achieved after SARS-CoV-2 vaccinationTotalP value2DAfter 2 doses3DAfter first booster4DAfter second boosterNo seroconversionn (%)29 (26.6)35 (31.2)15 (13.8)31 (28.4)109 (100)Vaccine schemeHeterologous, n (%)23 (79)28 (82.5)15 (100)26 (84)92 (84)0.326Homologous, n (%)6 (21)6 (17.5)5 (16)17 (16)Age, yr, median (IQR)55 (40–62)54 (45–77)59 (54–65)54 (45–77)58 (45–72)0.245Sex, men, n (%)17 (57)25 (73)8 (28)18 (62)68 (64)0.564Diabetes mellitus, n (%)3 (10)8 (23)6 (40)7 (23)24 (22)0.160Type of transplant, n (%)Kidney28 (97)33 (97)15 (100)30 (90)106 (98)0.687Kidney-pancreas1 (3)1 (3)——2 (2)Time of transplant, mo, median (IQR)113 (30–148)84 (32–174)68 (34–155)45 (32–112)68 (32–146)0.415Patients in the first year of transplant, n (%)2 (7)2 (6)1 (7)2 (7)7 (7)0.998Triple immunosuppression, n (%)17 (59)22 (65)11 (73)26 (87)76 (71)0.097Antimetabolite, n (%)0.000None10 (35)4 (12)1 (7)1 (3)16 (15)Mycophenolate13 (45)28 (85)13 (87)29 (96.7)83 (78)Azathioprine6 (20)1 (3)1 (7)0 (0)8 (7)CNI, n (%)0.312None3 (11)5 (15)1 (7)1 (3.3)10 (9)Tacrolimus23 (79)22 (65)9 (60)25 (83)79 (73)Cyclosporine3 (10)7 (21)5 (33)4 (13)19 (17)Prednisone, n (%)28 (97)32 (94)13 (87)28 (9%)101 (94)0.654Everolimus, n (%)10 (35)10 (30)1 (6.7)1 (3)22 (21)0.006Rituximab, n (%)02 (6)002 (2)0.224Thymoglobulin, n (%)15 (47)5 (15)3 (20)6 (21)19 (18)0.931Rejection in last 3 mo, n (%)001 (6.7)08 (2.9)0.103Lymphocyte count, cells/μl, median (IQR)2400 (1800–2552)2010 (1560–2570)2600 (1800–3440)1900 (1288–2286)2108 (1560–2600)0.073Lymphocyte count < 1400 cells/l, n (%)2 (7)6 (18)3 (20)10 (36)21 (20)0.058Serum creatinine, μmol/l, median (IQR)109 (90–134)113 (96–141)114 (87–122)119 (87–138)114 (92–134)0.886eGFR ml/min per 1.73 m2, mean ± SD62 ± 1856 ± 2159 ± 1757 ± 2058 ± 190.661IgG anti-RBD SARS-CoV-2 (BAU/ml), median (IQR)After 2D42 (23–173)0000 (0–8)0.000After 3D2453 (1255–3505)179 (41–412)02.525 (0–1770)0.000After 4D4659 (2778–13,394)621 (254–1913)74 (33–283)0254 (7–2775)0.0002D, 2 doses; 3D, 3 doses; 4D, 4 doses; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate using CKD-EPI Formula; IQR, interquartile range; RBD, receptor binding domain; triple immunosuppression: antimetabolite + calcineurin inhibitor + prednisone.Homologous vaccination: 3 doses of BNT162b2 mRNA vaccine 30 days apart; heterologous vaccination: 2 doses of inactivated SARS-CoV-2 vaccine (CoronaVac) and 2 BNT162b2 mRNA boosters 30 days apart. Lymphocyte counts were assessed previous to vaccination. The threshold to define seroconversion was 10 BAU/ml (black dotted line). Open table in a new tab Serum levels of anti-RBD IgGs depend on the number of doses to achieve seroconversion (Figure 1b; green, pink, blue, and gray). Anti-RBD IgG serum levels were significantly higher in the Seroconversion-2D group (Figure 1b; green) in comparison with Seroconversion-3D and -4D (Figure 1b; pink and blue) at the 3-time point measurement (after 2, 3, and 4 doses) in both vaccine schedules.In the heterologous regimen, anti-RBD IgG increased with each dose. For example, in the Seroconversion-2D-group, we observed a 14-fold after the third dose and 1.7 after the fourth dose (from a median of 186 to 2682 and 4659 BAU/ml respectively, P = 0.000). Meanwhile, with the homologous scheme, antibody titer increased only 5.5-fold after the third dose (from a median of 223 to 1225 BAU/ml, P = 0.000).A comparison between the heterologous and homologous schemes at each point of measurement can be found in Figure 1b. Antibody levels were higher in patients after 2 doses of BNT162b2 mRNA than with 2 doses of CoronaVac (median 223 BAU/ml [interquartile range (IQR) 124–390] vs. 35 BAU/ml [IQR 16–99], P = 0.003). After the third dose of the heterologous scheme, the antibody response was enhanced compared with the homologous (seroconversion 2D, median 2682 [IQR 1741–4460] vs. 1225 BAU/ml [724–2627], P = 0.158 and in seroconversion 3D group median186 BAU/ml [IQR 61–624] and 37 BAU/ml [IQR 18–188], P = 0.074). Although not significant, these differences have a tendency to be considered when defining possible booster dose regimens.Only patients in the seroconversion-2D group after the third and fourth doses in heterologous vaccination achieved antibody titers comparable with 2 doses of BNT162b2 mRNA in the healthy group. Meanwhile, only 26.7% of patients with seroconversion after the fourth dose have levels of antibody >264 BAU/ml (Supplementary Figure S2 and Supplementary Material).In multivariate analysis, variables associated with low antibody level (<264 BAU/ml) after heterologous or homologous vaccination were mycophenolate and time post-KT (Supplementary Table S3). We used antibody level of 264 BAU/ml, suggested by other authors as a correlate of protection in healthy individualS15 and KTR,S17 to compare the number of patients in each group that reached this level, making it easier to visualize the response to vaccines. We are not suggesting a specific level of anti–SARS-CoV-2 antibodies that correlates with protection in KTR. During the 5-month follow-up, 10 KTRs had COVID-19 confirmed by polymerase chain reaction during the second wave of the pandemic in Uruguay (between December 2021 and February 2022). Two rejection episodes and 1 IgA nephropathy recurrence were reported. The 2 patients with rejection belonged to the group with higher antibody titers (seroconversion-2D, heterologous: 17,413 and 3470 BAU/ml) (details in the Supplementary Material and Supplementary Table S4). No deaths were reported.We provided data that heterologous vaccination with 4 doses induced an enhanced humoral response in KTR. Although antibody titers are highly heterogeneous, it could be predicted by taking into consideration the number of doses to achieve seroconversion. We described the following 4 different groups: seroconversion-2D, -3D, -4D, and no response associated with the magnitude of humoral immune response after vaccination. These differences in humoral response were associated with the use of mycophenolate or everolimus and time since kidney transplant.Patients with seroconversion after 2 doses of either vaccination scheme have higher antibody titers than those who seroconverted after the third or fourth dose and were similar to healthy control after 30 days of 2 doses of mRNA vaccines.Although nonstatistically significant, heterologous vaccination achieved higher antibody titers than homologous. In the healthy population, heterologous vaccination had a better immunologic response than homologous,6Reynolds C.J. Gibbons J.M. Pade C. et al.Heterologous infection and vaccination shapes immunity against SARS-CoV-2 variants.Science. 2022; 375eabm0811https://doi.org/10.1126/science.abm0811Crossref Scopus (21) Google Scholar,S18 but in KTR, this effect cannot be found.S16 This difference could be since prior studies included groups of patients with heterogeneity in immunologic response.S19Potential biases for this study are the low number of patients in the homologous vaccination group; most patients were approximately 50 years old and lacked evaluation of cellular immunity.Our results provide additional evidence of heterogeneous humoral response among KTRs after 4 doses of heterologous vaccination or 3 doses of mRNA homologous. Although there is information of response to the third and fourth dose vaccinations, as far as we know, it is the first work to study immune response after 4 doses of SARS-CoV-2 vaccines administered to every kidney transplant patient without considering seroconversion status. Patients who seroconverted after 2 doses achieved higher levels of SARS-CoV-2 antibody than those with seroconversion after the third or fourth dose and reached antibody titer similar to healthy control. Meanwhile, most patients with seroconversion after the fourth dose have low levels of antibodies. These differences in immune response depend on mycophenolate use and time since kidney transplant. This stratification strategy according to the number of doses to achieve seroconversion could be used to personalize boosters in KTR.Heterologous vaccination seemed to induce an enhanced humoral response compared with homologous vaccination. However, future studies with larger size groups stratified by the magnitude of humoral response are needed to settle this debate.DisclosureAll the authors declared no competing interests. Vaccination is the cornerstone in the fight against the ongoing COVID-19 pandemic.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar, 2Benotmane I. Gautier-Vargas G. Cognard N. et al.Low immunization rates among kidney transplant recipients who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine.Kidney Int. 2021; 99: 1498-1500https://doi.org/10.1016/j.kint.2021.04.005Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar, 4Benotmane I. Gautier G. Perrin P. et al.Antibody response after a third dose of the mRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients with minimal serologic response to 2 doses.JAMA. 2021; 326: 1063https://doi.org/10.1001/jama.2021.12339Crossref Scopus (163) Google Scholar, 5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Kidney transplant recipients (KTRs) had a weak immunologic response after 2 doses of either vaccine scheme.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar, 2Benotmane I. Gautier-Vargas G. Cognard N. et al.Low immunization rates among kidney transplant recipients who received 2 doses of the mRNA-1273 SARS-CoV-2 vaccine.Kidney Int. 2021; 99: 1498-1500https://doi.org/10.1016/j.kint.2021.04.005Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar,5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,S1–S9 Severe COVID-19 breakthrough infection has been reported in KTR with 2 doses of mRNA vaccine.3Quiroga B. Soler M.J. Ortiz A. et al.Safety and immediate humoral response of COVID-19 vaccines in chronic kidney disease patients: the SENCOVAC study.Nephrol Dial Transplant. 2021; : gfab313https://doi.org/10.1093/ndt/gfab313Crossref PubMed Scopus (11) Google Scholar,S10,S11 On the basis of these data and evidence that boosters improve immunogenicity, international societies recommended a third dose in KTR and recently a fourth dose in a case-by-case basis.4Benotmane I. Gautier G. Perrin P. et al.Antibody response after a third dose of the mRNA-1273 SARS-CoV-2 vaccine in kidney transplant recipients with minimal serologic response to 2 doses.JAMA. 2021; 326: 1063https://doi.org/10.1001/jama.2021.12339Crossref Scopus (163) Google Scholar, 5Westhoff T.H. Seibert F.S. Anft M. et al.A third vaccine dose substantially improves humoral and cellular SARS-CoV-2 immunity in renal transplant recipients with primary humoral nonresponse.Kidney Int. 2021; 100: 1135-1136https://doi.org/10.1016/j.kint.2021.09.001Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 6Reynolds C.J. Gibbons J.M. Pade C. et al.Heterologous infection and vaccination shapes immunity against SARS-CoV-2 variants.Science. 2022; 375eabm0811https://doi.org/10.1126/science.abm0811Crossref Scopus (21) Google Scholar, 7Caillard S. Thaunat O. Benotmane I. et al.Antibody response to a fourth messenger RNA COVID-19 vaccine dose in kidney transplant recipients: a case series.Ann Intern Med. 2022; 175: 455-456https://doi.org/10.7326/L21-0598Crossref PubMed Scopus (38) Google Scholar, 8Prendecki M, Thomson T, Candice L, et al. Comparison of humoral and cellular responses in kidney transplant recipients receiving BNT162b2 and ChAdOx1 SARS-CoV-2 vaccines. medRxiv. Published July 14, 2021. https://doi.org/10.1101/2021.07.09.21260192Google Scholar,S12–S14 Different methods to assess immune response after vaccines, such as humoral and T cell function, have been used. However, many of these studies cannot be compared because of the lack of standardized assays.9Ikizler T.A. Coates P.T. Rovin B.H. Ronco P. Immune response to SARS-CoV-2 infection and vaccination in patients receiving kidney replacement therapy.Kidney Int. 2021; 99: 1275-1279https://doi.org/10.1016/j.kint.2021.04.007Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The World Health Organization International recommended standardization of antibody measurement by using binding antibody units per milliliter (BAU/ml) for binding serologic tests.S9 A cutoff value of 264 BAU/ml anti-receptor binding domain (RBD) IgG had been correlated with protection of symptomatic disease in the healthy population,S15 whereas Barrière et al.S16 proposed a 3-zone classification of anti–SARS-CoV-2 antibody levels for patients with cancer: a group of nonresponders with specific antibody levels <40 BAU/ml, a group that responds to vaccines with levels >260 BAU/ml, and a third area of uncertainty between these 2 values. Although they may be approximate values, the idea of this kind of classification can also be useful for its application in KTR patients. We are carrying on a multicenter, prospective, observational study for monitoring anti–SARS-CoV-2 specific IgG in KTR.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar Here, we present a second report on antibody levels after 4 doses of heterologous vaccination and 3 doses of homologous scheme stratified by the number of doses required to achieve seroconversion compared with healthy control. In Uruguay, 90% of KTRs received inactivated SARS-CoV-2 (CoronaVac, Sinovac Biotech Ltd., Beijing, People’s Republic of China) and 10% BNT162b2 mRNA (Pfizer, Manhattan, NY/BioNTech, Mainz, Germany), the most used vaccines worldwide, according to definitions of the Ministry of Public Health (details in the Supplementary Material). After the demonstration that only 29% of KTR seroconverted,1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar the Ministry of Public Health approved heterologous and homologous booster/s in all KTRs, irrespectively of seroconversion status. The heterologous vaccination group received 2 doses of inactivated SARS-CoV-2 (CoronaVac) and 2 BNT162b2 mRNA boosters 30 days apart, and the homologous vaccination group received 3 doses of the BNT162b2 mRNA vaccine (Supplementary Figure S1). Among all 1400 KTRs in follow-up in Uruguay (90% received 2 doses of CoronaVac and 10% 2 doses of mRNA-based vaccines), all were invited to participate in the study. Of these, 289 patients accepted to participate and were enrolled to study immunologic response to SARS-CoV-2 after 2 doses of vaccines.1Seija M. Rammauro F. Santiago J. et al.Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.Clin Kidney J. 2021; 15: 527-533https://doi.org/10.1093/ckj/sfab291Crossref PubMed Google Scholar From this initial cohort, we only included for the present analysis patients who had blood samples available after 30 days of 2 initial doses and boosters according to the vaccination scheme (heterologous n = 92; homologous n = 17). A total of 180 patients who did not have all the samples available were excluded. Patients included in the study had no prior confirmed COVID-19 diagnosis until homologous or heterologous vaccination scheme was completed. All individuals declared not to have had any sign or symptom compatible with COVID-19 before vaccination. All patients underwent serologic testing for anti-RBD specific IgG 30 days after 2 initial doses and posteach booster. Patients were stratified into the following 4 groups according to the number of doses required to achieve seroconversion: seroconversion achieved after 2 doses (seroconversion-2D), 3 doses (seroconversion-3D), 4 doses (seroconversion-4D), and no seroconversion. In addition, a subanalysis between homologous or heterologous vaccine schemes was performed. Control group was constituted of 82 healthy individuals, fully vaccinated, with 2 doses of CoronaVac or BNT162b2 between April 1 and May 31, according to the recommendations of the Uruguayan National Health Authority considering age and comorbidities. No specific anti-RBD IgG antibodies were found before vaccination in the serum samples of the healthy control group (baseline seronegative). We do not had information on this regard in the KTR group. Seroconversion was defined as anti–RBD-SARS-CoV-2 specific IgG >10 BAU/ml. Baseline patients’ characteristics according to seroconversion groups are detailed in Table 1 and according to vaccine scheme in Supplementary Tables S1 and S2. Seroconversion after 2 doses was 26% globally, and it improved an additional 31.2% with the third dose (30.4% heterologous and 34.7% homologous vaccination, respectively) (Figure 1a). In the heterologous regimen, 21% of the patients seroconverted after the fourth dose of BNT162b2 mRNA (Figure 1a). At the end of heterologous and homologous vaccination, 71.7% and 70.6% of the patients seroconverted, respectively. In the seronegative group, a greater proportion of patients were treated with mycophenolate (96.7% vs. 45%, 85% and 87% in Seroconversion-2D, -3D, and -4D groups: respectively, P = 0.000) (Table 1); they also had less time since transplantation (median of 45 months vs. 113, 84, and 68 months in Seroconversion-2D, -3D, and -4D, respectively, P = 0.415) and lower lymphocyte count (median 1900/μl vs. 2400, 2010, and 2600/μl inSeroconversion-2D, -3D, and -4D, P = 0.415), statistically nonsignificant. 2D, 2 doses; 3D, 3 doses; 4D, 4 doses; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate using CKD-EPI Formula; IQR, interquartile range; RBD, receptor binding domain; triple immunosuppression: antimetabolite + calcineurin inhibitor + prednisone. Homologous vaccination: 3 doses of BNT162b2 mRNA vaccine 30 days apart; heterologous vaccination: 2 doses of inactivated SARS-CoV-2 vaccine (CoronaVac) and 2 BNT162b2 mRNA boosters 30 days apart. Lymphocyte counts were assessed previous to vaccination. The threshold to define seroconversion was 10 BAU/ml (black dotted line). Serum levels of anti-RBD IgGs depend on the number of doses to achieve seroconversion (Figure 1b; green, pink, blue, and gray). Anti-RBD IgG serum levels were significantly higher in the Seroconversion-2D group (Figure 1b; green) in comparison with Seroconversion-3D and -4D (Figure 1b; pink and blue) at the 3-time point measurement (after 2, 3, and 4 doses) in both vaccine schedules. In the heterologous regimen, anti-RBD IgG increased with each dose. For example, in the Seroconversion-2D-group, we observed a 14-fold after the third dose and 1.7 after the fourth dose (from a median of 186 to 2682 and 4659 BAU/ml respectively, P = 0.000). Meanwhile, with the homologous scheme, antibody titer increased only 5.5-fold after the third dose (from a median of 223 to 1225 BAU/ml, P = 0.000). A comparison between the heterologous and homologous schemes at each point of measurement can be found in Figure 1b. Antibody levels were higher in patients after 2 doses of BNT162b2 mRNA than with 2 doses of CoronaVac (median 223 BAU/ml [interquartile range (IQR) 124–390] vs. 35 BAU/ml [IQR 16–99], P = 0.003). After the third dose of the heterologous scheme, the antibody response was enhanced compared with the homologous (seroconversion 2D, median 2682 [IQR 1741–4460] vs. 1225 BAU/ml [724–2627], P = 0.158 and in seroconversion 3D group median186 BAU/ml [IQR 61–624] and 37 BAU/ml [IQR 18–188], P = 0.074). Although not significant, these differences have a tendency to be considered when defining possible booster dose regimens. Only patients in the seroconversion-2D group after the third and fourth doses in heterologous vaccination achieved antibody titers comparable with 2 doses of BNT162b2 mRNA in the healthy group. Meanwhile, only 26.7% of patients with seroconversion after the fourth dose have levels of antibody >264 BAU/ml (Supplementary Figure S2 and Supplementary Material). In multivariate analysis, variables associated with low antibody level (<264 BAU/ml) after heterologous or homologous vaccination were mycophenolate and time post-KT (Supplementary Table S3). We used antibody level of 264 BAU/ml, suggested by other authors as a correlate of protection in healthy individualS15 and KTR,S17 to compare the number of patients in each group that reached this level, making it easier to visualize the response to vaccines. We are not suggesting a specific level of anti–SARS-CoV-2 antibodies that correlates with protection in KTR. During the 5-month follow-up, 10 KTRs had COVID-19 confirmed by polymerase chain reaction during the second wave of the pandemic in Uruguay (between December 2021 and February 2022). Two rejection episodes and 1 IgA nephropathy recurrence were reported. The 2 patients with rejection belonged to the group with higher antibody titers (seroconversion-2D, heterologous: 17,413 and 3470 BAU/ml) (details in the Supplementary Material and Supplementary Table S4). No deaths were reported. We provided data that heterologous vaccination with 4 doses induced an enhanced humoral response in KTR. Although antibody titers are highly heterogeneous, it could be predicted by taking into consideration the number of doses to achieve seroconversion. We described the following 4 different groups: seroconversion-2D, -3D, -4D, and no response associated with the magnitude of humoral immune response after vaccination. These differences in humoral response were associated with the use of mycophenolate or everolimus and time since kidney transplant. Patients with seroconversion after 2 doses of either vaccination scheme have higher antibody titers than those who seroconverted after the third or fourth dose and were similar to healthy control after 30 days of 2 doses of mRNA vaccines. Although nonstatistically significant, heterologous vaccination achieved higher antibody titers than homologous. In the healthy population, heterologous vaccination had a better immunologic response than homologous,6Reynolds C.J. Gibbons J.M. Pade C. et al.Heterologous infection and vaccination shapes immunity against SARS-CoV-2 variants.Science. 2022; 375eabm0811https://doi.org/10.1126/science.abm0811Crossref Scopus (21) Google Scholar,S18 but in KTR, this effect cannot be found.S16 This difference could be since prior studies included groups of patients with heterogeneity in immunologic response.S19 Potential biases for this study are the low number of patients in the homologous vaccination group; most patients were approximately 50 years old and lacked evaluation of cellular immunity. Our results provide additional evidence of heterogeneous humoral response among KTRs after 4 doses of heterologous vaccination or 3 doses of mRNA homologous. Although there is information of response to the third and fourth dose vaccinations, as far as we know, it is the first work to study immune response after 4 doses of SARS-CoV-2 vaccines administered to every kidney transplant patient without considering seroconversion status. Patients who seroconverted after 2 doses achieved higher levels of SARS-CoV-2 antibody than those with seroconversion after the third or fourth dose and reached antibody titer similar to healthy control. Meanwhile, most patients with seroconversion after the fourth dose have low levels of antibodies. These differences in immune response depend on mycophenolate use and time since kidney transplant. This stratification strategy according to the number of doses to achieve seroconversion could be used to personalize boosters in KTR. Heterologous vaccination seemed to induce an enhanced humoral response compared with homologous vaccination. However, future studies with larger size groups stratified by the magnitude of humoral response are needed to settle this debate. DisclosureAll the authors declared no competing interests. All the authors declared no competing interests. This work was supported by Fondo para la Convergencia Estructural del Mercosur (FOCEM, COF 03/11); Agencia Nacional de Investigación e Innovación ( ANII ), Uruguay; and Fondo de Investigación en Nefrología (FOINE), Hospital de Clínicas, Uruguay. Supplementary Material Download .pdf (.83 MB) Help with pdf files Supplementary File (PDF)Supplementary Methods.Figure S1. Types of vaccination schemes and IgG anti-RBD measurement.Figure S2. Percentage of patients with IgG anti–SARS-CoV-2 RBD above 264 BAU/ml after 4-dose heterologous and 3-dose homologous vaccination scheme.Table S1. Clinical characteristics of patients according to number of doses to achieve seroconversion after 4-dose heterologous SARS-CoV-2 vaccination.Table S2. Clinical characteristics of patients according to number of doses to achieve seroconversion after 3-dose homologous SARS-CoV-2 vaccination.Table S3. Multivariate analysis for IgG anti-RBD SARS-CoV-2 < 264 BAU/ml as a surrogate of protection.Table S4. Adverse events reported post-vaccination.Supplementary References.STROBE Statement. Download .pdf (.83 MB) Help with pdf files Supplementary File (PDF) Supplementary Methods. Figure S1. Types of vaccination schemes and IgG anti-RBD measurement. Figure S2. Percentage of patients with IgG anti–SARS-CoV-2 RBD above 264 BAU/ml after 4-dose heterologous and 3-dose homologous vaccination scheme. Table S1. Clinical characteristics of patients according to number of doses to achieve seroconversion after 4-dose heterologous SARS-CoV-2 vaccination. Table S2. Clinical characteristics of patients according to number of doses to achieve seroconversion after 3-dose homologous SARS-CoV-2 vaccination. Table S3. Multivariate analysis for IgG anti-RBD SARS-CoV-2 < 264 BAU/ml as a surrogate of protection. Table S4. Adverse events reported post-vaccination. Supplementary References. STROBE Statement.

ABSTRACT Background Antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after mRNA or adenoviral vector-based vaccines is weak in kidney transplant (KT) patients. However, few studies have focused on humoral response after inactivated virus-based vaccines in KT. Here, we compare antibody response following vaccination with inactivated virus (CoronaVac®) and BNT162b2 mRNA. Methods A national multicentre cross-sectional study was conducted. The study group was composed of patients from all KT centres in Uruguay, vaccinated between 1 and 31 May 2021 (CoronaVac®, n = 245 and BNT162b2, n = 39). The control group was constituted of 82 healthy individuals. Participants had no prior confirmed coronavirus disease 2019 (COVID-19) test. Blood samples were collected between 30 and 40 days after the second dose. Serum-specific immunoglobulin G (IgG) antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein were determined using the COVID-19 IgG QUANT ELISA Kit. Results Only 29% of KT recipients showed seroconversion (36.5% BNT162b2, 27.8% inactivated virus, P = 0.248) in comparison with 100% in healthy control with either vaccine. Antibody levels against RBD were higher with BNT162b mRNA than with inactivated virus [median (interquartile range) 173 (73–554) and 29 (11–70) binding antibody units (BAU)/mL, P < 0.034] in KT and 10 times lower than healthy control [inactivated virus: 308 (209–335) and BNT162b2: 2638 (2608–3808) BAU/mL, P < 0.034]. In multivariate analysis, variables associated with negative humoral response were age, triple immunosuppression, estimated glomerular filtration rate and time post-KT. Conclusion Seroconversion was low in KT patients after vaccination with both platforms. Antibody levels against SARS-CoV-2 were lower with inactivated virus than BNT162b mRNA. These findings support the need for strategies to improve immunogenicity in KT recipients after two doses of either vaccine.