key: cord-0897520-5w20c4td
authors: Crespo, Marta; Barrilado‐Jackson, Antoni; Padilla, Eduardo; Eguía, Jorge; Echeverria‐Esnal, Daniel; Cao, Higini; Faura, Anna; Folgueiras, Montserrat; Solà‐Porta, Eulàlia; Pascual, Sergi; Barbosa, Francesc; Hurtado, Sara; Ribera, Laura; Río‐No, Laura; Pérez‐Sáez, María José; Redondo‐Pachón, Dolores; Pascual, Julio
title: Negative immune responses to two‐dose mRNA COVID‐19 vaccines in renal allograft recipients assessed with simple antibody and interferon gamma release assay cellular monitoring
date: 2021-10-07
journal: Am J Transplant
DOI: 10.1111/ajt.16854
sha: 52be871ad7dd4605f8f8cb608a514d1ccfcd9119
doc_id: 897520
cord_uid: 5w20c4td

Studies are urgently needed to characterize immunogenicity, efficacy, and safety of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) mRNA vaccines in kidney transplant (KT) recipients, excluded from major clinical trials. Complex ELISPOT and other cellular response techniques have been applied, but simpler tools are needed. An easy‐to‐use real‐world monitoring of SARS‐CoV‐2 IgG antibodies against the Spike protein and QuantiFERON(®) SARS‐CoV‐2 IFNγ release assay (IGRA) were performed at baseline and 28 days after the second dose in KT recipients and controls (dialysis patients and healthy ones). All healthy controls and >95% dialysis controls became positive for anti‐S IgG antibodies, while only 63.3% of KT patients seroconverted with a very low antibody level. A positive IGRA was documented in 96.9% of controls, 89.3% peritoneal dialysis, 77.6% hemodialysis, 61.3% of KT patients transplanted more than 1 year ago and only 36% of those transplanted within the previous 12 months. Overall, 100% of healthy controls, 95.4% of dialysis patients and 78.8% KT recipients developed any immune response (humoral and/or cellular) against SARS‐CoV‐2. KT patients showed low rates of immune responses to mRNA Coronavirus infectious disease 2019 vaccines, especially those with recent transplantations. Simple humoral and cellular monitoring is advisable, so that repeated doses may be scheduled according to the results.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus infectious disease 2019 (COVID- 19) , which has caused the worst pandemic in the last decades. COVID-19 cases can be asymptomatic or mild in around 80% of individuals, especially in young adults and children. However, patients over 60 years old and with comorbidities are in major risk, requiring intensive respiratory support and presenting more frequently complications such as multiorganic failure or death. 1, 2 Several studies indicate that chronic kidney disease (CKD) is the most common comorbidity in severe COVID-19. [3] [4] [5] [6] Furthermore, patients on renal replacement therapy, on dialysis or with a kidney transplant (KT), have shown the highest morbidity and mortality. 3, [7] [8] [9] A functional immune system is essential to overcome SARS-CoV-2 infection. In the acute phase, activation of CD4 and CD8-T cells is observed in most infected patients. CD4-T cells conduct T helper 1 (Th1) responses, expressing cytokines like interferon gamma (IFNγ) that contribute to viral clearance. CD8-T cells directly destroy infected cells through cytotoxicity. 10, 11 Regarding humoral response, SARS-CoV-2 antigen-specific antibodies are detected in the first weeks since symptoms onset, reaching peak levels in the third week.

Neutralizing antibodies, which bind to the Spike (S) protein and prevent interaction with the cellular receptor ACE2 are also generated, granting immune protection against SARS-CoV-2 by disabling viral entry. 11 Antibody durability has not yet been uniformly established, although some studies suggest that levels decrease just after reaching a peak. 12, 13 Despite this, as antibody levels that offer protection have not been determined, this decrease may not imply loss of immunity against reinfections. 10 Two studies have shown persistence of antibody and cellular responses 6 months after COVID-19 in over 100 adult hemodialysis (HD) patients. 14, 15 Transplant recipients can also show robust although delayed humoral and cellular responses after infection. 16, 17 Since the beginning of the pandemic, governments of states affected by COVID-19 implemented sanitary measures to limit virus propagation and reduce high morbidity and mortality rates. The most suitable way to achieve these objectives is generating herd immunity with vaccines. 18 Vaccines must induce antibody production and T cell activation to prevent infection and spread to others. 19 Cellular immunity stimulated by mRNA-1273 is characterized by activation of S-specific CD4-T cells with Th1 profile, while BNT162b2 additionally induces a considerable CD8-T cell response, 20, 21 and both induce antibodies against the S protein.

Due to the increased risk of severe and fatal COVID-19 in KT patients, they have been prioritized for COVID-19 vaccination.

Because the response to several vaccines is recognized to be poorer in these patients, 22, 23 it is imperative to assess the proportion of responders, the quality of the response as well as the best time for vaccination across the lifetime of the CKD patient with simple and reliable immune response tools. In 104 heart and liver transplant recipients, 64% developed antibodies and 79% T cell responses measured with ELISPOT against the S protein 1 month after completing mRNA-1273 vaccination. 24 

Patients were called by telephone and answered a standardized questionnaire 7 days after receiving both the first and second dose of the mRNA vaccine. This questionnaire, created from previous data, 27, 28 included solicited local (pain, redness and swelling at injection site) and systemic (fever, fatigue, headache, chills, vomits, diarrhea, myalgia, and arthralgia) adverse events (AE) and their severity. Patients were also followed through electronic chart records 28 days after the second dose to detect unsolicited adverse reactions, serious AE or medically attended side effects. Factors associated with lack of seroconversion in KT recipients in univariate analysis were darbepoetin need for anemia management, a KT performed during the previous 6 months, high serum creatinine or low estimated glomerular filtration rate (eGFR). At multivariate analysis, only KT <6 months showed marginal association with a negative antibody response to vaccination according to the manufacturer cut-off point (p = .05) ( Table 3) .

Establishing good response in the 25th percentile of antibody production, risk factors for not reaching this cut-off in the univariate analysis were darbepoetin treatment, recent KT, and low eGFR.

In the multivariate analysis, a recent KT was the only significant factor associated with a lack of substantial production of antibodies (Table 4 ). Every month of posttransplant period increased by 1% the possibilities of achieving the 25th percentile of antibody production.

Including only seropositive patients, median (IQR) antibody levels were 412 (165-704) AU/ml ( Table 5 ). As expected, healthy con- 

Blood levels, ng/ml (mean ± SD) 6.12 ± 2.06 -

Bold values indicate statistically significant p values (p < .05).

Abbreviations: eGFR, estimated glomerular filtration rate; IQR, interquartile range; RRT, renal replacement therapy; SD, standard deviation. Regarding the type of vaccine, individuals of the healthy control group immunized with Moderna elicited higher antibody levels than with Pfizer. Conversely, no significant differences were observed in HD controls (Table 5) . (Table 5) .

Factors associated with IGRA response are described in Table 6 .

Roughly two thirds (65.9%) of IGRA(+) individuals were also positive for anti-S IgG, while only 11.2% were negative for assays. We performed subgroup analyses comparing patients and controls. Data are shown in Figure 6 . Bold values indicate statistically significant p values (p < .05).

Abbreviations: CI, confidence interval; eGFR, estimated glomerular filtration rate; KT, kidney transplant; OD, odds ratio. a Impossible calculation, as patients without prednisone and a negative response were = 0.

b Different models, using either serum creatinine or eGFR.

recipients is less frequent and significantly less intense, especially in those transplanted within the previous year.

Almost all HD and PD controls generated antibodies against SARS-CoV-2 S protein, but antibody levels were significantly lower than in our healthy controls. Seroconversion rates in this study resemble those described in other ones. [31] [32] [33] [34] KT recipients had discouraging antibody levels after vaccination, indicating a poor response to mRNA vaccines. They are in a more However, studies involving patients on dialysis report similar rates even when using different kits. [31] [32] [33] [34] KT with worse kidney graft function were less likely to seroconvert in univariate analysis, as reported previously. 36, 37 However, multivariate analyses showed that the association was not significant. Older age has been associated with lower antibody levels in dialysis patients 31, 39 and this correlation was seen in our HD and KT patients, but again the multivariate adjustment diluted the effect.

A short time since transplantation became the only detectable risk factor for a negative response to vaccination, and a lower production of antibodies, as seen in transplant recipients. 24, 34 This finding suggests that the capacity to produce antibodies is impaired early after KT, probably related to the amount of immunosuppression administered, independently of recipient age. 22, 23 It is worth noting, however, that immunosuppressive drug class, dose and levels were not associated to a negative response.

T cell responses showed trends like humoral responses across all groups. Lower T cell responses were observed in the KT patients than in healthy and dialysis controls, especially in those who had received the kidney graft <1 year before vaccination. A previous smaller study reported that 57.8% of KT recipients elicited SARS-CoV-2 cell-mediated immunity in contrast to all their HD patients using an ELISPOT assay. 25 We employed a new and simple kit that assesses T cell immunity through IGRA, looking for a simpler tool for monitoring.

Using this assay, Stumpf et al. described similar positive rates in their KT and dialysis patients (30% and 78%). 34 In contrast to ELISPOT or intracellular cytokine staining, QuantiFERON ® SARS-CoV-2 allows the processing of a larger number of samples without requiring much effort. 40 Moreover, concordance between detection of IFNγ-expressing cells by intracellular cytokine staining and quantification of soluble IFNγ through ELISA was high in SARS-CoV-2 convalescent patients 41, 42 and vaccinated renal patients. 34 This format has been used in tuberculosis detection for over a decade. Several societies recommend their use, and the latest generation of QuantiFERON ® -TB has proved to increase sensitivity even in immunocompromised patients. 43 In addition, tuberculosis and cytomegalovirus QuantiFERON ® assays have shown a good correlation with ELISPOT in renal patients. 44 51 The incorporation of IGRA, as an easier way to assess cellular immunity, may be key for the complete follow-up of many patients at risk of COVID-19.

Vaccination is the best way to prevent SARS-CoV-2 infection.

Nevertheless, KT patients develop weaker responses than healthy individuals and dialysis patients, probably related to the immunosuppression associated with anti-rejection treatment. 52 Precautions still need to be taken in order to protect this vulnerable population, as COVID-19 cases in vaccinated KT recipients have already been described. 53, 54 Our findings have direct clinical implications. It is mandatory to find a suitable strategy to improve their immunological responses. In France, authorities have approved the administration of a third mRNA vaccine dose in immunocompromised individuals, which includes transplant recipients and patients on dialysis. 55 Following this recommendation, a study with 396 solid organ transplant recipients observed an increase of the seropositive rate from 41.4% to 67.9% 4 weeks after receiving a third BNT162b2 dose. 56 Another alternative, known as the cocoon strategy, would be prioritizing vaccination of patients' relatives to create a safe environment with a lower risk of SARS-CoV-2 infection. 26 This is of particular interest in recent transplant recipients, who unfrequently develop immune responses after vaccination. In this regard, it is of utmost importance to find out if the response to the vaccine obtained on the waiting list persists despite the intense initial immunosuppression at transplantation.

In summary, a small percentage of KT recipients developed humoral responses 28 days after vaccination with mRNA vaccines, F I G U R E 6 Safety assessment in vaccinated individuals using a standardized questionnaire | 13 AJT CRESPO Et al. and the median antibody level was considerably low in responders, with a shorter time since transplantation associated to impaired response. Regarding cell-mediated immunity, a similar trend was observed, in which patients with higher antibody levels were more likely to mount T cell responses. KT patients, especially within the first year after transplantation, must be followed to prevent possible infections as we cannot ensure that they are fully protected against SARS-CoV-2. In addition, the immunological memory endurance in renal patients must be evaluated so that other approaches can be carried out if the vaccine effects wane.

The authors thank Laura Claramunt, Rosa Causadias, and Anna Bach for their assistance with patients. The authors also thank the staff of Bold values indicate statistically significant p values (p < .05).

Abbreviations: CI, confidence interval; eGFR, estimated glomerular filtration rate; OD, odds ratio; RRT, renal replacement therapy. a Different multivariate models maintain the three different variables within the model, marginal significance with KT of more than a year. b Not significant either including serum creatinine or eGFR.

c Impossible to calculate, as patients negative without tacrolimus or cyclosporine were = 0.

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This study was performed with partial funding from grants FIS-FEDER PI19/00037 and PI20/00090. The manuscript was produced as part of the Master degree of AB-J.

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0001-6992-6379Antoni Barrilado-Jackson https://orcid.org/0000-0002-7376-6440María José Pérez-Sáez https://orcid.org/0000-0002-8601-2699Julio Pascual https://orcid.org/0000-0002-4735-7838