Th1 Cytokines Signature in 2 Cases of IgA Nephropathy Flare after mRNA-Based SARS-CoV-2 Vaccine: Exploring the Pathophysiology

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as one of the three most pathogenic human coronaviruses of the last century. Although it is less deadly than SARS-CoV or the Middle East respiratory syndrome coronaviruses, its transmissibility has led to an unprecedented worldwide health crisis with more than 200 million cases diagnosed to date [1].

Coronavirus disease-19 (COVID-19) is the denomination of the clinical syndrome associated with SARS-CoV-2 infection. It mainly manifests with mild to severe respiratory disease, but multiorgan involvement has been reported. The spike (S) glycoprotein is key to the fusion of the viral and host cell membranes after recognizing angiotensin-converting enzyme 2 (ACE2). The occurrence of acute kidney failure (AKI) among infected patients ranges between 1 and 42% of cases depending on the series and the definition of AKI [2]. Some authors suggest that SARS-CoV-2 can damage the different kidney compartments [3, 4], despite tubular cells being the most frequently affected. This may be due to their high expression of ACE2 [5].

Among the glomerular diseases reported after SARS-CoV-2 infection, collapsing glomerulopathy and IgA nephropathy (IgAN) flares are among the most frequent [6]. It has been hypothesized that hyperproduction of interferon gamma (IFNγ) due to viral infection leads to APOL1 gene risk variant overexpression, thus resulting in podocyte dysregulation and collapsing glomerulopathy. In a case series of 240 patients with SARS-CoV-2-associated AKI reported by May et al. [6], 2.9% of biopsied cases were diagnosed with IgAN. Zhang et al. [7] suggest that IgAN flares may be related to an aberrant and long-lasting IgA response to the infection, with high levels of specific anti-SARS-CoV-2 IgA antibody levels in serum.

Administration of mRNA vaccines targeted against the S glycoprotein, mRNA-1273 and BNT162, have been associated with the occurrence of IgAN flares (summarized in Table 1). Despite IgA production being majorly triggered by mucosal infections, mRNA vaccines also elicit serum S-specific IgA response that may be related to these IgAN flares. In preclinical studies, mRNA vaccines trigger CD4+ lymphocyte production of IFNγ, tumor necrosis alpha (TNFα), and interleukin-2 (IL-2) [8]. Sahin et al. [9] reported a predominance of the T-helper (Th) 1 over the Th2 response in patients immunized with BNT162b1 mRNA vaccine based on the cellular higher production of IFNγ and IL-2 but not interleukin-4 or interleukin-5. Some authors reported the polarization towards a Th1 phenotype in IgAN [10], and Han et al. [11] demonstrated higher IFNγ serum levels in IgAN compared to healthy controls. Thus, vaccination with mRNA-based vaccines and IgAN flares may share humoral and cellular pathways [12].

Herein, we report 2 cases of IgAN flare that occurred after vaccination in patients previously diagnosed with the disease. We compare the clinical features of these vaccination-related flares to their first episode of IgAN. Thereafter, we analyze a Th1-related cytokine panel in serum and urine of these cases compared to a cohort of nonvaccine-related IgAN cases and healthy controls in order to deepen the knowledge of the pathophysiology of these vaccine-related flares.

A 36-year-old female was diagnosed with IgAN in January 2020. At the moment of the diagnostic biopsy, her creatinine was 115 µmol/L, her 24-h albuminuria was 1.4 g/day, and she presented microhematuria. Treatment with prednisone and mycophenolate mofetil was prescribed. In May 202,1 her creatinine and 24-h albuminuria had improved to 80 µmol/L and 0.7 g/day, respectively, but microhematuria persisted. In July 2021, she was admitted to the emergency room claiming gross hematuria, fever, and malaise few hours after the administration of the second dose of mRNA-1273 SARS-CoV-2 vaccine. Her serum creatinine had risen from 80 µmol/L to 159 µmol/L. Her urinalysis showed an increase in her pre-existing persistent hematuria, and her proteinuria rose from 0.7 g/day to 1.5 g/day at admission. Under the clinical assumption of IgAN flare, a new renal biopsy was performed. Light microscopy showed diffuse moderate mesangial hypercellularity and extracapillary proliferation. A mild interstitial inflammatory infiltrate was present. Immunofluorescence staining was predominantly IgA positive with a mesangial pattern. Those findings confirmed IgAN flare. At the moment of the admission, she was being treated with mycophenolate mofetil 250 mg twice a day and 2.5 mg of prednisone/day. After the kidney biopsy, prednisone dose was increased to 0.5 mg/kg body weight. Two months later, kidney function improved to creatinine 96 µmol/L and proteinuria decreased under 0.5 g/day. Currently, she is being treated with mycophenolate mofetil 250 mg twice a day and 5 mg of prednisone per day.

A 50-years-old male had been diagnosed with IgAN in 2001 by kidney biopsy. He did not receive immunosuppressive treatment. He was infected with SARS-CoV-2 in 2020, presenting mild symptoms. In March 2021, his creatinine was 88 µmol/L and his albuminuria was 0.7 g/day. He referred gross hematuria 72 h after the first dose of BNT162 SARS-CoV-2 vaccine on a routinary outpatients visit in June 2021. At that moment, his renal function showed a mild deterioration to serum creatinine 107 µmol/L, and his albuminuria severely increased to 3.3 g/day without associated hypoalbuminemia. His urinalysis showed an increase in his pre-existing persistent hematuria. A new kidney biopsy was performed. Light microscopy showed mesangial hypercellularity affecting more than 50% of the glomeruli, associated with mild interstitial infiltrate. Immunofluorescence staining demonstrated IgA deposition. Serum creatinine and proteinuria spontaneously recovered to their baseline values.

We therefore compared the clinical and analytical parameters of both patients at the moment of the diagnosis and at the moment of the postvaccination flare. In both cases, the clinical presentation was similar, without extrarenal organ involvement. Interestingly, during the flare, peak serum creatinine, urinary albumin to creatinine ratio, and urinary erythrocyte count were higher compared with the values of these variables at diagnostic in the 2 cases reported. Tables 2 and 3 show the analytical and clinical features of the diagnostic and the post-vaccine IgA flares of these patients.

Regarding the kidney biopsy, the histological lesions found at the biopsy performed during the post-vaccine flare were similar to those present at the diagnostic, as shown in Table 4. Figure 1 shows light microscopy images of the kidney biopsies performed in the post-vaccine IgAN flare cases.

We obtained serum and urine samples from the 2 patients who presented with an IgAN flare temporarily associated with mRNA SARS-CoV-2 vaccine. As diseased controls, we included n = 13 patients diagnosed with IgAN who had available serum and urine samples at the moment of the diagnostic stored in our biobank. Exclusion criteria were as follows: documented urinary tract infection, sepsis, or evidence of other superimposed glomerular or autoimmune disease. We also recruited n = 6 healthy controls. Healthy controls were volunteers from our institution without any known medical condition.

Serum and urine samples were obtained at the moment of the diagnostic and prior to the initiation of the treatment, centrifuged at 2,000 rpm 10 min and stored at −80°C until cytokine assessment. Based on the literature review, we selected three cytokines associated to the Th1 response: IFNγ, IL-2, and TNFα. We determined the serum and urinary concentration of these cytokines using a customized Multiplex Immunoassay commercial kit (Invitrogen ProcartaPlex; Thermofisher Scientific, Waltham, MA, USA). We used a Luminex MAGPIX® reader to retrieve the results.

Low levels of IL-2 were detected in serum and urine from patients diagnosed with IgAN, which were similar to the levels noticed in healthy controls. Serum and urine IFNγ and TNFα were undetectable both in IgAN patients and healthy controls. Interestingly, patient 1 showed an increase in serum IL-2 and TNFα compared to the IgAN cohort and healthy controls. In contrast, patient 2 showed similar levels of cytokines compared to IgAN patients and healthy controls. Table 5 shows mean serum and urine concentration of the cytokines among groups.

The availability of effective vaccines has dramatically decreased the severity and mortality of COVID-19. Nonetheless, the massive vaccination of worldwide population has led to the notification of multiple adverse effects. It is necessary to identify mechanism of these supposed adverse effects in order to establish the individual risk profile of IgAN flare.

There is scarce data about the incidence of de novo IgAN associated with mRNA vaccination. According to the United Kingdom Medicines and Healthcare products Regulatory Agency’s report, 1.4 million first doses and approximately 1.2 million second doses of the mRNA-1273 vaccine, and 19.4 million and 24.0 million second doses of the BNT162 vaccine have been administered until September 24^th, 2021, in the UK. Among the individuals who received the mRNA-1273 vaccine, 2 IgAN have been notified. Regarding the ones receiving the BNT162 vaccine, there have been 2 IgAN cases notified, 18 nephrotic syndromes, 6 minimal change disease, and 2 rapidly progressive glomerulonephritis cases [13]. This registry does not include information regarding the previous diagnostic of glomerular disease in these cases.

Although being the most frequently reported renal adverse effect of mRNA-based SARS-CoV-2 vaccines in the medical literature, the overall incidence of flares among patients previously diagnosed with IgAN is low. Lim et al. [14] reviewed 145 patients diagnosed with IgAN in the past 5 years in active follow-up at their institution. Of those, none of the 61.5% of vaccinated patients was diagnosed with a flare and did not present de novo gross hematuria.

Regarding the severity of the flares, our 2 cases presented higher peak serum creatinine and albuminuria compared to their first episode. The findings of the kidney biopsy performed on patient 1 at the moment of the post-vaccine flare were similar to those in her first diagnostic biopsy, including active inflammatory lesions such as cellular crescents that required the intensification of the immunosuppressant treatment. In contrast, post-vaccine biopsy in patient 2 showed low inflammatory activity, which was consistent with the mild elevation of the creatinine and with the self-limiting clinical course of the flare in this patient. Importantly, at 3 months both patients recovered to their baseline creatinine. The clinical picture of the vast majority post-vaccine IgAN flares cases published (summarized in Table 1) consists of the occurrence of gross hematuria with scarce elevation of serum creatinine. Only in 3 of the cases, cellular crescents were present in the kidney biopsy. Nevertheless, kidney biopsy was performed only in 7 out of the 19 cases of IgAN flares reported.

IgAN is a mesangioproliferative glomerulonephritis characterized by the presence of immunocomplex deposits composed of a form of aberrantly glycosylated IgA1 and antibodies specific against these forms. Mesangial deposition of IgA1-contaning immune complexes activates the complement system and leads to glomerular damage. Aside from elevated aberrantly glycosylated IgA1 serum levels, genetic, dietetic, and environmental factors are involved in the pathogenesis of the disease [15]. IgA is the most profusely produced immunoglobulin isotype, and the main one involved in mucosal defense. Thus, IgA dominates the early neutralizing response to SARS-CoV-2 [16]. Wisnewski et al. [17] longitudinally monitorized the IgA response after mRNA SARS-CoV-2 vaccine in 4 healthcare workers without prior history of infection. The vaccine elicited a specific anti-S IgA response in addition to the IgG response with a similar kinetic but a faster decline after the first dose [17]. Some authors expressed their concern that a nonmucosal trigger such as mRNA vaccine could be able to develop an IgA response that could lead to an IgAN flare, but the results of the study from Wisnewski et al. [17] confirm that this link is possible. IgA serum levels were only over the normal range in the case of patient 2 (cutoff for the normal values in our laboratory is 4,400 mg/L), as seen in Table 2. Whether the elevation of IgA levels is in the setting of the IgAN flare or a response to the vaccine remains inconclusive. Nevertheless, galactose-deficient IgA1 and glycan-specific IgG and IgA autoantibodies are the antibodies involved in IgAN pathogenesis, and their serum levels have been proposed as disease biomarkers, aside from the total IgA serum levels [18].

After a review of the literature, we centered our cytokine studies on a set of Th1 cytokines: IL-2, IFNγ, and TNFα. IL-2 controls the differentiation and homeostasis of T cells [19]. Schena et al. [20] reported higher IL-2 production in IgAN patients compared to controls. Zhang et al. [21] also found a significant increase in IL-2 and IFNγ in serum from IgAN patients at the moment of the diagnostic kidney biopsy before treatment. IFNγ is critical for the Th1 response to infection: enhances antigen presentation, participates in macrophage activation, and increases lymphocyte recruitment. Some authors suggest the role of the hyperproduction of IFNγ in IgAN [22]. TNFα is mainly produced by macrophages, but in inflammatory situations can be produced by Th1 lymphocytes, and has a role in the orchestration of the inflammatory immune response. Circulating TNFα levels have been associated with the severity of the disease course of IgAN [23-25]. In contrast, Ruszkowski et al. [26] found higher proportions of circulating Th2, Th17, and Th22 with lower Th1 and Treg cell populations.

Thus, some authors found a predominance of the Th1 activity, meanwhile other authors described a preponderance of the Th2 subset in IgAN. We were not able to confirm the elevation of the Th1 cytokines in the patients with IgAN. The predominance of these subsets may vary depending on the activity of the disease, the course of the disease, and the received treatment. There is a myriad of factors that can influence the level of the evaluated cytokines. The bulk of the studies do not take into account the temporal changes in the Th1/Th2 cytokines, and the cohorts of patients studied are majorly heterogenous. One must also take into account the plasticity of the Th subsets, which can acquire different phenotypes and produce different cytokines depending on the signals present in the cell environment. Another source of variability may be the different methodology used in the evaluation of the levels of the cytokines, which include ELISA, CLIA and in our case, bead-based assays in the Luminex platform.

mRNA-based COVID-19 vaccine’s response is characterized by a Th1 and also a Th17 increased response [27]. Bergamaschi et al. [28] evaluated the serum concentration of 52 cytokines and chemokines in healthy individuals receiving the BNT162 vaccine and found a rise in IFNγ and TNFα after administration, and no differences in IL-2 levels. This finding is in line with the reports from Taborska et al. [29], who reported the presence of spike-specific IFN and TNF producing T cells after BNT162 vaccination.

In patient 1, we found serum IL-2 and TNFα slightly elevated in comparison to healthy controls and patients with IgAN flare not related to the vaccine. We were not able to detect IFNγ in the serum or urine in any cohort. Differences with the findings reported by Bergamaschi et al. [28] may be due to the different techniques used to evaluate the concentration of the analytes, together with the potential effects of the immunosuppressive therapy in the case of patient 1.

The existing evidence is insufficient to confirm a causal association between mRNA-based SARS-CoV-2 vaccines and IgAN. Studies based on international multicentric registries are necessary in order to establish the real incidence of IgAN flares among previously diagnosed patients, but also the real incidence of IgA de novo diagnosis in general population after vaccine administration. At this moment of the pandemic, we consider that the benefits of vaccination are greater than the risk of IgAN flares based on the number of notified cases and their severity. The administration of a third vaccine dose or the combination of the different available vaccines may be a potential source of IgAN flares in the future. Finally, there is a lack of mechanistic studies that could help in the understanding of the disease process. We here suggest that hyperactivation of the Th1 pathway may be involved, but larger studies with more refined methods for numerical and functional Th1 lymphocytes subpopulations are needed to ascertain this hypothesis.

We acknowledge HUB-ICO-IDIBELL Biobank, funded by Instituto de Salud Carlos III (PT17/0015/0024) and by Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de Catalunya (XBTC).We thank CERCA Programme/Generalitat de Catalunya for institutional support. We thank ISCIII RETICS RedinRen RD16/0009/0003. We also thank Silvia Barceló, head of Molecular Interactions IDIBELL, for her support in Luminex procedures.

The Clinical Research Ethics Committee of Bellvitge University Hospital approved the study protocol. Patients signed informed consent prior to inclusion. The patients included in the diseased controls cohort provided informed written consent to have data from their medical records used in research and stored samples for this purpose.

The authors have no conflicts of interest to declare.

This study has been funded by Instituto de Salud Carlos III through the Grant CM19/00107, CM21/00170 and JR21/00059 co-funded by the European Social Fund (ESF investing in your future). This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain) (Grants FIS-ISCIII PI20/00812 and PI17/00277, co-funded by the FEDER funds/European Regional Development Fund-a way to build Europe).

Laura Martinez Valenzuela and Juliana Draibe conceptualized the study. Laia Oliveras, Paula Antón, and Francisco Gómez reviewed the medical records. Laura Martinez Valenzuela performed cytokine determination and elaborated the manuscript. Montserrat Gomà and Eugenia Quiros evaluated and captured the light microscopy images. Xavier Fulladosa, Josep Maria Cruzado, Juan Torras, and Juliana Draibe critically supervised the study.

All data are available under request to the corresponding author.