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Anti-Factor B Antibodies and Acute Postinfectious GN in Children

Sophie Chauvet,1,2,3 Romain Berthaud,3,4 Magali Devriese,5 Morgane Mignotet,5 Paula Vieira Martins,5 Tania Robe-Rybkine,1 Maria A. Miteva,3,6 Aram Gyulkhandanyan,7 Amélie Ryckewaert,8 Ferielle Louillet,9 Elodie Merieau,10 Guillaume Mestrallet,11 Caroline Rousset-Rouvière,12 Eric Thervet,2,3 Julien Hogan,13 Tim Ulinski,14 Bruno O. Villoutreix,3,15 Lubka Roumenina,1 Olivia Boyer,3,4,16 and Véronique Frémeaux-Bacchi1,5,3

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT Background The pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that, unlike acute postinfectious GN, has a poor prognosis. Methods This retrospective study investigated mechanisms of complement activation in 34 children with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement protein autoantibodies, carried out related functional characterization, and compared results with those of 60 children from the National French Registry who had C3 glomerulopathy and persistent hypocomplementemia. Results All children with acute postinfectious GN had activation of the alternative pathway of the com- plement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3 convertase) were found in a significantly higher proportion of children with the disorder versus children with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase activity in vitro,confirming their pathogenic effect. We also identified crucial antibody binding sites on factor B, including one correlated to disease severity. Conclusions These findings elucidate the pathophysiologic mechanisms underlying acute postinfectious GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as C3 glomerulopathy, and to help determine therapy.

JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019080851

Acute postinfectious GN (APIGN) is the leading Received August 28, 2019. Accepted December 26, 2019. 1,2 cause of acute nephritis in children. Streptococcal Published online ahead of print. Publication date available at skin or throat infections are still the main triggers of www.jasn.org. the disease in children. After a latency period after Correspondence: Veronique Frémeaux-Bacchi, Department infection, the typical presentation is an acute ne- of Immunology, European Hospital Georges Pompidou, 20 rue phritic syndrome with low C3 levels sometimes Leblanc 75015 Paris, France. Email: veronique.fremeaux-bacchi@ associated to a nephrotic syndrome, and rarely to aphp.fr a rapidly progressive GN. The diagnosis is mainly Copyright © 2020 by the American Society of Nephrology

JASN 31: ccc–ccc,2020 ISSN : 1046-6673/3104-ccc 1 CLINICAL RESEARCH www.jasn.org based on clinical and biologic features and confirmed by nor- Significance Statement malization of C3 levels within a few days or weeks and by the resolution of nephritic syndrome either with supportive care Acute postinfectious GN, the leading cause of acute nephritis in only, or with a short corticosteroid therapy.3 Kidney biopsy is children, associates with transient complement activation of un- notrequiredinmostchildren.3 It is only indicated in case of determined mechanism. Its clinical features overlap considerably i.e. with those of C3 glomerulopathy, a severe chronic condition. In this atypical presentation or course ( , persistent hypocomple- retrospective study, the authors demonstrated that in more than mentemia beyond 3 months) to rule out differential diagnosis 90% of children with acute postinfectious GN, complement over- or to assess prognosis in severe forms. Using light micros- activation results from activation of the alternative pathway of the copy, APIGN is characterized by glomerular cell proliferation complement system, driven by transient presence of autoanti- bodies targeting factor B, a component of the alternative C3 con- with endocapillary hypercellularity due to varying degrees of fi fl vertase. They also identi ed crucial antibody binding sites on factor neutrophil recruitment. Characterization by immuno uo- B, including one correlated to disease severity at onset. The pres- rescence microscopy involves bright glomerular C3 staining ence of anti-factor B antibodies was highly specific to acute post- with typical subepithelial deposits associated or not with infectious GN, suggesting that screening for these antibodies might Ig staining. Mechanisms underlying the glomerular lesions help clinicians distinguish the disorder during its acute phase from remain unclear. It was first suggested that the formation C3 glomerulopathy. of immune complexes, containing specific antigens (i.e., streptococcal antigens), and their deposition within the glo- Methods). For comparison, we used clinical and biologic merular tuft drive complement activation and immune cell data of 60 children from the National French Registry with infiltration.4 Later, it was proposed that the alternative pathway biopsy-proven C3G and persistent hypocomplementemia of the complement system has a major contribution in APIGN defined by low C3 levels for at least 1 year. In this group, as assessed by decreased plasma C3 and normal plasma C4 only plasma samples collected at the acute phase, available in levels in a large majority of patients and the demonstration 28 of 60 hypocomplementemic C3G (H-C3G) patients, were that streptococcal antigens were able to directly activate the used for antibody screening (Supplemental Figure 1). His- complement alternative pathway.5 The main differential diag- tologic criteria used for diagnosis of APIGN and C3G are nosis in a child with postinfectious acute nephritic syndrome provided in the Supplemental Methods. The local ethical and low C3 levels is C3 glomerulopathy (C3G), a glomerul- committee approved the study and written informed consent opathy of poor prognosis that warrants prompt specific was obtained from all patients’ legal guardians for genetic treatment.6,7 C3Gismediatedbyacquiredorinheriteddys- analysis. regulation of the complement alternative pathway, resulting in prolonged low plasma C3 levels in half of the patients Assays for Complement Component and Complement and predominant C3 deposition in glomeruli.8,9 Consider- Gene Analysis able overlapping in the clinical, biologic, and histopatho- C3, C4, and soluble C5b-9 (sC5b-9) were measured in patients’ logic features of APIGN with C3G hampers appropriate EDTA plasma samples. Screening for anti-factor H, anti-C3b, management.7 anti-C3bBb, anti-FB autoantibodies in patients’ plasma This study was designed to investigate the mechanism of and study of antibody isotype specificity were performed complement pathway activation in a cohort of patients with by ELISA (Supplemental Methods).10–12 Genetic variation APIGN. We discovered pathogenic anti-factor B (anti-FB) screening of complement factor H (CFH), complement factor I, autoantibodies that drive complement overactivation in C3, complement FB (CFB), and complement factor H related APIGN in contrast to C3G. The screening of anti-FB anti- 5 (CFHR5) genes and complex rearrangement between bodies may become a useful tool for guiding diagnosis and CFH/CFHRs were performed as previously described.12 Results therapeutic strategies in patients with nephritic syndrome of genetic analysis were compared with that of 45 of 60 patients and low C3 levels. with H-C3G with available DNA, 80 French volunteers who were healthy,13 and European individuals from the 1000 Genomes Project.14 METHODS Epitope Mapping and Functional Characterization of Study Population Anti-FB Antibodies We conducted a multicentric, retrospective study on 34 chil- By ELISA, we tested the ability of anti-FB autoantibodies to dren diagnosed with APIGN between April 2008 and August bind to 32 linear peptides of FB or 15 recombinant FB proteins 2017. The children’s plasma was sent to the French Reference carrying selected amino acid changes generated by site- Laboratory in the Georges Pompidou European Hospital directed mutagenesis as previously described (Supplemental (Paris) for complement analyses. Patients were eligible if Methods).15 Peptides and amino acid changes were mapped they were ,18 years of age, with clinical and/or biopsy- on the structure of FB, C3bB, and C3bBb using the Pymol proven diagnosis of APIGN with low plasma C3 levels followed software and the atomic coordinates of FB,16 C3bBD,17 and by C3 normalization within 90 days (Figure 1, Supplemental C3bBb18 (Supplemental Methods).

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To determine the functional consequences of anti-FB au- Suspected APIGN toantibodies on complement activation, total IgG purified N=62 from EDTA plasma of patients with APIGN were tested for their capacity to enhance fluid-phase and cell-surface alternative pathway convertase activity. In fluid phase, IgG Incomplete clinical data from patients with APIGN were incubated in normal N=5 human serum as a source of complement in the presence of EGTA-magnesium to block the classic/lectin pathways. APIGN diagnosis ruled out The release of cleavage fragments C3a, Bb, C5a, and sC5b-9 was N=10 then measured by ELISA (Supplemental Methods). We studied the surface activation of the complement alternative pathway by ELISA and hemolytic assays. We tested both the ability of Clinically confirmed APIGN anti-FB autoantibodies to enhance formation of C3bB and N=47 C3bBb complexes on plates, and we tested the ability of anti- FB antibodies to enhance formation of C3 convertase or to Persistent low C3 level after stabilize a preformed C3 convertase on sensitized sheep 90 days erythrocytes. Lysis of erythrocytes measured by spectrome- N=6 try (OD 414 nm) directly reflectstheconvertaseactivity (Supplemental Methods). No available control sample before 90 days of follow up N=7 Statistical Analyses Data are expressed as number and percentage for categoric variables and as median and interquartile range for continu- APIGN with transient low C3 N=34 ous variables. Differences among the APIGN and C3G groups were evaluated using the Mann–Whitney and Kruskal–Wallis tests, as appropriate, for comparison of continuous variables. Chi-squared or Fisher exact tests were used for comparison of Biopsy-proven APIGN categoric variables. The correlation coefficient between two N=20/34 normally or non-normally distributed variables was calculated using the Pearson method and the Spearman correlation co- Figure 1. Thirty-four children with APIGN were included in the study. Among the 62 patients with suspected APIGN, five pa- efficient, respectively. Statistical analyses were generated using tients had incomplete data for making a final diagnosis and in ten Prism 5 GraphPad software. P values ,0.05 were considered fi patients APIGN diagnosis was ruled out. Six patients had anam- signi cant. nestic, clinical, and biologic data that supported the diagnosis of APIGN but plasma C3 levels remained low for .90 days. In seven other cases with APIGN, normalization of C3 levels was not as- RESULTS sessed within 90 days after the diagnosis.

Clinical Features of Patients at the Acute Phase and during Follow-Up polymorphonuclear cell infiltration and subepithelial deposits A total of 34 children with APIGN were included in the study by light microscopy or C3 deposition by immunofluorescence (Figure 1). Main clinical data at diagnosis are detailed in were also identified in patients with H-C3G. Chronic lesions Table 1. The median (interquartile range) age at diagnosis (i.e., sclerotic glomeruli, interstitial fibrosis, or tubular atro- was5.6years(4.7–7.6). All 34 children had nephritic syn- phy) were only found in patients with C3G (Supplemental drome. A total of 15 of 34 patients had criteria of severe Results, Supplemental Table 1). A total of 13 children (39%) disease with nephrotic syndrome and/or dialysis requirement. received corticosteroids. After a median follow-up of 316 days Of the 34 patients, 82% had a clear history of recent infection (154–496), recovery from nephritic syndrome was observed in and renal disease occurred after a median latency period of all 32 children with complete available data. Notably, none had 5(1–10) days. This result is consistent with data from a large chronic renal insufficiency. One child had persistent micro- pediatric study reporting that 16% of children had no recent scopic hematuria and proteinuria at a follow-up of 57 days, history of infection at the time of APIGN diagnosis.19 At another patient had normal urinary sediment while receiving the time of renal diagnosis, the infection site was available for enalapril after a follow-up of 864 days, and one had persistent 23 patients and Streptococcus was the likely responsible patho- hypertension 127 days after the onset. In all three patients, a gen in 20 of 23 (87%) patients. Kidney biopsy performed in biopsy had been performed and kidney histology was com- 20 of 34 patients (59%) was compatible with APIGN in all cases patible with the diagnosis of APIGN (Table 1, Supplemental (Supplemental Table 1). Endocapillary proliferation with Results).

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Table 1. Clinical data for treatment of children with APIGN at admission and than in the H-C3G group (22 of 27 [81%] last follow-up versus 31 of 55 [56%], P50.02) (Table 2). Number At the acute phase, anti-FB autoanti- Characteristic Value (n534) bodies were identified in 31 of 34 (91%) Median age, yr (IQR) [min–max] 34 5.6 (4.7–7.7) children with APIGN and in four of [3.2–13.2] 28 (14%) children with H-C3G (P50.001; Sex ratio, n, male/female 34 23/11 odds ratio, 62.00; 95% confidence interval Anamnesis [CI], 12.65 to 303.8; relative risk, 7.97) History of recent infection, n (%) 33 27 (82%) (Figure 2A, Table 2). Sensitivity and speci- Median time between infection and first renal 27 5 (1–10) ficity of anti-FB antibodies for APIGN symptom, d (IQR) diagnosis were 95% (95% CI, 85% to Type of infection, n (%) 23 99%) and 82% (95% CI, 66% to 93%) Tonsillitis 4 (17%) (Figure 2B). Frequency of anti-FB anti- Peritonsilar abscess 1 (4%) fi Retropharyngeal abscess 1 (4%) bodies remained signi cantly higher Acute otitis media 2 (9%) even when comparing subgroups of Pneumonia 1 (4%) biopsy-proven APIGN (19 of 20 [95%]) Cutaneous infections 0 (0%) and H-C3G with infectious trigger (0 of Other 14 (61%) 10 [0%], P,0.001) (Supplemental Likely responsible pathogens, n (%) 28 22 (79%) Figure 2A, Supplemental Table 2). Anti- Streptococcus 20 (71%) FB antibodies were not found in 15 pa- Mycoplasm pneumoniae 2 (7%) tients with IgA nephropathy and 26 with Other 4 (14%) lupus nephritis used as control groups fi No pathogen identi ed 28 6 (21.4%) (Supplemental Figure 2B). A total of No screening for infectious pathogen 34 6 (18%) 24 of 31 patients with APIGN were Clinical presentation at diagnosis, n (%) Macroscopic hematuria 34 24 (71%) tested for anti-FB IgG subclasses. In Microscopic hematuria 34 5 (15%) 18 of 24 (75%) samples, anti-FB antibodies Edema 32 14 (44%) were of the IgG1 isotype (Supplemental Hypertension 34 18 (53%) Results, Supplemental Table 3). Biology at diagnosis At disease onset, plasma C3 levels were Proteinuria, n (%) 34 34 (100%) inversely correlated with anti-FB autoanti- Nephrotic syndrome, n (%) 33 11 (33%) body titer, and plasma sC5b-9 levels were ARF, n (%) 34 27 (79%) positively correlated with them (P50.01, – – – Median serum creatinine, mmol/L (IQR) [min max] 32 70 (48 106) [31 609] R250.21 and P50.003, R250.41, respec- 2 – – – Median eGFR, ml/min per 1.73 m (IQR) [min max] 29 74 (43 96) [7 122] tively) (Figure 2, C and D). Correlation be- RRT, n (%) 33 5 (15%) tween C3 level and anti-FB titer tend to be Kidney biopsy, n (%) 34 20 (59%) fi Evolution, n (%) signi cant and correlation between sC5b-9 fi Treatment 33 13 (39%) level and antibody titer remained signi cant Corticosteroids 13 (39%) in patients with isolated anti-FB antibodies Nephritic syndrome recovery 32 32 (100%) (Supplemental Figure 3, A and B). In con- Persistent isolated microscopic hematuria 31 4 (13%) trast, C3 and sC5b-9 levels did not correlate Renal abnormalities at last follow-up 32 4 (12%) with anti-C3bBb antibody titer in patients Persistent proteinuria 32 2 (6%) with both anti-FB and anti-C3bBb anti- No proteinuria under ACEI 32 1 (3%) bodies (Supplemental Figure 3, C and D). Persistent hypertension 32 1 (3%) During the follow-up, the anti-FB an- Persistent renal failure 32 0 (0%) tibody levels became negative in 23 of 31 Death 34 0 (0%) or decreased in six of 31 patients with Median follow-up duration, d (IQR) [min–max] 34 316 (154–496) [90–1671] APIGN (Figure 3, A and B, Supplemental IQR, interquartile range; ACEI, angiotensin-converting enzyme inhibitor. Table 4). At the last anti-FB antibody screening, all patients except three had nor- mal C3 levels. None of the 23 tested patients Complement Activation Biomarkers and Identification with APIGN had any anti-FH autoantibodies, whereas ten of of Anti-FB Antibodies in Children with APIGN 48 (21%) children with H-C3G did have anti-FH autoantibodies At diagnosis, all patients had low C3 levels and normal C4 (P50.02). Ten of 31 (32%) children from the APIGN group had levels. The proportion of tested children with sC5b-9 above anti-C3bBb autoantibodies versus 24 of 41 (59%) in the H-C3G the normal range was significantly higher in the APIGN group group (P50.033) (Table 2).

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Table 2. Immunologic findings of patients with APIGN and amount of C3a, Ba, C5a, and sC5b9 released in normal human H-C3G at onset of disease serum by ELISA after incubation with IgG from patients or Measure APIGN (n534) H-C3G (n560) P value healthy donors. After incubation, Bb and C3a were signifi- ’ C3 level, mg/L (660–1250) 217 (138–310) 257 (138–449) 0.12 cantly increased by four of 13 tested patients IgG, and C5a Low C3 level (,660 mg/L) 100% 100% 1 and sC5b-9 levels were significantly increased by five of C4 level, mg/L (93–380) 247 (189–316) 176 (138–231) 0.003 13 tested patients’ IgG compared with IgG from healthy donors Low C4 level, mg/L 0 (0%) 3/60 (5%) 0.55 (above mean12 SD of healthy donors), suggesting that anti- sC5b-9 level (,460 ng/ml) 877 (659–1700) 634 (368–1565) 0.1 FB antibodies are able to induce overactivation of fluid-phase Elevated sC5b-9 22 of 27 (81%) 31 of 55 (56%) 0.02 C3 and C5 convertases (Figure 5, A–D). (.460 ng/ml) By ELISA assays, we found that C3b binding to FB and C3bBb Anti-FB Abs, n (%)a 31 of 34 (91%) 4 of 28 (14%)b ,0.001 fi a formation on plates were signi cantly enhanced in the presence Anti-C3bBb Abs, n (%) 10 of 31 (32%) 24 of 41 (59%) 0.033 of purified IgG from 15 of 28 (54%) and from four of 20 (20%) Anti-FH Abs, n (%)a 0 of 23 (0%) 10 of 48 (21%) 0.02 a c d patients with APIGN compared with IgG from healthy donors, Anti-C3b Abs, n (%) 4of34(12%) 3of41(9%) 0.7 respectively (Supplemental Figure 9, A and B). Results are expressed as median (interquartile range), unless otherwise specified. C3, C4 and sC5b-9 levels are reference values obtained from 100 On sheep erythrocytes, anti-FB antibodies enhance C3 french healthy donors. Abs, antibodies; FH, factor H. convertase activity, resulting in a significant increase of eryth- a Autoantibodies were screened by ELISA. rocyte lysis compared with lysis obtained with Ig from healthy bAnti-FB antibodies were screened at acute phase of H-C3G. cAnti-C3b antibodies were combined with anti-FB antibodies in four out of donors (P50.02) (Figure 5E, Supplemental Figure 10). To test four cases. the capacity of anti-FB antibodies to stabilize a preformed C3 d Anti-C3b antibodies were isolated in three out of three cases. convertase, sheep erythrocytes bearing C3bBb were incubated in EDTA, in the presence of total purified IgG from patients Epitope Mapping of Anti-FB Antibodies in APIGN with APIGN. We used as positive control Ig from patients with Reactivity against 32 linear peptides of FB (Supplemental H-C3G who were positive for anti-C3bBb antibodies and Ig Table5)inplasmafrom29of34patientswithAPIGNwas from healthy donors was used as negative control. The lysis tested by ELISA. Nine of these 32 peptides were recognized change between baseline and after 20 minutes of incubation, fl fi in more than four patient samples and were considered hot- re ecting the dissociation of C3 convertase, was signi cantly in- spot sequences (Figure 4A, Supplemental Figure 4). Peptide creased in patients with APIGN compared with patients with P5 624–637, located on the serine protease (SP) domain, was H-C3G ( 0.002). These results suggest that anti-FB antibodies more frequently recognized by IgG from children with non- are not able to stabilize a preformed C3 convertase (Figure 5F). severe disease onset (as defined by absence of nephrotic syn- drome and/or dialysis requirement) (five of 15 [33%] versus Complement Gene Analysis in Patients with APIGN zero of 13 [0%]; P50.04) (Supplemental Figures 5 and 6). In and H-C3G the same area of peptide 624–637, two peptides (591–606 Genetic analysis was performed in 23 of 34 patients with and 660–674) were only recognized by IgG from patients APIGN and 45 of 60 patients with H-C3G. Rare complement fi , with nonsevere renal presentation, suggesting that the bind- gene variants (de ned by a minor allele frequency 0.1%) fi ing of anti-FB antibodies in the vicinity of the catalytic triad, were detected in 22% ( ve of 23) of patients with APIGN, fi in the SP domain, could modulate the pathogenic effect of which was signi cantly higher than in healthy donors (6%, P5 anti-FB antibodies. 0.04) but similar to the control group of patients with To confirm the importance of these hotspots for anti-FB H-C3G (six of 45, 13%) (Supplemental Table 6). Among the fi fi antibody binding, we produced 15 recombinant FB mutants, ve rare variants identi ed in patients with APIGN, one is including nine recombinant FBs bearing amino acid changes located in the C-terminal portion of CFH which is involved on the identified hotspots. By ELISA, we showed that six of in the binding to surface-bound C3b and to negatively charged fi in these nine recombinant FBs were associated with significant cellular structures and is classi ed as probably damaging by silico CFH CFB modification in anti-FB antibody binding compared with that prediction scores. Two variants in and two in with wild-type recombinant FB (Figure 4B). Among the six (without published functional study or without any demon- fi recombinant FB mutants with mutations outside the hotspot strated functional effect) are classi ed as variants of undeter- fi 24,25 CFH regions, only one showed modified IgG binding (Supplemental mined signi cance. The three variants in the gene fi fi Figure 7). Positions of the seven amino acid changes of interest identi ed in the H-C3G cohort are classi ed as pathogenic 26,27 and the nine hotspot peptides are indicated on the molecular and/or previously published (Supplemental Table 7). structure of FB (Figure 4C) and FB within C3bB and C3bBb complexes (Supplemental Figure 8). DISCUSSION Functional Consequences of Anti-FB Autoantibodies To test the capacity of patients’ IgG to induce activation of the We demonstrated that the pathophysiology of APIGN in fluid-phase alternative pathway convertase, we measured the children implies an activation of the complement alternative

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A B <0.0001 <0.0001 0.61 100 2800 2200 1600 1400 75 1200 1000 50 800 600 Sensitivity 25 400

Anti-FB antibodies UA/ml 200 AUC 0.94 [95% CI 0.88-1] 0 0 APIGN H-C3G HD 0255075100 n=34 n=28 n=85 100 - Specificity

C D 2500 500

2000 400

300 1500

200 1000 C3 (mg/L)

100 sC5b-9 level (ng/ml) 500

0 0 0 1000 2000 3000 0 500 1000 1500 2000 Anti-FB Antibodies (UA) Anti-FB Antibodies (UA) N=34/34 children with APIGN N=27/34 children with APIGN

Figure 2. Anti-FB antibodies at acute phase of the disease are specific of APIGN and correlate with C3 and sC5b-9 levels. (A) Reactivity of autoantibodies against FB was tested by ELISA in plasma samples from 34 patients with APIGN, 28 patients with H-C3G (collected at acute phase of disease), and 84 healthy donors. Results of antibodies are expressed in arbitrary units (UA). The cutoff of positivity was determined with plasma samples from the 84 healthy donors (HD). (B) Receiving operater characteristic curve of the positivity of anti-FB autoantibodies in APIGN. The area under the receiving operater characteristic curve for the use of anti-FB antibodies titer to dis- criminate APIGN from H-C3G patients was 0.93 (95% CI, 0.88 to 0.99). The optimal cutoff point was 88.5, which had a sensitivity of 0.91 (95% CI, 0.76 to 0.98) and a specificity of 0.85 (95% CI, 0.71 to 0.94). (C and D) Correlation between titers of anti-FB antibodies and C3 levels and sC5b9 investigated in 34 and 27 tested patients, respectively. (C) Plasma C3 levels inversely correlate with titers of anti-FB antibodies (P50.01; 95% CI, 20.68 to 20.142; R520.46). (D) Soluble C5b9 levels in plasma correlate with anti-FB antibody titers (P50.001; 95% CI, 0.35 to 0.82; R50.64). AUC, area under the curve. pathway resulting from the presence of transient autoanti- C3 and sC5b-9 in patients’ plasma. In vitro, we demonstrated bodies targeting FB, a component of the alternative C3 con- that anti-FB IgG enhances C3 convertase formation. vertase. This is a major breakthrough in understanding the Pathogenic variants in CFB have been described in atypical mechanisms underlying this frequent disease. hemolytic uremic syndrome, another complement-mediated Of the patients in this study who were clinically diagnosed disorder.16,20–23 These variants, located on the FB vWdomain, with APIGN, 90% had IgG autoantibodies that reacted with result in C3 convertase overactivation. Interestingly, we FB. Upon activation of the alternative pathway, FB is cleaved identified hotspots for anti-FB autoantibody binding that by factor D, yielding the noncatalytic portion Ba and the active encompass the majority of the FB variants identified in atypical fragment Bb. The Bb fragment comprises two protein do- hemolytic uremic syndrome, suggesting that anti-FB anti- mains: a von Willebrand (vW) type A domain, which associates bodies binding on vW-domain hotspots could increase the to C3b to form the C3 convertase C3bBb, and an SP domain formation of the C3 convertase. We also identified hotspots that cleaves C3. In APIGN, we found that anti-FB autoanti- in the SP domain of FB that are in immediate proximity to bodies are transient, mainly of the IgG1 subclass and drive the catalytic triad, including one hotspot associated with activation of the complement alternative pathway. Autoanti- nonsevere disease onset (i.e.,withnonephroticsyndrome body titers correlated with the level of hypocomplementemia nor dialysis requirement). Binding of anti-FB antibodies on and their disappearance was associated with normalization of the SP domain could modulate the C3 convertase activity

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A B 3000 34 33 32 31 30 2500 29 28 27 26 25 24 2000 23 22 21 P7 20 19 18 1500 17 16 15 14 13

12 Anti-FB antibodies UA/ml 1000 11 10 9 P24 8 P28 7 6 500 5 P6 4 P32 3 P22 2 P29 P25 1 0 0 30 60 90 120 150 180 210 240 270 300 500 600 700 800 900 At acute phase last sample days after first renal symptoms

Figure 3. Anti-FB antibodies in APIGN patients are transient. (A) Kinetics of anti-FB antibody positivity, C3 levels, and renal symptoms in patient with APIGN according to renal symptoms. Bars in gray indicate the presence of renal symptoms (high BP and/or hematuria and/or proteinuria and/or renal failure). Samples positive for anti-FB antibodies and low C3 levels are indicated by a red asterisk. Samples positive for anti-FB antibodies with normal C3 levels are indicated by a blue asterisk. Samples negative for anti-FB antibodies with low C3 levels are indicated by a yellow asterisk. Samples negative for anti-FB antibodies and/or normal C3 levels are indicated by a green asterisk. In patients 2, 9, 11, 20, 21, and 27, measurements of C3 levels confirming normalization of C3 within 90 days after renal diagnosis were performed in an external biologic center and were not indicated on the figure. (B) Titer of anti-FB autoantibodies at acute phase and on the last available sample. In 31 of 34 patients with anti-FB antibodies at diagnosis, control of anti-FB reactivity was available in 29 patients. For two patients (P7 and P24), no sample was available during follow-up. In six of 29 patients, antibody titer remained positive during the follow-up in patient P28 (titers 622 UA at day 852), P6 (titer 364 UA at day 28), P22 (titer 189 UA at day 30), P29 (titer 123 UA at day 75), P32 (titer 260 at day 32), and P25 (titer 103 at day 31). Dotted line indicates the positivity threshold (.88 UA/ml). enhanced by anti-FB antibodies binding to the vW domain. majority of children with APIGN. We can hypothesize two Given the usually favorable outcome of APIGN, our findings different mechanisms: (1) because streptococcal surface is are unlikely to modify the classic management of APIGN. able to directly activate the alternative pathway,29 it may Indeed, a strategy based on removal of anti-FB antibodies by lead to the formation of FB neoepitopes; and (2) streptococcal plasma exchange seems unnecessary in most patients but proteins and FB may share common conformational and/or may be discussed in the rare cases requiring RRT. Our find- cryptic epitopes. In both cases, streptococcal infection may ings also improve our understanding of the mechanisms of promote the emergence of transient anti-FB autoantibodies, action of immunosuppressive therapies. Corticosteroids contributing to complement overactivation and deposition of could not only act as antiproliferative agents but could also complement C3 breakdown products in the glomeruli. In this decrease the production of anti-FB antibodies when the use hypothesis, the control of infection and clearance of strepto- of long-acting immunosuppressors seems less appropriate coccal antigens from glomeruli may result in anti-FB autoan- in the setting of APIGN. Moreover, one can assume that a tibody disappearance, leading to the decrease of complement transient inhibition of C5 may improve the outcome in se- activation followed by resolution of nephritic syndrome. Anti- vere cases of APIGN that require dialysis, although further FB antibodies are exceptional in C3G, identified in 3% of the confirmation is required.28 French cohort of adult patients with C3G11,30 and in 14% of The mechanism of anti-FB autoantibody emergence re- children with H-C3G in this study where they remain detect- mains unclear. Streptococcal infection was identified in a large able over years.30 Anti-FB antibodies have also been described

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A 10 * 8 * * * * 6 * * * * 4

patients (Nb) 2 0 251-265 261-277 268-283 290-307 300-314 310-326 331-347 342-356 366-380 376-391 386-397 407-421 417-431 429-443 439-451 456-468 471-485 498-511 509-523 520-534 532-546 556-569 575-589 584-597 591-606 603-619 624-637 634-654 660-674 680-686 693-705 719-739 vW domain SP domain

Catalytic triad H501 D551 S674 D251- MIDAS S253- T328 D364 S255

B 1.5

1.0 m+2SD

0.5 OD(450nm)

m-2SD 0.0

WT K312A N318A D321A D378A E379D P407A D506A E509A

D345A-D346A vW domain SP domain

D378A C E379D 376-391

D254G K298E

F261L 342-356 300-314 P407A 310-326

M433I 429-443

K704A

624-637 K701A

719-739 520-534

498-511 E509A K508R E541A D506A

Figure 4. Anti-Fb antibodies recognize hotspots on serine protease and von Willebrand type A domains of FB. (A) Reactivity of au- toantibodies against 16 linear peptides of vW type A domain and 16 linear peptides of SP domain of FB was tested by ELISA in plasma samples from 29 patients with APIGN. A total of 27 of 32 peptides were recognized by at least one patient sample positive for anti-FB antibodies. Five hotspot peptides of the vW type A domain and four hotspot peptides of the SP domain recognized by more than four patient samples (indicated by an asterisk) were identified. Location of the amino acids forming the catalytic triads and MIDAS site are indicated below the graph. (B) Reactivity against recombinant FB (wild-type and nine recombinant FBs bearing mutations on hotspot peptide sequences [K312A, N318A, D321A, D378E, E379D P407A, D506A, E509A, and the double mutant D345A-D346A]) in plasma samples from 13 patients with APIGN was tested by ELISA. Results are expressed in OD. Means62SD(m12SD, m-2SD) of binding to the wild type are indicated in dotted lines. Five mutations were associated with a significant decrease of anti-FB antibody binding to recombinant FB (D321A, D378E, E379D, P407A, and D506A), whereas one mutation was associated with a significant increase (E509A). (C) Localization of the hotspots on the FB structure. The five hotspot peptides of the vW domain are indicated by red ribbon, and the four hotspot peptides of SP domain are indicated in green. The hotspot amino acid changes are in dark blue. Amino acids of the MIDAS are indicated in magenta and amino acids of the catalytic triads are indicated in yellow. Cyan indicates amino acids referred to in the literature as being associated with hyperfunctional C3 convertase.20–23 MIDAS, magnesium ion–dependent, metal ion–dependent adhesion site.

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A B 600 100 80 400 60 40 200 Bb ( μ g/ml)

C3a (ng/ml) 20 0 0 HD-Ig APIGN-Ig anti-C3bBb HD-Ig APIGN-Ig anti-C3bBb Ab/C3G Ab/C3G n=7 n=13 n=8 n=7 n=13 n=8

C D 50 400 40 300 30 200 20

C5a (ng/ml) 100

sC5b9 ( μ g/ml) 10 0 0 HD-Ig APIGN-Ig anti-C3bBb HD-Ig APIGN-Ig anti-C3bBb Ab/C3G Ab/C3G n=7 n=13 n=8 n=7 n=13 n=8

E F 0.02 0.001 0.31 0.002 100 50

80 40

60 30

40 20 % of lysis 20 10

0 0

HD-Ig APIGN Δ Lysis between T0 and T20 (%) HD-Ig APIGN H-GC3 n=16 n=17 n=7 n=6 n=12

Figure 5. Anti-FB antibodies from children with APIGN enhance activity of fluid-phase and cell-surface C3 convertase but do not stabilize solid-phase C3 convertase. Activation of the fluid-phase complement alternative pathway in the presence of anti-FB anti- bodies. After incubation of normal human serum (EGTA-magnesium) in the presence of total purified IgG from 13 patients with APIGN, eight patients with C3G positive for anti-C3bBb antibodies, and seven healthy controls, C3a, Bb, C5a, and sC5b9 generation was quantified by ELISA. In the experimental condition, at baseline, median C3a, Bb, C5a, and sC5b9 levels in normal human serum are 6ng/ml,18mg/ml, 9 ng/ml, and 1.8 mg/ml, respectively. After 30 minutes of incubation, median levels obtained with IgG from healthy donors (HD-Ig) are 253.9, 40.6, 159 ng/ml, and 18.6 mg/ml, respectively. (A-B) C3a and Bb releases were significantly increased in patients with APIGN (four of 13 and four of 13, respectively) and in patients with C3G positive for anti-C3bBb antibodies (four of eight and four of eight patients, respectively) compared with IgG from healthy donors (upper mean12 SD). (C-D) C5a and sC5b9 were significantly increased in patients with APIGN but not in the presence of anti-C3bBb antibodies from patients with C3G. (E-F) Activation of the solid-phase complement alternative pathway in the presence of anti-FB antibodies. The capacity of total purified IgG from patients and controls (healthy donors and patients with C3G positive for anti-C3bBb antibodies) to enhance C3/C5 convertase for- mation and stabilization activity was tested using hemolytic assays. Results are expressed as the percentage of the maximum lysis of erythrocytes (osmotic lysis with water). (E) To evaluate the capacity of anti-FB antibodies to enhance cell-surface C3 convertase formation, C3 convertase was formed on sensitized sheep erythrocytes bearing C3b in the presence of Ig from patients with APIGN or controls. In the presence of Ig from healthy donors, the median of the maximum lysis is 20%. Lysis was significantly increased in presence of Ig from patients with APIGN. (F) To evaluate the capacity of anti-FB antibodies to stabilize a preformed surface-bound C3 convertase, total purified IgG from patients with APIGN, diluted in gelatin veronal buffer-EDTA, were incubated on sheep erythrocyte bearing preformed C3bBb. IgG from patients with H-C3G positive for anti-C3bBb antibodies were used as positive control and Ig from healthy donors as negative controls. At baseline (T0), the median percentage of maximum lysis is 43% (40%–53%), 79% (43%–100%), and 75% (42%–100%) in the presence of Ig from those with HD-Ig, APIGN, or H-C3G, respectively. At 20 minutes (T20), median percentage of maximum lysis is 13% (15%–19%), 48% (15%–65%) and 65% (31%–100%) in the presence of Ig from those with HD-Ig, APIGN, or H-C3G, respectively. The change in lysis (D) between T0 and T20, reflecting dissociation of C3 convertase, is significantly increased in patients with APIGN compared with those with H-C3G. Results are expressed as DLysis, representing the difference of lysis between baseline and after 20 minutes of incubation. An increase of DLysis suggests a decay of the enzyme. Ranges are the extrems. HD-Ig, Ig from healthy donors.

JASN 31: ccc–ccc,2020 Anti-Factor B and Postinfectious GN 9 CLINICAL RESEARCH www.jasn.org in rare adult cases with immune complex GN in the setting of syndrome with low C3 levels to differentiate children with concomitant infection of nonstreptococcal origin, suggesting anti-FB antibodies, who would likely have a good renal the potential role of other microorganisms in the emergence prognosis without major support, from children without of such autoantibodies.11 any detectable anti-FB antibody, who would be at risk for The contribution of rare variants in CFH and CFB genes H-C3G requiring long-term immunosuppression and may significantly enriched in children with APIGN needs to be have worse renal prognosis. determined. It may signify that a specific genetic background predisposes children to a complement-mediated disease or to the emergence of anti-FB antibodies after a streptococcal infection. ACKNOWLEDGMENTS At the acute phase of the disease, anti-FB autoantibodies may be of crucial help to distinguish APIGN from H-C3G, in We thank clinicians who referred patients: Prof. Bernard Boudaillez particular when H-C3G is triggered by infection. Both diseases (Department of Pediatrics, University Hospital Center of Amiens, may share similar clinical features— i.e., present as acute Amiens, France), Dr. Pascal Saunier (Department of Pediatrics, nephriticsyndromewithlowC3levels—and cannot be dif- Fontainebleau Hospital Center, Fontainebleau, France), Dr. Gwenaelle ferentiated by renal biopsy alone.31 The distinction of both, Roussey-Kesler (Department of Pediatric Nephrology, University based on a bundle of arguments, remains crucial because the Hospital Center of Nantes, Nantes, France), Dr. Sophie Taque (De- two diseases have completely different outcomes. Complete partment of Pediatrics, University Hospital Center of Rennes, Rennes, remission in APIGN is the rule, whereas H-C3G has poor renal France), and Dr. Robert Novo (Department of Pediatric Nephrology, – outcome despite intensified immunosuppression.32 34 In vitro, University Hospital Center–Jeanne de Flandre University, Lille, we demonstrated that anti-FB autoantibodies are not able to France). We thank Prof. Chantal Loirat for her expert advice. stabilize a C3 convertase formed on red blood cells. Therefore, anti-FB antibodies cannot play the role of C3 nephritic factor that per definition are antibodies that recognize a neoepitope DISCLOSURES of C3bBb without binding FB or C3b alone and result in 35 stabilization of C3 convertase. C3 nephritic factors are Dr. Frémeaux-Bacchi received fees from Alexion Pharmaceuticals, Apellis, foundinmostpatientswithC3Gandarecorrelatedwith and Roche for invited lectures and/or board membership and is the recipient complement biomarkers.12 In APIGN, anti-C3bBb anti- of a research grant from Alexion Pharmaceuticals. All remaining authors have bodies were found in less than a third of patients, were mainly nothing to disclose. of the IgG3 subclass, and did not correlate with C3 and sC5b- 9 levels. Taken together, these findings suggest they are not FUNDING relevant in the APIGN process and, unlike anti-FB antibodies, cannot help clinicians to distinguish between APIGN and H-C3G. This work was supported by a European Union Seventh Framework Pro- 1 gramme grant 2012-305608 (EURenOmics), Kidneeds research grant 2015, The limitations of this study are the following: ( )pa- Agence Nationale de la Recherche research grant ANR-16-CE18-0015-01, tients with APIGN were recruited from hospitals and this CompC3, and the Fondation pour la Recherche Médicale (FDM may have led to recruitment bias toward more severe cases of 20130727355). APIGN36;(2) its retrospective nature has hampered exten- sive/standardized complement exploration—we are now de- signing a prospective study that will include incident cases of SUPPLEMENTAL MATERIAL both APIGN and H-C3G to confirm the specificity of the anti-FB antibody assay; and (3)theroleofStreptococcus This article contains the following supplemental material online at or other microorganisms on the emergence of anti-FB http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019080851/-/ antibodies requires further investigation. First, compari- DCSupplemental. son of the amino acid sequence of identified peptides with Supplemental Methods. Streptococcus sequences did not identify any sequence ho- Supplemental Results. mology; second, APIGN occurs after a latent period, render- Supplemental Table 1. Histopathological data of APIGN patients ing the identification of a specific bacterial strain at onset of and hypocomplementemic C3 glomerulopathy (with or without in- renal symptoms difficult. fectious trigger). In conclusion, our results provide new insights into the Supplemental Table 2. Immunological findings of patients pathophysiologic mechanisms underlying complement acti- with acute post-infectious GN and hypocomplementemic-C3 vation in APIGN in children. We identified anti-FB antibodies glomerulopathy. as new players in the disease contributing to overactivation of Supplemental Table 3. Isotype specificity of anti-Factor B and anti- the complement alternative pathway. Therefore, anti-FB anti- C3bBb antibodies by ELISA in patients with acute post-infectious bodies may be of great help at the acute phase of nephritic GN and related C3 and sC5b-9 levels.

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Supplemental Table 4. Complement assays at disease onset and at 7. Al-Ghaithi B, Chanchlani R, Riedl M, Thorner P, Licht C: C3 Glomerul- last anti-FB antibody screening. opathy and post-infectious glomerulonephritis define a disease spec- – Supplemental Table 5. List of the 32 linear peptides of FB. trum. Pediatr Nephrol 31: 2079 2086, 2016 8. Pickering MC, D’Agati VD, Nester CM, Smith RJ, Haas M, Appel GB, Supplemental Table 6. Number and proportions (%) of children et al.: C3 glomerulopathy: Consensus report. Kidney Int 84: 1079–1089, with APIGN, of French controls and of controls from the 1000 Genomes 2013 project database, who carried at least one rare variant (MAF ,0.1) in 9. Hou J, Markowitz GS, Bomback AS, Appel GB, Herlitz LC, Barry Stokes one of the four tested complement genes (C3, CFB, CFH, CFI). M, et al.: Toward a working definition of C3 glomerulopathy by im- fl – Supplemental Table 7. Rare variants identified in 5 of 23 (21.7%) muno uorescence. Kidney Int 85: 450 456, 2014 10. Blanc C, Togarsimalemath SK, Chauvet S, Le Quintrec M, Moulin B, patients with acute post-infectious GN and in 6 of 45 (13%) patients Buchler M, et al.: Anti-factor H autoantibodies in C3 glomerulopathies with hypocomplemntemic-C3 glomerulopathy among C3, CFB, and in atypical hemolytic uremic syndrome: One target, two diseases. CFH, CFI genes. JImmunol194: 5129–5138, 2015 Supplemental Figure 1. Population study of 60 patients with 11. Marinozzi MC, Roumenina LT, Chauvet S, Hertig A, Bertrand D, Olagne hypocomplementemic-C3 glomerulopathy. J, et al.: Anti-factor B and anti-C3b autoantibodies in C3 glomerulop- Supplemental Figure 2. Titers of anti-Factor B antibodies in athy and Ig-associated membranoproliferative GN. J Am Soc Nephrol 28: 1603–1613, 2017 patients with acute post infectious GN and hypocomplementemic 12. Marinozzi MC, Chauvet S, Le Quintrec M, Mignotet M, Petitprez F, C3 glomerulopathy. 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Position on C3bBb molecular structure of genetic variation. Nature 526: 68–74, 2015 peptide 624-637 associated with non severe renal presentation. 15. Marinozzi MC, Vergoz L, Rybkine T, Ngo S, Bettoni S, Pashov A, et al.: Complement factor B mutations in atypical hemolytic uremic syndrome- Supplemental Figure 7. Anti-Factor B antibodies binding to re- disease-relevant or benign? J Am Soc Nephrol 25: 2053–2065, 2014 combinant Factor B. 16. Milder FJ, Gomes L, Schouten A, Janssen BJ, Huizinga EG, Romijn RA, Supplemental Figure 8. Molecular structure of C3bB and C3bBb et al.: Factor B structure provides insights into activation of the central with peptides and amino-acid changes of interest for anti-Factor B protease of the complement system. Nat Struct Mol Biol 14: 224–228, antibody binding. 2007 17. Forneris F, Ricklin D, Wu J, Tzekou A, Wallace RS, Lambris JD, et al.: Supplemental Figure 9. 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AFFILIATIONS

1Inflammation, Complement and Cancer Team, Cordeliers Research Center, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) S1138, Paris, France; Departments of 2Nephrology and 5Immunology, Assistance Publique- Hôpitaux de Paris (AP-HP), Georges Pompidou European Hospital, Paris, France; 3Paris University, Paris, France; 4Department of Pediatric Nephrology, AP-HP, Necker Hospital – Sick Children, Paris, France; 6INSERM U1268 Medicinal Chemistry and Translational Research, Cibles Thérapeutiques et Conception du Médicament UMR8038 Centre National de la Recherche Scientifique (CNRS), Paris, France; 7University of Paris Diderot, Sorbonne Paris Cité, Molécules Thérapeutiques In Silico, INSERM UMR S973, Paris, France; 8Department of Nephrology, Rennes Hospital, Rennes, France; 9Department of Pediatric Nephrology, Rouen Hospital, Rouen, France; 10Department of Pediatric Nephrology, Tours Hospital, Tours, France; 11Department of Pediatry, Villefranche sur Soane Hospital, Villefranche sur Soane, France; 12Department of Pediatric Nephrology, Timone Hospital, Marseille, France; 13Department of Pediatric Nephrology, AP-HP, Robert Debré Hospital, Paris, France; 14Department of Pediatric Nephrology, AP-HP, Trousseau Hospital, Paris, France; 15Laboratory of cristallography and biological Nuclear magnetic resonance, UMR 8015 CNRS, Paris, France; and 16Reference Center for Hereditary Kidney and Childhood Diseases (MARHEA), Imagine Institute, Paris, France

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