Mapping autoantigen epitopes: molecular insights into autoantibody-associated disorders of the nervous system

Anti-NMDAR encephalitis is the most commonly recognized autoimmune encephalitis (reviewed in [1]). It is associated with anti-NMDAR antibodies, has been reported in females and males, and has been observed in all age groups, from young children to the elderly [13]. Due to the severity of anti-NMDAR encephalitis, as well as its rising prevalence, several studies have strived to uncover the target epitope of anti-NMDAR antibodies and also to explore the mechanisms of anti-NMDAR antibody pathogenicity. NMDARs are composed of two GluN1 (also called NR1) and two GluN2/3 (also called NR2 and NR3) subunits that form a central ion channel (Fig. 1). Importance of the conservation of conformational antigen has been shown by the lack of immunoreactivity of anti-NMDAR antibody on immunoblots [10]. In 2012, the epitope of anti-NMDAR antibodies was mapped using a series of NMDAR mutants expressed in human embryonic kidney (HEK)293 cells that were incubated with CSF from patients with and without anti-NMDAR encephalitis [35]. The first step was to distinguish which subunit was involved in antibody binding. Anti-NMDAR antibody-positive CSF did not recognize the GluN2 variant of NMDAR by immunocytochemistry, but they did recognize GluN1 subunit and a splice variant of GluN1 [35]. GluN subunits are made up of two extracellular domains; the amino (N)-terminal domain (ATD) and the ligand-binding domain (LBD) consisting of segment 1 (S1) and segment 2 (S2), followed by three transmembrane-spanning domains (TM1, 3, 4), a transmembrane loop (TM2), and an intracellular carboxyl (C)-terminal domain (Fig. 1a). To determine whether it was the extracellular ATD or S1/S2 domains that were important for antibody binding, two different constructs were created. The first mutant included only the ATD domain and missed the S1/S2 domains, which resulted in intense patient antibody staining. The second construct excluded ATD and contained S1/S2 only, which abolished antibody binding [35]. These results suggested that the ATD of GluN1 is necessary for antibody recognition. Additional deletion constructs were created, including single-point mutations within the ATD, to define specific antibody binding. Among these, the most important mutant was N368D/G369I double mutant, which prevented patient antibodies from binding. Therefore, residues N368/G369 were found to be important targets of anti-NMDAR antibodies. As expected, control patients without anti-NMDAR antibodies did not stain for NMDAR mutants. Interestingly, the ATD of NMDAR includes seven putative N-linked glycosylation sites, one of which found at position N368. Analysis of N368Q mutant with patient samples showed reduced immunoreactivity but did not abolish antibody binding to the same extent as the double mutation of N368/G369 [35]. This result is interesting because it suggests that N-glycosylation sites are involved in creating the ideal epitope conformation. Pathogenic studies, discussed below, have also been carried out using this mutant.

Anti-AMPAR antibodies are detected in patients with anti-AMPAR encephalitis in a small number of described cases [11, 36]. AMPAR is an ionotropic glutamate receptor comprising of four subunits, GluR1-4 (Fig. 1). The extracellular domain contains ATD, LBD, three transmembrane domains, a transmembrane loop, and a C-terminal cytoplasmic tail. In 2014, the epitope of AMPAR was mapped [32] (Fig. 1c). Two different methods were employed to test antibody specificity in patient serum and CSF: immunocytochemistry following expression of mutated AMPAR in HEK293 cells and western blots using mutant AMPAR fused to thioredoxin tag expressed in bacteria. Anti-AMPAR antibodies were found to react to both GluR1 and GluR2 subunits of AMPAR. Deletion of S1 domain resulted in little to no immunoreactivity, and mutants lacking the bottom lobe of ATD prevented antibody binding. These results indicate that the main immunogenic targets of anti-AMPAR antibodies are located within the bottom lobe of ATD, but the precise residues were not mapped (Fig. 1c). In the case of AMPAR, although its tertiary structure is similar to the one of NMDAR and the AMPAR epitopes were located within the same glycosylation-prone region, the influence of glycosylation was not demonstrated. Anti-AMPAR antibodies have also been shown to influence receptor function (please see below for pathogenicity).

Anti-Caspr2 antibody epitope was mapped in 2015 [37, 38]. Caspr2 is an axonal protein located at the juxtaparanodes of myelinated axons and, along with Lgi1, is part of the VGKC complex. Caspr2 is made up of eight distinctive subdomains. From the N- terminus, these include the following: discoidin (Disc), lamininG (Lam1), lamininG (Lam2), epidermal growth factor 1 (Egf1), fibrinogenC (FibC), lamininG (Lam3), Egf (Egf2), and lamininG (Lam4) (Fig. 1d). The specificity of anti-Caspr2 antibody was also investigated using a series of deletion constructs, and then antibody-positive patient sera were tested by cell-based assay followed by immunocytochemistry [37] or flow cytometry [38]. A variety of different constructs were created involving single and multiple domain deletions to uncover the subdomain(s) involved in antibody binding. Autoantibody immunoreactivity was found to be specific to the extracellular N-terminal domain of Caspr2 and that autoantibodies target multiple subdomains, with the Disc domain containing the major target epitope. Indeed, the ΔDisc single domain deletion construct was the only one that showed significantly reduced immunoreactivity, but without a complete abrogation of binding [37]. Therefore, Olsen et al. concluded that while a major epitope is contained within the Disc domain of Caspr2, additional unknown epitopes also exist [37, 38]. The second study supports these conclusions [38] and also showed that N-terminal Disc and Lam1 domains are major antigenic targets in sera and CSF [38] (Fig. 1d). Furthermore, deglycosylation by tunicamycin or Peptide-N-Glycosidase F (PNGase F) did not prevent anti-Caspr2 antibody recognition, suggesting that N-linked glycosylation sites on Caspr2 are not important for antibody binding [37].

Anti-GAD65 antibodies have been associated with various syndromes including encephalitis, stiff person syndrome (SPS), cerebellar ataxia, epilepsy, and type 1 diabetes [25, 39‐42]. GAD65 belongs to the pyridoxal 5′-phosphate (PLP)-dependent enzymes, involved in the synthesis of the neurotransmitter GABA [43, 44]. GAD65 consists of a PLP-binding domain, C-terminal domain (CTD), catalytic loop (CL), and N-terminal domain (NTD). In type 1 diabetes, the major target of anti-GAD65 antibodies have been mapped to PLP- and C-terminal regions [45, 46]. However, using competition binding with recombinant monoclonal antibodies, anti-GAD65 antibodies from stiff person syndrome were found to recognize a linear epitope on residues 4–22 and two conformational epitopes spanning residues 308–365 and 451–585 in ~30 and ~70 % patients, respectively [47] (Fig. 1e). These antigenic targets were not recognized by serum antibodies from patients with type 1 diabetes [47]. This suggests that epitopes could be different between diseases. Furthermore, differences in autoantibody epitope may also explain variations between patient clinical phenotype even though they are all anti-GAD65 antibody-positive. A more recent study aimed to examine epitope specificity among all anti-GAD65 antibody-positive patients with different neurological syndromes [48]. They found that patients predominantly recognized the middle domain, corresponding to the catalytic region of GAD65 (aa221-444) (Fig. 1e). N- and C-terminals were also recognized, but to a lesser extent, and there was no GAD-specific epitope that defined any neurological disorder [48].

Patients with demyelinating diseases, including acute disseminated encephalomyelitis (ADEM), neuromyelitis optica spectrum disorder (NMOSD), bilateral optic neuritis (BON), and chronic relapsing inflammatory optic neuropathy (CRION), have been found to be associated with antibodies against conformationally intact MOG [21, 49‐55] (recently reviewed in [51]). MOG is a glycoprotein from the immunoglobulin superfamily, localized to the outermost layer of myelin (Fig. 1f), allowing easy access for potential pathogenic autoantibody binding. To gain precise insight into anti-MOG antibody recognition, 111 patient sera derived from MS, NMOSD, ADEM, or CRION patients were analyzed by live cell-based assay and flow cytometry after binding on MOG mutated by point mutagenesis within its extracellular immunoglobulin (Ig)-like domain [56]. Overall, seven epitopes were distinguished, and all were located within the ß strands of MOG, with the most frequently recognized epitope positioned in the CC’-loop within the extracellular Ig-like domain. Interestingly, the major epitope was a proline at position 42 within the CC’-loop, and the second most commonly recognized epitope was at positions 103 and 104 within the FC’-loop (Fig. 1f). The epitope specificity was similar in all of the disorders analyzed. Interestingly, position 42 is occupied by a serine in mouse MOG (mMOG). Due to mutation at P42, patient sera that recognized human MOG did not recognize mMOG, suggesting that the use of a mouse model to study anti-MOG antibody pathogenicity may be difficult due to failure of human autoantibody to recognize mMOG.

Arguably, the most recognized autoantibodies in demyelination are anti-AQP4 antibodies that are associated with neuromyelitis optica (NMO), a severe demyelinating disorder affecting the optic nerve and spinal cord. Anti-AQP4 antibody-positive patients present with longitudinal extended transverse myelitis and recurrent optic neuritis [57, 58]. Serum anti-AQP4 antibodies are polyclonal and have been shown to bind to conformational epitopes and linear peptides [59]. Epitope mapping has been complicated by the tendency of AQP4 to multimerize into membrane-bound homo- or heterotetramers and higher order structures called orthogonal arrays of particles (OAPs) [60]. A study screened for anti-peptide antibodies by ELISA and found that anti-AQP4 antibody-positive sera were reactive against three different peptides of AQP4, including aa1-22 (42.9 % of patients), aa88-113 (33 %), and aa252-275 (23.8 %). All epitopes identified were found to localize to the intracellular domains of the isoform M23 of AQP4 (AQP4-M23) [61] (Fig. 1g). However, when conformational APQ4-M23 was used, other epitopes were determined (Fig. 1g), and it is likely that these drive disease-relevance and pathogenicity. Two main conformational epitopes were found in extracellular loop A (aa61-64) and loop C (aa146-150) [62] (Fig. 1g). Mutations at W227 in the third extracellular loop E also influenced autoantibody binding in experiments using double loop A/E and C/E mutants. The importance of the conformation was also confirmed by a further study in which a point deletion at D69 within the second transmembrane domain altered the structural rearrangement of extracellular loop A and impaired antibody binding in 85.7 % of NMO patients [63]. These studies emphasize once again how the role of the conformation into epitope assembly can be teased out by the choice of methodology. Interestingly, another recent study focused on the epitope bound by recombinant anti-AQP4 antibodies (rAb) derived from CSF plasmablasts of NMO patients and had the added advantage of dealing with monoclonal rAb rather than patient polyclonal serum. They found that the amino acids within loop C (T137/P138, Val150, and N153) and loop E (H230/W231) were important for antibody binding (Fig. 1f), as well as an influence of loop A on rAb binding [64]. These epitopes are also bound by human serum autoantibodies as it has recently been confirmed by cell-based assay [65].

Neuropsychiatric symptoms such as cognitive dysfunction, mood disorders, and/or psychosis develop in 17–75 % of systemic lupus erythematosus (SLE) patients [66], manifesting as neuropsychiatric systemic lupus erythematosus (NPSLE). A broad range of autoantibodies has been implicated in NPSLE, including those directed against native DNA, ribosomal P proteins, phospholipids, a proliferation-inducing ligand, and histones. The hallmark anti-DNA antibodies in SLE have generated substantial interest in the context of neuropsychiatric syndromes due to their cross-reactivity with the GluN2 (NR2) subunits of NMDAR. This connection was uncovered by screening a phage display peptide library with the R4A antibody which targets murine double-stranded DNA. From the phage library, a five amino acid consensus sequence, DWEYS, was identified on the GluN2a (NR2a) and GluN2b (NR2b) subunit of NMDAR in residues 283–287 [67] (Fig. 1b). Indeed, screening of serum and CSF identified a proportion of SLE patients that harbor anti-GluN2 (NR2) antibodies [67‐70]. While several attempts have been made to correlate anti-GluN2 antibodies with clinical symptoms in patients, the data are inconsistent. Interestingly, anti-NR2 antibodies have also been detected in Rasmussen’s encephalitis and chronic progressive epilepsia using western blot [71]. Recently, serum and CSF from patients with focal NPSLE and peripheral involvement were tested against peptide sequences of GluN1 (NR1) subunit of NMDAR [72]. Antibodies recognized an epitope within the N-terminal extracellular domain at aa19-44 and aa56-81 using ELISA (Fig. 2f). However, the relevance of anti-GluN1 antibodies to NPSLE pathogenicity and their conformational epitopes have yet to be determined.

Antibody-mediated diseases have also been associated with the PNS. Identification of the main autoantigens involved in PNS disorders has preceded many of the autoantigens in CNS disorders, possibly due to the greater accessibility to the PNS or to the now-refuted dogma that the brain was impenetrable to antibodies. However, a number of new targets have been identified for a number of disorders, highlighting ongoing development of autoimmune neurology.

Currently, the mechanism by which an autoimmune response is initiated is unclear, and where exactly CNS autoantibodies are generated has been a subject of much interest. Studies have shown that disease-associated B cells are found both within the periphery and CNS and that the cells are clonally related, for example in MS [125]. Palanichamy et al. demonstrated that an exchange of immunologically active clusters of related B cells occurs between the CNS and peripheral blood compartments. Also, the majority of B cell maturation occurs outside the CNS [126]. These recent discoveries highlight the relevance of using patient sera for autoantibody detection and epitope analysis studies, since B cell maturation can occur in the periphery, specifically in the cervical lymph nodes [126].

While mapping of epitope will likely help us understand autoantibody function, conversely it may also be true that an understanding of antibody role, which could influence antigenic function, will help to predict or uncover the epitopes. Overall, knowledge of the autoantibody epitope target may provide valuable insight into the earliest immune mechanisms of autoimmunity, lead to improved diagnosis, allow more precise testing, and enable development of epitope-specific therapies. In addition, determining whether the target epitope is conserved between species, for example, between human and rodent, may have relevance to pathogenic studies in animal models.

Some of the autoantigens detected in the antibody-mediated disorders described above can have two or more isoforms. Knowledge of autoantibody isoform specificity may be important for diagnosis and will ensure precise testing. For example, AQP4 has two isoforms, AQP4-M1 and AQP4-M23. Inconsistent diagnostic results have emerged due to the isoform selected for anti-AQP4 antibody testing [127‐129]. Untagged AQP4-M23 isoform used in a live cell-based assay was the most ideal method, yielding a specificity of 100 % and sensitivity of 74.4 % [130]. Another study found that AQP4-M1 cell-based fluorescence assay was the most reliable method for antibody testing and serological diagnosis [131], and another large study did not find significant differences between AQP4-M1 and AQP4-M23 [132]. However, recently, a multi-center study further confirmed the value of using AQP4-M23 isoform for optimal sensitivity [133].

Autoantibodies targeting extracellular, rather than intracellular, domains of an antigen have a higher probability of being pathogenic by modulating receptor function, which can be studied in vitro and in vivo. However, since epitopes may vary between species, matching epitope targets between human autoantibodies and murine models is important for animal studies. For instance, the majority of patients with anti-MOG antibodies did not recognize conformational intact mMOG [56], whereas epitopes recognized by anti-NMDAR antibodies are similar between the two species [35], or at least share some cross-reactivity as in the case of anti-AQP4 antibodies [62, 134]. Longitudinal studies of autoimmune neurological disorders in humans are necessary to substantiate findings from animal models and determine whether the same mechanisms are relevant to human disease. Based on these results, decisions can be made as to whether the therapies that have proved effective in animal models are translatable to human disorders.

Understanding the antibody epitope will enable inquiry into mechanisms of antibody pathogenic potential. In the case of NMDAR, residues N368/G369 are located within the bottom lobe of ATD, in close proximity to the hinge of the two ATD lobes that are important for receptor physiology. Outside-out single channel recordings were measured, which showed that anti-NMDAR antibody binding prolongs the open time of the receptor. These results suggest that antibodies influence channel function, and knowing where antibodies bound also helped to hypothesize on how they can hinder their target function. Previous studies support anti-NMDAR antibody pathogenicity as they can induce receptor internalization [135, 136] and alter receptor trafficking [137]. Recently, two studies have endeavored to assess anti-NMDAR antibody pathogenicity in animal models [138, 139]. Planaguma et al. used dialyzed patient and control CSF samples before cerebroventricular infusion using osmotic pumps. Upon injection of patient samples, but not control samples, mice developed progressive memory deficits and depressive-like behaviors over the next 18 days but did not have other affected behavioral or locomotor activities [138]. Wright et al. investigated the epileptogenic potential of anti-NMDAR antibodies. C57BL/6 mice were also injected intracerebroventricularly, but with purified IgG from CSF of anti-NMDAR antibody-positive patients or healthy controls. Anti-NMDAR IgG antibodies alone did not produce spontaneous seizures. However, when mice were challenged intraperitoneally with chemo-convulsant pentylenetetrazol, mice injected with anti-NMDAR antibodies had more convulsive seizures than healthy controls within 2 days [139]. Although these studies are complementary and commendable, the animal models did not entirely recapitulate human disease phenotype. For example, the movement disorders observed in most anti-NMDAR encephalitis patients were not present in either models, and this illustrates the ambiguity and difficulty in assessing antibody pathogenic mechanisms in vivo.

To evaluate the functional effect of anti-AMPAR antibodies, purified anti-AMPAR IgG antibodies from patients or controls were incubated on primary rat cerebrocortical neurons, and miniature excitatory postsynaptic current (mEPSCs) recordings were measured. Compared to control IgG, it was found that anti-AMPAR antibodies altered mEPSCs in cultured neurons [32]. Therefore, binding of anti-AMPAR antibodies to the bottom lobe of ATD may alter the electrophysiology of AMPAR and affect normal function of neuronal receptor upon binding.

Different amino acid recognition can lead to epitope-specific pathogenic effects as in the case of anti-GAD65 antibody, for which pathogenic evidence is mounting [47, 140, 141]. Indeed, antibodies directed to residues 308–365 induce intense and rapidly weaning effects on motor and cognitive functions, and antibodies to residues 451–585 induce sustained medium-sized effects in cerebellum-injected rats [142]. These results have to be discussed in the context of the intracellular location of GAD65, and although the antibody access has not been fully explained yet, the uptake of immunoglobulins into neurons could occur via antigen-dependent or antigen-independent mechanisms [142, 143].

Due to their IgG isotype, some antibodies have the capacity to activate immunological cytotoxic mechanisms. This has been the case for anti-Caspr2 and anti-MOG antibodies in encephalitis and demyelinating disorders, respectively. Indeed, both human anti-Caspr2 and anti-MOG antibodies have been shown to activate complement in vitro [50, 144]. However, evidence of anti-MOG antibody pathogenicity in demyelinating disorders is still limited. In vivo and in vitro animal studies indicated that anti-MOG antibodies mediate complement-mediated lysis [145, 146]. One study suggested a mechanism of cell-mediated cytotoxicity, as sera from pediatric samples were found to induce natural killer cell activation to surface-expressing MOG [21]. Using MOG-transfected HEK293-A cells, another study demonstrated that anti-MOG antibodies, at high titers, are able to activate complement to cause lysis of MOG-expressing cells, whereas antibodies at low titers did not [50]. A more recent study observed loss of cytoskeletal organization in MOG-expressing MO3.13 cells incubated with demyelinating pediatric purified IgG, which contained anti-MOG antibodies [147]. Although the above studies have started to uncover the effector mechanisms of anti-MOG antibodies, how exactly autoantibody binding to its epitope contributes to these mechanisms is yet to be established.

Antibody pathogenicity has also been demonstrated in PNS antibody-mediated disorders. For instance, pathogenicity of anti-AChR antibodies is evidenced by the passive transfer of experimental autoimmune myasthenia gravis (EAMG) following anti-AChR monoclonal antibody injection. Antibodies targeting the AChR main immunogenic region appear to be particularly potent, with one study showing that several different monoclonal antibodies targeting this region of AChR efficiently induced myasthenic symptoms in rats [148]. The monoclonal antibodies used in this study were rat IgG1 or IgG2 against AChR from fish electric organ or mammalian muscle, and each antibody caused an approximate 50 % reduction in AChR quantal content. In contrast, antibodies directed against intracellular portions of the α1 subunit or δ subunit, and monoclonal antibodies against the extracellular portion of the β1 subunit of AChR did not cause EAMG. Further to this, the effects of monoclonal antibodies against the main immunogenic region were not as potent as anti-AChR sera, suggesting that other serum factors play a role in enhancing myasthenic symptoms. These factors may be other IgG subclasses or polyclonal antibodies that target other regions of AChR. As mentioned above, anti-MuSK antibodies target the Ig-like domain 1. Interestingly, that region contains residue I96 that is essential for interaction between MuSK and Lrp4 [149]. Anti-MuSK antibodies could be pathogenic via the prevention of MuSK-Lrp4 binding, either by hindering binding or by modifying MuSK topology. Passive transfer of MuSK MG patient sera or IgG [84, 150, 151] and the extracellular domain of Lrp4 [152] have demonstrated similar pathogenic effects in mice. In the context of LEMS, injection of a peptide sequence of synaptotagmin containing residues 20–53 stimulated antibody production in rats and caused electrophysiological defects at the NMJ, including reduced acetylcholine release [153]. Similarly, animal transfer studies involving autoantibodies against Shaker-like VGKC [102] cause aberrations in synaptic transmission. There is further evidence for the autoimmune etiology of NMT in that it may be associated with myasthenic syndromes and SLE.

Purified SLE anti-DNA antibodies following injection into mouse brain also have neurotoxic effects. This effect might occur via their binding to NMDAR as GluN2 blocker MK-801-treated mice had no neuronal injury after antibody injection [67]. Conversely, not all antibodies show pathogenic features, as seen in LEMS. Antibodies are raised against the VGCC β subunit in some patients but are unlikely to be a pathogenic factor in LEMS as rats immunized with recombinant β subunit fail to develop neuromuscular defects, despite exhibiting high antibody titers [99].

Currently, it is unclear what initiates autoimmunity. One proposed hypothesis is that ectopic expression of antigens, for example by tumors, would activate autoimmune responses. Ovarian teratoma, found in 50 % of anti-NMDAR encephalitis adult patients, have been shown to express NMDAR [10, 135, 159]. However, these tumors are seldom detected in all patients, especially younger ones, who represent an important group of patients. Another proposed hypothesis is molecular mimicry, a phenomenon whereby infectious microbes express antigens which share structural homology with self-antigens, so immune responses initiated towards microbes may result in a reaction against the body’s own antigens [160, 161]. One study reports the mechanism of cross-reactivity between AQP4 and human T-lymphotropic virus 1 (HTLV-1). After matching one intracellular AQP4 epitope (described in [61]) to a highly similar sequence in the human TAX1BP1 protein involved in HTLV-1 replication, the investigators detected anti-AQP4 antibodies in mice immunized with the TAX1BP1-derived peptide [162]. These results suggest that a latent HTLV-1 infection could lead to TAX1BP1 antigen presentation and the production of anti-AQP4 antibodies, perhaps through T cell-mediated mechanisms [162]. Further experiments are required to validate these hypotheses in NMO.

Additionally, several studies report a progression to anti-NMDAR encephalitis following herpes simplex virus encephalitis (HSVE). In some cases, patients were initially diagnosed with HSVE, but later progressed to develop anti-NMDAR encephalitis or anti-D2R antibody-associated movement and psychiatric disorders that tested positive for anti-NMDAR and anti-D2R antibodies [163‐167]. Although these studies have identified a link between HSV and development of autoimmune disorders, whether or not molecular mimicry is involved is still unclear. High neuronal antigen levels could be released during damage to the brain parenchyma after infection by neurotropic herpes simplex virus (HSV), therefore contributing to an activation of an autoimmune response. Additionally, anti-NMDAR encephalitis has also been observed to follow a varicella zoster brain infection in one patient [168]. On the other hand, some autoantibodies, for example anti-D2R antibodies, have been detected in disorders, such as group A Streptococcus-associated Sydenham chorea, that are clearly induced by bacterial infection, and molecular mimicry has been suggested between bacterial antigens and D2R using a rAb isolated from a patient [169]. There is a longstanding hypothesis of molecular mimicry in GBS, as about one quarter of cases are preceded by infection with Campylobacter jejuni [170]. Evidence for this hypothesis arose from a study in rabbits, where animals immunized with C. jejuni-like lipopolysaccharides (LPS) developed antibodies against a panel of ganglioside antigens [171]. The same study found that serum IgG from these rabbits and GBS patients bound gangliosides at the nodes of Ranvier. Molecular mimicry between gangliosides on the surface of neurons and C. jejuni LPS has since been supported by mass spectrometry and serological studies. LPS of the C. jejuni strains that cause GBS harbor motifs that resemble GM1, GD1a, and GQ1b gangliosides [172, 173]. Antibodies against other combinations of gangliosides are thought to induce related disorders like Miller-Fisher syndrome [110]. However, the fact that not all GBS patients are reactive to C. jejuni suggests that molecular mimicry might not be the only mechanism at play. Overall, determination of possible molecular mimicry relies on a linear comparison between pathogen and autoantigen sequences, and multiple confounders, such as conformational non-linear epitopes and polyclonality, exist, making it a challenging concept to prove.

Additionally, patients with rheumatoid arthritis, MG, LEMS, Crohn’s disease, SLE, systemic vasculitis, and IgA nephropathy have all been shown to have altered immunoglobulin glycosylation [174‐178]. It has been recently suggested that these N-glycans present within Fc region of the antibody molecule could influence antibody effector function and therefore play a role in autoimmunity, although this concept would need further validation [179].

Mapping of autoantibody epitopes has shown that patients, individually or in clusters, can have different recognition patterns. They can recognize either a single dominant epitope, a combination of major and minor epitopes, or multiple different epitopes of the same antigen. The presence of multiple epitopes may suggest that a polyclonal antibody response is active.

The studies discussed in this review have generally reported single autoantibody immunoreactivity associated with its respective disorder. For example, anti-NMDAR antibodies are detected in anti-NMDAR encephalitis. However, several recent studies have reported the presence of double autoantibody positivity in a small number of patients. The phenomenon of double antibody positivity remains an understudied subject and has been observed in both CNS and PNS antibody-mediated disorders. The presence of autoantibodies against multiple antigens, including anti-ganglionic AChR antibody, was first reported in patients with paraneoplastic disorders and may be a potential biomarker to predict cancer [180]. In 2010, a case report of a 56-year-old man with non-paraneoplastic limbic encephalitis was found to be associated with anti-NMDAR and anti-VGKC antibodies [181]. A subgroup of limbic encephalitis patients positive for anti-GABA_BR antibody was also found to be positive to other antigens including thyroid peroxidase (TPO), GAD65, sex determining region Y-box 1 (SOX1), or N-type VGCC [16]. Anti-GABA_BR antibodies have also been observed in anti-GAD65 antibody-positive patients with paraneoplastic syndromes [182]. Multiple antibody positivity associated with cancer has been reported in several studies [182, 183]. Petit-Pedrol et al. (2014) recently reported five cases of anti-GABA_AR and anti-GAD65 antibodies co-occurring in CSF and serum [17], and a single case report of a female with encephalitis was found to harbor anti-GABA_AR and anti-GAD65 antibodies in CSF and serum [184]. Other double antibody positivity combinations that have been identified are anti-NMDAR and anti-GAD antibodies [185], anti-NMDAR and anti-D2R [165, 186], anti-GABA_BR and anti-Hu [187], and anti-Caspr2 and anti-Lgi1 in a patient with Morvan syndrome [188]. Recently, patients positive for anti-MOG and anti-NMDAR antibodies were reported [189], and later on, double antibody positivity for anti-NMDAR and anti-GlyR and anti-MOG and anti-GlyR antibodies were also shown in two patients [190]. One study even reported triple antibody positivity, including anti-GABA_BR, anti-Hu, and anti-NMDAR antibodies [187].

The occurrence of MG in LEMS patients is a widely accepted phenomenon [191]. Although diagnosis of MG in LEMS individuals has mainly been based on clinical presentation, tests for serum autoantibodies against AChR and VGCC revealed a number of double seropositive cases [192‐194]. A case of triple antibody seropositivity has also been identified with anti-AChR, anti-VGKC, and anti-MuSK antibodies in a patient with MG and Morvan’s syndrome which is commonly associated with VGKC [195]. In SLE, one third of patients have anti-NMDAR antibodies [196]. In addition, a cancer-positive LEMS patient double positive for anti-VGCC and anti-GABA_BR antibodies has been reported [197]. The coexistence of multiple anti-ganglioside antibodies is not uncommon, for instance, anti-GM1 and anti-GM1b in GBS patients [198]. Additionally, examples of seropositivity exist for anti-ganglioside antibodies together with other antibodies. One study indirectly detected immunoreactivity against nicotinic AChR in GBS patient serum, with the finding that serum interfered with the postsynaptic transmission of nicotinic AChR [199]. Anti-VGKC antibodies have been found along with antibodies against ganglioside GQ1b in variants of GBS [200].

These cases of double or multi-antibody positivity are interesting, although mechanisms behind this poly-immunoreactivity have not been elucidated. Underlying tumors could drive the generation of additional antibody seropositivity [180, 197]. Another possibility is the concurrence of two single antibody-specific disorders in the same patient, which is more likely the case for double anti-NMDAR and anti-MOG antibody and anti-AChR and anti-AQP4 antibody positivity [166, 201]. Multi-antibody positivity may be explained by the concept known as epitope spreading, in which persistent recognition and activation to self-antigens lead to chronic immune system activation [202]. Epitope spreading within the same antigen (intramolecular) or to different antigens (intermolecular) has predominantly been observed in animal models, particularly experimental autoimmune models of EAE and EAMG [203, 204]. Early reports of intermolecular epitope spreading date from the 1990s. Epitope spreading was reported in several mice models of MS, including (SJLxPL)F1, relapsing EAE (R-EAE), SJL/J, B10.PL, and Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) [204‐208]. The mechanism behind epitope spreading is unknown, but it has been suggested that local antigen presenting cells, such as dendritic cells, would initiate epitope spreading in CNS by activating naïve T cells [204]. EAMG can be induced by immunization of organisms with AChR, yielding humoral and cellular responses. When rats were immunized with a recombinant fragment of the extracellular AChR α subunit to induce EAMG, antibodies directed against a hidden intracellular loop of the subunit were detected in two of the immunized animals several weeks after an initial response to the extracellular portion [203]. The fact that the presence of antibodies against the intracellular epitope corresponded to increased disease severity in these animals suggests that epitope specificity may be an important determinant of disease outcomes.

In humans, epitope spreading has also been observed in pediatric MS [209]. Serum samples were collected in children with acquired CNS demyelinating syndromes (ADS) at time of initial attack, and at follow-up at 3 months when children were categorized to either have pediatric MS (ADS-MS) or a monophasic illness (ADS-mono). Immunoreactivity of patient serum samples was tested against a panel of 48 CNS antigens, including MOG, myelin basin protein (MBP), proteolipid protein, and CNPase. At initial attack, serum antibody pattern between ADS-MS and ADS-mono patients reacted to similar number of CNS antigens. After 3 months, ADS-MS patients reacted against a greater number of CNS antigens and bound to different epitopes within these antigens, while ADS-mono samples demonstrated a reduction in CNS antibody response [209]. This evidence is suggestive of intra- and intermolecular epitope spreading in patients with ADS-MS. Epitope spreading has also been observed in a third of anti-AQP4 antibody-positive patients (7/20) over a 15-year follow-up, whereas the majority (13/20) maintained a stable antibody binding pattern [65]. Additionally, in a 5-year longitudinal study on 11 patients harboring anti-MOG antibodies, there was constant epitope recognition with little evidence of intramolecular epitope spreading [56]. This was also observed in MG patients. A recent study examining anti-MuSK antibody-positive MG sera found that intramolecular epitope spreading was relatively uncommon within a minimum of 5 years of follow-up. However, when it occurred, immunoreactivity “spread” from the first and second Ig-like domains to the Fz-like domain [34].

Whether epitope spreading occurs over time and in which time frame is therefore important to determine. Such knowledge may prove useful in designing appropriate immunotherapies, like monoclonal antibodies or blocker peptides that bind specific epitopes at different stages of progression.