Neuritogenic Peptides Derived from

Equine P2 Basic Detect Circulating Antibodies in Ataxic Horses Siobhan P Ellison Tom J Kennedy Austin Li

Corresponding Author: Siobhan P. Ellison, DVM PhD 15471 NW 112th Ave Reddick, Fl 32686 Phone: 352-591-3221 Fax: 352-591-4318 e-mail: [email protected]

KEY WORDS: Need Keywords nosis of EPM. No cross-reactivity between the antigens was observed. An evaluation of the agreement between the assays (McNe- ABSTRACT mar’s test) suggests as CRP values increase, the likelihood of a positive MPP ELISA also Polyneuritis equi is an immune-mediated increases. Clinical signs of EPM may be neurodegenerative condition in horses that is due to an immune-mediated polyneuropathy related to circulating demyelinating anti- that involves complex in vivo interactions bodies against equine with the IL6 pathway because MPP antibod- 2 (MP ). The present study examined the 2 ies and elevated CRP concentrations were presence of circulating demyelinating anti- detected in some horses with S. neurona bodies against neuritogenic peptides of MP 2 sarcocystosis. in sera from horses suspected of equine pro- tozoal encephalomyelitis (EPM), a neurode- INTRODUCTION generative condition in horses that may be Polyneuritis equi is a neurodegenerative immune-mediated. The goals of this study condition in horses that is related to circulat- were to develop serum ELISA tests that may ing demyelinating antibodies against equine identify neuroinflammatory conditions in myelin basic protein 2 (MP2). The clinical horses with EPM and indirectly relate the signs of polyneuritis equi (PE) are simi- pathogenesis of inflammation to IL6 by se- lar to equine protozoal myeloencephalitis rum C-reactive protein (CRP) concentration. (EPM).1,2,3,4 Equine protozoal encephalo- Circulating antibodies against MPP, recom- myelitis (EPM) is also a neurodegenerative binant equine myelin P2 protein (rMP2), condition in horses that may be due to an and CRP were assayed in 52 neurologically immune-mediated pathogenesis of disease abnormal horses by ELISA in this retrospec- as indicated by infection studies.5Muscle tive study. We detected antibodies against wasting, an abnormal gait, and paralysis/ MPP (40%), rMP2 (33%), and CRP (65%) paresis of cranial nerves are signs associated in these horses that had a presumptive diag- with both neurodegenerative diseases. The

Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015. 175 etiology of PE is unknown but is Table 1. Antigen sequence for MPP, SAG 6, SAG 1, generally considered an immune- and IL6 that are homologous to residues on equine mediated peripheral polyneuritis. myelin basic protein 2. The clinical signs associated with EPM are due to inflammation asso- Sequence Name ciated with infections by pathogenic GFKNTEISFKLGQEFEETTADN MPP protozoa, in most cases Sarcocystis EFEETTAD SAG 6 neurona. The histopathological FEET SAG 1 lesions of both diseases have been described.1,4 It was suggested that TTAD IL6 alterations in cytokine levels may contribute to neuropathology seen the mechanism of disease is attributable to in horses with EPM, particularly T cell- cytokine IL6 pro-inflammatory pathways. A mediated immunity.5 It is possible that both BLAST (NCBI) analysis reveals that there diseases are linked by a common disease are amino acid homologies between the mechanism that stimulates proinflammatory neuritogenic peptide MPP contained within pathways via the IL6 cytokine. PE and some S neurona surface antigens. Clinical signs and histopathological Sarcocystis neurona SAG 4 and SAG 5 lesions that are similar to PE can be ex- share homology with MPP residues that are perimentally induced in rats.5 Induction of in the non-disease producing regions of the experimental autoimmune neuritis (EAN) peptide. However, the S neurona SAG’s 6 in susceptible animals is accomplished by and 1 have in the neuri- togenic region of the MPP peptide. injection of MP2 or neuritogenic peptides Cytokine IL6-dependent stimulation of (MPP) derived from MP2, or by passive transfer of lymphocytes from syngeneic ani- the acute phase C-reactive protein (CRP) results in detectable serum CRP levels in mals sensitized to MP2. The neuritogenic site for experimental induction of EAN is lo- horses.7 The IL6 plasma buffering system cated on 26 amino acids of MP2. Induction in horses may preclude sensitive measure of disease in rats by adoptive transfer into of IL6 in plasma, however, the serum CRP naive rats suggested that the antigenic site concentration may provide an indirect for the disease process may due to at least measure of IL6 activity in equine disease.8 one T cell epitope identified on MPP.6 The The purpose of this study was to develop neuritogenic peptide, MPP, contains a puta- a serum ELISA to evaluate the presence of tive T cell epitope that corresponds to equine MPP antibodies in horses and implicate the cytokine IL6. Equine IL6 shares amino acid pathogenesis of disease to IL6 pro-inflam- sequence homology with residues within the mation. A serum MPP ELISA may be useful EAN-inducing site of MPP.6 Cytokine IL6 to elucidate the complex in vivo interactions is a proinflammatory cytokine related to the associated with neuroinflammation present pathology associated with adoptive transfer in PE and EPM. of EAN that can be inhibited by anti-IL6 in MATERIALS AND METHODS vivo. Levamisole HCl decreases the synthe- Antigens sis of IL6 in dogs and rats.9,10 Levamisole HCl was shown to alleviate clinical signs A peptide (MPP) that corresponds to amino of ataxia in horses with suspected EPM acid residues 57-78 of equine myelin pro- and suggests the pathogenesis of disease tein was synthesized (United BioSystems, may be IL6 related if the mechanism of Herndon, VA). Recombinant myelin action of levamisole HCl in horses also P2 protein (MP2) was produced in E coli blocks IL6 production.6,7 Antibody reactiv- (MyBiosource, San Diego, CA). Sarcocystis ity in an ELISA that uses MPP as antigen neurona 1, 5, 6 antigens were produced as 6 is not pathogen specific, but may indicate previously described. The sequence of the

176 Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med. common amino acids Table 2. Results showing the reactivity of sera against antigens are shown in Table 1. used in the ELISA. Rabbit α PMP2 was positive against MPP and Sera MP2 and rSAG 1 and rSAG 6, but not rSAG 5. Control positive A positive control S sera CP bound antigens MPP, MP2, rSAG’s 1, 5, and 6. neurona SAG 1, 5, 6 MPP MP2 rSAG 1 rSAG 5 rSAG 6 serum sample (CP) Rabbit α PMP pos pos pos neg pos was obtained from 2 a 12-year-old thor- CP pos pos pos pos pos oughbred mare with a 5-week history of tion plates coated with approximately 0.1µ severe ataxia (grade 4). Negative control grams of protein in carbonate buffer. Plates sera was pooled from 10 clinically normal were read on a Molecular Devices automatic yearling thoroughbred horses that did not plate reader. Optimum concentrations for have antibodies against S neurona. antigen coating and the detecting antibodies Sera from untreated horses (n=52) that were determined by standard checker-board had clinical signs of ataxia with or without titration. muscle wasting determined by veterinar- The equine P2 myelin protein ELISA ians that suspected EPM were analyzed for described by Fordyce was modified by

S neurona antibodies and retrospectively using MPP or MP2 coated and blocked assayed for antibodies against MP2, MPP, plates.6 Briefly, the plates were incubated and CRP. Antibodies were assayed against for 40 minutes (MP2) or 2 hours (MPP) at o S neurona SAG 1, 5, 6, and MP2 in 52 sera. 37 C with diluted serum. The plates were Anti-MPP antibodies and serum concentra- washed and alkaline phosphatase conju- tion of C-reactive protein was determined gated anti-horse whole molecule (Sigma, St. in 49 sera. Rabbits were immunized with Louis MO) was added and incubated for 30 a synthetic 14 amino acid peptide within minutes at 37oC. Diluted control sera bind- amino acids 17-77 of human PMP2 protein, ing to antigens was detected with alkaline affinity purified, and lyophilized (Antibody phosphatase conjugated anti-rabbit whole Verify; Las Vegas, NV). Rabbit anti-PMP2 molecule (Sigma, St. Louis MO). The plates antibodies were diluted to a concentration of were washed and reacted with 4-nitrophenyl 1 mg/ml as a stock reagent. Alkaline phos- phosphate in carbonate buffer (pH 9) for 15 phatase anti-rabbit was obtained from Sigma and 30 minutes. A positive result was re- Aldrich (St. Louis, MO). corded if the OD was > 0.518. The recipro- Assay cal of the last dilution positive (OD 0.518 or The ELISA was performed in 96 well greater at 405nm) was recorded as the titer. (HyBind, Greiner, Monroe, NC) microtitra- The serum concentration of C-reactive

Table 3. Raw OD 405 showing the binding of rSAG 1, 5, 6, MPP, and MP2 by S. neurona positive sera (CP) in serial 2 fold dilutions 1:8 through 1:64, wells A-E. The raw OD 405 after reacting rSAG 1 and rSAG 6 wells with rabbit anti-PMP2 followed by ELISA using S. neurona positive control sera, CP, are shown, wells F, G, and H.

rSAG 1 rSAG 5 rSAG 6 MPP rMP2 rSAG1 rSAG5 rSAG6 A B C D E F G H 1:8 1.937 1.944 1.935 1.935 1.921 1.509 1.944 0.841 1:16 1.922 1.929 1.916 1.898 1.890 1.500 1.929 0.680 1:32 1.897 1.896 1.540 1.824 1.865 1.296 1.896 0.753 1:64 1.894 1.893 1.505 1.632 1.640 1.473 1.893 0.619

Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015. 177 Table 4. Results of sera tested by antigen showing the number of sera Rabbit anti- that were positive and the percent of total sera tested. PMP2 slightly blocked binding of Number of sera % S neurona positive MP2 positive 18 33% sera to rSAG 1 and MPP positive 21 40% significantly blocked S. neurona positive 31 60% binding to rSAG 6 antigens (Table 3). S. neurona SAG 1 positive 29 56% This antibody did S. neurona SAG 6 positive 25 48% not block binding to CRP positive 32 65% rSAG 5 antigens. MPP and MP positive 7 13% The results of an 2 indirect ELISA using MPP positive and MP negative 14 27% 2 rMP2, MPP, and S

MPP negative and MP2 positive 11 21% neurona SAG’s as antigen are shown in MPP negative and MP2 negative 20 38% Table 4. The serum CRP and MPP positive 12 24% concentration of CRP positive and MPP negative 20 41% C-reactive protein CRP negative and MPP positive 7 14% was determined, a CRP and MPP negative 10 20% positive result was a value >10 µ grams MP and S. neurona positive 17 33% 2 per ml (data not S. neurona negative 21 40% shown). The distri- SAG 6 and MPP positive 12 23% bution of seroposi- tive ataxic horses by SAG 6 positive and MPP negative 13 25% antigen is shown in SAG 6 negative and MPP positive 9 17% Chart 1. SAG 6 and MPP negative 18 35% Assuming MPP

SAG 1 and MPP positive 13 25% or MP2 indicate a neuropathy, a SAG 1 positive and MPP negative 15 29% McNemar’s test was SAG 1 negative and MPP positive 8 15% used to evaluate SAG 1 and MPP negative 16 31% agreement between the MPP and SAG 1, protein was measured by capture ELISA as SAG 5, and SAG 6 per manufacturer recommendations (Gen- results. For each of these comparisons, the way, San Diego CA). P value is not significant, indicating a lack of agreement/lack of cross-reactivity between RESULTS these antigens. There was no statistically Rabbit anti-PMP2 antibody was positive significant relationship between MPP and against MPP and MP2, and rSAG 1 and CRP concentration, however the compari- rSAG 6, but not rSAG 5 (Table 2). Control son suggests as CRP values increase, the positive horse sera (CP) was obtained from likelihood of a positive MPP ELISA also an ataxic horse with a presumptive diagno- increases, Chart 2. sis of EPM, and was positive against MPP, MP2, rSAG’s 1, 5, and 6. Negative control DISCUSSION sera from horses without disease did not The neuritogenic peptide MPP may be useful to detect IL 6-mediated neurological bind any of the antigens MPP, MP2, rSAG 1, 5, or 6 (data not shown). dysfunction in horses with suspected EPM.

178 Vol. 13, No.3, 2015 • Intern J Appl Res Vet Med. Circulating antibodies Chart 1. Antibody distribution by antigen binding in horses against MP2 and the neuri- with suspected EPM. togenic peptide MPP of

MP2 were detected in 32 of 52 horses with suspect- ed EPM in this study. Not all horses were positive for antibodies against both myelin (Table 4). Continued stimula- tion with whole myelin protein induces disease tolerance in rats without loss of activity against the neuritogenic peptide MPP. Therefore, chronically diseased animals may Sarcocystis neurona challenged horses also be MP2 negative/MPP positive on antibody produce antibodies when challenged, how- tests.5 It was not possible to correlate dura- ever, antibody levels are not directly related tion of disease in these horses because onset to clinical signs or prognosis. It was reported of disease was not clearly identified. that as clinical disease progressed, the serum titer increased in S neurona induced infec- A diagnostic test that detects MP2 antibodies in horses with polyneuritis was tions.12 Individual responses to infections suggested by Fordyce.2 Not surprisingly, an- are possible with S neurona infections and an absence of circulating antibodies can be ti-MP2 antibody titer was not a useful prog- nosticator of disease outcome in the Fordyce due to the variable expression of S neurona study. Individual responses to immune surface antigens.12,13,14 stimulation were observed in experimen- The MPP peptide contains a putative T tally induced autoimmune neuritis in rats.5 cell epitope that allowed adoptive transfer of experimental neuritis in rats. Chart 2. The relationship between MPP and CRP se- Homology with equine IL6 amino rum concentration is charted. The line predicting the acids at MPP residues 74-77 may probablity of an MPP seropositive result indicates that implicate IL6 as a T cell marker as the serum concentration of CRP increases there is a for the disease inducing cells in highr probablity of a positive MPP test indicating that horses. The cytokine IL6 induces neuroinflammation (MPP positive) may be related to the production of CRP by the liver. (IL6-induced) CRP. We found no statistically signifi- cant correlation (95% confidence) between MPP seropositive samples and serum CRP concentration in this group of horses. However, the analysis suggested a linear relation- ship. In this study, as CRP value increased, the probability that the sample is MPP seropositive increased, Chart 2. Antibodies (against MPP) that cross-react or bind IL6 may be expected to reduce

Intern J Appl Res Vet Med • Vol. 13, No. 3, 2015. 179 the cytokine dependent production of CRP. active disease producing residues of the anti-

That was not seen. Cytokine IL6 is a multi- gen. The anti-PMP2 sera did not completely functional cytokine that regulates pleiotropic block binding of CP sera to MPP, suggesting roles in inflammation.16 The soluble IL6 anti-PMP2 does not bind the same regions receptor (sIL6r) is an IL6 agonist, and the on MPP as the sera from a diseased horse control of many IL6 mediated events are but may react with multiple epitopes of MPP regulated via sIL6r. Possibly CRP affects that include the non-disease producing resi-

IL6 mediated inflammatory events by releas- dues of MPP or additional antigens of MP2. ing sIL6r into the plasma.7 If this is true, one Anti-PMP2 antibody did not completely would expect to measure IL6 in the plasma block CP binding to rSAG 6, indicating in acute disease. The elevated CRP values that the antibodies anti-PMP2 and CP bind in the MPP seropositive horses may indicate different epitopes of rSAG 6 that may that the neuritis is acute, and is mediated include the neuritogenic region of MPP at through the IL6 cytokine pathways. How- residues EFEETTAD in this assay. The test ever, we did not measure IL6 in this study. results using sera from these clinically ill A sustained elevation in anti-MPP in a horse horses were evaluated by McNemar’s test that is CRP negative may indicate a chronic and showed insignificance between MPP or neuritis as seen in the hyper-immunized MP2 ELISA and SAG 1, 5, or 6 ELISA test EAN rats. results. The lack of agreement between The The EPM-suspect horses were seroposi- ELISA tests indicate the antigens S neurona tive against antibodies to S neurona SAG SAG 1, 5, or 6 and MPP are not cross- 1, 5, or 6. There is amino acid sequence reactive. homology between S neurona SAG’s 1, 4, 5, IL6-mediated disease can be assayed us- and 6 and MPP.6 In order to investigate the ing MPP and MP2. Assays that use MPP and proteomics of SAG proteins in the neuroin- MP2 may be useful to elucidate the patho- flammatory disease process, we identified genesis of disease associated with EPM, but sequence homology between S neurona not the etiology. Possibly, early infections SAG’s and the neuritogenic peptide MPP with S neurona induces peripheral polyneu- and MP2. We determined the binding of ritis in horses that confounds a diagnosis of equine sera to the MP2 and MPP proteins EPM. It is possible that the proteomics of and the relationship to SAG 1, 5, and 6. parasites antigens facilitate inflammatory Control horse sera was seropositive for all disease. However, the SAG 1, 5, 6 ELISA the proteins used in this study. A mono- can indicate the etiology is due to S neu- specific polyclonal antibody against amino rona because these assays measure different acid residues was specific to MP2 bound antigens. If the inflammatory component of MPP, rSAG 1, and rSAG 6, but not rSAG 5 EPM is IL6-mediated, blocking IL6 is a vi- antigens. Based on structural and antibody able therapeutic strategy for diseased horses. binding data, anti-PMP antibodies presum- 2 REFERENCES ably reacted with the neuritogenic peptide 1. Antemortem diagnosis of polyneuritis equi. Aleman MPP and not the non-disease producing resi- M, Katzman SA, Vaughn B, Hodges J, Crabbs TA, dues of the equine myelin basic protein 2. Christopher GC, Shelton GD, Higgins RJ. 2009, J We showed that anti-PMP could Vet Intern Med, Vol. 23, pp. 665-668. 2 2. Use of an ELISA in the differential diagnosis of partially block binding of CP sera to rSAG cauda equina neuritis and other equine neuropa- 1 and rSAG 6, but not rSAG 5. Blocking thies. Fordyce S, Edington N, Bridges GC, Wriht the binding of CP sera to antigens that had JA, Edwards GB. 1987, Equine Veterinary Journal, Vol. 19, pp. 55-59. homology in the disease-producing regions 3. Cauda Equina Neuritis: A Chronic Idiopathic Poly- of MPP, but not the antigens with homol- neuritis in Two Horses. Rousseaux CG, Futcher ogy to the non-disease producing regions of KG, Clark EG, Nayloor JM. 1984, Can Vet J, Vol. MPP, may support that anti-PMP binds the 25, pp. 214-218. 2 4. An update on Sarcocystis neurona infedctions in

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