Microbial Pathogenesis 1990 ; 8 : 151-156
A neutralizing monoclonal antibody against Coxsackievirus B4 cross-reacts with contractile muscle proteins Kirk W . Beisel,' Javaraiah Srinivasappa, Z Margaret R . Olsen,' Anne C . Stiff,' Karim Essani z and Bellur S . Prabhakar 2 'Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, U .S .A ., and 'Laboratory of Oral Medicine, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, U .S .A . (Received September 17, 1989 ; accepted in revised form December 29, 1989)
Beisel, K . W . (Dept of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, U .S .A .), J . Srinivasappa, M . R . Olsen, A . C . Stiff, K . Essani and B . S . Prabhakar . A neutralizing monoclonal antibody against Coxsackievirus B4 cross-reacts with contractile muscle proteins . Microbial Pathogenesis 1990 ; 8: 151-156 . A panel of Coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies (mAbs) were tested against a panel of normal mouse tissues . One mAb, 356-1, reacted specifically with murine heart tissue . Immunohistochemical studies revealed an A band pattern of staining of the heart . Examination of sequential differential extracts of heart by Western immunoblotting showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain . A rather weak crossreaction was found with actin . These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin . This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin . Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein . These studies imply that molecular mimicry is one mechanism by which autoimmunity could develop during CVB4 induced myocarditis . Key words : Coxsackievirus B4 ; monoclonal antibodies ; molecular mimicry ; heart muscle .
Introduction A viral etiology in the development of autoimmune pathogenesis has been proposed for a number of diseases including myocarditis .' Mechanism of virus-induced autoimmunity is influenced by the genetic make up of both the virus and the host- 1-3 From experimental and clinical observations several mechanisms have been proposed to .1,4 explain the development of autoimmunity as a sequela to infection In virus-induced myocarditis the chronic inflammation of the myocardium is thought to be due to an autoimmune response . Several studies suggest that there is a direct relationship between certain viral infections (i .e . group B, Coxsackieviruses, influenza and mumps) and the induction of autoantibodies to heart .' In fact, in some of these studies the cytolytic activity of the sera from patients with viral myocarditis against myocytes could be adsorbed with respective viral agents .' These studies suggest that a shared antigenic determinant between the virus and the heart tissue (molecular mimicry) is responsible for myocardial autoimmunity . Recent studies from a number
0882-4010/90/020151 +06 $03 .00/0
© 1990 Academic Press Limited
K . W . Beisel et a/ .
VP-IVP- 2 1VP-3-
Fig . 1 . Reactivity of antibody 356-1 with purified CVB4 . Purified CVB4 virions were run on SDS-PAGE and then electroblotted on to nitrocellulose . The blot was reacted with polyclonal rabbit anti-CVB4 serum (A) and 356-1 (B) and then the blots were treated with the appropriate 125 1-labeled anti-Ig reagent . Polypeptides were identified based on their molecular weights . VP-4 polypeptide is present in a very small amount in CVB4 capsid, therefore, the reaction of polyclonal anti-CVB4 serum with VP-4 in the gel is not usually visible .
of laboratories have used monoclonal antibodies (mAbs) to demonstrate molecular mimicry between infectious agents and normal host tissues ."' The current study was initiated to characterize the nature of the antigens recognized by a mAb, 356-1, which neutralizes Coxsackievirus B4 (CVB4) and cross-reacts with the heart tissue . 6 ' The data presented here demonstrates that 356-1 binds murine cardiac myosin and the VP-1 capsid protein of CVB4 and establishes a molecular relationship between these two proteins . Results Reactivity of 356-1 with CVB4 In order to determine the viral polypeptide recognized by 356-1, the viral proteins from purified CVB4 were separated by SDS-PAGE and were then electrophoretically transferred to nitrocellulose . As shown in Fig . 1, hyperimmune rabbit anti-CVB4 serum detected all four proteins . However, mAb 356-1 appears to react with the viral capsid protein, VP-1 . This, coupled with the ability of 356-1 to neutralize CVB4, strongly
Cross-reactive CVB4 antibody
Table 1 Immunohistochemistry of monoclonal antibodies with muscle tissues Reactivity Species
Mouse Atrium + Ventricle + Skeletal muscle Rat Atrium + Ventricle + Skeletal muscle + Hamster Atrium Ventricle + Skeletal muscle + Rabbit Atrium Ventricle + Skeletal muscle + Guinea-pig Atrium Ventricle Skeletal muscle + Chicken Atrium Ventricle Skeletal muscle Monkey Atrium Ventricle Skeletal muscle Human Atrium Ventricle Skeletal muscle
6C1 + + + + + + + + + + + + + + + + + + + + + + + +
suggests that it is recognizing an antigenic determinant on the VP-1 capsid protein of CVB4 . Characterization of heart muscle proteins that react with 356-1 The muscle reactivity and species specificity of 356-1 was then examined and compared with another mAb which binds to muscle tissues . Monoclonal antibody 6C1, was found to react with all muscle tissues . As shown in Table 1, the 6C1 reacted with atrial, ventricular and skeletal muscle tissues from mouse, rat, hamster, rabbit, guineapig, chicken, monkey and human, mAb 356-1 reacted with atria from all species tested except monkey and human . However, it only reacted with ventricles of mouse, rat, hamster and rabbit . The atrial, ventricular and species staining pattern differences exhibited by 356-1 suggest that 356-1 binds with a contractile muscle protein(s) which has various isoform variants . Both the mAbs had some similarities in their reactivity patterns with the high ionic strength salt extract of mouse heart . A wide band of approximately 198 kDa and a minor 99 kDa band were observed to react with 6C1 (not shown) . However, primarily a 198 kDa band was observed when stained with 356-1 [Fig . 2(A), lane b] . In addition, several weak reactive bands were also observed with both antibodies . Previously, we had demonstrated that the 198 kDa protein band is the myosin heavy chain and that many of the associated lower molecular weight bands probably represent breakdown products of myosin ." A second protein with a relative molecular weight of approximately 46 kDa similar to that of actin was stained by 356-1 [Fig . 2(A), lane b] . This mAb was also found to react weakly with purified muscle a-actin (data not shown) thereby suggesting that 356-1 can bind, but weakly, to actin . Further testing of protease digested heart extract on Western blots showed that 356-1 can
K . W . Beisel et al.
1 54 (B)
Fig . 2 . (A) Cross-reactivity of monoclonal antibody 356-1 with murine muscle : a high ionic-strength extract of heart muscle from A/J mice was run on SDS-PAGE and then electrophoretically transferred to nitrocellulose . Lanes a and b, reactivity with a high ionic-strength heart tissue extract ; lanes c and d, reactivity of protease digested heart extract . The reactivity of 356-1 was detected by an appropriate immunoperoxidase labeled goat anti-mouse IgG heavy chain-specific reagent . An IgG2a mouse monoclonal antibody to K k haploype 22 was used as a negative control . This antibody gave results similar to that with the second antibody alone (not shown) . Lanes a and d, anti-IgG conjugate only . (B) Reactivity of 6C1 and 356-1 with cardiac and skeletal muscle myosin . Monoclonal antibodies diluted in PBS were titered using an ELISA assay for reactivity against murine cardiac (o o) and skeletal (o---9) muscle myosins . Titers shown in logs .
primarily react with 130 kDa and 90 kDa bands [Fig . 2(A), lane c] . To test the specificity of 356-1 further, it was reacted with myosins purified from murine heart and skeletal muscle [Fig . 2(B)] . Monoclonal antibody 356-1 was found to react strongly with the cardiac myosin and relatively weakly with the skeletal muscle myosin suggesting that 356-1 has a greater affinity for cardiac myosin . As predicted from the immunoperoxidase staining of normal muscle tissues, no difference in the binding of 6C1 with cardiac and skeletal muscle myosins was noted [Fig . 2(B)] .
Discussion This study demonstrates that the VP-1 capsid polypeptide of CVB4 and the cardiac myosin share a common antigenic determinant . The mAb, 356-1, was originally selected for its ability to neutralize CVB4 virus and had been shown to react with a moderately conserved epitope on the viral capsid ." The A-band immunostaining pattern of heart muscle but not the skeletal muscle 10 had indicated that a heart-specific contractile protein(s) was recognized by 356-1 . The present study shows that the 356-1 binds primarily to myosin heavy chain [Fig . 1(A), lane b] and unlike 6C1 (not shown) does not react with a 99 kDa band which is thought to be myosin rods . In addition, protease digestion of murine cardiac myosin supported this observation in that 356-1 bound both the heavy meromyosin (130 kDa) and head (90 kDa) fragments [Fig . 2(A), lane c] . Data from the immunostaining experiment suggested that 356-1 predominantly reacts with the cardiac myosin heavy chain isoform . In rodents, the V1 myosin is the predominant chain found in the atrium . However, the ventricles contain
Cross-reactive CVB4 antibody
varying quantities of both V1 and V3 . For example, rabbits predominantly express the cardiac V3 isoform, rats have equal proportion of isoform V1 and isoform V3 and mice exclusively express the myosin heavy chain isoform V1 . 18 This along with the staining pattern observed with hearts from these species using 356-1 suggests that antibody 356-1 primarily reacts with V1 isoform . Lack of 356-1 reactivity with the atrial tissue of the higher vertebrates (e .g . monkey and man) suggests that a mutational event or a gene conversion in the a-myosin heavy chain gene may have altered the epitope . Earlier studies have shown antigenic mimicry between the myocardium and other infectious agents . In Chagas disease, cross-reactivity between Trypanosoma cruzi and a myocardial sarcoplasmic reticulum antigen, the Ca t ' dependent ATPase, has been identified ." This phenomenon has also been demonstrated to occur in streptococcalinduced rheumatic heart disease . 20 Cunningham et al. 21 have found that the group A streptococcal mAbs reacted with the rod of the myosin heavy chain . In contrast 3561 recognizes a determinant in the head portion of myosin heavy chain . This distinction indicates that myosin heavy chain has a variety of epitopes which are similar to determinants on various pathogens . Recently, Neu et al." demonstrated that autoimmune myocarditis could be induced by immunization of mice with cardiac myosin mixed with complete Freunds adjuvant . It would be of interest to examine whether 356-1 recognizes the antigenic epitope of cardiac myosin heavy chain that produces autoimmune disease . Further studies should also reveal whether the shared epitope between CVB4 and cardiac myosin is a result of a common primary amino acid sequence or spatial conformation . Nevertheless, our studies suggest that molecular mimicry could be a component in the development of autoimmune inflammatory heart disease as a sequela to CVB4 infection .
Materials and methods
Monoclonal antibodies . Monoclonal antibody 351 -1 (IgG2a) was affinity purified on a protein-A sepharose column and was resuspended in PBS, pH 7 .4, at a concentration of 2 mg/ml . Monoclonal antibody 6C1 (IgM), was produced by fusing normal splenocytes with myeloma cells . $ Immunohistochemistry . Reactivity of the mAbs with normal tissues was determined by the indirect immunofluorescence or immunoperoxidase techniques as previously described .' Immunochemistry . Polyacrylamide gel electrophoresis was carried out in the presence of SDS ."' Blots stained with horseradish peroxidase-labeled (HRP) affinity as previously described purified goat anti-mouse IgG or IgM (heavy chain specific) were developed using a substrate solution containing 0 .03% DAB and 1 % H 20 2 . Whereas blots with 125 1-labeled rabbit and mouse Ig, were exposed at -80°C to Kodak XR-5 X-ray film with intensifying screen . ELISA . The mAbs were tested for their reactivity to murine skeletal and cardiac myosins in an ELISA described earlier ." The titer is expressed in log s dilutions and the endpoint is the highest dilution giving an OD value above twice that of negative control . Differential muscle tissue extractions . Cardiac and skeletal muscle extracts were prepared using a modification of the protocol as described by Alvarez et a/ . 10 Briefly, homogenized tissue was extracted sequentially in a low-ionic strength (LS) buffer (20 mm Tris-HCI, 1 mm EDTA, pH 9 .0), high-ionic strength (HS) buffer [20 mm Tris-HCI, 0 .6 M KCI, 50 mm Na 4 P 2 0 7 . 10H 20, 2 mm ATP, 2 mm MgC1 2 , 1 mm dithiothreitol (DTT), pH 7 .4] and a detergent buffer (10% SDS, 0 .5 M Tris-HCI, 25% v/v glycerol, pH 6 .8) . The low and high ionic strength extracts were treated with protein-A sepharose beads at pH 8 .0 to remove immunoglobulin . Purification of myosin and actin . Myosin and actin from murine cardiac and skeletal muscle were prepared as previously described . 12
K . W. Beisel et al.
Purification of CB4. The prototype JVB strain of CVB4 was grown and purified as previously described ." The purified virus was subjected to SDS-PAGE 14 and Western blot analysis- 15 This work was supported in part by U .S Public Health Service grant HL-38276 from the National Heart, Lung and Blood Institute . The authors acknowledge Dr Abner Louis Notkins for his support and critical review of the manuscript, the technical assistance of Patricia Pepler, Laura Poole and Sara Sells in preparation of the various murine cardiac and skeletal muscle tissue and Karen Hardman and Eloise Mange for their skill in preparing the manuscript .
References 1 . Notkins AL, Onodera T, Prabhakar B . Virus-induced autoimmunity . In : Notkins AL, Oldstone MBA, eds . Concepts in viral pathogenesis . New York : Springer-Verlag, 1984 ; 210-5 . 2 . Gauntt CJ, Trousdale MD, LaBadie DRL, Paque RE, Nealon T. Properties of Coxsackievirus B3 variants which are amyocarditic or myocarditic for mice . J Med Virol 1979 ; 3 : 207-20 . 3 . Wolfgram LJ, Beisel KW, Herskowitz A, Rose NR . Variations in the susceptibility to Coxsackievirus B3-induced myocarditis among different strains of mice . J Immunol 1986 ; 136 :1846-52 . 4 . Oldstone MBA, Notkins AL. Molecular mimicry . In : Notkins AL, Oldstone MBA, eds . Concepts in viral pathogenesis II . New York: Springer-Verlag, 1986 ; 195-202 . 5 . Maisch B, Trostel-Soeder R, Stechemesser E, Berg PA, Kochsiek K . Diagnostic prevalence of humoral and cell-mediated immune reactions in patients with viral myocarditis . Clin Exp Immunol 1982 ; 48 : 533-45 . 6 . Srinivasappa J, Saegusa J, Prabhakar BS, Gentry MK, Buchmeirer MJ, Wiktor TJ, Koprowski H, Oldstone MBA, Notkins AL . Molecular mimicry : frequency of reactivity of monoclonal antiviral antibodies with normal tissues . J Virol 1986; 57 : 397-401 . 7 . Saegusa J, Prabhakar BS, Essani K, McClintock PR, Fukuda Y, Ferrans VJ, Notkins AL . Monoclonal antibody to Coxsackievirus B 4 reacts with myocardium . J Infect Dis 1986; 153 : 372-3. 8 . Hartman AB, Mallett CP, Srinivasappa J, Prabhakar BS, Notkins AL, Smith-Gill SJ . Organ reactive autoantibodies from non-immunized adult Balb/c mice are polyreactive and express non-biased V„ gene usage. Mol Immunol 1989 ; 26 : 359-70 . 9 . Clark WA Jr, Chizzonite RA, Everett AW, Rabinowitz M, Zak R . Species correlations between cardiac myosin isomyosins . A comparison of electrophoretic and immunological properties . J Biol Chem 1987 ; 257 :5449-54 . 10 . Alvarez FL, Neu N, Rose NR, Craig SW, Beisel KW . Heart-specific autoantibodies induced by Coxsackievirus B3 : identification of heart autoantigens . Clin Immunol Immunopathol 1987 ; 43: 12939 . 11 . Neu N, Rose NR, Beisel KW, Herskowitz A, Gurri-Glass G, Craig SW . Cardiac myosin induces myocarditis in genetically predisposed mice . J Immunol 1987 ; 139: 3630-6 . 12 . Neu N, Craig SW, Rose NR, Alvarez FL, Beisel KW . Coxsackievirus induced myocarditis in mice : cardiac myosin autoantibodies do not react with the virus . Clin Exp Immunol 1987 ; 69 : 566-74 . 13 . Prabhakar BS, Haspel MV, McClintock PR, Notkins AL . High frequency of antigenic variants among naturally-occurring human Coxsackie B 4 isolates identified by monoclonal antibodies . Nature 1982 ; 300: 374-6 . 14 . Laemmli UK . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970 ; 227 : 680-5 . 15 . Towbin HH, Staehelin T, Gordon J . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications . Proc Natl Acad Sci USA 1979; 76 : 4350-4 . 16 . Beisel KB, Rose NR . Relationship of Coxsackievirus to cardiac autoimmunity . In : Friedman H, Bendinelli M, eds. Coxsackieviruses : a general update . New York : Plenum Press, 1988 ; 271-92 . 17 . Prabhakar BS, Srinivasappa J, Ray UR . Selection of Coxsackievirus B 4 variants with monoclonal antibodies results in attenuation . J Gen Virol 1987; 68 : 865-9 . 18 . Clark WA, Everett AW, Chizzonite RA, Eisenberg BR, Zak R . Classification and characterization of cardiac isomyosins . Eur Heart J 1984; 5 [Supplement F] : 69-75 . 19 . Sadigursky M, Acosta AM, Santos-Buch CA . Muscle sarcoplasmic reticulum antigen shared by a Trypanosoma cruzi clone . Am J Trop Med Hyg 1982 ; 31 : 934-41 . 20 . Zabriskie JB, Freimer EH . An immunological relationship between the group A streptococcus and mammalian muscle . J Exp Med 1966 ; 124 : 661-78 . 21 . Cunningham MW, Hall NK, Krisher KK, Spanier AM . A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin . J Immunol 1986; 136: 293-8 . 22 . Ozato K, Mayer N, Sachs DH . Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and la antigens. J Immunol 1980; 124: 533-40 .