Immunology 1991 72 426-433

ADONIS

001928059100072D

Murine anti-cytomegalovirus monoclonal antibodies with autoreactivity C. M. LAWSON, H. L. O'DONOGHUE, H. E. FARRELL,* G. R. SHELLAM* & W. D. REED Departments Of Medicine and *Microbiology, The University of Western Australia, Nedlands, Western Australia

Acceptedfor publication 31 October 1990

SUMMARY Certain murine monoclonal antibodies (mAb) raised against structural proteins of mouse cytomegalovirus (MCMV) display distinct patterns of multiple organ-autoreactivity in addition to their viral specificities. We analysed the autoreactivity of five such mAb by immunoperoxidase histochemistry, western immunoblot and enzyme-linked immunosorbent assay (ELISA). Four mAb recognized cellular autoantigens in the salivary gland, lung, heart, liver, kidney, ileum, striated muscle and brain, as detected by immunoperoxidase histochemistry. However, the mAb showed different specificities for nuclear, cytoplasmic and surface membrane antigens on various cell types in addition to common autoreactivities. Immunoblot analyses showed that some of the mAb recognized polypeptides of various molecular weights obtained from l00,000g supernatants of normal BALB/c liver, brain, striated and cardiac muscle homogenates. Reactivity of the mAb with a 200,000 molecular weight (MW) polypeptide was similar to our previous finding of the reaction of late immune polyclonal sera with a 200,000 MW polypeptide, the heavy chain of myosin. The mAb reacted to the cardiac isoform of myosin as determined by ELISA and immunoblot. Reactivity of mAb with cardiac myosin, as detected by immunoblot, was removed by absorption with cardiac myosin and recovered in the eluate. However, cardiac myosin used in a competitive inhibition ELISA did not abrogate the reactivity of the mAb with MCMV antigens. These anti-MCMV mAb appear to be multispecific for both virus and self-antigens, including cardiac myosin, and possibly recognize these different antigens through partly similar or distinct antigen-binding sites.

INTRODUCTION Human cytomegalovirus (HCMV) infection has been associated with several diseases with autoimmune components, including myocarditis,' acute inflammatory polyneuropathy2 and Sjdgren's syndrome.3 The humoral response produced during HCMV infection is not completely virus specific. Rheumatoid factors, anti-nuclear4 and anti-smooth muscle antibodies5 are found in patients with HCMV infection. In the murine model, mouse cytomegalovirus (MCMV) infection induces the production of virus-specific antibodies6-8 in addition to multiple organ-autoreactive antibodies of the IgM and IgG class.9 Late immune sera (Day 100 post-infection) react predominantly with a 200,000 molecular weight (MW) polypeptide from 100,000 g supernatants of normal mouse organ homogeAbbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; HCMV, human cytomegalovirus; mAb, monoclonal antibody; MCMV, mouse cytomegalovirus; MEF, mouse embryonic fibroblast; MOBS, phosphate-buffered saline with mouse osmolarity; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel

electrophoresis. Correspondence: Dr C. M. Lawson, Dept. of Medicine, The University of Western Australia, Nedlands, 6009, Western Australia.

nates.'0 This autoreactivity, shown to be directed against the heavy chain of myosin, may be of pathological significance in mice of the susceptible BALB/c strain which develop persisting myocarditis associated with high titres of anti-cardiac myosinspecific IgG antibody. The mechanisms of induction of the autoimmune response following CMV infection are not known. Polyclonal B-cell activation," molecular mimicry of virus antigenic epitopes with self proteins,'2"13 incorporation of host cell proteins into the virion during virus assembly,'4 and fusion of genomic and viral DNA'5 have been raised as possible explanations for the occurrence of autoreactivity following CMV infection. Our previous finding of cross-reaction of the anti-MCMV monoclonal antibody (mAb) ACl with a ribonucleoprotein'6 prompted us to further screen for immunological autoreactivity of other mAb raised to MCMV antigens. Five anti-MCMV mAb capable of neutralizing the virus'6 were studied. mAb 1 E8, 3B2 and 4F9 are of the IgG2b isotype and display perinuclear fluorescence when applied to MCMV-infected mouse embryonic fibroblasts (MEF). mAb 1E8 immunoprecipitated viral proteins of 56,000 MW and 60,000 MW, and 4F9 immunoprecipitated predominantly a 60,000 MW polypeptide but also 28,000, 34,000, 36,000, 48,000-50,000, 68,000, 76,000, 83,000 and 92,000 MW polypeptides of MCMV. Immunoblot analysis

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Autoreactive monoclonal antibodies to cytomegalovirus found 3B2 to react with a 50,000 MW polypeptide of MCMV. mAb of the IgM isotype, EA4 and 1B4, displayed perinuclear and cytoplasmic fluorescence, respectively, on MCMV-infected fibroblasts. mAb EA4 reacted with multiple viral polypeptides by the immunoprecipitation technique only, and mAb IB4 reacted with 87,000, 92,000, 98,000, 102,000 and 110,000 MW proteins of MCMV. In this paper we have analysed the auroreactivity of these five virus-neutralizing mAb by immunoperoxidase histochemistry, immunoblot and ELISA. The mAb displayed autoreactivity to normal mouse organs and tissues, as assessed by immunoperoxidase histochemistry. Furthermore, the mAb reacted with a 200,000 MW polypeptide identified as the heavy chain of myosin. These observations raise the possibility of common epitopes on self-antigens, in particular the myosin heavy chain, and MCMV. MATERIALS AND METHODS Mice

Specific pathogen-free inbred female BALB/c mice of 8 weeks of age were supplied by the Animal Resources Centre, Murdoch, Western Australia and were seronegative for anti-MCMV antibodies, as previously reported.7 Monoclonal antibodies The production and characterization of the mAb used in this study are described elsewhere.'6 Briefly, BALB/c mice were inoculated with 1 pg of purified MCMV antigen (Smith virus strain passaged once in MEF) emulsified in Freund's complete adjuvant by the subcutaneous route and 30 days later with 0-1 pg of MCMV antigen in saline by the intraperitoneal and intravenous routes. After 4 days, spleen cells were fused with non-secreting myeloma line P3X63Ag.8.653 using polyethylene glycol. Hybridomas were selected in HAT medium, subcultured twice by limiting dilution and injected into pristane-treated mice. Serum and ascites proteins were removed by 30% ammonium sulphate precipitation and then immunoglobulins precipitated with 50% ammonium sulphate and dialysed against saline. The mAb 1 E8, EA4, 1 B4, 4F9 and 3B2 used in this study all neutralized MCMV. 16 The mAb were adjusted to 5 mg/ml. As a control, immunoglobulins were similarly precipitated from pristane-primed BALB/c mice that had been injected with the myeloma cell line alone.

Immunoperoxidase histochemistry Immunoperoxidase histochemistry was carried out with composite paraffin-embedded blocks of Bouin's fluid-fixed normal, uninfected BALB/c organs and tissues, as previously described,9 using a 1/20 dilution of primary antibody. The rabbit antibody to mouse immunoglobulins conjugated to peroxidase (Dakopatts, Glostrup, Denmark) applied to normal uninfected tissue sections did not result in immunoperoxidase staining. Preparation of tissue homogenate supernatants Striated and cardiac muscle, liver, and brain tissue homogenate supernatants were prepared from normal, uninfected BALB/c mice as described previously.'0

427

Polyacrylamide gel electrophoresis (PAGE) and immunoblot Tissue homogenate supernatants adjusted to 1 mg/ml were dialysed against sample buffer (10 mm Tris-HCI, I mm EDTA, 1% sodium dodecyl sulphate, SDS, 40 mm DTT, 10 M urea, pH 8), denatured for 10 min at 100° and applied to 10% polyacrylamide gels with a 3% polyacrylamide stacking gel. Purified mouse cardiac myosin and MCMV antigen8 were similarly treated without the addition of urea. Identical gels were run, one of which was stained with Coomassie blue. Polypeptides from the remaining gels were transferred to nitrocellulose and immunoblot performed, as previously described.9 Standard proteins (BioRad, Richmond, CA) of defined MW ranging from 14,000 to 200,000 were included in each gel.

Isolation of mouse myosin Myosin was isolated from hearts of mice as previously described.'0 Myosin-enriched fractions were precipitated by dialysis against an equal volume of saturated ammonium sulphate solution, dissolved in 50 mM Na4P207* 10H20, pH 8 5, at pH 8 5, at 1 mg/ml, and stored at -20°. The purity of the heavy chain of myosin was checked by SDS-PAGE and was represented by a single band of approximately 200,000 MW. The protein preparation reacted by immunoblot and ELISA with a commercial anti-myosin heavy chain mAb (RPN. 1169; Amersham, Amersham, Bucks, U.K.).

Coupling of myosin to Sepharose beads The coupling of cardiac myosin to beads was performed as described previously.'0 Bovine serum albumin (BSA) was similarly coupled to the beads. Absorption of mAb with myosin mAb at 5 mg/ml were incubated with 2 volumes of cardiac myosin-coupled Sepharose or BSA-coupled Sepharose. After continuous mixing for 18 hr at room temperature, the beads were centrifuged at 100 g for 5 min and the absorbed sera saved. The beads were washed five times with phosphate-buffered saline adjusted to mouse osmolarity (MOBS, 330 mOsmol) and then once with cold double distilled water. Myosin-reactive antibodies were eluted from the beads by incubation for 15 min at 40 with 0-05 M acetate buffer (equivalent to the original volume of sera), pH 2-8, with constant mixing. Following centrifugation at 100 g for 5 min, the eluted antibodies were dialysed overnight against MOBS at 4°. Enzyme-linked immunosorbent assay (ELISA) Myosin preparations were diluted in carbonate-bicarbonate buffer with 50 mM Na4P207 10H20, pH 9-6, and 0 25 pg/I00 p1/ well incubated overnight at 4° in polystyrene microtitre plates before use in the ELISA, as described previously.9 ELISA for determining anti-MCMV antibody reactivity was performed as previously reported.7 mAb at 5 mg/ml were serially diluted twofold before use and the specificity of the conjugate used corresponded to the isotype of the mAb, as previously characterized. 16

Competitive inhibition assay Serial log 4 dilutions of mouse cardiac muscle myosin were made in 50 mm Na4P207 10H20, pH 7.4, with 1% BSA. A constant amount of the mAb was mixed with the above dilutions of cardiac myosin and buffer only for 1 hr at room temperature,

C. M. Lawson et al.

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Figure 1. Autoreactivity of MCMV neutralizing mAb with normal, uninfected mouse tissues detected by immunoperoxidase histochemistry. (a) Cross-section of villi of small intestine and mAb 1 B4 showing strong reaction with nuclei of absorptive cells and fibroblasts ( x 213). (b) Villi of small intestine and normal BALB/c serum ( x 213). (c) Cardiac muscle and mAb 4F9 showing antinuclear and anti-myofibrillar autoreactivity ( x 213). (d) Cardiac muscle and normal BALB/c serum ( x 213). (e) Salivary gland and mAb 4F9 reacting with nuclei of cells lining the ducts and serous alveoli (x213). (f) Cardiac muscle and mAb 1B4 showing autoreactivity with myocardium ( x 213). (g) Brain tissue and mAb 4F9 showing peri-nuclear reactivity ( x 213). (h) Normal cardiac muscle and polyvalent immune serum from a BALB/c mouse infected with 104 plaque-forming units of MCMV 10 days previously (x213).

clarified by centrifugation and the supernatants tested in the ELISA. In a separate experiment, the mAb were incubated with serial log 2 dilutions of MCMV antigen in MOBS with 1% BSA in the competitive inhibition assay. RESULTS

The autoreactivity of anti-MCMV mAb was assessed by

immunoperoxidase histochemistry using composite sections of normal, uninfected mouse organs and tissues, including salivary gland, thymus, lung, heart, liver, kidney, spleen, ileum, striated muscle and brain, fixed in Bouin's fluid. No autoantibody activity was detected in normal BALB/c mouse serum obtained from uninfected animals (Fig. lb, d), although immunoglobulin-positive lymphocytes and plasma cells were stained in lymphoid tissue. As a further control, we tested immunoglobulins prepared from mice injected with the myeloma cell line alone. In concordance with the result of normal mouse serum,

Autoreactive monoclonal antibodies to cytomegalovirus Table 1. Organ and tissue autoreactivity of anti-MCMV monoclonal antibodies detected by immunoperoxidase histochemistry

Anti-MCMV mAb Tissue

1E8

EA4

1B4

4F9

3B2

Myeloma alone*

Salivary gland Thymus Lung Heart Liver Kidney Spleen Ileum Striated muscle Brain

+

+

+

+

-

-

+ + + + + + +

+ + + +

+ + + + + + + +

+ + + +

+ + +

+

-

-

+ +

+

+

* Immunoglobulins prepared from mice injected with the myeloma cell line alone.

no autoantibody reactivity was detected (Table 1). However, the anti-MCMV mAb showed positive autoreactivity, with four out of five mAb showing multiple organ-autoreactivity by immunoperoxidase histochemistry (Fig. la, c, e-g), with various specificities as summarized in Table 1. mAb 1 E8 recognized the cytoplasm of epithelial cells lining

the intra- and interlobular ducts within the salivary glands but did not react with mucous or serous alveolar cells. A strong reaction with bronchial epithelial and alveolar cells in the lung was found, although most cell types within the lung were recognized. 1 E8 also reacted with the cytoplasm of most cell types within the myocardium, liver, brain and adrenal gland. In the kidney, a positive reaction was seen with cells lining tubules but not glomeruli. Muscle fibres in striated muscle and ileum were positively stained, and epithelial cells and the lamina propria of the duodenum reacted strongly. However, 1 E8 did not react with cells of the thymus, spleen or connective tissues of the lung, liver, adrenal gland or kidney. A second mAb, EA4, displayed reactivity with epithelial cells lining the intra- and interlobular ducts and scattered basket cells within the salivary glands. Cytoplasmic staining of the bronchial epithelium of the lung and all cell types of the myocardium, liver, striated muscle and brain was observed. In the kidney all cell types except those of glomeruli were stained. EA4 also reacted with the epithelial cells, stroma and smooth muscle of the ileum. As with 1E8, mAb EA4 did not react with the thymus or spleen. Unlike the two mAb previously described, mAb 1 B4 strongly reacted with the nuclei and perinuclear regions of some alveolar cells in the salivary gland in addition to cells lining the ducts. 1 B4 reacted with the nuclei of most cell types in the myocardium (Fig. I f), brain, lung, liver and striated muscle. For comparison, heart autoreactivity of polyvalent serum from a MCMV-infected mouse is shown in Fig. 1(h). In the kidney, cells in glomeruli and tubules were positive for nuclei staining by 1 B4. mAb IB4 also reacted with most cell types in the spleen and small intestine (Fig. Ia) but did not react with cells of the thymus. mAb 4F9 displayed cytoplasmic and strong nuclear staining

429

of the epithelial cells lining the ducts and nuclei staining only of the basket cells of acini within the salivary glands (Fig. le), alveolar cells of the lung, myocytes (Fig. lc), hepatocytes and cells of the ileum and kidney. However, 4F9 did not react with the spleen or thymus. Perinuclear staining of cells of the brain was observed (Fig. ig). However, only weak cytoplasmic reactivity was seen with striated muscle. In contrast to the above autoreactions, mAb 3B2 reacted weakly with muscle fibres only as assessed by immunoperoxidase histochemistry. Autoreactivity of mAb with polypeptides from normal mouse organ homogenates The reactivity of anti-MCMV mAb with polypeptides from 100,000 g supernatants of homogenized normal, uninfected brain, liver, striated and cardiac muscle and with MCMV polypeptides was analysed by immunoblot (Fig. 2). Approximately 20 to 30 different MW polypeptides were observed by SDSPAGE of the organ preparations (Fig. 2a, some bands have been lost in photographic reproduction). No reaction was observed by immunoblot using the secondary antibody alone and proteins of the brain, liver, striated or cardiac muscle (data not shown). mAb 1 E8 reacted with a common polypeptide of approximately 200,000 MW within brain, liver, striated and cardiac muscle preparations (Fig. 2b). In addition, a positive reaction with a few polypeptides of MW greater than 200,000 MW was seen in the striated muscle extract. mAb EA4 reacted with a single 200,000 MW polypeptide in brain, liver, striated and cardiac muscle preparations (Fig. 2c). mAb 1 B4 reacted strongly with many polypeptides in brain, liver, striated and cardiac muscle (Fig. 2d). Several bands were common to all organ preparations tested, including two polypeptides of approximately 132,000 and 87,000 MW. mAb 4F9 did not react with the available polypeptides in the liver and brain homogenates but reaction with a 200,000 MW polypeptide in striated and cardiac muscle was evident (Fig. 2e). mAb 3B2 was seen to react with a polypeptide of 87,000 MW common to liver and brain, and to a 200,000 MW polypeptide common to both the muscle preparations (Fig. 2f). Some reactivity to the 200,000 MW polypeptide was occasionally seen in normal mouse serum. Immunoglobulins prepared from mice injected with the myeloma cell line reacted with a polypeptide of 200,000 MW present in all organ homogenate preparations. To clarify the possible contribution of normal mouse immunoglobulins precipitated down with the mAb produced in ascites fluid, we tested by immunoblot immunoglobulins precipitated from the supernatant of the 3B2 hybridoma grown in vitro in the absence of normal mouse serum. This preparation of 3B2 gave" a similar pattern of reactivity to polypeptides obtained from liver, brain, cardiac and striated muscle preparations, as shown in Fig. 2f. Thus in the absence of normal mouse immunoglobulins, the anti-viral mAb crossreacted with self polypeptides, including the 200,000 MW

polypeptide. As previously demonstrated by immunoblot, no visible reaction of 1E8 was seen to polypeptides in the virus preparation; however, immunoprecipitation techniques demonstrated reactivity with MCMV.'6 Reactivity of the mAb EA4, 1 B4, 4F9 and 3B2 with MCMV proteins of various MW was similar to our previous findings.'6 Additional reactivities to

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C. M. Lawson et al.

EE

200,000

116,000

92,000 66,t00

45,000

(d)

(e)

(f)

Figure 2. SDS-PAGE and immunoblots of purified MCMV and 100,000 g supernatant proteins of normal, uninfected mouse tissues and organs showing antibody reactivity of MCMV neutralizing monoclonal antibodies 1 E8 (b), EA4 (c), 1 B4 (d), 4F9 (e) and 3B2 (f). SDS-PAGE of molecular weight standards and proteins is shown in (a).

Table 2. Reactivity of anti-MCMV mAb with cardiac myosin mAb

Isotype*

ELISA titret

1E8 EA4

IgG2b

1 B4 3B2 4F9

IgM IgG2b IgG2b

Anti-myosin mAb Normal BALB/c sera Normal BALB/c sera

IgM IgM

256 16 8 64 32 2560 4 (8t) 8 (16t)

IgM

IgG

*mAb isotype was characterized previously'6 and the selected conjugate for each isotype, IgG or IgM used in the ELISA. t ELISA titre (reciprocal endpoint dilution) of antibody reactivity. t ELISA titre for immunoglobulins prepared from pristane-primed BALB/c mice immunized with the myeloma line P3X63Ag.8.653 only.

proteins, including the 200,000 MW polypeptide of MCMV, were observed in this study. The high MW viral proteins were not present in the gels used in our earlier study due to different polyacrylamide concentrations (5-15%) and denaturing conditions of immunoblot to those used here.

Autoreactivity of mAb with cardiac myosin As polyclonal immune serum from BALB/c mice infected with MCMV reacts strongly with the heavy chain of cardiac myosin, a 200,000 MW polypeptide,"' the reactivity of the mAb with cardiac myosin was titrated in the ELISA. Several mAb showed positive reactivity to myosin (Table 2). Low titres of anti-myosin antibodies were found in normal mouse serum and in the immunoglobulin preparation from pristane-primed mice injected with the myeloma cell line alone. A commercial mAb with specificity for the heavy chain of myosin reacted with the mouse cardiac myosin antigen used in the ELISA (Table 2). The myosin reactivity of the five anti-MCMV mAb was confirmed by immunoblot using electrophoresed mouse cardiac myosin (Fig. 3). The commercial anti-myosin mAb also reacted by immunoblot with the purified mouse myosin preparation and the 200,000 MW polypeptide from normal mouse heart homogenate (Fig. 3*).

431

Autoreactive monoclonal antibodies to cytomegalovirus e

c

100

(a)

0~~~~~~ 0

821% E

a

E

E

c

e

FL ca E

75 50

co

25

L w

m

200,000

116,00C 92,00O

50 25

66,000

25

45,000 SOS-PAGE

*

Immunoblbt

Figure 3. SDS-PAGE and immunoblot of cardiac myosin purified from normal, uninfected BALB/c mice using anti-MCMV mAb and a commercial anti-myosin mAb. SDS-PAGE of molecular weight standards and the myosin preparation is shown on the left and the nitrocellulose immunoblot developed with the mAb is shown on the right. In addition, the reactivity of the commercial anti-myosin mAb with the normal mouse heart homogenate preparation is shown on the far left of the nitrocellulose*.

c

,0 4

co0)

O M

20000 116,000

92,000 66,000

45,000 Immunoblot SDS-PAGE Figure 4. SDS-PAGE and immunoblot ofcardiac myosin from normal, uninfected BALB/c mice using anti-MCMV mAb absorbed with either cardiac myosin or BSA-coupled Sepharose 4B.

Reactivity of the anti-MCMV mAb with the heavy chain of myosin could be removed by overnight absorption with cardiac myosin coupled to Sepharose (Fig. 4) and recovered following elution (data not shown), as demonstrated by immunoblot. As a control, the mAb were similarly absorbed with BSA coupled to Sepharose and were observed to retain their reactivity with the heavy chain of myosin (Fig. 4). Inhibition of the binding of the mAb to myosin and MCMV was tested by preincubating a constant concentration of the five anti-MCMV mAb, and as a control the commercial anti-myosin mAb, with various concentrations of mouse cardiac myosin.

1.5

0

Myosin

(,"g)

0 006

0

Figure 5. Competitive inhibition analysis of myosin reactivity of antiMCMV IgG mAb (a) and IgM mAb (b). Inhibition of antibody binding to mouse cardiac myosin was determined using log 4 dilutions of myosin. mAb reactivity to myosin is shown for 1E8 (0), 3B2 (0), 4F9 (A), EA4 (-), 1B4 (0), and the commercial anti-myosin mAb (-).

The remaining reactivity with myosin (Fig. 5) and MCMV was then assessed by ELISA. As expected, the myosin reactivity of the commercial anti-myosin mAb was inhibited in a dosedependent manner (Fig. 5b). In contrast, maximum inhibition of myosin reactivity was only about 40% for mAb EA4 and 1 B4, and merely 20% for mAb 3B2. No significant inhibition of myosin reactivity was observed for mAb 1E8 or 4F9. These results may suggest that the mAb have a low affinity binding site for myosin or alternatively that absorption is incomplete after 1 hr incubation at room temperature. Also, myosin did not inhibit the MCMV reactivity of the anti-MCMV mAb as detected by ELISA (data not shown). Perhaps the binding of the mAb to myosin is through a separate and unrelated antigen binding site to that of MCMV. Furthermore, when MCMV was used instead of myosin as the inhibitor, the myosin reactivity of the mAb EA4 and 1 B4 was similarly inhibited in a dose-dependent manner (55% inhibition was observed with 25 Mg of MCMV inhibitor), confirming their cross-reaction with myosin. As expected, MCMV inhibited the reactivities of the mAb to MCMV antigen (data not shown), indicating that the mAb have a higher affinity for MCMV than for myosin.

DISCUSSION In this paper, we report the autoreactivity of five virus neutralizing mouse mAb produced to structural proteins of MCMV. The mAb recognized normal tissues and cell types with varying specificities as detected by immunoperoxidase histochemistry using Bouin's fluid-fixed sections of normal mouse organs and tissues. These mAb were found to react by immunoblot assay with various polypeptides in normal, uninfected mouse organ homogenate preparations. The antiMCMV mAb were originally screened by indirect immunofluorescence for reactivity with MCMV-infected MEF and were found to display no reactivity with uninfected MEF.'6 This apparent lack of autoreactivity may possibly be due to the relatively limited number of self antigenic determinants represented in MEF cultures. Screening mAb against composite blocks of normal organs and tissues, as outlined in this paper, allows exposure to a substantial part of the host's 'antigenic repertoire'.

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The observation of the anti-MCMV mAb cross-reaction with myosin, albeit of low affinity for some mAb, is noteworthy in view of the finding that polyvalent immune sera from MCMV-infected mice markedly reacts with this major contractile protein.'0 Indeed, BALB/c mice produce cardiac isoformspecific antibodies in parallel with persisting myocarditis. Furthermore, polyclonal immunoglobulins from post-infection sera, affinity purified for cardiac myosin, cross-react with MCMV polypeptides.'7 Passive transfer of these anti-cardiac myosin antibodies from late immune sera of BALB/c mice induces myocarditis in uninfected BALB/c mice.'7 Thus antimyosin antibodies produced following MCMV infection appear to contribute to the pathogenesis of autoimmune myocarditis. mAb of the multiple organ-autoreactive type have been obtained from virus-immunized mice, mice with autoimmune disease'8 and normal uninfected mice.'9 There are three possible scenarios for multiple organ-autoreactivity of mAb.20 Firstly, the mAb may recognize the same epitope in the same proteins in various organs. Secondly, the mAb may recognize the same epitope in different proteins in certain organs and, thirdly, a low affinity mAb may bind to structurally different epitopes on different proteins. Further complex situations may arise if the antigen contains an epitope only partly homologous to the epitope on the cross-reacting antigen.2"22 That is, one mAb binding site might be polyfunctional and accommodate more than one epitope, as 'idiotype-specific antibody' and antigen can bind simultaneously to the same mAb molecule.23 Therefore, the simple interaction of a mAb with two unrelated proteins does not establish molecular identity. In this study, even though the mAb bound myosin they were unhindered in their reactivity with MCMV, suggesting possibly different antigen-binding sites or affinities for the two antigens. Myosin is a structurally complex molecule and most likely presents a wide range of possible antibody-forming sites. Since mAb may recognize antigenic determinants as short as four to six amino acids, a similar and not necessarily identical sequence may be shared between MCMV and myosin. Murine mAb derived from spleen cells of normal, uninfected BALB/c mice have been described to react with myosin. '9 These workers suggest that the antibody reactivity is directed towards the alpha-helical conformational structure of the light meromyosin fragment. Here, we have also found anti-myosin reactivity in normal mouse serum and in immunoglobulins prepared from pristane-primed BALB/c mice injected with the myeloma cell line; however, the titres were low relative with most of the mAb and post-infection polyvalent sera from mice infected with MCMV.'0 Infection with MCMV, in this setting, appears to trigger an autoantibody response with higher titres to those found in the normal, uninfected host. The possible reactivity of the anti-MCMV mAb to the alpha helical conformation of the rod region of the myosin heavy chain rather than to a linear sequence was not distinguished in this study. Apparent homologies in viral and self proteins may lead to the development of autoimmune responses in genetically predisposed individuals.24 Other viruses have been shown to induce the production of mAb specific for the virus but also with reactivity to nuclear and cytoplasmic constituents of uninfected cells. Antibodies against the glycine-alanine copolymer domain of the Epstein-Barr virus nuclear antigen cross-react with a normal cellular protein.25 A mouse neutralizing mAb produced against coxsackievirus B4 has been shown to react with normal

mouse myocardium.26 These workers suggest that there may be common antigenic determinants on normal tissues and coxsackievirus leading to the phenomenon of molecular mimicry. Mice infected with reovirus develop polyendocrine disease with autoantibodies reactive to pancreas, thymus, gastric mucosa and anterior pituitary.27 mAb raised from reovirus type 1infected SJL mice were shown by immunoperoxidase staining to react with certain cells of normal mouse organs. One mAb was shown to react with cells of the pancreatic islets but not with acinar tissue, glucagon, insulin or growth hormone.'8 Srinivasappa et al.28 have screened over 600 mAb made against 11 different viruses using 14 different Bouin's fluid-fixed organs and tissues from normal uninfected mice in a similar manner to that outlined in this study. They found that 3 5% of anti-viral mAb reacted with normal uninfected cells and some were of the multiple organ-reactive type. Therefore, it appears that crossreactivity of anti-viral mAb with self is a phenomenon common to many virus groups. Human CMV encodes host protein homologues.29'30 The coating of CMV with a ubiquitous host protein and/or the incorporation of homologous self molecules in the structure of the virion may in certain individuals facilitate the induction of autoantibodies to that 'host protein' which may potentially lead to autoimmune disease. Although the mAb raised against MCMV described in this study display autoreactivity, their potential significance in eliciting autoimmune disease is unknown.

ACKNOWLEDGMENTS This investigation was supported in part from the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia. Dr C. M. Lawson and Dr H. E. Farrell were supported by W. A. and M. G. Saw Medical Research Fellowships of the University of Western Australia.

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21. 22. 23.

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Murine anti-cytomegalovirus monoclonal antibodies with autoreactivity.

Certain murine monoclonal antibodies (mAb) raised against structural proteins of mouse cytomegalovirus (MCMV) display distinct patterns of multiple or...
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