CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 1994, p. 445-449 Vol. 1, No. 41071-412X/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Antineurofilament and Antiretinal Antibodies in AIDS Patientswith Cytomegalovirus Retinitis

DANIEL F. ROSBERGER,l* SONAM L. TSHERING,1' 2 BRUCE POLSKY,3 M. H. HEINEMANN,RENATE F. KLEIN,4 AND SUSANNA CUNNINGHAM-RUNDLES2

Department of Ophthalmology, 1 The Immunology Laboratory, Department of Pediatric Hematology/Oncology, 2 andDepartment of Microbiology,4 Cornell University Medical College, New York, New York 10021, and Infectious

Disease Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 100213

Received 25 October 1993/Returned for modification 10 January 1994/Accepted 14 April 1994

Sera obtained from AIDS patients with cytomegalovirus (CMV) retinitis before and after treatment withfoscarnet, AIDS patients with human immunodeficiency virus (HIV) retinopathy, AIDS patients without retinaldisease, and normal healthy controls with and without positive CMV serologies were assayed for the presenceof antibodies against the 200-kDa outer, 160-kDa middle, and 68-kDa core subunits of the neurofilamenttriplet. Additional studies were performed to determine the presence of antibodies reactive with proteinsextracted from crude human retinal antigen preparations. Antibodies against the 200-, 160-, and 68-kDaproteins of the neurofilament triplet were detected in 15 of 15 AIDS patients with CMV retinitis. The expressionof these antibodies was unaffected, qualitatively, by successful treatment with foscarnet. In contrast, only 30%oof patients with HIV retinopathy unrelated to CMV, fewer than 35% ofAIDS patients with positive CMV titersbut without evident retinitis, and fewer than 25% of healthy controls with positive or negative CMV titerspossessed antibodies against any of the triplet proteins (P < 0.001). Antibodies against several clusters ofretinal antigens were also identified in the sera of patients with CMV retinitis. In summary, the data indicatethat retinal elements damaged by CMV infection induce an antibody response against the 200-, 160-, and68-kDa components of the neurofilament triplet as well as other, as yet undefined retinal antigens.

Retinal necrosis produced by cytomegalovirus (CMV) infec-tion has become the leading cause of visual field loss anddecreased visual acuity among American AIDS patients, with aprevalence rate reportedly being between 15 and 46% (3, 5,10). Currently, diagnosis of CMV retinitis, progression ofretinal necrosis, and response to antiviral therapy are largelydetermined by clinical examination (8, 22). For the experi-enced examiner, the lesions of CMV retinitis are characteristicand are usually distinguishable from other forms of chorioreti-nitis (caused by other herpesviruses, Candida albicans, Toxo-plasma gondii, and Cryptococcus neoformans), sarcoid, or neo-plastic processes. These, however, may occasionally mimicCMV infection.With the emergence of effective, albeit toxic, therapy for

CMV retinitis (4, 9, 11, 12, 16, 19, 21), the importance oftimely, accurate diagnosis has become clear. Because of con-cerns about treatment toxicity, studies are under way toevaluate the advisability of deferring treatment (20). Since, atpresent, diagnosis, and therefore treatment, must await thevisualization of characteristic retinal lesions, one of the goals ofthe study described here was the demonstration of a serologicmarker of the pathologic process which might indicate early,nonfunduscopically visualizable infection and identify those athigh risk for the development of CMV retinitis.For some years it has been suggested that antibodies di-

rected toward lung small-cell carcinoma and ovarian carci-noma might cross-react with neurofilament proteins and mightbe involved etiologically in the development of the paraneo-plastic syndrome cancer-associated retinopathy (7, 13, 14, 25).Recently, the presence of antineurofilament antibodies has

* Corresponding author. Mailing address: Cornell University Med-ical Center, Box 268, 525 East 68th St., New York, NY 10021. Phone:(212) 746-3414. Fax: (212) 746-8609.

been reported in retinitis pigmentosa (6). These antibodies arepresumed to result secondarily from the release of necroticneural elements which are piled up and which are then capableof stimulating B lymphocytes to produce antibodies reactiveagainst neurofilament proteins.We were interested in whether retinal necrosis associated

with CMV infection might release retinal and/or neural anti-gens capable of stimulating autoreactive B lymphocytes toproduce antibodies against retinal and neurofilament proteins.We examined sera from AIDS patients with CMV retinitis forthe presence of antibodies directed against the 200-, 160-, and68-kDa components of the neurofilament triplet before and 3weeks following treatment with foscarnet. Comparative studieswere performed with sera from AIDS patients with humanimmunodeficiency virus (HIV) retinopathy not associated withCMV, AIDS patients without retinal disease, and healthycontrols with positive and negative CMV titers.

MATERIALS AND METHODS

Patient and control sera collection. AIDS patients withCMV retinitis were recruited from a clinical trial evaluatingfoscarnet treatment of CMV retinitis at the Memorial Sloan-Kettering Cancer Center. AIDS patients with HIV retinopathybut without evidence of CMV retinitis were recruited frompatients seen at The New York Hospital ophthalmology clinic.Diagnosis and evaluation of disease progress were performedroutinely by two of us (M.H.H. and D.F.R.). For patientsparticipating in the foscarnet trial, blood was drawn immedi-ately prior to the initiation of foscarnet treatment and approx-imately 3 weeks thereafter. AIDS patients without retinitiswere recruited from the AIDS Clinical Trial Unit at theMemorial Sloan-Kettering Cancer Center and were monitoredregularly by one of us (B.P.). The medical conditions of allAIDS patients were monitored closely by one of us (B.P.).

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446 ROSBERGER ET AL.

None had prominent neurologic complaints at the time of thestudy. However, routine neuroimaging to rule out occultlesions was not performed.

Healthy controls were recruited from normal volunteers,and CMV titers were determined by enzyme-linked immu-nosorbent assay (Sigma Chemical, St. Louis, Mo.). All patientsreceived a complete ophthalmologic examination, includingdetailed fundus examination, by two of us (M.H.H. andD.F.R.). Only the CMV retinitis patients had the typicalcomplaints of retinal dysfunction, that is, blurring of vision andfloaters. None of the control patients, including those withHIV retinopathy, had visual complaints beyond refractiveerror.

Analysis of neurofilament antibodies. A total of 125 ng eachof the 200-, 160-, and 68-kDa purified neurofilament proteins(ICN Biochemicals, Lisle, Ill.) was spotted separately onto1-cm squares of nitrocellulose paper (Schleicher & Schuell,Keene, N.H.), and the squares were allowed to air dry.Blocking of unbound sites was then performed by using a 5%milk (Carnation Co., Los Angeles, Calif.) solution in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl [pH 7.5]).Following extensive washing in TBS, blots were incubated for2 h in either patient or control sera diluted 1:50 in TBS. Theblots were again washed extensively, but this time in TBS with0.05% Tween 20 (Sigma), and were then incubated for 2 h witha peroxidase-labelled goat anti-human immunoglobulin GAM(1:200 in TBS; Sigma). Following washes in TBS with 0.05%Tween 20, blots were developed by using 50 mg of 4-chloro-1-naphthol (Sigma) in 20% methanol (100 ml) with 0.02%hydrogen peroxide. Monoclonal mouse anti-200-, anti-160-,and anti-68-kDa antibodies (Boehringer Mannheim, Indianap-olis, Ind.) were used as controls.Western blot (immunoblot) analysis. Human cadaveric eyes

(<24 h postmortem) were obtained from the New York EyeBank for Sight Restoration. Each retina was dissected andhomogenized in 0.5 ml of distilled, deionized water by using aDounce homogenizer. Homogenized retinas were then boiledfor 2 min in 2.5 ml of sample buffer (0.0625 M Tris HCl, 2.3%sodium dodecyl sulfate, 10% glycerol [pH 6.5]). The proteinsfrom the equivalent of one retina were resolved on a 1.5-mm12.5% polyacrylamide gel by using a discontinuous buffersystem (15). Gels were then stained with Coomassie brilliantblue (Eastman Kodak, Rochester, N.Y.). Alternatively, theresolved proteins were electroblotted onto nitrocellulose paper(28). Blots were then cut into 1-cm strips and then treated asdescribed above: blocked with 5% milk, washed, incubatedwith patient or control sera, washed, incubated with peroxi-dase-labelled goat anti-human immunoglobulin GAM,washed, and developed.

RESULTS

Presence of antineurofilament antibodies. Sera from Amer-ican Caucasian male homosexual AIDS patients with clinicallydefined CMV retinitis (Fig. 1) who were involved in a clinicaltrial of foscarnet were assayed by dot blotting for the presenceof antibodies reactive with proteins of the neurofilament triplet(proteins of 200, 160, and 68 kDa). In 15 of 15 (100%) patientsinvestigated prior to foscarnet treatment, antibodies against allthree neurofilament proteins were present (Fig. 2; Table 1).Sera from 12 of the original 15 patients were assayed atapproximately 3 weeks after treatment with foscarnet. Serafrom all 12 (100%) patients had retained the antineurofilamentantibodies.

In contrast, in 25 AIDS patients with positive CMV titers butwithout evidence of retinitis, 32% possessed antibodies against

at least one of the triplet (P < 0.001). All of these (32%)possessed anti-200-kDa antibodies, but only six (24% of theoriginal 25 patients) had anti-160-kDa antibodies and only one(4% of the original) had antibodies directed against the68-kDa neurofilament protein. Similar results were noted inthe 10 patients with HIV retinopathy studied. Three (30%)had anti-200-kDa antibodies (P < 0.001) and two (20%) hadanti-160-kDa antibodies. None of the patients with HIV reti-nopathy, but without CMV retinitis, had antibodies against the68-kDa neurofilament subunit.

Healthy controls with and without antibody titers againstCMV were also assayed. Of the 35 healthy controls withpositive anti-CMV titers, 7 (30%) had anti-200-kDa antibod-ies, 2 (5.7%) had anti-160-kDa antibodies, and only 1 (2.9%)had anti-68-kDa antibodies (P < 0.001). Of the 24 healthycontrols with negative CMV titers, 5 (20.8%) had the anti-200-kDa antibodies and none (0%) had anti-160-kDa antibodies (P< 0.001). None of the healthy controls with negative CMVtiters had antibodies directed against the 68-kDa neurofila-ment protein.

Presence of antiretinal antibodies. Further studies wereperformed to investigate the presence of antiretinal antibodiesin American Caucasian male homosexual AIDS patients withCMV retinitis. Sera from six such patients were analyzed byWestern blot analysis by using crude preparations of retinalproteins extracted from human cadaveric eyes (obtained <24 hpostmortem). All six demonstrated the presence of antiretinalantibodies. Antibodies were noted against a high-molecular-mass (120 to 140 kDa) and low-molecular-mass (27 to 30 or 30to 33 kDa) cluster of retinal proteins (Fig. 3). Patients 1 and 2had antibodies against retinal proteins with molecular massesof approximately 140, 120, and 30 kDa. Patient 3 had antibod-ies against only the 33-kDa retinal protein. Patient 4 hadantibodies against the 27-kDa protein as well as antibodiesagainst a retinal protein in the range of 40 kDa. Patient 5 hadthe anti-120-kDa antibodies as well as the 27-, 30-, and 33-kDacluster of proteins. Finally, patient 6 had antibodies directed atthe 30- and 33-kDa retinal proteins. Two normal healthyvolunteers with positive CMV titers and two with negativeCMV titers were used as controls. No antiretinal antibodies ineither the high- or low-molecular-mass clusters were noted inany of the controls. Data for one control with negative CMVtiters are shown in Fig. 3.

DISCUSSION

CMV retinitis is currently the leading cause of acuity andvisual field loss in American AIDS patients, and a largeproportion of these patients progress to blindness. Recently,treatment with ganciclovir (4, 9, 19, 21) and foscarnet (11, 12,16) has produced some very encouraging results. However, theinitiation of therapy must be delayed until the lesions of retinaldamage are already present. Moreover, because of the renaltoxicity of foscarnet and the myelosuppression of ganciclovir(2, 9, 11), controversy surrounds the issue of early treatment(20). The ability to identify high-risk groups is therefore ofgreat importance, especially as less toxic therapies becomeavailable.

In the current study, all 15 (100%) AIDS patients withclinically evident CMV retinitis possessed antibodies againstthe 200-, 160-, and 68-kDa proteins of the neurofilamenttriplet. The presence of these antibodies was unchanged 3weeks following treatment with foscarnet, despite clear clinicalimprovement. This suggests that the antibody response devel-ops secondarily to the release of necrotic retinal antigens in amanner similar to that previously proposed with retinitis

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NEUROFILAMENT ANTIBODIES IN CMV RETINITIS 447

FIG. 1. Fundus photographs of patient 1, before (a) and after (b) foscarnet treatment, demonstrating the characteristic brushfire lesions andvessel sheathing of CMV retinitis and near-total resolution following therapy. The pattern of antineurofilament antibodies, however, remainedunchanged (Table 1).

pigmentosa (6). This is in contrast to cancer-associated reti-nopathy, in which antibody originally directed against ovariancarcinoma or lung small-cell carcinoma (7, 13, 14, 25) cross-reacts with the 200-kDa neurofilament protein and is pre-sumed to have an etiologic role.

It does remain possible that the presence of antineurofila-ment antibodies in CMV retinitis patients might be explainedby the dysregulation of the humoral immune response seen inAIDS which results in generalized, nonspecific B-cell hyperre-activity. However, this seems unlikely, given that only 8 (32%)of 25 AIDS patients without retinitis had anti-200-kDa neuro-

200 160 68 200 160 68 200 160 68 200 160 68

--1 4 7 10

2 5 5 1

6 9 1 2

FIG. 2. Representative dot blot analysis of antineurofilament anti-bodies. In this example, blots 1 to 9 contain sera from the nine AIDSpatients with CMV retinitis prior to foscarnet therapy demonstratingthe presence of antibodies against the 200-, 160-, and 68-kDa compo-nents of the neurofilament triplet. Blots 10, 11, and 12 contain serafrom controls stained with mouse monoclonal anti-200-, anti-160-, andanti-68-kDa antibodies, respectively.

filament antibodies and only 1 patient (4%) had antibodiesagainst the 68-kDa neurofilament protein. We also cannot ruleout the possibility that this patient might be in the earlieststages of retinitis, before funduscopic changes are evident.The 68-kDa protein forms the internal core of the filament,

whereas the 160- and 200-kDa proteins appear to be moreperipherally located (17, 24, 29). It may be that the 68-kDacomponent is exposed to autoreactive B lymphocytes only withnecrosis of or severe damage to the neurofilament. However,antibodies that cross-react with the outer 200-kDa protein, asis seen in cancer-associated retinopathy, may be sufficient tointerfere with vision. We also studied sera from patients withHIV retinopathy, a disease characterized by cotton wool spots,indicative of retinal infarction but without the severe retinalnecrosis seen in CMV retinitis. None of the patients with HIVretinopathy had anti-68-kDa antibodies, suggesting that exten-sive damage to neurofilaments such as that seen in CMVretinitis may be necessary to expose these determinants.

In our study, approximately 20% of normal healthy controlswith or without CMV titers had anti-200-kDa antibodies. Thisis somewhat higher than that in some previous studies (6, 26,27) and lower than that in one other study (24). This mayreflect the increased sensitivity of our method compared withthat of the former study and the increased purities of ourantigen preparations compared with those used in the latterstudy. Antibodies against intermediate filaments have previ-

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448 ROSBERGER ET AL.

TABLE 1. Presence of antibodies reactive with neurofilament triplet proteins

No. of subjects with antibodies/total no. of subjects (%)

Patient group200 kDa 160 kDa 68 kDa Any of thetriplet

AIDS patients with CMV retinitis, pretreatment 15/15- (100) 15/15a (100) 15/15a (100) 15/l15 (100)(ni = 15)

AIDS patients with CMV retinitis, posttreatment 12/12 (100) 12/12 (100) 12/12 (100) 12/12 (100)(n = 12)

AIDS patients without CMV retinitis but with HIV 3/10a (30) 2/10a (20) 0/5 (0) 2/5 (30)retinopathy (n = 10)

AIDS patients with CMV titer but no CMV retinitis 8/25a (32) 6/25a (24) 1/25a (4) 8/25a (32)and normal fundus (n = 25)

Healthy controls with CMV titers (n = 35) 7/35a (20) 2/35a (5.7) 1/35- (2.9) 7/35a (20)Healthy controls without CMV titers (n = 24) 5/24" (20.8) 0/24a (0) 0/24a (0) 5/24a (20.8)

a P < 0.001 by Pearson chi-square test, Yates-corrected chi-square, and Fisher exact test for CMV-positive AIDS patients with CMV retinitis versus CMV-positiveAIDS patients with and without non-CMV retinitis and controls.

ously been reported in sera from patients following systemicviral infections (hepatitis, varicella, rubeola, mumps, and Ep-stein-Barr viruses) (18, 26) as well as in sera from patients withneurologic diseases (1, 27). This may, in part, explain thepresence of antineurofilament antibodies in the general pop-ulation and the marginally increased percentage of AIDSpatients with anti-200-kDa antibodies seen in the currentstudy.None of the controls with negative CMV titers and only one

(2.9%) of the controls with positive CMV titers possessedanti-68-kDa antibodies. If these results are confirmed in largerstudies by using quantitative analysis of antibody titers, thepresence of autoreactive antineurofilament antibodies mayprovide adjunctive criteria for the diagnosis of CMV retinitisand objective criteria for the evaluation of progression andresponse to treatment. It may also facilitate the earlier insti-tution of treatment should less toxic modalities become avail-able (e.g., oral ganciclovir). We have previously reported, in asmall retrospective study, evidence that suggests that anti-68-kDa antibodies may be present preceding the development of

l8OkD*

84kD E

48kD

36kD E

14kD-)N-

AIDS Patients withCMV-retinitis

1 2 3 4 5 6 c

FIG. 3. Western blot demonstrating the presence of antibodiesagainst retinal proteins in the sera of six American male homosexualAIDS patients with CMV retinitis (lanes 1 to 6). One representativeWestern blot with sera from a patient with AIDS but without retinitisis included as control.

CMV retinitis (23). Prospective evaluation of AIDS patientswithout retinitis to determine when antineurofilament antibod-ies become evident in relation to the development of CMVretinal lesions is under way and may help to determine whetherthe presence of anti-68-kDa antibodies defines a high-risksubgroup of AIDS patients likely to develop CMV retinitis.This may permit the initiation of therapy (ganciclovir orfoscarnet) before significant retinal damage has occurred.We also evaluated sera from six AIDS patients with CMV

retinitis for the presence of antibodies against crude retinalantigen preparations. All of the patients had antibodies reac-tive with either a high-molecular-mass (120 to 140 kDa) or alow-molecular-mass (27 to 30 and 30 to 33 kDa) cluster ofproteins. These antibodies were not seen in healthy controls.Additional study is warranted to define the significance of thisfinding, which appears to provide an additional marker ofdisease.

In conclusion, our studies suggest that retinal elementsdamaged by CMV infection induce an antibody responseagainst the 200-, 160-, and 68-kDa components of the neuro-filament triplet as well as other as yet undefined retinalantigens, possibly before grossly evident retinal damage hasoccurred. The presence of these antibodies may provide auseful marker for the diagnosis of CMV retinitis, may assist inidentifying high-risk groups requiring closer monitoring, andmay facilitate the identification of more appropriate treatmentregimens.

ACKNOWLEDGMENTS

We gratefully acknowledge David Le Beck of the Cornell Ophthal-mic Diagnostic Center for photographic assistance.

This work was supported, in part, by The Helena RubensteinFoundation, grant NIAAID N01-A1-62533, The Dyson Foundation,Research to Prevent Blindness, grant NIH HL 437-81, grant NIH NCICA90259, and The Children's Blood Foundation.

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