Disease Virus-Infected Rats Neutralizing Antibodies in Borna

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Neutralizing antibodies in Borna disease virus-infected rats

CG Hatalski, S Kliche, L Stitz, et al.

1995. Neutralizing antibodies in Borna disease jvi.ASM.ORG - virus-infected rats. J. Virol. 69(2):741-747.

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Neutralizing Antibodies in Borna Disease Virus-Infected Rats

1,2 1 3 1 CAROLYN G. HATALSKI, STEFANIE KLICHE, LOTHAR STITZ, AND W. IAN LIPKIN * Laboratory for Neurovirology, Department of Neurology,1 and Department of Anatomy and Neurobiology,2 University of California, Irvine, California 92717, and Institut fu¨r Virologie, jvi.ASM.ORG - Justus-Liebig-Universita¨t, D-35392 Giessen, Germany3

Received 8 August 1994/Accepted 2 November 1994

Borna disease is a neurologic syndrome caused by infection with a nonsegmented, negative-strand RNA

virus, Borna disease virus. Infected animals have antibodies to two soluble viral proteins, p40 and p23, and a at COLUMBIA UNIVERSITY July 22, 2010 membrane-associated viral glycoprotein, gp18. We examined the time course for the development of neutralization activity and the expression of antibodies to individual viral proteins in sera of infected rats. The appearance of neutralizing activity correlated with the development of immunoreactivity to gp18, but not p40 or p23. Monospeciﬁc and monoclonal antibodies to native gp18 and recombinant nonglycosylated gp18 were also found to have neutralizing activity and to immunoprecipitate viral particles or subparticles. These ﬁndings suggest that gp18 is likely to be present on the surface of the viral particles and is likely to contain epitopes important for virus neutralization.

Borna disease virus (BDV) is a nonsegmented, negative- Virus titration and neutralization assay. Viral infectivity in 20% brain homo- strand RNA virus (5, 7, 11, 13) that causes an immune-medi- genates was determined by the method of Pauli et al. (44). Virus neutralization was performed with a modification of the method of Danner et al. (12). Briefly, ated neurologic illness (53) in a wide variety of naturally in- 50 FFU of BDV was incubated with serial dilutions of antibodies or sera for 1 h fected animal hosts, including birds, cats, sheep, and horses at 37ЊC, added to rabbit fetal glial cells, and incubated for 5 days. Serum was heat (19, 34, 36–38, 42, 63). Experimental infection in rats (40) inactivated at 56ЊC for 30 min. In selected assays, mouse complement (1:50) results in a multiphasic syndrome characterized by hyperactiv- (Sigma, St. Louis, Mo.) was added to the virus concurrent with the addition of ity, stereotyped behaviors, dyskinesias, and dystonias (52). MAbs to determine the effects of complement on neutralization activity. The The viral antigenome contains five open reading frames in dilution of serum or antibody required to reduce the number of FFU by 50% was defined as the neutralization titer (NT50). As controls for each neutralization the following 5Ј-to-3Ј order: p40, p23, gp18, p57, and pol (7, assay, rabbit fetal cells were exposed to medium without virus, treated with virus 11). mRNAs (25, 30, 39, 46, 56, 58, 61) and proteins (3, 18, 31, in medium alone (no antibodies), or treated with virus incubated with sera from 49, 57) corresponding to three of these open reading frames, normal rats. Pilot studies showed that approximately 8% of normal rat sera p40, p23, and gp18, have been found in infected cells and interfered with BDV infectivity at dilutions up to 1:16. Therefore, sera were considered to be neutralizing only if the NT50 exceeded 1:32. Supernatant from tissues in a 5Ј-to-3Ј expression gradient (p40 Ͼ p23 Ͼ gp18) (7, nonproducing myeloma cell lines, as well as MAbs directed against BDV-p23 11, 17, 26, 47). Antibodies to p40 and p23 (soluble antigens) (24/36F1) and BDV-p40 (38/17C1) (57), were found to neutralize infectivity at are readily detected in both sera and cerebrospinal fluid of dilutions of 1:2. Thus, MAbs were considered to be neutralizing only if the NT50 naturally and experimentally infected animals (31, 33, 35). An- exceeded 1:4. tibodies to gp18, a membrane-associated glycoprotein (previ- Preparation of proteins (recp40, recp23, recp18, and gp18). Plasmids encoding p40 (pBDV-40 [39]), p23 (pBDV-23 [56]), and gp18 (pBDV-gp18 [25]) were ously described as 14.5 kDa), have been reported less fre- subcloned (6) into the prokaryotic expression vector pet15b (Novagen, Madison, quently (31, 48). Although neutralization activity has been Wis.). Recombinant proteins (recp40, recp23, and recp18) were expressed in found in sera of animals infected with BDV (12, 22, 31, 32), Escherichia coli and purified according to the manufacturer’s protocol (Nova- the antibodies responsible for neutralization activity have not gen). Purity and antigenicity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (immunoblot) analysis been investigated. An enzyme-linked immunosorbent assay with sera from infected rats. Native, glycosylated gp18 was prepared from in- (ELISA) based on recombinant BDV proteins has been estab- fected rat brain as described previously (49). lished (6) that provides a sensitive method for detection of ELISA. ELISA was performed as described in reference 6. Briefly, plates antibodies to gp18. We find that the appearance of neutralizing coated with recombinant protein were incubated with serially diluted sera or antibodies in infected rats correlates with immunological reac- MAbs. Bound horseradish peroxidase (HRPO)-coupled secondary antibody [goat anti-mouse F(abЈ)-HRPO, goat anti-rat immunoglobulin G (IgG), and tivity to gp18. Furthermore, monospecific and monoclonal an- IgM-HRPO; Sigma] was quantified on a microplate reader (Thermo max; Mo- tibodies (MAbs) directed against gp18 neutralize BDV infec- lecular Devices, Menlo Park, Calif.) by using the chromagen 3,3Ј-5,5Ј-tetrameth- tivity and immunoprecipitate viral particles or subparticles. ylbenzidine (Sigma). The ELISA end point titer was defined as the serum or antibody dilution that generated an optical density of 0.3. SDS-PAGE, Western blot and immunoprecipitation (IP). Recombinant or MATERIALS AND METHODS native BDV proteins were subjected to SDS-PAGE (27) and transferred to nitrocellulose (Schleicher & Schuell, Inc., Keene, N.H.) or Immobilon-N mem- BDV-infected animals. Sixty-thousand focus-forming units (FFU) of BDV branes (Millipore Corp., Bedford, Mass.) (59). Membranes were blocked and He/80-1 (8, 20, 50) was used to intranasally (i.n.) infect each of 70 6-week-old incubated with primary antibody as described by Briese et al. (6). After incuba- Lewis rats. Rats were observed at 3-day intervals for weight loss, ruffled fur, or tion with secondary antibody (goat anti-mouse IgG-alkaline phosphatase, goat postural abnormalities consistent with acute disease. Sera were collected at the anti-rat IgG-alkaline phosphatase, or goat anti-rat IgG and IgM-HRPO [Sig- time of sacrifice. Under metofane anesthesia, rats were perfused with buffered ma]), immune complexes were visualized with Western Blue (Promega Madison, 4% paraformaldehyde; brains were fixed overnight in perfusate at 4ЊC. Twenty- Wis.) for alkaline phosphatase or chemiluminescence (ECL kit; Amersham, micron sagittal sections were collected onto gelatin-coated slides and stained Arlington Heights, Ill.) for HRPO according to the manufacturer’s instructions. with hematoxylin and eosin. Inflammation was scored by using the scale of Stitz, gp18 and recp18 were precipitated by using sera from infected rats, monospecific Sobbe, and Bilzer (54). antibodies, or MAbs and protein A-Sepharose (Pharmacia Biotech Inc., Piscat- away, N.J.) as described by Persson et al. (45) and then were assayed by Western blot. MAbs. MAbs to gp18 were generated according to the method of Thiedemann * Corresponding author. Phone: (714) 824-6193. Fax: (714) 824- et al. (57). Briefly, BALB/c mice were immunized intraperitoneally with 5 ␮gof 2132. Electronic mail address: [email protected]. gp18 in complete Freund’s adjuvant. Three and 6 weeks after the initial immu-

741 DOWNLOADED FROM 742 HATALSKI ET AL. J. VIROL. nization, mice were given intraperitoneal booster immunizations with 5 ␮gof gp18 in incomplete Freund’s adjuvant. Four days before fusion of spleen cells with cells from the mouse myeloma cell line X63-Ag8.653 (24), mice were given an intravenous booster immunization with 15 ␮g of gp18. All hybridomas were initially screened for reactivity to gp18 by ELISA. Tissue culture supernatants from positive hybridomas were concentrated by ammonium sulfate precipitation (23) and tested by Western blot and IP for reactivity with gp18 and recp18. The Ig isotype was determined with an agglutination isotyping kit (Serotec, Oxford, England) according to the manufacturer’s instructions. MAb 24/36F1, directed against BDV-p23 (57), was used as a negative control in Western blot and IP jvi.ASM.ORG - experiments. Generation of polyclonal sera against the 18-kDa protein. To produce antibodies against recp18, two 2-month-old Lewis rats were injected subcutaneously with 25 ␮g of protein in Freund’s complete adjuvant and were given booster injections 3 weeks later with 25 ␮g of protein subcutaneously in Freund’s incomplete adjuvant. After 6 weeks, animals received intraperitoneal injections of 25

␮g of protein in phosphate-buffered saline (PBS) with 20 ␮g of lipopolysaccha- at COLUMBIA UNIVERSITY July 22, 2010 ride (Salmonella typhimurium; Difco, Detroit, Mich.) at 2-week intervals for a total of three injections. Serum was collected every 2 weeks during weeks 7 through 14 for analysis by ELISA and Western blot and for determination of NT. Mouse antibodies to native gp18 have been described previously (25). Affinity adsorption of BDV-specific serum antibodies. Antibodies that bound to recp23 and recp40 were sequentially removed from the serum of an infected rat according to the method of Crabb et al. (10). Serum (D2) from an adult BDV-infected Lewis rat (15 weeks post-i.n. infection), was diluted 1:10 in TBS (Tris-balanced saline, consisting of 50 mM Tris [pH 7.4] and 100 mM NaCl) and incubated overnight at 4ЊC with membrane-bound recp23. The anti-recp23 antibody-depleted serum (D2 ⌬␣recp23) was removed, the membrane was washed with TBS, and adsorbed anti-recp23 antibodies were eluted (recp23 eluant) by incubation with 1 ml of 0.1 M glycine–0.15 M NaCl (pH 2.7) for 3 min. The pH of the eluant was adjusted by addition of 300 ␮l of 10 mM Tris-HCl (pH 7.5). The anti-recp23 antibody-depleted serum was then incubated with membrane-bound recp40 (D2 ⌬␣recp23, ⌬␣recp40) and purified as before (recp40 eluant). Anti- body depletion from serum and antibody elution from membrane-bound proteins were monitored by Western blot and ELISA. At each step during the purification, antibody-depleted sera and eluted antibodies were analyzed for neutralizing activity. Antibodies to gp18 or recp18 were also adsorbed (D2 ⌬␣gp18, D2 ⌬␣recp18) and eluted (gp18 eluant, recp18 eluant) by this method. These adsorption and elution experiments were repeated with serum (B3) from an additional adult BDV-infected rat (15 weeks post-i.n. infection). IP of BDV particles or subparticles and analysis by RT-PCR. Forty-thousand FFU of BDV in a volume of 200 ␮l was treated with 50 ␮g of DNase I and RNase A per ml (Boehringer Mannheim Corp., Indianapolis, Ind.) for 30 min at 37ЊC and then was incubated for2hatroom temperature with 100 ␮l of one of the following: (i) serum from acutely or chronically infected rats at 1:10 dilution in PBS, (ii) purified serum antibodies at a 1:10 dilution, (iii) mouse anti-gp18 sera or rat anti-recp18 sera at 1:20 dilution, or (iv) MAbs against gp18 at a 1:5 dilution. Next, 100 ␮l of 1-mg/ml protein A-Sepharose (Pharmacia) in PBS was added, and the mixture was incubated overnight at 4ЊC. The protein A-Sepha- rose-antibody-virus complex was washed three times in PBS and then resus- pended in 100 ␮l of water. Total RNA was extracted (9) and used for reverse transcription (RT)-PCR amplification of a 693-nucleotide region of the viral genome (nucleotides 753 to 1446) according to the method of Schneider et al. (primers 7 and 9) (50). PCR products were analyzed by agarose gel electrophoresis. PCR products were cloned and sequenced to confirm that they represented the predicted region of the genomic RNA (50). Negative controls for RT-PCR included the omission of virus from IP reaction mixtures and the use of genomic sense primers during first-strand cDNA synthesis.

RESULTS

Time course of disease and appearance of antibodies to BDV FIG. 1. Time course for the appearance of antibodies to BDV proteins in in infected rats. Rats developed Borna disease within 5 weeks serum samples from individual rats after i.n. infection. (A) Neutralization activity postinfection (p.i.). The acute phase of the disease, 4 to 8 in sera from BDV-infected rats at three time points (5, 10, and 15 weeks p.i.). weeks p.i., was associated with marked weight loss (data not Each serum is represented by a circle. Bars indicate mean NTs for each group (5, shown), disheveled fur, dystonic posture, hind limb paresis and 10, or 15 weeks p.i.). The asterisk represents sera with NTs less than or equal to 1:16. (B) Plot of mean recp18 ELISA titers (open columns) with neutralization paralysis, mortality of 35%, and prominent inflammatory cell titers (hatched columns) at three time points (5, 10, and 15 weeks p.i.). Sera infiltrates in the brain. In the chronic phase of disease, 10 to 15 analyzed were the same as those in panel A. Mean values for neutralization weeks p.i., signs of disease stabilized and inflammation reactivity were determined as described for panel A. Arrows indicate threshold for ceded. Virus titers in the brains of animals acutely (5 weeks significance in the neutralization assay (1:32) and recp18 ELISA (1:250). These values were selected because normal rat sera reacted in the neutralization assay p.i.) and chronically (15 weeks p.i.) infected were (2.4 Ϯ 0.4) ϫ and recp18 ELISA at titers of 1:16 and 1:125, respectively. (C) Time course for 5 4 10 and (4.4 Ϯ 0.2) ϫ 10 FFU/ml, respectively. the appearance of antibodies to recp40, recp23, and gp18 by Western blot Sera were monitored for virus neutralization activity (Fig. analysis. Proteins were size fractionated by SDS-PAGE and transferred to ni- 1A and B) and the presence of antibodies reactive with recp40, trocellulose membranes. Membranes were incubated first with sera and then with HRPO-coupled goat anti-rat IgG. Bound secondary antibody was detected by recp23, recp18, or native gp18 in Western blot (Fig. 1C) and chemiluminescence. Results shown are from serum of one representative animal ELISA. Neutralization activity was first detected in sera (28% at several different time points post-BDV infection. of the animals) at 5 weeks p.i. By week 15 p.i., all sera had DOWNLOADED FROM VOL. 69, 1995 NEUTRALIZING ANTIBODIES IN BORNA DISEASE 743

TABLE 1. Characterization of serum antibodies

Reciprocal Reciprocal Serum IP-RT-PCRa rp18 ELISA NT titer Chronic (15 wk p.i. [D2]) 1,000–1,500 ϩ 4,300–5,000 D2 ⌬␣recp23, ⌬␣recp40b 1,000–1,500 ϩ 4,000–5,000 D2 ⌬␣recp18b 600–700 ϩ NSc jvi.ASM.ORG - D2 ⌬␣gp18b 160–200 ϩ 800–840 recp18 eluantd 60–100 ϩ 2,400–3,000 gp18 eluantd 240–400 ϩ 1,200–2,000 recp23 eluantd NS Ϫ NS recp40 eluantd NS Ϫ NS Rat␣recp18 320–480 ϩϾ5,000

Mouse␣gp18 160–320 ϩϾ5,000 at COLUMBIA UNIVERSITY July 22, 2010

a IP of BDV and detection of genomic RNA by RT-PCR. b Chronic rat sera (D2) adsorbed with recombinant (recp23, recp40, recp18) or native (gp18) protein. c NS, not significant. The NT was considered to be not significant below 1:32; the recp18 ELISA titer was considered to be not significant below 1:250. d Antibodies in chronic rat sera (D2) eluted from recombinant or native protein. FIG. 2. MAb detection of gp18. (A) IP of gp18 with MAbs. gp18 was first incubated with MAbs or sera from infected or noninfected rats and then was precipitated with protein A-Sepharose, size fractionated by SDS-PAGE (12% neutralization activity with a mean titer of 1:977 Ϯ 1:246. polyacrylamide), and transferred to Immobilon-N membranes. Precipitated gp18 Antibodies to recp18 were first detected by ELISA at week 5 was visualized with rat anti-recp18 sera, goat anti-rat IgG-AP, and Western Blue. Lanes: 1, serum from infected rat (15 weeks p.i.); 2, serum from noninfected rat; p.i. and showed a marked increase in titer by 15 weeks p.i. 3, MAb 14/29A5; 4, MAb 14/26B9; 5, MAb 14/8E1; 6, MAb 14/13E10; 7, MAb (1:4,610 Ϯ 1:1,463) (Fig. 1B). In contrast, antibodies reactive 14/18H7; 8, MAb 24/36F1 (MAb directed against the BDV 23-kDa protein with recp40 and recp23 were detected by ELISA within 4 [negative control]); 9, no antibody. The arrow indicates gp18; H and L represent weeks of infection, reached a titer greater than 1:20,000 by 8 heavy and light chains of Ig, respectively. (B) MAbs were analyzed for binding to native gp18 in Western blot. gp18 was separated by SDS-PAGE (12% polyacryl- weeks p.i., and remained elevated through 15 weeks p.i. (6). amide) and transferred to an Immobilon-N membrane. Strips were incubated Antibodies reactive with recp40 and recp23 were detected by with MAbs or sera from infected or noninfected rats. Bound antibodies were Western blot between weeks 4 and 5 p.i., whereas antibodies to detected with alkaline phosphatase-conjugated goat anti-rat IgG or goat anti- mouse F(ab)-specific antibodies and Western Blue substrate. Lanes are as de- gp18 were detectable only after week 10 p.i. (Fig. 1C). 3 Affinity adsorption of neutralizing sera. To determine scribed for panel A. Molecular mass markers (10 Da) are shown at the right. whether the presence of antibodies to gp18 correlates with neutralization activity, two rat serum samples (D2 and B3, 15 weeks p.i.) were tested in the neutralization assay after succes- tibodies eluted from proteins recp40 (recp40 eluant) and sive depletions of antibodies to individual BDV proteins. An- recp23 (recp23 eluant) had no neutralization activity (Table 1). tibodies to BDV-specific proteins were removed from D2 rat In contrast, the NT50 of the D2 serum was 1:1,000 to 1:1,500 serum by adsorption with membrane-bound protein. The effi- prior to adsorption with recp18 (D2 ⌬␣recp18) and decreased ciency of antibody depletion from serum was monitored by to 1:600 to 1:700 after adsorption with recp18 (D2 ⌬␣recp18) Western blot (data not shown) and ELISA. Prior to adsorp- and to 1:160 to 1:200 after adsorption with gp18 (D2 ⌬␣gp18) tion, the titers to recp40 and recp23 were each greater than (Table 2). The NTs of antibodies eluted from recp18 (recp18 1:20,000. After adsorption with recp23, the titer to recp23 eluant) and gp18 (gp18 eluant) were 1:60 to 1:100 and 1:240 to decreased to 1:200. After adsorption with recp40, the titer to 1:400, respectively (Table 1). Similar results were obtained recp40 decreased to 1:150. Eluted antibodies were reactive by with serum from rat B3 (data not shown). ELISA with the proteins used for adsorption: recp23 eluant Monospecific antibodies to recp18 and gp18. Sera from rats titer, 1:5,000; recp40 eluant titer, 1:15,000. Serum antibodies and mice immunized with recp18 and gp18, respectively, were remaining after adsorption and eluted antibodies were then tested for neutralization activity. Neutralizing antibodies in tested for neutralizing activity. The NT of the D2 serum (NT50, both rats and mice were detected at 12 weeks postimmuniza- 1:1,000 to 1:1,500) did not change after adsorption with recp23 tion. Sixteen weeks after immunization with recp18, two rats and recp40 antigens (D2 ⌬␣recp23, ⌬␣recp40) (Table 1). An- had NTs between 1:320 and 1:480 (Table 1); serum samples

TABLE 2. Characterization of anti-gp18 MAbs

Characterization by: Reciprocal Reciprocal rp18 MAb Ig class Western blot IP IP-RT-PCRa NT ELISA titer gp18 rp18 gp18 rp18 14/29A5 IgG2b Ͼ400–1,000 ϩϩϩϩ 500–1,000 Ϫ 14/26B9 IgM 8–16 ϪϪϩϩ 4–8 ϩ 14/8E1 IgM 50 ϪϪϩϩ 200–300 ϩ 14/13E10 IgM 50–100 ϪϪϩϩ 16–64 ϩ 14/18H7 IgG3 100–200 ϪϪϩϩ 128–256 ϩ

a IP of BDV and detection of genomic RNA by RT-PCR. DOWNLOADED FROM 744 HATALSKI ET AL. J. VIROL. jvi.ASM.ORG - at COLUMBIA UNIVERSITY July 22, 2010

FIG. 3. Neutralization proﬁle of sera and MAbs. BDV (50 FFU [ffu]) was preincubated with serial dilutions of serum or MAb and then added to 10,000 rabbit fetal glial cells. After 4 days of incubation, the infected cells were visualized as described by Pauli et al. (44). The number of infected cell foci per well was counted. (A) Serum from noninfected rat. (B) serum from infected rat (15 weeks p.i.; D2). (C) MAb 14/13E10. (D) MAb 14/29A5.

from two mice immunized with gp18 had NTs between 1:160 (data not shown). Serum from noninfected (normal) rats was and 1:320 (Table 1). not neutralizing at dilutions greater than 1:16 (Fig. 3A). MAbs to gp18. MAbs were generated against gp18. Five IP of BDV with neutralizing antibodies. BDV stock was positive clones were identified by ELISA with gp18 as antigen. treated with nucleases to eliminate free nucleic acids and then The MAbs represented three different Ig isotypes, yet all con- was incubated with sera or MAbs and protein A-Sepharose. tained the kappa light chain (Table 2). Although each of the RNA was extracted from immunoprecipitated viral particles or MAbs immunoprecipitated gp18 (Table 2 and Fig. 2A) and subparticles and subjected to RT-PCR for amplification of recp18 (Table 2), only MAb 14/29A5 reacted by Western blot viral genomic RNA. Neutralizing rat sera (Fig. 4A), monospe- (Table 2 and Fig. 2B). cific sera to recp18 (Fig. 4B) or gp18 (Fig. 4B), and D2 serum Concentrated supernatants from all five MAbs neutralized antibodies eluted from recp18 or gp18 precipitated BDV par- BDV infectivity (Table 2). Similar to sera from chronically ticles. Removal of antibodies to recp23, recp40, recp18, or infected rats (Fig. 3B), the NT of four MAbs was greatest at gp18 did not affect the capacity of neutralizing sera to precip- the highest antibody concentration (Fig. 3C). In contrast, one itate viral particles. Four MAbs also precipitated BDV (Fig. MAb, 14/29A5, neutralized BDV only when used at a dilution 4B). One MAb, 14/29A5, did not precipitate viral particles at of 1:400 to 1:1,000 (Fig. 3D). Neutralizing sera from chroni- any dilution (1:5, 1:100, 1:200, or 1:500). Serum samples from cally infected rats, mice immunized with gp18 (23), or rats noninfected or two acutely infected rats (5 weeks p.i.) (Fig. 4A) immunized with recp18 (Table 1) had the capacity to inhibit did not precipitate BDV. Experiments with sera and MAbs are 100% of BDV infectivity (Fig. 3B). In contrast, MAbs to gp18, summarized in Tables 1 and 2. Negative controls for RT-PCR used individually or in concert, inhibited a maximum of 68% of included the omission of virus from IP and the use of genomic BDV infectivity (Fig. 3C and D). Supernatants of two MAbs, sense primers for first-strand cDNA synthesis.

14/18H7 (NT50, 1:16) and 14/13E10 (NT50, 1:32), showed co- operativity in neutralization assays; pooling of these MAbs DISCUSSION resulted in a higher NT (NT50, 1:100 to 1:150). To determine the extent to which neutralization was complement dependent, The presence (or absence) of neutralizing antibodies in neutralization activity of MAbs was tested with addition of BDV-infected animals has been controversial. Some reports either active or heat-inactivated mouse complement. No in- have not shown evidence for neutralizing antibodies (8, 21, 41); crease in NT was detected with addition of mouse complement however, this may reﬂect different time points for collection of DOWNLOADED FROM VOL. 69, 1995 NEUTRALIZING ANTIBODIES IN BORNA DISEASE 745

says, indicating that it bound to a continuous epitope. Unlike the other MAbs, 14/29A5 neutralized infectivity only after dilution (Fig. 3D), a profile consistent with neutralization by virus aggregation as reported in other viral systems (15, 43). Although all of the gp18 MAbs detected recp18 (nonglycosylated protein), it is possible that there are additional epitopes for neutralization which include the carbohydrate portion of gp18 (55). jvi.ASM.ORG - Sera from chronically infected rats had greater neutralization activity than monospecific sera or MAbs directed against gp18. Higher neutralization activity in sera from infected animals could reflect factors that influence epitope presentation, such as interactions between gp18 and other proteins or the

virion envelope. Alternatively, gp18 may not be the only BDV at COLUMBIA UNIVERSITY July 22, 2010 protein that elicits neutralizing antibodies. Sera from chronically infected animals retained partial neutralizing activity and the capacity to precipitate virus after adsorption with gp18. Although this may be due to incomplete subtraction of anti- FIG. 4. RT-PCR of immunoprecipitated BDV. Virus was treated with nucle- bodies to gp18 (Table 1), neutralizing antibodies may be di- ases to eliminate nucleic acid not contained within virions and then was immu- rected against other viral proteins as well. For example, an noprecipitated with sera or MAbs and protein A-Sepharose. RNA was extracted additional candidate for a virion surface protein that may elicit and subjected to RT-PCR to amplify a 693-nucleotide viral genomic sequence. PCR products were visualized in an ethidium bromide-stained 1% agarose gel. neutralizing antibodies is p57. This putative protein contains (A) Precipitation of BDV with sera from infected rats. Lanes: 1, serum from multiple potential N-glycosylation sites and, as the product of infected rat (15 weeks p.i.); 2, serum from infected rat (5 weeks p.i.); 3, serum the fourth open reading frame on the BDV genome, is in the from infected rat (15 weeks p.i. [no BDV]); 4, serum from infected rat (15 weeks gene position generally occupied by glycoproteins in members p.i.), genome sense primer used for first-strand cDNA synthesis. (B) Precipita- tion of BDV by using anti-recp18 or anti-gp18 antibodies. Lanes: 1, monospecific of the Mononegavirales family (7). rat antisera to recp18; 2, monospecific mouse antisera to gp18; 3, anti-gp18 MAb There are several plausible explanations for the late appear- 14/13E10; 4, anti-gp18 MAb 14/29A5. DNA markers (base pairs) are shown to ance of neutralizing antibodies in Borna disease. One possibil- the right. ity is that, in early disease, gp18 is expressed at lower levels than the 40- and 23-kDa viral proteins, which elicit high-titer antibodies. A second possibility is that gp18, which is mem- sera or variation in the assay system for neutralization. Al- brane-associated (49), may be restricted to the central nervous though there are reports of neutralizing antibodies in serum system until late in disease. In contrast, the soluble antigens and cerebrospinal fluid of both naturally and experimentally p40 and p23 may diffuse outside of the central nervous system infected animals (12, 22, 31, 32), neither the time course for in early disease to serve as immunogens. Support for this hy- development of neutralizing antibodies nor their target anti- pothesis comes from studies that show accumulation of soluble gens have been characterized. Here we show that the neutral- antigens (p40 and p23) in distal renal tubule cells 10 days after izing activity of BDV-infected rat sera increases dramatically i.n. infection (4). Two alternative explanations for the later from the acute (5 weeks p.i.) to the chronic (15 weeks p.i.) appearance of neutralizing antibodies have been proposed for phase of disease and provide evidence to indicate that neutral- mice infected with lymphocytic choriomeningitis virus, on the ization activity is due, at least in part, to antibodies that react basis of the observation that CD8ϩ T-cell depletion accelerates with a BDV glycoprotein, gp18. The time course for the ap- the appearance of neutralizing antibodies. Battegay et al. sug- pearance of neutralizing antibodies seems to correlate with gest either that (i) CD8ϩ T cells play a role in inhibiting or immunoreactivity to gp18. Furthermore, removal of antibodies deleting B cells specific for neutralizing antibodies or (ii) to gp18 or recp18 dramatically decreased the NT of BDV- CD8ϩ T cells effect generalized immunosuppression after B infected rat sera. In contrast, subtraction of antibodies to two cells specific for nonneutralizing epitopes are converted to other viral proteins, p40 and p23, had no effect. plasma cells (2). Neutralization activity was detected with monospecific anti- In some neurotropic viral infections, neutralizing antibodies serum against both gp18 and recp18, as well as with MAbs have been implicated in viral clearance and protection from against gp18. These MAbs represent three different isotypes, fatal disease (1, 14, 16, 28, 29, 51, 60, 62). The role of neutral- IgM, IgG2b, and IgG3, indicating that multiple isotypes are izing antibodies in Borna disease is less clear, given that central capable of virus neutralization. Addition of complement did nervous system viral titers remain elevated in the presence of not enhance neutralization activity of the MAbs, suggesting neutralizing antibodies in serum and cerebrospinal fluid. Fu- that the mechanism for neutralization was neither comple- ture work will be directed toward determining whether passive ment-mediated inactivation of virus nor steric hindrance by a administration of neutralizing antibodies or immunization with complement-MAb-virus complex. gp18 and other virion surface proteins can alter BDV patho- It is likely that at least three different antibody binding sites genesis. on gp18 were involved in neutralization. Four MAbs, which immunoprecipitated both gp18 and recp18 but did not detect ACKNOWLEDGMENTS protein in Western blots, presumably bound to discontinuous epitopes. The observation that use of MAbs 14/13E10 and C. Hatalski and S. Kliche contributed equally to this work. We thank E. Robinson, K. Tyler, T. Briese, and members of the Neurovirology 14/18H7 in combination resulted in greater neutralization ac- Laboratory for criticism of the manuscript. tivity than use of either MAb alone suggests that these MAbs Support was provided by NS-29425, Deutsche Forschungsgemein- recognized either different discontinuous epitopes or different schaft (Forschergruppe ‘‘Pathogenita¨tsmechanismen von Viren’’), and binding sites on a single discontinuous epitope. One MAb, the American Foundation for Aging Research. W.I.L. is the recipient 14/29A5, detected protein in Western blots, as well as IP as- of a Pew Scholars award from the Pew Charitable Trusts. L.S. is a DOWNLOADED FROM 746 HATALSKI ET AL. J. VIROL. recipient of a Schilling Professorship for Clinical and Theoretical Med- in rats. J. Virol. 64:706–713. icine. 30. Lipkin, W. I., G. H. Travis, K. M. Carbone, and M. C. Wilson. 1990. Isolation and characterization of Borna disease agent cDNA clones. Proc. Natl. Acad. REFERENCES Sci. 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