JOURNAL OF VIROLOGY, Apr. 1997, p. 3105–3113 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 71, No. 4
Major Linear Antibody Epitopes and Capsid Proteins Differentially Induce Protective Immunity against Theiler’s Virus-Induced Demyelinating Disease HIROYUKI YAHIKOZAWA,1 ATSUSHI INOUE,1,2 CHANG-SUNG KOH,2 YONG-KYUNG CHOE,1,3 AND BYUNG S. KIM1* Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, Illinois 606111; Department of Medicine (Neurology), Shinshu University of Medical School, Matsumoto 390, Japan2; and Korea Research Institute of Bioscience and Technology, Korea Institute of Science and Technology, Taejon, Korea3 Received 19 August 1996/Accepted 13 January 1997
Theiler’s murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEVIDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VP1 and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1262–276), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection. TMEV: one on each external capsid protein, i.e., VP1233–244 (54), VP274–86 (16), and VP324–37 (53). Among the T-cell epitopes, VP1233–244 and VP274–86 appear to be responsible for the pathogenesis of TMEV-IDD as CD41 T cells specific for these epitopes are found in the inflammatory lesions of the CNS (54). Therefore, cell-mediated immunity is likely to set off a chronic immunologically mediated inflammatory response leading to demyelination. For continuous stimulation of inflammatory immune responses, viral persistence in the CNS seems to be critical (33). This is consistent with the observation that only susceptible mice, but not resistant mice, develop a life-long chronic infection in the CNS (28). Moreover, the conversion from resistance to susceptibility to TMEV-IDD mediated by treatment with lipopolysaccharides results in increased viral persistence in the CNS, suggesting a correlation between susceptibility and viral persistence (44). Many virally induced diseases can be prevented by vaccinations of the host prior to viral infection. Clinical and epidemiological studies indicate that humoral immunity plays a major role in the protection against picornaviruses (20, 38). Recently, determinants on viral capsid proteins of many different picornaviruses which are recognized by antibodies neutralizing viral infection in vitro have been identified. Such neutralizing antibodies are found to protect the host from the corresponding picornaviruses (5, 32, 36, 55). Antibody epitopes which are involved in neutralization of TMEV apparently include the regions of VP1101 (56), VP1268 (40), and VP1225–276 (50). Using fusion proteins and synthetic peptides, we have previously identified six major linear antibody epitopes on the capsid proteins of TMEV BeAn 8386 strain (21, 23): A1A (VP112–25), A1B (VP1146–160), A1C (VP1262–276), A2A (VP22–16), A2B (VP2165–179), and A3A (VP324–37). Among the antibodies specific for these epitopes, only antibodies to A1C, A2A, and A2B
Theiler’s murine encephalomyelitis virus (TMEV) belongs to the picornavirus family. Like other picornaviruses, TMEV has an icosahedral capsid which consists of four capsid proteins, VP1, VP2, VP3, and VP4 (39, 42). The VP1, VP2, and VP3 capsid proteins are external, and VP4 is internal (19, 31). Consequently, the major immune responses are directed to the external capsid proteins (16, 21, 53, 54). TMEV is divided into two subgroups (26, 30, 49): one subgroup includes GDVII and FA strains, which produce fatal acute encephalitis, and the other is the Theiler’s original subgroup, including the BeAn 8386 and Daniel strains, which result in chronic progressive demyelinating disease in the central nervous system (CNS) of susceptible mouse strains (26, 30). The results of epidemiological studies (1, 9) have suggested a viral etiology for human multiple sclerosis (MS), and the clinical and histopathological features of TMEV-induced demyelinating disease (DD) are very similar to those of MS (8). In addition, susceptibility to TMEV is associated with genotypes of the major histocompatibility complex (22, 29, 45) and T-cell receptor (22, 35). Thus, TMEV-IDD is considered to be a relevant model of MS. Although the mechanism of TMEV-IDD is not well understood, several lines of evidence indicate the importance of T cells in the pathogenesis of demyelination. For example, depletion of the CD41 T-cell population decreases disease incidence (14, 17, 52), and susceptibility to TMEV-IDD strongly correlates with the level of virus-specific delayed type hypersensitivity (DTH) response (6, 44). Three predominant T-cell epitopes have recently been identified on the capsid proteins of * Corresponding author. Mailing address: Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-8693. Fax: (312) 503-1339. E-mail:
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FIG. 1. Schematic representation of GST fusion proteins containing the major viral capsid proteins. The major linear B-cell epitopes (A1A, A1B, A1C, A2A, A2B, and A3A) and the T-cell epitopes (VP1233–244, VP274–86, and VP324–37) are indicated.
were able to neutralize viral infection in vitro (21). We have also shown that preimmunization of susceptible mice with UVinactivated virus protects susceptible mice against TMEVIDD, whereas postimmunization results in acceleration of the clinical disease (7). In addition, several studies by others have suggested the critical role of antibodies in protecting susceptible (15, 24, 46) as well as resistant (48) mice from TMEVIDD. However, the potential role of antibody responses to these individual B-cell epitopes in the protection and/or the progression of TMEV-IDD has not yet been investigated. To determine the role of antibodies specific for individual capsid proteins and linear epitopes in the protection from TMEV-IDD, we have utilized synthetic peptides as well as fusion proteins for immunization. We report here that preimmunization of susceptible mice with fusion proteins containing VP1 or VP2 (but not VP3) results in protection, suggesting that certain viral capsid proteins are more efficient in the induction of protective immunity. In addition, antibody responses to individual antibody epitopes within a single capsid protein, without involvement of T-cell responses to TMEV epitopes, also provided differential protection. Interestingly, the appearance of antibodies to A1C, exhibiting the highest neutralizing activity in vitro, was delayed, and the antibody levels corresponded to the disease progression. However, such antibodies were able to protect the mice from TMEV-IDD when the antibodies were present at early stages of viral infection. Therefore, our results strongly suggest that antibodymediated protection from virally induced demyelination is most effective at the early stage of viral infection but not during the progression of the disease. These results may provide useful information for the development of efficient vaccines against various pathogenic picornaviruses by using viral epitopes and/or capsid proteins. MATERIALS AND METHODS Animals. Female SJL/J mice were purchased from the National Cancer Institute, Frederick, Md. All mice were housed at the Animal Care Facility of Northwestern University. Viruses. The BeAn 8386 strain of TMEV was propagated in BHK cells grown in Dulbecco’s modified Eagle medium supplemented with 7.5% donor calf serum and purified by isopycnic centrifugation on Cs2SO4 gradients, as previously described (27). Fusion proteins. Glutathione S-transferase (GST)-based fusion proteins of three major capsid proteins of TMEV (VP1, VP2, and VP3) were generated as described previously (53, 54). Briefly, VP1, VP2, and VP3 coding sequences were amplified from the full-length BeAn cDNA clone by PCR with appropriate primers. The amplified sequences for the capsid proteins were inserted at the multicloning site of pGEX-5X-1 (Pharmacia Biotech, Uppsala, Sweden) and expressed as described previously for the production of GST-VP1, GST-VP2, and GST-VP3 (Fig. 1). Purity of affinity-purified fusion proteins in glutathioneagarose columns (Sigma Chemical Co., St. Louis, Mo.) was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.
J. VIROL. Synthetic peptides. Six peptides representing previously defined linear antibody epitopes of TMEV (21) were synthesized by using the RAMPS system (DuPont Co., Wilmington, Del.) with 9-fluorenylmethyloxycarbonyl reagents. A major single peptide (.95%) was present in each of the peptide preparations according to the reverse-phase high-pressure liquid chromatography analysis. The relative positions of these antibody epitopes on individual capsid proteins are shown in Fig. 1. Conjugation of synthetic peptides and carrier proteins was done as described previously (21). Briefly, synthetic peptides representing linear epitopes (1 mg) were conjugated with either bovine serum albumin (BSA) (10 mg) or keyhole limpet hemocyanin (KLH) (15 mg) with 0.1% glutaraldehyde followed by extensive dialysis. Induction of demyelinating disease. Five- to six-week-old mice were injected intracerebrally (i.c.) with 106 PFU of TMEV BeAn 8386 in a volume of 30 ml in the right cerebral hemisphere. This inoculum consistently induces demyelinating disease in more than 90% of SJL/J mice. Clinical symptoms include a waddling gait, extensor spasm, paralysis, loss of the righting reflex, incontinence, and hunched posture. The clinical signs of demyelination for individual mice were examined weekly by allowing mice to walk on a polyethylene (Dynalab) walking board. Analysis of TMEV-specific antibodies. Antibodies specific for viral epitopes were measured by an adaptation (21) of the indirect enzyme-linked immunosorbent assay (ELISA) as described previously (11). Briefly, either 0.3 mg of TMEV virus or 0.1 mg of synthetic peptides conjugated to BSA was used to coat microtiter plates, and then the plates were blocked with 1% Blotto. Twofold serial dilutions of sera starting from a 1:100 dilution were added, washed, and then allowed to react with alkaline phosphatase-conjugated goat anti-mouse secondary antibody. The enzyme reaction was developed with p-nitrophenyl phosphate and measured colorimetrically with an ELISA reader at 405 nm. For the determination of immunoglobulin G (IgG) isotypes, alkaline phosphataseconjugated isotype-specific goat antibodies (anti-mouse IgG1, IgG2a, and IgG2b; Fisher Co., Itasca, Ill.) were used as secondary antibodies after titration with the corresponding isotype controls. In addition, relative levels of antibodies to individual linear epitopes were assessed by immunodot assays as described previously (21). Briefly, synthetic peptides conjugated to BSA (0.1 mg in 1 ml) were applied onto nitrocellulose paper. After a brief incubation (2 to 5 min), the filters were then blocked in 1% Blotto and incubated with a 1:1,000 dilution of immune sera. The filters were then incubated with alkaline phosphatase-conjugated goat antimouse IgG antibody (Promega), and the color reaction of the enzyme was developed by adding a mixture of nitroblue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate. T-cell proliferation assay. Spleen cells (6 3 105) were cultured in 96-well, microculture plates in RPMI containing 0.5% syngeneic mouse serum and 5 3 1025 M 2-mercaptoethanol. Triplicate cultures were stimulated with UV-inactivated TMEV (10 mg), VP1233–250 (1 mM), VP274–86 (1 mM), or VP324–37 (1 mM) and incubated for 72 h at 378C under 5% CO2. A hen egg lysozyme peptide (HEL34–45) (1 mM) was used as a negative control. Cultures were then pulsed with 1.0 mCi of [3H]TdR and harvested 18 h later. Measurements of [3H]TdR uptake by the proliferating cells were determined in a scintillation counter. Statistical analysis. The significance (two-tailed P value) of the differences between the results for experimental animal groups with various treatments and the control group was analyzed based on the unpaired, nonparametric test by using the InStat Program (GraphPAD Software, San Diego, Calif.).
RESULTS Preimmunization with individual capsid proteins differentially protects from TMEV-IDD. We have previously observed that the subcutaneous immunization of mice with UV-inactivated TMEV prior to i.c. injection with virus is able to protect susceptible mice from demyelinating disease (7). To determine the individual viral capsid proteins involved in the protection, susceptible SJL/J mice were immunized with fusion proteins containing individual capsid proteins. Mice were immunized subcutaneously with fusion proteins (GST-VP1, GST-VP2, and GST-VP3) or control GST at 14 and 5 days prior to viral infection (Fig. 2). Preimmunization with GST-VP1 resulted in a very significant protection (P of 0.0078) compared to that of the control mice similarly preimmunized with GST alone. The date of 50% disease onset (DO50) in mice preimmunized with GST-VP1 was 51 days post-i.c. inoculation, while that in control mice was 39 days. Mice preimmunized with GST-VP2 also showed very significant protection (P of 0.0072), with a DO50 of 70 days. In contrast, preimmunization with GST-VP3 did not show any significant effect (P of 0.528) on the clinical course of demyelination, with a DO50 of 40 days (Fig. 2). These
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FIG. 2. Preimmunization with GST-VP1 or GST-VP2 (but not GST-VP3) protects mice from TMEV-IDD. Female SJL/J mice (11 or 12 mice per group) were subcutaneously immunized with 30 mg of either control GST or GST fusion proteins (GST-VP1, GST-VP2, or GST-VP3) in complete Freund’s adjuvant and incomplete Freund’s aduvant at 14 and 5 days, respectively, prior to i.c. viral inoculation. Preimmunization with either GST-VP1 or GST-VP2 resulted in significantly delayed onset and low incidence of TMEV-IDD compared to results for the immunization with control GST. In contrast, preimmunization with GSTVP3 did not change the clinical course. The two-tailed probability was determined by the unpaired, nonparametric test with data obtained between days 28 and 98.
results indicate that immunization of susceptible SJL/J mice with either VP1 or VP2, but not VP3, can evoke protective immunity against TMEV-IDD, suggesting the differential efficiencies of individual capsid proteins for the induction of such immunity. Immunization with fusion proteins containing individual capsid proteins induces antibody responses to selective linear epitopes. To correlate immunity parameters involved in the differential protection by immunization with individual capsid proteins (Fig. 2), antibody (Fig. 3 and 4A) and T-cell responses of the immune mice to TMEV epitopes were analyzed (Fig. 4B). Antibody responses on immunizations with GST-fusion proteins prior to and immediately after viral infection were examined to assess the level of antibodies specific for the major linear epitopes (Fig. 3). Immunization with GST-VP1 primarily induced a relatively high level of antibodies to A1A while immunization with GST-VP2 and GST-VP3 induced low levels of antibodies to A2A (less than A2B) and A3A, respectively (Fig. 3). The induction of antibodies to TMEV by the GST fusion proteins was specific because similar immunization with GST alone did not induce any detectable levels of anti-TMEV antibodies. Interestingly, however, the levels of antibodies to A1B in GST-VP1-immunized mice and to A2B in GST-VP2immunized mice were detected only after viral infection (Fig. 3). Therefore, immunizations with GST-VP1 and GST-VP2 may effectively induce antibodies to only certain epitopes, although these may be able to prime lymphocytes involved in antibody production to additional epitopes following viral infection. Capsid protein-induced protective immunity results in enhanced production of antibodies to TMEV and elevated T-cell responses to the major T-cell epitopes. Pooled serum from each group in Fig. 2 was also examined at 42 days after viral infection in order to correlate the antibody levels with disease progression (Fig. 4). The experimental groups immunized with GST-VP1 and GST-VP2 displayed high titers of antibodies reactive to TMEV compared to those for the control group immunized with GST alone. However, the levels of antibody in
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mice immunized with GST-VP3 were lower than those in mice immunized with either GST-VP1 or GST-VP2 (Fig. 4A). These results suggest that the delay of TMEV-IDD is associated with the ability of the immunogens to induce high levels of antibodies to whole virus rather than antibodies to the linear epitopes in the specific capsid proteins. Further to correlate the antibody levels and T-cell responses to TMEV or T-cell epitopes, splenic T-cell proliferative responses were examined (Fig. 4B). T-cell proliferative responses to UV-inactivated TMEV did not appear to correspond to the levels of antibody production, although relatively high levels of T-cell responses to VP1233–250 or VP274–86 appear to correlate with the elevated antibody responses to TMEV in mice preimmunized with GST-VP1 or GST-VP2, respectively (Fig. 4). Interestingly, mice immunized with GST-VP3 showed very little enhancement of T-cell proliferative response to VP324–37 over the background level in GST-immunized control mice infected with TMEV (Fig. 4B). These results suggest that immunization with GSTVP3 does not induce a similar level of T-cell response to the major VP3 epitope, unlike GST-VP1 or GST-VP2. The levels of antibodies to most of the major linear epitopes inversely correlate with the degree of demyelinating disease. To further analyze the antibody response to TMEV involved in
FIG. 3. Antibody responses to major linear antibody epitopes in mice immunized with GST fusion proteins. At 10 days after the second immunization with GST, GST-VP1, GST-VP2, or GST-VP3 prior to viral infection and at 12 days after viral infection, the antibody levels in the pooled serum (three mice) for each group were analyzed by indirect ELISA.
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mice, regardless of the capsid fusion proteins used for immunization, displayed levels of anti-A1C antibodies relatively higher than those in healthy mice (Fig. 5). Low but detectable levels of anti-A1C antibodies in healthy mice immunized with GST-VP1 and GST-VP2 may reflect the fact that some of these mice may already have developed subclinical demyelination. In addition, the increase in the levels of anti-A1C antibodies in clinically affected mice immunized with GST-VP1 was less impressive than that of the other VP1 epitope (A1A) or in other experimental groups. These results may represent the prior priming effects by immunization with GST-VP1, which contains the VP1 epitopes. To confirm the correlation between the elevated levels of anti-A1C antibody and onset of TMEV-IDD observed in pooled sera (Fig. 5), antibody levels of individual mice from selected groups were assessed 42 and 100 days after viral infection (Fig. 6). The levels of antibody to A1C were extremely low or undetectable when the mice were clinically healthy,
FIG. 4. Comparison of antibody and T-cell proliferative responses to TMEV in mice preimmunized with GST fusion proteins. (A) At 42 days after viral inoculation, the levels of antibody to TMEV in pooled serum (12 mice) from each group of mice were analyzed by indirect ELISA. The sera from mice immunized with GST-VP1 or GST-VP2, but not GST-VP3, show markedly high levels of antibodies to TMEV compared to those in control mice. (B) At 114 days after viral infection, splenocytes from each group of clinically affected mice were cultured for 4 days in the presence of UV-inactivated TMEV, synthetic peptides bearing the major T-cell epitopes (VP1233–250, VP274–86, and VP324–37), or control peptide (HEL34–45). Results are expressed as the difference in counts per minutes: the counts per minute of [3H]TdR in cultures stimulated with TMEV peptides 2 the counts per minute in cultures stimulated with HEL34–45.
the protection from virally induced demyelination, we compared the levels of antibodies reactive to the major linear B-cell epitopes in the pooled sera of healthy and clinically affected mice on day 42 after viral infection (Fig. 5). Antibodies from mice free of clinical signs which were preimmunized with GST-VP1, GST-VP2, and GST-VP3 recognized predominantly A1B, A2A, and A3A, respectively. This finding is largely consistent with the results of our previous study demonstrating that the major linear antibody epitopes in SJL/J mice immunized with UV-inactivated virus include A1B, A1C, A2A, and A3A (21). In addition, the antibody response to the major linear epitope on VP3 (A3A) in mice immunized with GSTVP3 was comparable to the levels of antibodies to the epitopes on VP1 and VP2 in mice immunized with the respective fusion proteins. Therefore, the failure of protection in mice immunized with GST-VP3 is not due to the lack of antibodies to VP3. However, preimmunization of susceptible SJL/J mice with GST-VP1 did not efficiently induce antibody response to A1C. Surprisingly, however, the sera from clinically affected
FIG. 5. Comparison of levels of antibodies to linear epitopes in healthy and clinically affected mice. All mice preimmunized with GST or GST fusion proteins were bled at 42 days after viral infection and sera were pooled based on the clinical status, i.e., healthy groups (4 to 11 mice per group) and clinically affected groups (1 to 8 mice per group). IgG antibodies to linear B-cell epitopes were determined by an indirect ELISA. Healthy mice preimmunized with GST-VP1, GST-VP2, and GST-VP3 display relatively higher levels of antibodies to the respective epitopes (A1B, A2A, and A3A) than clinically affected mice. In contrast, clinically affected mice show significantly elevated levels of antibodies to A1C, regardless of the fusion proteins immunized.
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FIG. 6. Comparison of levels of antibodies to A1C in the same individual mice at two separate time points, 42 and 100 days postinfection. Individual healthy mice in each group at 42 days after viral infection were further examined at 100 days postinfection. Only mice clinically affected at 100 days postinfection produced increased levels of antibodies to A1C. The antibody levels in a single representative mouse in each category are shown.
except for the mice preimmunized with GST-VP1 which developed clinical symptoms later. However, only the mice which became clinically affected at day 100 postinfection displayed drastic increases in the levels of antibody to A1C, in contrast to the low or undetectable levels of anti-A1C antibodies in mice which remained healthy. Therefore, the increase in the levels of antibodies to A1C clearly correlates well with the progression of TMEV-IDD, even in individual mice examined during the course of demyelination. Antibodies to only certain linear epitopes provide protection from TMEV-IDD. Since the fusion proteins containing individual capsid proteins used in this study include both B-cell and T-cell epitopes (Fig. 1), it is difficult to rule out the role of T cells in the protection induced by the preimmunization (Fig. 2). To confirm the protective role of antibodies specific for the linear antibody epitopes, we similarly immunized SJL/J mice, prior to viral infection, with KLH conjugates of synthetic peptides containing each linear B-cell epitope of TMEV (Fig. 7A). Interestingly, preimmunization with all the B-cell epitopes (A1A, A1B, and A1C) within the VP1 capsid protein resulted in significant (P of #0.0058) protection against TMEV-IDD. The incidences of disease in mice preimmunized with A1AKLH, A1B-KLH, and A1C-KLH were 40, 80, and 50%, respectively, at 124 days after viral infection. However, preimmunization with a cocktail of A2A, A2B, and A3A conjugates did not result in significant protection (Fig. 7B). The lack of protection by immunity towards these individual peptide conjugates was verified with separate animal groups immunized
with single peptide-KLH conjugates (data not shown). Since these immunizations resulted in similarly high levels of specific antibodies to the corresponding linear epitopes (Fig. 7C), antibodies to only certain epitopes must be more effective in the antibody-mediated protection. To test whether the pre-existence of antibodies to the linear epitopes at an early stage of viral infection is necessary for the protection from TMEV-IDD, SJL/J mice were immunized with KLH conjugates of synthetic peptides containing the linear VP1 epitopes at 5 and 15 days post-viral infection (Fig. 8A). TMEV-infected mice immunized with A1C and A1A conjugates displayed significant levels of protection, although mice immunized with A1B conjugates produced only a trend but not a significant level (P of 0.2) of protection. It is interesting to note that this A1B epitope also induced the least protection in mice immunized prior to viral infection (Fig. 7A). Similarly, immunization of virus-infected mice with A2A or A3A conjugates, which do not provide protection on preimmunization, did not confer protection from the disease development (data not shown). The lack of protection after immunization with certain peptide conjugates was not due to the poor induction of antibody responses to the epitopes as the antibody levels to the epitopes are similar to each other (Fig. 8B). These results clearly indicate that antibodies specific for only certain linear epitopes are able to protect susceptible mice from further developing TMEV-IDD when such antibodies are present before the onset of disease.
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DISCUSSION Effective means to protect the host from diseases induced by various picornaviruses have been extensively investigated, including vaccine development against poliovirus as well as footand-mouth disease virus (18, 32, 38). In addition, efforts have been made to identify the capsid proteins and/or epitopes most effective in inducing protective immunity (2, 5, 13, 18, 51, 55). Interestingly, immunization with the VP1 capsid protein appears to provide the best protection from several picornavirus infections, such as poliovirus and foot-and-mouth disease virus (2, 5, 18, 55). Since antibodies directed to VP1 most strongly neutralize viral infection in vitro, such antibodies have been considered to play an important role in the protection. Similarly, VP1 epitopes of TMEV, including the A1C region, are found to exhibit a strong neutralization activity in vitro (21, 37, 50). However, it is not yet clear whether such a neutralizing antibody response is critical for protection of the host in vivo from virally induced disease, particularly immunologically mediated chronic inflammatory disease such as TMEV-IDD. In addition, the role of individual capsid proteins in the protection and/or pathogenesis of this demyelinating disease has not been well studied. It has been demonstrated in this study that immunization with certain individual capsid proteins of TMEV (VP1 and VP2 but not VP3) prior to viral infection results in significant protection of susceptible SJL/J mice from TMEV-IDD (Fig. 2). Preimmunization with VP1, VP2, and VP3 as GST fusion proteins induced high levels of antibodies to selective linear epitopes, A1A/A1B, A2A, and A3A, respectively. However, antibodies produced in mice immunized with UV-inactivated TMEV recognize A1B, A1C, A2A, and A3A (21, 23), differing in reactivities to A1A and A1C. Thus, the pattern of epitope recognition by antibodies induced with individual capsid proteins is somewhat different from that of antibodies raised against whole virus. Therefore, induction of the antibody responses to A1A and A1C may be dependent on the tertiary structure of the viral protein on the virion, which may differ from the isolated VP1 protein. Taken together, the utilization of individual capsid proteins as a vaccine may induce immune responses qualitatively as well as quantitatively different from those induced by inactivated whole virus. To further assess the role of antibodies specific for individual epitopes, synthetic peptides representing linear epitopes were also utilized (Fig. 7 and 8). Such peptides were conjugated to a highly immunogenic carrier protein, KLH, in order to induce the epitope-specific antibody responses without invoking T-cell responses to other viral epitopes which might be involved in the pathogenesis of TMEV-IDD. Interestingly, antibodies to all three VP1 linear epitopes (A1A, A1B, and A1C) were able to protect the mice, although antibodies to A1B were only marginally effective. Thus, the efficient protection by antibodies induced by these VP1 linear epitopes is consistent with the protective immunity induced by the VP1 fusion protein (Fig. 2). The lack of protective immunity induced by A3A-KLH conjugates (Fig. 7B and data not shown) also corresponds well with the failure of such immunity induction by the VP3 fusion protein. It is interesting that the A3A antibody epitope contains an overlapping T-cell epitope as well (21, 53). Therefore, it is not yet clear whether antibodies to this epitope are unable to provide protection or whether the protection is nullified by the increased, potentially pathogenic T-cell response to this region. Immunizations with A2A-KLH and A2B-KLH conjugates also failed to induce significant protection (Fig. 7B and data not shown), in contrast to the induction of protective immunity by whole VP2 protein (Fig. 2). It is thus conceivable
FIG. 7. Preimmunization with synthetic peptides containing the major VP1 linear antibody epitopes selectively protects mice from TMEV-IDD. The twotailed P values were determined by the unpaired, nonparametric test between experimental groups and the control group, with data obtained between 34 and 124 days after viral infection. (A) Female SJL/J mice (10 mice per group) were preimmunized with 100 mg of KLH or KLH-conjugated peptides (KLH-A1A, KLH-A1B, and KLH-A1C) in complete Freund’s adjuvant and incomplete Freund’s adjuvant at 15 and 6 days, respectively, prior to viral inoculation. Preimmunizations with the VP1 epitope conjugates resulted in significant protection (P of #0.0058) from TMEV-IDD. (B) Female SJL/J mice (10 mice per group) were preimmunized with a cocktail of KLH-conjugated peptides (100 mg each of A2A-KLH, A2B-KLH, and A3A-KLH). These mice were not significantly (P of 0.07) protected from TMEV-IDD. (C) The relative levels of antibodies to major linear antibody epitopes in each group of mice prior to onset of disease (21 days after viral infection) were determined by the immunodot method.
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FIG. 8. Postimmunization with synthetic peptides containing certain VP1 linear antibody epitopes protects mice from TMEV-IDD. The two-tailed P values were determined by unpaired, nonparametric test between experimental groups and the control group, with data obtained between 34 and 124 days after viral infection. (A) Female SJL/J mice (10 mice per group) were postimmunized with 100 mg of KLH or KLH-conjugated peptides (A1A-KLH, A1B-KLH, and A1C-KLH) in complete Freund’s adjuvant and incomplete Freund’s adjuvant at 5 and 15 days after viral inoculation, respectively. Postimmunizations with A1AKLH and A1C-KLH (but not A1B-KLH) conjugates significantly (P of ,0.05) protected mice from TMEV-IDD. (B) The relative levels of antibodies to major linear antibody epitopes in each group of mice prior to onset of disease (28 days after viral infection) were determined by the immunodot method.
that the major antibody populations involved in the protection following immunization with the VP2 fusion protein may not include antibodies to A2A and A2B. Perhaps, minor linear antibody epitopes may function as major epitopes in the protection induced by immunization with the GST-VP2 fusion protein. Alternatively, the protection induced by the fusion protein may represent an immunity other than antibody response to viral epitopes. The relationship between the neutralizing activity of antibodies and their ability to protect the host from viral infection and/or virally induced disease is not yet clear. In other virus systems, antibodies capable of neutralizing virus in vitro do not necessarily protect the host and, in some cases, nonneutralizing antibodies are capable of protecting the host (3, 25, 43). Two major neutralizing antibody epitopes of the Daniel strain have previously been identified on VP1 by using escape mutants
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against neutralizing monoclonal antibodies in vitro. One is at the region with residue 101, which is not cross-reactive to the BeAn strain (56). The other includes the C-terminal region with residue 268 (40, 47). This region also corresponds to the A1C linear epitope of the BeAn strain (21, 23, 50). Furthermore, antibodies against two additional linear epitopes (A2A and A2B) of VP2 are also able to neutralize the virus in vitro (21). However, results of immunization with peptide conjugates in this study indicate that antibodies to A1A and A1C are the most effective in protecting from TMEV-IDD (Fig. 7 and 8), although antibodies to A1A are not able to neutralize the virus (21). Interestingly, a similar induction of protective immunity against the closely related Mengo virus has previously been observed with the corresponding C-terminal sequence (VP1259–277) of VP1 (36). Furthermore, antibodies to other linear epitopes (A2A, A2B, and A3A) are not effective in inducing such protective immunity in vivo (Fig. 7), despite the fact that antibodies to A2A and A2B neutralize the virus in vitro (21). Therefore, our results are consistent with previous observations that the protective function of antibodies in vivo does not correlate with the in vitro neutralizing activity (10, 25, 43). Since the major T-cell population involved in the immunologically mediated inflammatory response of TMEV-IDD appears to be Th1, the predominance of Th1-dependent, IgG2a anti-TMEV antibodies was expected (12). However, the major IgG subclass of anti-TMEV antibodies has been determined as IgG2a (4, 41) or IgG2b (46), depending on the investigator. Our previous (21) and present (data not shown) studies have shown that IgG2b is the major IgG subclass of antibodies against the linear antibody epitopes of TMEV. The production of IgG2b is known to be influenced by transforming growth factor b, which is produced by a variety of cell types including astrocytes, macrophages, and certain Th2 cells (34). Therefore, perhaps the production of antibodies to TMEV during the development of disease may be heavily influenced by the presence of transforming growth factor. We have previously shown that the antibody to A1C is a predominant anti-TMEV antibody population in the sera and cerebrospinal fluids of virus-infected SJL/J mice (21). In the present study, the predominance of this antibody was confirmed in mice with TMEV-IDD. Interestingly, the levels of such antibodies appear to be correlated with the progression of TMEV-IDD (Fig. 5 and 6). This increase in the level of antiA1C antibody during the course of disease is unique for this epitope. Our studies revealed that the anti-A1C antibody is capable of protecting the host from the induction and/or progression of TMEV-IDD if such antibodies are present at the early stages of viral infection (Fig. 7 and 8). Therefore, it would be advantageous for the virus to induce a delayed antibody response to A1C until chronic viral infection is well established in the CNS, at which time the antibodies are no longer protective. Limitations in the quantity and/or the restricted access by antibodies to intracellular virions may provide only partial protection from TMEV-IDD. However, it is not yet clear how the production of such a potent protective antibody is delayed and parallels the progression of disease. Although B cells producing antibodies to VP1 and VP2 proteins as well as antibodies to A1C have been previously identified in the demyelinating lesions (4, 21), the role of antibodies in the demyelination process is not yet known. It is conceivable that the selective elevation of antibodies to the A1C epitope in TMEV-infected SJL/J mice (Fig. 5 and 6) contributes to pathogenesis rather than the protection of the host. This possibility is consistent with the facts that protection by such antibody is efficient in only the early stages of viral
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infection and viral persistence is not reduced in the presence of high levels of anti-A1C antibodies in SJL/J mice with TMEVIDD (21). Once chronic viral infection is established, antibodies to TMEV may be inefficient in eliminating or reducing viral persistence in the CNS, perhaps due to the lack of accessibility to the virion. However, it is premature to exclude the roles of such antibodies in clearance of viral persistence, in light of the recent finding that certain antibodies may be able to control intracellular viral replication (10, 25). The role of these antibodies in pathogenesis is also not yet clear. Such antibodies may not be essential to the pathogenicity of demyelination. This possibility is consistent with the observation that TMEVIDD can be developed without producing high levels of antiA1C antibodies (unpublished observation). Nevertheless, the production of high levels of anti-A1C antibody may be consequential to the elevated pathogenic T-cell response involved in TMEVIDD. Interestingly, the A1C antibody epitope (VP1262–276) is located close to a major T-cell epitope (VP1233–250) of VP1 (21, 54), which appears to be associated with the pathogenesis of TMEV-IDD (unpublished data). This close proximity between the major pathogenic T-cell epitope and the antibody epitope may provide a synergistic effect for T-cell stimulation as well as antibody production via potential T-cell–B-cell interactions (35a). Further investigations regarding the role of antibodies towards individual epitopes in protection and/or development of virally induced disease will help to elucidate the pathogenic mechanisms and may provide potential means of protecting the host from various pathogenic picornaviruses. ACKNOWLEDGMENTS This work was supported by Public Health Service grants NS-28752 and NS-33008 from the National Institute of Neurological Disorders and Stroke. We acknowledge Kathleen Kerekes for excellent technical support.
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