Immunization with Nonstructural Proteins ... - Journal of Virology

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lent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and ... alitis that is potentially fatal. ..... Monoclonal antibody cure.
JOURNAL OF VIROLOGY, May 1997, p. 3415–3419 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 71, No. 5

Immunization with Nonstructural Proteins Promotes Functional Recovery of Alphavirus-Infected Neurons MARK D. GORRELL,1† JULIE A. LEMM,2‡ CHARLES M. RICE,2‡

AND

DIANE E. GRIFFIN1,3*

1

Department of Neurology, Johns Hopkins University School of Medicine, and Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health,3 Baltimore, Maryland 21205, and Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 631102 Received 30 August 1996/Accepted 21 January 1997

The encephalitic alphaviruses are useful models for understanding virus-neuron interactions. A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in mice by infecting motor neurons and inducing apoptosis of these nonrenewable cells. Antibodies to the surface glycoproteins suppress virus replication, but other recovery-promoting components of the immune response have not been recognized. We assessed the effect on the outcome of NSV-induced encephalomyelitis of immunization of mice with nonstructural proteins (nsPs) by using recombinant vaccinia viruses. Mice immunized with vaccinia virus expressing nsPs and challenged with NSV initially developed paralysis similar to unimmunized mice but then recovered neurologic function. Mice preimmunized with vaccinia virus expressing structural proteins were completely protected from paralysis. Mice immunized with vaccinia virus alone showed paralysis with little evidence of recovery. Vaccinia virus expressing only nsP2 was as effective as vaccinia virus expressing all the nsPs. Protection provided by immunity to nsPs was not associated with a reduction in virus replication or with improved antibody responses to structural proteins. Protection could not be passively transferred with nsP immune serum. The depletion of T cells at the time of NSV infection decreased protection. The data show that antiviral immune responses can improve the ability of neurons to survive infection and to recover function without altering virus replication. Alphaviruses cause sporadic and epidemic meningoencephalitis that is potentially fatal. Neurons are the primary cells in the nervous system that are infected (12). The clearance of infectious virus from the brain is immune response mediated (19, 24), and surviving animals are immune to reinfection. Sindbis virus (SV), the prototype alphavirus, has a singlestrand message-sense RNA genome of 11,703 nucleotides which encodes three major structural proteins (C, E2, and E1) and four nonstructural proteins (nsPs; nsP1, nsP2, nsP3, and nsP4). The E2 glycoprotein, which heterodimerizes with E1 to form the envelope spikes, is an important target of the immune response. Antibodies to E2 can both prevent infection and clear infectious virus from the brain and spinal cord (19, 25, 40). Antibody to E1 can also prevent fatal infection (25, 40). No role for immune responses to the nsPs of alphaviruses has yet been identified. nsPs of other plus-strand RNA viruses elicit an immune response which can be protective. Poliovirus, hepatitis A virus, and foot-and-mouth disease virus routinely stimulate antibodies to the nsPs of these picornaviruses (7, 13, 28). Immunization with the NS1 protein of the dengue and yellow fever viruses expressed in recombinant vaccinia viruses protects against fatal infection of mice with these flaviviruses (5, 8, 34–36, 46). NS3 contains several dominant dengue virusspecific T-cell epitopes (16). Alphavirus nsPs are translated from the 59 two-thirds of the genomic RNA immediately upon infection to make polypro-

tein precursors which are cleaved co- or posttranslationally to produce intermediate polyproteins and finally nsP1, nsP2, nsP3, and nsP4 (42). nsP1 has methyl- and guanylyltransferase activities and is important for the initiation of minus-strand synthesis. Sequences in nsP1 modulate substrate specificity of the nsP2 protease. nsP2 encodes an NTPase and putative RNA helicase in the N-terminal domain and a papain-like cysteine protease responsible for cis and trans nonstructural polyprotein cleavages in the C-terminal domain. nsP3 is phosphorylated and appears to participate in both minus-strand and subgenomic mRNA synthesis. nsP4, located downstream of an opal termination codon and produced in small amounts by readthrough, is the viral RNA-dependent RNA polymerase (42). Immune responses to SV structural proteins and nsPs were generated by infecting mice with recombinant vaccinia viruses expressing individual and combined SV proteins. Protection from encephalomyelitis was assessed by intracerebral challenge with neuroadapted SV (NSV), a virulent strain of SV (11). A unique form of protection was observed; infected mice became paralyzed initially but were more likely to recover and had a more rapid recovery from NSV-induced paralysis if previously immunized with SV nsPs. This effect was not due either to enhanced virus clearance or to improved antibody responses to SV structural proteins, suggesting a unique mode of protection.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205; Phone: (410) 955-3459. Fax: (410) 955-0105. E-mail: [email protected] .edu. † Present address: Centenary Institute, Royal Prince Alfred Hospital, Sydney, Australia. ‡ Present address: Dept. of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492.

Viruses. The recombinant vaccinia viruses have been described previously. VSIN-S (formerly VV3S-7) expresses the three structural proteins C, E2, and E1 from the HRSP strain of SV as a polyprotein (30). VSIN-NS expresses the four nsPs from the toto 1101 strain of SV as a polyprotein (22). The VSIN-S and VSIN-NS recombinant viruses are derivatives of the WR strain of vaccinia virus. SV protein expression is under the control of the 7,500-molecular-weight (7.5K) promoter, and the integration site is the tk locus. vv1234 expresses the four nsPs together as a polyprotein, and vv1, vv2, vv3, and vv4 express each of the four nsPs individually (18). The vv recombinant viruses were derived by marker rescue of various pTM3 derivatives with the WR strain of vaccinia virus (27). Insertions

MATERIALS AND METHODS

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were made at the tk locus and have the following genomic structure (59 to 39): tk early promoter, 59 part of the tk coding region, T7 promoter, encephalomyocarditis virus internal ribosomal entry site, SV nsP coding region, T7 terminator, 7.5K promoter, gpt coding region, 39 tk gene, and tk termination signal. Coinfection with the vTF7-3 helper virus, which expresses the T7 polymerase, was used to drive nsP expression by the recombinant vv viruses (9). The WR strain of vaccinia virus was used for control infections. Vaccinia viruses were grown and assayed by plaque formation in Vero cells. Stocks of NSV, a neuroadapted strain of SV (11), and HRSP, a tissue cultureadapted strain of SV (1, 41), were grown in BHK-21 cells. Stock viruses were assayed, and the amounts of NSV in brain were determined by plaque formation on BHK-21 cells of serial 10-fold dilutions of tissue culture supernatant fluid of brain homogenized in Hanks’ balanced salt solution (HBSS). Mouse immunizations and challenge. Groups of 6 to 14 female 3- to 5-weekold BALB/cJ mice (Jackson Laboratories, Bar Harbor, Maine) were immunized either with 105 PFU of vaccinia virus (WR strain) or recombinant vaccinia virus in 10 ml of HBSS injected subcutaneously and intradermally into the base of the tail or with 107 PFU of the HRSP strain of SV injected subcutaneously. vv recombinant viruses (105 PFU) were mixed with 105 PFU of vTF7-3 before injection since expression of the nsP coding region is under control of the T7 promoter and coinfection of cells with a virus expressing the T7 polymerase is required for optimal expression of the nsP. Immunized mice were challenged by intracerebral inoculation with 1,000 PFU of NSV in 0.03 ml of HBSS 15 days after completion of the immunization protocol. Antibody treatment and T-cell depletion. Immune serum samples were collected from mice 3 weeks after immunization with vaccinia virus alone or vaccinia virus expressing the structural proteins (VSIN-S) or nsPs (VSIN-NS and vv1234) of SV. Serum (0.2 ml) was given to each recipient mouse by intraperitoneal inoculation. Monoclonal antibodies (MAb) against mouse CD4 (MAb GK 1.5; American Type Culture Collection, Rockville, Md.) (45) or CD8 (MAb 2.43; American Type Culture Collection) (31) were produced as ascitic fluid, and 0.1 ml of ascitic fluid was injected intraperitoneally 1 day before and 4 and 8 days after challenge with NSV. This regimen resulted in 90 to 95% depletion of the respective cell populations (18a, 29). Clinical assessment. Paralytic disease due to NSV-induced encephalomyelitis was monitored by clinical score. Disease progressed from ruffled fur to evidence of paralysis beginning with a limp tail, followed by hind leg paralysis and death 5 to 10 days after infection. Not all infections, even in unimmunized mice, were fatal. Therefore, the condition of mice was scored and expressed as the percent alive and well. Up to 10 days postchallenge, “unwell” was defined as a limp tail, limb paralysis, or death. After day 10, “unwell” was defined as paralysis or death because the tail was an unreliable indicator of disease during the convalescent phase. Mice were monitored for 15 to 30 days after challenge. Antibody measurement. Antibodies to structural proteins were measured as previously described by enzyme immunoassay with 3 mg of polyethylene glycolprecipitated SV per ml (39). Horseradish peroxidase-conjugated rabbit antimouse immunoglobulin (Dako Corp., Carpenteria, Calif.) diluted 1:200 was the secondary antibody. Color was developed with orthophenyldiamine (Sigma Fine Chemicals, St. Louis, Mo.) as the substrate, and plates were read at 450 nm. Assays were performed with pools of sera from all the mice in each treatment group. Each titer reported is the log of the highest dilution of serum that gave a reading which was double the background optical density.

J. VIROL.

FIG. 1. Outcomes of NSV-induced encephalomyelitis in unimmunized (Untreated) mice and mice immunized with HRSP, vaccinia virus, or recombinant vaccinia virus expressing the structural proteins (VSIN-S) or nsPs (VSIN-NS) of SV. Mice were immunized at 25 days of age and challenged intracerebrally with 1,000 PFU of NSV 15 days later. There were five or six mice in each group (x2 5 8.6; P , 0.01 for outcome at 14 days in VSIN-NS-immunized mice compared to that in vaccinia virus-immunized mice). Mortality was 80% for untreated mice, it was 60% for vaccinia virus-immunized mice, and it was 0% for HRSP-, VSIN-S-, and VSIN-NS-immunized mice.

paralysis, but immunization with vaccinia virus did not affect the outcome (Fig. 1). Immunization with VSIN-NS, the recombinant vaccinia virus that expresses the four nsPs, produced an intermediate outcome. After the challenge infection with NSV, VSIN-NS-immunized mice initially became paralyzed to an extent similar to that of control mice but recovered rapidly (x2 5 8.6; P , 0.01 at day 14). In this experiment, mice were monitored only for 2 weeks since an alteration in the rapidity of recovery was not an anticipated effect of immunization. Therefore, similar experiments with longer follow-up after NSV challenge were performed to determine whether recovery occurred only in immunized mice or just occurred earlier than in control mice (Fig. 2). Recovery occurred in small numbers of control vaccinia virus-immunized mice 14 to 21 days after infection, but recovery was more rapid and more complete in mice immunized with recombinant vaccinia virus expressing the nsPs (VSIN-NS) (x2 5 34.7; P , 0.001 at day 21).

RESULTS NSV-induced disease. The severity of SV encephalomyelitis in mice is a predictable and reproducible biologic characteristic which is dependent on the route of inoculation, the strain of virus used for infection, and the age of the mouse infected (14, 43). HRSP is an avirulent SV strain which causes no disease in mice older than 1 week (38), but NSV is a virulent SV strain which causes 80 to 100% mortality in 3- to 4-week-old BALB/cJ mice inoculated intracerebrally (11). These differences in virulence are associated in part with amino acid differences in the E1 and E2 glycoproteins (23). The mice in this study were 6 to 10 weeks old at the time of NSV infection because of the time needed for immunization prior to challenge. All unimmunized control mice either died or became paralyzed after infection with NSV. In those paralyzed mice that survived, there was little or no recovery of neurologic function (Fig. 1). Immunization with recombinant vaccinia viruses. Immunization of mice with the HRSP strain of SV or with VSIN-S, which expresses the SV structural proteins C, E2, and E1 from HRSP, resulted in complete protection from NSV-induced

FIG. 2. Outcomes of NSV-induced encephalomyelitis in mice immunized with vaccinia virus or with recombinant vaccinia virus expressing the structural proteins (VSIN-S) or nsPs (VSIN-NS) of SV. Mice were immunized at 30 to 32 days of age and challenged intracerebrally with 1,000 PFU of NSV 15 days later. The data were pooled from three separate experiments (x2 5 34.7; P , 0.001 for outcome at 21 days in VSIN-NS-immunized mice compared to that in vaccinia virus-immunized mice). The mortalities for immunized groups were as follows: vaccinia virus, 40%; VSIN-NS, 4%; and VSIN-S, 0%.

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FIG. 3. Amounts of virus in the brains of mice immunized with vaccinia virus or recombinant vaccinia virus expressing either the structural proteins (VSIN-S) or nsPs (VSIN-NS) of SV at various times after intracerebral infection with 1,000 PFU of NSV. Data are the geometric means and standard errors of the means of virus assays from three mice at each time point.

To determine whether this improved recovery was due to limited replication of virus or to more rapid clearance of virus from the brains of nsP-immunized mice, the amounts of virus in the brain were measured (Fig. 3). Although the amounts of virus were generally lower in mice immunized with recombinant vaccinia virus expressing the structural proteins (VSIN-S), there was no discernible change in virus replication in the brains of mice immunized with recombinant vaccinia virus expressing the nsPs (VSIN-NS) compared to that of control mice immunized with vaccinia virus. To determine which nsP(s) elicited an immune response that accelerated recovery, groups of mice were immunized with recombinant vaccinia viruses expressing individual nsPs (vv1, vv2, vv3, or vv4) or all four nsPs as a polyprotein (vv1234) (Fig. 4). Inoculations included an equal amount of the helper virus vTF7-3. Vaccinia virus expressing nsP2 (vv2) was as effective in promoting recovery as was the recombinant vaccinia virus expressing all the nsPs (vv1234), and vv1, vv3, and vv4 all provided some protection compared to vaccinia virus alone. Mechanism of protection. The mechanism by which VSIN-NS improves recovery was investigated by using serum

FIG. 4. Outcomes of NSV-induced encephalomyelitis in mice immunized with recombinant vaccinia viruses expressing individual SV nsPs or all nsPs as a polyprotein. Groups of seven mice received a single immunization at 32 days of age and were challenged intracerebrally with 1,000 PFU of NSV 15 days later. The mortalities for immunized groups were as follows: vaccinia virus, 23%; vv1234, 29%; vv1, 29%; vv2, 0%; vv3, 0%; and vv4, 32%.

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FIG. 5. Effects of serum transfer on the outcome of NSV-induced encephalomyelitis. Groups of 12 mice were injected intraperitoneally 1 day before challenge with 0.2 ml of serum from mice infected 3 weeks before with vaccinia virus or recombinant vaccinia virus expressing the structural proteins (VSIN-S) or nsPs (VSIN-NS and vv1234) of SV. The mortalities for passively immunized groups were 0% for anti-vaccinia virus and 8% for anti-VSIN-S, anti-VSIN-NS, and anti-vv1234.

transfers and T-cell depletions. Serum transfers from VSIN-Sinfected mice to naive recipients the day before NSV challenge conferred protection (Fig. 5). However, similar serum transfers from VSIN-NS-infected mice did not alter the outcome of NSV challenge. After the collection of serum samples, the mice used as donors for serum transfers were challenged with NSV. All VSIN-S-immunized mice were protected, and VSINNS-immunized mice recovered more rapidly than did the vaccinia virus-infected controls (data not shown), indicating that the serum donors had been successfully immunized. T cells were depleted by injecting MAb to mouse CD4 and CD8 together or alone beginning 1 day before NSV infection (Fig. 6). This treatment did not diminish the protection conferred by VSIN-S (Fig. 6A), which is known to be due to antibody. In contrast, VSIN-NS-induced protection was reduced by T-cell depletion (Fig. 6A). Depletion of either CD4 or CD8 T-cell subsets slowed, but did not prevent, recovery (Fig. 6B). Since antibodies to the E1 and E2 glycoproteins can clearly affect the outcome from NSV infection (19, 37, 40), we examined the possibility that mice immunized with nsPs make antibodies to the structural proteins of SV more rapidly or in larger amounts through cognate help from nsP-specific immune T cells present at the time of infection (32). The time course of antibody production to structural proteins in immunized mice with various degrees of protection (Fig. 4) was examined (Fig. 7). As expected, mice immunized with VSIN-S (complete protection) had antibody at the time of challenge and this was increased by intracerebral inoculation of NSV. Mice immunized with vv2 (almost complete recovery), vv4 (partial recovery), or vaccinia virus (no protection) developed antibodies to SV structural proteins at similar times and in similar amounts. DISCUSSION The ability of antibody to the SV envelope glycoprotein E2 both to prevent disease and to promote recovery from NSVinduced encephalomyelitis is well established (19, 37, 40). Thus, observations that vaccinia virus expressing the structural

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FIG. 6. Effects of T-cell depletion of NSV-induced paralysis and recovery in mice preimmunized with vaccinia virus, VSIN-S, and VSIN-NS. (A) Groups of 10 mice preimmunized with VSIN-S or VSIN-NS were (2T cells) or were not depleted of T cells with antibody to CD4 and CD8. (B) Groups of 10 mice preimmunized with VSIN-NS were depleted of CD4 (2CD4) or CD8 (2CD8) T cells or were not T cell depleted. The mortalities for these groups were 39% for vaccinia virus-immunized controls, 0% for VSIN-S-immunized mice with and without T-cell depletion, 4% for VSIN-NS-immunized mice without depletion, 17% for VSIN-NS-immunized mice with CD4 depletion, 6% for VSIN-NS-immunized mice with CD8 depletion, and 5% for VSIN-NS-immunized mice with both CD4 and CD8 depletion.

proteins of SV (VSIN-S) and passive transfer of serum from VSIN-S-immunized mice fully protect mice from NSV-induced fatal encephalomyelitis are in accord with previous work. However, our discovery that recombinant vaccinia virus expressing the nsPs of SV (VSIN-NS) can influence the course of recovery from disease was not expected and suggests that immune responses also assist animals to recover from encephalitis. Furthermore, this protection appears to be mediated by T cells rather than by antibody and protects mice not by decreasing virus replication in the central nervous system (CNS) but by promoting recovery of neuronal function after infection. Protection from flavivirus-induced encephalitis of mice is provided by immunization with the nonstructural NS1 protein of yellow fever and dengue viruses. NS1 is expressed on the surfaces of infected cells and appears to play a role in virus maturation (3). Protection from fatal disease can be induced by immunizing mice with expressed or purified NS1 alone (2, 5, 34, 35, 46) or with a recombinant vaccinia virus expressing NS1 (8). Monkeys can also be protected in this way from yellow fever virus-induced hepatitis (33). Protection is provided by antibody (10) and is dependent on the presence of the Fc piece of immunoglobulin (36). Protection correlates with production

FIG. 7. Antibodies to the structural proteins of SV produced after intracerebral infection with NSV. Mice were immunized with vaccinia virus or recombinant vaccinia virus expressing the structural proteins (VSIN-S), nsP2 (vv2), or nsP4 (vv4) of SV, and antibody was measured by enzyme immunoassay (EIA).

of complement-fixing antibody and can be passively transferred with complement-fixing MAb to NS1 (34). Anti-NS1 antibody appears to act directly by decreasing virus replication in the CNS (36). Since NSV replication in the CNS was not affected by immunization and antibody could not transfer protection, the immune response to the nsPs of alphaviruses appears to provide protection by a different, non-antibody-dependent mechanism compared to the immune response to NS1 of flaviviruses. Alphavirus nsP protection is dependent on the presence of T cells at the time of challenge. T cells can contribute to recovery from viral infection in many ways. The most clearly documented effect of T cells in virus infection is the ability of CD4 or CD8 cytotoxic T cells to lyse and therefore eliminate virus-infected cells in tissue (6). There are a number of reasons to postulate that this may not be the mechanism of protection from NSV-induced encephalitis. The cells predominantly infected by NSV in the CNS are neurons (12). Neurons express little if any major histocompatibility complex class I or class II antigens, making them poor targets for T cells (4, 15, 17). Furthermore, immune-cell-mediated death of infected neurons would result in permanent paralysis as surely as virus-induced death of neurons. We examined the possibility that the nsP-induced mechanism of protection is cognate T-cell help for B cells (32). If this were the case, the levels of antibody to SV structural proteins would be higher in nsP recombinant vaccinia virus-immunized mice after NSV challenge, but the antibody levels were similar to those in vaccinia virus-immunized mice. An alternative possibility is that T cells infiltrating the CNS (26) produce a cytokine that improves the chance of neuronal survival after NSV infection. SV induces apoptosis in neurons in vivo and in vitro (20, 21). Both the virulence of the virus and the maturity of the neurons infected determine whether neurons die as a consequence of infection (44). These studies suggest that there is also an intermediate fate, neuronal dysfunction (manifest as paralysis) followed by immunologically mediated control of virus replication and subsequent recovery of neuronal function. We suggest that cytokines produced by T cells early in infection improve the chance of neuronal survival and recovery of function. Since ependymal cells, which do express major histocompatibility complex antigens, are infected early after inoculation, T cells may interact with these cells or with microglia to induce local cytokine synthesis. These protective responses may or may not be unique to the immune response

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to alphavirus nsPs. It is most likely that the protective functions of T cells during viral encephalitis occur in response to structural proteins as well but that in alphavirus encephalomyelitis they are masked by the protective effects of antibody to E1 and E2. ACKNOWLEDGMENTS This work was supported by Javits Neuroscience Investigator Award NS29234 (D.E.G.) and research grant AI24134 (C.M.R.) from the National Institutes of Health. REFERENCES 1. Burge, B., and E. Pfefferkorn. 1966. Isolation and characterization of conditional lethal mutants of Sindbis virus. Virology 30:204–213. 2. Cane, P. A., and E. A. Gould. 1988. Reduction of yellow fever virus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment. J. Gen. Virol. 69:1241–1246. 3. Cardiff, R. D., and J. K. Lund. 1976. Distribution of dengue-2 antigens by electron immunocytochemistry. Infect. Immun. 3:1699–1709. 4. Daar, A. S., S. V. Fuggle, J. W. Fabre, A. Ting, and P. J. Morris. 1984. The detailed distribution of HLA-A, B, C antigens in normal human organs. Transplantation 38:287–292. 5. Despres, P., J. Dietrich, M. Girard, and M. Bouloy. 1991. Recombinant baculoviruses expressing yellow fever virus E and NS1 proteins elicit protective immunity in mice. J. Gen. Virol. 72:2811–2816. 6. Doherty, P. C. 1993. Cell-mediated cytotoxicity. Cell 75:607–612. 7. Ehrenfeld, E., D. Brown, X. Y. Jia, and D. F. Summers. 1995. Antibodies against viral nonstructural proteins in response to infection with poliovirus. J. Infect. Dis. 171:845–850. 8. Falgout, B., M. Bray, J. J. Schlesinger, and C.-J. Lai. 1990. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J. Virol. 64:4356–4363. 9. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83:8122–8126. 10. Gould, E. A., A. Buckley, A. D. Barrett, and N. Cammack. 1986. Neutralizing (54K) and non-neutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus protein confer immunity in mice. J. Gen. Virol. 67:591–595. 11. Griffin, D. E., and R. T. Johnson. 1977. Role of the immune response in recovery from Sindbis virus encephalitis in mice. J. Immunol. 118:1070–1075. 12. Jackson, A. C., T. R. Moench, B. D. Trapp, and D. E. Griffin. 1988. Basis of neurovirulence in Sindbis virus encephalomyelitis of mice. Lab. Invest. 58: 503–509. 13. Jia, X. Y., D. F. Summers, and E. Ehrenfeld. 1992. Host antibody response to viral structural and nonstructural proteins after hepatitis A virus infection. J. Infect. Dis. 165:273–280. 14. Johnson, R. T., H. F. McFarland, and S. E. Levy. 1972. Age-dependent resistance to viral encephalitis: studies of infections due to Sindbis virus in mice. J. Infect. Dis. 125:257–262. 15. Joly, E., and M. B. A. Oldstone. 1992. Neuronal cells are deficient in loading peptides onto MHC class I molecules. Neuron 8:1185–1190. 16. Kurane, I., M. A. Brinton, A. L. Samson, and F. A. Ennis. 1991. Dengue virus-specific, human CD41 CD82 cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones. J. Virol. 65:1823–1828. 17. Lampson, L. A., and W. F. Hickey. 1986. Monoclonal antibody analysis of MHC expression in human brain biopsies: tissue ranging from “histologically normal” to that showing different levels of glial tumor involvement. J. Immunol. 136:4054–4062. 18. Lemm, J. A., and C. A. Rice. 1993. Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34. J. Virol. 67:1905–1915. 18a.Levine, B. Unpublished data. 19. Levine, B., J. M. Hardwick, B. D. Trapp, T. O. Crawford, R. C. Bollinger, and D. E. Griffin. 1991. Antibody-mediated clearance of alphavirus infection from neurons. Science 254:856–860. 20. Levine, B., Q. Huang, J. T. Isaacs, J. C. Reed, D. E. Griffin, and J. M. Hardwick. 1993. Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. Nature 361:739–742. 21. Lewis, J., S. L. Wesselingh, D. E. Griffin, and J. M. Hardwick. 1996. Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence. J. Virol. 70:1828–1835. 22. Li, G., B. M. Pragai, and C. M. Rice. 1991. Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant. J. Virol. 65:6714–6723.

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23. Lustig, S., A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss. 1988. The molecular basis of Sindbis virus neurovirulence in mice. J. Virol. 62:2329–2336. 24. McFarland, H. F., D. E. Griffin, and R. T. Johnson. 1972. Specificity of the inflammatory response in viral encephalitis. I. Adoptive immunization of immunosuppressed mice infected with Sindbis virus. J. Exp. Med. 136:216– 226. 25. Mendoza, Q. P., J. Stanley, and D. E. Griffin. 1988. Monoclonal antibodies to the E1 and E2 glycoproteins of Sindbis virus: definition of epitopes and efficiency of protection from fatal encephalitis. J. Gen. Virol. 70:3015–3022. 26. Moench, T. R., and D. E. Griffin. 1984. Immunocytochemical identification and quantitation of mononuclear cells in cerebrospinal fluid, meninges, and brain during acute viral encephalitis. J. Exp. Med. 159:77–88. 27. Moss, B., O. Elroy-Stein, T. Mizukami, W. A. Alexander, and T. R. Fuerst. 1990. Product review. New mammalian expression vectors. Nature 348:91– 92. 28. Neitzert, E., E. Beck, P. Auge de Mello, I. Gomes, and I. E. Bergmann. 1991. Expression of the aphthovirus RNA polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections. Virology 184:799–804. 29. Newell, C. K., S. Martin, D. Sendele, C. M. Mercadal, and B. T. Rouse. 1989. Herpes simplex virus-induced stromal keratitis: role of T-lymphocyte subsets in immunopathology. J. Virol. 63:769–775. 30. Rice, C. M., C. A. Franke, J. H. Strauss, and D. E. Hruby. 1985. Expression of Sindbis virus structural proteins via recombinant vaccinia virus: synthesis, processing, and incorporation into mature Sindbis virions. J. Virol. 56:227– 239. 31. Sarmiento, M., A. L. Glasebrook, and F. W. Fitch. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing LYT 2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125:2665–2672. 32. Scherle, P. A., and W. Gerhard. 1986. Functional analysis of influenzaspecific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J. Exp. Med. 164:1114–1128. 33. Schlesinger, J. J., M. W. Brandriss, B. Cropp, and T. P. Monath. 1986. Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. J. Virol. 60:1153–1155. 34. Schlesinger, J. J., M. W. Brandriss, and E. E. Walsh. 1985. Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48. J. Immunol. 135:2805–2809. 35. Schlesinger, J. J., M. W. Brandriss, and E. E. Walsh. 1987. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J. Gen. Virol. 68:853–857. 36. Schlesinger, J. J., M. Foltzer, and S. Chapman. 1993. The Fc portion of antibody to yellow fever virus NS1 is a determinant of protection against YF encephalitis in mice. Virology 192:132–141. 37. Schmaljohn, A. L., E. D. Johnson, J. M. Dalrymple, and G. A. Cole. 1982. Non-neutralizing monoclonal antibodies can prevent lethal alphavirus encephalitis. Nature 297:70–72. 38. Sherman, L. A., and D. E. Griffin. 1990. Pathogenesis of encephalitis induced in newborn mice by virulent and avirulent strains of Sindbis virus. J. Virol. 64:2041–2046. 39. Stanley, J., S. J. Cooper, and D. E. Griffin. 1985. Alphavirus neurovirulence: monoclonal antibodies discriminating wild-type from neuroadapted Sindbis virus. J. Virol. 56:110–119. 40. Stanley, J., S. J. Cooper, and D. E. Griffin. 1986. Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice. J. Virol. 58:107– 115. 41. Strauss, E. G., C. M. Rice, and J. H. Strauss. 1984. Complete nucleotide sequence of the genomic RNA of Sindbis virus. Virology 133:92–110. 42. Strauss, J. H., and E. G. Strauss. 1994. The alphaviruses: gene expression, replication, and evolution. Microbiol. Rev. 58:491–562. 43. Tucker, P. C., E. G. Strauss, R. J. Kuhn, J. H. Strauss, and D. E. Griffin. 1993. The age-dependent neurovirulence of Sindbis virus for mice is influenced by a single amino acid change at position 55 of the E2 glycoprotein. J. Virol. 67:4605–4610. 44. Ubol, S., P. C. Tucker, D. E. Griffin, and J. M. Hardwick. 1994. Neurovirulent strains of alphavirus induce apoptosis in bcl-2-expressing cells; role of a single amino acid change in the E2 glycoprotein. Proc. Natl. Acad. Sci. USA 91:5202–5206. 45. Wofsy, D., D. C. Mayes, J. Woodcock, and W. E. Seaman. 1985. Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice. J. Immunol. 135:1698–1701. 46. Zhang, Y.-M., T. C. Hayes, T. C. McCarty, D. R. Dubois, P. L. Summers, K. H. Eckels, R. M. Chanock, and C.-J. Lai. 1988. Immunization of mice with dengue structural proteins and nonstructural protein NS1 expressed by baculovirus recombinant induces resistance to dengue virus encephalitis. J. Virol. 62:3027–3031.