Antibody Prevents Virus Reactivation Within the Central Nervous System

Mice

Breeding pairs of C57BL/6-Igh-6^tm1Cgn (IgM^−/−) mice (30) and syngeneic C57BL/6 (wt) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Homozygous IgM^−/− mice were bred at the University of Southern California School of Medicine (Los Angeles, CA) under pathogen-free conditions. The absence of Ig in IgM^−/− mice was confirmed by testing sera for murine IgG by ELISA, as previously described (31). All mice used were between the ages of 7–8 wk of age.

Viral infection and titer determination

Mice were infected via intracerebral inoculation with 50 plaque-forming units of the 2.2v-1 mAb-derived variant of JHMV in 32 μl of Dulbecco’s PBS, pH 7.4, as previously described (32). The 2.2v-1 strain of JHMV was kindly supplied by J. Fleming (University of Wisconsin, Madison, WI). Tissue levels of infectious virus were determined by plaque assay on monolayers of DBT cells, as previously described (6). Briefly, clarified homogenates from one-half of the brains were serially diluted in serum-free-PBS containing 10% tryptose phosphate broth, and virus was adsorbed for 90 min at room temperature. Plaque numbers were determined following 24–48-h incubation at 37°C. Data represent the average of triplicate samples for groups of four or more mice.

Clinical disease

Clinical disease was graded as previously described (6, 28): 0, healthy; 1, ruffled fur and hunch back; 2, slow mobility and inability to upright; 3, paralysis and wasting; 4, moribund and death. Data represent the average for four or more mice per group.

CTL assays

Spleen cell suspensions were prepared at 7, 11, and 21 days postinfection (p.i.). Splenocytes from three mice per group were pooled, and 1 × 10⁸ cells were cultured for 6 days at 37°C with irradiated (25 Gy) syngeneic JHMV-infected spleen cells in RPMI 1640 medium supplemented with 10% FCS (Gemini Biological Products, Calabasas, CA), 2 mM glutamine, 25 μg/ml gentamicin, 1 mM sodium pyruvate, 5 × 10⁻⁵ M 2-ME, and nonessential amino acids. Cytolytic activity was measured as previously described (6, 7). Briefly, syngeneic EL-4 (H-2^b) target cells were infected with either a recombinant vaccinia virus (rVV) expressing amino acids 510–518 of the JHMV spike (S) protein (vJS510) or a control rVV expressing the Escherichia coli lacZ gene (vSC8) at a multiplicity of infection of 5 (33). Following incubation for 14 h at 37°C, target cells were labeled with 100 μCi Na⁵¹CrO₄ (New England Nuclear, Boston, MA) for 1 h at 37°C. Effector cells were added to target cells at various E:T ratios. ⁵¹Cr release was measured after 4-h incubation at 37°C. Data are expressed as percent specific release defined as: ((experimental release) − (spontaneous release))/((total release − (spontaneous release)). Maximum spontaneous release values were ≤20% of total release.

RNA expression

Total RNA was prepared from one-half of spinal cords divided longitudinally by homogenization in guanidine isothiocyanate and isolated by centrifugation through 5.4 M cesium chloride at 100,000 × g for 18 h, as previously described (31). The cDNA were prepared using avian myeloblastosis reverse-transcriptase (Promega, Madison, WI) and oligo(dT) primers (Promega) for 1 h at 42°C. PCR was performed using AmpliTaq DNA polymerase (Perkin-Elmer, Branchburg, NJ) and primers specific for IFN-γ (31) and JHMV nucleocapsid protein (N) (viral nucleotides 527-1257): 5′-ATA GGA TCC ATG GCT ACT AGG TTT GCG CCC GGC-3′ and 5′-ACA GTT ACC TAC ATC TGC ACC ACC ATC TTG-3′. Amplification of IFN-γ mRNA was carried out using 35 cycles of one denaturing step for 45 s at 94°C, primer annealing for 45 s at 59°C, and extension for 1.5 min at 72°C. Amplification for JHMV N mRNA was conducted using 25 cycles of one denaturing step at 94°C (45 s), primer annealing at 72°C (45 s), extension step at 72°C (2 min), followed by a final extension step for 7 min. For quantification, PCR products were diluted in denaturing solution (0.4 N NaOH, 25 mM EDTA), neutralized with Tris-HCl (1 M; pH 8), and transferred to 0.45-μm Nytran membranes (Schleicher & Schuell, Keene, NH) using a Minifold I dot-blot apparatus. Membranes were hybridized overnight at 60°C with [³²P]ATP-labeled internal oligonucleotide probes for IFN-γ (31) and JHMV N mRNA: 5′-ATA GGA TCC ATG GTT TTG GCT AAG CTC GGT AAA G-3′. Membranes were washed, exposed to Storage Phosphor Screens (Molecular Dynamics, Sunnyvale, CA), and scanned using a PhosphorImaging Scanner (Molecular Dynamics). To adjust for differences in cDNA quantity, levels were normalized to the housekeeping enzyme hypoxanthine phosphoriboxyltransferase, as previously described (31). Mean values for three mice per group are presented.

Sequence analysis

Virus derived from brain samples was propagated once on DBT monolayers, and the cells were lysed by addition of guanidine thiocyanate solution when approximately 80% of the cells exhibited cytopathology. RNA was isolated by phenol/chloroform extraction. RNA from cells infected with parental virus and uninfected cells were used to control for mutations introduced by Taq polymerase and PCR contamination, respectively. RNA (5 μg) was reverse transcribed using avian myeloblastosis virus reverse-transcriptase and random hexanucleotide primers (Promega). A cDNA encompassing the immunodominant CTL epitope within the JHMV S protein (viral nucleotides 1528–1554) plus surrounding 500 nucleotides 1420–1890(1420–1890) was amplified using oligonucleotide primers JS1895 and JS1390, as previously described (8). The cDNAs were sequenced on an ABI Prism automated sequencing apparatus using oligonucleotide JS1390 as primer, as previously described (8).

Ab preparations

Serum-free supernatants from a hybridoma producing JHMV-neutralizing mAb J.2.2 (IgG2b) (34) were concentrated by precipitation with saturated ammonium sulfate. Anti-JHMV polyclonal Ab was obtained from mice hyperimmunized with JHMV (35). Anti-JHMV and control ascites were induced in immunized and naive mice, respectively, by injection of Sarcoma 180 cells (35). Ascites were defibrinated (36) and heat inactivated at 56°C for 30 min before use. Control ascites was tested by ELISA to insure the absence of JHMV Ab, as previously described (31).

Passive transfers

IgM^−/− mice received 150 μg of either JHMV-neutralizing mAb J.2.2 or an equal amount of IgG2b myeloma protein (Zymed Laboratories, San Francisco, CA). These Ab and the polyclonal anti-JHMV and control ascites were transferred into the peritoneal cavity of infected IgM^−/− mice at 9, 12, and 17 days p.i. Neither the isotype control myeloma protein nor the ascites obtained from naive mice had detectable anti-JHMV Ab, as determined by ELISA. Neutralization titers were determined by the plaque-reduction method (6, 31, 35). Briefly, 50–100 plaque-forming units of either DM (for mAb J.2.2) or the 2.2v-1 (for immune ascites) JHMV strains were mixed with serial 4-fold dilutions. After 1 h at 37°C, residual virus was determined by plaque assay. The dilution that reduced plaque numbers by 50% was designated as titer (35). The neutralizing titers of the J.2.2 mAb and anti-JHMV ascites were 1/1600 and 1/3200, respectively.

Histopathology

Brains, bisected in the mid-coronal plane, and spinal cords were prepared for frozen sections or embedded in paraffin. For paraffin preparations, tissues were fixed for 3 h in Clark’s solution (75% ethanol, 25% glacial acetic acid) before embedding. Sections were stained with either hematoxylin and eosin or luxol fast blue. Distribution of JHMV Ag was determined by immunoperoxidase staining (Vectastain-ABC kit; Vector Laboratories, Burlingame, CA) using the anti-JHMV mAb J.3.3 specific for the carboxyl terminus of the N protein as the primary Ab (34, 37) and horse anti-mouse mAb as secondary Ab (Vector Laboratories). CD4⁺ and CD8⁺ T cells were identified in frozen sections fixed with acetone for 2 min at room temperature and stained using anti-CD4 (L3T4; PharMingen, San Diego, CA) or anti-CD8 (Ly-2, PharMingen) mAb. Both primary Ab were detected with a biotinylated rabbit anti-rat serum preabsorbed with mouse serum (Vector Laboratories) and visualized using Vectastain-ABC kits. Apoptotic cells were identified using Oncor ApopTag Kit (Gaithersburg, MD), which utilizes TdT for extension of fragmented DNA, as previously described (6). Tissue processing and staining were performed according to the manufacturer’s instructions. For quantitative comparisons, four serial step sections of individual samples from groups of three to four mice were stained, and all positive cells counted. Student’s t test was used for statistical analysis.

Biphasic pathogenesis in IgM^−/− mice

To determine whether humoral immunity contributes to JHMV pathogenesis, clinical disease and mortality were compared in infected IgM^−/− and wt mice. Both wt and IgM^−/− mice developed clinical signs of disease (mild ruffled fur and hunched back) by 7–8 days p.i. (Fig. 1⇓A), which progressed to hind limb paralysis and wasting in both groups by approximately 13 days p.i. In contrast to the clinical recovery of wt mice, IgM^−/− mice showed signs of increasing lethargy and wasting between 14 and 40 days p.i. (Fig. 1⇓A). The increase in clinical signs coincided with an increase in mortality of IgM^−/− mice after 25 days p.i. (Fig. 1⇓B). By day 40 p.i., only 8% of JHMV-infected IgM^−/− mice survived and the remainder appeared moribund. By contrast, 80% of wt mice had completely recovered from infection and minor gait abnormalities.

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FIGURE 1.

IgM^−/− mice exhibit increased morbidity (A) and mortality (B) following JHMV infection. IgM^−/− and wt mice were infected with JHMV, and the morbidity and mortality were recorded as follows: 0, healthy; 1, hunched back and ruffled fur; 2, slow mobility and inability to upright; 3, paralysis and wasting; 4, moribund and death. Average scores of 4 or more mice per time point are shown. Error bars indicate the SE means.

The kinetics of viral replication in IgM^−/− and wt mice was compared to determine whether increased morbidity and mortality in the JHMV-infected IgM^−/− mice resulted from an altered ability to eliminate infectious virus from the CNS. Consistent with the clinical signs of disease, infectious JHMV was cleared from the CNS of both groups with similar kinetics between 3 and 11 days p.i. (Fig. 2⇓). However, by 14 days p.i., when no infectious JHMV could be isolated from the CNS of wt mice, infectious virus had reemerged in the IgM^−/− mice after 11 days p.i. Between 14 and 35 days p.i., infectious JHMV continued to increase in the CNS of the surviving IgM^−/− mice. By contrast, infectious virus remained undetectable in the CNS of the wt mice. These data confirmed the role of cellular immunity in JHMV clearance during acute CNS infection (6, 7) and suggest that the anti-viral Ab response is critical for maintaining the suppression of infectious virus, resulting in the establishment of a persistent CNS infection.

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FIGURE 2.

JHMV replication in the CNS of IgM^−/− and wt mice. Each time point is the mean of four to six samples and the SEMs shown as error bars. Student’s t test was used to test for significance between groups (*, p < 0.05).

CNS inflammation in IgM^−/− mice

To determine whether IgM^−/− mice exhibited altered inflammatory responses, the extent of mononuclear cell infiltration and frequency of apoptotic cells in the CNS was compared with wt mice. At day 7 p.i., the majority of the mononuclear cell infiltration and apoptotic cells were localized to the perivascular areas, with only a few CD4⁺ or CD8⁺ T cells detected within the parenchyma in both groups (data not shown). At day 21 p.i., the CNS of the IgM^−/− mice showed increased numbers of perivascular mononuclear cells (Fig. 3⇓). This distribution of inflammatory cells is reminiscent of the perivascular inflammation observed during acute JHMV infection (38). Indeed, a 10-fold increase in perivascular CD4⁺ T cells was observed in the IgM^−/− mice compared with the wt mice at day 21 p.i. (Fig. 3⇓). In both groups, the CD8⁺ T cells were present in a diffuse pattern within the parenchyma (Fig. 3⇓). Although the numbers of CD8⁺ T cells varied dramatically in individual microscopic fields, in contrast to the CD4⁺ T cells, the total number of CD8⁺ T cells in the IgM^−/− mice was not statistically different from wt mice. Fewer apoptotic cells were detected at 21 days p.i. compared with 7 days p.i.; however, no difference in the numbers or distribution of apoptotic cells was observed comparing the two groups at 21 days p.i. (Fig. 3⇓). These data suggest, in contrast to the absence of CD4⁺ T cells that dramatically increased the number of apoptotic lymphocytes (38), the absence of humoral immunity did not result in an increase in apoptosis of effector cells within the CNS. Both wt and IgM^−/− mice had prominent demyelination within the spinal cord (Fig. 4⇓). In contrast to wt mice, demyelinating lesions were detected in the brains of IgM^−/− mice in areas in which virus Ag was found (data not shown). These data suggest that the cell-mediated immune response is not compromised in the IgM^−/− mice, although the increase in infectious virus is associated with increased demyelination.

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FIGURE 3.

Immunopathology in the CNS of JHMV-infected IgM^−/− and wt mice at 21 days p.i. IgM^−/− mice (A) show increased cellular infiltrates localized to the perivascular areas (small arrows) compared with wt mice (B). IgM^−/− mice have increased numbers of perivascular CD4⁺ T cells (small arrows) (C) compared with wt mice (D), although both groups have similar total numbers of CD4⁺ T cells within the CNS. Although focal areas of the wt mice showed increased numbers of parenchymal CD8⁺ T cells (arrowheads), the total numbers present were similar in both groups (E and F). Few apoptotic cells (large arrows) were detected in either IgM^−/− (G) or wt mice (H). Sections in A and B were stained with hematoxylin and eosin. CD4⁺ and CD8⁺ T cells were detected by immunoperoxidase stain using the avidin-biotin-peroxidase complex method on acetone-fixed frozen sections (C–F). Apoptotic cells were detected using TUNEL stain on frozen sections. Magnification for A–F is ×125. For G and H, magnification is ×400.

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FIGURE 4.

Demyelination in the CNS of IgM^−/− and wt mice infected with JHMV. Demyelination in the spinal cords of IgM^−/− (A) and wt mice (B) at 21 days p.i. Sections were stained with luxol fast blue, and areas of demyelination are outlined by arrows.

Cellular tropism

IgM^−/− mice and wt controls were infected with the JHMV 2.2v-1 variant derived by selection from a highly virulent JHMV with neutralizing mAb J.2.2 (32). A possible explanation for the recrudescence of JHMV within the CNS of the IgM^−/− mice is reversion of the 2.2v-1 phenotype to a more virulent phenotype (39). To examine this possibility, mAb J.2.2 was passively administered to infected IgM^−/− mice at 9, 12, and 17 days p.i. and compared with infected IgM^−/− mice receiving an isotype-matched Ab with no detectable ability to recognize JHMV. These time points were chosen to parallel the initial detection of neutralizing Ab in JHMV-infected mice (6). Neither passive transfer of mAb J.2.2 nor the control Ab altered the clinical course of infection or prevented recrudescence of JHMV at 21 days p.i. (Table I⇓). Furthermore, no difference in the extent of inflammation or distribution of viral Ag was detected comparing the mAb J.2.2 recipients, the control Ab recipients, or untreated IgM^−/− mice (data not shown). These data suggest that reversion to the parental phenotype was not responsible for the reactivation of JHMV in the CNS of the IgM^−/− mice following initial clearance.

Altered cellular tropism of JHMV within the CNS as a contributing factor to the phenotype observed in IgM^−/− mice was examined by determining the distribution of viral Ag. At day 7 p.i., the majority of viral Ag was present in oligodendrocytes, microglia, and astrocytes in both groups (Fig. 5⇓). Only rarely were neurons positive for viral Ag in either group. At 11 days p.i., few Ag-positive cells were detected in the brains of either group (Fig. 5⇓), consistent with the reduction in infectious virus (Fig. 2⇑). A similar cellular distribution of viral Ag in both groups during the acute phase of disease (7–11 days p.i.) suggests that altered cellular tropism did not contribute to the recrudescence of infectious JHMV in the absence of Ab. Similarly, only focal areas containing viral Ag-positive cells were detected within the spinal cords of wt mice at 21 days p.i. (Fig. 5⇓), consistent with the inability to recover infectious virus at this time point (Fig. 2⇑). By contrast, the brains of IgM^−/− mice showed numerous Ag-positive cells (Fig. 5⇓), consistent with the presence of infectious virus (Fig. 2⇑), and the spinal cords showed a more diffuse distribution of Ag-positive cells. In both sites, virus Ag was predominantly found in the white matter; however, focal involvement of gray matter was also seen. The cellular distribution of virus Ag at 21 days p.i. in the IgM^−/− mice was similar to that seen at earlier time points with predominant localization to astrocytes, oligodendrocytes, and microglia. A small number of Ag-positive neurons was also present. With the exception of viral spread to a small number of neurons, the distribution of viral Ag was comparable in the two groups, suggesting no change in cell tropism in the absence of humoral immunity.

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FIGURE 5.

Distribution of JHMV Ag within the CNS. JHMV Ag (arrowheads) was detected in the brains of both IgM^−/− (A and C) and wt mice (B and D) at days 7 (A and B) and 11 (C and D) p.i. without apparent differences in the extent or distribution of virus-infected cells. Note the decrease in Ag-positive cells at day 11 p.i. At day 21 p.i., viral Ag was localized to cells within the white matter of the spinal cord (E), but not the brain (F) of wt mice. In IgM^−/− mice, viral Ag was present in the both the spinal cord (G) and brain (H). Immunoperoxidase stain used the avidin-biotin-peroxidase complex method for viral Ag (mAb J.3.3) with hematoxylin and eosin counterstain (magnification, ×125).

CTL responses in IgM^−/− mice

CTL responses in the spleens of IgM^−/− and wt mice were comparable at day 7 p.i. (Fig. 6⇓), consistent with the predominant role of these effectors in JHMV clearance from the CNS (6, 7). CTL activity in the spleens of wt mice was equivalent to the day 7 p.i. level at 21 days p.i. By contrast, peripheral CTL activity in IgM^−/− mice was reduced compared with the levels detected in wt mice (Fig. 6⇓), suggesting the retention of CTL within the CNS due to the presence of infectious virus and viral Ag within the CNS (Figs. 2⇑ and 5⇑). Accumulation of IFN-γ mRNA and the mRNA encoding the N protein of JHMV within the CNS of infected IgM^−/− mice was examined to demonstrate that the decreased peripheral CTL activity in IgM^−/− mice represented continuous recruitment and/or sequestration in the CNS due to the presence of infectious virus (40). The IFN-γ mRNA levels in the CNS of IgM^−/− and wt mice were approximately equivalent during acute infection and viral clearance (7, 9, and 11 days p.i.; Fig. 7⇓). The kinetics of accumulation of JHMV N mRNA paralleled the kinetics of virus replication within the CNS of both groups (Fig. 7⇓). However, while the levels of IFN-γ mRNA in wt mice decreased to below detection limits as infectious virus was eliminated (Fig. 5⇑), IFN-γ mRNA levels in the CNS of the IgM^−/− mice remained elevated (Fig. 7⇓). Although both CD4⁺ and CD8⁺ T cells could contribute to IFN-γ mRNA within the CNS, CD8⁺ T cells are a major source of this cytokine (41, 42), suggesting that reduced splenic CTL in the IgM^−/− mice is due to the presence of activated CTL within the CNS (Fig. 3⇑).

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FIGURE 6.

CTL activity following JHMV infection. CTL activity at 7 (A) and 21 (B) days p.i. Splenocytes from four infected mice/group were pooled and restimulated for 6 days. JHMV-specific CTL activity was tested in a 4-h ⁵¹Cr release assay. EL4 (H-2^b) target cells were infected with either a rVV expressing the Escherichia coli lacZ gene (vSC8) or an rVV expressing amino acids 510–518 of the JHMV S protein (vJS510).

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FIGURE 7.

Kinetics of IFN-γ and JHMV N mRNA accumulation in the CNS of mice during JHMV infection of IgM^−/− and wt mice. RNA was extracted from the spinal cords of JHMV-infected IgM^−/− and wt mice at various times p.i. Levels of IFN-γ (A) and N (B) mRNA are expressed as a relative amount value for comparison. Each point represents the mean value of at least three mice per group, and error bars represent the SEM.

The possibility that CTL escape variants contributed to viral reactivation in IgM^−/− mice was examined by sequence analysis of the region of the gene encoding the JHMV S protein, which contains the immunodominant H-2D^b-restricted CTL epitope (amino acids 510–518) (33, 43). The 500 nucleotides of the gene (nucleotides 1420–1890) surrounding the CTL epitope (nucleotides 1528–1554) were sequenced from viruses isolated from infected IgM^−/− mice. None of the viruses isolated at 7, 11, or 21 days p.i. had dominant base substitutions or deletions within the S gene region encoding the immunodominant CTL epitope. Two of three mice analyzed at 30 days p.i. had wt sequences. In a third mouse, however, sequence analysis suggested that approximately 50% of the S genes contained a mutation within the CTL epitope. Consistent with this estimate, five of nine cloned PCR products contained the identical mutation (G→T at nucleotide 1539), resulting in a W→C change at amino acid 513. None of the six cloned PCR products from another 30 day p.i. mouse had any detectable mutations within the S gene. To determine whether the W→C mutation altered CTL recognition, viruses with both the wt and mutant sequences were plaque purified from mice 30 days p.i., and the presence of wt and mutation was confirmed by sequence analysis. Target cells (IC-21 (H-2^b)) infected with both viruses were recognized by JHMV-specific CTL (data not shown). These data suggest that mutations within the immunodominant CTL epitope can indeed be obtained in the absence of humoral immunity. However, the paucity of mutants obtained only well after virus reactivation supports a functional cell-mediated immune response within the CNS of infected IgM^−/− mice.

Passive transfer eliminates recrudescence of infectious JHMV

Serum JHMV-neutralizing Ab is detected approximately 9 days p.i. in JHMV-infected wt mice (6). These data suggest that the Ab response contributes to preventing JHMV recrudescence. To demonstrate that the absence of an Ab response is responsible for the reactivation of infectious virus, anti-JHMV Ab was passively transferred to infected IgM^−/− mice at 9, 12, and 17 days p.i., and its ability to inhibit CNS virus reactivation was determined at 21 days p.i. Control IgM^−/− mice received an equal volume of ascites obtained from naive mice. IgM^−/− mice that received the passive transfer of anti-JHMV Ab had reduced clinical signs compared with IgM^−/− mice that received control Ab (Table II⇓). Consistent with the reduction in clinical signs, IgM^−/− mice treated with anti-JHMV Ab also had either undetectable or significantly (p < 0.01) reduced levels of infectious JHMV within the CNS (Table II⇓). By contrast, high titers of infectious virus were detected in IgM^−/− mice that received nonimmune ascites (Table II⇓). Anti-JHMV Ab-protected IgM^−/− mice showed little or no viral Ag in the brain at 21 days p.i. (Fig. 8⇓), while extensive demyelination and viral Ag in cells morphologically consistent with oligodendrocytes were found in spinal cords (Fig. 8⇓). By contrast, both the brain and spinal cord of IgM^−/− mice treated with nonimmune ascites had numerous Ag-positive cells and prominent demyelination (Fig. 8⇓) similar to untreated JHMV-infected IgM^−/− mice (Figs. 3⇑ and 4⇑). These data support a direct role of the anti-viral Ab response in controlling the reactivation of JHMV within the CNS and establishing persistent JHMV infection.

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FIGURE 8.

Immunopathology in anti-JHMV Ab-treated IgM^−/− mice. Brain and spinal cords from anti-JHMV Ab-treated and control IgM^−/− mice at 21 days p.i. were stained for JHMV Ag with J.3.3 mAb and counterstained with hematoxylin and eosin. Sections from IgM^−/− mice treated with control Ab show extensive cellular infiltrates and numerous Ag-positive cells (arrowheads) (A and B). By contrast, fewer viral Ag-positive cells (arrowheads) were detected in the brains (C) and the spinal cords (D) of IgM^−/− mice, which received the passive transfer of anti-JHMV Ab (magnification, ×125).