Molecular mimicry, anti-coxsackievirus B3 neutralizing monoclonal antibodies, and myocarditis.

Molecular mimicry has been suggested as one mechanism to explain chronic myocarditis in some murine strains in the postinfectious period following induction of acute myocarditis by coxsackievirus B3 (CVB3). To test this hypothesis, neutralizing mAbs were generated against a highly myocarditic CVB3 virus (CVB3m). These mAbs neutralized several myocarditic and amyocarditic CVB3 variants by cytopathic effects inhibition assays. Data from several experiments suggest that these mAbs recognize discontinuous epitopes on CVB3m capsid proteins. Several mAbs were found to induce cardiopathologic alterations subsequent to i.p. inoculation of normal adolescent male CD-1 or C3H/HeJ mice. Immunocytochemical assays demonstrated significant binding of two mAbs to the surface of normal cultured murine cardiac fibroblasts. Also, several mAbs were shown to participate in C-mediated lysis of normal cardiac fibroblasts, but this property did not correlate well with cardiopathogenic potential. The two properties of a mAb that were the best predictors for cardiopathogenic potential were the capacity for stimulation of normal murine fibroblasts to produce a chemoattractant activity for unelicted murine peritoneal macrophages, and the capacity for recognition of an epitopes(s) on murine or human cardiac myosins. These data show that some anti-CVB3m neutralizing mAbs can participate in proinflammatory reactions in vitro and induce cardiopathologic alterations in vivo, suggesting one mechanism by which CVB3-induced chronic inflammation in murine heart tissues can be sustained in the absence of continued virus replication.

Anti-Coxsackievirus B3 neutralizing antibodies with pathological potential.

Adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3m) will develop acute myocarditis with focal lesions by 7 days post-inoculation (p.i.). Administration of murine sera containing anti-CVB3m-neutralizing antibodies into CVB3m-inoculated mice at 3 days p.i. will exacerbate myocarditis, suggesting the presence of pathological antibodies. To study potential pro-inflammatory properties of virus-induced antibodies, a panel of anti-CVB3m-neutralizing monoclonal antibodies (mAbs) was generated. Several studies demonstrated shared epitopes between CVB3m particles and cultured murine cardiac or neonatal skin fibroblasts: (1) one or more mAbs bound to cultured cardiac fibroblasts; (2) several mAbs can participate in complement-mediated lysis of neonatal skin fibroblasts; and (3) at least one mAbs stimulated synthesis of a macrophage chemoattractant from cultured neonatal skin fibroblasts. Injection of one mAb in three doses, each of about 5 micrograms, into adolescent male CD-1 mice induced focal myocarditic lesions which were similar to CVB3m-induced lesions. One mAb induced a diffuse interstitial hypercellularity in most mice and two mAbs did not induce detectable cardiopathology. These data suggest that some anti-CVB3m neutralizing idiotypes (antibodies) which initially can provide protection via virus clearance mechanisms can also bind to cross-reacting epitopes on normal tissues. Binding of antibodies to normal heart tissues could stimulate proinflammatory reactions by several mechanisms and sustain myocarditis.

Characterization of the antibody response in vaccinated mice protected against Coxsackievirus B3-induced myocarditis.

Adolescent male CD-1 mice can be rendered resistant to coxsackievirus B3 (CVB3m)-induced myocarditis following inoculation as neonates with a single dose of a temperature-sensitive mutant virus (ts1), derived from the prototype parent virus (CVB3m). Previously, anti-CVB3 neutralizing antibodies were not detected in sera of adolescent ts1 vaccinees by a standard plaque-reduction assay (Gauntt et al 1983. Infect. Immun. 39:851). However, a more sensitive cytopathic effects-reduction assay permitted detection of low titers of anti-CVB3m neutralizing antibodies of the IgG class prior to challenge with CVB3m. Following CVB3m challenge, serum anti-CVB3m neutralizing antibody titers of ts1 vaccines declined on days 1-2 post-inoculation (p.i.) then increased over the next 6 days. The neutralizing antibodies were of both the IgG and IgM classes. Normal mice challenged with CVB3 did not produce detectable serum anti-CVB3m neutralizing antibody until day 4 p.i. and by 8 days p.i. the neutralizing antibody was only of the IgM class. Thus, adolescent murine ts1 vaccines mount a secondary antibody response to CVB3m in both neutralizing IgG and IgM, but resistance to CVB3m-induced myocarditis is due to the presence of low levels of anti-CVB3m IgG neutralizing antibody in serum at the time of challenge with CVB3m.

Antimyocarditic activity of the guanine derivative BIOLF-70 in a coxsackievirus B3 murine model.

Prophylactic administration of a nontoxic dose of 9-[[2-benzyloxyl-1-(benzyloxymethyl)ethoxy]methyl]-6-chlo roguanine (BIOLF-70) to mice reduced the number of myocarditic lesions induced by coxsackievirus B3 (CVB3). BIOLF-70 exhibited minimal antiviral activity against CVB3 in HeLa cells and murine neonatal skin fibroblasts and minimally reduced CVB3 yields in heart tissues. The drug had no effect on serum anti-CVB3 neutralizing antibody titers and did not induce the production of interferon. Flow microfluorometric analyses of splenic lymphocytes taken from BIOLF-70-treated, CVB3-inoculated mice at 7 days postinoculation showed that the proportion of T lymphocytes was increased, as measured by fluorescent staining of Thy-1 and Lyt-2 surface markers, compared with the proportion of T lymphocytes in splenic cells from virus-inoculated or BIOLF-70-treated or normal groups of mice. Splenic lymphocytes from BIOLF-70-treated, CVB3-inoculated mice showed reduced cytotoxic activity against CVB3-infected target fibroblasts. Splenic cells from BIOLF-70-treated, CVB3-inoculated mice had slightly higher natural killer cell activity than did those from the other three groups of mice, which had comparatively similar levels of natural killer cell activity. The data suggest that BIOLF-70 exerts antimyocarditic activity perhaps by some antiviral activity in heart tissues and by immunomodulatory mechanisms which appear to involve T suppressor or T cytotoxic lymphocyte subpopulations and natural killer cells.

Murine forebrain anomalies induced by coxsackievirus B3 variants.

Neonatal or 7-day-old mice inoculated intracranially with either of two temperature-sensitive mutants (ts1, ts6) or the parent coxsackievirus B3 (CVB3) subsequently developed porencephaly or hydranencephaly. The forebrain anomaly induced depended upon age of the animal at inoculation and virus variant inoculated. Sections of brains from hydranencephalic mice revealed severe meningeal reactions, necrotizing encephalitis, and liquifactive necrosis in the cerebrum. No pathology was found in the pons, medulla, or cerebellum. Immunofluorescence studies with hyperimmune anti-CVB3 antiserum showed a random distribution of virus-infected cells in the cerebrum. Virus was recovered from several organs but little to no interferon and no anti-CVB3 neutralizing antibody were present in brain tissues. Availability of cells for replication of virus at the time of inoculation and replicative properties of each virus likely contributed to the outcome. Thus, forebrain anomalies resembling those found in infants can be induced in a murine model by select variants of coxsackievirus B3.