Localization of a neutralization site of Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses that produce enteric and neurological diseases in mice. Subgroup TO strains of TMEV cause persistent infections with demyelination, while subgroup GDVII strains neither persist nor demyelinate. We produced neutralizing monoclonal antibodies (mAbs) to clarify the mechanisms of persistence and demyelination. Some of the neutralizing mAbs reacted with isolated VP1 on Western blots, while others were conformation specific. The neutralization site for the former TMEV mAbs was on the VP1 trypsin cleavage site of the intact virion. The neutralization site for the conformation-specific mAbs was distinct and was not affected by trypsin. Trypsin treatment of subgroup TO strains increased their infectivity for L cells, whereas the infectivity of subgroup GDVII strains was decreased by trypsin treatment. Subpopulations of virus in subgroup TO-infected tissue culture cells and in infected mouse brain homogenates contained VP1-cleaved virus; this VP1-cleaved virus gave rise to a large persistent fraction in neutralization tests when it was reacted with VP1-specific mAbs. These findings have implications regarding the pathogenesis of subgroup TO demyelinating disease. TMEV VP1 cleavage may be important for virus persistence because of disruption of a major neutralization epitope. The change in virus surface structure caused by VP1 cleavage may affect cell binding and lead to altered cytotropism. Immunocytes, which have been implicated in subgroup TO demyelination, may provide a source for proteases for VP1 cleavage.     

Trypsin-sensitive neutralization site on VP1 of Theiler''s murine encephalomyelitis viruses.

We generated Theiler's murine encephalomyelitis virus mutants resistant to several neutralizing monoclonal antibodies (MAbs) having their epitopes near a trypsin cleavage site of VP1. Neutralization and Western blot (immunoblot) studies suggest that two of the MAbs have identical epitopes that partly overlap the epitope of a third MAb. Sequencing of RNA of the mutants localized the epitopes to a site near the carboxyl end of VP1. The limited diversity of nucleotide changes seen in the mutants and the immunodominance of the site suggest that the carboxyl end of VP1 may have an important function.     

Alternative translation initiation site in the DA strain of Theiler''s murine encephalomyelitis virus.

Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus.     

Receptors for Theiler''s murine encephalomyelitis virus: characterization by using rabbit antiviral antiserum.

An immunological assay was developed to characterize the binding of Theiler's murine encephalomyelitis virus to BHK-21 cell receptors. After absorption of the virus and formaldehyde fixation, rabbit antibodies and Staphylococcus aureus protein A labeled with 125I formed a specific complex on the surfaces of the cells. The optimal multiplicity of infection in this system was 10 PFU per cell. The virus was internalized at 33 and 37 degrees C, but internalization did not take place at 25 or 4 degrees C. The binding was proportional to the number of cells and was significant within 30 s. Cell surface receptors were still active after fixation, and only intact viruses were bound, as demonstrated by the lack of binding of the purified, isolated virion proteins VP1, VP2, and VP3.     

Theiler''s murine encephalomyelitis virus neutralization escape mutants have a change in disease phenotype.

DA strain of Theiler's murine encephalomyelitis virus produces a persistent demyelinating infection. We previously produced escape mutant viruses that are resistant to a neutralizing monoclonal antibody and have a mutation in VP1 amino acid residue 268 in a neutralization site (Y. Ohara, A. Senkowski, J. Fu, L. Klaman, J. Goodall, M. Toth, and R.P. Roos, J. Virol. 62:3527-3529, 1988). In contrast to wild-type DA strain, these escape mutants produce little if any demyelinating disease after inoculation into weanling mice.     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

Survival of athymic (nu/nu) mice after Theiler''s murine encephalomyelitis virus infection by passive administration of neutralizing monoclonal antibody.

Little or no antiviral immune response is mounted in athymic nude mice infected with the Daniels strain of Theiler's murine encephalomyelitis virus. In these athymic mice, increasing levels of infectious virus could be detected in the central nervous system. Seventy-five percent (9 of 12) of the nude mice were moribund or dead by 4 weeks postinfection. In contrast, treatment of Theiler's virus-infected nude mice with a neutralizing monoclonal antibody (H7-2) against the viral protein VP-1 resulted in a dramatic reduction of infectious virus within the central nervous system. All antibody-treated nude animals survived beyond 4 weeks postinfection. Monoclonal antibody titers could be maintained by passive transfer in treated nude mice at levels comparable to those of polyclonal antibody titers found in heterozygous infected nu/+ littermates. Areas of demyelination were detected in the untreated animals as early as 7 days after infection with little or no remyelination present. In approximately one-half of the antibody-treated nude animals, no demyelinating lesions were found. However, the rest of these treated mice were found to have areas of both demyelination and remyelination. Thus, anti-Theiler's murine encephalomyelitis virus antibody against VP-1 can play a dramatic role in the survival of mice, clearance of virus, limiting viral spread, and altering the pattern of disease in the absence of a functional T-cell response.     

Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120.

Monoclonal antibodies (MAbs) were obtained by immunizing mice with synthetic peptides corresponding to the third variable (V3) or the third conserved (C3) domain of the external envelope protein (gp120) of human immunodeficiency virus type 2 (HIV-2ROD). One MAb, designated B2C, which was raised against V3 peptide NKI26, bound to the surface of HIV-2-infected cells but not to their uninfected counterparts. B2C was capable of neutralizing cell-free and cell-associated virus infection in an isolate-specific fashion. The antibody-binding epitope was mapped to a 6-amino-acid peptide in the V3 variable domain which had the core sequence His-Tyr-Gln. Two MAbs, 2H1B and 2F19C, which were raised against the C3 peptide TND27 reacted with gp120 of HIV-2ROD in a Western immunoblot assay. The C3 epitopes recognized by these two MAbs appeared inaccessible because of their poor reactivity in a surface immunofluorescence assay. Although partial inhibition of syncytium formation was observed in the presence of the anti-C3 MAbs, their neutralizing activity appeared weak. Finally, the effects of these MAbs against CD4-gp120 binding were assessed. Partial inhibition of CD4-gp120 binding was observed in the presence of high concentrations of B2C. On the other hand, no inhibition of CD4-gp120 binding was observed in the presence of anti-C3 MAbs. Since complete neutralization could be achieved at a concentration corresponding to that of partial binding inhibition by B2C, some different mechanisms may be involved in the B2C-mediated neutralization. These results, taken together, indicated that analogous to the function of the V3 region of HIV-1, the V3 region of HIV-2ROD contained at least a type-specific fusion-inhibiting neutralizing epitope. In this respect, the V3 sequence of HIV-2 may be a useful target in an animal model for HIV vaccine development.     

Neutralizing monoclonal antibodies to hepatitis A virus: partial localization of a neutralizing antigenic site.

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.     

Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates.

Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype.     

Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.     

Persistent and acute central nervous system infections are caused by Theiler''s murine encephalomyelitis viruses which differ in RNA composition but code for only slightly different proteins

The RNA and proteins for four representatives of the two subgroups of Theiler's murine encephalomyelitis viruses were studied. The large RNase T1-resistant oligonucleotides, when mapped along the RNA molecules, were found to be differently distributed in the two subgroups. Replicative form RNAs of two representatives were partially denatured, and the denaturation maps obtained were found to be similar but not identical. In addition, the analysis of the tryptic maps of the capsid proteins of all four isolates revealed that only small differences in the peptide map patterns exist among these viruses. The correlation of these findings with the pathogenicity of Theiler's viruses is discussed.     

Syngeneic anti-idiotypic antisera to murine anti-HLA class II monoclonal antibodies

BALB/c mice have been immunized with six anti-HLA Class II monoclonal antibodies (MoAb); the latter included MoAb CR11-462, Q5/6, and Q5/13 to monomorphic determinants, the anti-HLA-DR1,4,w6,w8,w9 MoAb AC1.59, the anti-HLA-DRw9 MoAb AB7ae9, and the anti-HLA-DQw3 MoAb AC6G. The six monoclonal antibodies markedly differ in their ability to induce anti-idiotypic antibodies, because the latter were not detected in the sera from the mice immunized with the MoAb AB7ae9 and with the MoAb AC6G. The MoAb AC1.59 and CR11-462 elicited antibodies to private idiotypes, and the MoAb Q5/6 and Q5/13 elicited antibodies to private and public idiotypes. The titer of the latter in the anti-MoAb Q5/6 antiserum appears to be markedly lower than that of the former ones; no marked difference was detected in the titer of the two types of antibodies in the anti-MoAb Q5/13 antiserum. Blocking experiments with a panel of monoclonal antibodies showed that the MoAb Q5/13 shares idiotypes with the anti-HLA Class I MoAb Q5/6, 127, and 441 and with the anti-HLA Class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, but does not share idiotypes with any of the nine anti-human melanoma-associated antigen MoAb tested. The spectrotypes of the anti-MoAb CR11-462 and anti-MoAb Q5/13 antisera comprise two major components in the pH 6.2 to 6.7 range, that of the anti-MoAb Q5/6 antiserum comprises two major components in the pH 6.5 to 6.8 range, and that of the anti-MoAb AC1.59 antiserum comprises a number of components in the pH 5.6 to 7.2 range.     

Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity.