key: cord-0004854-w2akghcp authors: Lecomte, J.; Cainelli-Gebara, V.; Mercier, G.; Mansour, S.; Talbot, P. J.; Lussier, G.; Oth, D. title: Protection from mouse hepatitis virus type 3-induced acute disease by an anti-nucleoprotein monoclonal antibody date: 1987 journal: Arch Virol DOI: 10.1007/bf01310740 sha: c5fb930bbae814ba9fbb56da1336ed900abc28f6 doc_id: 4854 cord_uid: w2akghcp Fusion of MHV-3-immune splenocytes from MHV-3-resistant A/J murine strain, with NS myeloma cells produced several hybridomas. Among eight hybridoma clones, the 1E7A4H1 clone secreted kappa IgG2a apparently directed against the nucleoprotein of the MHV-3 virion. The monoclonal antibody was able to neutralize the in vitro cytopathic effect of MHV-3 on cultured L2 cells, and was detected by indirect immuno-fluorescence on MHV-3-infected cultured YAC cells. In addition, it conferred a significant protection against MHV-3-induced acute disease, if injected intraperitoneally to C57BL/6 mice before inoculation with MHV-3. protein (N), that can neutralize the cytopathic activity of the virus on cultured cells and protect susceptible mice from the acute disease resulting from an infection with type 3 murine hepatitis virus (MHV-3). For hybridoma production, MHV-3-resistant A/J mice [7] , 10-12 week old, were infected intraperitoneally with in vivo propagated MHV-3 virus (103LDs0). Two weeks later, the mice received a second virus challenge, (105LDs0, intraperitoneally). Animals were killed 3 days later and the spleens removed. Fusions with the splenocytes and P3/NSI/1-Ag4-1 (' 'NS-1") myeloma cells, of BALB/c origin, were performed. Screening of rocheclonal antibodies specific for MHV-3 antigens was done, by both enzymelinked immunosorbent (ELISA) and dot-immunoblotting assays [3] . After two successive preliminary screenings with ELISA, 8 hybridomas positive in both ELISA and dot-immunoblotting assays, were selected. Eight cloned hybridoma lines were established after two clonings at limiting dilutions from two hybridomas: 1E7 (one clone) and 2F4 (seven clones). The clones were characterized and tested for reactivity with MVH-3 virus proteins. All were positive by ELISA, whereas only 1E7A4H1 was positive by ELISA and a dot immunoblotting test. All were of the IgG2a kappa subtype. All but one gave workable amounts of ascitic fluid, after transplantation of the hybridomas in (A/J X BALB/ e) F1 hybrid mice. Since all the seven subclones of the 2F4 series gave similar data by neutralization we report here results with the monoclonal antibodies 1E7A4H1 and 2F4H9F4. The polypeptide specificities of the monoclonal antibodies were determined by Western immunoblotting [14] using concentrated supernatants ofMHV-3-infected L 2 cells. Figure 1 shows that both tlhe 1E7A4H1 and 2F4H9F4 reacted with a protein of 51kDa with minor bands showing at 62kDa and 47kDa and that the 1E7A4H1 monoelonal antibody gave the most intense reaction (lane 3). The latter antibody gave a similar imunobtotting profile with the A59 strain of MHV and showed strong cross-reactivity by ELISA with MHV-A59 and MHV-4, whereas the ep-Rope recognized by monoclonaI antibody 2F4H9F4 appeared to be conserved on MHV-4 but lost on MHV-A59 (data not shown). It was therefore established that both monoelonal antibodies were most likely specific for two different epitopes on the structural iX protein. The viral specificity of the monoclonal antibodies was also established by indirect immunofluoreseence. Chromatographically purified immunoglobulins from ascitic fluid were used for reaction with MHV-3-infected live YAC cells [5] . The expression of MHV-3 antigens on live infected cells was detected with a FITC-conjugated rabbit anti-mouse immuneglobulin serum (Miles Laboratories Inc., Elkhart., Ind.). Figure 2A shows that the 1E7A4H1 ascitic fluid reacted with the surface of MHV-3-inleered YAC cells, but not with the membrane of non-infected cells In vivo neutralizations were pertbrmed by intraperitoneal injections of C57BL/6 mice with 0.5 mL of ascitie fluid, 4h before challenge with 1000 LDs0 of MHV-3 as described [7] . Mice surviving the acute, normally lethal disease, recovered healthy appearance within 2 weeks, and the ratios surviving mice to infected mice were determined at 15 days after infection. Only 1E7A4H1 provided significant protection when injected 4h before infection (Table 1) . No protection was obtained with the nonneutralizing ascitic fluid from the 2F4H9F4 hybridoma clone or with an irrelevant monoclonal antibody. It was of some interest to look Ibr histological evidence of the protect[on coni~rred by the 1E7A4H1 monoclonai antibody in susceptible * Non-neutralizing 2F4H9F4 anti-MHV-3 monoclonal antibody (5 mice) or unrelated (6D4-II) anti-herpes-1 virus monoclonal antibody (13 mice) C57BL/6 mice. For this, another group of 5 mice was injected as above with 0.5 mL of aseitie fluid, 4h before challenge with 1000 LD~0 of MHV-3. Five other C57BL/6 mice were injected with a similar amount of an irrelevant monoelonal antibody against the human herpes t y p e d virus. Intraperitoneally inoculated C57BL/6 mice are very sensitive to MHV-3. Infection of the liver results in rapidly lethal hepatitis within 6-7 days [16] . For histologic examination, brains and livers were removed by day 6, fixed in histologic fixative (Perfix, Fisher Scientific Co., Fair Lawn, NJ), sectioned (6 gm) and stained with hematoxilin and eosin. At this time two out of 5 mice, having received an irrelevant monoclonM antibody, had died from the MHV-3 challenge. For the three other surviving mice, multifoeal necrotizing hepatitis was evident in the liver of surviving mice sacrified six days post-inoculation. Numerous randomly distributed loci were present in which hepatoeytes had undergone eoagulative necrosis with a marked infiltration of mixed leukoeytes (Fig. 3A) . In contrast, at 6 days after challenge, in mice having received the 1E7A4Ht monoelonal antibody, small scattered lesions were observed consisting mainly of a mixed reaction of nuclear cells. No lesions were observed in the brain of either passively protected or infected mice (Fig. 3B) . To our knowledge, 1E7A4H1 is the first anti-MHV-3 monoclonal antibody reported to be specific for the N protein, and to confer in vivo protection. Furthermore, it was shown that it reacts with an epitope which is expressed on the surface of infeeted YAC cells. In contrast, the 2F4 monoclonM antibodies, although specific for the N protein, did not show any complement mediated neutralization in vitro or protective activity in vivo and did not react with infected cell plasma membranes. An anti-N protein monoclonal antibody without in vitro neutralizing activity against MHV-2, that could confer protection in MHV-2-infeeted mice, has been reported previously [9] . On the other hand, monoclonal antibody against the N protein of M H V -J H M (MHV-4), like our 2F4 monoelonal antibodies, did not have any protective activity against a lethal dose of virus [2] . It can be assumed that the 1E7A4H1 and 2F4 monoclonal antibodies are reacting against different epitopes, which would also explain the different patterns of cross-rcaetivities shown b y E L I S A with MHV-A59 and MHV-4. Obviously because of the small number of monoclonal antibodies in this series exhibiting different patterns of reaction it is not possible to analyse virus structures in relation to protection mechanisms, as has been done for other MHVs [2, 11, 12, 17] . However, the present s t u d y demonstrates that at least one epitope on the N protein is accessible to antibodies on the surface of infected cells. This has not yet been shown for MHVs b u t it has been shown for the N protein of influenza virus [18] . It can be envisaged that although an anti-N protein monoclonM antibody cannot prevent adsorption of the virus to initiate a first cycle of replication, it could confer protection by reacting with the surface of infected cells causing antibody-dependent complement or cell-mediated cytolysis, as has been shown for various virus infections [6, 10] . Mouse hepatitis virus strain-related patterns of tissue tropism in suckling mice Murine hepatitis virus-4 (Strain JHM)-induced neurologie disease is modulated in vivo by monoelonal antibody Production et caract6risation d'anticorps monoclonaux eontre le virus de l'h6patite murine type 3. MSc Dissertation Antigenic relationships of murine eoronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus Persistent infection with mouse hepatitis virus type 3 in mouse lymphoid cell line Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: distinct mechanisms of action in vivo Genetic study of mouse sensitivity to mouse hepatitis type 3 infection: influence of the H-2 complex Prevalence of natural virus infections in laboratory mice and rats used in Canada Protective effect of monoelonal antibodies on lethal mouse hepatitis virus infections in mice Destruction of virus-infected cells by antibo@ and complement Antigenic differentiation of mouse hepatitis viruses by neutralization test Topographical mapping of epitopes on the glycoproteins of murine hepatitis virus 4 (JHM) : correlation with biological activities Antigenic variation among murine coronaviruses: evidence for polymorphism on the peplomer glyeoprotein E2 Electrophoretie transfer of proteins from polyaerytamide gels to nitrocellulose sheets: procedure and some applications Genetic variation of neurotropic and non-neurotropie murine coronaviruses ter Meulen V (t982) The biology and pathogenesis of coronaviruses Hybridoma antibodies to the murine eoronavirus JHM: characterization of epitopes on the peplomer protein (E2) Expression of influenza A virus internal antigens on the surface of infected P815 eells This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada to D.O., Nr A3044. We thank Dr Jean-Marie Dupuy for continuous support and Dr. Valeriu Micusan fbr performing chromatographic purification of ascitic fluid. Authors' address: Dr. J. Lecomte, Institute Armand-Frappier, Centre de recherche en virologie, 531 boulevard des Prairies, Ville de Laval, Qu6bec, Canada H7N 4Z3.Received April 6, 1987