Adjuvant activity of muramyl dipeptide derivatives to enhance immunogenicity of a hantavirus-inactivated vaccine.

The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 micrograms mouse-1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3-9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76-118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.

Association of serine in position 1124 of Hantaan virus glycoprotein with virulence in mice.

Hantaan virus (HV) of the genus Hantavirus causes a fatal disease in suckling mice following intraperitoneal or intracerebral infection. HV cl-1, which was obtained from the 76-118 strain of HV by growth in Vero E6 cells, exhibited high mortality rates in mice whereas mice infected with HV cl-2 survived without any clinical signs. To determine the molecular basis for the marked difference in virulence, we compared the nucleotide sequences of the large (L), medium (M) and small (S) segments of HV cl-1 genome with those of HV cl-2 and found that there was only one predicted amino acid substitution. This amino acid substitution was in position 1124 of the glycoprotein encoded by the M genome segment, in which serine in HV cl-1 was replaced by glycine in HV cl-2. Although there were several nucleotide and amino acid differences between the parental 76-118 strain and HV cl-1, the serine in position 1124 of the glycoprotein was common to the pathogenic parent and the pathogenic mutant. These results suggest that this substitution may be responsible for the virulence of this hantavirus.

Production of nonstructural proteins of hepatitis C virus requires a putative viral protease encoded by NS3.

Hepatitis C virus (HCV) is a positive strand RNA virus with certain similarity to flaviviruses and pestiviruses. To examine the processing and possible assembly of HCV proteins, we constructed a recombinant vaccinia virus that expresses a full-length genomic RNA, infected chimp liver cells with the virus, and analyzed HCV-related protein products by immunofluorescent antibody staining and Western blot detection with mouse monoclonal antibodies. The putative core, envelope, and NS1 and NS3 proteins that yielded from this recombinant were 22, 32, 53 to 58, and 65 kDa in size, respectively. The NS4 protein was unexpectedly small, with an estimated molecular weight of 7 kDa, and the NS5 protein was found to be further cleaved into 52-kDa NS5a and 58-kDa NS5b proteins, the latter of which contains a hallmark of RNA replicase. A point mutation in the putative protease domain of NS3 resulted in a failure in the production of NS3, NS4, NS5a, and NS5b, but coexpression of NS3 restored the proper processing of these proteins, demonstrating that NS3, the putative viral protease, is essential for the production of these nonstructural proteins. Thus, HCV strikingly resembles pestiviruses in the size and the processing mode of the nonstructural proteins, particularly NS4 and NS5.

Pathogenesis of Hantaan virus in mice.

The virulence of two virus clones (HV cl-1 and HV cl-2) of Hantaan virus, which were plaque-purified on Vero E6 cells, were compared in suckling mice infected by the intraperitoneal or intracerebral route. HV cl-1 increased the mortality of the mice whereas HV cl-2 did not. Furthermore, virus of high titre was isolated from various organs of mice infected with HV cl-1 and high titres were maintained, whereas after infection with HV cl-2, virus was isolated from various organs only in low titre and only temporarily. HV cl-1 strongly induced cell-to-cell fusion, but the cell fusion activity was at a minimum level in cells infected with HV cl-2. However these two clones induced similar titres of antibodies in mice. Cytotoxic T lymphocyte assays against macrophages infected with homologous and heterologous virus showed that cytotoxic T cell activity was induced in mice infected with HV cl-2, but suppressed in mice infected with HV cl-1. These results suggest that an alteration in the cell fusion function and the cytotoxic T cell activity are important in the pathogenesis of Hantaan virus infection in newborn mice.

Development of inactivated vaccine against virus causing haemorrhagic fever with renal syndrome.

B-1 virus belonging to the hantavirus group was serially passaged in the brains of newborn mice. Inactivated vaccine was prepared from the brains after inactivation with formalin and then purification by ultracentrifugation. The antigenic potency of this vaccine in vitro was determined by antibody-bound enzyme-linked immunosorbent assay (ELISA) and serial diluted vaccine bound to an aluminium hydroxide gel was inoculated into Balb/c mice to test immunogenicity. After two injections of this vaccine preparation, antibodies were detected in the mice by immunofluorescent, neutralizing and haemagglutination inhibition antibody tests. When mice immunized with this vaccine were challenged with B-1 virus and Hantaan virus (KHF-83-61BL strain), the virus titres in their lungs and spleens were significantly less than those in non-immunized mice. These results suggest that inactivated B-1 virus vaccine is effective against virus challenge by homotypic (B-1 virus) and heterotypic (Hantaan virus) viruses.

Viruses of hemorrhagic fever with renal syndrome (HFRS) grouped by immunoprecipitation and hemagglutination inhibition.

Nine viruses antigenically related to Hantaan virus that were isolated from various rodents in several countries and from a patient were examined by immunoprecipitation and by hemagglutination inhibition (HI). The immunoprecipitation tests were done with a monoclonal antibody that reacted with glycoprotein G2 and an antibody against a nucleocapsid protein. The viruses were classified into four subtypes on the basis of the molecular weights of their precipitated polypeptides and the pattern of results of the HI tests.

Persistent infection of rats with haemorrhagic fever with renal syndrome virus and their antibody responses.

Newborn (within 24 h after birth), 1-week-old and 6-week-old (adult) rats were inoculated with a Hantaan-related virus (B-1) and attempts were made to isolate the virus from various organs. Virus-specific antigens were detected in various organs of newborn rats. Moreover virus could be isolated from almost all their organs even 25 weeks after infection. In contrast, in rats infected at 6 weeks of age the virus could be isolated from various organs but its concentration decreased progressively with time. The levels of haemagglutination-inhibiting (HI) and neutralizing antibodies to B-1 virus in the sera were measured. In adult rats, HI antibodies were first detected 2 weeks after infection and their titre rose to a maximum after 5 weeks. On the other hand, in newborn rats the levels of antibodies remained low until 5 or 6 weeks after infection and started to increase to a high level more than 9 weeks after infection. Furthermore, in rats infected soon after birth IgM antibodies predominated for a long time and these antibodies also neutralized infectivity at a high level. These data suggest that the induction of a high titre of neutralizing antibodies mainly of the IgM type does not result in virus clearance in newborn rats and that cellular immunity may be important for virus clearance in vivo.

Characterization of glycoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies.

Viruses causing hemorrhagic fever with renal syndrome (HFRS) encode two glycoproteins, G1 and G2. For determination of the biological functions of these glycoproteins, we isolated 15 hybridomas secreting monoclonal antibodies directed against the glycoproteins of the B-1 and Hantaan viruses (HV). From results of neutralizing and hemagglutination inhibition (HI) tests, and studies on the antigenic reactivities of the antibodies with other HV-related viruses by immunofluorescence, we classified these hybridoma clones into two groups producing antibodies to the G1 proteins of the B-1 virus, six groups producing antibodies to G2 proteins of the B-1 virus, and four groups producing antibodies to the G2 protein of HV. Of the antibodies to G2 produced by 12 clones, groups A and B had high HI activity with HV-related virus cross-reactivity and moderate neutralizing activity, group C had moderate HI activity with virus specificity but low neutralizing activity, group G had high neutralizing activity and low HI activity, and five other groups had little or no HI or neutralizing activity. Group A reacting with G1 protein had low level of both neutralizing and HI activity, while group B had no HI activity. One clone of monoclonal antibody had high neutralizing activity and no HI activity, but it did not react with either polypeptide by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by the immunoblotting method. These data suggest that both glycoproteins are the targets of neutralizing antibodies. Furthermore, the results indicate that the antigenic determinants with hemagglutination activity are mainly on the G2 protein, and that the domains related to neutralizing activity and to HI activity are separate.

Haemagglutination-inhibition test for haemorrhagic fever with renal syndrome using virus antigen prepared from infected tissue culture fluid.

Haemagglutinating (HA) antigens of four strains of virus related to that causing haemorrhagic fever with renal syndrome (HFRS) were prepared from infected tissue culture fluids by ultracentrifugation. The titres of the precipitated antigens were increased considerably by acetone extraction and sonication. Acetone extraction completely inactivated infectious virus in the antigen preparations. The antigens were pH-dependent, with pH optima at 5.8. Good correlations were observed in human and rat sera between the titres obtained by the haemagglutination-inhibition (HI) test and an indirect fluorescent antibody test. Moreover, strong cross-reactions among these strains were demonstrated by the HI test. The HI test has not been used previously with HFRS viruses because of the danger involved in preparing HA antigen, but these results indicate that a safe method is available for serological identification of HFRS.

Isolation of haemorrhagic fever with renal syndrome virus from leukocytes of rats and virus replication in cultures of rat and human macrophages.

Newborn rats were inoculated intraperitoneally with haemorrhagic fever with renal syndrome (HFRS)-related virus (B-1 strain), and virus isolation from their various organs was attempted between 1 and 25 weeks after inoculation. Virus could be isolated repeatedly from lung, brain, spleen and kidney and also from peripheral blood. When virus isolation was carried out on fractionated peripheral blood cells, virus was associated mainly with the macrophage fraction and to a lesser extent with granulocytes. Virus replicated well in peritoneal exudate cells of normal rats and it grew in the adherent mononuclear cells from normal human peripheral blood. These data suggest that macrophages, permissive for HFRS-related virus replication, might contribute to the spread of viral infection in vivo.

Evaluation of focus reduction neutralization test with peroxidase-antiperoxidase staining technique for hemorrhagic fever with renal syndrome virus.

Titers of hemorrhagic fever with renal syndrome virus were estimated by counting foci stained with peroxidase-antiperoxidase or by using immunofluorescence methods. Foci stained with PAP were clearer and easier to count. The peroxidase-antiperoxidase method showed a linear relationship between virus concentration and the number of foci. A focus-reduction neutralizing antibody test was performed with immune rat sera and sera from patients. Again, the peroxidase-antiperoxidase method proved to be more convenient and reliable than the immunofluorescence method. Antigenic differences between Hantaan virus, 76-118 and hemorrhagic fever with renal syndrome virus B-1 were clearly demonstrated by the neutralizing antibody test.

Antigenic differences between two viruses, isolated in Japan and Korea, that cause hemorrhagic fever with renal syndrome.

Hantaan virus (HV) 76-118, isolated from Apodemus agrarius coreae in Korea, and hemorrhagic fever with renal syndrome (HFRS) virus B-1, isolated from a rat in Japan, were examined for polypeptide compositions and for differences in immune responses in rats. In immunoprecipitation experiments, a major polypeptide of ca. 50 kilodaltons (K) was detected with antisera against HV 76-118 in cell extracts from Vero E6 cells infected with HFRS virus B-1, whereas three major polypeptides of 74 K (glycosylated), 57 K (glycosylated), and 50 K were detected with antisera against HFRS virus B-1. On the other hand, two polypeptides with molecular weights of 55,000 (glycosylated) and 50,000 were detected with either antiserum in cell extracts infected with HV 76-118. In neutralizing antibody tests with antisera prepared in rats, a remarkable difference in antibody titer (5 to 30 times higher to the homologous virus than to the heterologous virus) was observed between the two viruses. However, this difference was not so marked (1 to 4 times higher to the homologous virus) in the immunofluorescent antibody test. Twenty hybrid cell lines producing mouse monoclonal antibodies against HV 76-118 were isolated by fusion of spleen cells from BALB/c mice immunized against HV (strain 76-118) with mouse myeloma cells. The specificity of these monoclonal antibodies was established by immunofluorescent antibody, neutralizing antibody, and fluorescent antibody to membrane antigen tests and by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These hybrid cell lines were classified into three groups based principally on the IF staining pattern of the HV-infected cells: (i) antibodies which showed a discrete patch pattern in the cytoplasm by the immunofluorescent antibody test, reacting with the membrane antigen of infected cells and immunoprecipitating a 55-K glycoprotein from HV 76-118-infected cell lysates and a 57-K glycoprotein from the heterologous (strain B-1) HFRS virus-infected cell lysates. Among these, depending on the neutralizing antibody activity and the reaction with the heterologous antigen, three subgroups designated I-A, I-B, and I-C were established; (ii) antibodies which showed large granular dots in the cytoplasm, neither having neutralizing antibody activity nor immunoprecipitating any antigen; (iii) antibodies which showed defined granular dots throughout the cytoplasm, reacting with a 50-K polypeptide of both virus strains. These antibodies also classified into two subgroups based on the reactivity with the B-1 strain.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental infection in newborn mice and rats by hemorrhagic fever with renal syndrome (HFRS) virus.

Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76-118 strain) or HFRS-related virus (B-1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76-118 strain than in the groups infected with the B-1 strain, while the B-1 strain was more virulent in rats than the 76-118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B-1 strain, while it was recovered from only the brain and lungs of rats infected with the 76-118 strain. Viral antigen was readily detected in various organs of rats infected with the B-1 strain, but the amount and distribution of antigens were less in rats infected with the 76-118 strain. Our results suggest that the virulence of HFRS-related virus is variable, depending on the species of infected animals as well as on the virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.