Kumato Mifune

Linear and Conformation‐Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies‐related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus‐specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2‐mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation‐dependent epitopes, respectively.

Therapeutic Oral Vaccination Induces Mucosal Immune Response Sufficient to Eliminate Long‐Term Helicobacter pylori Infection

We examined the efficacy of therapeutic oral vaccination using Helicobacter pylori‐whole cell sonicate and cholera toxin (CT) in mice persistently infected with H. pylori. Efficacy was determined by bacterial culture and microscopic examination of gastric tissues for the persistence of bacteria at 6 weeks after the last vaccination. Vaccination of H. pylori‐whole cell sonicate combined with CT eradicated bacteria in 10/16 mice (62.5%). Interestingly, oral vaccination with CT alone also eliminated the bacteria in 8/17 mice (47.1%). However, a therapeutic intraperitoneally administered vaccine failed to eradicate H. pylori from the stomach (1/17 mice, 5.9%). Identification of the type of immunity involved in the eradication process showed that oral vaccination enhanced the antigen‐specific IgA in the feces and saliva. The efficacy of eradication of H. pylori correlated well with increases in IgA secretion in mucosal tissue and a higher labeling index of IgA‐positive lumina of pyloric glands. Moreover, the expression of IL‐4 mRNA in the stomach of mice with eradicated bacteria was higher than in the uneradicated group. Our results suggest that the efficacy of vaccination depends on the mucosal IgA response in the gastrointestinal tract against H. pylori via Th2 cell activation and that therapeutic oral vaccination induces a mucosal immune response sufficient to eradicate long‐term infection with H. pylori.

Protection against rabies in mice by a cytotoxic T cell clone recognizing the glycoprotein of rabies virus

By the use of liposomes containing the purified surface glycoprotein (G) of rabies virus and the haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus, the target antigen of anti-rabies virus cytotoxic T lymphocyte (CTL) clones isolated in a previous study was identified as the G protein. Recognition of the H-2K determinant of the class I major histocompatibility complex (MHC) was necessary for target lysis by the CTL clones. One of the CTL clones was examined for the ability to protect mice against a lethal rabies virus infection. CTL were transferred into syngeneic mice which had been infected in the hind footpad with the ERA strain of rabies virus. The infection was converted into a lethal infection by cyclophosphamide treatment 1 day after virus infection. Transfer of CTL 2 to 3 days after virus infection protected approximately 50% of mice during the observation period of 4 weeks. Greater protection was obtained in mice receiving both anti-rabies virus antibodies and CTL cells.

Hemolysis and cell fusion by rhabdoviruses

Fusion of certain enveloped viruses with cellular membranes is thought to be the mechanism by which the viral genome penetrates host cells. This has been clearly demonstrated with paramyxoviruses, where F protein activated by proteolytic cleavage is playing a critical role not only for the cell fusion and hemolysis, but also for infectivity [l-3]. Influenza viruses and togaviruses such as Semliki forest virus (SFV) Sindbis virus and rubella virus have been demonstrated capable of causing hemolysis and cell fusion at low pH [4-91. The virus may be taken into phagocytic vesicles and reach the secondary lysosome, where the viral envelope fuses with lysosomal membrane in its acidic environment, resulting in the release of viral genome into the cytoplasm. cells with Eagle minimum essential medium (MEM) containing 0.2% bovine serum albumin (BSA). The CVS strain of rabies virus was grown in murine neuroblastoma cells (N-l 8 clone) with serum free Eagle MEM. Viruses were pelleted by ultracentrifugation and the resulting pellets were resuspended in small amount of PBS (-) of pH 7.4 to yield 1 OO-fold concentration. Rabies virus was used for experiments after purification in 20-60% linear sucrose gradient centrifugation. Hemagglutination was carried out in an ice bath at final pH 6.4 as in [ 131 except that the concentration of erythrocytes increased to 1 .O% and the concentration of BSA in borate saline (pH 9.0) decreased to 0.2%.

A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine‐pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.

Rapid desensitization of serotonin 5-HT2C receptor-stimulated intracellular calcium mobilization in CHO cells transfected with cloned human 5-HT2C receptors

Abstract: Serotonin 5‐HT2C receptor‐mediated intracellular Ca2+ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5‐HT2C receptors. Fura‐2 acetoxymethyl ester was used to investigate the regulation of 5‐HT2C receptor function. CHO cells, transfected with a cDNA clone for the 5‐HT2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin‐sensitive [3H]mesulergine binding (KD = 0.49 nM). The addition of 5‐HT mobilized intracellular Ca2+ in a dose‐dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC50 value for 5‐HT of 0.029 µM. Exposure to 5‐HT, 1‐(3‐chlorophenyl)piperazine dihydrochloride (a 5‐HT2C agonist), and 1‐(4‐iodo‐2,5‐dimethoxyphenyl)‐2‐aminopropane (a 5‐HT2C and 5‐HT2A agonist) resulted in increased intracellular Ca2+ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5‐HT‐mediated intracellular Ca2+ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5‐HT in a time‐ and concentration‐dependent manner. Mezerein and phorbol 12‐myristate 13‐acetate, protein kinase C activators, weakly inhibited the intracellular Ca2+ mobilization induced by 10 µM 5‐HT. Furthermore, the protein kinase C inhibitor H‐7 partially prevented the protein kinase C activator‐induced inhibition of the 5‐HT‐mediated increase in intracellular Ca2+ concentration. The desensitization induced by pretreatment with 5‐HT was blocked by W‐7, added in conjunction with 5‐HT, and partially inhibited by W‐5, a nonselective inhibitor of protein kinases and weak analogue of W‐7. Therefore, the 5‐HT2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5‐HT2C receptor activation mobilizes Ca2+ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase‐mediated feedback.

Sequential analyses of the mutations in the core upstream and precore regions of hepatitis B virus genome in anti-HBe positive-carriers developing acute exacerbation.

The nucleotide sequences of the core upstream and precore regions (371 nucleotide length, nt. 1604‐1974) of hepatitis B virus (HBV) were analysed sequentially in three subjects who were positive serorogically for anti‐HBe and had acute clinical exacerbation after immunosuppressive treatment. These patients were asymptomatic HBV carriers before therapy. The results revealed that the mutant with an 8‐bp deletion (nt. 1768–1775) located in the basic core promoter region was dominant in the asymptomatic HBV carrier phase in two of three subjects. After exacerbation, however, such mutant clones possessing 8‐bp deletion disappeared or decreased in number and were replaced by the clones possessing a precore stop codon mutation G to A (nt. 1896) or by the clones possessing additional contiguous point mutations A to T (nt. 1762) and G to A (nt. 1764) and a new point mutation C to T (nt. 1653). Possible relationships between acute exacerbation of liver function accompanied by mutation and the transition of the dominant clones were discussed. J. Med. Virol. 53:266–272, 1997.

Essential Role of T cells in the postexposure prophylaxis of rabies in mice.

Athymic nude mice injected intramuscularly with a street strain of rabies virus were not protected against rabies by postexposure administration of beta‐propiolactone‐inactivated rabies vaccine. In contrast, their normal littermates were completely protected from death by the same vaccination regimens. Nude mice did not produce IgG antibody as a result of the vaccine during the test period of 15 days, whereas normal littermates produced IgG antibody from day 5 after vaccination. However, passive immunization with antirabies hyperimmune mouse ascites showed that antibody was completely ineffective in protecting either nude mice or their normal littermates against rabies when given later than 2 days after infection. No significant difference in the induction of circulating interferon by the vaccination was noted in these mice. Passive transfer of immune spleen cells to nude mice immediately after infection resulted in 30 to 37.5% protection of the mice. Passively transferred spleen cells did not produce detectable amounts of neutralizing antibody in the recipient mice except on day 2 after the transfer, when a low level of antibody was detected. These observations demonstrate the essential role of T cells in the postexposure prophylaxis of rabies in mice. The mechanisms of the failure of postexposure vaccination in nude mice are discussed.

Nucleotide sequence of the nucleoprotein gene of the RC.HL strain of rabies virus, a seed strain used for animal vaccine production in Japan.

By using a phage vector (lambda ZAP II) and the mRNA extracted from IMR-32 cells infected with the RC·HL strain of rabies virus, we constructed a cDNA library from which four nucleoprotein (N)-specific cDNA clones were obtained by Southern blot hybridization. These clones contained a cDNA insert of about 1.4 kb, in which the longest open reading frame was the same length as that reported for the N cDNA of three fixed strains, CVS, PV, and SAD B19. When the nucleotide and deduced amino acid sequences were compared between the RC·HL and the three strains, homology was within the range of 91.5–91.8% and 95.1–96.0%, respectively. Of 183 nucleotides of the RC·HL N-cDNA that were not identical to that of the corresponding site of at least one of the three strains, 41 were shared with the CVS strain, whereas only three were shared with either of the other two strains. In the amino acid sequence, we found 29 residues that were not shared in common with all of the four strains, 11 of which were the substitutions with radically different amino acids that might cause conformational changes of the protein, and, in addition, five of which were located in the region close to the C terminus. The number of such amino acid substitutions between the RC·HL and CVS strains was smaller than that of the other three strains. These results are not inconsistent with the presumption that the RC·HL and CVS strains originated from the same laboratory strain of the Pasteur viruses.

A Unique Mutation of Glycoprotein Gene of the Attenuated RC-HL Strain of Rabies Virus, a Seed Virus Used for Production of Animal Vaccine in Japan

Although the RC‐HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.