Genetic and antigenic characterization of newly isolated bovine toroviruses from Japanese cattle.

Torovirus, a member of the Coronaviridae family, is a gastrointestinal infectious agent that has been identified in humans, cattle, pigs, and equines. Toroviruses, except equine torovirus, are difficult to propagate in cell culture; indeed, to date, only the Aichi/2004 strain of bovine torovirus (BToV) has been isolated among the human, bovine, and porcine toroviruses. In the present study, four cytopathogenic BToVs were isolated from diarrheal feces of the cattle using the HRT-18 cell line, and their genetic and antigenic properties were compared. The cytopathogenic features of BToV isolates in HRT-18 cells were similar to those of the Aichi/2004 strain. However, none of the isolates showed cytopathogenic effects in the HRT-18 cells of different origin, suggesting that one significant factor contributing to the cytopathogenicity of BToV depends on properties of the HRT-18 cells themselves. All BToVs isolated were able to agglutinate mouse, but not chicken, erythrocytes, while they lacked receptor-destroying enzyme activity. Analysis of the N terminus of the spike gene showed that three isolates, but not the Gifu-2007TI/E strain, were phylogenetically located in cluster 1 and its analogs and revealed high cross-reactivity with each other, as demonstrated by neutralization (NT) and hemagglutination inhibition (HI) assays. The Gifu-2007TI/E strain was classified close to cluster 2 and exhibited relatively low cross-reactivity with these viruses; however, the difference was not sufficient to classify BToVs into serotypes, suggesting that at least two subtypes distinguishable by the structure of the N terminus of the spike gene and that both NT and HI tests may be exist.

Separation and characterization of adjuvant oligosaccharide oleate ester derived from product mixture of mannitol-oleic acid esterification.

Nearly 30 years after intense investigations of mannide monooleates for use as vaccine adjuvants, a novel adjuvant-active saccharide oleate ester was isolated and identified from the product mixture synthesized from mannitol and oleic acid. The mixture, which contained many kinds of mannide mono- and dioleates and their derivatives, was fractionated by liquid chromatography (LC), and the fraction with the highest adjuvanticity was obtained. Gel permeation chromatography (GPC) showed that it consisted of one major compound with an average molecular weight (MW) 2850. Infrared (IR) absorption and proton nuclear magnetic resonance spectra suggested it had oligosaccharide moieties and oleate domains. These findings suggested that it was an oligosaccharide oleate ester of the average MW 2850. The molecular ratio of oleate chains per monosaccharide unit was approximately 0.8. The ester induced both IgG1 and IgG2a antibody responses in mice in a dispersed form without base oil. This ester thus appears to be one of the adjuvant-active compounds largely contributing to the excellent adjuvanticity of mannide oleate mixture broadly used as vaccine emulsifier. These results and previous findings suggest that the fundamental adjuvanticity of this 'oligo' saccharide acylate ester was in accord with the hydrophil-lipophil balance (HLB) theory, similarly to other saccharide acylate esters. It is now expected that this compound will be useful as novel vaccine adjuvant which may induce both Th1 and Th2 type immune responses with low or no toxicity, not only as an vaccine emulsifier but in an aqueous suspension form.

Relationship between adjuvant activity and amphipathic structure of soyasaponins.

A correlation between adjuvant activity and amphipathic structure of saponin was first demonstrated on an experimental basis using structurally consecutive analogues. To clarify the physicochemical factors regulating the adjuvanticity of saponin, we compared the profile of the antibody response against chicken ovalbumin (OVA) in mice and hydrophile-lipophile balance (HLB) of eight purified soyasaponins. Soyasaponins bearing sugar chain(s) showed adjuvanticity stimulating anti-OVA total-IgG and IgG1 antibody responses, while their corresponding aglycones soyasapogenols A and B, did not. Among bisdesmosidic soyasaponins, soyasaponin A(1) (HLB: 26.9) with a long sugar side chain induced stronger total-IgG and IgG1 antibody responses than soyasaponin A(2) (HLB: 21.4). For monodesmosidic soyasaponins, the ranking in terms of antibody response was soyasaponin I (which has the highest HLB value (13.6) among the monodesmosidic soyasaponins) > soyasaponin II (HLB: 12.2) > soyasaponin III (HLB: 10.0). The adjuvant activity increased with the HLB value. The length, the number, and the composition of sugar side chains affecting the HLB value would give the overall conformation of each saponin molecule, and the amphipathic structure may define the fundamental adjuvanticity of saponins.

Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs.

The protective effect of an alpha-toxoid vaccine of Clostridium septicum purified alpha-toxin was investigated in guinea pigs. Purified alpha-toxin was treated with formalin to make toxoid, and alpha-toxoid vaccine was prepared by mixing alpha-toxoid (4 to 64 microg/dose) with an aluminum phosphate gel as adjuvant. Guinea pigs were immunized twice with different doses of alpha-toxoid vaccine, and challenged with spores of C. septicum. The guinea pigs surviving after challenge had been immunized with 8 microg/dose or more of alpha-toxoid. All these animals produced titers of 20 units or higher of antitoxin at the challenge. The results suggest that C. septicum alpha-toxin plays an important role in protection against challenge with spores in guinea pigs.