マウス脳増殖フォルマリン不活化デングウイルスの抗原性と免疫原性
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デングウイルス1,2,3,4型原株を乳呑マウス脳で増殖させた後10%乳剤を作成し,低速遠心の上清を硫酸プロタミン処理し,フォルマリンで不活化した.その低速遠心上清から活性炭処理と超遠心でウイルス粒子を精製し,乳離れした幼若マウスに接種して免疫原性を検討した.免疫血清の抗体価は間接ELISA,血球凝集抑制(HI)反応,及び中和試験で測定した.免疫原の濃度と免疫マウス血清のIgG-ELISA抗体価の間には用量反応関係が認められた.同型の免疫原と測定抗原に対しては免疫血清のHI価とIgG-ELISA価の相関係数は0.8-0.94と高かったが異型の免疫原と測定抗原の組み合わせでは0.76-0.88とやや低い値を示した.種々の血清反応における抗体価も同型の免疫原と測定抗原の組み合わせで最も高かったが異型の組み合わせの場合も可なりの交差反応がELISAとHIで認められ,IgM-ELISAがIgG-ELISAよりも特異性が高いとは云えなかった.IgG-ELISA抗体の産生状況から,用いたデングウイルスの中で3型が最も免疫原性が高く,次いで1型,4型であり,2型は最も低いと考えられた.この傾向は異なる型のデングウイルスを混合した2価及び3価の免疫原を用いた場合にも認められた.2型ウイルスを含まない免疫原で得られた抗血清は2型抗原に対する抗体価が有意に低かったが,1型と2型又は2型と3型の混合物で免疫すると凡ての型の測定抗原に対して十分高いIgG-ELISA抗体価が得られた.従って4つの型のデングウイルスに広く反応するIgG-ELISA抗体を産生するには免疫原に2型ウイルスを含める必要がある,一方,HI試験では免疫原に1型ウイルスを含まないと得られた抗血清の1型に対する抗体価が低く,2型又は4型を含まなかっても2型又は4型に対する抗体価は大差がなかった.Prototype strains of dengue viruses, type 1, 2, 3 and 4, were grown in brains of suckling mice (SMB) and 10% homogenates were prepared. After low-speed centrifugation, protamine sulfate treatment and formalin inactivation, virions were concentrated and partially purified by activated charcoal treatment and ultracentrifugation, and was innoculated to weaning mice to test their immunogenicities. Antibody titers in the immune sera were measured by indirect ELISA, hemagglutination-inhibition (HI) as well as neutralization (N) tests. Dose-response relationship was observed between the amount of immunogen inoculated and the IgG-ELISA titer of immunized mice. Correlation coefficients (r-value) between the HI and the IgG-ELISA titers of the immune sera were 0.89-0.94 for the homologous type of immunogen and assay anitigen combination, and less rvalues (0.76-0.88) were obtained for the heterologous combinations. Serological reactions gave the highest titer for the homologous type of antigen-immunogen combination, however, significant cross-reactions were observed for the heterologous types of dengue antigens especially by the ELISA or the HI test and IgM-ELISA did not give higher typespecificity than the IgG-ELISA. The IgG-ELISA and HI antibody production indicated that the immunogenicity was highest for type 3 (D3) followed by type 1 (D1), and then type 4 (D4), and least for type 2 (D2) antigens. This tendency was also observed when mice were given trivalent, or tetravalent types of combined dengue immunogens and immune sera were measured by the IgG-ELISA. Omission of D2 from the immunogen produced antisera with significantly reduced anti-D2 IgG-ELISA titer, while immunization with D1+D2 or D2+D3 produced antisera with sufficient cross-reactions for all types of dengue antigens in the IgG-ELISA. Thses results indicated the importance to include D2 immunogen in order to produce broadly reactive IgG-ELISA antibodies. On the other hand, results by the HI test on mouse sera obtained with multivalent immunogen showed that ommission of D1 from immunogen resulted in significant reduction in the anti-D1 HI antibody titers, while ommission of D2 or D4 from the immunogen did not affect the resulting anti-D2 or anti-D4 HI antibody titers.
- 1986-06-30
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