Department of Animal and Plant Pathology of The Rockefeller Institute for Medical Research, Princeton, New Jersey.
J Exp Med. 1945 Nov 30;82(6):385-410.
Studies on the biochemical, biophysical, and immunogenic properties of Japanese B type encephalitis virus and vaccines have been made in order to determine whether a purified vaccine suitable for human use could be obtained by means of differential centrifugation of extracts of infected mouse brains. Studies were also made on extracts of normal mouse brains, it was found that extracts of normal as well as of infected mouse brains contained fairly large amounts of several components of high molecular weight. Components having sedimentation constants near 5 and 40 Svedberg units were found in extracts of infected brains. However the rates of sedimentation of the different components were so similar that it was found impossible, from a practical standpoint, to secure a vaccine consisting largely of virus by means of differential centrifugation. It was also found that a considerable portion of virus was lost or destroyed in the centrifugation process so that it was impossible to secure an effective degree of concentration of immunogenic potency. Although vaccines possessing about twice the immunogenic potency of the starting material were obtained, it was concluded that it was not practical to purify and concentrate Japanese B type encephalitis virus in infected mouse brain extracts by means of differential centrifugation for the production of a vaccine on a large scale. The optimum pH stability range of Japanese B type encephalitis virus activity was found to be near pH 8.5. The virus is inactivated fairly rapidly at pH 7 and very rapidly at more acid reactions. The virus is inactivated rapidly near pH 10. Extracts of infected mouse brains with buffers near pH 8 containing disodium phosphate were found to possess slightly higher titers than saline extracts near pH 7. However vaccines prepared from such extracts were found to possess essentially the same immunogenic potency, hence, although extraction at the more alkaline reaction may perhaps remove more active virus, there was no indication that more immunogenic material was removed at the more alkaline reaction. The use of different diluents in the titration of virus activity and the use of different agents in the preparation and storage of virus suspensions were investigated. It was found that low titers were obtained when Ringer's solution, phosphate buffer at pH 7, or saline-phosphate buffer at pH 8.2 were used as diluents but that high titers were obtained when 10 per cent rabbit serum in saline or in phosphate buffer, 10 per cent skim milk in saline or in phosphate buffer, or 1 per cent arginine at pH 8.3 were used. Undiluted skim milk adjusted to pH 8.4 was found to be as satisfactory as undiluted rabbit serum for the preparation of infected brain suspensions for storage at -70 degrees C. A satisfactory neutralization test was conducted with virus stored in undiluted skim milk at -70 degrees C. and subsequently diluted with 10 per cent skim milk in saline. The demonstration that skim milk can be substituted for rabbit serum in the storage, titration, and neutralization tests of Japanese B type encephalitis virus is of practical importance, for skim milk is more convenient to prepare and more readily available in many localities.
为了确定是否可以通过对感染鼠脑提取物进行差速离心来获得适合人类使用的纯化疫苗，对乙型日本脑炎病毒的生化、生物物理和免疫原性特性以及疫苗进行了研究。还对正常鼠脑提取物进行了研究，结果发现正常和感染鼠脑的提取物均含有相当大量的几种高分子量成分。在感染脑的提取物中发现了沉降常数接近 5 和 40 Svedberg 单位的成分。然而，不同成分的沉降速率非常相似，因此从实际角度来看，不可能通过差速离心获得主要由病毒组成的疫苗。还发现，在离心过程中相当一部分病毒丢失或破坏，因此无法有效浓缩免疫原性效力。尽管获得了免疫原性效力约为起始材料两倍的疫苗，但结论是，通过差速离心从感染鼠脑提取物中纯化和浓缩乙型日本脑炎病毒用于大规模生产疫苗是不切实际的。发现乙型日本脑炎病毒活性的最佳 pH 稳定性范围在 pH8.5 附近。病毒在 pH7 时迅速失活，在更酸性的反应中迅速失活。病毒在 pH10 附近迅速失活。发现含有磷酸二钠的 pH8 附近缓冲液的感染鼠脑提取物比 pH7 附近的生理盐水提取物具有略高的效价。然而，从这种提取物制备的疫苗被发现具有基本相同的免疫原性效力，因此，尽管在更碱性的反应下提取可能会去除更多活性病毒，但没有迹象表明在更碱性的反应下会去除更多的免疫原性物质。研究了在病毒活性滴定中使用不同的稀释剂以及在病毒悬浮液的制备和储存中使用不同的试剂。发现当使用林格氏溶液、pH7 的磷酸盐缓冲液或 pH8.2 的生理盐水-磷酸盐缓冲液作为稀释剂时，获得的效价较低，但当使用 10%兔血清生理盐水或磷酸盐缓冲液、10%脱脂乳生理盐水或磷酸盐缓冲液或 pH8.3 的 1%精氨酸时，获得的效价较高。发现未稀释的调整至 pH8.4 的脱脂乳与未稀释的兔血清一样适用于制备用于在-70°C 下储存的感染性脑悬液。用在-70°C 下储存的未稀释脱脂乳储存的病毒进行了令人满意的中和试验，并随后用 10%的生理盐水稀释。证明在乙型日本脑炎病毒的储存、滴定和中和试验中可以用脱脂乳代替兔血清，这具有实际意义，因为脱脂乳更容易制备，在许多地方更容易获得。
