Laboratories of The Rockefeller Institute for Medical Research.
J Exp Med. 1936 Jul 31;64(2):223-32. doi: 10.1084/jem.64.2.223.
A study was undertaken on the effect in vivo, in the guinea pig, of equine encephalomyelitis virus antiserum upon the antigenic response to active, as compared with that to formolized, inactive virus. It was found that when animals were given subcutaneously a proper amount of hyperimmune serum 1 hour before inoculation, in the subcutis, of either active or of inactive virus, no immunity was induced against an intracerebral test of more than 1,000 and less than 10,000 M.L.D. of virus. This preventive power of the serum was lost by its dilution, the loss being proportional to the dilution, and, on the other hand, more serum was needed to obtain the blocking effect as the quantity of virus was increased. When an insufficient amount of serum was introduced into the animals along with the same quantities of active virus or formolized vaccine, a certain number of those receiving the untreated virus succumbed to virus infection in the course of the inoculations, but the survivors were rendered resistant to the intracerebral test; all the guinea pigs treated with higher dilutions of serum and with formolized material were brought safely to an immune state. The point to be stressed then is that antigenic stimuli present in untreated active virus and in formolized virus tissue suspensions in which no active virus is demonstrable by drastic tests (1) and which are wholly noninfective in animals (1), are completely inhibited from acting by the use of proper amounts of immune serum. The mechanism underlying this preventive power of adequate amounts of serum may be explained on the basis of facts deduced in preceding papers of this series (1, 3) and in the present article. We have shown that 3 x 10(7) m.i.u. of active virus contains a sufficient amount of antigen to induce immunity without the necessity of its multiplication in the animal body. This has been fully established by the similar degree of resistance brought about by 3 x 10(7) m.i.u. of virus formolized to a degree in which no active virus could be revealed (1). The assumption that the blocking effect of serum in the quantity employed prevents multiplication of the virus which is reflected in the production of inadequate amounts of antigen, is therefore untenable, since this effect was obtained when a sufficient amount of antigen was present in "living" as well as in "killed" virus. On the other hand, with insufficient amounts of immune serum (to be noted in higher dilutions shown in Table II), only the active virus could multiply-the formolized vaccine was not affected in respect to its antigenicity by these quantities of serum-and so produce more antigenic substance. This substance, in turn, brought about greater resistance in the host. The precise action of proper amounts of serum in preventing development of immunity by both active and inactive virus is not definitely known. However, two hypotheses are offered for consideration: the first implies that the action of the serum is direct, that is, by entering into combination with the antigens to bar antigenic capacity; the second ascribes to the serum an indirect action, on the cells of the body, in such a way as to make them unable to react to the antigenic stimuli present in the inoculated materials. The identity of these antigenic stimuli in virus suspensions containing the active, infective agent or this agent inactivated by formalin is at the present time undetermined. If virus were obtainable in pure state, free from extraneous material, the answer to this question might be readily given, but it is quite a different matter when the substance called virus is a mixture of the infective agent, of inflammatory tissue products, of tissue, etc. We have, however, shown that induced immunity is not due to the presence of "living" virus, but whether the antigenic action originates from "killed" virus or from another constituent of the suspension is not clear. On the other hand, Sabin (7) suggests the possibility that the virus may not be the direct antigenic stimulus but that some substance on which it acts and which becomes liberated from infected cells may be the factor responsible. While this subject awaits the results of further study, we believe that formalin inactivates the infective agent in virus suspensions and preserves the antigenic component therein, whatever its nature may be. It would be of interest if this phenomenon of prevention of antigenic capacity by proper amounts of immune serum might apply to such materials which by their very nature do not multiply in the body of the host, e.g., toxin and antitoxin. Theobald Smith (8) and later Park (9) demonstrated that in mixtures of diphtheria toxin-antitoxin, when smaller amounts of immune serum (antitoxin) are used, the toxicity of the mixture is retained and immunity results; if the serum is increased, toxicity is reduced and immunity occurs irregularly, and if more serum is added, no toxicity nor immunity results. This is supported by the experiments of Hartley (10) on washed precipitates from underneutralized, neutral, and overneutralized mixtures of antitoxin and toxin: those derived from underneutralized material are toxic and powerfully antigenic; those from neutralized, atoxic and of good antigenic action, and from overneutralized, atoxic and of low antigenicity. Hartley states, moreover, that the precipitate reactions of toxicity and antigenicity bear a close relationship to the nature of the mixture from which they are produced. There is, therefore, a connection between the preventive reactions of the serum on the two forms of virus and of antitoxin on toxin in respect to toxicity and antigenicity. Furthermore, the toxin is rendered atoxic with retention of immunizing capacity by formalin: the production of toxoid or anatoxin (Glenny and Hopkins (11), Ramon (12))-again a condition related to the effects following formolization of the virus. It has, however, been stated that "in an immunizing mixture prepared with modified [formolized, but partially detoxified] toxin the antitoxin present does not within wide limits affect the antigenic power" (Glenny, Hopkins, and Pope (13)). It is not known whether a preliminary injection of antitoxic serum could have prevented the antigenic power of fully detoxified toxin, that is, after the passive immunity induced by the serum disappears. If a preventive action of antitoxic serum could be shown under these circumstances, a remarkable correlation of the reactions of proper amounts of antitoxin to toxoid and of proper amounts of immune serum on the virus would be evident. Finally, the inhibition of antigenic power of both active and inactive virus by immune serum has been demonstrated to apply to the virus of equine encephalomyelitis in guinea pigs and no generalizations of the application of the phenomenon to other viruses are intended.
一项研究旨在探讨马脑炎病毒抗血清在豚鼠体内对活病毒和福尔马林灭活病毒的抗原性反应的影响。研究发现，当动物在皮下接种活病毒或福尔马林灭活疫苗前 1 小时皮下给予适量的高度免疫血清时，病毒对颅内 1000 倍和 10000 倍 MLD 的测试的免疫力均未诱导。血清的这种预防作用会因稀释而丧失，损失与稀释度成正比；另一方面，随着病毒数量的增加，需要更多的血清来获得阻断作用。当将适量的血清与相同数量的活病毒或福尔马林疫苗一起引入动物体内时，如果引入的血清量不足，一部分接受未处理病毒的动物在接种过程中会死于病毒感染，但幸存者对颅内测试具有抗性；所有用更高稀释度的血清和福尔马林材料处理的豚鼠都安全地进入免疫状态。因此，重点是，未处理的活病毒和福尔马林组织悬浮液中存在的抗原刺激物（1）在剧烈测试中未显示出任何活性病毒，并且在动物体内完全无感染性（1），使用适当量的免疫血清完全可以抑制它们的作用。这种足够数量的血清的预防能力的基础机制可以根据本系列（1、3）和本文中的先前论文中推导出的事实来解释。我们已经表明，3 x 10(7)m.i.u.的活病毒含有足够的抗原，可以诱导免疫，而无需在动物体内繁殖。这已经通过用福尔马林处理至无法揭示任何活性病毒的程度的 3 x 10(7)m.i.u.病毒形式所产生的相似程度的抵抗力得到充分证实（1）。因此，血清的阻断作用阻止了病毒的繁殖，从而导致产生的抗原量不足，这种假设是站不住脚的，因为在“活”病毒和“死”病毒中都存在这种阻断作用。另一方面，用较高稀释度（表 II 中注意到的）的免疫血清，只有活病毒才能繁殖——福尔马林灭活疫苗不会受到这些数量的血清的影响——因此产生更多的抗原物质。这种物质反过来又在宿主中引起更大的抵抗力。适当量的血清对活病毒和灭活病毒的免疫发展的预防作用的确切机制尚不清楚。然而，提出了两个假设供考虑：第一个假设是血清的作用是直接的，即通过与抗原结合来阻止抗原性；第二个假设是血清对身体的细胞具有间接作用，使其无法对接种材料中的抗原刺激做出反应。目前，尚不确定含有活性、感染性因子或用福尔马林灭活的此类因子的病毒悬浮液中的这些抗原刺激物是否相同。如果可以获得纯病毒，而没有外来物质，那么这个问题的答案可能很容易给出，但当所谓的病毒是感染性因子、炎症组织产物、组织等的混合物时，情况就完全不同了。然而，我们已经表明，诱导的免疫力不是由于“活”病毒的存在，而是抗原性作用是否源自“死”病毒或悬浮液中的其他成分尚不清楚。另一方面，Sabin（7）提出了一种可能性，即病毒可能不是直接的抗原刺激物，而是它作用的某种物质，并从受感染的细胞中释放出来，可能是负责的因素。在进一步研究结果出来之前，我们认为福尔马林可以使病毒悬浮液中的感染性因子失活，并保留其中的抗原成分，无论其性质如何。如果这种适当数量的免疫血清可以防止抗原能力的现象可能适用于其本身在宿主体内不会繁殖的物质，例如毒素和抗毒素，那么这将是很有趣的。Theobald Smith（8）和后来的 Park（9）证明，在白喉毒素-抗毒素混合物中，当使用较少量的免疫血清（抗毒素）时，混合物的毒性得以保留并且产生免疫力；如果血清增加，则毒性降低并且免疫力不规则发生，如果添加更多的血清，则既没有毒性也没有免疫力。这得到了 Hartley（10）对来自中和不足、中和、中和过度的抗毒素和毒素混合物的未洗沉淀的实验的支持：来自中和不足的材料的沉淀是有毒的和强有力的抗原性；来自中和的沉淀是无毒的且具有良好的抗原活性，来自中和过度的沉淀是无毒的且抗原性较低。此外，Hartley 还指出，沉淀反应的毒性和抗原性与它们产生的混合物的性质密切相关。因此，血清对两种形式的病毒和抗毒素对毒素的预防反应在毒性和抗原性方面存在联系。此外，毒素用福尔马林处理后变得无毒但保留了免疫能力：产生类毒素或抗毒素（Glenny 和 Hopkins（11），Ramon（12））-再次是与病毒福尔马林化后产生的效应相关的条件。然而，有人指出，“在用修饰的[福尔马林化但部分解毒]毒素制备的免疫混合物中，存在的抗毒素在很大程度上不会影响抗原性”（Glenny、Hopkins 和 Pope（13））。尚不清楚预先注射抗毒素血清是否可以防止完全解毒毒素的抗原能力，即在血清诱导的被动免疫力消失后。如果可以证明抗毒素血清的预防作用，那么适当数量的抗毒素对类毒素和适当数量的免疫血清对病毒的反应之间就会出现明显的相关性。最后，免疫血清抑制活病毒和灭活病毒的抗原性已被证明适用于豚鼠中的马脑炎病毒，并且不打算将该现象的应用推广到其他病毒。
