experiment, nonimmune serum was mixed with vaccinia virus-containing lymph and was compared with a mixture of vaccinia virus and immune serum. Skin lesions appeared at the site of inoculation of the mixture of virus and nonimmune serum but not at the site of inoculation of virus plus immune serum. While preliminary in scope, the prototype of neutralization was established. The marker system here was the skin of the calf, and in years to come it would be broadly implemented in experimental animals, in embryonated eggs, and in tissue culture. At the end of the published discussion, Sternberg commented, “I believe that there is something in the blood of the immune calf that neutralizes the vaccine virus.” The year of Sternberg’s paper, published in 1892, is usually marked as the start of the science of virology. Coincidentally, in a paper published in the same year in St. Petersburg, Dmitri Ivanowski described the passage of tobacco mosaic disease infectivity through a bacterial filter (20). These landmark events signify that virology and viral serology had their birth in the same year, albeit on different continents. Protection by neutralization was soon demonstrated in other virus-host systems. In April 1910 in Paris, A. Netter and C. Levaditi demonstrated protection of monkeys against experimental polio with serum from human subjects who had experienced the illness from 6 weeks to 3 years earlier (28). The monkeys revealed no signs of illness. In comparison, a monkey that received virus mixed with serum from a healthy person became paralyzed and died. The investigators noted that these studies demonstrated the identity of the human disease and the experimental disease in monkeys. In a following paper in May 1910, they also demonstrated antiviral activity from a subject who had not been paralyzed but had had symptoms of abortive illness (29). Thus, they identified the virus causing the abortive form of polio as well as the paralytic form. This supported the epidemiological findings of Ivar Wickman on the role of the abortive form in disseminating the illness (51). Also in May 1910, Simon Flexner and Paul A. Lewis in New York City demonstrated that serum from monkeys that had recovered from experimental poliovirus infection, when mixed with active virus, prevented paralysis in intracerebrally inoculated monkeys (14).
Similar findings were later reported for yellow fever. In their 1928 report from Nigeria on the experimental transmission of yellow fever to Macacus rhesus monkeys, Adrian Stokes et al. demonstrated that convalescent-phase human serum protected monkeys against experimental infection (46). Max Theiler greatly facilitated further laboratory studies of yellow fever infection by demonstrating the susceptibility of white mice to intracerebral inoculation of the virus (47). Virus passed in mice was neutralized by convalescent-phase serum from humans or monkeys. Parenthetically, in a later study of the protection test in mice, Theiler distinguished the terms “protection” as in vivo survival after inoculation of virus mixed with immune serum, whereas “neutralization” occurred in vitro (48) (Fig. 7). This seemed a useful distinction at the time, but it was frequently ignored in the literature. For example, in tabulating various serological reactions, Joseph Edwin Smadel listed “neutralization” for in vivo tests for many viruses (42). In the present context, Theiler’s assay was termed the mouse protection test, and it was an important step in laboratory diagnosis. The application of serology to the epidemiology of yellow fever was emphasized by W. A. Sawyer and W. Lloyd. They wrote of mapping “large areas with respect to endemicity, epidemicity, or absence of yellow fever . . .” (38). They pointed out the difficulty of adapting monkeys to such studies because of the great number of animals required. Instead, they modified Theiler’s use of white mice and found the technique useful for epidemiological studies.
Figure 7 Neutralization test in tissue culture. In the first stage, antibodies and live virus are mixed together. In the second stage, the mixture is added to susceptible cells in tissue culture. If antibodies specific for the virus are present, no cytopathic effects will occur; otherwise, the cells will be attacked and reveal cytopathic effects, as shown on the right (22a). (From Diagnostic Virology, courtesy of the author, Diane S. Leland, Indiana University School of Medicine.)
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Another major step in the laboratory measurement of antiviral immunity was the development of the hemagglutination inhibition assay. George Hirst of the Rockefeller Foundation first observed and reported hemagglutination of red cells by allantoic fluid of influenza virus-infected embryonated chicken eggs. He reported, “When the allantoic fluid from chick embryos previously infected with strains of influenza A virus was being removed, it was noted that the red cells of the infected chick, coming from ruptured vessels, agglutinated in the allantoic fluid” (17). The observation was also made by L. McClelland and R. Hare in the same year (24). M. Burnet had observed the phenomenon but rued that he had not followed up. “Then came a discovery which I should have made but did not” (9).
Inhibition of agglutination by serum from a recovered individual would be a demonstration of immunity that could be measured. In the first publication, Hirst described hemagglutination inhibition as an efficient and immune-specific method to determine antibody titers. Compared to the more cumbersome mouse neutralization tests, hemagglutination results were “of the same order of magnitude.” In a subsequent report, Hirst wrote, “. . . the mouse test is very complicated and involves the interplay of many forces over a period of 10 days, while the in vitro test is relatively simple. Because of the complexity of the mouse test, it seems probable that the agglutination inhibition test gives a more accurate picture of the in vitro combining ratios of virus and antibody” (18).
An assay related to hemagglutination, hemadsorption (Fig. 8), was later developed by Alexis Shelokov and his colleagues at the U.S. National Institutes of Health (39). Several viruses, particularly influenza virus, were isolated and identified by “selective attachment of erythrocytes onto the monolayer surface of tissue culture cells.” In the absence of cytopathic effect, infection was recognized by the specific attachment of red cells to the infected, intact cell monolayer. One of the methods reported by the investigators to verify the type of viral isolates was hemadsorption-inhibition.
Figure 8 Hemadsorption. Tissue culture cells infected with certain viruses produce receptors with an affinity for red blood cells which attach to the surface and are visible microscopically. In this illustration, influenza B virus has infected rhesus monkey kidney cells, facilitating the specific attachment of red blood cells which outline only the virus-infected cells. (Collection of Marilyn J. August.)
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Standardization
