Читать книгу To Catch a Virus - John Booss - Страница 29

Theories of humoral immunity, the basis of serological testing, were forged against Elie Metchnikoff’s (Fig. 4) theory of phagocytosis in host defense. Metchnikoff’s studies of phagocytosis evolved from his interest in digestion by invertebrates in the latter 1870s (40). In her biography of Metchnikoff, Olga, his second wife, gives a charming quote describing the inception of her husband’s phagocyte theory: “One day when the whole family had gone to a circus to see some extraordinary performing apes, I remained alone with my microscope, observing the life in mobile cells of a transparent star-fish larva, when a new thought suddenly flashed across my brain. It struck me that similar cells might serve in the defense of the organism against intruders” (27). Metchnikoff devised a simple experiment in which he “introduced them (rose thorns) at once under the skin of some beautiful star-fish larvae as transparent as water.” The following morning he confirmed that the thorns were surrounded by mobile cells. “That experiment formed the basis of the phagocyte theory, to the development of which I devoted the next twenty-five years of my life.”

Figure 4 Elie Metchnikoff. Metchnikoff’s studies of phagocytosis initiated the science of immunology, specifically cellular immunity. With Paul Ehrlich, who developed the theoretical basis for the action of antibodies or humoral immunity, Metchnikoff received the Nobel Prize in 1908. (Courtesy of Wikimedia Commons.)

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On showing his experiments to Rudolf Virchow, the father of modern pathology, Metchnikoff was advised to proceed with caution. As opposed to Metchnikoff’s view of inflammation as “a curative reaction,” contemporary medicine viewed leukocytes as supporting the growth of microbes (27). Metchnikoff left Messina and on subsequent travels through Vienna, the word “phagocytes” was suggested by zoologists as a Greek translation of “devouring cells” (27).

In 1884, Metchnikoff published a seminal work on phagocytosis in Virchow’s Archive (26). Regretfully, however, as his wife recorded, “. . . the memoir passed unnoticed; the full significance of it had not been grasped” (27). Over a century later, A. M. Silverstein, in his history of immunology, amplified that observation: “One may conclude that the cellular theory of immunity advanced by Elie Metchnikoff in 1884 did not constitute just one further acceptable step in a well-established tradition; rather it represented a significant component of a conceptual revolution with which contemporary science had not yet fully learned to cope” (40).

As described in the 1884 work, Metchnikoff investigated a fungal disease of Daphnia, a transparent water flea (22). Daphnia ingested asci, sac-like structures in which spores are formed. Upon release into the digestive tract, spores traversed the intestinal wall to the body cavity, where they were attacked by blood cells, ultimately disintegrating into granules. Host giant cells were seen to have formed from the fusion of ameboid cells; however, the host did not always win out.

Using Metchnikoff’s other studies of anthrax bacilli inoculated subcutaneously into frogs and rabbits as a take-off point, George Nuttall confirmed that phagocytosis was observed but also noted extracellular destruction of anthrax (reference 30, translated in reference 22). With blood and other body fluids in vitro from several different species of animals, either immunized or not, Nuttall demonstrated degeneration of free anthrax bacilli and other bacteria. Hence, phagocytosis was not the entire explanation for destruction of bacteria. “These investigations have shown that independently of leucocytes, blood and other tissue fluids may produce morphological degeneration of bacilli” (22). The studies of Metchnikoff on phagocytosis and Nuttall on humoral mechanisms demonstrated the two arms of host defense. However, they did not demonstrate immunization, specific host responses after particular challenges. That was most clearly achieved in studies of immunity to tetanus and diphtheria toxins by Emil Behring and Shibasaburo Kitasato.

In his masterful The History of Bacteriology, published in 1938, William Bulloch succinctly described the stepwise series of discoveries leading up to the landmark work of Behring and Kitasato (8): “F. Loeffler (1884) discovered the diphtheria bacillus and Kitasato (1889) proved that the tetanus bacillus is the cause of lockjaw. Roux and Yersin (1889) proved that the diphtheria bacillus operates by virtue of a poison (toxin) which it elaborates, and K. Faber (1889) showed that the tetanus bacillus acts in a similar manner.” The stage was set for Behring and Kitasato to describe the mechanism of immunity to diphtheria and tetanus (4). Observing that blood from immune animals neutralized diphtheria toxin, they used this concept to devise experiments to demonstrate that treated animals would be insensitive to tetanus. These experiments are described in Behring and Kitasato’s paper translated in Brock’s collection of landmark papers in microbiology (7). They present experiments in support of four conclusions: (i) blood of a rabbit immune to tetanus neutralizes or destroys tetanus toxin, (ii) that property is found in cell-free serum, (iii) the property is stable in other animals when used for therapy, and (iv) the property does not occur in animals not immune to tetanus. The importance of the paper can be found in Brock’s comment, “The science of serology can be said to have begun with this paper.” Behring followed this paper with a similar report focusing on diphtheria (3). He won the Nobel Prize in 1901.

While Silverstein characterized the 1890 Behring and Kitasato paper as “[t]he most telling blow to the cellular theory of immunity . . .” (40), the interplay of phagocytic and humoral mechanisms was studied by A. E. Wright and S. R. Douglas. They presented experiments allowing them to state, “We have here conclusive proof that the body fluids modify the bacteria in a way which renders them ready prey to the phagocytes . . . We may speak of this as an opsonic effect.” They termed the elements in the blood exerting this effect as “opsonins” (52).

The theoretical basis for the activity of antibodies was developed by Paul Ehrlich in his side chain theory (31). With the stimulation of an antigenic challenge, host cells produced side chain receptors that specifically combined with the antigen. With sufficient stimulation, side chain receptors could be released into the blood as circulating antibodies (12). Perhaps more to the point in the current context, Ehrlich established principles of quantitation (8). Paul Ehrlich and Elie Metchnikoff shared the Nobel Prize in 1908, an early recognition of the importance of both humoral and cellular immunity (1).

The above-described studies, which were of great importance to establishing mechanisms of host defense, were heavily dependent on microscopic observations of the infecting organism. There was little direct application to submicroscopic viruses until the studies of Jules Bordet (Fig. 5), another Nobel Prize winner (1919); he was recognized for his work on complement fixation, which became one of the key mechanisms with which to document antiviral immunity. During the period in which this work was done, Bordet worked in the laboratory of Elie Metchnikoff at the Pasteur Institute in Paris. There were two preliminary steps which Bordet addressed in an 1895 paper in Annales de l’Institut Pasteur (translated and condensed in reference 7). Building on the work of R. F. J. Pfeiffer, he confirmed that the granulation followed by lysis of Vibrio cholerae bacteria exposed to serum of immunized rabbits is strain specific (Pfeiffer phenomenon). Testing several strains of vibrios in vitro, he showed that the greatest destruction was of bacterial strains against which the rabbits had been vaccinated. Such immunological specificity is a cornerstone of serological diagnosis.

Figure 5 Jules Bordet. With his brother-in-law, Octave Gengou, Bordet demonstrated the fixation of complement by reacting with bacteria and immune serum. The complement was then no longer available to participate in a hemolysis reaction. The assay, known as complement fixation, became a mainstay of diagnostic virology by demonstrating the development of antibodies in serum after infection. Bordet received the Nobel Prize in 1919. (Courtesy of the National Library of Medicine.)

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Bordet demonstrated that there were separate immunizing and bacteriolytic components. While the bactericidal property is destroyed by heating, he built on the work of C. Fraenkel and G. Sobernheim, who showed that the immunizing component is resistant to heating. In a system in which counts of Vibrio cholerae were the markers, he demonstrated that neither heated immune serum from goats nor fresh serum from nonimmune guinea pigs alone would inhibit growth. However, the combination of heated immune goat serum and unheated guinea pig serum abolished bacterial growth. This phenomenon occurred whether or not cells were present in the guinea pig serum. From a diagnostic perspective, two features deserve emphasis: (i) the source of complement, then known as alexine, need not be the species tested for immunity, and (ii) complement, but not the immune function of serum, is destroyed by heating to 60°C. Therefore, the components can be added separately. The dissection of the bacteriolytic system into two components was a remarkable accomplishment on its own merits. The experimental challenge had been made further complicated by at least two other factors. Some nonimmunized animals possessed bacteriolytic capacity in their serum, as shown in Nuttall’s work. In addition, not all bacteria were equally susceptible to immune-mediated bacteriolysis. Hence, the clarity of Bordet’s experimental design is all the more remarkable.

Next came the crucial paper in the scientific foundation of the complement fixation reaction. With his brother-in-law Octave Gengou, Bordet demonstrated the deletion or fixation of complement, as currently understood, by a combination of immune serum and the target bacilli (reference 6, translated in reference 22). With this combination, the complement was unavailable to facilitate hemolysis as a marker system. As Bordet and Gengou pointed out, the work was dependent on the previous demonstration of two concepts. First, red cells and microbes could each delete alexine (complement). Second, the same alexine could participate in either hemolysis or bacteriolysis. With appropriate controls, in a two-step experimental setup, they first mixed plague antiserum and a suspension of plague bacilli with alexine-containing serum from animals. In the second step, into the first mixture they introduced heated guinea pig serum immunized against rabbit’s blood together with rabbit’s blood. Hemolysis occurred in all tubes except those containing the bacilli, the specific antiserum, and alexine (Fig. 6). Hence, the antiserum conferred the capacity to fix alexine (complement), making it unavailable to participate in hemolysis of sensitized red cells. At a stroke, Bordet and Gengou had demonstrated a marker system that did not require microscopic examination for the presence of the pathogen. Complement fixation to detect viral antibodies became the cornerstone of viral diagnosis for many years.

Figure 6 Complement fixation. In stage 1, complement, antigen, and antibodies are mixed together. If antibody is present for the antigen, complement will be bound (fixed). In stage 2, if the complement has been fixed in the first stage, it will be unavailable to combine with antibody-coated erythrocytes. Therefore, a bull’s eye pellet of cells will appear in the bottom of the tube, as shown on the bottom left. However, if complement has not combined with antigen and antibody in the first stage, it will be available to lyse antibody-coated red cells. In that case, no bull’s eye pellet will be seen at the bottom of the tube, as shown on the bottom right (22a). (From Diagnostic Virology, courtesy of the author, Diane S. Leland, Indiana University School of Medicine.)

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There was an interesting parallel of scientific reports in 1901, each of importance to the beginnings of clinical virology. In that year, Bordet and Gengou demonstrated the experimental basis of the complement fixation reaction, while W. Reed and J. Carroll demonstrated that the submicroscopic pathogen for yellow fever passed through a filter which blocked bacteria (33).