Development and Initial Field Trials of Yellow Fever 17D Vaccine

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Research efforts at the Rockefeller Institute in New York were directed towards the development of an attenuated vaccine that had no neurovirulence properties. The French strain "fixed" by passage in mouse brain was considered too dangerous for use in humans, since it was capable of producing yellow fever encephalitis in monkeys and was associated with neurological accidents in humans. There was also a concern that the virus had residual viscerotropism, since Findlay and Clarke had shown reversion on repeated direct liver passage in monkeys [42].

Theiler and his colleagues decided to utilize recently described methods for cultivation of tissue in attempts to induce attenuation of wild-type yellow fever virus. Virus growth in minced tissue cultures prepared from mouse and chicken embryos had been developed at the Rockefeller Institute by Alexid Carrell and Thomas M. Rivers [43, 44]. In 1932, Theiler and Eugen Haagen demonstrated that the neurotropic French virus could also be propagated in chick-embryo tissue cultures [45], but attempts to grow unadapted virus strains failed. To avoid the danger of the neurotropic virus, it was important to derive a method adaptation in mouse brain. The operating principal was that the virus propagated under conditions that were generally unfavorable or restrictive, would lead to the selections of variants with altered phenotypic characteristics. This principle appeared to explain Findlay and Stern's observation that viserotropic yellow fever virus passaged in a transplantable mouse carcinoma had partially lost its virulence [46].

In 1936, Wray Lloyd (Fig. 1), Theiler, and technician Nelda Ricci reported the first successful in vitro cultivation of the Asibi strain [47]. After 240 passages, the virus became progressively less viscerotropic for rhesus monkey, although it retained its capacity to produce encephalitis after inoculation. The virus (designated

Fig. 1 Dr Wray Lloyd played a key role in the adaptation of yellow fever virus to tissue culture during studies in the early 1930s at the Rockefeller Foundation in New York. He investigated the use of the 17E virus (grown in mouse embryo tissue culture) with immune serum from human immunization. Lloyd took the 17E virus to Brazil in 1935, where he conducted further clinical studies. He died of an accidental fall in 1936. (Photo courtesy of the Rockefeller Archive Center, North Tarrytown, New York)

Fig. 1 Dr Wray Lloyd played a key role in the adaptation of yellow fever virus to tissue culture during studies in the early 1930s at the Rockefeller Foundation in New York. He investigated the use of the 17E virus (grown in mouse embryo tissue culture) with immune serum from human immunization. Lloyd took the 17E virus to Brazil in 1935, where he conducted further clinical studies. He died of an accidental fall in 1936. (Photo courtesy of the Rockefeller Archive Center, North Tarrytown, New York)

17E) was deemed too virulent for human inoculation without coadminstration of immune serum. The 17E virus replaced the mouse brain virus for sero-immunization of laboratory workers [48]. In November, 1935, speaking at the Annual Meeting of the American Society of Tropical Medicine in Baltimore, Sawyer noted that " ... a safer strain has supplanted the original neurotropic strain for use with immune serum in vaccination and it is confidently expected that a strain of virus safe for use without protective immune serum will finally be achieved." [49].

Between 1934 and 1936, multiple attempts were made to cultivate the Asibi virus in other substrates that might favor selection of attenuated variants, including minced tissue culture of mouse and guinea pig testicle, and of chick embryo [49]. After initial propagation in whole mouse embryo tissue culture, passage to these alternative substrates was achieved, in each case with an attendant decrease in vis-certotropism of the virus. Since neurotropism was not markedly diminished it was decided to attempt sequential passages in chicken embryo tissues from which brain and spinal cord had been removed before mincing. The most important experiment, designated 17D, was initiated after 18 subcultures in whole mouse embryo cultures, as which point the virus as passed to whole minced chick-embryo cultures. After 59 subcultures in the latter tissue, the virus was then passed in minced chick embryo devoid of nervous tissue, each subpassage being checked for virus intracerebral inoculation of mice. Hugh H. Smith took responsibility for the oversight of these subculture experiments.

After 100 passages in chick embryo without nervous tissue (i.e., at the 176th passage since initiating in vitro culture), Smith noted a decrease in the neurovirulence of the virus, with mice surviving or developing nonlethal paralysis. Theiler and Smith confirmed that this virus induced minimal viremia and no hepatitis in rhesus monkeys after subcutaneous inoculation and, most importantly, that it had a markedly diminished neurovirulence for mice [50] (Table 2). The loss of neurovirulence for monkeys diminished between the 89th and 114th passage subcultures, and

Table 2 Biological characteristics of wild-type yellow fever virus and attenuated vaccine viruses derived empirically by serial passage (after Theiler and Smith [54])

Virus

Virulence for

Mice AST (days)

ic

Monkeys sc or ip

Wild-type

8-10

Fatal hepatitis

Fatal hepatitis

French neurotropic

4-10

Fatal encephalitis (100%)

Viremia, fatal

encephalitis (30%)

17D

8-20

Encephalitis (<10%)

Minimal viermia, no

illness

the neurovirulence in mice diminished between the 114th and 176th passage. Monkeys inoculated with the attenuated virus were protected against lethal peripheral challenge with the Asibi virus. At last, a strain was at hand that could be tested in humans without the addition of protective immune serum! In March 1937, Theiler and Smith submitted landmark papers to the Journal of Experimental Medicine, describing the development of and the first clinical trials with 17D viruses [51].

The vaccine was prepared from infected chick embryos ground with normal human serum to stabilize the virus, centrifuged, sterilized by filtration, and lyo-philized. Bacterial sterility was checked and a potency test was performed by intracerebral inoculation of mice. The first two subjects to take the vaccine were Theiler and Smith themselves, both being immune (Theiler by virtue of an accidental infection while at Harvard in 1929, Smith by immunization with the French neurotropic virus plus immune goat serum) [31]. Two other immune subjects received minor febrile reactions were recorded, but all subjects had increases in serum protective antibody levels. Theiler and Smith had reached a critical milestone in vaccine research.

Theiler concluded that the secret to success had been the elimination of nervous tissue from minced chick embryos used to propagate the virus, since parallel passage series carried out over several hundred subcultures in minced whole chick embryos (experiment 17D WC) and in chick-embryo brain tissue (17D CEB) had not led to a decrease in viscerotropism or neurotropism. Theiler attempted to confirm that the absence of neural tissue had been responsible for attenuation. Starting with the virus at the 212th subculture, he established a new series of passages in which 17D WC and CEB viruses were passed in chick-embryo brains only. No modification occurred in the pathogenic properties of any of the viruses. Thus he was neither able to reproduce the level of the attenuation of 17D nor revert 17D to neurovirulence.2 The reasons for the rapid change in 17D between the 89 th and

2 Later, Theiler passes the 17D virus sequentially by intracerebral inoculation of mice. Starting with the attenuated virus (176th subculture), 195 mouse brain passages were made, with period checks for monkey neurovirulence. After 106 passages, the virus causes encephalitis in monkeys. A parallel since 120 passages in chick embryo showed no phenotypic change. The virus thus appeared to be stable when maintained in chick embryo tissue, but with selective pressure could revert to neurovirulence.

114th subcultures in the original series remained unexplained [52]. The mutational events (or selection of preexisting variants) responsible for attenuation of 17D had occurred by chance during the course of systematic experiment that could not be readily duplicated. Virus attenuation by serial passage was an unpredictable procedure. Theiler and Smith's achievements were the result of a systematic and meticulous application of empirical process and keen, continuous observation by prepared minds, but they also had been extraordinarily lucky!

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