American Journal of Epidemiology 2005 161(3):207-212; doi:10.1093/aje/kwi033
Copyright © 2005 by the Johns Hopkins Bloomberg School of Public Health
David Bodian’s Contribution to the Development of Poliovirus Vaccine
Neal Nathanson
From the Departments of Microbiology and Neurology, School of Medicine, University of Pennsylvania, Philadelphia, PA.
Received for publication September 22, 2004; accepted for publication October 18, 2004.
Abbreviations:
MV, mixed virus.
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INTRODUCTION
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David Bodian spent almost his whole scientific career (1942–1983)
at the Johns Hopkins University, first in the Department of
Epidemiology at the School of Hygiene and Public Health and
then in the Department of Anatomy at the School of Medicine.
Arguably, his most important research contribution was the elucidation
of the pathogenesis of poliomyelitis—a contribution that
played a major role in the development of inactivated poliovirus
vaccine (Salk vaccine), the first successful vaccine against
poliomyelitis. In this historical review, I have chosen to focus
on those aspects of Bodian’s research that were relevant
to vaccine development. This retelling of the story is necessarily
biased, since it emphasizes the work of one investigator while
acknowledging that less attention is paid to the work of many
others who made important contributions. In addition, I distort
history somewhat by presenting the research results in an apparently
logical sequence, while in truth the story more resembled a
jigsaw puzzle put together somewhat randomly to reveal an orderly
whole when completed. Furthermore, I have deliberately chosen
some examples from my own collaborations with David Bodian,
even though the experiments were performed after 1955. Finally,
as Dave Bodian’s last trainee in virology, I write this
with fond memories of an inspiring role model.
I begin with a quotation from a talk that David Bodian gave in 1976, at the time of his retirement as chair of the Department of Anatomy (1): "In 1945, Professor Burnet of Melbourne wrote, ‘While I was in America recently I had good opportunity to meet with most of the men actively engaged on research in poliomyelitis... The part played by acquired immunity to poliomyelitis is still completely uncertain, and the practical problem of preventing infantile paralysis has not been solved. It is even doubtful whether it ever will be solved.’ ... Most of us doing research on poliomyelitis in 1945 were mainly motivated by curiosity, rather than by the hope of a practical solution in our lifetime." And yet, on April 12, 1955, just 10 years after Burnet’s 1945 letter, Thomas Francis announced the successful field trial of inactivated poliovirus vaccine (2). What explains this abrupt transition from a state of confusion and dismay to the triumphant optimism of 1955? The explanation lies in a set of discoveries that were made regarding the pathogenesis of poliovirus infection and the role of antibody in its control. This story is the subject of the following essay. 6
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PATHOGENESIS OF POLIOMYELITIS
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The key discoveries leading to the formulation and testing
of the first poliovirus vaccine are summarized in table
1. The
first breakthrough was the finding that primary cultures of
human cells could be used to grow poliovirus and that the virus
produced a rapid, consistent, and readily detected cytopathic
effect (
3). This finding provided a system for the ready isolation
of poliovirus from patients—a method of growing virus
stocks, quantitating infectious virus, and measuring neutralizing
antibody. Simple as it may have been, the cell culture system
had profound implications for research on poliomyelitis. First,
it led to the isolation of "wild-type" strains of poliovirus
directly from the stools and throat swabs of infected patients;
these low-passage isolates were used to prepare virus stocks
that could be used for experimental studies. Prior to this time,
experimental virus stocks had been prepared from the spinal
cords of monkeys infected by intracerebral injection of the
virus, and the "standard" virus used for most studies was the
mixed virus (MV) strain. The MV strain had been developed at
the Rockefeller Institute in the laboratory of Simon Flexner
and had undergone many serial intracerebral passages.
When virus isolates obtained by Enders’ cell culture
method were compared with the MV strain, they were found to
differ dramatically in their biologic properties. Multiple intracerebral
passages of the MV strain had selected for a virus that was
highly neurotropic and did not cause viremia, while fresh wild-type
isolates were viremogenic.
Table 2 illustrates these differences and contrasts neuroadapted (MV strain) and viremogenic (Mahoney strain) poliovirus (4). In this experiment, cynomolgus monkeys were divided into two groups; one group underwent a nerve block by freezing of the sciatic nerve and the other group had a sham operation. Both groups were then given poliovirus injections in the gastrocnemius (calf) muscle. Nerve block protected against the neurotropic MV strain but not against the Mahoney strain, indicating that the Mahoney virus spread via the bloodstream—bypassing the neural block—while the MV strain could not bypass the neural block, since it was an obligatory neurotrope.
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TABLE 2. Difference between neuroadapted poliovirus (MV* strain) and viremogenic poliovirus (Mahoney strain) in the route of spread from a peripheral site of infection to the spinal cord
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Concepts of the pathogenesis of poliomyelitis had been based
on monkey experiments with the MV strain, and those concepts
had to be radically revised on the basis of experiments with
wild-type isolates. Central to the revised view of poliovirus
pathogenesis was the role of viremia.
Figure 1 shows an experiment in which cynomolgus monkeys were inoculated intravascularly with four wild-type isolates (5). Each of the isolates induced viremia, but there was marked difference in the viremogenic potential of different isolates. Three caused minimal viremia that was detectable only through testing at multiple time points after infection and led to a low frequency of paralysis. By contrast, the Mahoney strain—which produced the highest-titer viremia—paralyzed about 50 percent of infected monkeys. The correlation of viremia level and paralysis rate provided important circumstantial evidence for the role of viremia in the path to the central nervous system (5).
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FIGURE 1. Causation of viremia by wild-type isolates of poliovirus (see text for discussion). Data were obtained from the paper by Bodian (5). TCD50, 50% tissue culture infectious dose.
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Parallel investigations conducted during outbreaks of paralytic
poliomyelitis showed that viremia also occurred in human infection.
Table
3 shows results from a study in which contacts of patients
with paralytic poliomyelitis were bled twice, shortly after
the onset of the index case and about 1 month later (
6). The
paired serum samples were tested for antibody, and the first
serum samples taken were also tested for virus. Contacts were
classified in several categories: those previously infected
with poliovirus; those who were not infected; those with recent
infection (converters) who had antibody in the first serum sample;
and those converters with no detectable antibody in the first
serum sample. There were nine subjects in the last category,
and virus was isolated from five of those nine, which is a significant
success rate considering the single sample tested and the low
level of viremia produced by many wild-type poliovirus strains
(figure
1).
The reconstructed view (figure
2) of pathogenesis indicated
that poliovirus was an enterovirus that initially infected lymphoid
tissues in the tonsil and intestine (Peyer’s patches)
and then spread to draining lymph nodes, and—via lymphatic
drainage—entered the circulation (
7). Bloodborne virus
invaded the central nervous system, either directly across the
blood-brain barrier or via peripheral nerve ganglia, and resulted
in destruction of lower motor neurons in the spinal cord, producing
the flaccid paralysis characteristic of poliomyelitis. The critical
point was that viremia was an essential step on the pathway
from the portal of entry to the central nervous system.
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FIGURE 2. David Bodian’s scheme of the pathogenesis of poliovirus infection based on studies in monkeys, chimpanzees, and humans. Reproduced with permission from the original article by Bodian (7). (Copyright 1955, American Association for the Advancement of Science (http://www.sciencemag.org).) CNS, central nervous system.
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PROTECTIVE ROLE OF ANTIBODY AND FORMULATION OF A VACCINE
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The revised view of the pathogenesis of poliomyelitis set the
stage for studies on the role of antibodies in recovery from
poliovirus infection and protection against paralytic poliomyelitis.
A necessary preliminary to these studies was a simple, quick,
and reproducible test for neutralizing antibodies. This was
the metabolic inhibition test developed by Jonas Salk, based
on Enders’ cell culture system. As practiced in the Bodian
laboratory in the 1950s (
8), the metabolic inhibition test utilized
small test tubes in which serial dilutions of serum samples
were set up in nutrient medium plus phenol red indicator dye.
A virus inoculum of 100 tissue culture infectious doses was
added; the mixture was left at room temperature for 1 hour to
permit neutralization to occur; and a freshly prepared suspension
of monkey kidney cells was added. After 5 days of incubation
at 37°C, the test was read. In the absence of virus, the
cells produced metabolic acid and the phenol red dye turned
yellow; if virus was present, the cells were destroyed and the
color remained red; and if antibody neutralized the virus, the
cells metabolized and the culture was yellow. Using this system,
it was possible to obtain very clear endpoints within a few
twofold dilutions of the test serum.
The other critical prerequisite for the rational development of a vaccine was determination of whether there was a single antigenic type of the virus (as with measles and smallpox viruses) or several antigenic types (as with adenoviruses and rhinoviruses). For this purpose, antigenic similarity or difference was best defined as cross-protection in humans. In other words, if a single infection with wild-type poliovirus consistently protected an individual against the paralytic consequences of subsequent exposure to poliovirus, it could be considered that all strains of the virus were part of a single antigenic type. On the other hand, if there were several different antigenic types, a putative efficacious vaccine formulation would need to include each of these types.
There were reported anecdotal instances of patients who had experienced more than a single paralytic attack from poliomyelitis, but they were rare and had not been well documented in the absence of the ability to isolate virus and test patients for neutralizing antibody. Therefore, the most rigorous approach to this question was to infect monkeys with a given strain of poliovirus and, a few months later, challenge them with a second strain.
Table 4 provides an extract from such an experiment, which differentiated two antigenic types of poliovirus (9). In this experiment, rhesus monkeys were first infected with one of four viral isolates. Many of the animals developed paralysis but survived, and the surviving paralyzed monkeys were then challenged by a second intracerebral injection with either the same isolate or a different isolate. Homologous challenge (reinfection with the same virus) provided 100 percent protection, indicating absolute protection with viruses in the same antigenic group. Using this standard, if the first infection conferred 100 percent protection against a different virus isolate (heterologous challenge), the two viruses were classified in the same antigenic group. If the second virus caused a second paralytic attack in some of the animals (partial cross-protection), it was classified in a different antigenic group. On these grounds, the four strains compared in table 4 could be placed into two antigenic groups.
Experiments of this kind, done by Bodian and others, indicated
clearly that wild-type polioviruses could be classified into
three types, as defined by cross-protection. Parallel results
were obtained using Salk’s neutralization test. These
data implied that a protective vaccine would have to be multivalent—that
is, it would have to immunize against each of three antigenic
types.
The similarity of results using cross-challenge of monkeys and tests of serum neutralizing antibody led to the question of the role of antibody in recovery from poliovirus infection. Figure 3 models acute poliovirus infection of humans, using a virulent wild-type virus in monkeys (8). In this experiment, cynomolgus macaques were infected intramuscularly with the Mahoney strain of poliovirus and were followed for viremia and the appearance of neutralizing antibodies in their serum. Mahoney virus induced viremia that had its onset a few days after infection and lasted less than 1 week. Importantly, immediately following the disappearance of virus from the blood, neutralizing antibody was first detected and rose in titer thereafter. The detection of antibody on the first day after viremia disappeared was consistent with the interpretation that it was responsible for ending the viremia phase.
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FIGURE 3. Apparent termination of viremia by neutralizing antibodies (see discussion in text). Data were obtained from the paper by Nathanson and Bodian (8). TCD50, 50% tissue culture infectious dose.
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Evidence that antibodies played a critical role in the termination
of viremia suggested that circulating antibodies might be able
to prevent viremia if they were present prior to infection.
Figure
4 shows results from an experiment in which this hypothesis
was tested (
10). Cynomolgus monkeys were given graded doses
of antibody (pooled human gamma-globulin) by intramuscular injection;
24 hours later, the animals were bled and the serum was tested
for the level of passive antibody. Right after bleeding, the
monkeys were given an injection at a different intramuscular
site with a paralyzing dose of Mahoney virus. Assays of serum
obtained prior to viral infection showed that each twofold increase
in the dose of antibody produced a corresponding twofold increase
in the level of antibody in the recipient monkeys. Furthermore,
there was a strong correlation between passive antibody level
and degree of protection against paralytic poliomyelitis. Thus,
in the control group given no antibody, the rate of paralysis
was approximately 95 percent; an antibody titer of 3 conferred
little protection; a titer of 6 conferred about 50 percent protection;
and a titer of 11 conferred about 90 percent protection. This
result implied that neutralizing antibody could provide protection
against potentially paralytic infection with poliovirus.
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FIGURE 4. Protection against potentially paralytic poliovirus infection provided by neutralizing antibodies (see text for description of experiment). Data were obtained from the paper by Nathanson and Bodian (10).
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Data of this type made available a "correlate of protection,"
one that could be quantified. By inference, if an immunogen
could be prepared that would induce neutralizing antibodies
in the circulation, it might protect immunized subjects against
the paralytic consequences of subsequent exposure to wild-type
poliovirus. It was now timely to consider various ways in which
poliovirus or its antigens could be formulated as a vaccine,
using the correlate of protection to evaluate potential efficacy.
Jonas Salk (and later Albert Sabin) relied heavily upon this
correlate as he evaluated different experimental vaccine formulations.
Salk found that a simple chemical, formaldehyde, could be used
to inactivate poliovirus and render it noninfectious in both
cell cultures and animals. Furthermore, when injected intramuscularly,
this inactivated poliovirus vaccine would elicit neutralizing
antibodies.
Figure 5 shows results from a trial of the immunogenicity of an experimental batch of inactivated poliovirus vaccine (11). The subjects had preexisting antibody to none, one, or two types of poliovirus prior to immunization with a tri-valent vaccine. Responses shown are responses only to the serotypes that were negative prior to immunization. The experimental vaccine induced an immune response in all individuals and to all three serotypes of the virus; furthermore, antibody responses in most participants exceeded the critical level of 10 by a considerable margin. Data of this kind provided significant promise of the efficacy of such a vaccine. (This brief account bypasses all of the complex issues surrounding safety and production of inactivated poliovirus vaccine.)
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FIGURE 5. Neutralizing antibody response of normal human volunteers to immunization with an experimental batch of inactivated poliovirus vaccine (see text for details). Reproduced with permission from the original article by Salk (11). TCID50, 50% tissue culture infectious dose.
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To test the protective efficacy of inactivated poliovirus vaccine,
a large field trial was conducted in 1954 (
12). This trial involved
hundreds of thousands of children (selected as the highest-risk
group) and included a vaccinated group and a placebo control
group. No attempt is made here to describe this massive trial,
which stands as a landmark in human experimentation. I will
only refer to one data set extracted from the published results
(
12). From the trial data, it was possible to test the hypothesis
that the putative correlate of protection (a neutralizing antibody
titer of about 10) would protect against paralytic poliomyelitis.
Table 5 shows that, in the placebo control group, there were 40 cases of paralytic poliomyelitis (12). The hypothesis was formulated that vaccinees with a neutralizing antibody titer of 8 or greater would be protected and those with a titer of less than 8 would not. Among vaccinees, 35.4 percent had titers less than 8, leading to the prediction of 14.2 paralytic cases among vaccinees (35.4 percent of 40 placebo cases). In fact, 14 cases were observed in the vaccinated group, confirming the hypothesis and validating neutralizing antibody as a correlate of protection.
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TABLE 5. Correlation between titer of neutralizing antibody and protection against paralytic poliomyelitis in recipients of inactivated poliovirus vaccine*
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REPRISE
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This account is admittedly both brief and oversimplified. However,
I believe it documents the critical contribution of research
on the pathogenesis of poliomyelitis to the development of an
efficacious poliovirus vaccine. I have tried to indicate the
central role that David Bodian played in elucidating the pathogenesis
of poliomyelitis and how his discoveries moved the field forward
in a logical progression toward the formulation and testing
of inactivated poliovirus vaccine. Once again, I emphasize that
David Bodian was part of a larger group of investigators whose
aggregate contributions were essential to the success of the
undertaking. However, I would venture, as an admittedly biased
observer, to assert that Dave was clearly one of the intellectual
leaders of this effort, a scientist to whom others turned because
of his keen mind, analytic prowess, and recognized integrity.
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NOTES
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Reprint requests to Dr. Neal Nathanson, 1600 Hagys Ford Road,
Narberth, PA 19072 (email:
nathansn{at}mail.med.upenn.edu).
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REFERENCES
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- Bodian D. Poliomyelitis and the sources of useful knowledge. Johns Hopkins Med J 1976;138:130–6.[ISI][Medline]
- Laurence WE. Salk polio vaccine proves success: millions will be immunized soon. New York Times 1955;104(April 13):1.
- Enders JF, Weller TH, Robbins FC. Cultivation of the Lansing strain of poliomyelitis virus in cultures of various human embryonic tissue. Science 1949;109:85–9.[Free Full Text]
- Nathanson N, Bodian D. Experimental poliomyelitis following intramuscular virus injection. I. The effect of neural block on a neurotropic and a pantropic strain. Bull Johns Hopkins Hosp 1961;108:308–19.[ISI][Medline]
- Bodian D. Viremia after intravascular inoculation of poliovirus. I. General aspects of infection after intravascular inoculation with strains of high and of low invasiveness. Am J Hyg 1954;60:339–57.
- Bodian D, Paffenbarger RS Jr. Poliomyelitis infection in households: frequency of viremia and specific antibody response. Am J Hyg 1954;60:83–98.[ISI]
- Bodian D. Emerging concept of poliomyelitis infection. Science 1955;122:105–8.[Free Full Text]
- Nathanson N, Bodian D. Experimental poliomyelitis following intramuscular virus injection. II. Viremia and the effect of antibody. Bull Johns Hopkins Hosp 1961;108:320–33.[ISI][Medline]
- Bodian D. Differentiation of types of poliomyelitis viruses. I. Reinfection experiments in monkeys (second attacks). Am J Hyg 1949;49:200–24.[Medline]
- Nathanson N, Bodian D. Experimental poliomyelitis following intramuscular virus injection. III. The effect of passive antibody on paralysis and viremia. Bull Johns Hopkins Hosp 1962;111:198–220.[ISI][Medline]
- Salk JE. Studies with noninfectious poliovirus vaccines. Poliomyelitis: papers and discussions presented at the third international poliomyelitis conference. Philadelphia, PA: J B Lippincott Company, 1955:167–85.
- Francis TJ Jr, Korns RF, Voight RB, et al. Evaluation of the 1954 field trial of poliomyelitis vaccine. Final report. Ann Arbor, MI: School of Public Health, University of Michigan, 1957.
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INTRODUCTION
PATHOGENESIS OF POLIOMYELITIS