LIVE POLIO IRUS VACCINES

536
Efficacy-Field Evidence
frigerator depending on shipping difficulties to
Helsinki. Sera were separated from clots, inactivated
at 56 ° C., and stored at 4 ° C. until
tested.
Stool specimens were collected in about 50 ml.
screw cap bottles by the persons themselves and
brought by them to the sampling place. They
were usually stored at outdoor temperature (in
summertime about +200 C. and in wintertime
about -30 ° C.) for one or two days before
shipping to Helsinki where they were stored
at -200 C.
Testing of Specimens
Virus isolations and identification from stool
specimens were done at the Department of
Virology, University of Helsinki, and the monkey
virulence tests were done by the Virus and
Rickettsial Research Section of the Lederle
Laboratories.
Of the 1,280 sera collected, 500 sera representing
the first four blood specimens from a
total of 125 persons, were tested at the Lederle
Laboratories for neutralizing antibodies against
the three types of polioviruses. The rest of the
sera collected in 1958 as well as those collected
in 1959 were tested at the Department of
Virology, University of Helsinki.
Virus isolations and identification technic.
Ten per cent stool suspensions were prepared in
bovine amniotic fluid containing 500 units of
penicillin, 500 vig. of streptomycin and 100 IU
of mycostatin per ml. The stool suspensions
were shaken in centrifuge tubes containing glass
beads and were then centrifuged in a PR 1
angle centrifuge at 2,500 rpm. at 50 C. for 20
minutes. The supernatant fluid was centrifuged
once more in a Spinco Model L preparative
centrifuge at 18,000 rpm. for 15 minutes. The
supernatant from the second centrifugation was
used for inoculation of three HeLa cell-culture
tubes, each tube receiving 0.5 ml.
The tubes were inspected microscopically
every third day for cytopathic changes. As soon
as such changes occurred second passages were
made by inoculating 0.2 ml. of tissue-culture
fluid into each of three HeLa cell tubes. Agents
from the second passage were typed with specific
polio immune sera. A pool of the three type sera
was included to each test. The sera used were
hyperimmune rabbit or guinea pig sera.
Serum neutralization tests. Neutralization
tests at the Department of Virology, University
of Helsinki, were done as tube tests based on
inhibition of the cytopathic effect, using about
100 TCD,, of virus and fourfold serum dilutions
beginning with a dilution of 1:4. Corresponding
tests at the Lederle Laboratories were
done by using the color tests.* Serum titers are
expressed as the inverse value of the last neutralizing
serum dilution.
Virulence tests in monkeys. Ten percent
stool suspensions were made in distilled water
and then shaken in the cold for about 12 hours.
Centrifugation at 5,000 rpm. for one hour followed.
To the supernate 10,000 units of penicillin
and 10 mg. of streptomycin per ml. were
added. Further centrifugation for two hours at
12,000-14,000 rpm. was then carried out and the
pH of the supernate adjusted to seven.
Each stool preparation was inoculated into
groups of two to three Cynomolgus monkeys,
each animal receiving 2 X 0.5 ml. injections
intrathalamically. The animals were observed
for 21 days for clinical signs of the disease, at
which time they were sacrificed for histologic
examination of brain and spinal cord specimens.
RESULTS
Immunity status of the population before the
trial. Result of determinations of neutralizing
antibodies of the Sottunga population against
all three types of polioviruses prior to the trial
(Table 2), shows that most of the children under
10 years of age were triple negative as estimated
by the method used. Also some of the adults
were triple negative and the poliovirus antibody
pattern corresponded to that in the whole archipelago
before the great 1953 polio epidemic in
this region, and is thus one of low infection rate.
Accordingly, Sottunga island must have escaped
the infection in 1953 and apparently, there has
been no polio on the island during the last 10
years.
Presence of poliovirus in the community at
the start of the trial. Since HeLa cells were
used for the isolations, probably only polioviruses,
Coxsackie B viruses, and a few Coxsackie
* Cox, H. R., Cabasso, V. J., Markham, F. S.,
Moses, M. J., Moyer, A. W., Roca-García, M., and
Ruegsegger, J. M. "Immunological Response to
Trivalent Oral Poliomyelitis Vaccine." Brit. M. J.,
2:591-597, 3 October 1959.