LIVE POLIO IRUS VACCINES

Laboratory Investigations of Attenuated Vaccine Strains
125
sons fed trivalent Lederle vaccine were incubated
for 1 hour at 37 ° C. with divalent rabbit, antipoliovirus
serum (50 MIT units of each antibody
per 0.1 ml.) so that single poliovirus types could
be studied.
Viral titrations were performed in Rhesus kidney-cell
culture tubes containing Earle's balanced
salt solution (0.22 per cent sodium bicarbonate)
and 0.5 per cent lactalbumin hydrolysate. Plaque
assays were done in 2 oz. bottles using the same
medium containing 20 per cent skim milk. 4
Temperature (T) characteristics were determined
by incubating half the inoculated culture
tubes or plaque bottles in a 36.0° - 0.3 incubator
and half at an incubator temperature of
39.5 ° C. or 40.0 ° C. A final reading was done
on the sixth or seventh day. Tests included the
appropriate controls with seed virus strain as
well as the virulent Mahoney Type 1 strain.
T character is expressed as log,, titer at 360 C.
minus log,, titer at 39.5 ° C. or 40 ° C. Plaque
diameter T character is a ratio of the diameter
at 39.5 ° C. over diameter at 36 ° C. expressed
as per cent.
Neurovirulence of virus preparations for monkeys
was determined by inoculating amounts of
virus calculated to be at the end-point of infectivity
for the homotypic seed virus. Three Rhesus
monkeys were inoculated by each route, intrathalamically
(I TH), intraspinally (IS), and intramuscularly
(IM)-a total of nine monkeysobserved
for paralysis, sacrificed 14 to 21 days
after inoculation and portions of the CNS were
examined histopathologically by methods previously
described. 2 ' 3 Monkey neurovirulence results
are expressed as the maximum plaqueforming
units (PFU) of virus causing no lesions
(threshold) .
RESULTS
Comparison of T Character and Monkey Neurovirulence.
The T character may be measured by
several methods, including monkey-kidney cell
culture tube titration, plaque count assay and
the measurement of plaque diameter as determined
at 36 ° C. and 39.5 ° C. To determine
which of these techniques best correlates with
neurovirulence for monkeys, a comparison was
made with the results of monkey studies previously
reported from this laboratory. 2 ' In the
results presented in Fig. 1 each T characteristic
is the average of three separate determinations.
A shorter line for T character indicates increased
ability to grow at 39.5 ° C. and a shorter line for
monkey neurovirulence indicates increased virulence.
No determination is indicated by absence
of a line. The plaque count determination of
T character of Types 1 and 2 appears to be correlated
better with monkey neurovirulence following
CNS inoculation than do the tube titration
or plaque diameter methods. This correlation is
especially striking with the Lederle Type 2 strain
which shows higher neurovirulence following
CNS inoculation; however, this strain shows a
tube titration T characteristic suggestive of low
virulence, whereas the plaque count T character
indicates greater virulence. There was poor correlation
between all T determinations and monkey
virulence in the case of each Type 3 strain.
In addition, correlation is lacking between the
T characteristic and neurovirulence following
IM inoculation. The latter observations are consistent
with other data indicating that neurovirulence
following IM inoculation appears to
measure different properties than does direct
central nervous system (CNS) inoculation. 5 '
During the course of this study it was noted
(Fig. 2) that there was a considerable differential
of virus growth when temperature of incubation
was raised from 39.5 ° C. to 40 ° C. - 0.3.
While the indicated temperature difference was
0.5 ° C., we could not exclude a somewhat greater
difference due to a temperature variation of
-0.3 ° C. within the incubators and due to a
variation of --0.2 ° C. between thermometers. The
higher temperature permitted better differentiation
of virus strains but the overall ranking of
virulence by T character remained the same.
In addition the ranking by T character of the
Type 3 strains appeared better correlated with
neurovirulence for monkeys following CNS inoculation
when the higher temperature of incubation
was 40 ° C. rather than 39.5 ° C.
As a result of these experiments, plaque count
assay was adopted as a reasonable screening test
for the detection of changes in neurovirulence
of monkeys inoculated I TH or IS.
Effect of Laboratory Manipulation on the T
Character and Monkey Neurovirulence. Alteration
of the T character and monkey neurovirulence
has been reported to be associated with
laboratory manipulation of attenuated viruses.?