LIVE POLIO IRUS VACCINES

DISCUSSION
CHAIRMAN ANDERSON: Thank you very much,
Dr. Hodes. The papers which have been presented
are now open for discussion.
Dr. Lépine.
DR. LÉPINE: I was very much interested in
Dr. Hodes' paper because in Paris we have been
carrying out a similar piece of work, the results
of which are about to be published.
More than one year ago, in the purification
of poliovirus, we started using cellulose columns
of the DEAE type, on which the virus was passed
and then eluted with solutions of NaCl of increasing
molarity. Fractions were collected by
an automatic collector. We started by using the
virulent Mahoney strain and we obtained a pattern
in which most of the virus, more than 95
per cent, was eluted in one of the first fractions.
Next, we tried an attenuated strain, the strain
1342 which is included in the French inactivated
vaccine, and to our great surprise we
obtained quite a different pattern of elution of
the strain. Most of it was eluted in the fraction
which we call fraction 50; then we wondered
whether there was a correlation between the
different patterns with attenuation, or whether
it was only a characteristic of a given strain.
It so happened that we had in store the 1342
strain, which, since its isolation from samples,
was kept in the deep freeze at different stages
of attenuation.
Starting from the non-attenuated original
strain, we found that it gave a pattern which is
almost similar to the Mahoney strain. Gradually,
as the different degrees of attenuation
were used, we obtained a shift to the right, that
is, a shift from fraction 1 to fraction 50.
The same was done with the Type 3 strain
which we have and we obtained an almost similar
evolution of the pattern.
In connection with the Type 2 strain, the
work is at present in progress.
Surprising as it may be, we were expecting
to obtain fractions which would give a good
complement fixation, but so far we have failed.
It seems that the complement-fixing fraction is
lost on the column, or is not eluted; in our tests,
the virulence was evaluated by inoculation into
the monkey and the titrations were made using
the tissue culture and CPE technique.
Thus we can characterize a strain by its specific
pattern of elution from the cellulose
column, and there seems to be a great degree of
correlation between the shape of the elution pattern
and the degree of attenuation of the strain.
In the present stage of our research, we think
that such elution patterns are significant markers
which could be used for the selection or the
characterization of strains. As an example of
selection, we have grown in tissue culture the
fraction number 50, which is present only in
small amounts in virulent strains; then we collected
the tissue-culture fluid and passed it again
on the cellulose column. Repeating this three
times, after three passages, we were able to
obtain a substrain which had the same elution
pattern as the attenuated strain obtained after
40 to 50 passages in tissue culture with our
maximal dilution technique. We therefore believe
that the elution pattern technique may be
a help in selecting the attenuated component of
the strain, as well as in characterizing the strain
itself.
PROFESSOR CHUMAKOV (through an interpreter):
My remarks with reference to the problem
of markers, under discussion, concern only
the methodological aspects. They are related to
a recent announcement by our colleagues at the
Institute of Poliomyelitis in Moscow, Drs. V. I.
Agol and M. Ya. Chumakova, on the mechanisms
of reaction of the so-called d marker.
Until now it has been generally accepted that
the ability of cell cultures to fully differentiate
attenuated strains from the virulent strains of
poliovirus is due to the differences in multiplication
of viruses in culture media with variable
concentration of bicarbonate (NaHCO3).
Therefore, not infrequently the d marker is also
referred to as the bicarbonate marker.
However, in 1960, Agol and Chumakova, while
studying fractions of infectious RNA derived
from attenuated and virulent strains of polioviruses,
unexpectedly discovered that the d-
marker can be completely reduplicated if, in-
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