KVÄLLSSYMPOSIUM 2008 Vaccinering av hund och katt

elated to the PM and Flury strains of rabies viruses that
are used in the production of human rabies vaccines
(Figs. 1 and 2). These differences are in some instances
located in areas that are in close enough proximity to the
antigenic sites (and also in the transmembrane region) to
potentially affect the conformation of the antigenic sites.
It is possible that the differences between the strains of
seed virus used in the production of rabies vaccines are
enough to cause slight conformational changes in the
antigen-binding site of the antibodies that are induced
after vaccination. These slight differences in the antigenbinding
site could cause the antibody to have a higher
affinity for a challenge virus used in an in vitro assay
that more closely resembles the antigen that caused its
production in the first place. The results of our study
indicate that the degree of homology between the strain of
challenge virus used in the RFFIT to measure the immune
response after vaccination and the strain of seed
virus used to produce the vaccine that subjects received
profoundly affects the reported RVNA values. Indeed,
the use of challenge virus strains with equivalent titers in
RFFIT assays resulted in approximately 30% higher
RVNA values in two-thirds of the serum samples we
analyzed when a homologous testing system was used. In
addition, the level of the RVNA titer (high, medium or
low) had no obvious or consistent effect on the percentage
of titer difference reported between the testing systems.
In most cases the choice of the challenge virus strain
used in a rabies virus neutralization assay does not play
a critical role in the evaluation of RVNA titers; for
example, periodic titer evaluations and the determination
of an immune response after post-exposure prophylaxis
where the exact titer level is less important
than the actual detection of neutralizing antibody. In
addition, the strain of rabies virus used in a rabies virus
neutralization assay is unlikely to be a determining
factor in the measurement of RVNA titers in persons
whose pre-exposure series may be from one vaccine
source and subsequent booster(s) from another source.
Similarly, persons who have had a rabies exposure will
have an immune response to the rabies antigens in the
exposure strain and to the vaccine strain confounding
the mix of antibodies produced. For all of the above
mentioned reasons it would provide little benefit to
routinely measure the RVNA response using separate
rabies virus challenge strains. In contrast, the measurement
of the humoral immune response after vaccination
for the specific purpose of evaluating a rabies vaccine
makes the choice of the challenge virus used in a rabies
virus neutralizing assay extremely important. When the
RVNA levels produced against vaccines made from two
different parent strains are compared using an assay that
employs a particular challenge virus strain in the testing
system, the combined effect of the quantity, functionality
and specificity of the respective antibody response
is measured. As demonstrated by this study, if the
S.M. Moore et al. / Biologicals 33 (2005) 269e276
challenge virus used in the assay is more closely related
to one parent virus strain than to the other, the titer
results obtained will be biased toward the homologous
vaccine. Most importantly, the evaluation methods used
to confirm an absence of significant difference between
the immune response produced by two vaccines involve
statistical comparisons of the GMT by the geometric
mean ratio (GMR). The Food and Drug Administration
(FDA) defines bio-equivalence as ‘‘pharmaceutical
equivalents whose rate and extent of absorption are
not statistically different when administered to patients
or subjects at the same molar dose under similar
experimental conditions’’ [13]. In comparing the statistical
evaluation of each vaccine, the confidence intervals
(CI) of the GMR are examined. When the lower limit of
the 95% CI is greater than 50% and the interval
includes 100%, ‘‘non-inferiority’’ is achieved. To determine
the stricter standard of ‘‘bio-equivalence’’, 90%
CI of the GMR must lie within 80e125%. If this
equivalence test is applied for the day 90 results in our
study, the GMTs obtained for PCECV are inferior to
PVRV when serum samples from subjects vaccinated
with PCECV are tested in a heterologous testing system
using the CVS-11 strain of challenge virus. Conversely,
the GMTs obtained for PVRV are inferior to PCECV
when serum samples from subjects vaccinated with
PVRV are tested in a heterologous testing system using
the Flury-LEP strain of challenge virus (Fig. 5). However,
when a homologous testing system is used to test
the serum samples for subjects in each vaccination group,
not only are the two vaccines non-inferior, they are
equivalent.
This report ascertains that the choice of challenge
virus strain used in rabies virus neutralization assays to
evaluate the production of RVNA titers after vaccination
should be taken into consideration when the titer
values will be used for the evaluation of new or existing
vaccines. Clearly, if quantifying the immune response to
the vaccine is the objective, then using a homologous
rabies virus strain in the testing would most appropriately
reflect this goal. Finally, it is important to
remember that modern cell culture rabies vaccines are
highly effective and cross-protection between strains has
been demonstrated [14,15]. The use of a heterologous or
homologous testing system to evaluate the level of
RVNA as a measure of complete ‘protection’ against
rabies infection is incorrect. To date, the level of RVNA
required to be ‘protective’ against infection in humans
is not known for an obvious reason: it is unethical to
conduct challenge experiments in humans to determine
the level of RVNA required for protection. On the
other hand, the use of rabies virus neutralizing antibody
testing systems to measure the immune response to
specific rabies antigens and the response to rabies
vaccines should not only be accurate and precise, but
also meaningful.
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