Several different vaccinia virus-based smallpox vaccines were used in the eradication effort and nearly all were associated with complications, ranging from general side effects such as regional lymphadenopathy, myalgia, fever, nausea, and malaise to more severe complications such as post-vaccinal encephalitis, encephalomyelitis, encephalopathy, myopericarditis, vaccinia necrosum, and eczema vaccinatum. Following smallpox eradication, routine vaccination was discontinued, a move supported by the risk benefit ratio of use of reactogenic vaccines against a disease no longer in existence. However, in 2002 the U.S. and British governments called for the resumption of the manufacture of smallpox vaccine to create national stockpiles as a precautionary measure in the event of a bioterrorist attack using smallpox 32
. Following similar concerns voiced by several other countries, in 2004 the WHO proposed the establishment of a global smallpox vaccine reserve 31
. In response to this need, the manufacture of “new generation” smallpox vaccines commenced. It is hoped that the many improvements in vaccine manufacture and control since the production of early traditional smallpox vaccines will result in the production of safer, less reactogenic, vaccines 33
One of the most serious adverse events following vaccination is post-vaccinal encephalitis. Rates of post-vaccinal encephalitis vary by vaccine strain, ranging from 44.9 cases per million vaccinations using the Bern strain (used in Germany and Austria in the 1950’s and 1960’s) to zero cases using the replication deficient MVA strain 8,18,24,33
National regulatory authorities require neurovirulence safety testing of live vaccines derived from viruses that target the central or peripheral nervous system 17
, although the methods have not been defined. To address this need, we previously developed a prototype assay for vaccinia virus neurovirulence assessment 20
. Here, we have furthered this work, providing data in support of use of the assay as a pre-clinical tool in which the neurovirulence of candidate vaccinia virus strains can be assessed. In our studies, four vaccinia virus strains previously used in smallpox vaccine campaigns were tested: the Copenhagen strain, reportedly associated with 33.3 cases of post-vaccinal encephalitis per million vaccinations; the Lister strain, associated with 26.2 cases of post-vaccinal encephalitis per million vaccinations; and Dryvax® strain, associated with 2.9 cases of post-vaccinal encephalitis per million vaccinations; and the MVA strain, which has not been causally associated with neurology adverse events 18,33
. Our data show that based on an assessment of mouse morbidity and mortality following intracerebral inoculation with 10 or 100 pfu of virus, the rank order of vaccine virus neurovirulence in humans, namely Copenhagen > Lister > Dryvax® > MVA, was identical to the rank order of vaccine virus neurovirulence in our mouse model. Also included in the assessment were the WR and IHD-J laboratory strains 12,20,25,28,29
, which, as expected, were more neurotoxic in mice than any of the vaccine strains. Of the two doses, neurovirulence discrimination among the virus strains appeared slightly better when 10 pfu of virus was used.
Based on virus replication kinetics in mouse brain, speed of replication, not peak titers, appeared to be the key factor responsible for virus strain-specific differences in mortality. Replication speed advantage at early stages of infection may allow the virus to briefly evade the host antiviral response, resulting in more rapid virus spread from the site of injection to other brain regions, resulting in increased virulence. While we saw clear evidence of increased early (day 2 post inoculation) virus spread in mouse brain upon comparing viruses at the more extreme ends of the virulence spectrum (e.g., IHD-J and WR vs. Dryvax), such differences were not as clear for more intermediate comparisons (e.g., Copenhagen vs. Lister or Lister vs. Dryvax), although the data did trend towards intermediate levels of spread by Copenhagen and Lister. In earlier comparative studies of vaccinia virus neurovirulence in intrathalamically inoculated monkeys, vaccinia virus strain-specific LD50
’s correlated with the spread of virus in brain at early time points 22
. Whether or not our hypothesis that speed of replication and early virus dissemination are key factors in neurovirulence remains to be proven. Nonetheless, for the purpose of developing and using a pre-clinical neurovirulence safety assay, the use of virus growth kinetics in the brain or virus spread in the brain do not appear to be practical endpoints.
In conclusion, our data support the use of mice to assess the human neurovirulence potential of vaccinia virus-based smallpox vaccines. We propose for this assay that mortality be assessed following intracerebral inoculation of 3-day old CD-1 mice with 10 to 100 pfu of virus. The neurovirulence of a candidate vaccine strain should be assessed based on its performance in the assay relative to a “low” virulence replication competent reference virus, such as Dryvax®, using an appropriate confidence interval for the comparison, e.g., in a “no worse than” scenario. A “high” virulence replication competent reference virus, such as WR or IHD-J should be used as a positive assay control. Validation of this assay by independent laboratories would provide the basis for use of this model to support the development, licensure and use of safer new-generation vaccinia virus-based smallpox vaccines.