Prime-boost immunization protocols using different expression systems have proven to be successful tools in protecting experimental animals against various important human diseases [1] including tuberculosis [2], AIDS [3], and hepatitis C [4]. The success of such vaccination schemes depends upon the efficiency of the expression systems, of which, recombinant vaccinia virus(VV) and bacterial plasmid vectors are among the more common systems studied [6–8]. Moreover, it has been shown that when at least one component in a prime-boost vaccination scheme includes a plasmid vector, there is a strong response from both pathways of the host immune system [8]. There is no unique mechanism to account for the efficiency of prime-boost vaccination protocols, but it is well known that synergy of priming and boosting in the host using different expression vectors evokes high-avidity CD4 and CD8 cells [5]. Consequently, several such vaccination protocols are now in human clinical trials [1, 6]. Here we present the results of experiments employing the sequential use of recombinant VV and a bacterial plasmid, both expressing non-structural protein, NS1, of tick borne encephalitis (TBE) virus, in protecting mice against lethal virus infection. It has previously been shown that both expression systems independently can partially protect mice against lethal challenge with the highly pathogenic TBE virus strain but only after repeated revaccinations [9, 10]. Despite the efficiency of commercially available whole-virion TBE vaccines [11], concerns have been expressed about the development of non-neutralizing anti-envelope antibodies that could enhance the infection [12] if vaccination protocols are not followed, as well as if individuals are subclinically infected with other flaviviruses or immune compromised. The use of another protective TBE virus antigen, such as the NS1 non-structural protein, presented by different expression systems may help to address these concerns in some circumstances [9, 10, 13].

Detection of anti-NS1 and anti-E (envelope protein) antibodies in vaccinated and challenged mice was carried out by radioimmuno precipitation analysis (RIPA). The analysis did not detect anti-NS1 antibodies in the blood of mice after primary vaccination with the recombinant vaccinia virus (W-NS1) (Fig. 1, lanes 5 and 6) but they were detected after booster vaccination with the recombinant plasmid pMV45 (Fig. 1, lanes 7 and 8). Anti-NS1 antibodies were also detected in vaccinated animals after subsequent challenge with the TBE virus (Fig. 2, lanes 7 and 8). However, sera from surviving mice did not contain antibodies against TBE virus E protein, suggesting that pronounced virus infection was not present in vaccinated and challenged animals (Fig. 2, lanes 7 and 8). In separate experiments, neither the W-NS1 recombinant nor the pMV45 plasmid, introduced alone, induced detectable amount of anti-NS1 antibodies in mice (data not shown).)Antibodies against NS1 protein could protect mice against the lethal challenge with homologous flaviviruses including TBE virus [15, 16]. Involvement of Th₁ immune response in protection elicited by the combined vaccination with two recombinant systems used in this study can not be excluded.

These studies are the first to demonstrate that the combined use of well-characterised expression systems employing recombinant vaccinia viruses and bacterial plasmids (DNA vaccination) can protect experimental animals against a lethal flavivirus infection. Both these expression systems could be rapidly deployed to provide simple methods of constructing vaccines against several dangerous emerging diseases that have caused severe threats to public health in many different countries during the past few years.

In our experiments we used the poxvirus recombinant based on the WR laboratory strain which replicates better in cell cultures and mice than other strains of VV. Although more convenient for our experiments, this strain could not be used directly for vaccination of humans due to its relatively high virulence. However, it can easily be used to prepare the analogous recombinants on the basis of vaccinal VV strains (Lister, Copenhagen, Praha and others) by inserting the NS1 gene into their tk gene. These strains have been broadly used for immunization against smallpox in the past.

Although vaccination against smallpox was stopped many years ago, it could be reinstated if a bioterrorist attack is thought to be imminent. Indeed, the USA, UK and several other European countries have stockpiled traditional vaccines as a precaution against such an event. However, it could be argued that it is more sensible to vaccinate humans, not with the traditional vaccinal VV strains, but with their recombinants carrying genes encoding protective antigens from other pathogens, inserted into the VV thymidine kinase (tk) gene [17]. Despite the fact that VV tk^- recombinants have never been used for vaccinations against smallpox in the past, there are many reasons that they could protect against the disease as with the original VV strains: 1) they have the same antigenic structure; 2) a tk^- recombinant constructed on the basis of the Lister strain that expressed hepatitis B virus HBsAg antigen [18] could efficiently replicate in calf skin, which enabled the production of dermal recombinant vaccine against hepatitis B and to conduct human trials [19]; 3) this recombinant induced a specific reaction in rabbit, calf and human skin and evoked neutralizing antibodies against orthopoxviruses [19, 20]. Such recombinants would have substantial advantages over traditional VV. Not only would they possess reduced neurovirulence due to destruction of the tk gene [18, 21, 22] (residual neurovirulence of VV is a main cause of severe postvaccinal complications), but they would also be able to offer protection against smallpox and another pathogen. For instance, VV recombinants expressing protective antigens of TBE virus, including NS1 protein, could be used in TBE endemic regions. In this case, prime-boost vaccination protocols, which include recombinant bacterial plasmids as a booster, could provide protection against TBE without raising concerns that pre-existing immunity to vaccinia virus could reduce the immune response to TBE viral proteins. Such strategies could also help to cut the cost of the vaccination procedure, thus making them attractive public health interventions.

A combined prime-boost vaccination protocol, including a VV recombinant and a bacterial plasmid, both containing the NS1 non-structural TBE virus protein gene, gave high levels of protection in mice against lethal challenge with the highly pathogenic TBE virus strain. This prime-boost vaccination protocol could be employed in the development of novel flavivirus vaccines for use in TBE endemic areas and in situations where a reintroduction of smallpox may occur.