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Abstract Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines.

Two-fold immunization of Balb/c mice with a vaccinia virus recombinant expressing the NP protein of influenza A/PR8/34 (H1N1) virus under the control of a strong synthetic promoter induced specific antibodies and protected animals against low-dose challenge by mouse-adapted heterosubtypic variants of human A/Aichi2/68 (H3N2) and avian A/Mallard/Pennsylvania/10218/84 (H5N2) influenza virus strains. The surviving immunized animals had lower anti-hemagglutinin antibody titers compared to non-immunized mice. There was no difference in viral titers in lungs of immunized and non-immunized animals that succumbed to the infection. In order to try to increase immune system presentation of NP-protein-derived peptides, and thereby increase their immunogenicity, we constructed another vaccinia-based NP-expressing recombinant containing a rapid proteolysis signal covalently bound to the NP protein. This sequence, derived from the mouse ornithine decarboxylase gene has been shown to increase degradation of various proteins. However, we found that when used as part of a recombinant NP, this signal neither increased its proteolytic degradation, nor was it more efficient in the induction of a protective response against influenza infection.

It was for the first time that complementation between the human and simian adenoviruses in human cells as well as the ability of the human adenovirus Ad2 (HADv2) genome to transform completely into the simian adenovirus SA7(C8) (SADv15) capsid (transcapsidation) was demonstrated. A defective adeno-adeno hybrid (recombinant) between the above viruses is described; the recombinant has the SA7(C8) capsid and Ad2 genome with a 10% insertion of SA7(C8) in the central region. Defective hybrid virions are able to replicate both in human and simian cells by using the SA7(C8) virus as helper. The hybrid virions help the above virus to replicate in human cells: they form a mutually complementing virion pair.

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

Fifteen vaccinia virus (VV) recombinants derived from VV strains Praha, LIVP and DD (i.e. Dryvax Wyeth vaccine-derived) and expressing genes for S, preS2-S or c antigens of hepatitis B virus (HBV) were tested in monkey CV-1 cells and human diploid LEP cells. The production of infectious virus was found to be alike in all the recombinants and parental viruses as well. However, several recombinants produced markedly lesser amounts of S and preS2 antigens in LEP cells than in CV-1 cells. This reduction was independent of the parental virus used. There was, however, a relationship between the production of preS2 in CV-1 cells and the production of S and preS2 antigens in LEP cells; in general, recombinants efficiently inducing preS2 antigen formation in CV-1 cells produced markedly reduced amounts of S and preS2 antigens in LEP cells. Reduction of HBV antigen production in LEP cells was not apparent in recombinants expressing only S or c antigens of HBV, and the production of c antigen by double recombinants was not influenced by simultaneous expression of preS2 and S. The various recombinants also differed in the ratio of S:preS2 antigen formation. This difference seemed to be associated with the length of the untranslated leader sequence preceding preS2 but not with the parental virus or cell type used. The titers of antibodies against S and preS2 antigens induced in mice immunized with different recombinants differed markedly. The differences in the ratio of S:preS2 antigen production in vitro were not reflected in vivo by S:preS2 antibody ratio.

The ability of different recombinant vaccinia viruses (RVV) expressing hepatitis B virus surface antigen (HBsAg) to induce anti-HBs and anti-vaccinia virus responses has been analyzed in mice. The RVVs tested differed with regard to the original vaccinia virus strain used as vector and the site of insertion of a foreign gene. It was found that the immunological responses to RVVs based on the WR strain were higher than those to RVVs based on the Lister strain. The immunological response was also quantitatively affected by the viral TK phenotype. The presence of the preS2 region in HBsAg gene, inserted into the RVV genome, led to the induction of anti-preS2 antibodies but decreased the antibody response to the S-portion of HBsAg.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.