Founded in 1975 as the all Union Research Institute of Applied Enzimology, currently,  the Institute of Biotechnology is mainly involved in research and training in the fields of biotechnology and molecular biology, including research and development of recombinant biomedical proteins, genetic and molecular studies of restriction modification phenomenon, developing of viruses diagnostics, epigenetic study of small RNA, drug design and synthesis, bioinformatics.

Hamster polyomavirus-derived virus-like particles particles

Spausdinti

Milda Žilinskaitė, Aistė Bulavaitė, Jonas Dabrišius, Rasa Petraitytė, Alma Gedvilaitė, Raimundas Ražanskas, Agnė Petuškaitė

The use of virus-like particles (VLPs) for vaccine development was stimulated by the successful introduction of the first human recombinant HBV vaccine based on yeast-expressed HBsAg-derived VLPs. VLPs can be generated by heterologous expression of viral capsid and envelope proteins and their subsequent spontaneous self-assembly in vivo or in vitro. VLPs mimick infectious viruses in their structural and immunological features but are non-infectious and highly safe because of the lack of a viral genome. Due to the repetitive antigenic structure, VLPs are highly immunogenic making them promising vaccine candidates against different pathogens.

The hamster polyomavirus (HaPyV) major capsid protein VP1 belongs to a family of polyomavirus VP1 proteins containing highly conserved structure motifs and functional domains. HaPyV VP1-derived VLPs offer a large panel of advantages making them a promising platform for vaccine development. These VLPs have been used as carriers for a variety of different foreign peptides, protein segments and entire proteins of different origin including virus- and cancer associated. A major reason for generation and using of chimeric VLPs is to transfer the intrinsic strong immunogenicity of the VLP carrier to per se low immunogenic peptide sequences. This was evidenced for a MUC1 peptide presented on HaPyV-derived VLPs; whereas the MUC1 peptide complexed to BSA did not induce a specific antibody response, chimeric HaPyV-VP1 VLPs harboring two copies of the peptide induce MUC1-specific antibodies (Zvirbliene et al., 2006a, b).

The localization of potential insertion sites for foreign peptide sequences is essential for the generation of chimeric VLPs as well as evaluation of foreign peptide size limits that can be inserted. In fact, fusion of longer or multiple copies of peptides/epitopes to VLPs usually introduces structural difficulties in protein folding and VLP formation. Comparative studies using defined model epitopes inserted into different positions of the HaPyV VP1-derived VLP carrier have demonstrated that the insertion site in the carrier determines the surface exposure, antigenicity and immunogenicity of the inserted foreign epitope on VLPs (Gedvilaite et al., 2006a; Aleksaite & Gedvilaite, 2006; Lawatscheck et al., 2007). These investigations demonstrated that insertion site #1 between aa positions 80 – 89) and #4 (between aa positions 288 – 295) of HaPyV-VP1 are superior in terms of insertion capacity and induction of a B-cell immunity. The large insertion capacity of site #4 was confirmed by the generation of chimeric VLPs with an eGFP insertion (Gedvilaite et al., 2006a). We studied also the possibility to use for presentation of foreign protein segments the simultaneous insertion of the same or different foreign peptides into the same VP1 molecule (Lawatscheck et al., 2007; Aleksaite & Gedvilaite, 2006). A limitation of the insertion capacity of HaPyV-VP1 was observed for VP1 fusion proteins harboring foreign insertions at four different sites (Lawatscheck et al., 2007). The potential influence of a flexible linker insertion on both sides of foreign nonamer peptides (few CTL epitopes of human tumor-associated antigens) on the assembly of VLPs was also studied (Lawatscheck et al., 2007; Aleksaite & Gedvilaite, 2006). The insertion of a flexible GSSG linker seems to improve the immunogenicity of a HaPyV-VP1 harboring two insertions of a CEA epitope, but not of the corresponding VLPs harboring a single insert. Chimeric HaPyV-derived VLPs were demonstrated to be highly immunogenic, even when applied without adjuvant, especially chimeric HaPyV-VLPs harbouring a CEA epitope at insertion site #1 which induced a long lasting B-cell immunity in mice (Lawatscheck et al., 2007).

In addition to the humoral immunity, we have demonstrated that HaPyV VP1 VLPs were able to induce a T-cell immune response. Yeast-expressed VP1-VLPs originating from rodent, primate and human polyomaviruses were tested for their interaction with human monocyte-derived dendritic cells and murine spleen cell-derived dendritic cells and the induction of an in vitro T-cell response. HaPyV- and MPyV-derived VLPs were found to induce maturation of the dendritic cells as evidenced by increased levels of surface maturation markers and a reduced uptake of FITC dextran and Lucifer Yellow. Moreover, the dendritic cells stimulated with these VLPs produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro (Gedvilaite et al., 2006a; Samonskyte et al., 2006).

A major drawback for the application of chimeric VLPs as vaccines in humans may represent a pre-existing immunity directed against the VLP carrier as the majority of the human population world-wide is persistently infected by the human polyomaviruses JCPyV and BKPyV. Few different strategies may solve the problems associated with a carrier-specific pre-existing immunity. We observed that the insertion of larger foreign protein segments into the VLP carrier demonstrated the reduction the intrinsic antigenicity and immunogenicity of the carrier itself. The level of reduction is related to the size of the foreign insertion; i.e. the larger the insert the lower the remaining VP1 antigenicity and immunogenicity. The generation of hantavirus nucleocapsid protein-specific mAbs by alternating immunization of mice with HaPyV-VP1 VLPs harboring a 120 aa-long segment of the N protein at sites #1 or #4, may underline the possibility to overcome the potential inhibitory effect of a pre-existing carrier-specific immunity on the antibody response against the foreign insertion (Zvirbliene et al., 2006). On the other hand, deletion mutagenesis analysis on the carboxy-terminal region of HaPyV-VP1 suggested that partial deletion within or truncation of this immunodominant region may also be a way to solve problems associated with the pre-existing immunity (Gedvilaite et al., 2006b).

EM of virus-like particles (VLPs)

An important basic research application of HaPyV VP1-derived VLPs was an investigation on the structure and assembly processes. VLPs were used to identify protein domains that are essential for VLP formation and responsible for shape and size determination using deletion mutagenesis approaches for HaPyV VP1 protein. Yeast-expressed truncation variants of HaPyV-VP1 lacking 35, 45 and 56 carboxy-terminal amino acid residues failed to form VLPs but those lacking 21, 69 and 79 amino acid (aa) residues at its carboxy-terminal region efficiently formed VLPs similar to those formed by the unmodified VP1 (diameter 40-45 nm). HaPyV-VP1 mutants with a single A336G aa exchange or internal deletions of aa 335 to aa 346 and aa 335 to aa 363 resulted in the formation of VLPs of a smaller size (diameter 20nm) (Gedvilaite et al., 2006b). Insertional mutagenesis using defined foreign insertions of different size and origin also allowed to confirm the structural flexibility of surface-exposed regions in VP1 (Zvirbliene et al., 2006).

Very successful was employment of HaPyV-VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. The immunizations of mice were performed in Laboratory of Immunology and the results of this study are presented there.

In conclusion, HaPyV VP1-derived VLPs were useful tools for basic research applications, i.e. in structural and assembly studies and immunological investigations. HaPyV VP1-derived VLPs harboring foreign peptides or proteins may represent promising vaccines not only for viral infections but also for cancer and auto-immune diseases and are successfully employed for hybridoma technology.