DNA VACCINES: BASIC VECTOR REQUIREMENTS, COMPONENTS THAT INFLUENCE

DNA vaccines are part of next-generation vaccines, an example of the reverse vaccinology approach. The shift from conventional vaccines toward next-generation vaccines ocurred in parallel with advances in bioinformatics. By using the resources available with genome databases, it is now possible to compare and analyse possible ORFs within existing annotated genomes, leading to identification and functional annotation of unknown hypothetical genes. In this manner, possible antigens can be identified based on homology searches with other known antigens, instead of laboriously expressing and analysing each protein from organisms with incomplete and/or unannotated genomes. Thus, reverse vaccinology refers to the development of a vaccine where one starts with identification of antigens in silico, and works backwards toward cultivation in vitro (Rappuoli, 2001).

4.1.2.1. Basic requirements for a DNA vaccine vector

The different components of the DNA vaccine vector will determine the level of expression, and subsequently the immune response elicited. Most plasmids that are used as vaccines have a shared set of characteristics, including: (i) a bacterial origin of replication with a high copy number for high production of plasmid DNA in transformed bacteria; (ii) a selection marker to allow for plasmid selection during bacterial growth; (iii) a strong eukaryotic promoter for expression in mammalian cells; (iv) a cloning site downstream of this promoter for insertion of the gene(s) of interest (GOI); (v) and lastly a polyadenylation sequence to stabilise and direct expression of the mRNA (Garmory et al., 2003; Gurunathan et al., 2000; Webster and Robinson, 1997). Sometimes introns are also included in the vector, since a lot of mammalian gene expression is dependent on, or increased by, the presence of an intron. Multicistronic vectors may be created to express more than one antigen, or to express an antigen together with an immunostimulatory element (Lewis and Babiuk, 1999).

4.1.2.2. Vector components that influence expression

The structure of a vector can be divided into two conceptual parts: the plasmid backbone unit and the transcription complex unit.

4.1.2.2.1. The plasmid backbone unit

The plasmid backbone unit consists of prokaryotic elements such as an origin of replication, multiple cloning site (MCS), a selectable marker, as well as optional immunostimulatory sequences with adjuvant activity. DNA vaccine vectors may contain an origin of replication from viral origin, such as from the Simian virus 40 (SV40) and the polyoma virus. The MCS is designed with unique and convenient restriction sites to allow insertion of the GOI, and designed to avoid hairpin formation in the 5’ end of the mRNA, since this reduces the level of translation in higher eukaryotes (Fu et al., 1991; Kozak, 1986; Kozak, 1989; Rao et al., 1988). The selectable

marker can either be a drug-resistant marker, enabling cells to detoxify an exogenously added toxic substance, or be an auxotrophic marker, enabling cells to synthesise an essential compound not present in the media. Krieg et al. (1994) demonstrated that certain DNA sequences induce cytokine secretion and lymphocyte activation. Certain bacterial species-dependant unmethylated cytidine-phosphate-guanosine (CpG) motifs are particularly immunostimulatory. These sequences were found to activate B cells in vitro and act as an adjuvant in vivo (Donnelly et al.,1997), as well as to activate monocytes, natural killer cells (NKC) and dentritic cells (DC) (van Drunen Littel-van den Hurk et al., 2001). Elimination of the CpG motifs from the plasmid backbone reduced the immunogenicity of the vaccine, an effect that was reversed by co-administering exogenous CpG-containing DNA, although not to the magnitude of including the motifs in the plasmid backbone (Klinman, 1998). Even the selectable marker can have an influence. The human cytomegalovirus (hCMV)-based vectors containing the ampicillin resistance gene (ampR) instead of the kanamycin resistance gene (kanR), produced a stronger immune response (Sato et al., 1996). This was thought to be the effect of a certain immunostimulatory sequence (ISS), a palindromic CpG hexamer 5’ AACGTT 3’, which is present twice in the ampR gene and absent in the kanR gene.

4.1.2.2.2. The transcription complex unit

The transcription complex unit (that drives the synthesis of the GOI) contains a promoter/enhancer region, an intron with functional splicing sites, the GOI, and a polyadenylation signal.

Mammalian expression plasmids mostly carry immediate early promoter/enhancer regions from pathogenic viruses, due to their high transcription initiation ability in mammalian tissues (Harms and Splitter, 1995). The hCMV enhancer/promoter is used most often, due to its induction of strong and consecutive expression in various cell types (Boshart et al., 1985; Schmidt et al., 1990; Thomson et al., 1984). Other promoter/enhancement regions include those from the SV40, the Rous Sarcoma Virus (RSV), the murine leukemia virus SL3-3, the mouse mammary tumour virus (MMTV), and the human immunodeficiency virus (HIV). A study done by Galvin et al. (2000) concluded that the level of humoral and cellular immunity induced by a DNA vaccine is directly correlated to the strength of its promoter.

Intervening sequences (introns) have a positive effect on antigen expression, possibly due to an enhanced rate of RNA polyadenylation, or nuclear transport, that is linked to RNA splicing (Huang and Gorman, 1990). Most gene vaccination vectors contain the intron A of hCMV. Chimeric introns, constructed from different donor and acceptor sites, can be optimised at the branchpoint site to match consensus sequences of splicing ,and lead to an increase in the level of expression (Senapathy et al., 1990).

High levels of gene expression are dependent on efficient termination and polyadenylation of RNA transcripts. Polyadenylation, the addition of 200-250 adenosine residues to the 3’ end of the RNA transcript, contributes to the stability and translation of the transcript (Bernstein and Ross, 1989; Jackson and Standart, 1990).

For DNA vaccine development, the GOI should be inserted into the vaccine vector to have the maximum immune response. Boyle et al. (1998) showed that both cellular and humoral immune responses were enhanced when using an antigen-targeting strategy, by vaccinating with DNA than encodes for an antigen-ligand fusion protein. Another factor to take into consideration is the target of the gene product. Different cellular

compartments will elicit different immune responses, for instance secreted or membrane-bound proteins induce antibodies more effectively than cytosolic antigens. On the other hand, cytotoxic T cell responses are greatly improved by cytosolic degradation and subsequent presentation on the MHC class I (Robinson and Pertmer, 2000).

4.1.2.3. The DNA vaccine vectors pCI-neo, VR1012 and VR1020

In this study the three mammalian expression vectors: pCI-neo, and two Vical vectors, VR1012 and VR1020 were used for DNA vaccine development. All three contain an expression cassette under control of the hCMV-IE enhancer/promoter, an intron, and a polyadenylation signal to regulate expression. To date only VR1020 has been approved by the FDA for human use.

In the pCI-neo vector (Figure 4.1), the chimeric intron is composed of the 5’ donor site of the first intron from the human β-globin gene, as well as the branch and 3’ acceptor site from an intron of an immunoglobulin heavy chain variable region gene (Bothwell et al., 1981). The sequences were modified to match the consensus sequences for splicing (Senapathy et al., 1990). Both the Vical vectors contain intron A from hCMV.

The pCI-neo vector contains the ampR gene, conferring resistance to ampicillin, while the two Vical vectors contain the kanR gene, conferring resistance to kanamycin. This allows for plasmid selection during growth in bacteria. Based on a study by Sato et al. (1996), this difference could cause the pCI-neo vector to produce a stronger immune response than the Vical vectors.

The VR1012 and VR1020 vectors contain a hCMV untranslated region, which contains intron A and a bovine growth hormone terminator element. VR1020 also contains a secretion signal, the human tissue plasminogen activator (tPA) signal peptide sequence. This is situated at the amino terminus of the expressed protein to facilitate secretion of the expressed recombinant protein to the outside of the cell, when the foreign gene is inserted downstream of and in-frame with the tPA signal. Smooker et al. (1999) demonstrated that their VR1020 construct, and not the VR1012 construct, directed expression so that the protein was exported from the cell. They found that when immunising mice with a Fasciola hepatica glutathione-S-transferase, an increased humoral response was achieved when the VR1020 vector was used, if compared to the VR1012 vector.

Figure 4.1 The mammalian expression vector pCI-neo (Promega), as an example to illustrate the important vector

components. In addition, the two restriction sites that were used for insertion of the oppA gene are indicated with striped arrows.