III. ABSTRACTS AND POSTERS / ORAL PRESENTATIONS
6. Mice deficient in interferon regulatory factor 4 (IRF4) are more susceptible to infection with mouse-adapted influenza A/Aichi/2/68
2.1.3.1. DNA vaccines against respiratory viruses and use of integrin to enhance its efficacy
DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches (Kim and Jacob 2009). Its production speed, simplicity, ability to elicit humoral as well as cellular immune responses against native antigenic proteins without the need for live vectors, makes this an attractive vaccination technique. A vast delivery methods exist which includes needle injection, fluid jet injection, injection followed by electroporation, bombardment with gold-particles coated-DNA, and topical administration to various mucosal sits such as the guy, respiratory tract, skin and eye. The most commonly used plasmid DNA vaccine consists of an origin of replication, a bacterial antibiotic resistance gene acting as a selectable marker, a promoter such as cytomegalovirus promoter or simian virus 40 promoter that is active in eukaryotic cells and RNA transcripts stabilized by polyadenylation signal sequences (van Drunen Littel-van den Hurk, Gerdts et al. 2000).
DNA-based vaccines have been shown to induce significant immune responses against several viral agents, including human immunodeficiency virus (Wang, Ugen et al. 1993), bovine herpesvirus (Cox, Zamb et al. 1993), hepatitis B virus (Davis, Michel et al. 1993), influenza virus (Fynan, Webster et al. 1993), rabies virus (Xiang, Spitalnik et al. 1994) and hepatitis virus (Lagging, Meyer et al. 1995).
Gene gun-delivered DNA initiates responses by transfected or antigen-bearing epidermal Langerhans cells that move in the lymph from the bombarded skin to the draining lymph nodes (Arnon 2011). The intramuscular injection is a more common
19 administered route for DNA vaccines. Following immunization, cells transfected with the plasmid DNA encode the protein of interest. This protein is then processed and presented to the immune system in the context of MHC Class I and/or Class II to activate antigen-specific CD8+ and CD4+ cells. Direct priming by somatic cells such as myocytes and keratinocytes provide a possible mechanism for this process.
Alternatively after intramuscular injection, functional DNA appears to move as free DNA which then enters the bloodstream to reach the spleen (Robinson and Torres 1997). The direct transfection of professional APCs such as DCs and cross-priming where secreted protein while taken up by professional APCS are presented to T cells through the MHC Class I-dependent pathways. Muscles cells have been shown to be critical in the protein expression and cellular immunity initiation of direct priming (Wolff, Malone et al. 1990). Factors as such cell-associated or secreted DNA- expressed antigens, DNA inoculation and delivery route (Arnon 2011) determine the nature of immune response. CD4+ T-helper cells involvement would encompasses either a type I or type 2 response where the former would promote a cellular mediated response involving cytotoxic CD8+ T cells whereas the latter would promote a humoral immune response involving B-cells and antibody production. Despite the absence of costimulatory molecules, muscle cells are able to initiate cellular immunity by secreting proteins that are taken by DCs to cross-prime CD8+ T cells at the DNA immunization site (Kim and Jacob 2009).
DNA vaccines offer the unique advantage over conventional protein-based vaccines/killed vaccines, in that the DNA is non-infectious and non-replicating.
Unlike live attenuated vaccines, DNA vaccines only encode the protein of interest, not viral antigens. This would reduce unwanted side effects but still enabling the use of multiple vaccinations to be administered to individuals without provoking an
20 immune-dampening vector-specific response. Unlike conventional vaccines employing either killed virus or purified antigens, DNA vaccination efficiently elicits cellular immune responses including cytotoxic T-lymphocyte (CTL) responses in addition to humoral immunity (Poh, Narasaraju et al. 2009).
Most protein-based and killed vaccines do promote a good humoral immune response but fail to induce a significant CMI response, as these are cleared up by professional APC, processed through the MHC Class II pathway and presented to CD4+ T cells, which in turns helps in the production of high-affinity antibodies by B cells. Live attenuated vaccines are able to induce both CMI and humoral responses.
There are concerns that some live vaccines may be associated with virus shedding and genetic mutation, causing the reversion to a wild-type phenotype (Cinatl, Michaelis et al. 2007).
However, DNA vaccines are not without shortcomings. The mechanisms by which DNA vaccines generate immune responses are complex. For intramuscular delivery of DNA, the majority of plasmids are thought to transfect muscle cells, which are poorly or only partially effective at presenting antigen and priming nạve immune cells (Nagaraju 2001). Instead, these cells are thought to produce antigen, which then transfer the antigen in some form to professional antigen-presenting cells (APCs) via a mechanism of cross-presentation such that MHC class I-restricted cytolytic T-cell responses can be elicited. In addition, plasmid DNA appears not to be simply an inert vector for delivering the gene (Liu, Wahren et al. 2006, Ulmer, Wahren et al. 2006). There is a possibility of integration of the DNA vaccine into the host genome, resulting in malignancy (Kim and Jacob 2009). Plasmid DNA encoding a small amount of protein may also induce autoimmunity as well as cause tolerance
21 rather than immunity due to the persistent production of this small amount of foreign antigen.
Among the approaches used to improve DNA vaccine efficacy includes the use of better promoter/enhancer (Harms and Splitter 1995, Garg, Oran et al. 2004), increasing the proteins availability in the cytosol, coadministration of immunomodulatory cytokines (Chow, Huang et al. 1997, Sailaja, Husain et al. 2003), optimizing vaccine administration and delivery (Babiuk, Baca-Estrada et al. 2002, Sharpe, Lynch et al. 2007), protein boosting following DNA vaccination (Epstein, Kong et al. 2005) (Richmond, Lu et al. 1998), use of adjuvants (Ozaki, Yauchi et al.
2005), direct targeting of DNA vaccines to APCs (Deliyannis, Boyle et al. 2000, Lew, Brady et al. 2000) and vectors encoding antigens fused to molecules that facilitate antigen spread and cross-priming (Ross, Xu et al. 2000, Hung, Cheng et al. 2001).
Targeting moieties have been explored as a means to enhance DNA vaccination, including various ligands such as transferin, antibody fragments, sugars, insulin and folate (Harbottle, Cooper et al. 1998).
Integrins are a class of related heterodimeric transmembrane surface receptors involved in cell-cell adhesion, and in the promotion of interactions between cells and components of the extracellular matrix glycoproteins (e.g. fibronectin and vitronectin), while their intracellular domains interact with the cytoskeleton. Integrin receptors mediate adhesive events that are critical for specific and effective immune responses to foreign pathogens. Integrin-dependent interactions of lymphocytes and APCs to endothelium regulate the efficiency and specificity of trafficking into secondary lymphoid organs and peripheral tissues. Within these sites, integrins function to facilitate cell movement through interactions with the extracellular matrix, and to promote and stabilize antigen-specific interactions between T-lymphocytes and APCs
22 that are critical for initiating T-cell activation events (Pribila, Quale et al. 2004). The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the Arg-Gly-Asp (RGD) motif. Such peptides promote cell adhesion when insolubilized onto a surface, but inhibit adhesion when presented to cells in solution (Ruoslahti 1996).