Efficacy of DNA vaccination by different routes of immunisation in sheep
Introduction
The immune response to a vaccine in an outbred population is influenced by many factors. These include, the type of vaccine used (live attenuated, inactivated, whole killed, subunit or DNA), the dose, the number of (and interval between) vaccinations, the peculiarities of the Ag, the genetic background of the individual and the route of immunisation. The route of immunisation can be a particularly important consideration in any vaccination strategy with different routes capable of inducing immune responses ranging from protective through to ineffective and even disease-exacerbating (Liew et al., 1985). In mice, intramuscular DNA vaccination induces a predominantly Th1 response while gene gun DNA vaccination induces a mainly Th2 response (Oliveira et al., 1999, McCluskie et al., 1999). One way the route of immunisation could influence the outcome of the induced immune response is the interaction between the vaccine and different APC at the site of injection (Shroff et al., 1990). Different routes of immunisation also result in the induction of immune responses by fundamentally different mechanisms. This was emphatically demonstrated in studies examining immune responses following excision of muscle bundles or skin following DNA vaccination (Torres et al., 1997). In this study, excision of muscle bundles shortly after intramuscular DNA injection did not affect the immune response induced, however, the removal of skin vaccinated by gene gun delivery of DNA-coated microparticles within the first 24 h resulted in abrogation of the immune response, highlighting the importance of tissue-resident antigen presenting cells in the immune response.
Gene gun vaccination has shown broad range applicability with demonstrated efficacy in mice (Bennett et al., 1999), livestock animals (Macklin et al., 1999, Braun et al., 1999), primates (McCluskie et al., 1999, Roy et al., 2000) and several other species (Kodihalli et al., 1997, Sundaram et al., 1996) and has the added advantage that only small amounts of DNA are required. However, the structure of skin differs between species resulting in the expression of DNA vaccine antigens in different cell types and at variable levels following gene gun deposition (Hengge et al., 1996), requiring optimisation of gene gun conditions for each species.
The protection afforded by the formalin-inactivated phospholipase D Ag from Corynebacterium pseudotuberculosis forms the basis of the Glanvac™ series of commercial vaccines against caseous lymphadenitis (CLA) in sheep. In its native form this Ag is not suitable as a DNA vaccine as it is toxic to sheep. However, the protective efficacy of genetically inactivated PLD (ΔPLD) protein has been demonstrated (Hodgson et al., 1999). In addition, we have previously shown that the ΔPLD Ag can confer protection when delivered i.m. as a DNA vaccine (Chaplin et al., 1999). This study also demonstrated that expressing ΔPLD as a C-terminal fusion to CTLA-4Ig, for targeting to the B7 complex on APC, increased the efficacy of this vaccine. In this paper we show that the immune response induced and the protection conferred, by this Ag in sheep is highly dependent on the route of immunisation.
The characterisation of immune responses to DNA vaccines in different tissues in mice can serve as a useful guide for the design of comparative vaccination trials in alternative species. However, since responses in rodents are often not predictive of responses in other animal species, the development of effective vaccines usually requires optimisation of the various factors affecting vaccine performance in the target species (McCluskie et al., 1999). This study addresses this aspect of vaccine design in sheep.
Section snippets
DNA constructs
The extracellular domain of bovine (bo) CTLA-4 (Parsons et al., 1996), the hinge, CH2 and CH3 domains of human IgG1 (Boyle et al., 1998) and the mature protein coding sequence from ΔPLD (Tachedjian et al., 1995) were linked, in-frame, to encode boCTLA4-Ig-ΔPLD as a single open reading frame in the plasmid pCI (Promega, Madison, WI, USA) as described previously (Chaplin et al., 1999). The DNA sequence of the insert was verified using an Applied Biosystems automated sequencer. The CTLA-4-Ig-ΔPLD
Optimisation of gene gun delivery of DNA in sheep
A systematic in vivo optimisation of the gene gun delivery pressure for ovine skin was undertaken prior to the vaccine trial. Optimal pressure was selected based on the intensity of EGFP expression within transfected keratinocytes since there appears to be a correlation between the level of Ag expressed from DNA vaccines and the level of immunity induced (Barry and Johnston, 1997). As shown in Fig. 1, EGFP fluorescence was associated with the presence of gold microparticles within cells.
Discussion
While vaccination can induce both effector and memory responses, only effector responses are usually measured. However, for most vaccines the capacity to generate memory is more important than their ability to induce effector responses. Indeed, the potential to rapidly respond to a pathogen with a strong and rapid response in the early stages of an infection will, in many cases, confer protection. The capacity for induction of memory is therefore often a better indicator of vaccine performance
Acknowledgements
This work was supported by the Cooperative Research Centre for Vaccine Technology, Australia. The Australian Research Council supports J-P.Y.S.
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