Infections of the respiratory tract and enteric disorders have significant economical impact on poultry production worldwide. Respiratory diseases may result in dramatic losses in parental stocks, layers and grow-out poultry. In the last decades the industrialization of poultry production combined with veterinary prophylactic measures and hygiene has continuously increased. Due to that the control of the classical monocausal diseases has been more successful. With a decrease in monocausal infections new emerging syndromes and factorial diseases of complex or unknown etiology have become a thread to the poultry’s health status.
Turkey Rhinotracheitis (TRT) was first observed in 1978 (BUYS u. DU PREEZ 1980) and was thought to be a syndrome of multicausal etiology for many years. Although the causative agent was later identified as the avian Metapneumovirus (aMPV) (MCDOUGALL u. COOK 1986), TRT still should be considered as a syndrome. The impact of aMPV-infection on turkey’s health is significantly influenced by a variety of additional factors, such as secondary bacterial infections and environmental stress.
The causative agent aMPV may induce immunosuppression. aMPV-induced lesions in the upper respiratory tract affect the mucosal clearance and destroy the integrity of the epithelial layer. These irritations caused by aMPV may facilitate secondary bacterial infections. Infections with aMPV may also reduce vaccination efficacy against other pathogens. Thus, the most effective way to prevent TRT and the losses associated with subsequent disorders is to protect birds against aMPV infection.
Today protection against aMPV infection is achieved by live and inactivated vaccines. But these vaccines often induce only short-lived immunity. Additionally, live vaccines may display residual immunosuppressive and pathogenic effects with its consequences. Live attenuated vaccines also display the potential risk of reversion to virulence. New vaccination strategies need to be explored to overcome problems associated with current aMPV vaccines. New vaccination strategies should combine
the following cardinal characteristics: easy, save and cost effective production and the potential of climate-independent storage and application. Furthermore future aMPV vaccines should not have residual immunosuppressive or pathogenic effects.
They should efficiently induce local protective immunity at the site of viral entry and replication. For this purpose genetically engineered vaccines may be promising candidates.
Some genetically engineered vaccines are already licensed for commercial use, such as a canarypox vector-based vaccine against West Nile Virus infection of horses (RECOMBITEK©, Merial) and a recombinant fowlpox-avian influenza vaccine for poultry (MICKLE et al. 1997). For humans plasmid-DNA vector vaccines already are under evaluation in clinical trials, for instance vaccines against Human Immunodeficiency Virus infection (ROBINSON 2007). For many other human or animal infectious pathogens vaccination strategies based on live viral vector vaccines, live recombinant bacterial vector vaccines, plasmid DNA vector vaccines or recombinant protein vaccines are under experimental development. Although live viral vectors, such as fowlpox virus and turkey herpesvirus, were shown to induce protection (FUCHS et al. 2006), their use in poultry production is problematic due to high prevalence of immunity against the most viral vectors used (SHARMA 1999).
Plasmid DNA vectors that encode for immunogenic proteins of respective pathogens and recombinant proteins were also shown to induce specific protection, but in most studies the vaccines were given parenterally. This route is not efficient for economic application of vaccines in large poultry operations. Additionally, the genetically engineered vaccines are sensitive to enzymatic degradation in vivo. A delivery system is needed that allows mass application of these genetically engineered vaccines. The delivery system should protect these sensitive inocula against degradation in vivo. Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) microparticles (MP) meet these requirements. PLGA-MP are biocompatible microscopic capsules that efficiently protect the encapsulated or bound sensitive plasmid-DNA or recombinant protein vaccines against degradation in vivo. They are suitable for economic mass production and application, and can be administered by all possible routes.
Additionally PLGA-MP were shown to have powerful adjuvans-effects, such as induction of non-specific immune reactions and stimulation of MP-uptake by phagocytic cells.
For the development of new vaccination strategies knowledge about the immunopathogenesis of the pathogen of interest is essential. The importance of humoral and cellular, local and systemic immune reactions during infection and their contribution to protective immunity needs to be known. It has been shown that for aMPV it is important to specifically induce local cellular immunity in the upper respiratory tract at the site of viral entry and propagation.
The goal of this project was to develop a novel vaccination strategy against aMPV-infection in turkeys. The following two objectives were addressed:
1. To understand the immunopathogenesis of aMPV-A and aMPV-B in turkeys with emphasis on local cellular immune reactions.
The pathogenesis of infections with virulent and attenuated vaccine strains and also the pathogenesis of infections with subtypes A and B were compared.
A challenge model for the evaluation of local and systemic, humoral and cellular immune reactions to aMVP infection was established.
Application of this defined challenge model for the testing of the inductive and protective effect of a new vaccination strategy.
2. To develop a prime-boost vaccination strategy against aMPV-infection in turkeys, based on genetically engineered vaccines combined with PLGA-MP.
A plasmid-DNA-vaccine against aMPV was produced by insertion of the gene sequence encoding for the aMPV-A Fusion (F) protein into an eukaryotic expression vector.
F-specific DNA vaccine was evaluated for expression and immunogenicity in vitro and in vivo.
A recombinant aMPV-A F protein was evolved homologue to the plasmid-encoded protein.
The recombinant F protein was characterized in vitro.
Protocols for the preparation of PLGA-MP were established.
MP-based, genetic engineered vaccines were produced by adsorption of the plasmid-DNA to cationic MP and by encapsulation of recombinant F protein into MP.
The MP-based vaccines were tested for safety and efficacy in vitro.
The MP-based vaccines were tested in a DNA vaccine-prime and recombinant F protein-boost regime for safety, efficacy and protective power against challenge with virulent aMPV in vivo.