Generation of monospecific antibodies for identification and characterization of viral gene products of ILTV and KHV
Unlike several pathogens of humans and other mammals most herpesviruses of birds, reptiles, amphibians and fish are poorly characterized beyond genome sequences. This also applies to economically relevant pathogens like infectious laryngotracheitis virus of chickens (ILTV), or koi herpesvirus (KHV), which causes mass mortality of kois and common carps. The investigation of expression kinetics of viral proteins, their localization in infected cells and virus particles, as well as detection of post-translational modifications and protein interactions can contribute substantially to deeper insight in the viral replication cycle, and to elucidation of the mechanisms of pathogenesis. Since this requires antibodies against defined viral gene products, predicted protein encoding open reading frames identified in the genome sequences of ILTV and KHV are cloned in bacterial or eukaryotic expression vectors and expressed in transformed cells. The purified expression products are used for immunization of rabbits or mice to obtain polyclonal, but monospecific antisera, or monoclonal antibodies. Using such antibodies we have been able to detect more than 20 viral gene products of ILTV, and 4 KHV proteins for the first time, many of them representing glycoproteins of the viral envelope (Fuchs et al., 2007; Rosenkranz et al., 2008). Since immunogenic envelope proteins are major herpesvirus antigens, our sera and antibodies are also suitable for virus diagnostics, and the generated expression constructs might be useful for development of recombinant vaccines.
Generation of attenuated gene deletion mutants of ILTV and KHV as potential live virus vaccines
Attenuated live virus vaccines have frequently proven to be the most efficacious and economic instruments for prophylaxis of herpesvirus infections in farm animals. Such live vaccines have been also prepared against infectious laryngotracheitis virus (ILTV) and koi herpesvirus (KHV) by serial in vitro passages and/or irradiation of pathogenic viruses, and are used in practice. However, many of these conventional vaccines possess considerable residual virulence. Furthermore, reversion to an even more virulent phenotype cannot be excluded, since the viruses are genetically not characterized, and the molecular reasons for attenuation are unknown. Therefore, we generate ILTV- and KHV-mutants possessing irreversible gene deletions by homologous recombination in cells cotransfected with infectious herpesvirus DNA and plasmids containing modified subgenomic fragments. To permit efficient production of the putative vaccines, genes are chosen for deletion which are dispensable for replication cell culture, but have proven to be virulence factors in other herpersviruses, e.g. the viral homologues of ubiquitous genes encoding enzymes of nucleotide metabolism like thymidine kinase (TK) or dUTPase. After in vitro characterization of the obtained virus mutants virulence in their natural hosts, chickens or carps, is evaluated in cooperation with the laboratories for recombinant viral vaccines (Dr. Veits), for viral fish diseases (Dr. Fichtner), and for pathology and bacteriology (Prof. Dr. Teifke). To test, whether the attenuated virus mutants confer protective immunity, animals surviving primary infection are challenged with high doses of pathogenic ILT or KHV. In all experiments the parental virus strains and rescuants of the investigated mutants are used as controls to verify that the observed attenuation has been indeed caused by the introduced gene deletions, and not by additional, accidental mutations. In this manner we have hitherto identified 6 ILTV gene products (pUL0, pUL47, dUTPase, gC, gG, gJ), whose deletion leads significant reduction of virulence without affecting the protective efficacy of live vaccination (Fuchs et al., 2007, Pavlova et al., 2010). Two of these gene products are highly antigenic envelope glycoproteins (gC, gJ) which induce specific antibodies in wild type ILTV-infected chickens. Thus, the corresponding deletion mutants should be suitable as marker vaccines, which permit serological differentiation of vaccinated from naturally infected animals (DIVA strategy). First promising results have been also obtained with respect to recombinant KHV vaccines.
Utilization of attenuated ILTV- and PrV-mutants as vectors for expression of immunogenic proteins of other animal pathogens (e.g. influenza viruses)
Attenuated live virus vaccines are in use for economic and efficacious prevention of infectious laryngotracheitis of chickens caused by ILTV as well as of Aujeszky’s disease of swine caused by pseudorabies virus (PrV). The attenuated viruses might be also used as vectors for immunogenic proteins of other pathogens, and applied as polyvalent vaccines. Therefore we attempt to modify licensed ILTV vaccine strains, or viruses which had been attenuated by deletion of virulence-determining genes (e. g. dUTPase gene, UL0, gG gene), for versatile insertion of foreign genes into nonessential genome regions. For transgene expression the strong immediate-early promoters of human or murine cytomegalovirus are utilized, since they are active in many cell types and viruses. To achieve sufficient protein expression it was further necessary to insert synthetic introns into the 5’-nontranslated parts of several of the genes. Using these methods, major antigens of different pathogens of poultry, like the fusion protein of Newcastle disease virus“, glycoprotein B of Marek’s disease virus, as well as hemagglutinin (H5, H7) and neuraminidase (N1) of highly pathogenic avian influenza viruses (HPAIV) were already expressed in recombinant ILTV (Fuchs et al., 2007; Pavlova et al., 2009). Animal experiments performed in collaboration with the laboratory for recombinant viral vaccines am IMB (Dr. Veits) revealed that a single ocular immunization with hemagglutinin-expressing ILTV-mutants protected chickens completely against lethal challenge infections with homologous and also with several heterologous HPAIV including recent Asian H5N1 viruses. In contrast to conventional inactivated influenza virus vaccines the H5- or H7-expressing ILTV-mutants further permitted convenient serological differentiation of vaccinated and naturally infected animals based on absence or presence of antibodies against other influenza virus proteins (DIVA strategy).
In a similar manner we are now using an approved PrV vaccine strain (Bartha) as vector for expression of immunogenic proteins of African swine fever, and of hemagglutinin and neuraminidase of porcine influenza viruses (pandemic H1N1 virus). Experimental infection of pigs has to reveal, whether these potential vector vaccines are similarly efficacious like corresponding ILTV recombinants in chickens.
Comparative functional analyses of conserved virus genes in the replication cycle of PrV and HSV-1
Deeper insight in fundamental mechanisms of herpesvirus replication requires unambiguous elucidation of the functions of those genes, which are conserved within the entire virus family. For unique properties of single subfamilies, like the neurotropism of most alphaherpesviruses, subgroup-specific genes might be also relevant. However, in the past distinct functions have been attributed to several structurally conserved genes in different herpesviruses. To verify, whether such contradictory results are evident, or only suggested by different experimental approaches, we have started to construct and to analyze isogenic deletion mutants of typical species of two alphaherpesvirus-genera, the human herpes simplex virus type 1 (HSV-1, genus Simplexvirus), and the animal pathogen pseudorabies virus (PrV, genus Varicellovirus) in parallel. These investigations are facilitated by the possibility to propagate both viruses in the same mammalian cell lines, and to study them in the same rodent animal models. Furthermore, the infectious full-length genomes of different PrV and HSV-1 strains have been cloned as bacterial artificial chromosomes (BAC) permitting fast and precise mutagenesis in E. coli. Comparative investigations of the generated PrV and HSV-1 mutants are performed in collaboration with the laboratories for herpesvirus host cell interactions (Dr. Klupp), for electron microscopy (Dr. Granzow), and for pathology and bacteriology (Prof Dr. Teifke). Besides replication in epithelial cell lines and in primary neuron cultures, virulence is studied in experimentally infected mice, and for evaluation conventional virological and histopathological methods are used, as well as electron microscopy and confocal fluorescence- and video microscopy.
Up to now, the functions of several conserved herpesvirus proteins were investigated in parallel, which are either involved in DNA packaging and capsid maturation in the cell nucleus (pUL25, pUL32), or in tegumentation and envelopment of virus particles in the cytoplasm (pUL37, pUL11, gM). All isogenic deletion mutants of PrV and HSV-1 exhibited similar phenotypes, and in several cases heterologous trans-complementation of PrV mutants by the homologous HSV-1 protein (pUL25, pUL32), or of HSV-1 mutants by the homologous PrV protein (pUL11) could be demonstrated (Fuchs et al., 2009; Kuhn et al., 2008; Leege et al., 2009). At present we focus on comparison of the still controversially discussed mechanisms of virion morphogenesis and intra-axonal transport of PrV und HSV-1 in neurons, during which the roles of the alphaherpesvirus-specific membrane proteins gE and pUS9 are of particular interest.