Dr. Nikolaus Osterrieder
This proposal is a continuation and extension of our long-term efforts to analyze the contributions of individual gene products of Equine Herpesvirus Type 1 (EHV-1) that are involved in pathogenesis and are important for immune protection. More specifically, our research has focused on the differences in membrane (glyco)proteins between virulent and avirulent strains, with a special emphasis on glycoproteins that determine host cell or tissue specificity and/or can be secreted from infected cells. Recent research in our and other laboratories has shown that multifunctional herpesviral glycoproteins, which represent (i) constituents of the virus envelope, and (ii) can be secreted from infected cells, are crucially involved in cellular tropism and play important roles in virus replication and pathogenesis. We have concentrated on how the knowledge of EHV-1 virulence factors can be exploited for the rational design and generation of efficacious and safe modified live vaccines that provide long-term immunity. The presented proposal aims at two major goals. Firstly, studies are planned to analyze the function in vitro and in vivo of individual immunomodulatory proteins of more recent EHV-1 strains that represent the neuropathogenic “biotype” of EHV-1. These investigations will be performed using a recent isolate from a premise in New York State (New York 2005, NY05) as a model organism. It is important to note that EHV-1 strains of the neurovirulent biotype still cause respiratory disease and abortions but are also able to induce severe neurological disease. Secondly, we shall investigate the functions of these immunomodulatory, secreted EHV-1 glycoproteins and their effect on the efficacy of modified live virus vaccines (MLV). We have recently shown that MLV vaccines have clear advantages over inactivated vaccines with respect to efficacy; yet, the degree and duration of immune protection induced by MLV vaccines is far from being perfect. Therefore, we will concentrate on rationally modifying attenuated EHV-1 by modifying secreted gene products, which are likely involved in immune modulation, and elucidate the effects of these modifications on the longevity and degree of immune protection against one of the economically most important and devastating viral diseases of the horse.
EHV-1 affects horse populations worldwide. Within the large family of the Herpesviridae it has been classified under the subfamily of the Alphaherpesvirinae . Owing to its biological properties and genomic organization, it forms the genus Varicellovirus together with its close relatives Equine Herpesvirus Type 4 (EHV-4), Varicella zoster virus, Bovine Herpesvirus Type 1 and Suid Herpesvirus Type 1 (Pseudorabies Virus).
EHV-1 targets three organ systems in the natural host: the respiratory tract, the reproductive organs, and the central nervous system (CNS). The affection of these three systems determines the clinical signs observed after EHV-1 infection, which are respiratory distress including nasal discharge, dyspnoea, and coughing, late-time abortions in pregnant mares, and neurological disease. The latter is usually characterized by a paralysis of the hind limbs and the organs that are innervated by the lumbar nerves (e.g., the bladder), but other manifestations of neurological disease resulting in opisthotonus, head pressing, and circling have also been observed.
More recently, the neurological form of the disease has been predominant and caused considerable losses to the equine industry. Large-scale epidemics of EHV-1 paralytic disease have been documented in race, performance, show, or companion horses in Maryland, Kentucky, Michigan, Pennsylvania, Texas, Iowa, Florida and New York (2005), Maryland (2004), Oregon, Ohio, Pennsylvania, Kentucky and Vermont (2003), Virginia (2002), Wyoming (2001), and Montana (2000). Across the recent outbreaks, high neurological attack- and case-fatality rates of approximately 30% and 50%, respectively, were observed. These numbers are alarming, because in the past the neurological form of EHV-1 disease used to be sporadic and restricted to individual animals of an affected herd. The recent reports, however, suggest the emergence of an epidemiological picture that resembles the well known “abortion storms” caused by EHV-1, i.e. that large numbers of animals per affected premise are developing severe clinical disease. For example, in the case of EHV-1 infection at the University riding school in Findlay, OH, more than 90% of the 138 horses became clinically sick, 35% developed neurological disease and 9% (12 horses) died or had to be euthanized. Another striking feature is that recent neurological cases of EHV-1 infection occurred despite regular vaccinations in very short intervals, indicating the possibility of the circulation of EHV-1 variants that are able to break vaccine protection. Alternatively, as suggested primarily by the outbreak at the University of Findlay, extensive use of inactivated vaccines may result in significantly increased susceptibility to the neurological form of the disease upon exposure to the agent.
Generally speaking, the pathogenesis of EHV-1 abortions and neurological disease is still somewhat enigmatic, but it is now generally accepted that CNS affection is probably a consequence of an infection and damage of the endothelia of small blood vessels, which results in inflammation (vasculitis) and thrombosis. The thrombosis in turn causes impaired microcirculation in affected areas, including oxygen deprivation of predominantly the white matter (hypoxia), axonal swelling, and ultimately neuronal death. Abortions and stillbirths can also be caused by the same pathogenic mechanism of vasculitis, thrombus formation, and hypoxia, with a complete or partial detachment of the placenta and malnutrition of the fetus. A matter of considerable controversy and an active area of research is the identification of correlates of virulence of individual strains and to identify cellular and tissue tropisms of various strains. In addition, efforts have been made – so far unsuccessfully – to identify clear correlates of immune protection against EHV-1-induced disease.
Objectives and Experimental Approach.
Our experimental approach will rely on the generation and analysis of mutant EHV-1 that will be engineered from infectious clones of two EHV-1 strains. In the beginning, the project will be focused on examining how two major EHV-1 virulence factors, gp2 and gG, exert their functions. For the planned in vitro studies, we will generate a recombinant NY05 virus in which the capsid is fluorescently labeled. This modification will allow easy identification of infected cells. Based on this mutant virus, we will first insert various forms of gp2 into the highly neurovirulent EHV-1 strain. We were able to show that both expression of full-length gp2 as well as secretion of the protein are necessary for full pathogenicity in a murine infection model. Secondly, we will assess the role of the chemokine-binding factor and membrane protein gG. While EHV-1 gG was shown to bind to various chemokines from a broad spectrum of species, including the mouse, deletion of gG in virulent strain RacL11 did not result in significantly reduced virulence in BALB/c or CBA mice. The properties of the recombinant viruses with respect to cellular tropism will be tested in cultured primary equine cells. Finally, recombinant NY05 viruses harboring various forms of gp2 as well as gG-negative NY05 will be tested in horses for their ability to induce EHV-1 disease. Both clinical parameters as well as nasal virus shedding and viremia levels will be determined by virus co-cultivation and quantified by real-time PCR.
In a related but distinct part of the proposal we shall concentrate on testing vaccine candidates (i.e. selected gp2 and/or gG mutants) in horses. Groups of horses shall be inoculated with mutant gp2 or gG viruses generated from vaccine strain RacH. The rationale of these studies is that we expect to induce a more effective and longer lasting immunity if immunomodulatory molecules (i.e. gp2 and gG) are either modified or absent. It is planned to immunize groups of 6 horses by the intramuscular route with three vaccine candidates that will be chosen based on the results from preliminary studies performed in cultured cells, mice, and horses, which are currently ongoing. As described above, we shall examine the induction of clinical disease after vaccination and challenge as well as viremia levels and nasal shedding. In addition, the quantity and quality of the immune response will be determined by examining the magnitude of secretory IgA levels and that of the circulating immunoglobulin isotypes (IgGa, IgGb, IgG(T)). Preliminary results strongly suggest that the ratio of different immunoglobulin isotypes has a high predictive value with regard to vaccine protection against EHV-1 cause d disease.