Dr. Gillian Perkins and Dr. Nikolaus Osterrieder
Dr. Gillian Perkins
Dr. Nikolaus Osterrieder
Streptococcus equi, a highly contagious and specialized bacterium, causes abscessation of lymph nodes (strangles) in horses. Acute infections and chronic complications, such as abdominal abscesses, accumulation of purulent material in the guttural pouch, and airway obstruction occur in about 20% of the cases, leading to sustained loss of performance and animal health and can even be fatal. All breeds are affected and infection seems to be maintained in the population through chronically infected carriers, which harbor the organism in their guttural pouches from where bacteria are intermittently shed. Once S. equi abscesses have formed, antibiotics have reduced efficacy, are generally not indicated, and can even lead to exacerbation of disease. Symptomatic therapy such as anti-inflammatory drugs and lancing of abscesses are often the only option. Adjuvanted intramuscular S. equi vaccines often cause muscle soreness and injection site abscesses, while commercial intranasal vaccines (modified live) can have residual virulence or contaminate remote injection sites resulting in abscess formation. Thus, current strangles vaccines are not without risks and efficacy data are limited.
Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortions and neurological disease. Recent work has shown that two genotypes differing in their ability to induce neurological disease circulate in the horse population in the US and worldwide. Modified live virus vaccines have been proven superior to inactivated preparations with respect to duration of immunity and completeness of protection, although vaccine efficacy still needs improvement. The goal of this work is to induce a long-lasting immune response to EHV-1 and S. equi by using EHV-1 as a vehicle to deliver S. equi antigens to avoid the huge problems associated with both diseases.
Equine herpesvirus type 1 (EHV-1) affects horse populations worldwide (10). 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 has been grouped into the genus Varicellovirus together with its close relative equine herpesvirus type 4 (EHV-4) (10, 15). EHV-1 targets three organ systems in the natural host: the respiratory tract, the reproductive organs, and the central nervous system (CNS). Consequently, the clinical signs observed after EHV-1 infection are respiratory disease including nasal discharge, respiratory problems and coughing, late-time abortions in pregnant mares, and neurological disease. The latter is usually characterized by paralysis of the hind limbs and the organs that are innervated by the lumbar nerves (e.g., the bladder, tail) (39). More recently, the neurological form of the disease has been predominant and caused considerable suffering of horses. Large-scale epidemics of EHV-1 paralytic disease have been documented in the past years, and more than 30 outbreaks have been recorded all over the United States since 2001, 11 of which occurred in 2006 and 6 in 2007. This latest surge has resulted in the classification of paralytic equine herpesvirus infection or equine. herpesvirus-induced myeloencephalopathy (EHM) as a potentially emerging disease by the United States Department of Agriculture. Concern has been raised because these recent outbreaks have resulted in large numbers of animals developing severe clinical disease. For example, in EHM outbreak at the University riding school in Findlay, OH in 2003, more than 90% of the 138 horses became clinically affected, 35% developed neurological disease and 9% (12 horses) died or had to be euthanized. Another striking feature of this outbreak is that recent neurological cases of EHV-1 infection occurred despite regular vaccinations in very short intervals. These observations indicate the possibility circulating EHV-1 variants, which can be maintained in the equine population despite frequent vaccination, which is based primarily on inactivated vaccines in the US (4).
The pathogenesis of EHV-1 abortions and neurological disease is thought to be 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, axonal swelling, and 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. The recent discovery of a polymorphism in the DNA polymerase and proof that this mutation is important for the neurologic potential of the virus support the notion of two different EHV-1 pathotypes circulating in the equine population (4).
Regardless of the existence of the EHV-1 pathotype, however, vaccination remains the cornerstone in the control of this viral disease. Evidence has accumulated that vaccine protection requires the induction of a robust cytotoxic T-cell response, while neutralizing antibodies are no correlate for protection. For the rational design of more efficacious modified live vaccines, it is likely crucial to modify the immunomodulatory capacity of EHV-1 (20) and we recently have identified a unique activity in EHV-1: ORF1 is predicted to encode a type 2 transmembrane protein that, through mutational analysis, was unequivocally shown to have strong MHC class I down regulating activity (Damiani, Van de Walle, Noronha, Osterrieder, unpublished). We will utilize this knowledge for this proposal and base our constructs on ORF1-negative viruses because we hypothesize that robust CTL responses will be required to control EHV-1, one of the long-term goals of this vaccine approach.
Streptococcus equi subsp. equi (S. equi) is a bacterial infection of horses that causes fever, mucopurulent nasal discharge and lymph node abscessation (29). High morbidity is not unusual in outbreak situations, with reports from nearly 50% to 100% developing clinical signs. (9). Secondary complications have been documented in as many as 20% of affected horses in a single outbreak and include abscesses in the abdomen or brain, persistent infection of the guttural pouches, purpura hemorrhagica, pneumonia and upper airway obstruction (thus the term “strangles”) (18, 19, 30). The overall mortality rate associated with S. equi infection in one large outbreak was approximately 8%, while 40% of horses with complications died (30). Persistence of the disease in the equine population is thought to be the result of intermittent shedding of the organism by chronically infected, asymptomatic carriers. Nearly 20% of horses have been observed to shed bacteria for some period of time following the resolution of clinical signs, and shedding up to 39 months was reported (9, 29).
Medical therapy for primary disease is often limited to supportive care and relief of symptoms such as nonsteroidal anti-inflammatory drugs and temporary tracheostomy while the illness runs its course. The use of antimicrobial therapy in horses with lymphadenopathy is thought to result in protracted disease (29). High morbidity and lack of definitive treatment to eliminate the organism result in a significant economic impact on all facets of the horse industry through veterinary costs, loss of performance time and sometimes death of the horse. In this situation, prevention of disease with a safe, effective and reliable vaccine is highly desirable. Attempts have been ongoing by researchers to provide the equine community with an S. equi vaccine that stimulates long-lasting immunity, which is observed in up to 75% of horses surviving natural infection (17). The vaccine should have minimal risk of adverse complications. This goal has been elusive, however, and complications associated with administration of commercially available vaccines have been documented from the earliest through the most recent products. One of the first intramuscular bacterins was reported to cause stiffness, local irritation, abscessation, skin sloughing and purpura hemorrhagica (11). Adverse reactions to currently available live attenuated vaccines include induction of clinical disease and abscessation after both intranasal and oral submucosal administration (6, 8). More attempts at novel preparations and routes of administration have resulted in similar complications in research and field trial settings, and hence are deemed inappropriate for further use (7, 8).
Challenges to the development of an ideal vaccine for S. equi include incorporation of significant antigens, stimulation of an adequate mucosal immune response, and avoidance of complications. Three proteins associated with the ability of S. equi to avoid phagocytosis are suggested to be involved in the mucosal immune response, including SeM, Se18.9 and IdeE (28, 31, 32, 35). Delivery of these antigens and subsequent immune response may be more safely achieved by a live viral vector such as EHV-1, an experimental approach that will be tested here in both a murine model and in horses.