Dr. Nikolaus Osterrieder
This research proposal is designed to generate and evaluate modified live Equine Herpesvirus Type 1 (EHV-1) vaccine viruses that express potent immunogens of Equine Influenza Virus (EIV) or West Nile Virus (WNV). The rationale is to generate a multivalent vaccine that can be administered to horses by the intranasal route and may lead to a better immune response that includes the induction of a mucosal and cell-mediated immunity besides a humoral response. This goal can be achieved because a live EHV-1-based vaccine should be able to induce both local and systemic immune responses. Ultimately, the generated vaccines shall be tested in horses for their innocuity and for the induction of EHV-1-, EIV-, and WNV-specific immune responses.
In recent years, EHV-1 outbreaks in the US have become more common and this tendency is continuing despite regular vaccinations of large numbers of horses mainly using inactivated vaccines such as Pneumabort K TM ( Fort Dodge ). Vaccination intervals are as short as two months in areas with high pathogen pressure due to large equine populations or movements (Dr. Y. Zhang, Ohio State Department of Agriculture, personal communication). Despite these vaccinations but consistent with the pathological picture associated with EHV-1 infections, respiratory symptoms in affected horses are observed, which then lead to abortions in the late stages of infection or to neurological symptoms that range from mild ataxia to fatal paraplegia. Only recently, major and devastating outbreaks of the neuronal form of the disease have occurred in several states, including Pennsylvania , Iowa , and Kentucky . The probably most severe cases were reported from the State University 's English Riding School in Findlay , Ohio , where more than 90% of the 138 horses on the affected premise were affected, and 30% exhibited neurological signs after the initial appearance of respiratory symptoms. Of these more than 40 horses with neurological symptoms, 12 died or were euthanized. This EHV-1 outbreak occurred despite a history of regular vaccinations, indicating the possibility of the circulation of new EHV-1 variants that are able to break vaccine protection ( http://www.thehorse.com , article number #4104 and #4272).
Equine Influenza Virus is also controlled by regular immunizations, which are usually done in six months intervals. Two subtypes of EIV are recognized in horses, namely H7N7 and H3N8. Because outbreaks of EIV are associated with high morbidity and malperformance, extensive vaccination programs are employed in horses, similar to those to protect against EHV-1. While viruses of the H7 subtype (A/equi-1) only rarely have been associated with disease during the last 15 years, they may still be circulating and cause subclinical infections. Much more frequently, however, infections are observed with viruses of the EIV H3 subtype (A/equi-2), the virus subtype that is predominant worldwide. Most of the EIV vaccines that are currently in use are based on formalin-inactivated virus preparations. The protective capacity of these inactivated vaccines, however, is highly questionable, much like that of inactivated EHV-1 vaccines. Modified live (MLV) EIV vaccines are available and show promising results with regards to protection, but their use may be problematic in pregnant or immunocompromised animals. The use of the cold-adapted FluAvert TM strain and the possibility of a local administration of the vaccine certainly has some advantages over the conventional inactivated vaccines. Safety issues that arise by using MLV influenza virus vaccines, including the remote possibility of a role of horses as carriers for infections of humans, are not entirely resolved. Therefore, the search for novel vaccines against equine influenza is still ongoing and includes DNA vaccine approaches using one of the viral outer envelope glycoproteins, the hemagglutinine, as the target for the antiviral immune response in the host. These approaches have also yielded some stimulating results after both intramuscular and intranasal application of the DNA vaccine, but the efficacy of the DNA vaccines under development is not yet comparable to that of the MLV.
In the summer and fall of 1999, cases of human encephalitis of unknown cause were reported in New York City . Shortly after the first case, a virus that was not endemic in the New World , West Nile Virus (WNV) was isolated and identified as the cause of the observed disease. The causative agent, which is a close relative of other encephalitis viruses like Yellow Fever Virus, Kunjin Virus, Japanese Encephalitis Virus, and St. Louis Encephalitis Virus, was initially isolated in the name-giving West Nile region in Uganda in 1937. Until the 1999 outbreak in New York , WNV was restricted to the Old World . Like many of its relatives within the large RNA virus family of the Flaviviridae , WNV is an arbovirus and is transmitted to warm-blooded animals by mosquitoes or – extremely rarely - ticks. In the special case of WNV, the mosquito, usually a member of the Culex species, gets infected while feeding on birds harboring the virus and then transmits virus to other animal species, mainly horses and man. The first isolate in the U.S. stems from a flamingo that succumbed to the disease in 1999, and the virus has since started its conquest of the American continent. WNV has been following mainly the routes of migrating birds and has reached the western coast of the US . Because the number of horses affected by the virus is continuing to grow and approximately 15,000 equine WNV cases were reported in 2002, vaccination of equines against the disease was started in 2001. The longevity of vaccine protection and the safety of the vaccine, however, are under debate and experimental vaccines, again including DNA vaccines, are being tested.
Objectives and Experimental Approach
The experiments planned in this proposal aim at the generation of a bi- or trivalent vaccine, which is able to protect against disease caused by three important viral pathogens of the horse, namely EHV-1, EIV and/or WNV. The experimental approach is driven by the notion that horses in the US are regularly vaccinated against each of the pathogens and that a more efficient vaccine protection may be achieved by (i) using a modified live rather than inactivated vaccines, and (ii) applying the vaccine intranasally, which may result in a more efficient protection especially in the case of EHV-1 and EIV. In a first step, recombinant EHV-1 are generated based on a vaccine strain that expresses immunogenic proteins of EIV (hemagglutinine) or WNV (the matrix preM and the envelope E proteins). Subsequently, these generated vaccines are tested for their potential to induce a virus-specific and protective immune response in mice. The recombinant vaccines will be based on a glycoprotein gM-negative virus derived from the RacH vaccine strain. The gM-negative virus is used as a basis for transgene expression, because it was shown to be a more potent inductor of anti-EHV-1 immunity than its parental virus in both mice and in preliminary studies in horses. Both, the intranasal and the subcutaneous route of immunization will be used for administration of EIV- or WNV-transgenic viruses, and the quantity and quality of the immune response will be measured. In the long run, we aim at testing the generated vaccine viruses in horses or ponies.