MX2008005634A - Prime boost vaccine for the protection of equines against equine influenza - Google Patents

Abstract

The present invention is concerned with vaccinating equines against the equine influenza virus (EIV). It has now been found that adequate protection against equine influenza in equines can be achieved when vaccination with a live equine influenza vaccine (prime) is followed by vaccination with an inactivated influenza vaccine (boost), wherein the two shots are given no longer than 8 weeks apart. The present invention therefore provides a method for vaccination of animals against equine influenza, wherein an animal is first vaccinated with a (prime) vaccine comprising an attenuated equine influenza virus, followed by a vaccination with a (boost) vaccine comprising an inactivated equine influenza virus, and wherein the boost vaccine is administered no longer than eight weeks after the prime vaccine. Preferably the two shots are given no longer than 8 weeks apart, for example, between 3-6 weeks apart, preferably between 4-6 weeks apart.

Description

CEVING AND REINFORCEMENT VACCINE FOR THE PROTECTION OF HORSES AGAINST THE EQUINE INFLUENZA Description of the Invention The present invention relates to the vaccination of equines against equine influenza virus (EIV). Equine influenza is an important respiratory viral disease that causes flu-like symptoms in horses. This disease is present throughout Europe, North America and parts of Asia. The symptoms of the disease caused by the equine influenza virus can be severe, and are often followed by secondary bacterial infections that can lead to pneumonia and other problems. Horses of all ages are susceptible but infection is more common in young horses without vaccination. Most horses exposed to the virus will show symptoms within a period of 1-5 days and will recover after 2-3 weeks. Explosive outbreaks have been observed in susceptible populations. The virus can be easily spread from horse to horse as a result of droplets and also from nasal discharge and from objects such as infected brushes and blankets. The disease is very contagious and there is an infection rate of almost 100% in a population that has not been previously exposed to the virus. This often follows the importation of infected horses from endemic areas that show no clinical symptoms, and is exacerbated by the fact that international transport of horses is increasing. Equine influenza virus was discovered in horses around 1956 when it was recovered during an epidemic of respiratory disease among horses in Eastern Europe (Sovinová O. et al., Acta.Virol., 2, 51-61, 1958) the virus, A / Equine / Prague / 1/56, (H7N7), is now designated as the prototype of the virus for equine influenza subtype-1. In 1963 other influenza viruses, now designated subtype-2, were discovered during a major outbreak in the United States (Waddell G. H. et al., J. Am. Vet. Med. Assoc, 143, 587-590, 1963). For subtype-2, the prototype virus is A / Equine / Miami / 1/63 (H3N8). The H3N8 subtype has meanwhile spread throughout the world and, currently, is the predominant subtype of the virus (subtype H7N7 has not been isolated since 1980). The subtype H3N8 is prone to antigenic drift. The many variants of the H3N8 subtype co-circulate. Especially the insulators circulating in Europe and the US. they were antigenically distinguishable, the European lineage is represented by the viruses A / eq / newmarket1 / 93 (N / 1/93) and the lineage of the E.E.U.U. is represented by A / eq / Newmarket / 2/93 (N / 2/93) (both Newmarket viruses were isolated from samples taken on the same day from vaccinated 2-year-old thoroughbred horses that had pyrexia and occasional cough) (Daly et al., Vaccine 22, 4101-4109, 2004).

The prevention of equine influenza depends largely on vaccination. Vaccines based on the virus need to be updated regularly to reflect the most recent epidemiological situation. It has been recommended that vaccines for equine influenza contain an H3N8 representative of both American and European lineages. Most vaccines for the protection of equines against infection with equine influenza virus are adjuvanted with inactivated vaccines based on the whole virus. Reasonably effective vaccines, based on the two most important types of this virus, are available, but equines need to be vaccinated 2-3 times per year to ensure their immune status. However, the efficacy of inactive (dead) virus vaccines is not always sufficient, and sometimes does not provide adequate protection for equines.

Some inactivated vaccines may even produce undesirable side effects, for example, inflammatory reactions at the injection site. In addition, inactivated vaccines can often not overcome maternal immunity in young foals, and can induce tolerance in a younger animal. Inactivated vaccines contain viral strains that represent the "American type" equine influenza virus as the "European type" of the virus and need to be updated annually with new strains as recommended annually by the WHO / OIE. A live attenuated vaccine for equine influenza was developed by Heska. This Flu Avert I N vaccine was introduced by Heska in the United States in November 1999. The flu vaccine Avert I .N. it is a "live modified" vaccine that incorporates a virus "adapted" to the cold that reproduces only in the upper regions of the horse's respiratory system, but the virus does not reproduce at the highest temperatures found in the lungs or in the respiratory tract inferior of the animal. The Heska vaccine can be administered using a nasal applicator, instead of a needle. Virus strains adapted to the cold were developed at the University of Pittsburgh by Drs. Patricia W. Dowling and Julio S. Youngner (US patent number 6, 177,082 B 1) However, there is a continuing need for still improved vaccines to protect equines against infection with equine influenza. It has now been found that adequate protection against equine influenza in equines can be achieved when vaccination with a live equine influenza vaccine (priming) is followed by vaccination with an inactivated influenza (booster) vaccine, where Two injections are applied with no more than 8 weeks of spacing. The present invention therefore provides a method for vaccination of animals against equine influenza, wherein an animal is first vaccinated with a vaccine (primed) comprising an attenuated equine influenza virus, followed by vaccination with the vaccine ( booster) comprising an inactivated equine influenza virus, and wherein the booster vaccine is administered no more than eight weeks after the priming vaccine. Preferably the two injections are applied with no more than 6 weeks of spacing, for example, between 3-6 weeks of spacing, preferably between 4-6 weeks of spacing. The invention additionally relates to the use of an inactivated equine influenza virus to prepare a booster vaccine for the vaccination of equines that have been vaccinated with a priming vaccine containing a live attenuated influenza virus, with no more 8 weeks before vaccination with the booster shot. It has been found that when equines are vaccinated with this priming and booster vaccine regimen according to the invention, equines are protected against clinical symptoms after challenge with an equine influenza virus.

On the other hand, equines are completely protected against the presence of the virus and no virus could be isolated from any vaccinated equine at any time. Vaccination for priming and booster according to the invention provides sterile immunity, which until now, could not be demonstrated by any vaccine against equine influenza. Even when the animals were challenged with a very recent strain of influenza, the priming and booster vaccination according to the invention provided sterile immunity against challenge with this recent virus. The priming vaccine used in the present invention contains the viral pathogen in live attend form, which means that the viral pathogen has been modified in a way that does not cause the disease, but still causes an immune response in the vaccinated animal that is attributed to protection against infection with the pathogen. The priming vaccine, comprising a live attend equine influenza virus, may, for example, contain a thermosensitive mutant of equine influenza virus. The priming vaccine also contains the normal components of a modified live vaccine, such as a convenient pharmaceutical carrier which is usually a buffered diluent, optionally a condom, etc. , or any other convenient component known to the skilled person. The modified live vaccine can be administered via any convenient route of administration. If the vaccine is based on a thermosensitive mutant of equine influenza virus, for example a ts mutant that only reproduces at (lower) temperatures in the upper respiratory tract, the vaccine is preferably administered via the intranasal route. Equine influenza viruses adapted to cold and vaccines based on them, for example, those described in US 6436408. An example of a vaccine that can be used as the vaccine for priming in the priming and booster regime as the invention is the commercially available modified live vaccine Flu Avert IN. (Heska Corp.) The booster vaccine comprises an inactive equine influenza virus. Vaccines based on inactivated influenza are known in the art. An inactivated vaccine may contain the virus as a whole virus (inactivated viral particles) or as subunits (a vaccine containing subunits of hemagglutinin and neuraminidase of the virus) in a convenient amount. Suitable amounts of the inactivated virus are known in the art. An inactive equine influenza virus may contain an adjuvant. Suitable adjuvants are known in the art. For example, a convenient adjuvant may be based on one or more saponin fractions. The saponin fractions are produced from Quillaja bark extracts (Quil A) (Morein et al., Clin.lmmunother., 3 (6), 461-475.1995: "Immunostimulating Complexes, clinical potential in Vaccine Development"). The saponin fractions can be used as such, or in the form of an immunostimulatory complex such as ISCOM or an ISCOM matrix, based on saponins, a sterol and a lipid. Examples of suitable saponin fractions, and ISCOMs and matrices based thereon are given in Morein et al. (Supra) and in WO96 / 11711. Useful fractions are for example "fraction A" or "fraction C" of Quil A or mixtures thereof. Good results were obtained when the booster vaccine was the "Equilis Prequenza" vaccine as developed by (Intervet), which is adjuvanted with an adjuvant based on the Iscom matrix. The vaccine for priming, or the booster vaccine (or both) may contain, or may be combined with, the derived immunogens, and which provide protection against infection with, other pathogens, such as equine herpes virus (EHV-1). and / or EHV4), equine encephalitis virus (EEE, WEE and / or VEE), the West Nile virus, tetanus, etc. Especially inactivated vaccines (which are used as a booster vaccine in the present invention) may contain a combination of antigens derived from various pathogens. EXAMPLES: Example 1: Comparison of different vaccination programs The purpose of this study was to compare different vaccination programs, using the modified live vaccine Flu Avert IN against a challenge with A / equine / 2 / South-Africa / 04/03 considering the OIE recommendation to update new influenza virus vaccines with the South African strain. Twenty-four year old Fjord horses were obtained and housed in a pasture. Seven horses were vaccinated twice with a dose of Flu Avert IN in a four week interval (group A). Seven horses were vaccinated with a dose of Flu Avert IN and four weeks later with a dose of Equilis Prequenza Te (group B) Six horses were vaccinated with a dose of Flu Avert IN to determine the onset of immunity (group C). Four animals were left unvaccinated to serve as control (group D). The Flu Avert IN contains equine influenza virus strain P821 which is a cold-adapted, thermosensitive mutant of equine influenza type A2 derived from the precursor virus A / equine / 2 / Kentucky / 1/91. The vaccine was registered by Heska Corporation and is distributed in the US. by Intervet inc. Euqilis Prequenza Te is a suspension for injection that contains: Active substances: Subunits of purified hemagglutinin of equine influenza virus: A / equine-1 / Prague / 1/56 100 AU (antigenic units) A / equine-2 / Newmarket / 1/93 50 AU / equine-2 / Newmarket / 2/93 50au Adjuvants: Purified saponins 375 ug (microgram) Cholesterol 125 ug Phosphatidylcholine 62.5 ug Excipient: Thiomersal traces the vaccine was registered by Intervet International BV. Three weeks after the second vaccination (groups A and B) or one week after vaccination (group C) all horses were sprayed with the A / equine-2 / South Africa / 04/03 virus. After the provoked horses were supervised for clinical symptoms of influenza, body temperature, virus excretion and serology. The blood samples were taken during the course of the vaccination and provocation to determine the levels of antibodies (Hl test) against various vaccinal strains. At the time of the provocation, the horses in group A had an average Hl title of 6.0 and 5.7 against Newmarket / 1/93 and Newmarket / 2/93 respectively, the horses in group B had an average Hl title of 6.1, 11.1 and 10.3 against Prague / 1/56, Newmarket / 1/93 and Newmarket / 2/93 respectively. The horses in group C had no Hl antibody at the time of challenge. After provocation all horses responded well against the Newmarket / 1/93 strain, the mean Hl titers in group A, B, C and D two weeks after the challenge were: 10.9, 10.3, 10.3 and 9.5 respectively . After the challenge, unvaccinated animals and horses of group C showed characteristic symptoms of influenza such a marked mucopurulent discharge and fever.

The animals vaccinated in group A and B showed only mild symptoms. The virus was isolated from some horses in group A and none of the horses in group B. The virus was isolated from all horses in group C within 3 days after the challenge (dpp) while all horses in the group D yielded viruses from day 1 to 6 dpp. All the parameters examined in the statistical analysis such as the temperature record, the total clinical records and the duration of virus excretion were significantly lower in the vaccinated animals of group A and B compared to the non-vaccinated group. It is concluded that the priming and booster vaccination course, of Flu Avert IN followed by Equilis Prequenza 4 weeks later, strongly reduces clinical symptoms and induces a sterile immunity when it is provoked with the recent strain of equine influenza virus isolated SA / 04/03. Twice Flu Avert IN with an interval of 4 weeks also gave good protection against SA / 04/03 comparable to the protection achieved by the recommended basic course of Equilis Prequenza vaccination. In addition, the onset of immunity to Flu Avert IN is very fast, horses without previous contact were partially protected against the provocation of SA / 04/03 7 days after vaccination. It is interesting to investigate the initiation of immunity to Flu Avert IN in previously primed animals.

Example 2: Provocation with the recent influenza strain after vaccination for priming and reinforcement In a previous study, reflected in example 1, it was shown that the horses showed a sterile immunity when they were first vaccinated with the Flu Avert IN vaccine and reinforced 4 weeks later with Prequenza. The purpose of this study was to reconfirm this observation using another virus challenge. Eight year old Fjord horses were obtained and housed in a pasture. Four horses were vaccinated with a dose of Flu Avert IN and four weeks later with a dose of Equilis Prequenza Te (group A) and four animals were left unvaccinated to serve as control (group B). Three weeks after the second vaccination all the horses were sprayed with the A / equine-2 / Newmarket / 05/03 virus. Afterwards, the provoked horses were supervised for the clinical symptoms of influenza, body temperature, virus excretion and serology. The blood samples were taken during the co of the vaccination and provocation to determine the levels of antibodies (Hl test) against various vaccinal strains. At the time of the provocation the horses in group A had an average Hl title of 6.0 and 5.7 against Newmarket / 1/93 and Newmarket / 2/93 respectively. After the provocation all the horses responded well against the strain Newmarket / 1/93, the mean Hl titers in group A and B two weeks after the challenge were: 10.9 and 9.5 respectively. After the challenge, unvaccinated animals showed characteristic symptoms of influenza such as marked mucopurulent discharge, cough and fever. The vaccinated animals showed only mild symptoms. No virus was isolated from the vaccinated horses. The virus was isolated from all control horses between 2 and 6 days after the challenge (dpp). All the parameters examined in the statistical analysis such as the temperature record, the total clinical records and the duration of virus excretion were significantly lower in the vaccinated animals compared to the unvaccinated group. It is concluded that the co of vaccination for priming and booster, Flu Avert IN followed by Equilis Prequenza 4 weeks later, strongly reduces clinical symptoms and induces a sterile immunity when it is provoked with the recently isolated equine influenza virus strain. Newmarket / 05/03. In general it is clear that when horses that are first vaccinated with Flu Avert IN and receive a booster 4 weeks later with Prequenza, a sterile immunity against equine influenza can be achieved after the challenge.

Claims (4)

1. Use of an inactive equine influenza virus to prepare a booster vaccine for vaccination of horses that have been vaccinated with a priming vaccine containing a live attenuated influenza virus, no more than 8 weeks prior to being vaccinated with the booster shot

2. Use according to claim 1, wherein the inactivated vaccine further contains an adjuvant.

3. Use according to claim 2, wherein the adjuvant is based on an ISCOM matrix.

4. Method to protect equines against equine influenza infection in which a horse is first vaccinated with a priming vaccine containing a live attenuated influenza virus, and not more than 8 weeks later is vaccinated with a booster vaccine which contains an inactivated equine influenza virus. SUMMARY The present invention relates to the vaccination of equines against equine influenza virus (EIV). It has now been found that adequate protection against equine influenza in equines can be achieved when vaccination with a live equine influenza vaccine (priming) is followed by vaccination with an inactivated influenza (booster) vaccine, where Two injections are applied no more than 8 weeks apart. The present invention therefore provides a method for vaccination of animals against equine influenza, wherein an animal is first vaccinated with a vaccine (primed) comprising an attenuated equine influenza virus, followed by vaccination with the vaccine. with a (booster) comprising an inactivated equine influenza virus, and wherein the booster vaccine is administered no more than eight weeks after the priming vaccine. The two injections are preferably applied no more than 8 weeks apart, for example, between 3-6 weeks spaced, preferably between 4-6 weeks apart.