The present invention is concerned with vaccinating equines
against viral infection.
Equines are susceptible to a variety of infectious diseases.
Due to the increasing international movement of horses the incidence of these diseases
Beside general sanitation, feeding, housing etc., vaccination of equines is therefore
an important tool in preventing of animal suffering as well as the economic losses
Infectious diseases for which horses can be vaccinated are, among others, equine
influenza virus (EIV) and Equine herpes virus (EHV1 and EHV4) and Equine encephalitis
(Western (WEE), eastern (EEE) and Venezuelan (VEE)) but also the West Nile virus,
African horse Sickness, rabies, tetanus, Equine Infectious Anemia etc.
The vaccinations a given horse should receive depends on whether the horse is, for
example, a domestic "backyard" horse, where insect borne diseases, such as EEE,
WEE and VEE, or WNV may be a problem, or a horse which travels a lot and may be
exposed to may other horses, where contagious viruses such as EIV may be a problem.
Most horses are vaccinated against tetanus, since tetanus causes against a deadly
disease in horses. In case the horse is, for example, a broodmare, the risk of abortion,
for example associated with EHV infection, should of course be minimized. Moreover,
some diseases may only be a problem in a particular part of the world, such as the
Potomac horse virus or rabies.
Vaccines may be based on either a modified live (attenuated),
an inactivated pathogen or on a subunit vaccine based on one or more important immunogens
derived from a pathogen. Vaccines exist for each pathogen separate, but also combination
vaccines, for example for protection against EIV, EHV and tetanus, exist.
Equine influenza is a major respiratory viral disease that
causes flu like symptoms in equines. This disease is present throughout Europe,
North America and parts of Asia. Disease symptoms caused by equine influenza virus
can be severe, and are often followed by secondary bacterial infections which can
lead to pneumonia and other problems. Horses of all ages are susceptible, but infection
is most common in young unvaccinated horses. Most horses exposed to the virus will
show signs within a period of 1-5 days and recover after 2-3 weeks.
Explosive outbreaks have been seen in susceptible populations. The virus can be
spread easily from horse to horse as a result of droplets and also from nasal discharge
and from things like infected brushes and rugs. The disease is very contagious and
there is almost 100% infection rate in a population that has been previously unexposed
to the virus. This often follows the import of infected horses from endemic areas
not showing clinical signs, and is worsened 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
virus for equine influenza subtype-1. In 1963 another influenza virus, now designated
subtype-2, was 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 over the world and, at present, is the predominant
virus subtype (The H7N7 subtype has not been isolated since 1980). The H3N8 subtype
is prone to antigenic drift. Various variants of the H3N8 subtype co-circulate.
Especially isolates circulating in Europe and the USA were antigenically distinguishable,
the European lineage is represented by A/eq/newmarket1/93 (N/1/93) and the US lineage
is represented by A/eq/Newmarket/2/93 (N/2/93) viruses (both Newmarket viruses were
isolated from samples taken on the same day from vaccinated 2 year old Thoroughbred
horses that has pyrexia and occasional coughing) (
Daly et al., Vaccine 22, 4101-4109, 2004
The prevention of equine influenza largely depends on vaccination. Vaccines based
on the virus need to be updated regularly in order to reflect the most recent epidemiological
situation. It has been recommended that vaccines for equine influenza contain a
representative H3N8 from both the American and European lineages.
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 inactivated (killed) virus vaccines is not
always sufficient, and some times does not provide adequate protection for equines.
Some inactivated vaccines can even produce undesirable side effects, for example,
inflammatory reactions at the site of injection. Furthermore, inactivated vaccines
are often not able to overcome maternal immunity in young foals, and can induce
tolerance in a younger animal. Inactivated vaccines contain viral strains representing
the "American type" equine influenza virus as well as the "European type" of the
virus and need to be updated yearly with new strains as recommended each year by
An attenuated live vaccine for equine influenza was developed by Heska. This vaccine
Flu Avert IN was introduced by Heska in the United States in November 1999. Flu
Avert I.N. vaccine is a "modified live" vaccine which incorporates a "cold adapted"
virus that replicates only in the upper regions of the horse's respiratory system,
but the virus does not replicate at the higher temperatures found in the lungs or
lower respiratory tract of the animal. Heska's vaccine can be administered using
a nasal applicator, rather than a needle. The cold-adapted virus strains were developed
at the University of Pittsburgh by Drs. Patricia W. Dowling and Julius S. Youngner
US Patent Number 6,177,082 B1
Equine herpes virus 1 (EHV-1) and equine herpes virus 4
(EHV-4) are an important cause of respiratory disease and abortion in horses world-wide.
Especially when inactivated vaccines against EHV are used, the vaccine should contain
a representative of both subtypes in order to provide adequate protection.
Such vaccines are commercially available, either alone, or in combination with,
for example, influenza and tetanus, such as Equilis Resequin (Intervet International
BV), which contains inactivated EHV-1 strain racH, inactivated EHV-4 strain 2252,
and three inactivated EIV strains (representatives of the European and American
A modified live vaccine for vaccination of equines against EHV infection, based
on the attenuated EHV-1 strain racH, exist as well (Prevaccinol, Intervet International
It has now been found that adequate protection against
viral infection in equines can be achieved when vaccination with a live viral vaccine
(prime) is followed by vaccination with an inactivated vaccine (boost) for the same
virus, wherein the two shots are given no longer than 8 weeks apart.
The invention therefore provides a method for vaccination of equines against infection
with an equine viral pathogen, wherein an animal is first vaccinated with a (prime)
vaccine comprising an attenuated form of the pathogen, followed by a vaccination
with a (boost) vaccine comprising the same pathogen in inactivated form, and wherein
the boost vaccine is administered no longer than 8 weeks after the prime vaccine.
The invention further relates to the use of an inactivated equine viral pathogen,
to prepare a boost vaccine for vaccinating equines that have been vaccinated with
a priming vaccine, containing the same pathogen in an attenuated live form, no longer
than 8 weeks prior to being vaccinated with the boost vaccine.
Preferably the two shots are given no longer than 6 weeks apart, for example, between
3-6 weeks apart, preferably between 4-6 weeks apart.
With "equine viral pathogen" is meant a virus that causes
an infectious disease in equines. The method of the invention is especially useful
for vaccination against viral infections such as infections with EIV and/or EHV-1/EHV-4.
Especially for EIV, it has been found that when equines are vaccinated with such
a prime-boost vaccine regimen according to the invention, the equines are protected
against clinical signs after a challenge with a virulent EIV.
Moreover, the equines are completely protected against
viral shedding and no virus could be isolated from any vaccinated equines at any
time. The prime boost vaccination according to the invention provides sterile immunity,
which hitherto, could not be demonstrated for any vaccine against equine influenza.
Even when the animals were challenged with a very recent strain of influenza, the
prime boost vaccination according to the invention provided sterile immunity against
challenge with this recent virus.
The prime vaccine used in the present invention contains
the viral pathogen in attenuated live form, meaning that the viral pathogen has
been modified in such a way that it does not cause the disease, but still elicits
an immune response in the vaccinated animal that attributes to protection against
infection with the pathogen.
In case of EIV the prime vaccine comprises an attenuated
live equine influenza virus. The attenuated live virus may, for example, be a temperature
sensitive mutant of the equine influenza virus. If the vaccine is based on a temperature
sensitive mutant of the equine influenza virus, for example a ts mutant which only
replicated at the (lower) temperatures in the upper respiratory tract, the vaccine
is preferably administered via the intranasal route. Cold-adapted equine influenza
viruses and vaccines based thereon are, for example, disclosed in
. An example of a vaccine that can be used as the prime vaccine in the
prime-boost regimen according to the invention is the commercially available modified
live vaccine Flu Avert I.N. (Heska Corp).
In case of a prime vaccine for EHV1/ EHV-4 infection, the
vaccine contains an attenuated EHV strain. An example of an attenuated live EHV-1
strain that can be used in a prime vaccine as used with the method of the invention
is the racH strain. This strain has served as the basis for modified live vaccines
against EHV infection such as Prevaccinol (Intervet International BV). Such a vaccine
can be used as the prime vaccine in the present invention.
The prime vaccine further contains the normal constituents
of a modified live vaccine, such as a suitable pharmaceutical carrier which is usually
a buffered diluent, optionally a preservative, etc., or any other suitable constituent
known to the skilled person. The modified live vaccine may be administered via any
suitable administration route.
The boost vaccine used with the invention contains the
equine viral pathogen in an inactivated form.
In case the prime boost vaccination according to the invention is used to protect
equines against infection with EIV, the boost vaccine comprises an inactivated equine
influenza virus. Vaccines based on inactivated influenza are known in the art. An
inactivated vaccine may contain the virus as whole virus (inactivated viral particles)
or as subunits (a vaccine containing heamagglutinin and neuraminidase subunits of
the virus) in a suitable amount. Suitable amounts of the inactivated virus are known
in the art. Good results were obtained when the boost vaccine was the "Equilis Prequenza"
vaccine (developed by Intervet International BV), which is adjuvated with an Iscom
matrix based adjuvant.
A boost vaccine for use in a prime boost vaccination according to the invention,
for the protection against EHV-1, may contain inactivated strains of both EHV-1
and EHV-4. Such inactivated vaccines against EHV are known in the art.
At least the boost vaccine used with the invention may
contain at least two inactivated viral pathogens. For example, a combination vaccine
may be used that contains both inactivated strains of EIV, as well as inactivated
strains of EHV. Optionally the boost vaccine may contain further immunogens derived
from other pathogens such as, for example, tetanus.
Inactivated combination vaccines against EIV/EHV & tetanus are known in the art
(for example, Equilis Resequin and Equilis Resequin Plus (Intervet International
The prime boost vaccination according to the invention
can be applied to more than one pathogen at the same time.
For example, the prime vaccination for both pathogens may be given in the form of
separate priming vaccinations, one for each pathogen, or in the form of a one-shot
priming vaccination where attenuated live forms of different pathogens are combined
in a combined live vaccine. For example, attenuated live strains of EIV and EHV
may be combined in one and the same vaccine formulation, or may be give separately
in two separate formulations, both used as priming vaccine in a method of the invention.
The boost vaccination may then also be given in separate or combined form.
Either the boost or the prime vaccine may be combined in one and the same formulation
with other immunogens such as, for example, purified tetanus toxoid, also when these
other pathogens are not administered in a prime boost vaccine regimen according
to the invention. Thus, for example, purified tetanus toxoid may be part of the
boost vaccine. Thus, existing inactivated combination vaccines can be used as the
boost vaccine in the present invention, when combined with a priming vaccination
for one of the viral pathogens, which priming vaccination contains the pathogen
in attenuated live form.
An inactivated boost vaccine as used in the invention may
contain a suitable adjuvant. Suitable adjuvants are known in the art. For example,
a suitable adjuvant may be based on Quillaja bark extracts or one or more saponin
Saponin fractions are produced from Quillaja bark extracts
(Quil A) (Morein et al., Clin.Immunother., 3(6), 461-475,1995: "Immunostimulating
Complexes, clinical potential in Vaccine Development"). Saponin fractions may be
used as such, or in the form of a immunestimulatory complex such a s an ISCOM or
ISCOM matrix, based on the saponins, a sterol and a lipid. Examples of suitable
saponins fractions, and ISCOMs and matrices based thereon are given in Morein et
al. (supra) and in
. Useful fractions are for example "fraction A" or "Fraction C" of Quil
A or mixtures thereof. The "Equilis Prequenza" vaccine as developed by (Intervet
International BV), is adjuvated with an Iscom matrix based adjuvant. Equilis Equenza
(Intervet International BV) is adjuvated with Quil A.
Example 1: Comparison of different vaccination schedules
The purpose of this study was to compare different vaccination
schedules, using the modified live vaccine Flu avert IN against a challenge with
A/equine/2/South-Africa/04/03 considering the recommendation of the OIE to update
new influenza vaccines with the South-African strain.
Twenty-four Fjord yearlings were obtained and housed on a pasture.
Seven horses were vaccinated twice with one dose of Flu
Avert IN at four weeks interval (group A).
Seven horses were vaccinated with one dose of Flu Avert IN and four weeks later
with one dose of Equilis Prequenza Te (group B).
Six horses were vaccinated ones with one dose of Flu Avert IN to determine the onset
of immunity (group C).
Four animals were left unvaccinated to serve as control
Flu Avert IN contains the equine influenza virus strain P821 which is a cold adapted,
temperature sensitive mutant of equine influenza type A2 derived from parent virus
A/equine/2/Kentucky/1/91. The vaccine was registered by Heska Corporation and is
distributed in the USA by Intervet inc.
Te is a suspension for injection containing:
Purified haemagglutinin subunits
from equine influenza viruses:
100AU (antigenic units)
375 ug (microgram)
The vaccine was registered by Intervet
Three weeks after the second vaccination (groups A and
B) or one week after the vaccination (group C) all horses were challenged by aerosol
with A/equine-2/South Africa/04/03 virus. After challenge horses were monitored
for clinical signs of influenza, body temperature, virus excretion and serology.
Blood samples were taken during course of the vaccination and challenge to determine
the antibody levels (HI test) against different vaccine strains.
At moment of challenge the horses in group A had a mean HI titre of 6.0 and 5.7
against Newmarket/1/93 and Newmarket/2/93 respectively, the horses in group B had
a mean HI titre 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 HI antibodies at moment of challenge.
After challenge all the horses responded well against the Newmarket/1/93 strain,
mean HI titres in group A, B, C and D at two weeks after challenge were: 10.9, 10.3,
10.3 and 9.5 respectively.
After challenge the non-vaccinated animals and the horses of group C showed characteristic
signs of influenza such as a marked mucopurulent discharge and fever. The vaccinated
animals in group A and B showed only mild signs. Virus was isolated from a few horses
in group A and from none of the horses of group B. Virus was isolated from all the
horses of group C between at 3 days post challenge (dpc) while all the horses of
group D shed virus from day 1 till 6 dpc. All parameters examined in the statistical
analysis such as the temperature score, the total clinical scores 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 prime boost vaccination course,
Flu Avert IN followed by Equilis Prequenza 4 weeks later, strongly reduces clinical
signs and induces a sterile immunity when challenged with the recent isolated equine
influenza virus strain SA/04/03. Two times Flu Avert with 4 week interval gave also
a good protection against SA/04/03 comparable with the protection archived by the
recommended basic vaccination course of Equilis Prequenza. Furthermore, the onset
of immunity of Flu Avert IN is very rapid, naive horses were partially protected
against SA/04/03 challenge 7 days after the vaccination. It is interesting to investigate
the onset of immunity of Flu Avert in previously primed animals.
Example 2: Challenge with recent influenza strain after prime boost vaccination
In a previous study, reflected in Example 1, it was demonstrated
that horses showed a sterile immunity when they were primed with the live Flu Avert
IN vaccine and boostered 4 weeks later with Prequenza. The purpose of this study
was to reconfirm this observation using another challenge virus.
Eight Fjord yearlings were obtained and housed on a pasture. Four horses were vaccinated
with one dose of Flu Avert IN and four weeks later with one 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 horses were challenged by aerosol
with A/equine-2/Newmarket/05/03 virus. After challenge horses were monitored for
clinical signs of influenza, body temperature, virus excretion and serology. Blood
samples were taken during course of the vaccination and challenge to determine the
antibody levels (HI test) against different vaccine strains.
At moment of challenge the horses in group A had a mean HI titre of 6.0 and 5.7
against Newmarket/1/93 and Newmarket/2/93 respectively. After challenge all the
horses responded well against the Newmarket/1/93 strain, mean HI titres in group
A and B at two weeks after challenge were: 10.9 and 9.5 respectively. After challenge
the non-vaccinated animals showed characteristic signs of influenza such as a marked
mucopurulent discharge, coughing and fever. The vaccinated animals showed only mild
signs. No virus was isolated from the vaccinated horses. Virus was isolated from
all the control horses between 2 and 6 days post challenge (dpc). All parameters
examined in the statistical analysis such as the temperature score, the total clinical
scores and the duration of virus excretion were significantly lower in the vaccinated
animals compared to the non-vaccinated group.
It is concluded that the prime boost vaccination course, Flu Avert IN followed by
Equilis Prequenza 4 weeks later, strongly reduces clinical signs and induces a sterile
immunity when challenged with the recent isolated equine influenza virus strain
Newmarket/05/03. In general it is clear that when horses that are primed with Flu
Avert IN and receive a booster 4 weeks later with Prequenza, a sterile immunity
against equine influenza can be archived after challenge.