VACCINATION
AGAINST EQUINE INFLUENZA:
QUID NOVI?
R. Paillot
*, D. Hannant, J.H. Kydd, J.M. Daly
Animal Health Trust,
Centre for Preventive
Medicine, Lanwades Park, Newmarket,
Suffolk
CB8 7UU, UK
Corresponding
author. Tel.: +44 8700 502460x1247; fax: +44 8700 502461.
E-mail address: romain.paillot@aht.org.uk (R. Paillot).
R. Paillot et al. Vaccine 24 (2006) 4047–4061
Abstract
Equine
influenza virus is a leading cause of respiratory disease in the horse. Equine
influenza vaccines containing inactivated virus were first developed in the
1960s. Despite their intensive use, equine influenza outbreaks still continue
to occur and therefore new strategies of vaccination are necessary to improve
vaccine efficacy. Numerous methods of vaccination have been evaluated and
commercialised in the horse, the most
recent being the cold-adapted influenza virus and poxvirus-based vaccines. As a large animal model, the horse is also a
useful species in which to evaluate the potential of new generations of
influenza vaccine such as live-attenuated influenza virus engineered by reverse
genetics. This report details the equine immune responses conferring protection
against influenza. It then undertakes a selective review of different
strategies of vaccination against equine influenza that have been developed
over the last two decades and discusses factors that may influence the efficacy
of vaccination. Finally it outlines progress in the development of a novel
vaccination strategy against equine influenza using reverse genetics.
© 2006
Elsevier Ltd. All rights reserved.
Keywords:
Influenza; Vaccine; Horse species
Contents
1.
Introduction.......................................................................................................
4048
2. Equine influenza
virus.............................................................................................
4048
2.1. Viral
structure...............................................................................................
4048
2.2.
Disease ....................................................................................................
4048
2.3.
Epidemiology in
horses......................................................................................
4049
2.3.1. EIV
subtypes .......................................................................................
4049
2.3.2.
Interspecies
transmission.............................................................................
4049
2.3.3. Strain
selection for vaccination.......................................................................
4049
2.4.
Protective immune responses.................................................................................
4049
2.4.1.
Humoral immune esponse...........................................................................
4050
2.4.2.
Cellular immune
response............................................................................
4051
3. Vaccines
against equine influenza virus..............................................................4051
3.1. ‘Dead’
vaccines.............................................................................................
4051
3.1.1.
Inactivated whole influenza virus vaccines...................................................4051
3.1.2.
Subunit vaccines....................................................................................
4053
3.1.3. DNA vaccines......................................................................................
4053
3.2. Live
virus/vector vaccines....................................................................................
4055
3.2.1. Live
attenuated influenza virus vaccine................................................................
4055
3.2.2.
Virus-based vector vaccines..........................................................................
4056
3.2.3. Live
attenuated influenza virus designed by reverse genetics.............................4057
3.3.
Effectof maternal antibodies and age on equine influenza vaccination................
4057
4. Conclusion
.......................................................................................................
4058
References........................................................................................................
4058
1. Introduction
Equine
influenza A viruses (EIV; H7N7 and H3N8 subtypes) are a leading cause of
respiratory disease in the horse. Although
the H7N7 influenza virus subtype has not been detected recently, the H3N8
subtype of equine influenza virus has
not been controlled successfully by vaccination and remains today a serious threat to horse
welfare and an economic problem for the horse industry. Historically, equine influenza vaccines were mainly composed of
whole inactivated viruses, which provide
protection against influenza through the
induction of a short-lived humoral immunity. This is in contrast to immunity
stimulated by natural infection, which is more robust and longer lived due to
the stimulation of both humoral and
cellular immune responses. The development of new strategies of vaccination that mimic
more closely the stimulation of the immune system induced by EIV infection, has
been the focus of EIV vaccine development in the last two decades. Thus, modern
vaccines composed of either
live
attenuated influenza virus, DNA plasmids or poxvirusvectors coding for
influenza virus proteins were developed and some have been commercialised.
Today, a new approach to EIV vaccination using live-attenuated influenza virus
engineered by reverse genetics, is also under development. This report will
describe the equine influenza virus, its pathogenesis, epidemiology and
immunology in the horse and then review different vaccination approaches.
2. Equine influenza virus
2.1. Viral structure
Influenza
viruses belong to the family orthomyxoviridae and are classified as A, B and C
based on antigenic differences in their nucleoprotein (NP) and matrix (M1)
protein. EIV is a type A influenza virus similar to type A human influenza
virus. Type A influenza viruses contain segmented RNA (about 13.6 kb)
consisting of eight linear, single stranded genomic fragments of negative
polarity. Six segments code for single proteins haemagglutinin (HA),
neuraminidase (NA), nucleoprotein (NP), three subunits of viral polymerase (PA,
PB1 and PB2), one segment codes for matrix 1 and 2 (M1 and M2) and one segment
codes for nonstructural protein 1(NS1) and nuclear export protein (NEP) [1,2].
In some strains, the +1 reading frame of PB1 codes for the PB1-F2 protein [3].
Equine
influenza viruses may be spherical (with a diameter of 80–120 nm) or
filamentous. The virus is composed of a bilayer lipidic envelope containing HA
(involved in binding of the virus to host cells) and NA glycoproteins (controls
the release of virus from infected cells). The envelope is supported by
adherent matrix proteins (M1 and M2) and surrounds the nucleocapsid, which
consists of the genomic RNA segments, enclosed within a helicallyarranged NP.
PB1, PB2 and PA, which form the RNA-dependent RNA polymerase complex, are
associated with the nucleocapsid. In human influenza virus, NS1 is involved in
RNA transport, splicing and translation. NEP participates in the export of
virion RNAs out of the nucleus [4].
2.2. Disease
Like human
influenza virus, EIV is highly contagious and is contracted by inhalation. EIV
infects and replicates in the ciliated epithelial cells of the upper and lower
respiratory tract, causing de-ciliation of large areas within 4–6 days. After
an incubation period of 1–3 days, the first clinical sign of equine influenza is
an elevation of body temperature (up to 41 .C), which can last 4–5 days. A
harsh dry cough, which releases large quantities of virus, contributes to the
spread of infection. Coughing is commonly accompanied by serous and/or
mucopurulent nasal discharges. Other clinical signs of disease are myalgia,
inappetance and enlarged submandibular lymph nodes (reviewed in [1] and [5]).
However, the severity of the disease depends principally on the strain of EIV
and the immune status of the individual. Infected horses can develop
bronchitis, bronchiolitis, and interstitial pneumonia associated with
congestion and alveolar oedema.
Cases of
laryngitis, tracheitis and myocarditis have also been reported. Clinical signs
wane 2 weeks post-infection, unless there are complications due to secondary
bacterial infections
[5,6]. In
some rare cases (e.g. old or stressed horses), EIV may penetrate the basal
membrane of infected respiratory epithelial cells, a phenomenon that could
explain exceptional cases of myositis, myocarditis, oedema and encephalitis observed after
influenza infection [1]. As for human influenza, there is still some debate as
to whether systemic signs result from systemic infection or immuno-pathogenesis
induced by viral products or inflammatory cytokines. The severity of clinical
signs of disease induced by EIV infection has been correlated to the local
synthesis of interleukin-6 (IL-6) and interferon (IFN) [7]. Equine influenza
induces a high morbidity and mortality in foals, horses in poor health and donkeys.
In adults, mortality is generally associated with secondary bacterial
infections leading to pleuritis, pneumonia or purpura haemorrhagica.
2.3. Epidemiology in horses
2.3.1. EIV subtypes
Influenza A
viruses are subtyped according to the antigenicity of HA and NA. Currently, 16
HA and 9 NA subtypes have been identified among influenza A viruses [8]. All
these subtypes are found in avian species. However, influenza viruses infecting
mammals are limited to a few subtypes (reviewed in [9]). Two equine influenza
virus subtypes have been recognised, the H7N7 subtype (equi-1) and the H3N8
subtype (equi-2). Due to genome segmentation, genetic reassortment can occur in
cells simultaneously infected with two different subtypes of influenza A virus.
Reassortment of the HA or NA genes is the basis for antigenic shift but this
has not been observed in horses [2].
The H7N7
subtype was first isolated from horses in Czechoslovakia in 1956 (prototype
strain: A/eq/Prague/1/56) [10]. The last
confirmed outbreak occurred in 1979 in Italy although this subtype was isolated
in India in 1987 [11] and Egypt in 1989 [12]. Nevertheless, serological
evidence of continued circulation of this virus subtype was reported in Central
Europe and Asia as late as 1991 [13]. The H7N7 equine influenza virus may still
circulate in a subclinical form and persist at a very low level in some parts
of the world.
The H3N8
subtype of EIV was first isolated in Miami in 1963 (prototype strain:
A/eq/Miami/1/63) [14], and has caused outbreaks of disease (e.g. USA 1963; UK
1976; Europe and North America 1978–1981; South-Africa 1986; India 1987; China
1993–1994; UK 2003).
2.3.2. Interspecies transmission
Influenza A
viruses are known to have evolved from an ancestral avian precursor into
different lineages infecting specific species, including horses [9]. In 1989,
an equine influenza epidemic was reported in north-eastern China and resulted
in up to 20% mortality in some herds. The virus causing this outbreak
(A/eq/Jilin/89) was shown to be more closely related to avian H3N8 influenza
virus than to contemporary equine influenza virus [15]. Despite a successful
cross-species transmission, this strain did not spread beyond or persist in
China. However, this outbreak illustrates the role of birds, as a natural
reservoir of influenza A virus, in the potential emergence of new strains of
influenza virus able to infect mammals. Mammal-to mammal transmission of
influenza A virus also occurs (e.g. swine to human). In recent years, influenza
viruses isolated from dogs (e.g. greyhounds) suffering severe respiratory
disease with a high percentage of mortality, were identified as closely related
to equine influenza virus (H3N8) and could indicate a new case of inter-species
transmission [16].
2.3.3. Strain selection for vaccination
For
decades, commercial vaccines contained representative strains of equine H7N7
and H3N8 influenza virus. Although vaccination against H7N7 influenza virus was
successful, H3N8 influenza virus-targeted vaccines have repeatedly failed to
protect horses. This discrepancy has been attributed to differences in
virulence between the two types of equine influenza virus. Antigenic drift of
the H3N8 influenza virus also contributes to the reduced efficacy of vaccines
[17, 18]. Phylogenetic analysis revealed that since the late 1980s, H3N8 equine
influenza viruses have diverged into two different lineages (i.e. ‘American’
and ‘European’).
Antigenic
differences between ‘European’ and ‘American’ H3N8 lineages are sufficient to
compromise cross-protection [18,19] and as a result, current guidelines
recommend that vaccines against equine influenza contain a representative of
each (H3N8) lineage [20,21]. The current recommendation of the Expert
Surveillance Panel on equine influenza is that vaccines contain an A/eq/South
Africa/4/2003 (H3N8) like virus (American lineage) and an A/eq/Newmarket/2/93
(H3N8)-like virus (European lineage). The presence of H7N7 equine influenza
virus strains (i.e. A/eq/Prague/56 or A/eq/Newmarket/77) in vaccines is not
recommended any more.
Despite
widespread compliance with OIE recommendations, outbreaks of equine influenza
have continued to occur in Europe, America and other parts of the world since
1989 (e.g. Hong Kong in 1992; Dubai in 1995 and Philippines in 1997). In areas
previously free of the disease, the appearance or introduction of EIV induced
devastating outbreaks (e.g. South Africa 1986–1987, India 1987). Today, only
Australia, New Zealand and Iceland are known to remain free from equine
influenza.
2.4. Protective immune responses
The
infection of epithelial cells with EIV is likely to stimulate an innate immune
response in horses, as described in humans after influenza virus infection
[22]. However, this response has not been extensively studied in the horse. EIV
infection has been associated with the local synthesis of IL6 and IFN, which is
likely to be type I IFN (i.e. IFN/) (Fig. 1A) [7]. Both IL-6 and IFN/. are
pro-inflammatory cytokines. IL-6 is a cytokine showing a wide range of
activities on B and T cells and is involved in the development of mucosal IgA responses [23]. IFN/ presents a variety of
immunomodulatory activities affecting both innate (e.g. enhancement of natural
killer (NK) activity) and adaptive immunity (e.g. promotion of Th1 response).
The activation of NK cells, another component of the innate response to
influenza virus, is probably involved since the derivation in vitro of cells
exerting a genetically non-restricted cytotoxic activity to EIV infected target
cells has been reported [24].
Natural
infection with EIV confers a long-term immunity to re-infection.
Experimentally, ponies primed to EIV by a previous infection are protected
against clinical signs and by reverse genetics).
Fig. 1.
Immune response induced by infection with live EIV at the mucosal surface.
(A) Primary EIV infection (wild type,
reassortant, attenuated or produced virus shedding when re-challenged. Complete
clinical and virological immunity against infection with an homologous strain
was shown to persist at least 32 weeks after the primary infection, and partial
protection was still achieved after 1 year
Epithelial cells become infected inducing the
local synthesis of IFN and IL-6 . The presentation of EIV antigen in lymphoid
tissues induces the synthesis of serum or mucosal antibodies able to neutralise
excreted virus, and stimulates the development of virus-specific CTL that lyse
infected cells.
(B)
Secondary EIV infection. Virus-specific antibodies act as a first line of defence
in the case of re-infection with EIV . However, if the level of antibody is
insufficient, epithelial cell infection could occur and induce the activation
of memory responses (25)..
(Ab:
antibody; APC: antigen presenting cells; B: B cell; m: memory; NALT/MALT:
nasal/mucosal associated lymphoid tissue; Th: T helper lymphocyte; CTL:
cytotoxic T lymphocytes; CTLp: precursor CTL.)
Both humoral and cellular immune responses
have been shown to protect against EIV infection [26]. These responses are
likely to be initiated in the nasal associated lymphoid tissues (NALT) of the
upper respiratory tract, which possess a specialised epithelium known as
follicle-associated epithelium (FAE). FAE is characterised by membranous
microvillus (M) cells that deliver virus antigens to T and B cells of the underlying
lymphoid tissue [27].
2.4.1.
Humoral immune response
HA and NA
molecules are the main targets of the humoral immune response against influenza
virus. In na¨ive ponies, experimental infection with EIV induces high levels of
serum IgM (which generally decline within 50 days after infection) [25], and
virus-specific IgA, IgGa and IgGb antibody in both serum and nasal secretions
[28].
In mammals,
nasal IgA may be very important for neutralising intracellular influenza virus,
for preventing virus shedding following infection, and for cross-protection
against antigenically different influenza viruses [29,30]. Therefore, a mucosal
immune response is important to protect horses against EIV. In foals, IgA can
be induced locally in mucosa or transported from the serum to nasal secretions
in the case of dimeric IgA antibody [31]. After experimental infection of
ponies, serum and mucosal (i.e. nasopharynx and trachea) IgA rose during the
second week following infection with EIV and declined quickly (only 20% of
serum IgA remaining 2 months after the infection). The serum IgA anamnestic
response is more rapid and persistent than the primary response [25, 28].
Complement
fixing antibody levels measured by single radial hemolysis (SRH) also increased
significantly in serum 7 days after a primary infection of ponies. However, the
SRH antibody response was shown to decline rapidly (only 25% of the peak value
3 months after infection), and the majority of horses were negative for SRH
antibodies 62 weeks after exposure to EIV [25]. IgGa and IgGb isotypes are
effective in both complement fixation and antibody-dependant cellular
cytotoxicity assays in vitro [32, 33], but their effector function in vivo has
not been determined. Serum virus-specific IgGa/b antibody also increased by 7
days post-infection with EIV but was shown to decline to pre-challenge levels
over a period of 15 months [25].
Small
antigenic changes in HA and NA epitopes (antigenic drift) allow the virus to
evade the protective humoral immune system of the host [18]. There is no
cross-protection between antibodies to the H7N7 and H3N8 subtypes of equine
influenza [1].
2.4.2. Cellular immune response
EIV
infection induces a long-term immunity independent of circulating antibody.
Ponies with low or undetectable titres of HA-specific antibodies were
clinically and virologically protected from challenge infection more than 1
year after infection with EIV [25, 34]. In the absence of a detectable antibody
response, it was likely that a cell-mediated immunity (CMI) was responsible for
this protection. However, there are only a few reports on CMI after equine
influenza infection.
A major
histocompatibility complex (MHC) restricted cytotoxic T lymphocyte (CTL)
activity specific for equine influenza was demonstrated 14 days after
experimental infection [25] and a significant virus-specific CTL response was
still detectable 6 months after infection. This activity was increased by a
second influenza infection (Fig. 1B) [35].It therefore appears that, in the
absence of any detectable antibody, a CTL response is coincident with
protection against infection, although this hypothesis has not been tested in
horses. More recently, an up-regulation of mRNA encoding IFN, IL-4 and IL-2 was
shown in peripheral blood mononuclear cells (PBMC) and lymph nodes 14 days
after experimental infection [36], but the significance of this in relation to
protection is unknown. An assay measuring virus specific IFN-. synthesis has
been published recently [37] and it may be adaptable to EIV in the near future,
making measurement of cellular immunity against this virus easier.
3.
Vaccines against equine influenza virus
The
principal aim of influenza vaccination is to reduce clinical signs of the
disease, with subsequent improved animal welfare leading to a shortened
convalescent period and reducing secondary infections. Reduced shedding of
virus has important implications for the spread of infection and is certainly
the other major target that should be achieved by vaccination. Vaccination should
also provide long-term immunity, an efficient memory response and
cross-protection against influenza viruses of different strains. It has been
estimated that 70% of a given population of horses needs to be fully vaccinated
to prevent epidemics of influenza [38].
In
assessing vaccine efficacy in horses, clinical protection against equine
influenza is defined by the absence of pyrexia, and other clinical signs
induced by infection, such as nasal discharge and cough. Virological protection is defined by the
absence of virus in mucosal secretions as detected by titration of virus in
nasal swab extracts. ‘Seroconversion’ is defined as a significant increase of
antibody.
Vaccination
schedules can differ according to a country’s regulations, the type of vaccine
and the vaccine manufacturer’s recommendations. However, a standard schedule
for equine influenza vaccination requires that primarily vaccinated horses have
to receive a second vaccination within 3 months. Generally, booster
immunisations must be administrated within 6 months of the second vaccination
and at least annually thereafter, but in some cases, boosters are given more
frequently [39, 40].
Current
vaccination strategies can be divided into the administration of either ‘dead’
or ‘live’ vaccines. ‘Dead’ vaccines include killed whole virus, subunit
proteins and DNA vaccination. ‘Live’ vaccines include attenuated virus or
living virus-based vector vaccines.
3.1. ‘Dead’ vaccines
Since the
introduction of EIV vaccines in the 1960s, the majority of equine influenza
vaccines commercially available contained inactivated whole virus or subunits.
The main advantages of these vaccines are the absence of pathogenicity, virus
replication and subsequent spread between hosts. For the preparation of these vaccines, EIV has
been traditionally grown in embryonated hens’ eggs. To reduce the subsequent
reactogenicity that may occur to repeated immunisation with egg protein,
methods of tissue culture have been developed.
3.1.1. Inactivated whole influenza virus vaccines
Protection
from influenza disease conferred by conventional inactivated vaccines is
strongly associated with the levels of circulating antibodies against HA,
provided that the vaccine strain and the challenge infection strain are
genetically and antigenically similar. For example, in an early study, ponies
with a single radial haemolysis (SRH) antibody level >154 mm2 were resistant
to infection with EIV, and resistant to clinical signs of disease with SRH
antibody levels >85 mm2 [41]. In a second study, ponies were protected from
infection with an SRH antibody level >120 mm2, and from clinical signs of
disease with SRH antibody >90 mm2 [42]. It should be noted that the severity
of disease could vary depending on the method of infection (e.g. intranasal
instillation versus nebulised aerosol) and the titre of virus used for
challenge infection, and these factors
could influence the level of SRH antibody
required for protection. However, it is clear that high levels of SRH antibody
immediately prior to exposure to influenza
virus play an important role in protection (Fig. 2A).
Fig. 2.
Immune response induced by inactivated whole EIV or subunit vaccines. (A)
Vaccination by intramuscular injection induces a humoral immune response ,
producing serum antibodies able to neutralise incoming EIV . However, this
antibody response is short lived and infection could occur in the case of low
level of antibody or a significantly mistmach between the vaccine strain of EIV
and the infectious EIV . (B) Inactivated whole virus vaccine administrated
intranasally and adjuvanted with CTB induces a local immune response producing
serum and mucosal antibodies with VN activity. Ab: antibody; APC: antigen
presenting cells; B: B cell; CTB: cholera toxin B; NALT/MALT: nasal/mucosal
associated lymphoid tissue; Th: T helper lymphocyte; Tc: cytotoxic T lymphocytes.
The
antibody isotype induced by a conventional inactivated influenza virus vaccine
(A/eq/Kentucky/1/81) was analysed and shown to be composed only of short-lived
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Amorphous
aluminum hydroxyphosphate gel (alhydrogel or adjus-phos) are common adjuvants,
which, in mice, stimulate IL-4 synthesis and activate Th2 cells, with enhanced
IgG1 and IgE production [43]. Other adjuvants include polymers of acrylic acid
cross-linked with polyallylsucrose, or a lipidic, non-aluminum, dual phase
adjuvant (MetaStimTM). Carbomer, an emulsifying agent that is a polyacrylic
acid with an extremely high molecular weight, is also used as an adjuvant [44].
The intranasal inoculation of the inactivated influenza vaccine with cholera
toxin B (CTB) as adjuvant has been shown to induce a local immune response,
composed of virus-specific VN and IgA antibodies, which protects ponies from
infection with EIV (Fig. 2B) [45]. Conventional inactivated vaccines
administered intramuscularly can cause very occasional adverse reactions (e.g.
local pain and swelling, pyrexia) [1].
3.1.2. Subunit vaccines
Current subunit vaccines contains
purified HA and NA proteins. These membrane proteins (antigens) are generally
adjuvanted with Quillaia saponin (Quil A) or integrated into immuno-stimulating
complexes (ISCOMTM1) to improve their antigenicity. ISCOMsTM are formed from a
matrix (i.e. a combination of Quil A, phospholipids and cholesterol), which
incorporates membrane proteins to give stable, self adjuvanting particles, held
together by hydrophobic interactions. In other species, ISCOMTM vaccines have
shown successful protection against numerous pathogens [46].In the late 1980s,
an ISCOM vaccine containing HA antigen from A/eq/Solvalla/79 (H3N8) was shown
to induce IgGab antibody lasting about 25 weeks in trachea and nasopharynx of horses
[25].
In the mid 1990s, an ISCOMTM vaccine
(EquipTM; Pitman-Moore) containing HA protein from both A/eq/ Newmarket/77
(H7N7) and A/eq/Brentwood/79 (H3N8) was evaluated in ponies before a challenge
infection with A/eq/Sussex/89 (H3N8), 15 months after three immunisations (two
intramuscular vaccinations 6 weeks apart, and a booster immunisation 5 months
later). Vaccinated ponies showed an SRH antibody response against both virus
subtypes after immunisation. Only 3/7 vaccinates developed a mild and transient
pyrexia, but vaccinated ponies were nearly completely protected from virus
shedding after challenge
infection (six out of seven ponies)
compared with the control group (zero out of five ponies). Two vaccinates did
not even demonstrate a significant increase in antibody after infection
[47]. With an equivalent amount of
HA antigen, an ISCOM vaccine was shown to induce SRH antibodies more
efficiently than an inactivated whole virus vaccine [42]. More recently,
an ISCOM-based vaccine containing HA
antigen from Newmarket/77 (H7N7), Borl¨ange/91 (H3N8) and Kentucky/98 (H3N8)
(EQUIP F, Schering-Plough Animal Health) provided
strong
protective immunity against EIV challenge.
Ponies received two intra-muscular
immunisations, 6 weeks apart, and were challenge infected with Newmarket/1/93
influenza virus 4 weeks after the second vaccination. Serum
SRH antibodies increased after the
first immunisation and were boosted after the second vaccination.
Virus-specific antibodies were predominantly of the IgGa and IgGb isotypes
(the best mediators of complement
fixing (CF) antibody and antibody-dependent cell cytotoxicity). Virus-specific
IgGc and IgG(T) increased after the second immunisation. In nasal
secretions, only virus-specific IgG
antibodies were detected. Vaccinated ponies were completely protected against
clinical signs of the disease but 43% showed virus shedding after
challenge infection [48]. In another
study, the mucosal administration of this ISCOM-based vaccine in systemically
primed ponies increased the level of nasal IgA after challenge infection,
induced a full protection against
clinical signs of disease and reduced the duration of virus excretion compared
with unvaccinated controls [49]. However, immunity induced by
1 ISCOM is a trademark of CSL
limited. sub-unit vaccines is limited
and dependent on the role of the particular antigens in protection. Ponies
vaccinated with a subunit vaccine containing Quil A as an adjuvant failed to
induce an influenza-virus specific CTL activity [35].
In humans,
an experimental ISCOM based vaccine for influenza containing HA molecules has
been shown to increase the humoral immune response and could have some effects
on CMI after vaccination compared with a conventional inactivated vaccine [50].
In summary, the efficacy of
inactivated whole virus or subunit vaccines is based on their ability to
stimulate high levels of SRH antibody that are required for protection.
However, this antibody response is short-lived, could be highly specific, and
subsequent protection could be sensitive to antigenic variation of influenza
viruses. In horses, the stimulation of an influenza virus-specific CMI by these
vaccines has not been demonstrated. Comparison of the differences between
immune responses induced by immunisation with killed virus or subunit vaccines
and viral infection suggest that vaccine design can be improved.
3.1.3. DNA vaccines
Administration of DNA plasmids
offers a different form of vaccination
and efficient protection against influenza infection has been demonstrated in several species (e.g.
mice, ferrets, chicken [51,52]). DNA
vaccination results in the in vivo
expression of antigenic proteins, leading to the stimulation of both humoral and cellular immune responses.
This approach is therefore advantageous compared with inactivated whole virus
or sub-unit vaccines. Technically, plasmid DNA is inexpensive to produce, has
good stability and can be lyophilised for long-term storage.
In mice, Olsen et al. [53]
demonstrated that gene gun delivery of plasmid DNA encoding the
A/eq/Kentucky/1/81 (H3N8) HA protein provided full protection against challenge
infection, with induction of VN antibody, serum virusspecific IgG antibodies
and serum IgA [53]. The impact of skin and mucosal immunisation with a plasmid
DNA vaccine coding for the HA protein of A/eq/Kentucky/1/81 influenza virus was
therefore studied in the horse [36,54]. Ponies were immunised by gene gun
injection at skin and mucosal sites (60 sites of injection per immunisation, 3
immunisations in total, around 60 days between each one). Vaccinated horses
were partially to completely protected against clinical signs of the disease
and virus shedding induced by challenge infection with a homologous strain of
influenza virus, 30 days after the last vaccination. DNA vaccination induced
serum IgGa and IgGb antibody responses (i.e. an isotype response similar to EIV
infection) but no mucosal IgA responses were
detected. Virus-specific
lymphoproliferative responses and up-regulation of IFN-. mRNA were also observed
after three immunisations with the DNA vaccine but differed markedly from
responses induced by equine influenza vrius infection in terms of regional
distribution (i.e. peripheral blood, draining or hilar lymph nodes) [36].
Protection resulting from DNA vaccination was associated with the presence of
both or the intramuscular injection of live vector-based vaccines will induce
the expression of EIV antigens and stimulate both humoral and cellular immune
responses . Serum antibodies will neutralise infectious EIV
and
EIV-specific CTL will lyse infected epithelial cells . (B) The administration
of DNA vaccine via the nasal route with CTB as adjuvant, has been shown to
induce the synthesis of a mucosal antibody response . Ab: antibody; APC:
antigen presenting cells; B: B cell; CTB: cholera toxin B; NALT/MALT:
nasal/mucosal associated lymphoid tissue; Th: T helper lymphocyte; Tc:
cytotoxic T lymphoctes. serum IgGa/IgGb antibodies and mucosal IgG, supporting
the idea that mucosal IgA is not essential for protection (Fig. 3A) [36,54].
Absence of virus shedding was correlated with the presence of mucosal IgGb at
the time of challenge infection [54]. However, as IgA has been shown to be an
important part of the immune response induced by natural infection, DNA
vaccines were modified in an attempt to achieve such a response.
Co-administration of DNA coding for IL-6 failed to improve either IgA synthesis
or protection from infection but may have caused a shift to a Th-2 like
antibody response [36]. Another recent study showed nasal IgA synthesis after
two intranasal DNA-HA vaccinations (33 days apart, derived from strain
A/eq/Kentucky/1/81) followed by two skin/mucosal vaccinations (36 days apart). Co-administration of cholera toxin (CT) and
CTB, which stimulate both humoral and cellular immune responses, as adjuvant
increased nasal IgA titre after vaccination compared with the DNA-HA
vaccination alone (Fig. 3B). IgA and IgGb antibody responses were also
increased in CT + DNA-HA vaccinated ponies after challenge infection with EIV
(81 days after the last immunisation, strain A/eq/Kentucky/1/81) compared with
unvaccinated ponies. However, vaccinated ponies were only partially protected
against clinical signs of disease and virus shedding [55]. Interestingly, some horses
were clinically protected, despite very low levels of serum antibody at the
time of challenge infection.
In summary, DNA vaccination was
shown to mount a protective influenza-specific immune response in horses with
good longevity compared with conventional inactivated virus vaccines. However,
studies have shown some variability in the level of protection from clinical
signs of disease and prevention of virus shedding achieved. The method of
administration of DNA vaccines (multiple sites of inoculation and multiple
injections for each immunisation) is obviously impractical for veterinary
practice. A DNA vaccine against West Nile virus has been recently licensed in
North America and will allow the evaluation of DNA vaccination feasibility
in the field.
Fig. 3. Immune response induced by
DNA or live vector-based vaccines. (A) A skin DNA immunisation
3.2. Live virus/vector vaccines
Hannant et al. [34] demonstrated
that the immune response induced by experimental infection with equine
influenza virus protects horses against re-infection for up to 1 year
[34]. These results suggest that an
equine influenza virus vaccine mimicking natural infection should be more
efficient in affording protection than conventional inactivated vaccines.
The use of a modified/attenuated
live influenza virus or a live virus-vector coding for EIV proteins are the
main approaches investigated in the last 20 years to produce a live equine
influenza vaccine.
3.2.1. Live attenuated influenza virus vaccine
Immunisation with a live attenuated
influenza virus closely mimics natural infection. Antigens are presented to the
immune system via both exogenous and endogenous pathways, and vaccines are
therefore expected to stimulate an immune response similar to those induced by
infection (Fig. 1A).
3.2.1.1. Reassortant
influenza virus.
The genome of influenza virus
consists of eight single strand RNA segments. Genetic reassortment can occur
when a cell is infected simultaneously with two different subtypes of influenza
A virus. An influenza virus with a limited growth in mammals (e.g. avian
influenza virus) can be reassorted with a mammalian influenza virus to obtain a
live attenuated virus able to induce a protective immune response. A
reassortant expressing internal proteins of the avian influenza virus A/Duck/
New York/6750/78 (H2N2) and surface glycoproteins ofthe equine influenza virus
A/eq/Georgia/1/81 (H3N8) was evaluated for efficacy and safety in hamsters and
ponies. Exposure of ponies to this reassortant induced mild clinical signs
compared with the parental equine influenza virus. The reassortant induced an
antibody response and was shed after infection. Five-and-a half months after
the exposure to the reassortant, ponies were partially protected from a
challenge infection with the parental equine influenza virus, the duration of
virus shedding was also slightly reduced [56]. The limited protection observed
in this study may have originated from the immune response raised against
equine influenza surface glycoproteins born by the reassortant. The response
raised against avian internal proteins probably played at best a limited role
in protection
against the equine influenza virus.
Vaccines based on avian/equine reassortants certainly would be too risky to
use. Reassortants could be safe in horses but induced disease in birds [57].
Furthermore, avian influenza virus can transmit directly to horses and has
caused a severe outbreak in the past (i.e. equine influenza in China during
1989 cause by an avian influenza (H3N8) virus [15]). Also taking into
consideration recent infections of humans
with avian influenza virus in Asia,
the use of such reassortants for horse vaccination would raise a lot of safety
concerns.
3.2.1.2. Temperature-sensitive (Ts) or cold-adapted influenza virus.
A temperature-sensitive (Ts)
influenza virus is expected to show a significant decrease (up to 100-fold) in
titre at a restrictive temperature (e.g. 39 .C) compared with the titre
obtained at a permissive temperature (e.g. 34 .C). In vivo, Ts influenza virus
multiplies efficiently in the cooler environment of the upper respiratory tract
where they induce local and systemic immune responses. However, replication is
inefficient in the warmer environment of the lower respiratory tract in horses
(38–39 .C), where replication of wild type virus may induce bronchitis,
bronchiolitis, interstitial pneumonia, congestion and oedema [5]. It has also
been
speculated that the high body
temperature of horses might repress virus reversion [58].
In the late 1980s, the Ts clone 8B1
(bearing a Ts lesion on the PA gene) was derived from the re-assortment of a
human influenza A Ts donor virus (bearing Ts lesions on the polymerase
and NP genes) with a wild type
A/eq/Cornell/16/74 (H7N7) equine influenza virus. In the horse, intranasal
instillation of clone 8B1 did not cause detectable clinical signs or febrile
responses. Vaccine virus that was shed retained the Ts phenotype. When
challenge infected (28 days after the last vaccine exposure) with the parental
equine H7N7 influenza virus, ponies were partially to fully protected from
clinical signs of the disease and virus shedding (21 of 40
vaccinated ponies did not shed virus
after challenge infection) [59]. In the mid 1990s, clone 8B1 virus was used as
the donor virus with the wild type A/eq/Kentucky/1/81 (H3N8)
influenza virus to produce an H3N8
Ts reassortant virus (Clone 255). Ponies vaccinated by a nebulised suspension
containing the reassortant virus showed no clinical signs after
immunisation with Clone 255 but did
seroconvert for haemagglutination inhibiting (HI) antibody after exposure to
the reassortant, indicating antigenicity of the reassortant. When
they were challenge infected with
the A/eq/Kentucky/1/81 parental influenza virus 1 or 2 months after immunisation,
vaccinated ponies were fully protected against clinical signs
of disease but virus shedding was
observed in 20–33% of vaccinated ponies. Ponies remained protected against
clinical signs of disease when they were challenged 7 and 10 months
after immunisation, but the
percentage of ponies shedding virus increased to 55% [58].
Cold adapted, Ts live equine
influenza viruses were also produced by serial passage in embryonated hens’
eggs at temperatures gradually reduced to 26 .C [60]. More recently,
a cold-adapted, Ts, modified-live
equine influenza virus vaccine (Flu Avert®2 I.N. Vaccine; Intervet) derived
from the wild-type A/eq/Kentucky/1/91 (H3N8) influenza virus, has been
evaluated and is available commercially in north America. With one nasal mucosal
immunisation, vaccinated ponies were fully protected from clinical signs of the
disease
when challenge infected 5 weeks
later with the parental A/eq/Kentucky/1/91 influenza virus. Vaccinates showed
significant clinical protection when re-challenged 6 months
(2 Avert is a registered trademark
of Heska corporation. ) post-vaccination and lower rectal temperatures when
challenged 12 months after vaccination. Virus shedding was also strongly
reduced after vaccination. Only 20% of vaccinates shed virus when challenge
infected 5 weeks post-vaccination. Six and 12 months post-vaccination, the
duration of virus shedding had decreased significantly compared with
unvaccinated control ponies. Although no correlation between SRH antibody
response and protection was observed, vaccinated ponies developed an anamnestic
SRH response after challenge [61]. This vaccine has also been shown to protect
ponies against infection with an heterologous influenza virus [62]. Vaccination
with these live attenuated vaccines induced longterm immunity that clearly
limits the duration and severity of
clinical signs and the shedding of
virus after challenge infection with equine influenza virus. However these
vaccines did not provide a sterile immunity.
The use of modified/attenuated-live
influenza virus vaccines has always raised concerns due to the high mutation
rate and potential reassortment of influenza virus. The segmented nature of the
influenza virus genome could allow for reassortment with a cocirculating
wild-type virus and subsequent loss of attenuation or/and emergence of a new
highly pathogenic influenza virus. In humans however, a live attenuated
influenza virus vaccine (mixture of two type A viruses (H1N1 and H3N2) and a
type B virus) has been approved and is currently in use (FluMist®3). Moreover,
Russia and Eastern European countries have been using live human influenza
vaccines for decades in widespread vaccination programmes, without the
emergence of new influenza virus reassortants. In horses, studies have revealed
the phenotypic stability of
the cold adapted, Ts modified live
equine influenza virus used as vaccine, its low spontaneous transmission to
unvaccinated ponies [62], and the absence of clinical signs of disease induced
by vaccination in ponies with exercise-induced immunosuppression [63].
3.2.2. Virus-based vector vaccines
Live recombinant vector vaccines are
constructed by inserting selected genes from the pathogen of interest into
live, infectious, but non-disease-causing viruses. With virusbased
vector vaccines, viral antigens are
expressed and synthesised de novo within the infected cell. Thus, because they
are presented via MHC class I (endogenous) and class II (exogenous)
antigen-processing routes, the selected viral antigens can stimulate both humoral
and cellular immune responses (Fig. 3A). This strategy of vaccination has been
the focus of intense scientific investigation in many species, including the
horse.
Recombinant poxviruses have been
widely used for vaccination [64,65].
Poxviruses are genetically stable and allow the insertion of a large segment of
foreign DNA coding for
selected antigens. Recombinant
poxviruses derived from vaccinia or avipoxvirus (i.e. canarypox virus) are
commercially (3 FluMist is a registered trademark of MedImmuneVaccines Inc.)
available and several recombinant
canarypox-based vaccines have been developed for the horse [66,67].
In the late 1980s, immunisation of
horses with recombinant vaccinia virus expressing HA and NA proteins of equine
influenza virus induced antibody responses successfully (VN, HI and SRH) [68].
More recently, Breathnach et al. [69] investigated the efficiency of a priming
immunisation with a DNA-vaccine coding for HA and NP proteins (Kentucky/1/81)
and booster vaccinations, 6 and 10 weeks later with a modified vaccinia Ankara
(MVA) virus encoding the
same proteins. MVA virus is a highly
attenuated strain of vaccinia virus showing a defect in replication, which is
frequently used in prime-boost vaccination protocols. Following extensive in
vitro passage, the parental vaccinia virus became replication-deficient in
virtually all mammalian cells [65]. Primary DNA vaccination did not induce
detectable virus specific humoral or cellular immune responses, but booster
administration of recombinant MVA strongly induced virusspecific serum IgGa and
IgGb as well as a virus-specific lymphoproliferative response and up-regulation
of IFN-. mRNA. Interestingly, both HA and NP antigens stimulated humoral and
cellular immune responses. The administration of a second booster vaccination
had little effect on virus specific antibodies or lymphoproliferative responses
but up regulated IFN-. mRNA. The efficiency of such constructs against EIV
infection was investigated in a recent study. Ponies were vaccinated with
MVA-HA alone (three immunisations) or as part of a prime boost regimen
including a DNA priming and two subsequent boosts with MVA-HA or MVA-NP (e.g.
DNA-NP plus MVA-NP). Ponies vaccinated
with MVA-HA alone or associated with
a DNA prime were significantly protected against clinical signs of disease and
virus shedding after infection with EIV when compared with unvaccinated ponies.
Vaccination stimulated both humoral and cellular immune responses, including an
IgA antibody response in nasal secretion. Protection induced by vaccination
with MVA-NP was lower but could be
explained by the absence of a VN antibody response, as NP is an internal
protein of EIV [70]. These studies provided evidence that recombinant MVA
vectors coding for EIV proteins efficiently stimulated the immune system and
afford a good level of protection.
In 2003, a modified live canarypox
virus-vectored influenza vaccine (ProteqFlu®4; Merial Ltd., UK) was licensed in
the European Union for use in horses. This live, vectored vaccine is safe, as canarypox
virus undergoes an abortive infection in mammalian cells [71]. This vaccine
contains
two canarypox recombinant viruses
coding for the HA proteins from Newmarket/2/93 and Kentucky/94 EIV strains
(European and American representative strains, respectively) adjuvanted with
Carbormer 974P. The efficacy of this new commercialised vaccine has been
recently studied [72,73]. Na¨ive ponies received one or two intramuscular
immunisations
with ProteqFlu® and were challenged
14 days after (4 ProteqFlu is a registered trademark of Merial Ltd. )the last
immunisation with a virulent strain of EIV (Newmarket/5/03; isolated recently
in the UK that induced severe disease after field infection). After the first
immunisation, all vaccinated ponies developed detectable levels of SRH
antibodies and a few ponies mounted an anamnestic response after the second
immunisation. Singly vaccinated ponies did not shed virus and were protected or
developed only mild signs of the disease after challenge infection with EIV and
similar results were obtained with ponies vaccinated twice.
Virus excretion was almost
completely suppressed in vaccinates [72]. An increased IFN-. protein synthesis
was also measured in vaccinated ponies after challenge infection with
EIV [73]. ProteqFlu® is therefore
successful in preventing infection with related strains of EIV. The immunity
induced by this vaccine is directed against HA proteins that are subject to
antigenic drift. Therefore regular updating of the vaccine will be required to
ensure continued efficacy. However, if there is a significant mismatch between
vaccine HA and circulating virus, the first line of defence (i.e. antibody) may
fail and a virus-specific T cell response would become important in promoting
recovery and virus clearance. Further work is required to evaluate the benefit
of including additional recombinant live vectors coding for the more conserved
NP or M proteins, which are postulated to contain immunodominant CTL epitopes
(and shown to induce a good level of protection in the case of NP [70]), to
reinforce the virus-specific T cell response provided by vaccines such as
ProteqFlu®.
Systemic inoculation is the most
frequent route of poxvirus-based vaccination. Few studies have evaluated the efficacy
of recombinant poxviruses delivered by mucosal routes. In mice, oral
immunisation with a MVA-based influenza vaccine afforded complete protection
against a
lethal influenza challenge [74]. In
humans, canarypox virus is not considered as a strong mucosal immunogen
[75].However, intranasal immunisation with canarypox virus-based
vectors coding for the canine
distemper virus (CDV) haemagglutinin and fusion genes protected juvenile
ferrets against an intranasal challenge with virulent CDV [76]. In horses,
mucosal influenza vaccination with
poxvirus-based vectors remains to be evaluated.
One concern with poxvirus-based
vaccines is the presence of pre-existing immunity specific to poxvirus and its impact
on subsequent vaccination with the same recombinant. Suppression of the
antibody response to recombinant antigen encoded by a vaccinia-based vector has
been
reported [77,78]. However, at least
in horses, vaccination with canarypox-based vaccines had no discernable effect
on the efficiency of subsequent immunisation using the same vector coding for
homologous or heterologous antigens (Dr. J.H. Minke, personal communication).
3.2.3. Live attenuated influenza virus designed by reverse genetics
Reverse genetics allows the
generation of entirely artificial recombinant influenza viruses from cloned DNA
plasmids [79]. In the mid 1990s, application of the reverse genetics method to
equine influenza virus was discussed after the generation of an attenuated
influenza virus by the insertion of a mutation in the cytoplasmic tail of NA
protein [80]. Ten years elapsed before the generation by reverse genetics of
attenuated equine influenza viruses encoding a carboxy-terminus truncated NS1
protein [81]. Briefly, an infectious recombinant virus was recovered after the
transfection of a dog kidney cell line (MDCK) with plasmids coding for each of
the eight influenza RNAs (PB1, PB2, PA, HA, NP, NA, M and NS) simultaneously
with protein expression plasmids (PB1, PB2, PA, NP and NS1). Derived from the
A/eq/Kentucky/5/02
(H3N8) influenza virus, the 3
recombinant viruses obtained showed a low replication capacity both in vitro in
MDCK cells, and also in vivo in embryonated hens’ eggs (9 to 11-dayold) or in
mice after an intranasal infection. The recombinant viruses ability to inhibit
the production of IFN/. by infected cells (previously associated with the NS1
protein in wild type virus [82]), was also decreased [81]. However, recombinant
virus replicated well in 7-day-old embryonated hens’ eggs, which lack a
competent IFN system compared with 9 to 11-day-old eggs. An unaltered IFN/. synthesis
induced by cellular infection with recombinant viruses is interesting
because these cytokines possess a
powerful adjuvant activity when co-administered with a human influenza vaccine,
inducing a Th1 immune response and protection against challenge
infection in mice [83]. An equine
influenza virus with an altered NS1 protein could be a good master strain for
live attenuated viral vaccines, reverse genetics allowing the insertion of HA
and NA genes of epidemic equine influenza virus strains. The same approach has
been suggested in the context of human vaccination by Schickli et al. [79],
Reverse genetics also allows the
generation of ‘cocktail’ virus vaccines as exemplified by the recent live
bivalent vaccine for parainfluenza and influenza virus. This recombinant
influenza virus (A/WSN/33, H1N1)
possesses the coding regions for the hemagglutinin/neuraminidase ectodomain of a
murine parainfluenza virus type 1 (Sendai virus) instead of the influenza virus
neuraminidase. Vaccinated mice were protected from a lethal challenge infection
with both parental viruses [84]. However, such combination needs attention in
the gene selection. In this case, the sialidase activity of the hemagglutin/neuraminidase
ectodomain of the parainfluenza virus compensated for the lack of influenza
virus neuraminidase necessary for the bivalent virus to replicate.
Reverse genetics is an efficient
method to generate recombinant live attenuated influenza virus. However, such
recombinants for equine influenza virus remain to be tested in vivo
in horses to assess their
antigenicity and efficacy in protection against challenge infection.
3.3. Effect of maternal antibodies and age on equine
influenza vaccination
Foals are highly sensitive to
disease. Their only defence against pathogens are maternal antibodies acquired
through the ingestion of colostrum during the first hours of life. This
passive immunity can protect foals
against influenza. However, the duration of maternal antibodies in foals is
controversial [85–87], but antibodies specific for equine influenza
generally decrease to a low but
detectable level by 6 months [88,89]. Protection of foals can be improved by
increasing the titre of protective antibodies in the colostrum, mainly by
influenza vaccination of the mare a few weeks prior to foaling. Thoroughbred
foals are generally vaccinated early to avoid any windows of susceptibility
(when protection against
influenza is not anymore assumed by
maternal antibodies and not yet by vaccination). However, residual maternally derived
antibodies can reduce vaccine efficiency, at least in
the case of inactivated, sub-unit or
attenuated virus vaccines [87,89–91]. The development of tolerance in 3 months
old foals to influenza subunit vaccination has been reported [88]
but remains controversial [89].
Primary vaccination should therefore occur after the complete disappearance of
maternal antibodies (i.e. after 6 months of age). The use of virus-based
vectors for equine influenza in
foals could circumvent the neutralising effect of maternal antibodies on
vaccine efficacy. A second factor that influences vaccine efficacy is the
foal’s weak immune response to
vaccination, which results in poor protection, and have been associated with an
immature activity of antigen presenting cells compared with adult horses [92].
Thus, the use of vaccines specifically designed to induce a strong CMI could be
less effective than expected. Mechanisms driving the maturation of the
neonate’s immune
system are not yet fully understood,
and as a result, effective vaccination schedules in foals still need
investigation.
Another factor that influences the
efficacy of vaccination is old age. The immune senescence observed in human and
equine populations presents a few similarities [93]. B and T cell activity is
altered [94,95] and titres of EIV-specific antibody are lower in old animals
when compared with younger animals [94,96]. The use of live modified/attenuated
equine influenza virus as vaccine could be a problem in this context of
immunosuppression. Therefore, annual vaccination with a conventional
inactivated or sub-unit vaccines is likely to be safer in this particular
population. Further studies will be necessary to evaluate the effect of age on
other approaches to vaccination (e.g. DNA, poxvirus-based vectors).
4. Conclusion
Today, the main types of equine
influenza vaccines in use contain whole inactivated virus or subunits.
Protection afforded by this first generation of vaccines is based on high levels
of protective antibodies. Second generation vaccines (i.e. live attenuated and
poxvirus-based vaccines) are now available. These stimulate both humoral and
cellular immune responses and so mimic more closely the protective immunity induced
by natural infection with influenza virus. These vaccines are not yet widely
used and time is necessary to evaluate their performance in the field. The
delivery routes of DNA vaccines remain a major issue, despite their ability to
protect horses against influenza. It is anticipated that live attenuated
influenza virus engineered by
reverse genetics will almost certainly be the next generation of vaccines
against equine influenza. A large battery of weapons is therefore becoming
available to the equine clinician.
Several laboratory animal models (e.g. rodents, ferrets and monkeys) are used
to evaluate vaccine efficacy against influenza infection. However the horse
model allows field studies of a range of vaccine techniques in the natural host
population, and integration of natural external factors (e.g. population dynamics,
genetic diversity) that can influence the outcome of vaccination. Therefore,
the horse is an interesting model of vaccination in the battle against the
evolutionary strategy of influenza viruses.
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