METHOD OF ELICITING AN IMMUNE RESPONSE AGAINST PANDEMIC
INFLUENZA VIRUS
FIELD OF THE INVENTION
This invention relates to a method of eliciting or inducing an immune response in a subject
against pandemic subtypes of influenza virus, and more particularly to a method of eliciting
or inducing an immune response in the subject which protects the subject against subsequent
challenge with a pathogenic, pandemic subtype of influenza A, such as the avian influenza A
(H5N1) that has caused infections in humans.
BACKGROUND OF THE INVENTION
Influenza is a major cause of disease in humans and a source of significant morbidity and
mortality worldwide with large segments of the human population affected every year.
Influenza viruses can be subtyped into A, B and C. The majority of viruses that circulate in
the human population are influenza A and B.
Annual vaccination is the primary strategy for preventing infections. The A strain of
influenza can be further subtyped, based on the antigenic differences of the two viral surface
transmembrane proteins, Haemagglutinin (HA) and Neuraminidase (NA). To date 16 HA
(HAl-16) and 9 NA (NAl-9) glycoprotein subtypes of influenza A viruses have been
identified. At present, two subtypes of influenza A circulate in humans (HlNl and H3N2)
[1].
On occasion, an influenza pandemic can occur when a new influenza virus emerges for
which people have little or no immunity. In the past century, three influenza A strain
pandemic outbrealcs have caused significant human influenza-related fatalities (1918, HlNl;
1957, H2N2; 1968, H3N2) [2]. In Hong Kong in 1997, a highly pathogenic H5N1 avian
influenza virus was transmitted directly from chickens to humans, causing six deaths from
18 confirmed infections [3;4]. Since this time, concern regarding an influenza pandemic has
been heightened by sporadic outbreaks of pathogenic H5N1 viruses. These outbrealcs have
resulted in 258 cases with 153 deaths across six countries, with outbreaks from Asia through
to Eiurope (Cambodia, China, hidonesia, Thailand, Turkey, and Vietnam) [5].
Since 1997, viruses of several other subtypes, including H2N2, H9N2, H7N7, H7N3 and
H10N7, have also been implicated in human infections and consequently these subtypes also
represent a significant pandemic threat. Because it is not possible to predict which subtype of
influenza virus will cause the next pandemic, an ideal vaccine would protect the host from
severe disease or death by eliciting an immune response that protects the host against a broad
range of influenza viruses, from the same or different subtypes. However, for the reasons
outlined below, the available vaccines, which rely on the induction of a neutralising antibody
response (primarily against HA and NA), are highly influenza strain-specific.
The HA and NA glycoproteins of influenza viruses undergo antigenic variation as a means
to escape the host immune response [6]. The presence of virus neutralising antibodies
specific for the HA glycoprotein at systemic or mucosal sites protects against infection with
influenza. However, as a consequence of antigenic variability the antibody response to HA is
highly strain-specific, and does not recognise the HA from influenza viruses of different
subtypes, or even highly divergent strains within the same subtype [7]. Cell-mediated
immunity on the other hand, in particular CDS"*" cytotoxic T cells (CD8'^ CTL) is primarily
responsible for clearing virus-infected cells, and thus limits the severity of, and promotes
recovery from infection [8]. In confrast to HA, the internal protein targets of the cell-
mediated immune response - the key ones being PB2, PA, Nucleoprotein (NP) and Matrix
protein (M) - are not prone to antigenic drift and as a consequence are highly conserved. For
example, the NP and M proteins of the H5N1 strains A/Indonesia/5/05 and
A/Vietnam/1194/04 share approximately 94% amino acid identity with A/Puerto Rico/8/34
(as shown in Fig.l). AyPuerto/Rico/8/34 is an HlNl virus isolated in 1934, and the source of
the structural proteins for the engineered vaccine sfrains, fraditionally prepared by re-
assortment and more recently by reverse genetics. Furthermore, there is a high degree of
conservation of CTL epitopes between these different influenza subtypes. Extension of this
analysis to include other virus subtypes, including H7N7 and H9N2, which also pose a
potential pandemic threat, demonstrates a high degree of conservation of CTL epitopes
across all A-strain viruses. Therefore, unlike the HA antibody response which is highly
strain-specific, CTL responses have the potential to be broadly effective, irrespective of the
influenza A-strain subtype [9]. Therefore, the ability to induce a strong CTL response is a
highly desirable feature for a pandemic influenza vaccine.
The induction of CDS"*" CTL, particularly in humans, has to date proven to be a significant
hurdle for vaccine development. Delivery systems such as DNA and viral vectors have
offered some hope, but have potential safety concerns, and in the case of DNA, generally
elicit poor cellular responses, in particular CDS'*" CTL responses. Additionally, viral vectors
have the problem of inducing neutralising antibodies to the vector, which limits repeated use.
Prime-boost combinations of DNA and live viral vector delivery are currently being
evaluated, and although results have been promising in animal models, they are yet to be
proven in humans, ISCONF'^ vaccines have been shown in numerous animal models, to be
potent inducers of both T-helper (CD4'^ and CTL (CDS'*") T cell responses to a wide variety
of antigens, including naturally occurring immunogens and recombinant proteins [10]. An
HlNl influenza ISCOIVF^^ vaccine has been shown to confer cross protection in mice
against lethal challenge with heterologous viruses, including H2N2, H3N2, H5N1 and H9N2
viruses [11]. Furthermore, protection was shown to be dependent on both CDS"*" T cells and
antibody [11].
It is generally accepted that the ability of ISCOM'^^ vaccines to induce strong CD8"^ CTL
responses is largely due to the fact that the antigen is incorporated into the ISCOMi""^
particle [12], which results in efficient cellular uptake and subsequent access of the antigen
to the MHC Class I processing machinery [13]. However, the manufacture of ISCOlVfM
vaccines is complex, difficult to scale up, and there are significant problems associated with
manufacturing control and consistency. Therefore ISCOlVn''^ vaccines, despite
demonstrating protection against a range of pathogens in a wide variety of animal species,
have limited product potential, particularly for high volume products such as pandemic
influenza vaccine which would demand the production of hundreds of millions of doses in a
short time frame.
hi work leading to the present invention, the inventors have developed a vaccine formulation
in which preformed ISCOMATRDC^'^ adjuvant which is "immunogen-free" in that it has
essentially the same composition and structure as the ISCOM'^'** vaccine but without the
incorporated antigen[12;14], is combined or mixed with influenza immunogen(s) such as the
standard tri-valent seasonal influenza vaccine as described below. Thus, in contrast to
ISCOM"^" vaccines, the influenza immunogen(s) in the vaccine formulation of the present
invention are not incorporated into the ISCOMATRDC"'^ adjuvant structure. Accordingly,
the production of the vaccine formulations of the present invention is simple, robust and
reproducible, and can be performed at a large scale.
Furthermore, the work leading to the present invention has demonstrated the ability of the
vaccine formulation comprising standard endemic influenza immunogen(s) to protect against
lethal challenge with a highly pathogenic, pandemic (H5N1) subtj^^e of influenza A virus,
using ferrets as an animal model.
Throughout this specification and the claims which follow, unless the context requires
otherwise, the word "comprise", and variations such as "comprises" and "comprising", will
be understood to imply the inclusion of a stated integer or step or group of integers or steps
but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it),
or to any matter which is known, is not, and should not be taken as an acknowledgment or
admission or any form of suggestion that that prior publication (or information derived firom
it) or known matter forms part of the common general knowledge in the field of endeavour
to which this specification relates.
SUMMARY QF THE INVENTION
The present invention provides a method for eliciting or inducing a protective immune
response in a subject against a pandemic subtype of influenza virus, which comprises
administering to the subject a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant.
In another aspect, the present invention provides the use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immimogen-free immunostimulating complex as adjuvant,
in the manufacture of a medicament for administration to a subject to elicit or induce a
protective immune response in the subject against a pandemic subtype of influenza virus.
In yet another aspect, the invention provides the use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant,
to ehcit or induce a protective immune response in a subject against a pandemic subtype of
influenza virus.
In a further aspect, the invention provides an agent for eliciting or inducing a protective
immune response in a subject against a pandemic subtype of influenza virus, wherein said
agent is a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an amino acid sequence comparison of (A) Nucleoprotein (NP), and (B)
Matrix protein (M) from A/Puerto Rico/8/34 (HlNl), A/Indonesia/5/05 (H5N1) and
A/Vietnam/l 194/04 (H5N1). The bolded letters are the described human CTL epitopes
(adapted from Suzanne L. Epstein, Jonathan W. Yewdell, Jack R. Bennink,
http;//www.flu.lanl.gov/review/epitopes.htinl). The NP & M proteins of A/Indonesia/5/05
and A/Vietnam/1194/04 share approximately 93% amino acid identity with A/Puerto
Rico/8/34.
Figure 2 shows HI antibody (A) and Virus Neutralisation titres (B) of sera from ferrets
inoculated with either (i) tri-valent seasonal influenza vaccine (containing 15|j.g of A/New
Caledonia/20/99, A/Wisconsin/67/2005 and B/ Malaysia/2506/2004) combined with
ISCOMATRIX™ adjuvant (60fj,g); (ii) A/Vietnam/1194/2004 combined with
ISCOMATRIXTM adjuvant (60ng); or (iii) ISCOMATRIX™ adjuvant alone (adjuvant
control). Sera was collected at day 28 after second vaccination and titrated against
AA^ietnam/1203/2004 and A/Indonesia/5/2005. Data are presented as the average of four
animals per group. Standard deviation is indicated.
Figure 3 shows survival (A), change in body temperature (B), and change in weight
(C) of ferrets challenged with 10* 50% egg infectious dose (EIDso) of A/Vietnam/l203/2004
following immunization with either, (i) tri-valent seasonal influenza vaccine (containing
15 fig of A/New Caledonia/20/99, A/Wisconsin/67/2005 and B/ Malaysia/2506/2004)
combined with ISCOMATRIX™ adjuvant (60^g); (ii) AA^ietnam/1194/2004 combined with
ISCOMATRIX™ adjuvant (eO^g) or (iii) ISCOMATRIX™ adjuvant alone (adjuvant
control) as indicated. Data are representative values of the four animals per group. In Figure
3A, post viral challenge ferrets were weighed and examined physically daily. Ferrets that
had lost more than 10% of body weight or showed signs of distress such as tremor or
abdominal discomfort were euthanized for ethical reasons. In Figure 3B, ferret temperature
was monitored daily by use of a subcutaneous implantable temperature transponder. The
vertical line represents time of challenge. The mean body temperature of an uninfected
ferret is 38.8°C. Data are representative values of the four animals per group. In Figure 3C,
ferret weights are represented as the percentage of the weight of the animal at the time of
challenge. Data are representative values of the four animals per group.
Figure 4 shows morbidity scores obtained with ferrets immunised with either: (i) tri-
valent seasonal influenza vaccine (containing 15)ag of A/New Caledonia/20/99,
A/Wisconsin/67/2005 and B/ Malaysia/2506/2004) combined with ISCOMATRIX™
adjuvant (60ng); (ii) AA^'ietnam/ll94/2004 combined with ISCOMATRIX™ adjuvant
(60ng); or (iii) ISCOMATRIX™ adjuvant alone (adjuvant control) as indicated. Following
challenge with 10* 50% egg infectious dose (EID50) of A/Vietnam/l203/2004, ferrets were
monitored for behavior and given a morbidity score based on the following scale (0 =
playful and alert, 1 = alert but playful only when induced to play, 2 = alert but not playful
when stimulated, 3 = neither alert or playftil, 4 = exhibiting physical symptoms necessitating
euthanasia, as described Materials & Methods.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a method for eliciting or inducing a protective
immune response in a subject against a pandemic subtype of influenza virus, which
comprises administering to the subject a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant.
Preferably, the subject is a human. However, the method of the invention also extends to
eliciting or inducing a protective immune response in a non-human animal or bird subject
such as a livestock animal or bird, a laboratory test animal or bird, a companion animal or
bird, or a wild animal or bird.
In accordance with the invention, the composition administered to the subject comprises at
least one immunogen of an endemic influenza subtype. As noted above, the "endemic"
influenza A subtypes presently circulating in humans are the HlNl and H3N2 subtypes.
Accordingly, the composition of the invention preferably comprises immimogen(s) of one or
both of these subtypes. In one particular embodiment of the present invention, the
composition may comprise a standard tri-valent influenza vaccine comprising inactivated
endemic influenza virus types A (HlNl and H3N2) and B, such as the Fluvax® split virion,
inactivated influenza vaccine (CSL Limited, Melbourne, Australia).
As used herein, references to "pandemic" subtypes of influenza virus are to be understood as
references to subtypes to which the subject population, particularly the human population, is
considered to be naive, that is to have no or little resistance either as a result of prior
vaccination or prior exposure. As noted above, such pandemic subtypes include in
particular, the H5N1, H2N2, H9N2, H7N7, H7N3 and H10N7 subtypes.
Administration of the composition comprising immunogen(s) of endemic influenza subtypes
in accordance with the present invention has been shown to elicit or induce a heterologous
protective immune response against a pandemic subtype of influenza virus.
As used herein, references to "an immune response" are to be understood as broadly
referring to both a humoral response (such as induction of virus neutralizing antibodies) and
a cell-mediated immime response (such as induction of CD8"^ cytotoxic T cells). References
herein to "a protective immune response" include an immune response which protects the
subject from subsequent infection, or reduces the likelihood of subsequent infection, upon
challenge or exposure to the pandemic subtype of influenza virus, and includes amelioration
of the symptoms of any subsequent infection as well as reduction of the severity of any
subsequent infection.
The composition administered to the subject in accordance with the present invention
includes an immunogen-free immunostimulattng complex as adjuvant, which is combined or
mixed with the influenza immunogen(s). As used herein, the term "immimogen-free" means
that the immunostimulating complex is formed without any immunogen or antigen being
incorporated into the structure of the complex. Preferably, this adjuvant is a saponin-based
immunogen-free immunostimulating complex comprising saponin, a sterol (such as
cholesterol), and optionally a phospholipid (such as phosphatidylethanolamine or
phosphatidylcholine), fonned as typically rigid, hollow, spherical, cage-like particles
measuring about 40 nm in diameter, and known as "empty ISCOMs", ISCOM matrix, or
more recently as ISCOMATRDC'''^ adjuvant [23]. Most preferably, the immunogen-free
immunostimulating complex is ISCOMATRIXi'^* adjuvant.
Conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may be
included in compositions of this invention. Generally, a composition in accordance with the
present invention will comprise an immunologically effective amount of the influenza
immunogen(s) admixed with the adjuvant, in conjunction with one or more conventional
pharmaceutically acceptable carriers and/or diluents. As used herein "pharmaceutically
acceptable carriers and/or diluents" include any and all solvents, dispersion media, aqueous
solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying
agents and the like. The use of such media and agents for pharmaceutical active substances
is well laiown in the art and is described by way of example in Remington's Pharmaceutical
Sciences, 18'*^ Edition, Mack Publishing Company, Pennsylvania, U.S.A.
The influenza immunogen(s) included in the composition of the present invention are
administered in an effective amount. An "effective amount" means an amount necessary at
least partly to attain the desired immune response, in particular the desired protective
immune response. The amount varies depending upon the age, health and physical condition
of the individual to be treated, the racial background of the individual to be treated, the
degree of protection desired, the formulation of the composition, the assessment of the
medical situation, and other relevant factors. It is expected that the amount will fall in a
relatively broad range that can be determined through routine trials. If necessary, the
administration of an effective amount may be repeated one or several times. The actual
ainount administered will be determined both by the nature of the desired protective immune
response and by the rate at which the active inununogen is being administered.
In accordance with the present invention, the composition is preferably administered to the
subject by a parenteral route of administration. Parenteral administration includes any route
of administration that is not through the alimentary canal (that is, not enteral), including
administration by injection, infusion and the like. Administration by injection includes, by
way of example, into a vein (intravenous), an artery (intraarterial), a muscle (intramuscular)
and under the skin (subcutaneous). The composition is preferably administered
subcutaneously, intradermally or intramuscularly, in a dosage which is sufficient to obtain
the desired immune response.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous
preparation of the active component which is preferably isotonic with the blood of the
recipient. This aqueous preparation may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents. The sterile injectable
preparation may also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a solution in a polyethylene glycol
and lactic acid. Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose, trehalose, glucose)
and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently
employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic
acid find use in the preparation of injectables.
The present invention also provides the use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-fi^ee immunostimulating complex as adjuvant,
in the manufacture of a medicament for administration to a subject to elicit or induce a
protective irrunune response in the subject against a pandemic subtype of influenza virus.
The invention further provides the use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-fi-ee immunostimulating complex as adjuvant,
to elicit or induce a protective immime response in a subject against a pandemic subtype of
influenza virus.
In addition, the invention provides an agent for eliciting or inducing a protective immune
response in a subject against a pandemic subtype of influenza virus, wherein said agent is a
composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant.
In accordance with the present invention, it has been shown that immunization with standard
tri-valent seasonal influenza vaccine containing A/New Caledonia/20/99 (HlNl),
A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2005 admixed with immunogen-free
ISCOMATRIX™ adjuvant (referred to herein as "Influenza ISCOMATRIX™ Vaccine")
affords protection against lethal challenge with wildtype A/Vietnam/1203/04 (H5N1).
Influenza ISCOM'''^ vaccines have been shown to induce a CDS"*^ CTL response in a variety
of species including humans [10; 15; 16]. However, to date there are no reports of
ISCOMATRIX^^-containing vaccines providing protection against lethal challenge in any
animal models. Because the antigen in an ISCOMATRIX"™ vaccine is simply mixed with
and not incorporated into the ISCOMATRIX''''^ adjuvant structure, the induction of CDS'*^
CTL responses is generally believed to be less efficient than with an ISCOM'^'^ vaccine
containing the same amount of incorporated antigen [12;17-20].
For this reason, a range of strategies have been developed to associate proteins with
preformulated ISCOMATRIXtm adjuvant to produce associated ISCOMATRK™ vaccines.
These include methods that take advantage of the physical properties of the
ISCOMATRIX'^'^ adjuvant such as electrostatic interactions, where positively charged
proteins will associate witli the negatively charged adjuvant. Procedures for modifying either
the protein or the adjuvant to maximise this type of association have also been developed
[21]. Other methods for achieving association include modifications of the components of
the ISCOMATRIX"™ adjuvant to enable coupling of proteins to various exposed chemical
groups. One example of this type of modification is referred to as chelating
ISCOMATRIX"^"^ adjuvant, in which a metal chelating group is incorporated into the
structure, which can then bind proteins containing a metal affinity tag such as hexahistidine
[18].
Therefore, given the expected requirement for the antigen to be incorporated into the
ISCOMATRIX^"^ adjuvant (as in an ISCOM™ vaccine) for optimal induction of cellular
immime responses, it is highly surprising that immunisation of ferrets with Influenza
ISCOMATRIX'^''^ Vaccine (standard tri-valent seasonal influenza vaccine containing A/New
Caledoniay20/99 (HlNl), A/Wisconsin/67/2005 (H3N2), and B/ Malaysia/2506/2004 simply
admixed with immunogen-free ISCOMATRIX'^'" adjuvant) protected against lethal
challenge with wildtype A/Vietnam/1203/04 (H5N1). Further, this protection was observed
in the absence of a detectable neutralising antibody response to H5N1, indicating that the
cellular immune responses (most probably CD8"^ CTLs) induced by the Influenza
ISCOMATRIX''"^ Vaccine are capable of protecting a naive animal fi-om severe disease and
death following lethal challenge with a highly pathogenic H5N1 influenza virus.
The ferret has been the experimental animal of choice for many virologists who are
interested in studying human influenza. The ferret has been particularly useful to study: (i)
pathogenesis of influenza including H5N1 isolates; (ii) response to challenge infection after
vaccination; (iii) efficacy of antivirals; (iv) transmissibility of antiviral drug-resistant
mutants; (v) viral shedding and (vi) the febrile response to influenza. The alternate model is
the mouse, and the wealth of different genetically modified mouse strains and the large range
of readily available reagents for analysis of the immune response have made this a
compelling model and a great deal has been learnt from its use. However, mice are not
naturally infected by human influenza viruses and there are no known influenza viruses of
mice. This natural resistance may be due to the presence of the Mx gene, which confers type
1 interferon-dependent protection against influenza (in mice but not humans), and also due
to the presence of unique inhibitors of the virus in murine secretions [22]. Influenza viruses
suitable for growth in mice are limited and those that are routinely used have been "mouse-
adapted" by blind passage in this host. In contrast, ferrets are naturally susceptible to human
influenza so there is no restriction on the viral strains that can be studied.
Further features of the present invention are more fiilly described in the following Example.
It is to be understood, however, that this detailed description is included solely for the
purposes of exemplifying the present invention, and should not be understood in any way as
a restriction on the broad scope of the invention as set out above.
EXAMPLE
A. Materials and Methods
Ferrets: Juvenile female or male ferrets (3-5 month old), approximately 700-1500g in
weight were sourced from IMVS (SA). Ferrets were seronegative to currently circulating
influenza (HlNl, H3N2, B viruses). Blood samples were collected immediately prior to each
vaccination and prior to viral challenge. A further blood sample to check the antibody
response to challenge was taken 14 days after exposure to virus or at the time of euthanasia.
Bleeds were performed on anaesthetised (Ketamine/Medetomidine 50:50 0.1 ml/kg, reversed
with Apitemazole) animals from the jugular or axillary veins, 4 sites, 1 ml each, using 19 to
23 gauge needle depending on the size of the ferret. Clinically affected animals were
euthanased immediately following either a 10% body weight loss or exhibition of signs
consistent with involvement of other organ systems eg. tremor, or abdominal discomfort.
Vaccine: The mixture of seasonal influenza antigen used for injection included equal
amounts (15|ig) of A/New Caledonia/20/99 (HlNl), A/Wisconsin/67/2005 (H3N2) and B/
Malaysia/2506/2004 (CSL Limited, Melbourne, Australia), For pandemic strains,
monovalent antigen (A/Vietnam/1194/2004) was prepared in an identical manner to seasonal
strains. In brief, virus was propagated in embryonated eggs, inactivated with j3-
propiolactone (ICN Pharmaceuticals Inc., Costa Mesa, CA), subjected to zonal
centrifligation on a sucrose gradient, and treated with sodium taurodeoxycholate (Sigma, St.
Louis, MI) to yield a purified, inactivated and disrupted antigen preparation. The
concentration of viral antigen was expressed in terms of haemagglutinin (HA) protein, which
was determined by standard single radial immunodiffusion and compared to a known
standard of the relevant strain. ISCOMATRK™ adjuvant in PBS pH 6.2 [23] was added to
the influenza antigen immediately prior to dosing.
Vaccine dosing: Two 0.5 ml doses (21 days apart) delivered intramuscularly into the
quadriceps or posterior muscle of the hind legs, using a 1 ml syringe with a 27 gauge needle.
Viruses: The H5N1 human influenza viruses: A/Vietnara/1203/04 (wild-type)
AA^ietnam/1194/04 (reverse engineered vaccine strain), and A/Indonesia/5/2005 (wild-type)
were obtained from World Health Organisation Influenza Collaborating Laboratories. Stock
viruses were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs at
35°C for 24-36hr and stored at -70°C. All experiments with highly pathogenic viruses were
conducted in a BSL 3+ contaiimient facility (AAHL, CSIRO Geelong).
Viral challenge: Three weeks post Dose 2, the ferrets were inoculated intra-nasally
(both nostrils) with 10^ 50% egg infectious dose (EID50) of wild-type challenge virus
(A/Vietnam/l203/2004) as described by Govorkova et al [24].
Immunogenicity Tests: hnmunogenicity was assessed by haemagglutination inhibition
(HI), and virus neutralisation (VN) (as described in the WHO Collaborating Centre for
Influenza, Standard Operating Procedures) using two-fold dilutions of serum and a single
stock source of HA antigen. Geometric mean titres were determined and seroprotection
significance of viral titre and morbidity data was determined using a two-tailed, paired
Student's t-test. Statistical significance of mortality data was determined by Chi squared
analysis.
Clinical assessment:
Observations: Animals were visually monitored daily throughout the study and twice daily
following challenge if the animals show signs of disease. General clinical observations were
made prior to challenge with specific records kept of any respiratory symptoms such as
coughing or sneezing. Reaction site observations (i.e. erythema, oedema) were noted at 2, 24
and 48hrs following each vaccination. Following challenge, activity scores were monitored
daily.
Weight: Animals were weighed while imder sedation at the time of dosing and
challenge (Day 0) and Days 3, 5, 7 and 14 post-challenge.
Temperature: Temperature was determined manually at sedation using digital thermometers
and continuously using a temperature transponder inserted subcutaneously with the aid of a
22 gauge needle approximately ten days prior to viral challenge.
Biological Samples: Nasal, oral swabs were taken on Days 3, 5 and 7 post-challenge for
virus isolation.
B. Results
Influenza ISCOMATRIXJ'^ Vaccine: Antibody Responses
Prior to the commencement of the ferret immunogenicity and challenge studies, sera was
collected fi-om the ferrets and tested by Enzyme Linked Immunoassay (ELISA), using
standard methodology for the presence of antibodies to A/New Caledonia/20/1999 (HlNl),
A/Wisconsin/67/2005 (H3N2) and B/ Malaysia/2506/2004. All ferrets tested were negative
for antibodies against all 3 strains, and were therefore considered as naive for influenza.
For the immunogenicity and challenge studies, ferrets were immxinised twice (days 0, 21),
with 3.75|ag or IS^g of HA from AA^ietnam/1194/2004 admixed with ISCOMATRIX™
adjuvant (60fig), or the current seasonal tri-valent influenza vaccine, containing 15 fig of HA
from each of A/New Caledonia/20/1999 (HlNl), A/Wisconsin/67/2005 (H3N2) and B/
Malaysia/2506/2004 admixed with ISCOMATRIX™ adjuvant (60ng) (referred to as
Influenza ISCOMATRIX'^'^ Vaccine). A group of control animals was similarly dosed with
ISCOMATRIX'^'^ adjuvant alone. Sera collected 28 days post the second dose was assessed
for the presence of influenza-specific antibody using haemagglutination inhibition (HI) (Fig.
lA) and viral neutralisation (VN) (Fig. IB) assays. As shown in Fig.2, ferrets immunised
with the A/Vietnam/1194/2004 ISCOMATRIX™ vaccine at both antigen doses (3.75^g &
15|ag) elicited strong antibody responses to both A/Vietnam/1203/2004 (H5N1, clade 1) and
A/Indonesia/5/2005 (H5N1, clade 2), demonsfrating that despite being from different H5N1
clades, A/Vietnam/1203/2004 and A/Indonesia/5/2005 are antigenically and serologically
closely related.
fri contrast, sera from the ferrets that were immunised with the Influenza ISCOMATRIX'^'^
Vaccine were negative by HI and VN against both A/Vietnam/1203/2004 and
A/Indonesia/5/2005. As expected, control ferrets that received 2 doses of ISCOMATRIX®
adjuvant alone were also negative in both assays.
Influenza ISCOMATRrX7'^ Vaccine: Protection against lethal challenge
Four weeks post the 2"^* vaccine dose, ferrets were inoculated intra-nasally (both nostrils)
with 10^ 50% egg infectious dose (EID50) of challenge virus (A/Vietnam/1203/2004) as
described by Govorlcova et al [24]. The ferrets were then monitored continuously for
temperature and daily for weight, physical appearance and morbidity.
As shown in Fig. 3A, all of the animals that had been immunised with the
A/Vietnam/1994/2004 ISCOMATRIX™ vaccines at both HA antigen levels (3.75^g &
15|ig) survived lethal challenge with wild-type (A/Vietnam/1203/2004) virus. This result is
not surprising, given that these animals developed high titre A/Vietnain/1203/2004-specific
antibody in response to these vaccines, as shown in Fig. 2. Surprisingly, however, the ferrets
that had been immunised with the Influenza ISCOMATRIX^m Vaccine also survived lethal
challenge, despite the absence of detectable neutralising antibody. As expected, all of the
control ferrets succumbed to the viral challenge and had to be euthanased for ethical reasons
either because they had lost more than 10% body weight or showed signs of distress such as
tremor or abdominal discomfort.
The temperature of the ferrets was monitored continuously using a subcutaneous temperature
transponder for 3 days pre-challenge to establish the base line and then for a further 7 days
post-challenge. As shown in Fig. 3B, the temperature of the control ferrets rose sharply by
2.5-3.0°C 12-24hrs post-challenge and remained at this elevated level until they were
euthanased for ethical reasons. Similarly, the temperature of ferrets immunised with the
Influenza ISCOMATRIX™ Vaccine rose sharply by 2.5-3.0°C 12-24hrs post-challenge,
however this rise was transient and temperatures returned to baseline 24 hrs later. One
animal in this group experienced a 2"'' slightly lower transient spike in temperature, which
again returned to baseline 24hrs later. In contrast, with the exception of one animal in the
high antigen dose group, the post-challenge temperature of all ferrets immunised with an
AA^ietnam/1994/2004 ISCOMATRIX™ vaccine did not at any stage rise above the pre-
challenge baseline level.
The weight of the ferrets was monitored daily throughout the study period (Fig. 3C). The
weight of all animals in the control group dropped by approximately 10% within 3 days of
challenge. Consistent v^th the static temperature profiles of the ferrets immimised with the
AA^ietnam/1994/2004 ISCOMATRIXtm vaccines, the weight of these animals increased
steadily post-challenge. In contrast, the weight of the ferrets immunised with the Influenza
ISCOMATRIX'^'^ Vaccine remained unchanged throughout the 7 day post-challenge
observation period.
Consistent with the weight and temperature data, all ferrets immunised with
AA^ietnam/1994/2004 ISCOMATRIXtm vaccine at both antigen levels (3.75|ag or 15|ig
HA) or the Influenza ISCOMATRIX™ Vaccine remained playful and alert post lethal
challenge with wild-type H5N1 virus (A/Vietnam/1203/2004). In contrast, control animals
that received ISCOMATRIX™ adjuvant alone demonstrated signs of morbidity, and by day
3 post-challenge, were neither playful or alert, and by day 5-7 their physical condition had
deteriorated to a level that necessitated euthanasia (Fig. 4).
C. Discussion
The key and surprising observation made during study is that the Influenza
ISCOMATRIX™ Vaccine, containing 15iag of HA from each of A/New Caledoma/20/1999
(HlNl), AAVisconsin/67/2005 (H3N2) and B/ Malaysia/2506/2004 combined with
ISCOMATRIX''''^ adjuvant protected ferrets against lethal challenge with a highly
pathogenic H5N1 virus (A/Vietnam/1203/2004) in the absence of a detectable neutrahsing
antibody response (HI and VN) to AA^ietnam/1203/2004. The transient rise in temperature
of ferrets in the Influenza ISCOMATRDC'''^ Vaccine group suggests that following challenge
these animals became infected, but that the extent of viral infection was limited by non-
neutralising-antibody-mediated immunological mechanisms leading to rapid clearance of the
virus and recovery from infection.
The observation that these animals did not lose weight and remained active and alert post
challenge supports this conclusion. In contrast, the control animals that received 2 doses of
ISCOMATRIX"^^ adjuvant alone experienced a rapid, prolonged temperature rise and their
health status deteriorated rapidly to a point that necessitated euthanasia.
At present, due to absence of assays to evaluate ferret cellular immune responses, in
particulai- CDS CTL responses, it is not possible to identify the immunological basis for
protection afforded by the Influenza ISCOMATRIX''''^ Vaccine against lethal challenge with
an H5N1 virus. Influenza ISCOM''''^ vaccines have been showoi to induce CDS"*" CTL
responses in a variety of species including humans [10;15;16]. Furthermore, an HlNl
ISCOM'™ vaccine has been shown in mice to protect against heterologous challenge in part
due to the induction of a cross-protective CDS"^ CTL response. However, as mentioned
above, it is widely accepted that the induction of optimal CD8"^ CTL responses requires the
antigen to be incorporated into the ISCOM™ or ISCOMATRIX™ adjuvant [17-19]. It is
therefore surprising to observe in this study that in the absence of a detectable neutralising
antibody response, the Influenza ISCOMATRDC'''^ Vaccine induced a cellular immxine
response, most likely although not fonnally proven a CDS"^ CTL response, that was potent
enough to protect ferrets against lethal challenge with a higlily pathogenic H5N1 virus.
Furthermore, given the high degree of sequence conservation between the internal proteins
(including the identified CD8"^ CTL epitopes) of all A-strain influenza viruses, it is
reasonable to assume that the Influenza ISCOMATRIXT'^ Vaccine would similarly protect
against other potential pandemic strains, including but not limited to: H7N7, H7N3, H9N2
andHlON?.
REFERENCES:
[ 1 ] Lin YP, Gregory V, Bennett M, Hay A. Recent changes among human influenza
viruses. Virus Res 2004 Jul;103(l-2):47-52.
[2] Oxford JS. hifluenza A pandemics of the 20th century with special reference to 1918:
virology, pathology and epidemiology. Rev Med Virol 2000 Mar;10(2):l 19-33.
[3] Subbarao K, Klimov A, Katz J, et al. Characterization of an avian influenza A (H5N1)
virus isolated from a child with a fatal respiratory illness. Science 1998 Jan
16;279(5349):393-6.
[4] Claas EC, Osterhaus AD, van BR, et al. Human influenza A H5N1 virus related to a
highly pathogenic avian influenza virus. Lancet 1998 Feb 14;351(9101):472-7.
[5] World Health Organization. Cumulative Number of Confirmed Human cases of Avian
hifluenza A/(H5N1) Reported to WHO. 2006. Ref Type: Data File
[6] Potter CW, Oxford JS. Determinants of immunity to influenza infection in man. Br
Med Bull 1979;35:69-75.
[7] Nozaki Y, Hasegawa Y, Takeuchi A, et al. Nitric oxide as an inflammatory mediator of
radiation pneumonitis in rats. Am J Physiol 1997;272(4 Pt 1):L651-L658.
[8] Epstein SL, Lo CY, Misplon JA, Bennink JR. Mechanism of protective immunity
against influenza virus infection in mice without antibodies. J Immunol 1998;160:322-
7.
[9] Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by
injection of DNA encoding a viral protein. Science 1993;259(5102):1745-9.
[10] Sjolander A, Drane D, Maraskovsky E, et al. Immune responses to ISCOM
formulations in animal and primate models. Vaccine 2001;19(17-19):2661-5.
[11] Sambhara S, Kurichh A, Miranda R, et al. Heterosubtypic immunity against human
influenza A viruses, including recenfly emerged avian H5 and H9 viruses, induced by
FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage
function. Cell Immunol 2001 Aug l;211(2):143-53.
[12] Morein B. The iscom antigen-presenting system. Nature 1988;332(6161):287-8.
[13] Villacres MC, Behboudi S, Nildcila T, LOvgren-Bengtsson K, Morein B. Internalization
of iscom-bome antigens and presentation under MHC class I or class II restriction. Cell
Immunol 1998;185(l):30-8.
[14] Lovgren K, Morein B. The requirement of lipids for the formation of
immunostimulating complexes (iscoms). Biotechnol Appl Biochem 1988; 10(2): 161-
72.
[15] Ennis FA, Cruz J, Jameson J, Klein M, Burt D, Thipphawong J. Augmentation of
human influenza A virus-specified cytotoxic T lymphocyte memory by influenza
vaccine and adjuvanted carriers (ISCOMS). Vii'ology 1999;259(2):256-61.
[16] Rimmelzwaan GF, Nieuwkoop N, Brandenburg A, et al. A randomized, double blind
study in young healthy adults comparing cell mediated and humoral immune responses
induced by influenza ISCOM vaccines and conventional vaccines. Vaccine 2000; 19(9-
10):1180-7.
[17] Cox J, Coulter A. Adjuvants - a classification and review of their modes of actions.
Vaccine 1997;15(3):248-56.
[18] Malliaros J, Quinn C, Arnold FH, et al. Association of antigens to ISCOMATRIX
adjuvant using metal chelation leads to improved CTL responses. Vaccine 2004;22(29-
30):3968-75.
[19] Lenarczyk A, Le TT, Drane D, et al. ISCOM based vaccines for cancer
immunotherapy. Vaccine 2004;22(8):963-74.
[20] Lovgren-Bengtsson K, SjOlander A. Adjuvant activity of iscoms; effect of ratio and co-
incorporation of antigen and adjuvant. Vaccine 1996;14(8):753-60.
[21] Le TTT, Drane D, Malliaros J, et al. Cytotoxic T cell polyepitope vaccines delivered
by ISCOMs. Vaccine 2001;19(32):4669-75.
[22] Horisberger MA. Interferons, Mx genes, and resistance to influenza virus. Am J Respir
Crit Care Med 1995;152(Suppl.):S67-S71.
[23] Drane D, Pearse M. The ISCOMATRIX adjuvant. In: Schijns VE, O'Hagan DT,
editors. Immunopotentiators in modem vaccines.Amsterdam ; Boston, Elsevier
Academic Press, 2006: p. 191-216.
[24] Govorkova EA, Rehg JE, Krauss S, et al. Lethality to ferrets of H5N1 influenza viruses
isolated from humans and poultry in 2004. J Virol 2005 Feb;79(4):2191-8.
CLAIMS:
1. A method for eliciting or inducing a protective immune response in a subject against
a pandemic subtype of influenza virus, which comprises administering to the subject a
composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant.
2. The method according to claim 1, wherein the subject is a human.
3. The method according to claim 1 or claim 2, wherein the composition comprises
imniimogen(s) of endemic influenza A HlNl and/or H3N2 subtypes.
4. The method according to any one of claims 1 to 3, wherein the immunogen-free
immunostimulating complex comprises saponin, a sterol and optionally a phospholipid.
5. The method according to claim 4, wherein the immunogen-free immunostimulating
complex comprises ISCOMATRIX''"'^ adjuvant.
6. The method according to any one of claims 1 to 5, wherein the pandemic subtype of
influenza virus is selected from the influenza A H5N1, H7N7, H7N3, H9N2 and HI ON?
subtypes.
7. The method according to claim 6, wherein the pandemic subtype is the influenza A
H5N1 subtype.
8. Use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free immunostimulating complex as adjuvant,
in the manufacture of a medicament for administration to a subject to elicit or induce a
protective immxme response in the subject against a pandemic subtype of influenza virus.
9. Use of a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-firee iramunostimulating complex as adjuvant,
to elicit or induce a protective immune response in a subject against a pandemic subtype o
influenza virus.
10. Agent for eliciting or inducing a protective immune response in a subject against ;
pandemic subtype of influenza virus, wherein said agent is a composition comprising
(i) at least one immunogen of an endemic influenza subtype, and
(ii) an immunogen-free iirununostimulating complex as adjuvant.

A method for eliciting or inducing a protective immune response in a
subject against a pandemic subtype of influenza virus comprises
administering to the subject a composition comprising (i) at least one
immunogen of an endemic influenza subtype, and (ii) an immunogen-free
immunostimulating complex as adjuvant.