CA2175719A1 - Compositions and methods for protection against immunologically diverse isolates of influenza virus - Google Patents

Abstract

A cross-protective cytotoxic T cell response to H1 HA and H2 HA subtypes of influenza virus is obtained by immunizing the host with a complex of solubilized influenza virus comprising haemagglutinin, specifically H1 HA, and an immune stimulating complex.

Description

217571~

TITLE OF THE INVENTION
C(L ~O~lllONS AND METHODS FOR PROTECTION AGAINST
._O~ICALLY DIVERSE ISOLATES OF INFLUENZA VIRIJS
FrRT~n OF T~R INVENTIO~
The present invention is related to the field of immunology and i8 particularly concerned with vaccines against immunologically diverse isolates of influenza virus .
BACKGROUND OF THE INVENTION
The development of improved vaccines against influenza virus is an active area of research. Current influenza (flu) vaccines are about 7096 effective at preventing illness in healthy adults (ref. 1 - various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these ref erences are hereby incorporated by ref erence into the present disclosure) and approximately 5096 effective at preventing hospitalization and rn~ in people aged 65 and over (ref. 2) . Desired properties of improved vaccines for influenza include the production of increased titers of virus neutralizing antibodies (IgG
and secretory IgA) against influenza antigens including HA in the serum and at mucosal surfaces to prevent infection (refs. 3 and 4), and induction of virus-specific cross-reactive T lymphocyte (CD8i and/or CD4') and cytokines to enhance recovery from infection (refs.
4, 5, 6).
Various strategies are being investigated to try and develop; ~_uved parenterally administered sub-unit vaccines. Approaches include the use of alternative delivery vehicles for ~nf~ n7a antigens, such as liposomes (ref. 7) and oil-in-water emulsions (ref. 8), new adjuvants (ref. 9) or; ;7at;on with DNA encoding various viral proteins (ref. 10, 11). Each of these ~17'~7~9 approaches is attempting to induce protective immune responses that are enhanced compared to current subunit preparations .
Immunostimulating complexes (ISCOMs) are an 5 adjuvanted particulate vaccine system comprising cholesterol, phospholipid, antigen and Quil A (ref. 12).
Studies in various animal species have demonstrated Pnh~n~-ed humoral and cell mediated immune responses against model antigens and microbial proteins when 10 formulated as ISCOMs (ref. 13).
Various properties oi ISCOMs contribute to the8e enhanced immune responses. Compared with soluble antigen, ISCOMs are more rapidly distributed from the site of inj ection to the draining lymph nodes and spleen 15 where they persist for longer periods of time (ref. 14) .
Following subcutaneous; i7~;0n, antigens formulated as ISCOMs are taken up by a subset of splenic macrophages distinct from those that take up soluble antigens (ref.
15). Within the APC, intact ISCOMs associate with 20 intracellular lipid membranes and localize within cytosolic and vesicular compartments (ref. 16), enabling processing for both class I and class II MHC-restricted T cell responses. ISCOMs have been shown to upregulate the expression of MHC class II expression on monocytes 25 (ref. 16), enhance the production of GM-CSF, TNF-~, IL-1 and IL-6 from macrophages and Thl (IL-2, IFN-~r) and Th2 (IL-4) cytokines from antigen-specific T cells (ref. 17) .
Both IgG1 and IgG2A responses are induced in mice following; In; 7~tion with antigens formulated as ISCOMs 30 (ref. 18).
Infection with influenza virus leads to serious disease. It would be desirable to provide improved immunogenic compositions including vaccines comprising influenza virus antigens for vaccination against disea8e 3 5 caused by inf luenza virus and f or the generation of diagnostic reagents.

SUMM~RY OF TH~ INVENTION
The present invention is directed towards the provision of compositions and methods for the protection again3t immunologically diverse isolates of influenza virus.
In accordance with one aspect of the invention, there is provided a method of protecting a host against disease caused by infection with an influenza virus, comprising administering to the host a complex of solubilized influenza virus comprising haemagglutinin (HA) or at least one fragment thereof and an immune stimulating complex (ISCOM) and effective to produce in the host cytotoxic T cells specific for HA of both H1 HA
and H2 HA subtypes of inf luenza virus .
The aolubilized influenza virus may be an H1 HA
subtype inf luenza virus strain . The H1 HA subtype against which cytotoxic T cells are produced may be an HlN1 subtype influenza virus strain, including A/Taiwan/1/86 and the H2 HA subtype may be an H2N2 2 0 subtype inf luenza virus strain, including A/~Japan/3 0 5/57 .
In a particular aspect, the immunizing influenza virus is purified substAnt;Ally free from non-viral proteins and may be solubilized by, for example, detergent extraction to provide solubilized influenza virus consisting substantially of HA. One suitable detergent is 296 Mega-10 .
Cytotoxic T-cells recognize a cytotoxic T-cell epitope comprising amino acids HA 189 to 199, which is conserved among H1 and H2 inf luenza viruses but not H3 influenza viruses.
In a particular aspect, the present invention provides a method of protecting a host against disease caused by infection with influenza virus, comprising administering to the host substantially purified haemagglutinin (HA) or fragment thereof retA;n;ng the immunological properties of HA inco--orated into 217~7~9 immunostimulating complexes (ISCOMs) and effective to produce in the host cytotoxic T-cells specific for HA of both of H1 XA and X2 HA subtypes of inf luenza virus .
The complexes for ; ; 7~tion provided herein contain an immunologically effective amount of haemagglutinin, for example, between about O . 05 ~g and about 10 ~g of haemagglutinin per dose.
The complexes for; In; 7ation provided herein may be administered by a variety of routes and a variety of ; ; 7;n~ schedules may be used. The 1 ; 7ation may comprise a single administration of the complex or a plurality (including two) administrations of the complex may be u3ed to produce the protective immune response.
Advantages of the invention include the ability to generate a cytotoxic immune response that protects against disease caused by infection with immunologically distinct isolates of influenza virus.
BRIEF DESCRIPTION OF FIGURES
The present invention will be further understood from the following General Description and specific Examples with reference to the Figures in which:
Figure 1, consisting of panels A, B, C and D, shows HAI and virus lung titers in BABL/c mice immunized with flu-ISCOMs or subvirion vaccine and challenged with live homologous virus;
Figure 2, consisting of panels A and B, shows a comparison between viral lung titers and weight 1088 following virus challenge of mice immunized with flu-ISCOMs or subvirion vaccine;
Figure 3 shows the induction of influenza H1 and X2 subtype cross-reactive cytotoxic T-cells in mice immunized with H1 XA flu-ISCOMs;
Figure 4, consisting of panels A, B, C and D, shows the mortality and weight 1088 following heterologous (X2) virus challenge of mice immunized with H1 flu-ISCOMs, subvirion vaccine or live virus; and ' 4 2l757l~
Figure 5, consisting of panels A, B, C and D, shows the mortality and weight 1088 following heterologous (H3) virus challenge of mice immunized with Hl flu-ISCOMs, 3ubvirion vaccine or live virus.
(~.T~ T, DESCRIPTION OF THE INVENTION
As discussed above, the present invention is concerned with the use of ISCOMs in combination with a solubilized f raction of inf luenza virus, including haemagglutinin, particularly derived from ~1 subtype lo inf luenza virus, ( " f lu- ISCOMS " ) to stimulate a cross-protective cytotoxic T-cell (CTL) response.
Immune stimulatory complexes or ISCOMs are a complex composed of typically 0.5 wt96 Ouillaia saponins, 0.1 wt96 cholesterol, O .1 wt~ ph ~nsphnl; ~id and antigen in PBS .
Occasionally, surfactants are used to prepare ISCOMs (such as, Mega 10) but are removed from the final formulation before use. The ad]uvant-active components of ISCOMs are derived by aqueous extraction of the bark of Quillaja sa~onaria and are further purified by chromatography. Quil A is a complex but purified mixture of Ouillai a saponins which are glycosides of quillaic acid and carbohydrates. ~urther chromatographic purification provides components with high adjuvant activity and ISCOM-forming properties.
In the data presented in the Examples below, we have demonstrated the ability of flu-ISCOMs to protect mice against challenge with both homologous and serologically distinct type A influenza virus. The protective efficacy of flu-ISCOMs was significantly better than that seen for commercial subvirion vaccine comprising 10-fold higher doses of HA. In addition, this increased efficacy of flu-ISCOMs over vaccine was demonstrated when protection was assessed in various way, including, virus load in the lung, mortality, morbidity and rate of recovery from infection. The immunological correlates associated with protection and recovery from influenza virus infection ~ ~ 2I7~719 have previously been elucidated from the results of studies performed in humans and animal models.
Pre-existing local secretory IgA and serum IgG specific for HA correlate with preventi4n against infection by 5 homologous virus (refs. 3, 4) . Recovery from infection with homotypic virus involves both serum IgG and class I
and class II MHC restricted cytotoxic T lymphocytes (CTL) (ref. 19). Cytotoxic T cells also appear to enhance the elimination of virus following heterotypic challenge, 10 thereby reducing morbidity and mortality (ref. 10). In the present invention, the ability of flu-ISCOMs to give improved protection in mice against inf ection with homologous virus correlated with the induction of higher serum HAI titers, ref lecting the presence of antibodies 15 specific for HA. At equivalent doses of HA, the serum HAI titers induced by flu-ISCOMs were at least 10-fold higher than those induced by vaccine . Similar f indings have been shown for antigens from laboratory strains of influenza and for other proteins formulated as ISCOMs 20 tested in various species (ref. 13). ISCoMs induce CD8' class I MHC restricted T cell responses against formulated soluble antigens derived from influenza virus and HIV (gpl60) (ref. 20) and ovalbumin (ref. 21) . In an earlier study, Jones and colleagues (ref. 22) reported 25 the induction of influenza specific CTL in mouse lungs following intranasal inoculation of flu-ISCOMs.
The data presented herein is the first disclosure of the antigen specificity and virus cross-reactivity of CTL
induced by flu-ISCOMs. Flu-ISCOMs comprising influenza 3 0 HA of the subtype H1 generated CTL that lysed target cells infected with H1 and H2, but not infected with H3 strains of influenza virus. In contrast, CTL from mice immunized with live H1 virus killed target cells infected with either H2 or H3 subtype viruses. No CTL were 3 5 generated in mice immunized with the subunit vaccine .
This dif f erentiaI virus subtype recognition by CTL

. ~, 2175719 generated by flu-ISCOMs and live virus correlated with their abilities to lyze target cells pulsed with known MHC class I H-2Kd-restricted peptides from influenza NP
(NP 147 to 158) and- HA (HA 189 to 199), previously shown to be recognized by CD81 CTI. generated after infection with live ;nf1llPn7~ virus (refs. 23, 24). CT~ generated by flu-ISCOMs recognised a synthetic peptide representing amino acids XA 189 to 199 that is conserved amongst H1 and H2, but not H3 flu viruses. In contrast, the ; t~rl( ;n~nt responge induced by infection with live virus was against NP 147 to 158, a sequence that is conserved among all three virus subtypes (ref. 25).
Flu-ISCOMs failed to generate CTL against NP despite the association of this protein with the ISCOM particles, as ~Pt~rTrinPd by polyacrylamide gel electrophoresis. There may be a number of explanations for the inability of flu-ISCOMs to induce CTL specific for NP, for example, NP
may not be present in sufficient amounts in the ISCOM
preparation. Alternatively, NP may not be associated with ISCOMs in a manner that allows efficient cytoplasmic processing of the protein for production of peptides that bind to MHC class I molecules.
It has been demonstrated that, following endocytosis, flu-ISCOMs accumulate intact, within the endosomes of antigen presenting cells and that these particles are exposed to the cell cytoplasm (ref. 16).
Viral membrane proteins, such as HA, that are able to associate with phospholipid on the surface of the ISCOM
particle may be more accessible to cytoplasmic proteases involved in processing for class I presentation. Weiss et al., (ref. 26) reported that lipidated, but not soluble NP, could be efficiently incorporated into ISCOMs. ISCOMs c~nti~inins the lipidated NP preparation did not induce CTl- in vivo.
In the data presented herein, we have shown that there was a correlation between the ability of , ~ 217~71~
flu(H1)-ISCOMs to induce cross-reactive CTL in mice against H1 and H2 inf luenza viruses and protection against mortality and morbidity due to infection with H1, H2 but not H3 inf luenza viruses .
In eontrast to the cross-protection induced by flu-ISCOMs described herein, mice immunized with live HlN1 inf luenza virus were poorly protected against mortality and morbidity following challenge with a H2N2 virus. This result was unexpected since live virus induced CTL that were specific for a known H-2Kd-restricted epitope within flu NP (NP 147 to 158) that is conserved amongst H1 and H2 viruses. One explanation for these observations is that the cross-protection observed may be mediated by 15 immunological me~n;P-"q other ~ than CD8+ T cells.
Indeed, in this mouse model, there may be qualitative and quantitative differences in the T lymphocyte subsets and their secreted cytokines following i ; 7ation with flu-ISCOMs compared with live virus that could contribute 20 to the enhanced clearance of virus after infection. For example, in the absence of CD8+ T cells, 2-/- mice can still clear virus after infection (ref. 27) and ean be proteeted against death following lethal virus ehallenge by; ; zation with vaeeinia expressing NP (ref . 6) . In 25 both studies, CD4+ eytotoxie T cells were implieated in mediating the proteetion.
No signifieant differenees have been seen in the quantity and type of eytokines produeed by virus and flu-ISCOMs. Both induee Thl and Th2 cytokines (ref. 17).
30 Another explanation for the differences in eross -proteetion observed between f lu virus and flu-ISCOMs is that the funetional aetivities of CTL
indueed by flu-ISCOMs and live virus differ and may depend on their speeifieities for the HA and NP proteins 35 respectively. Cytotoxic T-eells speeifie for HA have been implieated in mediating eross-proteetion in miee 217~71~
given a recombinant HA2 fragment from influenza HA
expressed as a fusion protein. Immunization with the HA2 protein of the Hl strain protected BAIB/c mice against Hl and H2, but not H3 strains of influenza virus (ref. 28).
5 Evidence for a protective role for CTL specific for NP
has been less conclusive and may depend on the form and ability of the antigen to r-;nti~;n long term memory T
cell responses (ref. 29). For example, while cross-protection has been observed in mice; ; ~7~1 with 10 cDNA encoding flu-NP (ref. 10), conflicting results have been reported for soluble NP (refs. 30, 31) and vaccinia-NP recombinants (refs. 32, 33) .
The flu-ISCOM preparations and vaccines provided herein are administered in a manner compatible with the 15 dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic.
The r1uantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual ' 8 immune system to synthesize antibodies and 20 to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner. However, suitable dosage ranges are readily detf~rrn; nA~le by one skilled in the art and may be of the order of micrograms 25 of the haemagglutinin. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subse~uent administrations. The dosage may also depend on the route of administration and will vary according to 30 the size o~ the host.
The concentration of HA protein in an immunogenic composition according to the invention may vary widely but may be in the range of micrograms. A vaccine which rr,nt~inc antigenic material of only one pathogen is a 35 monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and ~, 217~719 also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
EXAMPLES
The above disclosure generally describe3 the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may sugge3t or render exPedient. Although specific terms have been employed herein, such terms are ;nt,~nAf~l in a descriptive sense and not for purposes of limitations.
ExamPle 1:
This Example describes influenza viruses and vaccine preparations .
A/Taiwan/1/86 (HlN1) and A/Philippines/1/82 (H3N2) as live influenza viruses in egg-derived allantoic fluid, mouse-adapted A/Taiwan/1/86 and commercial A/Taiwan/1/86 monovalent subunit vaccine ('Fluzone') were obtained from t~r7nn~ t I,aboratories Inc., Swiftwater, PA, USA. Mouse adapted A/Japan/305/57 (H2N2) was generated by repeated passage of allantoic fluid virus in mouse lungs. A/Hong Kong/1/68 virus was obtained as allantoic f luid.
ExamPle 2:
This Example describes the preparation of complexes comprising influenza antigens and immunost; l~t;nS
complexes (ISCOMs).
ISCOMs were prepared according to a dialysis procedure (ref. 34). Briefly, influenza virus was grown in embryonated eggs and purified from allantoic fluid by sucrose density gradient centrifugation (20 to 6096 sucrose in PBS, 10, 000 x g for 60 min. ) . Purified virus was solubilized with 2~ Mega-10 (Bachem, Bubendorf, Switzerland) and the core pelleted through 30% (w/w) sucrose .
To the supernatant was added 1 mg each of phosphatidyl choline, cholesterol (Sigma, St ~ouis, MO) 5 and 5 mg Quil A (Spikoside, ISCOTEC Lulea, Sweden) per mg of total viral protein as determined by the Bradford assay (ref. 35). The mixture was incubated for 2 hours at room temperature, dialysed against PBS and layered onto a 10 to 5096 (w/w) sucrose gradient. After centrifugion at 40, 000 rpm for 18 hours, fractions were collected for characterization. Formation of ISCOMs was verif ied by negative staining electron microscopy and analytical sucrose density centrifugation. Cholesterol incorporation was ~l~tf~rm; nf~d using 3H-cholesterol .
Fractions showing co-migration of formed ISCOM particles, HA and cholesterol were pooled and dialyzed extensively against PBS. The HA content of the flu-ISCOM fractions and commercial sub-virion vaccine was determined by single radial immunodiffusion (SRID) after solubilization with 1~6 Zwittergent 314 non-ionic detergent (Calbiochem, La Jolla, CA) . The assays were performed using A/Taiwan/1/86 antigen and antiserum reference standards (Centers for Biologics Evaluation and Research) . The f inal Quil A content of the ISCOMs was estimated using the orcinol procedure (ref. 36). The ratios by weight of Quil A to HA in the final flu-ISCOM preparation was estimated as 7 :1.
Example 3:
This Example describes the; ; 7i~t; on of mice .
3 o Female BALB/c mice between 6 to 8 weeks of age were obtained from Charles River (Quebec, Canada). Mice were immunized subcutaneously on days 0 and 21 with A/Taiwan flu-ISCOMs or monovalent vaccine comprising 0.01 to 10 ~Lg HA in 0.1 mL PsS. Control mice received either PBS alone or 200 to 400 hemagglutination units (H~U) of live A/Taiwan virus as allantoic f luid . Fourteen days later 217~7~

the mice were challenged intranasally while under anaesthesia with 50 IlL of live mouse-adapted A/Taiwan/1/86 (5 LDso) or A/Japan/305/57 (5 LDso) or a 1 in 10 dilution of A/HK/1/68 in allantoic fluid.
Protection was assessed by either monitoring mortality daily and morbidity (weight changes in the mice) every 2 to 3 days up to 14 days post-challenge or by quantitation of virus in mouse lungs as follows. Four days following mouse challenge, lungs were dissected from individual animals and homogenized in 2 mL PBS/0 . 59~ gelatin using a Polytron homogenizer (Brinkman, Ontario). The lung suspensions were centrifuged at 3, 000 rpm for 15 min., supernatants filtered through a 0.45 ,um membrane (Millipore, Bedford, MA) and stored at -70 C until assayed. Serial dilutions of lung homogenates (100 ~L) were incubated in duplicate with washed confluent Madine Darby canine kidney cells in 24 well flat bottomed tissue culture plates for 1 hour at 37 C in 696 CO2. To each well was added 1 mL of overlay medium comprising 29~ Noble agar (DIFCO, Detroit, MI), 0.2g~ D(+) glucose (Sigma), 0.029 DEAE Dextran (Pharmacia, Quebec), 2.0~ BME vitamins (ICN, Ontario) and 20 ~g/mL TPCK trypsin (Sigma) in 2 x DMEM
medium (ICN) . Following incubation for 3 days at 37 C
(5~ CO2), 1 mL of a 296 neutral red solution in PBS was added to each well and, incubation r~nt; n~ overnight .
The stain was aspirated and the plaques counted and expressed as the mean logl0 plaque forming units/mL. The ability of mice sera to prevent influenza virus-mediated hemagglutination was ~ t~ n~d by a hemagglutination inhibition assay as follows. Six mice were bled at random via the orbital sinus vein for pre- and post-immunization bleeds. Sera samples were heated at 56 C for 30 min. to inactivate complement and pre-treated with trypsin/periodate to destroy endogenous inhibitors of hemagglutination. Serially diluted antisera were tested for their ability to inhibit the agglutination of 13 21757~
196 chick red blood by 4 HA units of A/Taiwan virus in a standard HAI assay (ref. 37) .
Exam~le 4:
This Example describes the determination of 5Cytotoxic T cell activity in; i z~d mice.
Peptides representing known H-2Kd-restricted cytotoxic T cell ~CTL) epitopes within A/PR8/34 influenza haemagglutinin, HA2 189 to 199 (I Y S T V A S S L V L) (SEQ ID NO. 1) (ref. 23) and nuclear protein (NP), NP 147 10 to 158 (R-) (T Y Q R T R A L V T G) (SEQ ID NO. 2) (ref .
24) were synthesized on an automated ABI 430A peptide synthe3izer using an optimized t-Boc chemistry according to the manufacturer' 8 instructions . Synthetic peptides were cleaved from the resin using hydrogen fluoride. The 15 purity of t~le peptides exceeded 85% as judged by reverse-phase HPLC. Amino acid analyses, performed on a Waters Pico-Tag 3ystem agreed with the theoretical amino acid compositions.
For CTL studies, mice were given a single 20 immunization with either 1 ~Lg HA as A/Taiwan flu-ISCOMs or 10 llg HA as monovalent subvirion vaccine in 0.1 ml PBS
via the subcutaneous route. Ten days later, spleens were removed and restimulated with live virus. Spleen cells (2.5 x 107) were incubated at 37 C, 69~ CO2 in an upright 25 flask in 20 mL RPMI/10% FCS with the same number of ~-irradiated (3000 Rads) normal syngeneic spleen cells previously infected for 1 hour at 37-C with A/Taiwan virus in ;~ n~o; c fluid at 5, 000 HAU/8 x107 cells .
After int~llhAti~n for 5 to 6 days, the cells were 30 tested in a standard Slchromium release assay. Briefly, washed effector cells were incubated with 10~ P815 mastocytoma target cells labelled with Slchromium at 50 to 100 ~LCi/106 cells in 96-well v-bottom tissue culture plates for 4 to 6 hours at 37 C in 6~ CO~. Target cells 35 were either untreated, pulsed with 10 IlM synthetic peptide or infected with live A/Taiwan/1/86 flu virus at 217571~

1000 HAU/106 cells, for 90 min. at 37 C. Plates were centrifuged at 1, 000 rpm for 5 min. and O .1 m~ of supernatant was removed for mea~u~ t of Slchromium content in a gamma counter. Spontaneous and total 5 release of S1chromium were determined by incubating target cells with either medium or 2 . 5g6 Triton-X100 respectively, in the absence of responder lymphocytes for the duration of the assay. Percentage specif ic chromium release was calculated as (counts - spontaneous 10 counts) / (total counts - spontaneous counts) x 100 . The spontaneous release of S1chromium in the absence of effector cells was between 5 to 15~ in these studies.
SUMMARY OF THE DISCI.OSURE
In summary of thi3 disclosure, the present invention 15 provides a novel immunization procedure for protecting hosts against disease caused by inf luenza virus which utili2es a complex of solubilized influenza virus and ISCOMs to produce cytotoxic T-cells specific for ~ of both H1 HA and H2 HA subtypes of influenza virus.
20 Modifications are possible within the scope of this invention .

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K~ ;N~;S
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Claims (10)

1. A method of protecting a host against disease caused by infection with an influenza virus, comprising administering to said host a complex of solubilized influenza virus comprising haemagglutinin (HA) or at least one fragment thereof and an immunostimulating complex (ISCOM) and effective to produce in said host cytotoxic T cells specific for HA of both H1 HA and H2 HA
subtypes of influenza virus.

2. The method of claim 1 wherein the solubilized influenza virus is an H1 HA subtype influenza virus strain.

3. The method of claim 2 wherein the H1N1 subtype influenza virus strain is A/Taiwan/1/86.

4. The method of claim 2 wherein the H2N2 subtype influenza virus strain is A/Japan/305/57.

5 . The method of claim 1 wherein the immunizing influenza H1 subtype virus is purified substantially free from non-viral proteins.

6. The method of claim 1 wherein the solubilized H1 subtype virus is solubilized by detergent extraction to provide solubilized influenza virus comprising HA.

7. The method of claim 1 wherein said cytotoxic T-cell recognize a cytotoxic T-cell epitope comprising amino acid HA 189 to 199 conserved among H1 and H2 influenza viruses.

8. A method of protecting a host against disease caused by infection with influenza virus, comprising administering to the host substantially purified haemagglutinin (HA) or at least one fragment thereof retaining the immunological properties of HA incorporated into immunostimulating complexes (ISCOMs) and effective to produce in said host cytotoxic T-cells specific for HA
of both of H1 HA and H2 HA subtypes of influenza virus.

9. The method of claim 8 wherein the purified HA is an H1 HA subtype HA.

10. The method of claim 1 or 8 wherein the complex comprises between about 0.05 µg and about 10 µg of haemagglutinin.