COMPOSITIONS AND METHODS FOR PROTECTION AGAINST IMMUNOLOGICALLY DIVERSE ISOLATES OF INFLUENZA VIRUS

FIELD OF THE INVENTION The present invention is related to the field of immunology and is 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 70% 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 references are hereby incorporated by reference into the present disclosure) and approximately 50% effective at preventing hospitalization and pneumonia in people aged 65 and over (ref. 2) . Desired properties of improved vaccines for ¹ 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 (CD8 ⁺ and/or CD4 ⁺ ) and cytokines to enhance recovery from infection (refs. 4, 5, 6) .

Various strategies are being investigated to try and develop improved parenterally administered sub-unit vaccines. Approaches include the use of alternative delivery vehicles for influenza antigens, such as liposomes (ref. 7) and oil-in-water emulsions (ref. 8), new adjuvants (ref. 9) or immunization with DNA encoding various viral proteins (ref. 10, 11) . Each of these

approaches is attempting to induce protective immune responses that are enhanced compared to current subunit preparations.

Immunostimulating complexes (ISCOMs) are an adjuvanted particulate vaccine system comprising cholesterol, phospholipid, antigen and Quil A (ref. 12) .

Studies in various animal species have demonstrated enhanced humoral and cell mediated immune responses against model antigens and microbial proteins when formulated as ISCOMs (ref. 13) .

Various properties of ISCOMs contribute to these enhanced immune responses. Compared with soluble antigen, ISCOMs are more rapidly distributed from the site of injection to the draining lymph nodes and spleen where they persist for longer periods of time (ref. 14) .

Following subcutaneous immunization, 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 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 (ref. 16), enhance the production of GM-CSF, TNF-α, IL-1 and IL-6 from macrophages and Thl (IL-2, IFN-γ) and Th2 (IL-4) cytokines from antigen-specific T cells (ref. 17) . Both IgGl and IgG2A responses are induced in mice following immunization with antigens formulated as ISCOMs (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 disease caused by influenza virus and for the generation of diagnostic reagents.

SUMMARY OF THE INVENTION The present in-^ntion is directed towards the provision of compositions and methods for the protection against 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 HI HA and H2 HA subtypes of influenza virus. The invention, in another aspect, provides the use of a complex of solubilized influenza virus comprising haemagglutinin (HA) or at least one fragment thereof and an immunostimulating complex (ISCOM) for the manufacture of a medicament for use in the stimulation in a host of cytotoxic T cells specific for HA of both HI HA and H2

HA subtypes of influenza virus.

The solubilized influenza virus may be an HI HA subtype influenza virus strain. The HI HA subtype against which cytotoxic T cells are produced may be an H1N1 subtype influenza virus strain, including A/Taiwan/1/86 and the H2 HA subtype may be an H2N2 subtype influenza virus strain, including A/Japan/305/57. In a particular aspect, the solubilized influenza virus is purified substantially 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 2% Mega-10.

Cytotoxic T-cells recognize a cytotoxic T-cell epitope comprising amino acids HA 189 to 199, which is conserved among HI and H2 influenza viruses but not H3 influenza viruses.

In a further 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 retaining the immunological properties of HA incorporated into immunostimulating complexes (ISCOMs) and effective to produce in the host cytotoxic T-cells specific for HA of both of HI HA and H2 HA subtypes of influenza virus. In an additional aspect of the invention, there is provided the use of substantially purified haemagglutinin (HA) or at least one fragment thereof retaining the immunological properties of HA incorporated into immunostimulating complexes (ISCOMS) for the manufacture of a medicament for use in the stimulation in a host of cytotoxic T cells specific for HA of both HI HA and H2 HA subtypes of influenzae virus. The complexes or compositions for immunization provided herein contain an immunologically effective amount of haemagglutinin, for example, between about 0.05 μg and about 10 μg of haemagglutinin per dose.

The complexes for immunization provided herein may be administered by a variety of routes and a variety of immunizing schedules may be used. The immunization may comprise a single administration of the complex or a plurality (including two) administrations of the complex may be used 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 loss following virus challenge of mice immunized with flu-ISCOMs or subvirion vaccine;

Figure 3 shows the induction of influenza HI and H2 subtype cross-reactive cytotoxic T-cells in mice immunized with HI HA flu-ISCOMs; Figure 4, consisting of panels A, B, C and D, shows the mortality and weight loss following heterologous (H2) virus challenge of mice immunized with HI flu-ISCOMs, subvirion vaccine or live virus; and

Figure 5, consisting of panels A, B, C and D, shows the mortality and weight loss following heterologous (H3) virus challenge of mice immunized with HI flu-ISCOMs, subvirion vaccine or live virus.

GENERAL DESCRIPTION OF THE INVENTION As discussed above, the present invention is concerned with the use of ISCOMs in combination with a solubilized fraction of influenza virus, including haemagglutinin (HA) , particularly derived from HI subtype influenza virus, ("flu-ISCOMS") to stimulate a cross-protective cytotoxic T-cell (CTL) response in a host.

Immune stimulatory complexes or ISCOMs are a complex composed of typically 0.5 wt% Quillaja saponins, 0.1 wt% cholesterol and 0.1 wt% phospholipid and antigen in PBS. In the present invention, the antigen is HA or a fragment thereof. Occasionally, surfactants are used to prepare ISCOMs (such as, Mega 10) but are removed from the final formulation before use. The adjuvant- active components of ISCOMs are derived by aqueous extraction of the bark of Quillaja saponaria and are further purified by chromatography. Quil A is a complex but purified mixture of Quillaja saponins which are glycosides of quillaic acid and carbohydrates. Further

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 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 prevention against infection by 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, thereby reducing morbidity and mortality (ref. 10) . In the present invention, the ability of flu-ISCOMs to give improved protection in mice against infection with homologous virus correlated with the induction of higher serum HAI titers, reflecting the presence of antibodies 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 findings have been shown for antigens from laboratory strains of influenza and for other proteins formulated as ISCOMs 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 the induction of influenza specific CTL m 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 HA of the subtype HI generated CTL that lysed target cells infected with HI and H2, but not infected with H3 strains of influenza virus. In contrast, CTL from mice immunized with live HI virus killed target cells infected with either H2 or H3 subtype viruses. No CTL were generated in mice immunized with the subunit vaccine. This differential virus subtype recognition by CTL 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 CD8 ⁺ CTL generated after infection with live influenza virus (refs. 23, 24) . CTL generated by flu-ISCOMs recognised a synthetic peptide representing amino acids HA 189 to 199 that is conserved amongst HI and H2, but not H3 flu viruses. In contrast, the immunodominant response 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 determined 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 containing 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 flu (HI) -ISCOMs to induce cross-reactive CTL in mice against HI and H2 influenza viruses and protection against mortality and morbidity due to infection with HI, H2 but not H3 influenza viruses.

In contrast to the cross-protection induced by flu-ISCOMs described herein, mice immunized with live H1N1 influenza 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 HI and H2 viruses. One explanation for these observations is that the cross-protection observed may be mediated by immunological mechanisms 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 immunization with flu-ISCOMs compared with live virus that could contribute 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 can be protected against death following lethal virus challenge by immunization with vaccinia expressing NP (ref. 6) . In both studies, CD4 ⁺ cytotoxic T cells were implicated in mediating the protection.

No significant differences have been seen in the quantity and type of cytokines produced by virus and flu-ISCOMs. Both induce Thl and Th2 cytokines (ref. 17) . Another explanation for the differences in cross-protection observed between flu virus and flu-ISCOMs is that the functional activities of CTL induced by flu-ISCOMs and live virus differ and may depend on their specificities for the HA and NP proteins respectively. Cytotoxic T-cells specific for HA have been implicated in mediating cross-protection in mice given a recombinant HA2 fragment from influenza HA expressed as a fusion protein. Immunization with the HA2 protein of the HI strain protected BALB/c mice against HI and H2, but not H3 strains of influenza virus (ref. 28) . 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 maintain long term memory T cell responses (ref. 29) . For example, while cross-protection has been observed in mice immunized with 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 dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies and 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 determinable by one skilled in the art and may be of the order of micrograms of the haemagglutinin. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of 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 contains antigenic material of only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and 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 describes 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 suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations. Example 1:

This Example describes influenza viruses and vaccine preparations. A/Taiwan/1/86 (H1N1) 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 Connaught Laboratories 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 fluid. Example 2:

This Example describes the preparation of complexes comprising influenza antigens and immunostimulating 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 60% 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 Louis, MO) 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 50% (w/w) sucrose gradient. After centrifugion at 40,000 rpm for 18 hours, fractions were collected for characterization. Formation of ISCOMs was verified by negative staining electron microscopy and analytical sucrose density centrifugation. Cholesterol incorporation was determined using ³ H-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% 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 Biologies Evaluation and Research) . The final 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 immunization of mice. 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 μg HA in 0.1 mL PBS. Control mice received either PBS alone or 200 to 400 hemagglutination units (HAU) of live A/Taiwan virus as allantoic fluid. Fourteen days later the mice were challenged intranasally while under anaesthesia with 50 μL of live mouse-adapted A/Taiwan/1/86 (5 LD ₅₀ ) or A/Japan/305/57 (5 LD ₅₀ ) 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/0.5% 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 μm membrane (Millipore, Bedford, MA) and stored at ' -70 ^° C until assayed. Serial dilutions of lung homogenates (100 μL) 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 6% C0 ₂ . To each well was added 1 mL of overlay medium comprising 2% Noble agar (DIFCO, Detroit, MI), 0.2% D(+) glucose (Sigma), 0.02% DEAE Dextran

(Pharmacia, Quebec), 2.0% BME vitamins (ICN, Ontario) and 20 μg/mL TPCK trypsin (Sigma) in 2 x DMEM medium (ICN) . Following incubation for 3 days at 37 ^° C (5% C0 ₂ ) , 1 mL of a 2% neutral red solution in PBS was added to each well and, incubation continued overnight. The stain was aspirated and the plaques counted and expressed as the mean logio plaque forming units/mL. The ability of mice sera to prevent influenza virus- mediated hemagglutination was determined 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 1% chick red blood by 4 HA units of A/Taiwan virus in a standard HAI assay (ref. 37) . Example 4: This Example describes the determination of

Cytotoxic T cell activity in immunized mice.

Peptides representing known H-2K ^d -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 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 synthesizer using an optimized t-Boc chemistry according to the manufacturer's instructions. Synthetic peptides were cleaved from the resin using hydrogen fluoride. The purity of the peptides exceeded 85% as judged by reverse-phase HPLC. Amino acid analyses, performed on a Waters Pico-Tag system agreed with the theoretical amino acid compositions. For CTL studies, mice were given a single immunization with either 1 μg HA as A/Taiwan flu-ISCOMs

or 10 μg 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 10 ⁷ ) were incubated at 37 ^° C, 6% C0 ₂ in an upright flask in 20 mL RPMI/10% FCS with the same number of γ-irradiated (3000 Rads) normal syngeneic spleen cells previously infected for 1 hour at 37 ^° C with A/Taiwan virus in allantoic fluid at 5,000 HAU/8 xlO ⁷ cells. After incubation for 5 to 6 days, the cells were tested in a standard ⁵¹ chromιum release assay. Briefly, washed effector cells were incubated with 10 ⁴ P815 mastocytoma target cells labelled with ⁵¹ chromιum at 50 to 100 μCι/10 ⁶ cells in 96-well v-bottom tissue culture plates for 4 to 6 hours at 37 ^° C in 6% C0 ₂ . Target cells were either untreated, pulsed with 10 μM synthetic peptide or infected with live A/Taiwan/1/86 flu virus at 1000 HAU/10 ⁶ cells, for 90 min. at 37 ^° C. Plates were centrifuged at 1,000 rpm for 5 mm. and 0.1 mL of supernatant was removed for measurement of ⁵¹ chromιum content in a gamma counter. Spontaneous and total release of ⁵¹ chromιum were determined by incubating target cells with either medium or 2.5% Tπton-XlOO respectively, in the absence of responder lymphocytes for the duration of the assay. Percentage specific chromium release was calculated as (counts - spontaneous counts) / (total counts - spontaneous counts) x 100. The spontaneous release of ⁵¹ chromιum in the absence of effector cells was between 5 to 15% m these studies. SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a novel immunization procedure for protecting hosts against disease caused by influenza virus which utilizes a complex of solubilized influenza virus and ISCOMs to produce cytotoxic T-cells specific for HA of both HI HA and H2 HA subtypes of influenza

virus. Modifications are possible within the scope of this invention.

Table 1 Specificity of CTL induced by flu-ISCOMs, subvirion vaccine and virus

% Lysis of Target Cells

Immunization Orig:in of CTL line

E:T Medium A/Taiwan A/Philippines HA peptide NP Peptide (H1N1) (H3N2) (189-199) (147-158)

A/Taiwan ISCOMs 30 1.6 24.0 3.0 40.6 0.7

60 2.6 31.8 5.7 46.2 0.6

A/Taiwan virus 24 1.8 32.3 60.5 11.0 53.8

A/Taiwan vaccine 30 11.1 13.7 nd 9.7 12.4 60 17.9 12.0 nd 16.7 18.0 CΛ

CTL cultures were generated and examined for lytic activity as described in Materials and Methods. Briefly, BALB/c mice were given one immunization with A/Taiwan flu-ISCOMs containing 1 μg of HA. Ten days later, spleen cell cultures were established by re-stimulation with γ-irradiated, A/Taiwan virus infected syngeneic spleen cells for 5 days. T cell cultures were tested for killing of ⁵¹ chromium-labelled P815 target cells infected with H1N1 (A/Taiwan) or H3N2 (A/Philippines/82) viruses or pulsed with synthetic peptides representing CTL epitopes from HI HA (amino acids HA2 189 to 199) or NP (amino acids 147 to 158) recognized by MHC class I (H-2K ^d ) restricted, CD8 ⁺ CTL. Data points represent the mean % lysis for triplicate tests for spleen cell cultures pooled from 2 mice tested at various effector to target ratio (E:T) .

Table 2 Serum HAI titers against Hi and H2 subtype flu viruses from BALB/c mice immunized with A/Taiwan (HI) ISCOMs, subvirion vaccine or virus

HAI Titer*

Immunization HA dose ⁽ μg ⁾ ag;ainst A/T,aiwan (HI) against A/Japan (H2) pc »st 1* post 2* post 1* post 2*

A/Taiwan ISCOMs 1 40 640 10 10

A/Taiwan vaccine 10 20 120 10 10

A/Taiwan virus 400 HAU 10 40 10 10

PBS - 10 10 10 10

BALB/c mice were immunized twice with A/Taiwan ISCOMs, subvirion vaccine or live virus at days 0 and 21. Bleeds were taken at days 13 and 34 and serum tested for HAI activity against homologous A/Taiwan/86 (HlNl) and heterologous a/Japan/57 (H2N2) viruses as described in Materials and Methods. *values are the mean titers for sera pooled from 6 mice.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A NAME: BURT, David (B STREET: 47 Dakin Drive (C CITY: Ajax (D STATE: Ontario (E COUNTRY: Canada (F POSTAL CODE (ZIP) : LIT 2S2

(A NAME: SAMBHARA, Suryaprakash (B STREET: 50 Harness Circle (C CITY: Markham (D STATE: Ontario (E COUNTRY: Canada (F POSTAL CODE (ZIP) : L3S 1Y1

(A NAME: UNDERDOWN, Brian (B STREET: 58 Mountain Avenue (C CITY: Hamilton (D STATE: Ontario (E COUNTRY: Canada (F POSTAL CODE (ZIP) : L8P 4G2

(A NAME: MOREIN, Bror (B STREET: Ollonstigen 3 (C CITY: Vreta (D STATE: Uppsala (E COUNTRY: SWEDEN (F POSTAL CODE (ZIP) : S-75590

(A NAME: KLEIN, Michel H (B STREET: 16 Munro Boulevard (C CITY: Willowdale (D STATE: Ontario (E COUNTRY: Canada (F POSTAL CODE (ZIP) : M2P 1B9

(ii) TITLE OF INVENTION: COMPOSITIONS AND METHODS FOR PROTECTION AGAINST IMMUNOLOGICALLY DIVERSE ISOLATES OF INFLUENZA VIRUS

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: lie Tyr Ser Thr Val Ala Ser Ser Leu Val Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Thr Tyr Gin Arg Thr Arg Ala Leu Val Thr Gly

1 5 10