Influenza (H1N1)–ISCOMs enhance immune responses and protection in aged mice

Mechanisms of Ageing and Development 96 (1997) 157 – 169

Influenza (H1N1)–ISCOMs enhance immune responses and protection in aged mice Suryaprakash Sambhara a, Samantha Woods a, Rita Arpino a, Anjna Kurichh a, Alan Tamane a, Karen Lovgren Bengtsson b, Bror Morein b, Brian Underdown a, Michel Klein a, David Burt a,* a

Pasteur Merieux Connaught Canada, 1755 Steeles A6enue West, North York, Ontario M2R 3T4, Canada b Department of Virology, National Veterinary Research Institute, Uppsala, Sweden Received 28 September 1996; received in revised form 15 January 1997

Abstract Aging is associated with a decline in immune function and the elderly are therefore more susceptible to infectious disease and less responsive to vaccination. Influenza antigens complexed as immunostimulatory complexes (ISCOMs) generate more potent protective immune responses compared with non-adjuvanted flu antigens in young adult mice. We report on the protective efficacy of flu – ISCOMs compared with the current split flu vaccine in an aged mouse model. DBA/2 mice aged 2 or 18 months were immunized with flu vaccine, ISCOMs or live virus, prior to challenge with the homologous virus. In aged mice, flu–ISCOMs induced significantly higher serum hemagglutination inhibition (HAI) titers compared to vaccine, similar to the levels obtained in young adult mice that received the split vaccine. Flu–ISCOMs but not vaccine induced cytotoxic T lymphocyte (CTL) responses in young and to a lesser degree in aged mice. In aged mice flu – ISCOMs significantly reduced illness and enhanced recovery from viral infection compared with vaccine. Our data suggests that flu–ISCOMs may offer an improved vaccine strategy for protection of elderly humans against the complications of influenza infection. © 1997 Elsevier Science Ireland Ltd.

Abbre6iations: Immunostimulatory complexes, ISCOMs; Hemagglutinin, HA; Hemagglutination inhibition, HAI. * Corresponding author. Present address: Connaught Center for Biotechnology Research, Connaught Laboratories Ltd., 1755 Steeles Avenue West, North York, Ontario, Canada, M2R 3T4. 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 1 8 8 9 - 7

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Keywords: ISCOMS; Immune response; Aging; Adjuvants

1. Introduction Influenza is responsible for illness, hospitalization and mortality in individuals aged 65 and over [1]. Indeed, while vaccination is of benefit to the elderly, the efficacy of current vaccines is generally lower (30–70%) compared to that observed for healthy adults and the young (70–80%) [2]. Studies in elderly humans [3] and aged mice [4,5] have shown reduced cellular and humoral immunity following exposure to influenza antigens compared to younger cohorts. Apart from the physiological changes in the elderly due to chronic diseases, aging in otherwise healthy individuals is associated with a decline in immune function. This reduced immune responsiveness is characterized by an increase in the proportion of T lymphocytes that are unable to respond to stimulation in vitro by specific antigen or mitogen due to defects in signal transduction and dysfunctional downstream events associated with T cell activation [6]. Such alterations of the T cell response result in lower levels of secretion of the Th1-type cytokine IL-2 from lymphocytes of aged humans [7] and mice [8]. In contrast, T cells from aged animals produced normal or enhanced levels of the Th2 cytokine IL-4 [9] compared to T cells from younger cohorts after non-specific stimulation. The inability of the elderly to mount adequate cell-mediated immune responses to viral antigen may result in delayed clearance of influenza virus from the lungs of infected individuals, diminished production of virus neutralizing antibodies and increased susceptibility to infection. Furthermore, current influenza vaccines do not elicit strong Th1 cytokine responses as evidenced by their inability to prime for MHC class I-restricted cytotoxic T lymphocyte responses (CTL) [10]. Vaccine formulations and adjuvants such as immunostimulatory complexes (ISCOMs) [11,12], free saponins [13] and hybrid yeast retrotransposon (Ty) particles [14] that promote both the induction of Th1-type cytokines and higher levels of virus neutralizing antibody may offer enhanced protection in the elderly against influenza. ISCOMs are particles approximately 40 nm in diameter, prepared by combining the saponin containing adjuvant Quil A, cholesterol, phospholipids and antigen [15]. ISCOMs have been shown to elicit antibody responses of both the IgG2a and IgG1 subtypes [16] and to enhance IL-2 and IFN-g synthesis [17] compared with soluble antigen. As a result of their ability to elicit strong Th1-type immune responses, ISCOMs induce enhanced antibody titers [11] and CTL responses [12] in experimental animals against a variety of immunogenic proteins. Recent studies in mice from our laboratory (paper in preparation) and from others [18] have demonstrated that influenza antigens presented as ISCOMs induce up to 10-fold higher titers of viral neutralizing antibodies and offer improved and more durable protection compared to current influenza vaccines. Antigens formulated as ISCOMs suggest that they may thus offer better protection against viral diseases in the elderly. To examine this

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possibility, we have compared flu–ISCOMs and current flu vaccine for their ability to induce protective immune responses in DBA/2 mice of 18 months of age to simulate the age and immune status of the human elderly population targeted for influenza vaccination.

2. Materials and methods

2.1. Materials Influenza A/Taiwan/1/86 (H1N1) live virus as allantoic fluid, commercial monovalent subunit vaccine (Fluzone) and mouse adapted virus (gift from M. Harmon) were from Connaught Laboratories, Swiftwater, PA. Female DBA/2 (H-2d) mice of 2 months and 18 months of age were purchased from the National Institutes of Aging (NIH, Bethesda, MA), where they are maintained in environmentally controlled, barrier facilities as virgin animals.

2.2. Analysis of cell surface markers Cell surface markers were identified and quantitated by flow cytometric analysis. Nylon wool non-adherent spleen cells (1 × 106) from non-immunized aged and young mice and viable spleen cells (1 ×106) isolated by centrifugation over Lymphoprep (Nycomed Pharma, Oslo, Norway) were resuspended in 100 ml PBS and stained for 30 min on ice with a panel of flurochrome-conjugated monoclonal antibodies. After staining, the cells were washed 3 times in PBS and analyzed on a Coulter Elite fluorescence activated cell sorter (FACS), (Coulter, Miami, Fl). The following monoclonal antibodies were used, R-PE-conjugated rat anti-mouse CD45RB, R-PE-conjugated rat anti-mouse CD44, PE-conjugated rat anti-mouse CD4, FITC-conjugated rat anti-mouse CD8, and FITC-conjugated rat anti-mouse ab T cell receptor (TcR) (Pharmingen, San Diego, CA). FITC-conjugated goat anti-mouse Ig was obtained from Jackson ImmunoResearch (West Grove, PA).

2.3. Preparation of flu– ISCOMs ISCOMs were prepared as previously described [19]. Briefly, live influenza virus grown in embryonated eggs was purified from allantoic fluid by sucrose density gradient centrifugation (20 – 60% sucrose in PBS, 10 000× 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 dialyzed supernatant was added 1 mg each 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. The mixture was incubated for 2 h at room temperature, dialysed against PBS, layered onto a 10–50% (w/w) sucrose gradient and centrifuged at 40 000 rpm for 18 h. Fractions tested for influenza hemagglutinin (HA) by single radial immunodiffusion (SRID) after solubilization with 1% Zwittergent

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314 non-ionic detergent (Calbiochem, La Jolla, CA) using A/Taiwan/1/86 antigen and antiserum reference standards (Centers for Biologics Evaluation and Research). Successful incorporation of protein into ISCOMs was verified by negative staining electron microscopy on fractions showing co-migration of HA and [3H]cholesterol.

2.4. Immunization protocol and 6irus challenge DBA/2 (H-2d) mice of 2 or 18 months of age (10 mice per treatment) were given two subcutaneous immunizations of either A/Taiwan/1/86 influenza vaccine (10 mg of HA) or flu – ISCOMs (1 mg of HA) on days 0 and 21. Influenza A/Taiwan ISCOMs and vaccine used in the present studies were quantified according to their HA content as measured by SRID. Six mice from each group were challenged fourteen days after the second immunization by giving 50 ml live mouse-adapted A/Taiwan/1/86 virus (5 LD50) intranasally while under anesthetic (Isoflurane). Mortality and weight changes in the mice were monitored daily and every 2–3 days respectively, up to 14 days post challenge. The data were analyzed by Student’s t-test.

2.5. Cytokine profiles Fourteen days after the final immunization the spleens were removed from inoculated mice and single cell suspensions were prepared. Spleens cells (3 × 106) were incubated in duplicate with 3 × 106 g-irradiated (3000 rads) normal syngeneic spleen cells infected with A/Taiwan/1/86 live influenza virus (5000 HA units (HAU)/8 ×107 cells) as antigen presenting cells (APC) in 2 ml cultures in a 24 well plate (Costar, Cambridge, MA). Control wells comprised immune spleen cells together with non-infected APC. After 48 h incubation the levels of g-interferon and IL-4 contained in the supernatant of each well was determined by sandwich ELISA. Briefly, 96 well Nunc Maxisorp microplates (Nunc, Kamstrup, Denmark) were coated overnight at room temperature with cytokine monospecific rat monoclonal antibodies (Pharmingen) diluted to a concentration of 2 mg/ml in 50 mM carbonate buffer pH 9.6. Recombinant IL-4 and IFN-g standards were obtained from Pharmingen and Genzyme respectively. Undiluted culture supernatants or the appropriate recombinant standards for each cytokine were added to the wells in duplicate and bound cytokines were detected using a biotinylated rat monoclonal antibody specific for each cytokine (Pharmingen) followed by the addition of a peroxidase conjugated streptavidin preparation (Vector Laboratories, Burlingame, CA) diluted to a concentration of 500 ng/ml. The plates were developed by the addition of the substrate 10% TMB (ADI Diagnostics, Toronto, Ontario) in 0.05% hydrogen peroxide (Fisher Scientific, Toronto, Ontario) in water. The absorbances of the reaction wells were read at dual wavelengths (450 and 540 nm) on a Multiskan MCC 340 Mk II (Flow Laboratories) microplate reader. The levels of cytokines present in the supernatant were derived by logit–log standard curve analysis using ELISA+ software (Meddata).

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2.6. Determination of cytotoxic T cell acti6ity For CTL studies, groups of 4 mice were immunized as described above with either 1 mg HA as A/Taiwan/1/86 flu–ISCOMs or 10 mg HA as monovalent subvirion vaccine in 0.1 ml PBS via the subcutaneous route. Two weeks later, spleens from two mice from each group were removed and pooled. Spleen cells 2.5 × 107 were incubated at 37°C, 6% CO2 in an upright flask with 10 ml RPMI – 10% FCS with the same number of g-irradiated (3000 Rads) normal syngeneic spleen cells previously infected for 1 h at 37°C with A/Taiwan/1/86 virus in allantoic fluid at 5000 HAU/8× 107 cells. After incubation for 5–6 days, the cells were tested in a 4 h standard 51chromium release assay for CTL activity. Briefly, washed cells (responders) were incubated with 2× 103 P815 (H-2d) cells labeled with 51chromium at 50 – 100 mCi/106 cells in 96-well v-bottom tissue culture plates for 4 h at 37°C in 6% CO2. Target cells were either untreated, or pulsed with 10 mM synthetic peptide spanning the HA2 sequence 189–197 from influenza virus PR/8 (H1)(I Y S T V A S S L), which is present in the transmembrane region of H1 and H2 but not in the H3 subtype of influenza viruses. The HA2 peptide is presented by the Kd allele and is known to be an immunodominant epitope. Plates were centrifuged at 1000 rpm for 5 min and 0.1 ml supernatant was removed to determine the degree of 51chromium release in a g-counter. Spontaneous and total releases of 51chromium were determined by incubating target cells with either medium or 2.5% Triton-X100 in the absence of responder lymphocytes. Percentage specific chromium release was calculated as (counts− spontaneous counts)/(total counts −spontaneous counts) × 100. The spontaneous release of 51chromium in the absence of effector cells was found to be between 5–15% in these studies.

2.7. Hemagglutination inhibition (HAI) assay Mice were bled via the orbital sinus vein 14 days after each immunization. Sera samples were heated at 56°C for 30 min to inactivate complement and pre-treated with trypsin – periodate to destroy endogenous inhibitors of HA. Serially diluted antisera were tested for their ability to inhibit the agglutination of 1% chicken red blood cells by 4 HA units of A/Taiwan virus in a standard HAI assay.

3. Results and discussion

3.1. Cell surface markers Analysis of various T and B cell surface markers demonstrated differences between the T cell populations of young and aged animals in non-primed mice (Table 1). Ninety percent of the nylon wool non-adherent cells were T cell receptor positive. Absolute numbers of splenic CD8 + and CD4 + cells were similar for both age groups, a finding consistent with other reports [6]. No difference in the level of CD4 expression was observed between T cells isolated from young and aged mice.

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However, the density of CD8 was : 50% lower on T cells from aged compared to young T cells, a finding that may explain why aging appears to impair the responsiveness of CD8 + more than that of CD4 + T cells [20]. There was an increase in the numbers of T cells positive for CD44 (Pgp-1) and a decrease in CD45RB + T cells, was observed for T cells from aged mice compared with those of the young animals. These changes in CD44 and CD45RB are consistent with an increase in the numbers of T cells exhibiting the memory phenotype [21,22], a characteristic of lymphocytes from aged animals [23,24]. No significant differences in the numbers of cells expressing a B cell marker, as assessed by surface immunoglobulin were detected. These results indicate that T cell populations from mice of 18 months of age used in this present study exhibit phenotype characteristic of aging.

3.2. Antibody Responses The HAI titers and viral specific total IgG titers induced by flu–ISCOMs in young and aged mice are presented in Table 2. An age related immune dysfunction is quite evident. The antibody responses in the aged mice in response to virus or vaccine were about 8 – 16 fold lower when compared to young mice. Flu–ISCOMs (1 mg of HA) induced flu specific antibody responses in the young mice over 8 fold when compared with the vaccine (10 mg of HA). Both flu vaccine and flu virus failed to induce detectable HAI and significant antigen specific IgG titers in the aged mice. However, in the aged mice flu–ISCOMs (1 mg of HA) induced flu specific antibody responses (HAI titer of 120 and antigen specific total IgG titer of 102 400) similar to the levels obtained with flu vaccine (10 mg of HA) in the young mice. Two important points emerge from these results: (1) flu–ISCOMS are highly immunogenic in young mice and induce significantly higher levels of HAI and flu antigen specific total IgG titers even at 1 log less dose of HA compared with vaccine; and (2) flu – ISCOMs can overcome the age related immunodeficiency in Table 1 FACS analysis of cell surface markers on splenic lymphocytes from non-immunized DBA/2 mice of 2 and 18 months (2 m and 18 m) of age Marker

Nylon wool adherent cells CD4 CD8 CD44 TcR CD45RB Spleen cells Surface Immunoglobulin

Positive cells (% total)

Fluorescence peak intensity

2 months

18 months

2 months

18 months

54.4 17.6 15.1 93.8 74.4

53.9 21.0 36.6 90.8 55.8

2.3 6.4 1.7 5.9 5.2

2.5 3.6 1.7 4.3 3.5

44.7

46.6

6.4

5.0

Data represent results from 1×106 labeled cells per marker, pooled from 2 mice.

51 200 6400 3200 1600 3200 800

960 120 120 B15 240 B15

The data represent the mean titer of 6 individual mice. HAI titers of PBS immunized 2 and 18 months old mice were B15 and total vaccine specific IgG titers were B100.

A/Taiwan-Iscoms 120 A/Taiwan-Iscoms 15 A/Taiwan-Vaccine B15 A/Taiwan-Vaccine B15 A/Taiwan-Virus 30 A/Taiwan-Virus B15

2 18 2 18 2 18

819 200 102 400 102 400 6400 51 200 3200

Antigen specific IgG titer

HAI titer

HAI titer

Antigen specific IgG titer

14 days post-secondary immunization

14 days post-primary immunization

Immunogen

Age of mice (months)

Table 2 HAI and flu antigen specific IgG (ELISA) serum titers of young and aged mice that were immunized with flu–ISCOMS, vaccine or virus

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aged mice and induce antibody titers comparable to those seen in younger animals that received influenza vaccine.

3.3. Morbidity To examine the relative protective efficacies of flu–ISCOMs and vaccine, immunized mice were monitored for changes in total body weight following challenge with live influenza virus. Significant differences were observed between the two immunogens in the degree of protection against illness and the rate of recovery. Young mice given flu – ISCOMs showed no significant loss of weight up to 14 days post challenge, whereas animals given vaccine lost almost 20% of their starting weight at day 5 (Fig. 1A). This was associated with an 8-fold decrease in the pre-challenge HAI titer for mice that received vaccine compared with flu–ISCOMs (Table 2). Aged mice given flu–ISCOMs fully recovered from signs of illness by day 14. However, mice that received vaccine were still 15% below their starting weight at this time point (Fig. 1B). The degree of weight loss and rate of recovery induced by flu – ISCOMs in aged mice was identical to that for vaccine at a ten-fold higher dose of HA in young mice (Fig. 1C) and the pre-challenge HAI titers of the mice in these groups are identical (Table 2). These data indicate that immunization with influenza HA in ISCOMs resulted in less morbidity and enhanced recovery in both young and aged mice compared with a 10-fold higher dose of HA as vaccine.

3.4. Cytotoxic T cells MHC class I-restricted CD8 + CTL are important in host recovery from various viral infections including influenza [25]. Previous studies have demonstrated a correlation between enhanced CTL activity and accelerated clearance of the virus from influenza infected lungs in the absence of virus neutralizing antibodies [26]. The enhanced ability of antigens associated with ISCOMs to induce CTL [12] compared to the vaccine may also account for the improved recovery from infection observed in both the young and aged mice immunized with flu–ISCOMs in the present study. Indeed we have shown that in young BALB/c mice, flu–ISCOMs, but not split flu vaccine induces MHC class I-restricted CD8 + CTL responses in vivo under immunization conditions identical to those used in this present study (in preparation). Hence, we investigated the ability of flu–ISCOMs to induce CTL in both young and aged mice. The results of a representative experiment are presented in Fig. 2. Only flu – ISCOMs but not vaccine induced significant CTL responses to the HA/2 peptide in the young mice (Fig. 2A). Although some CTL activity could be detected in aged mice that received flu–ISCOMs the magnitude of the response was reduced compared to that seen in young mice (Fig. 2B). The reduced CTL responses in the aged mice could be due to defects at various levels including processing and presentation of the HA/2 peptide or activation of precursor CTL leading to decreased clone size or decreased lytic activity or a combination of both. We are currently investigating these possibilities.

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Fig. 1. Changes in weight in young and aged immunized mice following challenge with flu – ISCOMs or vaccine. DBA/2 mice of 2 and 18 months of age were immunized with flu – ISCOMs (1 mg HA, ( )) or vaccine (10 mg HA, ( ) and challenged two weeks later with 5 LD50 live mouse-adapted A/Taiwan virus via the intranasal route. Weights of individual mice were determined at 2 – 3 day intervals for 14 days and expressed as a percentage of their weights immediately prior to challenge. Figures represent data for flu–ISCOMs vs. vaccine in mice of 2 (A) and 18 (B) months and flu – ISCOMS in mice of 18 months vs. vaccine in mice of 2 months of age (C). Starting weights (mean9S.E.M.) for young and aged mice were 23.89 0.52 and 24.79 0.68 g, respectively. Data are expressed as the means9S.E.M. for 6 mice per group. * PB 0.05 as determined by Student’s t-test.

3.5. Cytokine profiles IFN-g is produced in mouse lungs infected with influenza virus [27], however the enhanced recovery of aged mice primed with flu–ISCOMs in the present study does

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not correlate with the levels of this cytokine produced by stimulated splenocytes in vitro. Firstly, the amount of IFN-g produced by T cells from aged mice immunized with flu – ISCOMs was not significantly different to that generated in mice that received vaccine (Fig. 3A). Furthermore, aged mice primed with virus did not survive the live virus challenge (data not shown) despite producing significantly higher levels of IFN-g compared with either flu-ISCOM or vaccine groups following restimulation of splenic T cells in vitro (Fig. 3A). Results of recent studies using mice with disrupted IFN-g genes [28] also indicate that IFN-g is not essential for protection against lethal influenza virus infection. The secretion of Th2 cytokine IL-4 was enhanced significantly in in vitro restimulated spleen cells from young but not aged mice immunized with vaccine, flu–ISCOMs or live virus compared with animals given PBS (Fig. 3B). The pattern of cytokine release from T cells from young and aged animals observed in this study differs from those reported by others for naive T cells from aged mice stimulated in vitro with either mitogen or anti-CD3 antibody [9,29]. In those studies, secretion of both IL-4 [9] and IFN-g [29] was significantly increased in the aged group by non-specific activation. The reason for this discrepancy is unclear, however, the T cells used in the present study

Fig. 2. Cytotoxic T cell activity against HA/2 peptide. Two weeks post-secondary immunization, the spleens from two mice from each group were pooled and 2.5× 107 spleen cells were incubated with the same number of g-irradiated (3000 Rads) normal syngeneic spleen cells previously infected with A/Taiwan/1/86 virus as described in Section 2. On day 5, the cells were tested in a standard 4 h 51 chromium release assay for CTL activity. 2000 51chromium labeled P815 (H-2d) cells were either untreated, or pulsed with 10 mM synthetic peptide comprising of HA/2 peptide were used as target cells in all assays. The spontaneous release of 51chromium in the absence of effector cells was found to be between 5–15% in these studies. The HA2 peptide specific lysis was determined by subtracting the lysis obtained in the absence of peptide. Similar results were obtained in two separate independent experiments.

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Fig. 3. IFN-g and IL-4 secretion from immune murine spleen cells from young and aged mice restimulated in vitro with influenza virus. Spleen cells from two DBA/2 mice aged 2 (closed bars) and 18 (open bars) months immunized twice with flu – ISCOMs (1 mg HA), vaccine (10 mg HA), live virus (200 HAU) or PBS, were pooled and restimulated with APC infected with live influenza virus as described in Section 2. Values are the means9S.D. for duplicate samples taken from each pool.

represent an in vivo primed antigen-specific memory population generated with complex antigen; conditions that more closely mimic those occurring naturally. In those reports, where T cells from aged humans have been studied, no clear consensus has been established for changes in the secretion of IFN-g [30,31]. The possibility of using adjuvants and immunomodulators to overcome impaired immune responses in the elderly is attractive. For example, IL-12 has been shown to increase the activity of allospecific murine CTL derived from aged mice [32]. Co-administered IL-2 also enhanced the protective efficacies of influenza proteins against lethal virus challenge in young and aged mice [33]. However, the short half-life of cytokines administered in vivo may make this type of approach for vaccination impractical. Results of a recent report suggest that the reduced immune response to flu virus and increased susceptibility to flu infection can be overcome by immunization of mice with a high dose of a vaccinia recombinant expressing flu HA [5]. The findings were explained in terms of the broader tissue tropism of vaccinia compared with influenza virus, rather than due to increased cytokine production. However, an earlier study failed to demonstrate enhanced protection in aged mice immunized with a vaccinia – HA recombinant [34]. Our data indicates that in aged mice, flu – ISCOMs offer improved protection over live virus and commercial influenza vaccine. The reduced morbidity imparted by flu–ISCOMs correlate with enhanced production of HAI antibodies. This may be a result of: (1) upregulation of co-stimulatory molecules on APC and their ligands on responding T and B cells; (2) increased secretion of cytokines in the local environment; and (3) improved processing and presentation of antigens, or a combination of all these mechanisms.

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Our findings strongly indicate that flu–ISCOMs may offer improved protection over current influenza vaccines in humans, including the elderly.

Acknowledgements We thank Renu Vaish and Diane Gajewczyk (Connaught Center for Biotechnology Research, Connaught Laboratories Ltd.) for performing the flow cytometry and cytokine analysis respectively.

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