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Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice
Vaccine (2008) 26, 552—561
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/vaccine
Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice Andreas Wack a,∗, Barbara C. Baudner a, Anne K. Hilbert b, Ilaria Manini c, Sandra Nuti a, Simona Tavarini a, Hanno Scheffczik b, Mildred Ugozzoli d, Manmohan Singh d, Jina Kazzaz d, Emanuele Montomoli c, Giuseppe Del Giudice a, Rino Rappuoli a, Derek T. O’Hagan d a
Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy Novartis Vaccines, Emil-von-Behring-Street 76, 35041 Marburg, Germany c Department of Physiopathology, Experimental Medicine and Public Health,University of Siena, Italy d Novartis Vaccines, 4560 Horton Street, Emeryville, CA 94608, United States b
Received 11 January 2007; received in revised form 24 October 2007; accepted 9 November 2007 Available online 26 December 2007
KEYWORDS Adjuvants; Influenza vaccine; T-cell cytokine response
Summary Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a na¨ıve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in na¨ıve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains. © 2007 Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Novartis Vaccines Research Center, Via Fiorentina 1, 53100 Siena, Italy. Tel.: +39 0577 243469; fax: +39 0577 243564. E-mail address: [email protected] (A. Wack). 0264-410X/$ — see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.11.054
Combination adjuvants for the induction of antibody and T-cell responses
Introduction Influenza (commonly called flu) is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. In the US, influenza is responsible for approximately 40,000 deaths and 100,000 hospitalizations annually [1]. The occasional emergence of pandemic strains of influenza can produce a huge increase in the death toll, with the 1918 pandemic blamed for an excess of 50 million deaths worldwide [2]. It is widely acknowledged that the recent outbreaks of highly pathogenic avian influenza in humans, caused by H5N1 strains, may indicate that a new pandemic is imminent [3]. Annual epidemics typically have a high attack rate in children and cause significant illness, but mortality due to influenza is highest in adults aged greater than 65, or in individuals with pre-existing chronic diseases [4]. Inactivated injectable influenza vaccines have been available since 1940s and prevent influenza illness caused by antigenically matched strains in 60—90% of healthy individuals [5]. However, influenza vaccines are less effective at preventing flu in the elderly, mainly due to senescencerelated impaired immune responses. Both Ab titres and cell-mediated immunity in response to flu are reduced in the elderly [6,7]. In particular, influenza-specific responses by cytotoxic and IFN-␥-producing CD8 and CD4 T-cells are diminished [8—11], consistent with findings indicating a general age-related reduction of type-1 cytokines in humans [12,13]. Recently it was shown that in the elderly, the ratio of IFN-␥:IL-10 produced by PBMCs in response to in vitro influenza stimulation correlates better with protection than serum Ab titres [14]. As a consequence, it has been postulated that influenza vaccines for the elderly need to stimulate more effectively type-1 cytokines such as IFN␥ [15]. However, despite these limits, currently available influenza vaccines do offer significant protection of elderly individuals against influenza related mortality [16], and it remains to be determined to what extent these responses involve IFN-␥ production. Vaccine adjuvants are an attractive option to overcome impaired immune responses in the elderly and may offer the opportunity to enhance influenza vaccine efficacy [5]. However, in the US, no adjuvant has yet proven sufficiently safe for widespread use in humans since insoluble aluminium salts were introduced over 70 years ago [17]. Nevertheless, the MF59 adjuvant, an oil-in-water sub micron emulsion [18], has been extensively evaluated in clinical trials with antigens from influenza virus, herpes simplex virus (HSV-2), HIV, CMV, HBV and hepatitis C virus (HCV) [19,20]. The largest clinical experience with MF59 has been obtained with an adjuvanted influenza vaccine (Fluad® ), which is licensed in more than 20 countries, with more than 20 million doses distributed. Fluad, which comprises subunit flu antigens produced in chicken eggs in MF59, shows increased immunogenicity in particular in those elderly who have a higher risk of developing severe complications of influenza infection [21]. In addition, cross reactivity against heterovariant influenza strains was enhanced by the presence of MF59 [22]. Moreover, the enhanced immune responses with Fluad were achieved without affecting the overall safety profile of the vaccine, which was very well tolerated. MF59 was also evaluated as an adjuvant for a potential pandemic vaccine and induced significantly enhanced
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antibody responses in human subjects [23,24]. Importantly, MF59 also allowed a significant reduction in the antigen dose, while maintaining the potency of the vaccine, a finding that might be important to allow an increase in the number of people immunized when an influenza pandemic occurs, assuming vaccine is available [23]. In addition, Alum does not appear to be a potent adjuvant against pandemic strains [25]. In this context, the ability of MF59 to enhance cross neutralization against heterovariant potentially pandemic strains is a very encouraging observation [26]. All MF59 influenza vaccine studies described so far have involved flu antigens produced in chicken eggs. The studies described here represent the first evaluation of MF59 with an influenza vaccine derived from mammalian cell culture. The production of flu vaccine in cell culture (FCC) rather than eggs has a number of advantages, particularly in relation to the imminent threat posed by pandemic strains. FCC vaccines are not dependent on the availability of huge numbers of eggs, which might not be available if chickens are directly affected by avian flu. In addition, producing influenza vaccines in mammalian cell culture allows the possibility of the production of antigens that have greater similarity to the hemagglutinin actually produced during virus infection in vivo [27]. Here, we evaluated MF59 for its ability to adjuvant FCC vaccine and compared the potency of MF59 with a range of alternative adjuvants. The adjuvants chosen for evaluation have already been used in humans in approved vaccines, or represent promising new generation approaches. Alum is a safe and effective vaccine adjuvant that has been used in humans for more than 70 years [17]. Calcium phosphate (CAP) has previously been shown to be an effective adjuvant in man for a number of vaccines, including diphtheria and tetanus toxoids [28], but was gradually replaced with Alum in 1960s. Microparticles prepared from biodegradable and biocompatible polymers, poly-(lactide co-glycolides) (PLG), represent a new approach for vaccine delivery [17]. We recently showed that PLG microparticles with antigens adsorbed to their surface induced potent immune responses with a range of recombinant antigens [29,30]. Oligonucleotides containing unmethylated cytosine and guanosine dinucleotides, which are called CpG immunostimulatory sequences [31], have been shown to act as potent vaccine adjuvants in a range of species including humans and activate the innate immune response directly through TLR9 [17,32]. The ability of CpG to induce potent T helper 1 immune responses, characterized by T-cells secreting IFN␥ and TNF-␣, is in marked contrast to the Th2-response induced by the traditional adjuvant, Alum [33], and is likely to overcome Th1-deficits in the immune response of the elderly. A direct antiviral role against human influenza viruses has been demonstrated for both TNF-␣ and IFN-␥ [34], and, importantly, these cytokines strongly synergize in their effects against a number of viruses [35]. It has been shown for viruses such as herpex simplex, yellow fever and Ebola that the Th1-associated IgG2a immunoglobulin subclass is highly effective in protection [36—38]. For influenza, several publications indicate that the induction of the IgG2a subclass is protective [39], even in the absence of high virus-neutralizing activity [40], and that monoclonal Abs of this subclass can efficiently confer antiviral protection [41]. In some cases, it was shown that
554 this effect may be mediated by complement components [42]. Therefore, we were interested to see whether addition of CpG to a vaccine formulation might help induce a cytokine profile best suited for antiviral action and at the same time increase the titres of neutralizing Abs and of protective Abs of the IgG2a subclass.
Materials and methods
A. Wack et al.
Calcium phosphate (CAP) formulations Calcium phosphate suspension was homogenized at 15,000 rpm for 3 min using a 20-mm probe (ES-15 Omni International, Jarrenton, VA) to reduce particle size. FCC was then adsorbed to the homogenized calcium phosphate suspension at 1 mg/ml CAP, and 3 g/ml antigen, in 5 mM Histidine buffer pH 6.5 and 9 mg/ml Sodium Chloride overnight at 4 ◦ C. Each dose contains 100 g CAP.
Materials Preparation of PLG microparticles MDCK-cell derived trivalent flu vaccine (FCC) comprising the strains A/Wyoming/3/2003 H3N2 (Wyoming), A/New Caledonia/20/99 H1N1 (New-Caledonia) and B/Jiangsu/10/2003 (Jiangsu) as well as MF59 emulsion and aluminium hydroxide adjuvants, were obtained from Novartis Vaccines, Marburg, Germany. The FCC vaccine contains purified subunit antigens and is standardized for HA content by single-radialimmunodiffusion as recommended by regulatory authorities. MF59 was manufactured as previously described [18]. RG503, poly (lactide co-glycolide) 50:50 co-polymer composition (PLG), intrinsic viscosity 0.4 from manufacturer’s specifications, was obtained from Boehringer Ingelheim. Dioctylsulfosuccinate (DSS) was from Sigma Chemical (St. Louis, MO). The CpG oligonucleotide (5 -TCC ATG ACG TTC CTG ACG TT-3 ), previously described as 1826 was synthesized with a phosphorothioate backbone by Oligos etc. (Wilsonville, OR), ethanol precipitated, and re-suspended in 10 mM Tris (pH 7.0) 1 mM. Calcium phosphate was produced by Brenntag Biosector (DK-3600 Frederikssund, Denmark). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
Mice and immunizations Groups of 8—12 female 8-week-old Balb/c mice (Charles River) were used for experiments reviewed and approved by the institutional review committees. Animals were immunized intramuscularly at days 0 and 28 with 0.3 g of either soluble trivalent FCC antigen or mixed with MF59, or adsorbed on PLG microparticles, Alum or CAP, with and without CpG. Soluble CpG was added to the formulations prior to immunizations at 10 g/dose. Blood and spleen samples were collected at 2 weeks following the first immunization, and at 2 weeks, 3 months or 6 months following the second immunization.
MF59 vaccine preparation Preparation and composition of MF59 have been described previously [18]. FCC vaccine was prepared by mixing MF59 1:1 with trivalent FCC to a final concentration of 3 g/ml trivalent antigen and 1X Phosphate Buffered Saline.
Alum formulations FCC trivalent antigen was adsorbed to aluminium hydroxide at 1 mg/ml Alum, 3 g/ml antigen, 5 mM Histidine buffer pH 6.5 and 9 mg/ml sodium chloride at 4 ◦ C overnight. Each mouse dose contained 100 g Alum.
Microparticles were prepared by a solvent evaporation method as previously described [43]. Briefly, microparticles were prepared by homogenizing 10 ml of 6% w/v polymer solution in methylene chloride with 2.5 ml PBS using a 10mm probe (Ultra-Turrax T25 IKA-Labortechnik, Germany). The water-in-oil emulsion thus formed was then added to 50 ml of distilled water containing DSS, and the mixture was homogenized with a 20-mm probe (ES-15 Omni International, Jarrenton, VA) for 5 min in an ice bath. The emulsion that was then stirred at 1000 rpm for 12 h at room temperature; the methylene chloride was allowed to evaporate. The size distribution of the resulting microparticles was determined with a particle size analyzer (Master Sizer, Malvern Instruments, UK). The zeta potential was measured with a Malvern Zeta analyzer (Malvern Instruments, UK).
Adsorption of vaccine to microparticles Microparticles with adsorbed vaccine were prepared at a load of 0.03% w/w. Concentrated Histidine buffer solution was added to a final concentration of 10 mM pH 6.2. The suspension was allowed to mix on a lab rocker at 4 ◦ C overnight. Aliquots containing 3.6 g of trivalent FCC antigen were placed into small glass vials and lyophilized at −50 ◦ C and 90 × 10−3 mBar (FreeZone 4.5 L Benchtop freeze dry system, LABCONCO, Kansas City, Missouri), with Mannitol and Sucrose to affect a final concentration of 4.5% and 1.5% respectively upon re-constitution.
Determination of antibodies by hemagglutination inhibition assay The HI assay was carried out on individual sera taken 2 weeks after the first or second immunization. Briefly, 25 L of two-fold serially diluted samples are incubated with 25 L of strain-specific influenza antigen (Whole virus, containing four hemagglutinating units) for 60 min at room temperature. A 0.5% v/v suspension of red blood cells obtained from adult cocks are added and the mixture is incubated for another 60 min. Reactions are followed through visual inspection: a red dot formation indicates a positive reaction (inhibition) and a diffuse patch of cells a negative reaction (hemagglutination). All sera are run in duplicate. The titre is defined as the serum dilution in which the last complete agglutination inhibition occurs. The antibody concentration corresponds to the reciprocal value of the titre. Geometric mean titres (GMT) of at least six mice per group are shown.
Combination adjuvants for the induction of antibody and T-cell responses
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Figure 1 Enhanced HI antibody responses to adjuvanted influenza vaccine. Groups of eight Balb/c mice were immunized intramuscularly at weeks 0 and 4 with flu cell culture vaccine (FCC) containing 0.1 g each of Ag derived from influenza strain A-Wyoming H3N2, A-New-Caledonia H1N1 and B-Jiangsu, either alone (nil) or adjuvanted as indicated. Shown are the geometric means (and standard error) of serum hemagglutination inhibition (HI) titres against H3N2 (A), H1N1 (B) and against B (C) 2 weeks post first and second dose of vaccine containing single adjuvants. Three experiments with similar outcome were performed. Please note that scales are different in A, B, C.
Determination of antigen-specific antibody subclasses by ELISA
assayed in parallel. GMT of at least six mice was calculated, and IgG1:IgG2a ratios were calculated using these GMT.
Titration of HA-specific immunoglobulin G (IgG) subclasses 1 and 2a was performed on individual sera 2 weeks after the last immunization. Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 27—30 ◦ C with 0.2 g/well with H1N1, H3N2 or B in PBS and blocked for 1 h at room temperature with 300 L of 3% poly vinyl pyrolidine. Serum samples and serum standard were initially diluted 1:5,000—1: 20,000 in PBS, 1% BSA, 0.05% Tween-20, transferred into coated-blocked plates and serially diluted. Antigen-specific IgG1 and IgG2a was revealed with alkaline phosphataseconjugated goat anti-mouse IgG1 or IgG2a, respectively (Sigma Chemical Co., SA Louis, Mo.). Antibody titres are those dilutions that gave an optical density (OD) higher than the mean plus five times the standard deviation (S.D.) of the average OD obtained in the pre-immune sera. The titres were normalized with respect to the reference serum
Determination of virus-neutralizing titres To perform the virus neutralization assay, serum samples were diluted 1:10 in UltraMDCK-medium (Cambrex) and heat-inactivated for 30 min at 56 ◦ C. This 1:10 starting dilution of inactivated sera was serially diluted two-fold in UltraMDCK-medium in 96 well flat bottom plates and incubated with 50 L of A/New Caledonia/20/99 wild type virus (100 UI/50 L) for 90 min at 37 ◦ C in a 5% CO2 atmosphere. All sera were run in triplicate. Subsequently, 100 L of MDCKcell suspension were added to each well at a concentration of 48,000 cells/well. The plates were incubated for 4 days at 37 ◦ C in a 5% CO2 atmosphere. The neutralization titre of a serum is the dilution at which 50% of the wells are protected against virus infection. The titre is expressed as ND50 and calculated using the Reed and Muench method.
Figure 2 Addition of CpG to adjuvanted FCC vaccines further enhances HI antibody responses. Mice were immunized intramuscularly twice at weeks 0 and 4 with FCC containing 0.1 g each of Ag derived from influenza strain A-Wyoming H3N2, A-New-Caledonia H1N1 and B-Jiangsu, adjuvanted with the following combinations: none (FCC), alum, MF59, CAP or PLG, either alone or in combination with CpG, as indicated. Shown are the geometric means (and standard error) of serum HI titres against H3N2 (A), H1N1 (B) and against B (C) 2 weeks post-2 doses. Data is representative of three similar experiments. Please note that scales are different in A, B, C.
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Antigen-specific T-cell cytokine response Three mice per treatment were sacrificed, spleens were collected, and single cell suspensions were obtained. Red blood cells were lysed and splenocytes cultured in RPMI (Gibco) containing 2.5% FCS (Hyclone), beta-mercaptoethanol and antibiotics. Splenocytes were stimulated in the presence of anti-CD28 (1 g/ml) (Becton—Dickinson) and a mix of the three flu Ags (1 g/ml each), or with anti-CD28 alone (unstimulated, <0.1% total cytokinepositive cells), or with anti-CD28 plus anti-CD3 (0.1 g/ml) (Becton—Dickinson). After 4 h of stimulation, Brefeldin A (5 g/ml) was added for additional 12 h. Cells were washed, fixed in 2% paraformaldehyde, permeabilized in 0.5% saponin and stained with the following mAbs: FITCconjugated anti-CD4, PerCP-Cy5.5-conjugated anti-CD3, APC-conjugated anti-IFN-␥, PE-Cy7-conjugated anti-TNF␣, PE-conjugated anti-IL5 or PE-conjugated anti-CD154 (all Becton—Dickinson). Cells were acquired on a LSRII (Becton—Dickinson) and analyzed using DIVA software (Becton—Dickinson). Values displayed are means of groups of three mice. For each mouse, percentages of unstimulated samples were subtracted from the Ag-stimulated sample.
Statistical analysis Analysis of variance (ANOVA) was used to compare the treatment group means within a time point and to compare mean responses between time points. The statistical tests were performed in GraphPad Prism 4 software for PC. The ANOVA F-statistic was used to assess significance at the 5% level. If the overall F-statistic was significant, Tukey Multiple Comparison test between the groups were conducted (within the ANOVA model). The values plotted represent the geometric mean ELISA or HI titres for each group and error bars extend to 95% confidence upper limits.
Results Potency of individual adjuvants Fig. 1 shows the evaluation of HI antibody titres to each of the three influenza strains included in the FCC vaccine. MF59 was the most potent single adjuvant and induced significantly enhanced HI titres post-1 dose and post-2 doses (p < 0.05). For H3N2, MF59 induced a nine-fold increase in post-2 dose HI titres as compared to post-1 dose, while the other adjuvants induced only a 2—3-fold increase (Fig. 1A). Comparable effects are seen for the other two strains
Figure 3 Addition of CpG to adjuvanted FCC increases IgG2a antibody responses and virus neutralization. (A) Mice were immunized intramuscularly twice at weeks 0 and 4 with FCC vaccine alone or with MF59, CpG or the combination of the two, as indicated. IgG1 and IgG2a titres were determined by ELISA, and the ratios of IgG1:IgG2a isotype geometric mean titres against H1N1 two weeks post second dose were calculated. (B) Virusneutralizing titres were determined in sera pooled from the same mice as in (A) vaccinated as indicated. Sera were taken 2 weeks post second dose.
(Fig. 1B and C). The use of alternative adjuvants, including alum and the new generation immune potentiator, CpG, were not very effective and induced only marginally enhanced responses over FCC alone. There were no significant differences between the alternative approaches, only MF59 was able to induce significantly enhanced responses over FCC alone.
Figure 4 MF59 alone and in combination with CpG induces strong sustained Ag-specific T-cell responses. Mice were vaccinated with FCC adjuvanted as indicated. (A) dot plots of spleen CD4+ T-cell cytokine responses to FCC antigens at 2 weeks post second dose. The plots shown are electronically gated on live singlet CD3 + CD4+ cells, and percentages of cytokine-positive cells present in the regions are annotated. (B) histogram showing T-cell cytokine responses 2 weeks post second dose as obtained in (A). Cells gated as in (A) were further subdivided into TNF-a positive and negative, and percentages were calculated and plotted in a stacking histogram. (C) shows same analysis as (B), done at 6 months post second dose. (D) Time course of T-cell responses post-1 dose and post-2 doses as indicated. The total percentage of all cytokine positive T-cells is shown, corresponding to the overall height of the stacked columns in (B) and (C). (A) shows dot plots from individual but representative mice, each data point in (B—D) is a mean of three mice and representative of at least three experiments with similar results.
Combination adjuvants for the induction of antibody and T-cell responses
Evaluation of combination adjuvants The HI titres to single virus strains induced by combination adjuvants are shown in Fig. 2. For H3N2 and H1N1,
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adding CpG resulted in significantly (p < 0.05) enhanced HI titres in comparison to all the single adjuvants, including MF59 (Fig. 2A and B). Also for the B strain, addition of CpG enhanced the HI titres for all adjuvants except MF59 which
558 by itself induces a strong response, however the combination of MF59 and CpG gives a higher response to influenza B virus than any of the other adjuvant combinations tested (Fig. 2C). The addition of CpG to FCC vaccine alone or to FCC plus MF59 also enhanced the H1N1-specific IgG2a antibody isotype as measured by ELISA (Fig. 3A). Fig. 3A shows H1N1 specific isotypes, similar results were obtained for the other two strains contained in the vaccine. We also directly determined the virus-neutralizing titres in the same sera. As shown in Fig. 3B, while the addition of CpG increases neutralizing titres induced by FCC alone only slightly, MF59 leads to much higher titres, which are further increased when the combination of MF59 and CpG is used. In conclusion, it is clear that addition of CpG leads to a dramatic shift from the IgG1 to the IgG2a subclass, and both the IgG1 dominated titres induced by MF59 and the IgG2a-dominated Abs induced by MF59 plus CpG are highly efficient at neutralizing virus.
Evaluation of T-cell responses to adjuvanted vaccine The Ag-specific T-cell stimulation assays clearly indicate that MF59 was the most potent adjuvant for the induction of T-cell responses, estimated by the frequency of CD4 Tcells producing TNF-␣, IFN-␥, IL-5 or combinations thereof in response to antigenic stimulus (Fig. 4A and B). Even six months after vaccination, MF59-induced T-cell responses were clearly detectable and far above all other responses (Fig. 4C and D). The addition of CpG to MF59 induced a clear change in the profile of the T-cell response, switching it to a much more pronounced Th1 profile, dominated by a potent IFN-␥ response (Fig. 4A and B), which was also sustained up to six months (Fig. 4C and D). Recent publications have shown that CD154 is specifically up-regulated on most if not all CD4 T-cells responding to antigen [44]. As it is theoretically possible that with the cytokines we are checking for, only a fraction of the flu responsive T-cells are detected, while others respond by production of cytokines that are not part of our panel, we included this staining to ensure that all T-cells activated are counted. We assayed key vaccination groups for the percentages of CD154 positive CD4 T-cells in response to flu Ag and found values comparable to those in Fig. 4 (data not shown), confirming that the majority of Ag-specific T-cells were in fact detected by the three cytokines used in our assay. For all delivery systems, addition of CpG shifted the ratio of IL-5 to IFN-␥-producing cells towards IFN-␥, however, this effect was most dramatic for MF59. Overall, MF59 induces strong, long-lasting CD4 Tcell responses, and the combination MF59-CpG induced by far the strongest Th1 response of all adjuvant combinations tested.
Discussion The ability of MF59 to induce significantly enhanced immune responses with FCC vaccine is entirely consistent with previous results of pre-clinical and clinical studies using this adjuvant in combination with egg derived flu vaccine [20]. However, the current studies are the first to compare MF59 with a range of alternative adjuvants, including established
A. Wack et al. approaches (Alum and CAP), a new generation delivery system (PLG microparticles) and a new generation immune potentiator (CpG), which directly activates innate immunity through TLR9 [32]. The significant potency of MF59 observed in these studies in comparison to alternative adjuvants, including Alum, is in line with previous studies on a range of alternative antigens [18,20,45] and has important implications for the development of optimal flu vaccines against inter-pandemic strains and in preparations for an influenza pandemic. Hence, the accumulated data clearly establish that MF59 is a more potent adjuvant than Alum for a range of vaccines [18,20], while having a similarly acceptable safety profile in humans [19]. Also in the context of pediatric CMV and HIV vaccines, MF59 was shown to be a well tolerated, potent adjuvant [20,46]. Seronegative toddlers immunized with three doses of an MF59-adjuvanted CMV gB vaccine produced antibody titres higher than those found in adults naturally infected with CMV [46]. The MF59 adjuvanted HIV candidate vaccine was evaluated in newborns of HIV positive mothers and induced a specific antibody response in 87% of the immunized infants [47]. Additionally, the vaccine formulated with MF59 was significantly more potent than Alum for the induction of cell-mediated proliferative responses [48]. Hence, MF59 has significant potential for use as a broad range vaccine adjuvant in human vaccines [45] for a wide range of individuals of different ages. Although MF59 induced optimal HI titres as a single adjuvant and also induced potent T-cell responses, only the addition of CpG to MF59 allowed the induction of a potent Th1 response, as measured by the induction of IFN-␥. This finding is in line with previous ones showing that when combined with conventional vaccines, CpG shifts immune responses towards Th1 [33,49]. The antiviral role of interferons is well established, and experiments in vitro show anti-viral activity of IFN-␥ as well as TNF␣ against human influenza viruses on lung epithelial cells [34]. As these two cytokines strongly synergize in their antiviral effects [35], it is an important observation that a high proportion of T-cells induced by MF59 + CpG produce both TNF-␣ and IFN-␥ (Fig. 4B and C). Cytokine responses are known to be involved in the early and decisive stages of host defense against influenza infection [50]. We also show here that MF59 alone induces high titres of virus-neutralizing Abs which can be further increased by the addition of CpG. These high neutralizing titres are dominated by IgG1 in the case of MF59 and IgG2a in the case of MF59 plus CpG. While this confirms that both Ig subclasses can have strong neutralizing capacity, it remains to be seen whether there are additional protective effects of non-neutralizing IgG2a Abs as described previously [39—42]. As MF59 is reconfirmed as a safe adjuvant in millions of vaccinees and CpG has been used extensively in clinical trials, it is conceivable that this or similar combinations of MF59 with Th1-shifting immunopotentiators will be tested in clinical trials, most likely tailored to specific target groups with deficiencies in type-1 immune responses. In particular, as far as priming the immune response towards a type-1 profile is concerned, the vaccination strategy proposed here may come close to the situation of previous virus exposure, which is considered a very efficient way to induce protec-
Combination adjuvants for the induction of antibody and T-cell responses tion and is the rule rather than the exception in the human population. Studies are underway to address in depth which type of immune response is most efficient at protecting from flu. In mouse studies, a role for T-cells in protection from influenza infection is clearly established [51]. While the main effector T-cells appears to be the CD8 subset that kills virus-infected cells in a contact-dependent manner, CD4 Tcells have an important role in inducing and maintaining B cell and CD8 cell memory [52]. In addition, CD4 T-cells can also act as effector cells, as evidenced in mice lacking both B and CD8 T-cells [53]. Adoptive transfer of Th1 but not Th2 clones confers protection from influenza infection [54] to recipient mice. In IFN-␥ deficient mice, it was shown that IFN-␥ plays a role in protection from heterologous challenge, independently of the generation or recruitment of cytotoxic CD8 T-cells [55]. Studies in aged mice have provided support for the concept that age related susceptibility to influenza can be reversed by the induction of a more potent IFN-␥ response [56]. A number of publications indicate that age related susceptibility to influenza virus in humans is associated with a reduction in Th1 CD4 + responses and a diminished ability to kill virally infected cells [13,15]. Hence, the ability of CpG in combination with MF59 to induce an enhanced IFN-␥ response is a significant observation and may be important for the development of an optimal influenza vaccine for the elderly. In a recent clinical study, CpG was shown to be ineffective for the induction of enhanced HI titres against influenza vaccine, but induced an enhanced IFN-␥ response [57]. These observations are consistent with our own studies, in which CpG alone did not induce enhanced HI titres, but did result in a significantly enhanced Th1 response, when used in combination with MF59. Overall, we conclude that an optimal influenza vaccine for use in the elderly might comprise a combination of MF59 to induce the highest possible HI titres, with an immune potentiator (e.g. CpG) added to enhance the Th1 response, including the induction of IFN-␥ and TNF-␣. Further studies, both pre-clinical and clinical, will show whether these Th1-related cytokines confer enhanced protection or not. The vaccination approach described here may also be effective in children, since they are usually na¨ıve to the circulating strains. More generally, the rational combination of adjuvants is likely to offer opportunities for the development of vaccines for prevention and therapy of a range of important infectious diseases [17].
Acknowledgements We are grateful to Grazia Galli and Oretta Finco for help and advice on T-cell assays, to Michela Brazzoli for help with the artwork and to Marco Tortoli for excellent animal husbandry.
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Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice Andreas Wack a,∗, Barbara C. Baudner a, Anne K. Hilbert b, Ilaria Manini c, Sandra Nuti a, Simona Tavarini a, Hanno Scheffczik b, Mildred Ugozzoli d, Manmohan Singh d, Jina Kazzaz d, Emanuele Montomoli c, Giuseppe Del Giudice a, Rino Rappuoli a, Derek T. O’Hagan d a
Novartis Vaccines, Via Fiorentina 1, 53100 Siena, Italy Novartis Vaccines, Emil-von-Behring-Street 76, 35041 Marburg, Germany c Department of Physiopathology, Experimental Medicine and Public Health,University of Siena, Italy d Novartis Vaccines, 4560 Horton Street, Emeryville, CA 94608, United States b
Received 11 January 2007; received in revised form 24 October 2007; accepted 9 November 2007 Available online 26 December 2007
KEYWORDS Adjuvants; Influenza vaccine; T-cell cytokine response
Summary Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a na¨ıve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in na¨ıve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains. © 2007 Elsevier Ltd. All rights reserved.
∗
Corresponding author at: Novartis Vaccines Research Center, Via Fiorentina 1, 53100 Siena, Italy. Tel.: +39 0577 243469; fax: +39 0577 243564. E-mail address: [email protected] (A. Wack). 0264-410X/$ — see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.11.054
Combination adjuvants for the induction of antibody and T-cell responses
Introduction Influenza (commonly called flu) is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. In the US, influenza is responsible for approximately 40,000 deaths and 100,000 hospitalizations annually [1]. The occasional emergence of pandemic strains of influenza can produce a huge increase in the death toll, with the 1918 pandemic blamed for an excess of 50 million deaths worldwide [2]. It is widely acknowledged that the recent outbreaks of highly pathogenic avian influenza in humans, caused by H5N1 strains, may indicate that a new pandemic is imminent [3]. Annual epidemics typically have a high attack rate in children and cause significant illness, but mortality due to influenza is highest in adults aged greater than 65, or in individuals with pre-existing chronic diseases [4]. Inactivated injectable influenza vaccines have been available since 1940s and prevent influenza illness caused by antigenically matched strains in 60—90% of healthy individuals [5]. However, influenza vaccines are less effective at preventing flu in the elderly, mainly due to senescencerelated impaired immune responses. Both Ab titres and cell-mediated immunity in response to flu are reduced in the elderly [6,7]. In particular, influenza-specific responses by cytotoxic and IFN-␥-producing CD8 and CD4 T-cells are diminished [8—11], consistent with findings indicating a general age-related reduction of type-1 cytokines in humans [12,13]. Recently it was shown that in the elderly, the ratio of IFN-␥:IL-10 produced by PBMCs in response to in vitro influenza stimulation correlates better with protection than serum Ab titres [14]. As a consequence, it has been postulated that influenza vaccines for the elderly need to stimulate more effectively type-1 cytokines such as IFN␥ [15]. However, despite these limits, currently available influenza vaccines do offer significant protection of elderly individuals against influenza related mortality [16], and it remains to be determined to what extent these responses involve IFN-␥ production. Vaccine adjuvants are an attractive option to overcome impaired immune responses in the elderly and may offer the opportunity to enhance influenza vaccine efficacy [5]. However, in the US, no adjuvant has yet proven sufficiently safe for widespread use in humans since insoluble aluminium salts were introduced over 70 years ago [17]. Nevertheless, the MF59 adjuvant, an oil-in-water sub micron emulsion [18], has been extensively evaluated in clinical trials with antigens from influenza virus, herpes simplex virus (HSV-2), HIV, CMV, HBV and hepatitis C virus (HCV) [19,20]. The largest clinical experience with MF59 has been obtained with an adjuvanted influenza vaccine (Fluad® ), which is licensed in more than 20 countries, with more than 20 million doses distributed. Fluad, which comprises subunit flu antigens produced in chicken eggs in MF59, shows increased immunogenicity in particular in those elderly who have a higher risk of developing severe complications of influenza infection [21]. In addition, cross reactivity against heterovariant influenza strains was enhanced by the presence of MF59 [22]. Moreover, the enhanced immune responses with Fluad were achieved without affecting the overall safety profile of the vaccine, which was very well tolerated. MF59 was also evaluated as an adjuvant for a potential pandemic vaccine and induced significantly enhanced
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antibody responses in human subjects [23,24]. Importantly, MF59 also allowed a significant reduction in the antigen dose, while maintaining the potency of the vaccine, a finding that might be important to allow an increase in the number of people immunized when an influenza pandemic occurs, assuming vaccine is available [23]. In addition, Alum does not appear to be a potent adjuvant against pandemic strains [25]. In this context, the ability of MF59 to enhance cross neutralization against heterovariant potentially pandemic strains is a very encouraging observation [26]. All MF59 influenza vaccine studies described so far have involved flu antigens produced in chicken eggs. The studies described here represent the first evaluation of MF59 with an influenza vaccine derived from mammalian cell culture. The production of flu vaccine in cell culture (FCC) rather than eggs has a number of advantages, particularly in relation to the imminent threat posed by pandemic strains. FCC vaccines are not dependent on the availability of huge numbers of eggs, which might not be available if chickens are directly affected by avian flu. In addition, producing influenza vaccines in mammalian cell culture allows the possibility of the production of antigens that have greater similarity to the hemagglutinin actually produced during virus infection in vivo [27]. Here, we evaluated MF59 for its ability to adjuvant FCC vaccine and compared the potency of MF59 with a range of alternative adjuvants. The adjuvants chosen for evaluation have already been used in humans in approved vaccines, or represent promising new generation approaches. Alum is a safe and effective vaccine adjuvant that has been used in humans for more than 70 years [17]. Calcium phosphate (CAP) has previously been shown to be an effective adjuvant in man for a number of vaccines, including diphtheria and tetanus toxoids [28], but was gradually replaced with Alum in 1960s. Microparticles prepared from biodegradable and biocompatible polymers, poly-(lactide co-glycolides) (PLG), represent a new approach for vaccine delivery [17]. We recently showed that PLG microparticles with antigens adsorbed to their surface induced potent immune responses with a range of recombinant antigens [29,30]. Oligonucleotides containing unmethylated cytosine and guanosine dinucleotides, which are called CpG immunostimulatory sequences [31], have been shown to act as potent vaccine adjuvants in a range of species including humans and activate the innate immune response directly through TLR9 [17,32]. The ability of CpG to induce potent T helper 1 immune responses, characterized by T-cells secreting IFN␥ and TNF-␣, is in marked contrast to the Th2-response induced by the traditional adjuvant, Alum [33], and is likely to overcome Th1-deficits in the immune response of the elderly. A direct antiviral role against human influenza viruses has been demonstrated for both TNF-␣ and IFN-␥ [34], and, importantly, these cytokines strongly synergize in their effects against a number of viruses [35]. It has been shown for viruses such as herpex simplex, yellow fever and Ebola that the Th1-associated IgG2a immunoglobulin subclass is highly effective in protection [36—38]. For influenza, several publications indicate that the induction of the IgG2a subclass is protective [39], even in the absence of high virus-neutralizing activity [40], and that monoclonal Abs of this subclass can efficiently confer antiviral protection [41]. In some cases, it was shown that
554 this effect may be mediated by complement components [42]. Therefore, we were interested to see whether addition of CpG to a vaccine formulation might help induce a cytokine profile best suited for antiviral action and at the same time increase the titres of neutralizing Abs and of protective Abs of the IgG2a subclass.
Materials and methods
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Calcium phosphate (CAP) formulations Calcium phosphate suspension was homogenized at 15,000 rpm for 3 min using a 20-mm probe (ES-15 Omni International, Jarrenton, VA) to reduce particle size. FCC was then adsorbed to the homogenized calcium phosphate suspension at 1 mg/ml CAP, and 3 g/ml antigen, in 5 mM Histidine buffer pH 6.5 and 9 mg/ml Sodium Chloride overnight at 4 ◦ C. Each dose contains 100 g CAP.
Materials Preparation of PLG microparticles MDCK-cell derived trivalent flu vaccine (FCC) comprising the strains A/Wyoming/3/2003 H3N2 (Wyoming), A/New Caledonia/20/99 H1N1 (New-Caledonia) and B/Jiangsu/10/2003 (Jiangsu) as well as MF59 emulsion and aluminium hydroxide adjuvants, were obtained from Novartis Vaccines, Marburg, Germany. The FCC vaccine contains purified subunit antigens and is standardized for HA content by single-radialimmunodiffusion as recommended by regulatory authorities. MF59 was manufactured as previously described [18]. RG503, poly (lactide co-glycolide) 50:50 co-polymer composition (PLG), intrinsic viscosity 0.4 from manufacturer’s specifications, was obtained from Boehringer Ingelheim. Dioctylsulfosuccinate (DSS) was from Sigma Chemical (St. Louis, MO). The CpG oligonucleotide (5 -TCC ATG ACG TTC CTG ACG TT-3 ), previously described as 1826 was synthesized with a phosphorothioate backbone by Oligos etc. (Wilsonville, OR), ethanol precipitated, and re-suspended in 10 mM Tris (pH 7.0) 1 mM. Calcium phosphate was produced by Brenntag Biosector (DK-3600 Frederikssund, Denmark). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
Mice and immunizations Groups of 8—12 female 8-week-old Balb/c mice (Charles River) were used for experiments reviewed and approved by the institutional review committees. Animals were immunized intramuscularly at days 0 and 28 with 0.3 g of either soluble trivalent FCC antigen or mixed with MF59, or adsorbed on PLG microparticles, Alum or CAP, with and without CpG. Soluble CpG was added to the formulations prior to immunizations at 10 g/dose. Blood and spleen samples were collected at 2 weeks following the first immunization, and at 2 weeks, 3 months or 6 months following the second immunization.
MF59 vaccine preparation Preparation and composition of MF59 have been described previously [18]. FCC vaccine was prepared by mixing MF59 1:1 with trivalent FCC to a final concentration of 3 g/ml trivalent antigen and 1X Phosphate Buffered Saline.
Alum formulations FCC trivalent antigen was adsorbed to aluminium hydroxide at 1 mg/ml Alum, 3 g/ml antigen, 5 mM Histidine buffer pH 6.5 and 9 mg/ml sodium chloride at 4 ◦ C overnight. Each mouse dose contained 100 g Alum.
Microparticles were prepared by a solvent evaporation method as previously described [43]. Briefly, microparticles were prepared by homogenizing 10 ml of 6% w/v polymer solution in methylene chloride with 2.5 ml PBS using a 10mm probe (Ultra-Turrax T25 IKA-Labortechnik, Germany). The water-in-oil emulsion thus formed was then added to 50 ml of distilled water containing DSS, and the mixture was homogenized with a 20-mm probe (ES-15 Omni International, Jarrenton, VA) for 5 min in an ice bath. The emulsion that was then stirred at 1000 rpm for 12 h at room temperature; the methylene chloride was allowed to evaporate. The size distribution of the resulting microparticles was determined with a particle size analyzer (Master Sizer, Malvern Instruments, UK). The zeta potential was measured with a Malvern Zeta analyzer (Malvern Instruments, UK).
Adsorption of vaccine to microparticles Microparticles with adsorbed vaccine were prepared at a load of 0.03% w/w. Concentrated Histidine buffer solution was added to a final concentration of 10 mM pH 6.2. The suspension was allowed to mix on a lab rocker at 4 ◦ C overnight. Aliquots containing 3.6 g of trivalent FCC antigen were placed into small glass vials and lyophilized at −50 ◦ C and 90 × 10−3 mBar (FreeZone 4.5 L Benchtop freeze dry system, LABCONCO, Kansas City, Missouri), with Mannitol and Sucrose to affect a final concentration of 4.5% and 1.5% respectively upon re-constitution.
Determination of antibodies by hemagglutination inhibition assay The HI assay was carried out on individual sera taken 2 weeks after the first or second immunization. Briefly, 25 L of two-fold serially diluted samples are incubated with 25 L of strain-specific influenza antigen (Whole virus, containing four hemagglutinating units) for 60 min at room temperature. A 0.5% v/v suspension of red blood cells obtained from adult cocks are added and the mixture is incubated for another 60 min. Reactions are followed through visual inspection: a red dot formation indicates a positive reaction (inhibition) and a diffuse patch of cells a negative reaction (hemagglutination). All sera are run in duplicate. The titre is defined as the serum dilution in which the last complete agglutination inhibition occurs. The antibody concentration corresponds to the reciprocal value of the titre. Geometric mean titres (GMT) of at least six mice per group are shown.
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Figure 1 Enhanced HI antibody responses to adjuvanted influenza vaccine. Groups of eight Balb/c mice were immunized intramuscularly at weeks 0 and 4 with flu cell culture vaccine (FCC) containing 0.1 g each of Ag derived from influenza strain A-Wyoming H3N2, A-New-Caledonia H1N1 and B-Jiangsu, either alone (nil) or adjuvanted as indicated. Shown are the geometric means (and standard error) of serum hemagglutination inhibition (HI) titres against H3N2 (A), H1N1 (B) and against B (C) 2 weeks post first and second dose of vaccine containing single adjuvants. Three experiments with similar outcome were performed. Please note that scales are different in A, B, C.
Determination of antigen-specific antibody subclasses by ELISA
assayed in parallel. GMT of at least six mice was calculated, and IgG1:IgG2a ratios were calculated using these GMT.
Titration of HA-specific immunoglobulin G (IgG) subclasses 1 and 2a was performed on individual sera 2 weeks after the last immunization. Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 27—30 ◦ C with 0.2 g/well with H1N1, H3N2 or B in PBS and blocked for 1 h at room temperature with 300 L of 3% poly vinyl pyrolidine. Serum samples and serum standard were initially diluted 1:5,000—1: 20,000 in PBS, 1% BSA, 0.05% Tween-20, transferred into coated-blocked plates and serially diluted. Antigen-specific IgG1 and IgG2a was revealed with alkaline phosphataseconjugated goat anti-mouse IgG1 or IgG2a, respectively (Sigma Chemical Co., SA Louis, Mo.). Antibody titres are those dilutions that gave an optical density (OD) higher than the mean plus five times the standard deviation (S.D.) of the average OD obtained in the pre-immune sera. The titres were normalized with respect to the reference serum
Determination of virus-neutralizing titres To perform the virus neutralization assay, serum samples were diluted 1:10 in UltraMDCK-medium (Cambrex) and heat-inactivated for 30 min at 56 ◦ C. This 1:10 starting dilution of inactivated sera was serially diluted two-fold in UltraMDCK-medium in 96 well flat bottom plates and incubated with 50 L of A/New Caledonia/20/99 wild type virus (100 UI/50 L) for 90 min at 37 ◦ C in a 5% CO2 atmosphere. All sera were run in triplicate. Subsequently, 100 L of MDCKcell suspension were added to each well at a concentration of 48,000 cells/well. The plates were incubated for 4 days at 37 ◦ C in a 5% CO2 atmosphere. The neutralization titre of a serum is the dilution at which 50% of the wells are protected against virus infection. The titre is expressed as ND50 and calculated using the Reed and Muench method.
Figure 2 Addition of CpG to adjuvanted FCC vaccines further enhances HI antibody responses. Mice were immunized intramuscularly twice at weeks 0 and 4 with FCC containing 0.1 g each of Ag derived from influenza strain A-Wyoming H3N2, A-New-Caledonia H1N1 and B-Jiangsu, adjuvanted with the following combinations: none (FCC), alum, MF59, CAP or PLG, either alone or in combination with CpG, as indicated. Shown are the geometric means (and standard error) of serum HI titres against H3N2 (A), H1N1 (B) and against B (C) 2 weeks post-2 doses. Data is representative of three similar experiments. Please note that scales are different in A, B, C.
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Antigen-specific T-cell cytokine response Three mice per treatment were sacrificed, spleens were collected, and single cell suspensions were obtained. Red blood cells were lysed and splenocytes cultured in RPMI (Gibco) containing 2.5% FCS (Hyclone), beta-mercaptoethanol and antibiotics. Splenocytes were stimulated in the presence of anti-CD28 (1 g/ml) (Becton—Dickinson) and a mix of the three flu Ags (1 g/ml each), or with anti-CD28 alone (unstimulated, <0.1% total cytokinepositive cells), or with anti-CD28 plus anti-CD3 (0.1 g/ml) (Becton—Dickinson). After 4 h of stimulation, Brefeldin A (5 g/ml) was added for additional 12 h. Cells were washed, fixed in 2% paraformaldehyde, permeabilized in 0.5% saponin and stained with the following mAbs: FITCconjugated anti-CD4, PerCP-Cy5.5-conjugated anti-CD3, APC-conjugated anti-IFN-␥, PE-Cy7-conjugated anti-TNF␣, PE-conjugated anti-IL5 or PE-conjugated anti-CD154 (all Becton—Dickinson). Cells were acquired on a LSRII (Becton—Dickinson) and analyzed using DIVA software (Becton—Dickinson). Values displayed are means of groups of three mice. For each mouse, percentages of unstimulated samples were subtracted from the Ag-stimulated sample.
Statistical analysis Analysis of variance (ANOVA) was used to compare the treatment group means within a time point and to compare mean responses between time points. The statistical tests were performed in GraphPad Prism 4 software for PC. The ANOVA F-statistic was used to assess significance at the 5% level. If the overall F-statistic was significant, Tukey Multiple Comparison test between the groups were conducted (within the ANOVA model). The values plotted represent the geometric mean ELISA or HI titres for each group and error bars extend to 95% confidence upper limits.
Results Potency of individual adjuvants Fig. 1 shows the evaluation of HI antibody titres to each of the three influenza strains included in the FCC vaccine. MF59 was the most potent single adjuvant and induced significantly enhanced HI titres post-1 dose and post-2 doses (p < 0.05). For H3N2, MF59 induced a nine-fold increase in post-2 dose HI titres as compared to post-1 dose, while the other adjuvants induced only a 2—3-fold increase (Fig. 1A). Comparable effects are seen for the other two strains
Figure 3 Addition of CpG to adjuvanted FCC increases IgG2a antibody responses and virus neutralization. (A) Mice were immunized intramuscularly twice at weeks 0 and 4 with FCC vaccine alone or with MF59, CpG or the combination of the two, as indicated. IgG1 and IgG2a titres were determined by ELISA, and the ratios of IgG1:IgG2a isotype geometric mean titres against H1N1 two weeks post second dose were calculated. (B) Virusneutralizing titres were determined in sera pooled from the same mice as in (A) vaccinated as indicated. Sera were taken 2 weeks post second dose.
(Fig. 1B and C). The use of alternative adjuvants, including alum and the new generation immune potentiator, CpG, were not very effective and induced only marginally enhanced responses over FCC alone. There were no significant differences between the alternative approaches, only MF59 was able to induce significantly enhanced responses over FCC alone.
Figure 4 MF59 alone and in combination with CpG induces strong sustained Ag-specific T-cell responses. Mice were vaccinated with FCC adjuvanted as indicated. (A) dot plots of spleen CD4+ T-cell cytokine responses to FCC antigens at 2 weeks post second dose. The plots shown are electronically gated on live singlet CD3 + CD4+ cells, and percentages of cytokine-positive cells present in the regions are annotated. (B) histogram showing T-cell cytokine responses 2 weeks post second dose as obtained in (A). Cells gated as in (A) were further subdivided into TNF-a positive and negative, and percentages were calculated and plotted in a stacking histogram. (C) shows same analysis as (B), done at 6 months post second dose. (D) Time course of T-cell responses post-1 dose and post-2 doses as indicated. The total percentage of all cytokine positive T-cells is shown, corresponding to the overall height of the stacked columns in (B) and (C). (A) shows dot plots from individual but representative mice, each data point in (B—D) is a mean of three mice and representative of at least three experiments with similar results.
Combination adjuvants for the induction of antibody and T-cell responses
Evaluation of combination adjuvants The HI titres to single virus strains induced by combination adjuvants are shown in Fig. 2. For H3N2 and H1N1,
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adding CpG resulted in significantly (p < 0.05) enhanced HI titres in comparison to all the single adjuvants, including MF59 (Fig. 2A and B). Also for the B strain, addition of CpG enhanced the HI titres for all adjuvants except MF59 which
558 by itself induces a strong response, however the combination of MF59 and CpG gives a higher response to influenza B virus than any of the other adjuvant combinations tested (Fig. 2C). The addition of CpG to FCC vaccine alone or to FCC plus MF59 also enhanced the H1N1-specific IgG2a antibody isotype as measured by ELISA (Fig. 3A). Fig. 3A shows H1N1 specific isotypes, similar results were obtained for the other two strains contained in the vaccine. We also directly determined the virus-neutralizing titres in the same sera. As shown in Fig. 3B, while the addition of CpG increases neutralizing titres induced by FCC alone only slightly, MF59 leads to much higher titres, which are further increased when the combination of MF59 and CpG is used. In conclusion, it is clear that addition of CpG leads to a dramatic shift from the IgG1 to the IgG2a subclass, and both the IgG1 dominated titres induced by MF59 and the IgG2a-dominated Abs induced by MF59 plus CpG are highly efficient at neutralizing virus.
Evaluation of T-cell responses to adjuvanted vaccine The Ag-specific T-cell stimulation assays clearly indicate that MF59 was the most potent adjuvant for the induction of T-cell responses, estimated by the frequency of CD4 Tcells producing TNF-␣, IFN-␥, IL-5 or combinations thereof in response to antigenic stimulus (Fig. 4A and B). Even six months after vaccination, MF59-induced T-cell responses were clearly detectable and far above all other responses (Fig. 4C and D). The addition of CpG to MF59 induced a clear change in the profile of the T-cell response, switching it to a much more pronounced Th1 profile, dominated by a potent IFN-␥ response (Fig. 4A and B), which was also sustained up to six months (Fig. 4C and D). Recent publications have shown that CD154 is specifically up-regulated on most if not all CD4 T-cells responding to antigen [44]. As it is theoretically possible that with the cytokines we are checking for, only a fraction of the flu responsive T-cells are detected, while others respond by production of cytokines that are not part of our panel, we included this staining to ensure that all T-cells activated are counted. We assayed key vaccination groups for the percentages of CD154 positive CD4 T-cells in response to flu Ag and found values comparable to those in Fig. 4 (data not shown), confirming that the majority of Ag-specific T-cells were in fact detected by the three cytokines used in our assay. For all delivery systems, addition of CpG shifted the ratio of IL-5 to IFN-␥-producing cells towards IFN-␥, however, this effect was most dramatic for MF59. Overall, MF59 induces strong, long-lasting CD4 Tcell responses, and the combination MF59-CpG induced by far the strongest Th1 response of all adjuvant combinations tested.
Discussion The ability of MF59 to induce significantly enhanced immune responses with FCC vaccine is entirely consistent with previous results of pre-clinical and clinical studies using this adjuvant in combination with egg derived flu vaccine [20]. However, the current studies are the first to compare MF59 with a range of alternative adjuvants, including established
A. Wack et al. approaches (Alum and CAP), a new generation delivery system (PLG microparticles) and a new generation immune potentiator (CpG), which directly activates innate immunity through TLR9 [32]. The significant potency of MF59 observed in these studies in comparison to alternative adjuvants, including Alum, is in line with previous studies on a range of alternative antigens [18,20,45] and has important implications for the development of optimal flu vaccines against inter-pandemic strains and in preparations for an influenza pandemic. Hence, the accumulated data clearly establish that MF59 is a more potent adjuvant than Alum for a range of vaccines [18,20], while having a similarly acceptable safety profile in humans [19]. Also in the context of pediatric CMV and HIV vaccines, MF59 was shown to be a well tolerated, potent adjuvant [20,46]. Seronegative toddlers immunized with three doses of an MF59-adjuvanted CMV gB vaccine produced antibody titres higher than those found in adults naturally infected with CMV [46]. The MF59 adjuvanted HIV candidate vaccine was evaluated in newborns of HIV positive mothers and induced a specific antibody response in 87% of the immunized infants [47]. Additionally, the vaccine formulated with MF59 was significantly more potent than Alum for the induction of cell-mediated proliferative responses [48]. Hence, MF59 has significant potential for use as a broad range vaccine adjuvant in human vaccines [45] for a wide range of individuals of different ages. Although MF59 induced optimal HI titres as a single adjuvant and also induced potent T-cell responses, only the addition of CpG to MF59 allowed the induction of a potent Th1 response, as measured by the induction of IFN-␥. This finding is in line with previous ones showing that when combined with conventional vaccines, CpG shifts immune responses towards Th1 [33,49]. The antiviral role of interferons is well established, and experiments in vitro show anti-viral activity of IFN-␥ as well as TNF␣ against human influenza viruses on lung epithelial cells [34]. As these two cytokines strongly synergize in their antiviral effects [35], it is an important observation that a high proportion of T-cells induced by MF59 + CpG produce both TNF-␣ and IFN-␥ (Fig. 4B and C). Cytokine responses are known to be involved in the early and decisive stages of host defense against influenza infection [50]. We also show here that MF59 alone induces high titres of virus-neutralizing Abs which can be further increased by the addition of CpG. These high neutralizing titres are dominated by IgG1 in the case of MF59 and IgG2a in the case of MF59 plus CpG. While this confirms that both Ig subclasses can have strong neutralizing capacity, it remains to be seen whether there are additional protective effects of non-neutralizing IgG2a Abs as described previously [39—42]. As MF59 is reconfirmed as a safe adjuvant in millions of vaccinees and CpG has been used extensively in clinical trials, it is conceivable that this or similar combinations of MF59 with Th1-shifting immunopotentiators will be tested in clinical trials, most likely tailored to specific target groups with deficiencies in type-1 immune responses. In particular, as far as priming the immune response towards a type-1 profile is concerned, the vaccination strategy proposed here may come close to the situation of previous virus exposure, which is considered a very efficient way to induce protec-
Combination adjuvants for the induction of antibody and T-cell responses tion and is the rule rather than the exception in the human population. Studies are underway to address in depth which type of immune response is most efficient at protecting from flu. In mouse studies, a role for T-cells in protection from influenza infection is clearly established [51]. While the main effector T-cells appears to be the CD8 subset that kills virus-infected cells in a contact-dependent manner, CD4 Tcells have an important role in inducing and maintaining B cell and CD8 cell memory [52]. In addition, CD4 T-cells can also act as effector cells, as evidenced in mice lacking both B and CD8 T-cells [53]. Adoptive transfer of Th1 but not Th2 clones confers protection from influenza infection [54] to recipient mice. In IFN-␥ deficient mice, it was shown that IFN-␥ plays a role in protection from heterologous challenge, independently of the generation or recruitment of cytotoxic CD8 T-cells [55]. Studies in aged mice have provided support for the concept that age related susceptibility to influenza can be reversed by the induction of a more potent IFN-␥ response [56]. A number of publications indicate that age related susceptibility to influenza virus in humans is associated with a reduction in Th1 CD4 + responses and a diminished ability to kill virally infected cells [13,15]. Hence, the ability of CpG in combination with MF59 to induce an enhanced IFN-␥ response is a significant observation and may be important for the development of an optimal influenza vaccine for the elderly. In a recent clinical study, CpG was shown to be ineffective for the induction of enhanced HI titres against influenza vaccine, but induced an enhanced IFN-␥ response [57]. These observations are consistent with our own studies, in which CpG alone did not induce enhanced HI titres, but did result in a significantly enhanced Th1 response, when used in combination with MF59. Overall, we conclude that an optimal influenza vaccine for use in the elderly might comprise a combination of MF59 to induce the highest possible HI titres, with an immune potentiator (e.g. CpG) added to enhance the Th1 response, including the induction of IFN-␥ and TNF-␣. Further studies, both pre-clinical and clinical, will show whether these Th1-related cytokines confer enhanced protection or not. The vaccination approach described here may also be effective in children, since they are usually na¨ıve to the circulating strains. More generally, the rational combination of adjuvants is likely to offer opportunities for the development of vaccines for prevention and therapy of a range of important infectious diseases [17].
Acknowledgements We are grateful to Grazia Galli and Oretta Finco for help and advice on T-cell assays, to Michela Brazzoli for help with the artwork and to Marco Tortoli for excellent animal husbandry.
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