Vaccine 27 (2009) 20992107

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

A universal epitope-based inuenza vaccine and its efcacy against H5N1

Y. Adar a,1 , Y. Singer b , R. Levi b , E. Tzehoval c , S. Perk d , C. Banet-Noach d , S. Nagar d , R. Arnon c , T. Ben-Yedidia b,
a

Israel Institute for Biological Research, Ness Ziona, Israel BiondVax Pharmaceuticals Ltd., 14 Einstein st., Ness Ziona, Israel c Department of Immunology, the Weizmann Institute of Science, Rehovot, Israel d Kimron Veterinary Institute, Beit Dagan, Israel
b

a r t i c l e

i n f o

a b s t r a c t
Previous studies have shown that a recombinant vaccine expressing four highly conserved inuenza virus epitopes has a potential for a broad spectrum, cross-reactive vaccine; it induced protection against H1, H2 and H3 inuenza strains. Here, we report on the evaluation of an epitope-based vaccine in which six conserved epitopes, common to many inuenza virus strains are expressed within a recombinant agellin that serves as both a carrier and adjuvant. In an HLA-A2.1 transgenic mice model, this vaccine induced both humoral and cellular responses and conferred some protection against lethal challenge with the highly pathogenic H5N1 avian inuenza strain. Hence, it is expected to protect against future strains as well. The data presented, demonstrate the feasibility of using an array of peptides for vaccination, which might pave the way to an advantageous universal inuenza virus vaccine that does not require frequent updates and/or annual immunizations. 2009 Published by Elsevier Ltd.

Article history: Received 29 July 2008 Received in revised form 25 January 2009 Accepted 2 February 2009 Available online 13 February 2009 Keywords: Peptide Flagellin Inuenza H5N1 Toxicology

1. Introduction Inuenza is a highly infectious disease caused by frequently mutating inuenza viruses. It spreads rapidly around the world in seasonal epidemics, affecting 1020% of the total population. According to the WHO, 250,000500,000 people die annually worldwide of seasonal inuenza epidemic outbreaks [1]. Inuenza pandemic is one of the major concerns among health authorities due to recent bird u outbreaks, the increasing international travel, as well as over-population associated with poor sanitary conditions for humans and livestock living together in some developing countries. As a result there is a heightened risk for emergence of new and more violent inuenza virus strains for human as well as increased human infection by animal virus strains, as observed since 1997 with the appearance of avian inuenza strains (H5N1 and others) that infected humans. The currently used inuenza vaccines are comprised of three virus strains that are selected on an annual basis. There are four types of inuenza vaccines available: (a) whole virus vaccinesinactivated or live-attenuated virus; (b) split virus vaccines (virus fragments); (c) subunit vaccines or puried antigens (in which surface proteins hemagglutinin (HA) and neuraminidase

Corresponding author. Tel.: +972 8 9302529; fax: +972 8 9302531. E-mail address: Benyedidia@biondvax.com (T. Ben-Yedidia). 1 During sabbatical at the Weizmann Institute of Science. 0264-410X/$ see front matter 2009 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.02.011

(NA) are included); (d) virosomal vaccines: synthetic virus-like particles with embedded HA and NA virus surface proteins. All these vaccine types are strain-specic and their efcacy relies heavily on inclusion of antigens (viruses or their proteins) similar to those that are likely to infect during the following inuenza season. Frequent changes in inuenza viruses, as a result of antigenic drift or shift, limit protection due to low correlation between the vaccines antigens and the current circulating wild-type inuenza virus. Commercially available strain-specic vaccines lead to a relatively poor clinical efcacy of approximately 40% when there is not a match between vaccine and circulating strains [1,2]. Other shortcomings of inuenza vaccines include their complicated and time-consuming annual production cycles and the risk of allergy to egg products that they induce. These cumulative limitations are the driving force for the development of novel vaccines. One approach focuses on the identication of specic epitopes derived from infectious pathogens and has signicantly advanced the development of peptide-based vaccines. Improved understanding of the molecular basis of antigen recognition and human leukocyte antigen (HLA) binding motifs has resulted in the development of rationally designed vaccines based on motifs predicted to bind to human class I or class II major histocompatibility molecules. Many studies showed the immunological efcacy of peptide-based vaccines against infectious diseases in animal models, as reviewed in Ben-Yedidia and Arnon [3], as well as in clinical studies which demonstrated the responses to peptide vaccines candidates against various infectious diseases including malaria [4,5], hepatitis B [6]

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and HIV [7,8]. The use of synthetic peptides in vaccines offers practical advantages such as the relative ease of construction and production, chemical stability, and the avoidance of any infectious potential hazard. A CTL response to epitope-based vaccines is HLA dependent; the large degree of MHC polymorphism and the need for knowledge of HLA restrictions in the population to be vaccinated make it difcult to design a vaccine that will be efcient for all. A vaccine intended for a broad population should include T cell epitope(s) that will induce responses in the vast majority of the people; this can be achieved by selecting several T cell epitopes that are specic to the prevalent HLA genotypes in the population [9]. Peptides as such, are weak immunogens that are degraded fast within the body, and hence cannot serve as vaccines unless incorporated into a carrier protein. The epitope-based vaccine against inuenza, where the epitopes are inserted into agellin that serves both as a carrier and as an adjuvant was developed by Arnon et al. [1012]. The agellin-based vaccine was found to be safe and protective in several animal models [13,14]. The concept of utilizing agellin as a carrier is well documented in scientic literature [1518]. The intensive response to agellin is mediated by toll-like receptor 5, linking innate and adaptive immunity [19,20]. Consequently, the incorporation of TLR-ligands into vaccines could result in more potent and efcacious vaccines. We report herewith of pre-clinical studies with a vaccine containing a mixture of six recombinant agellin constructs, each expressing multiple copies of a single inuenza epitope. This vaccine administered intranasally or intramuscularly, induces cross reactive antibodies response as well as cellular immune responses. These responses and the ability of the vaccine to protect mice against challenge infection with a highly pathogenic avian inuenza strain are discussed. 2. Materials and methods All the experiments detailed in this paper were repeated at least 3 times except of the protection study against H5N1 that was performed twice. One experiment with intranasal administration and one experiment with intra muscular administration due to technical limitations of working under BSL3 conditions. 2.1. Animals HHD++2 transgenic mice were kindly provided by Dr. F. Lemonnier (Institute Pasteur, Paris, France). All animal procedures were approved by the Animal Research Committee at the Weizmann Institute of science (Rehovot, Israel). 2.1.1. Mice Db/x 2 microglobulin ( 2m) null mice, transgenic for a recombinant HLA-A2.1/Db/x 2 microglobulin single chain (HHD++2 mice [21]) at the age of 3 months were employed for challenge experiments and for the evaluation of the cellular and humoral immune response. Three-month-old female and male BALB/c and ICR mice were used throughout the study for evaluation systemic exposure (pharmacokinetic) and toxicology following a single intramuscular (IM) administration of the vaccine. 2.1.2. Rabbits Three-month-old female New Zealand white (NZW) rabbits were immunized 3 times at 3 weeks intervals with the vaccine suspended in PBS. Blood collections were performed 14 days after the last immunization.

2.1.3. Viruses Inuenza strains A/Texas/1/77 A/WSN/33, A/Wisconsin/67/06, A/PC/4/73 (H3N2), A/PR8/34, A/New Caledonia/20/99 (H1N1) and inuenza B (B/Lee/40) were employed. Viruses were grown in the allantoic cavity of 911-day-old embryonated hen eggs (Bar On Hatchery, Israel). Using sucrose gradient, the viruses were further puried and used for induction of cell proliferation responses and coating ELISA plates. Virus growth and purication, as well as its titration (by hemagglutination assay) were described by Barret et al. [22]. Titers were expressed as hemagglutination units (HAU). H5N1 virus used for coating plates was inactivated by a standard beta-propiolactone treatment. The highly pathogenic strain H5N1 used for infection was isolated in Israel as noted in Banet-Noach et al. [23]. This strain shares over 97% homology with the following H5N1 strains: A/duck/ Novosibirsk/56/2005, A/grebe/Novosibirsk/29/2005, A/whooper swan/Mongolia/3/05, A/egret/Hong Kong/757.2/03, and A/Duck/ Hong Kong/2986.1/2000. 2.2. Preparation of recombinant agellin Salmonella SL5928 bacteria is a agellin decient mutant, each of the plasmids pBVX01pBVX09 that code for agellin menchen with the inserted inuenza epitopes, are transformed into it, resulting in motile agellated bacteria. The agellin is puried from the growth medium using Akta chromatography system (Amersham BioScience, Uppsala, Sweden) containing Q Sepharose column (Amersham BioScience, Uppsala, Sweden). Next, the recombinant agellin is concentrated, dialyzed and concentrated. The resultant samples are then ltered for sterilization and kept frozen at 70 C. For vaccination, a mixture of the recombinant agellin containing equal quantities of the different recombinant agellin, each expressing a single inuenza epitope is prepared. Endotoxin concentration in the vaccine preparation is lower than 100 EU/ml as required for administration to human in inuenza vaccines. 2.3. Immunization procedures For determination of cellular immune responses, transgenic mice were immunized once sub-cutaneously with 200 g recombinant agellin emulsied in complete Freunds adjuvant. In protection experiments, the vaccine was administered to mice intranasally or intramuscularly (with 150 and 300 g, respectively). Immunization consisted of 3 administrations at 3-week intervals; bleeding 2 weeks post-immunization and infection 1 month after the last immunization. Infection with the H5N1 strain was performed in a bio safety level 3 (BSL3) facilities (Kimron Veterinary Institute, Beit Dagan, Israel). Rabbits were immunized with a single epitope HA 91108 expressed in agellin in 3 doses of 100, 300 and 600 g administered 3 times at 3 weeks intervals intramuscularly. For the safety study, a single dose, intramuscular toxicology study was performed in BALB/c and ICR mice. Mice were administered with a single intramuscular dose of 50 g/50 l/mouse on Day 0. One group of mice was sacriced on Day 2 and another group on Day 14 to establish short-term safety and recovery. 2.4. Proliferation and interferon gamma (IFN-gamma) determination The ability of cells to proliferate in vitro in response to incubation with the NP 335 peptides was monitored as previously described [24]. Briey, lymphocytes from the spleen and inguinal lymph nodes were removed aseptically 10 days after a single immunization, and a single cell suspension was prepared. The cells were cultured in 0.2 ml medium in the presence of the peptide (5, 10 and

Y. Adar et al. / Vaccine 27 (2009) 20992107 Table 1 Characteristics of epitopes included in the vaccine. Epitope HA 91108 SKAYSNCYPYDVPDYASL B-cell epitope HA 307319 PKYVKQNTLKLAT Th epitope HA 354372 PAKLLKERGFFGAIAGFLE B-cell epitope HLA DR 1,2,4,5,7,9 Homology to Flu strains H3N2 H3N2 B/HongKong/330/2001;B/Beijing/1/87;B/Singapore/222/79;B/Oregon/ 5/80;B/Shangdong/7/97;B/Memphis/13/03;B/Los Angeles/1/02;B/Nebraska/1/01;B/Hong Kong/548/2000 B/Hong Kong/156/99;B/Vienna/1/99;strain B/Lee/40 And many more (over 100)as found by Blast search in NCBI data base H1N2, H2N2, H3N2, H9N2 H1N1, H1N2, H2N2, H3N8, H5N1, H5N2, H5N9, H6N1, H6N2, H6N9, H7N7, H9N2, H9N2, H11N1, H11N8, H11N9, H14N5 H1N1, H1N2, H2N2, H3N2, H3N3, H4N2, H4N8, H5N1, H5N3NSA, H6N2, H7N1, H7N3, H7N3NSA, H7N7, H9N2, H11N1, H11N8, H11N9, H13N6, H13N8, H16N3

2101

Reference [49,50] [51,52] [33]

NP 335350 SAAFEDLRVLSFIRGY CTL epitope NP 380393 ELRSRYWAIRTRSG CTL epitope MI 212 SLLTEVETYVL B- and CTL-epitope

A2, A3, A3, B8, B27 A2, A3

[53] [5456]

20 g/well) or the virus (20, 100 and 500 HAU/well), and the proliferative response was evaluated 3 and 5 days later (when incubated with the virus or with the peptide, respectively) by pulsing the cells for 18 h with [3 H] thymidine and monitoring the incorporated radioactivity. Supernatant from proliferating cells was removed after 48 h for determination of IFN-gamma concentration using commercially available enzyme-linked immunosorbent assay kits (ELISA; DuoSet mouse IFN-gamma; R&D Systems, Minneapolis, USA) according to the manufacturers instructions. 2.5. IFN-gamma secretion from human peripheral blood mononuclear cells (PBMCs) Buffy coats from normal volunteers were layered onto FicollPaque Plus solution (GE Healthcare Bio-Science AB, Uppsala) and spun at 2000 rpm for 20 min. The interlayer containing PBMC was collected, washed twice with PBS, and incubated at 5 105 cells/well in the presence of 5 g agellin or individual peptides at concentrations of 1040 g/well. Incubation period with agellin was for 75 h whereas incubation with the peptides was for 120 h. Secreted IFN-gamma concentration was measured by ELISA kit (DuoSet human IFN-gamma; R&D Systems, Minneapolis, USA) according to the manufacturers instructions. 2.6. Measurement of peptide binding by stabilization of cell surface HLA Peptide binding to HLA A*0201 was measured by stabilization of HLA-A*0201 on T2 cells as determined by uorescence-activated cell sorting (FACS) analysis. The afnity of peptides for HLA-A*0201 molecules was measured by cell surface class I stabilization assay as described by Tourdot et al. [25] with minor modications. T2-A*0201 cells (106 ) were incubated in RPMI 1640 containing 100 ng/ml -2 human microglobulin (Sigma) and peptides (12.5, 25, 50 and 100 M) at 37 C overnight. Samples were washed with PBS supplemented with 0.5% BSA and 0.1% Azide and incubated with HLA-A2 specic Monoclonal antibodies (BB7.2, ATCC) at 4 C for 30 min, and then with FITC-conjugated F(ab )2 goat anti-mouse IgG Antibodies (Jackson, West Grove, PA) at 4 C for 30 min. After further washing, the cells were analyzed by FACScan with Cellquest software (BD Biosciences, Mountain View, CA). The afnity of each peptide for HLA-A*0201 molecule was determined by mean uorescence intensity (MFI) and calculated as: MFI of peptide incubated cellsMFI of cells incubated in absence of peptide (background). 2.7. ELISA Sera antibodies were detected in the enzyme-linked immunosorbent assay (ELISA). The virus (20 HAU/well) or the peptides (0.25 g/well) were used for overnight coating. Following

blocking of non-specic binding, sera in serial dilutions were added and incubated for 2 h at 37 C. After washing, peroxidaseconjugated secondary antibodies were added. Next, TMB peroxide substrate solution (DakoCytomation, Glostrup, Denmark) was added and the optical density was measured (450 nm). ELISA to measure IgE in sera following immunization with the vaccine was evaluated using a commercial murine IgE detection kit (Bethyl laboratories, Montgomery, TX).

2.8. Natural killers and cytotoxic T cells activity HHD++2 mice (4/group) were immunized with recombinant agellin vaccine or with the native agella (200 g in PBS, sub-cutaneously) twice on Days 0, 21. A third vaccination (in complete Freunds adjuvant) was administered on Day 42. Two mice of each group were sacriced 10 days after the second or the third immunizations to follow the cytotoxic T cell (CTL) and natural killing (NK) responses in their spleens. Lymphocyte suspensions were prepared. One-third of the splenocytes were pulsed with 100 M synthetic peptides for 2 h at 37 C and then added to the rest of the splenocytes. Cultures were incubated for 5 days in lymphocyte medium [RPMI medium supplemented with 10% FCS, 2 mM l-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 25 mM HEPES (pH 7.4), 50 M 2mercaptoethanol, and combined antibiotics]. CTL or NK assays were carried out as described previously [26]. Briey, viable cells (effectors cells) were separated by Lympholyte-M (Cedarlane Laboratories Ltd., Hornby, British Columbia, Canada) gradient centrifugation, washed, resuspended in lymphocyte medium, and admixed at four effectors-to-target (E:T) ratios ranging from 100:1 to 12.5:1 with 5000 [35 S]methionine-labeled target cells. Results are expressed as fold specic lysis above the lysis of agella alone.

2.9. Measurement of agellin concentration The recombinant agellin was highly puried and hence a standard Lowry assay [27] was used for evaluation of agellin protein concentration. To assess the pharmacokinetic prole of agellin in the serum, sandwich ELISA assay was performed in which the plates were coated with puried polyclonal rabbit antibodies against agellin and a secondary puried polyclonal guinea pig antibody anti-agella was added on top of the examined sera samples.

2.10. Measurement of endotoxin concentration A kinetic, chromogenic limulus amebocyte lysate (LAL) test [28] was employed to detect the level endotoxin in the vaccine preparation, the levels found in the puried vaccine constructs was 350 EU/dose using QCL-1000 kit (Lonza, Verviers, Belgium).

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Table 2 In silico binding predictions of the CTL epitopes included in the vaccine. T Cell Epitopesa NP 335350, 336 AAFEDLRVL 344 NP 380393, 380 ELRSRYWAI 388 MI 212, 3 LLTEVETYV 11
a b c d

HLA A2.1 B8 A2.1

SYFPEITHIb 23 29 23

BIMASc 0.849 80.00 2666

IC 50 (SMM)d 2586.2 2359.0 24.2

The exact CTL epitope within NP 335350 is NP 336344, the epitope within NP 380393 peptide is NP 380388 and the exact CTL epitope within MI 212 is MI 311. http://www.uni-tuebingen.de/uni/kxi/. http://www-bimas.cit.nih.gov/molbio/hla bind/. http://cagt.bu.edu/page/SMM submit.

2.11. Statistical analysis The Lifetest procedure using KaplanMeyer method and log rank test were applied for survival analyses curve between study groups (H5N1 survival study). All tests applied were two-tailed, and p-value of 5% or less was considered statistically signicant. The data was analyzed using the SAS Version 9.1 software (SAS Institute, Cary, NC). 3. Results 3.1. Selection of epitopes Based on in silico examination of the inuenza peptides and a literature review, the epitopes listed in Table 1 were included in the vaccine. Most of the epitopes confer a wide HLA coverage; they are linear and common to many inuenza strains. The name of each epitope denes its origin in terms of protein name (HA for hemagglutinin, NP for nucleoprotein) and the amino acids location within it. As shown in Table 2, all 3 peptides are classied as good binders with similar values according to the SYFPEITHI algorithm, Whereas according to BIMAS, which classies HLA-A*0201-restricted candidate peptides according to a theoretical score reecting the estimated dissociation half-life of the MHC/peptide complex [29], A high score of 2666 was calculated for the MI 212 peptide as compared to lower values for the NP peptides. The MI 311 epitope binds better than NP 380388 and the NP 336344 dissociates easily from the HLA molecule. SMM method is applied successfully to predict MHC binding, TAP transport and proteasomal cleavage [30]. Low values of IC 50 indicate of good binders and again, MI 311 seems to be better than the other two epitopes. 3.2. HLA-A*0201 peptide binding and stabilization assays To test the potential of the peptides to induce T-cell responses, we determined their ability to bind in vitro T2 HLA A*0201 cells
Fig. 1. Stabilization of HLA A*0201 as a result of peptide binding. T2 cells were incubated overnight with peptides (12.5100 M) MI 311, NP 336344 and HA 91108. Then stained with anti-HLA-A2 monoclonal antibodies and analyzed by FACS. Results are presented as MFI above background.

(decient for TAP transporters that express low and unstable amounts of HLA A*0201 molecules). Upon binding of peptides, stable and high levels of HLA A*0201 are expressed on the cell surface as demonstrated by ow cytometry. Fig. 1 shows a representative data of 3 repeated FACS measures. A high and dose-dependent binding of MI 311 peptide that is the specic CTL epitope within the MI 212 peptide was observed. The binding pattern for NP 336344 peptide is different: it is low at all doses. Both patterns are acceptable and may lead to efcient presentation of the peptide on human antigen presenting cells. The superior binding of the MI peptide over the Nucleoprotein NP 336344 peptide is also shown by the in silico analysis of their binding. The B cell epitope HA 91108 peptide, which is longer than the HLA groove, showed binding capacity only at the higher (100 M) concentration, probably due to the HLA motif in its N -terminal. NP 380388 serves as a negative control. 3.3. Cross-reactive antibodies in rabbits Sera from rabbits immunized 3 times with the recombinant agellin expressing the single B cell epitope HA 91108 were reacted

Fig. 2. Anti-inuenza viruses antibody response in pooled sera of vaccinated rabbits. Total Ig was assayed in serum samples diluted 1:500, 2 weeks after the third immunization with recombinant agellin expressing HA 91108 peptide. ELISA plates were coated with live inuenza A H3N2 strain (A/WSN/33, A/Wisconsin/67/06, A/Texas/1/77, A/PC/4/73), H1N1 strains (A/PR8/34, A/New Caledonia/20/99) and inuenza B (B/Lee/40).

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Fig. 4. IFN-gamma secretion from proliferating lymphocytes. HHD++2 transgenic mice (harboring the human A*0201 HLA) were immunized subcutaneously with recombinant agellin containing a single T cell epitope (NP 335350) or with PBS in complete Freunds adjuvant as a negative control. The lymphocytes cultures were stimulated with the NP 335 synthetic peptides and the secreted IFN-gamma in response to this stimulation was monitored. Only mice immunized with the recombinant agellin (F 335) responded to the NP 335 peptide by signicantly higher (p < 0.05) secretion of IFN-gamma. These data represent 3 repetitive studies of proliferation and IFN-gamma measures in the medium of proliferating lymphocytes.

Fig. 3. Lymphocytes proliferation following immunization with recombinant agellin containing NP 335350 epitope. Transgenic mice for HLA A*0201 were immunized once with agellin expressing NP 335350 epitopes. Proliferation of their splenocytes (SP) and lymph nodes lymphocytes (LN) was measured following in vitro stimulation with NP 335350 peptide (A) or with inactive inuenza virus (A/Texas/1/77, H3N2) (B): (white squares) splenocytes from mice administered with PBS; (lined squares) splenocytes from mice immunized with agellin expressing NP 335350 epitope; (black squares) lymph nodes lymphocytes from mice immunized with agellin expressing NP 335350 epitope. The proliferation of cells from immunized mice is signicantly higher than the proliferation observed in the control (PBS) group and in cells from the same group that were not incubated with the antigen (*p < 0.05). The proliferation stimulation index is calculated in comparison to the proliferation of splenocytes from each group when incubated without Ag.

HA 307319, HA 354372, NP 335350 and MI 212. Following incubation with 0.25 g of puried agellin approximately 9-fold elevations in the secretion of IFN-gamma as compared to the control (lymphocytes incubated with medium) was observed as shown in Table 3. Incubation of these cells with the recombinant agellin containing inuenza epitopes resulted in 9-fold elevation of IFN-gamma secretion; it is not clear if this elevation results from response to the specic peptides or to the agellin. According to the response to each of them separately, it can be concluded that both peptides and agellin has a potential to induce cellular immunity in human cells. 3.5. Activation of NK cells The immunogenicity of the peptides, namely their potential to induce specic CTLs, has been examined also in vivo. HHD++2 mice were immunized with recombinant agellin vaccine or with the native agella. Following in vitro re-stimulation with the mixture of peptides, the CTL-dependent lysis of single peptide-pulsed targets was evaluated. No CTL activity was observed following the second and the third immunization (data not shown). Natural killing (NK) lysis of sensitive YAC-1 cells was demonstrated after the second and third immunization of transgenic mice with a mixture of six recombinant agellin peptides in Freunds adjuvant. After the second immunization, the lysis of YAC-1 by NK cells from mice immunized with the recombinant agellin vaccine was higher than the lysis observed by NK cells from mice immunized with the native agellin. The epitope-based vaccine induced a natural killing activity which was 23-fold higher than the activity which was induced by the native agella. The lytic activity was augmented following the third immunization (Fig. 5).

in ELISA with several inuenza viruses. The results in comparison to pre-immune serum are described in Fig. 2. 3.4. Lymphocytes proliferation and IFN-gamma secretion In order to study cellular responses that is relevant for human subjects, the Db/x 2 microglobulin ( 2m) null mice, transgenic for a recombinant HLA-A2.1/Db/x 2 microglobulin single chain (HHD++2 mice) were tested. These mice combine classical HLA transgenesis with selective destruction of murine H-2 and show only HLA A*0201-restricted responses [21]. Splenocytes from mice sacriced 10 days after the 1st immunization were stimulated with 5, 10 and 20 g the NP 335350 peptide or with 20, 100 and 500 HAU of H3N2 A/Texas/1/77 virus per well. Representative data of three repetitive experiments of lymphocytes proliferation and IFN-gamma secretion that were evaluated are shown; signicant proliferation response was observed in both lymph nodes and splenocytes following in vitro incubation with the peptide (Fig. 3A) and with inuenza virus A/Texas/1/77 (Fig. 3B). In accordance, increasing IFN-gamma secretion was detected in the immunized group as compared to cells from non immunized mice that were stimulated with the same peptide antigen as shown in Fig. 4. The potential of human cells to mount a cellular response to the vaccine was examined in human PBMC (HLA A2; DR10 phenotype). Cells from healthy volunteers were incubated with the individual peptides that are included in the vaccine, at elevated concentrations of 1040 g/well. A signicant IFN-gamma secretion was detected in response to incubation with the peptides

Table 3 IFN-gamma secretion from human lymphocytes following incubation with the agellin and the peptides included in the vaccine. In vitro stimulator HA 91108 HA 307319 HA 354372 NP 335350 NP 380393 MI 212 Native agellin Stimulator concentration ( g/well) 20 40 40 40 40 40 5 Fold elevation in IFN-gamma secretion 2.00 12.87 3.87 5 1.61 8.87 9.26

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Fig. 5. Induction of NK activity by the recombinant vaccine. Mice were immunized with recombinant agellin vaccine or with the native agella 2 or 3 times. Spleens were removed on Day 10 after the last immunization (second or third) and stimulated by a mixture of 6 peptide included in the vaccine. NK activities as effectors cells were assayed on Day 5 with YAC-1 as target cells. The percentages of specic lysis at effector:target (E:T) ratios ranging from 100:1 to 12.5:1 was detected. Spontaneous release did not exceed 20% of maximal release. Results are shown as fold increase above NK activity induced by the native agellin.

Fig. 7. Antibodies against H5N1 virus. Pooled sera from 2 rabbits per antigen immunized 3 times with the vaccine containing the 6 epitopes reacted in a dose related pattern with H5N1 virus that was used as a coating antigen. The response is dened as the titer ratio of post/pre-immune serum. This ratio is higher in the rabbit immunized with 600 g of the vaccine than the response of sera from rabbits immunized with native agellin or with PBS.

3.6. Protection against highly pathogenic avian inuenza H5N1 To study the efcacy of the vaccine, the HHD++2 (HLA A*0201 transgenic) mice model was used. The mice were immunized intranasally (IN) or intramuscularly (IM) with 150 g (IN) or 300 g (IM) of the vaccine and challenged with a lethal dose (LD80 ) of H5N1 avian inuenza strain. Their survival was monitored for the following 3 weeks. Fig. 6 shows the survival curve following these immunizations, where the nal survival level of the intramuscularly immunized mice was 68% and the survival level of the PBS administered mice was 33%. Similar effect was found when the vaccine was administered via the intranasal route (81% survival of vaccinated vs. 43% survival of the control group). To evaluate the immune response to the H5N1 virus, rabbits serum after 3 immunizations with the vaccine was tested in ELISA for its ability to recognize this virus. The data demonstrate a dose related response (Fig. 7), where only immunization with the high dose of the vaccine resulted in a signicant elevation of H5N1 specic response. 3.7. Pharmacokinetic and systemic exposure To assess the pharmacokinetic prole of the vaccine following a single administration, BALB/c mice were injected intramuscularly with the vaccine and bled at 10 time points. Serum samples were monitored by ELISA for their agellin concentration, to calculate Cmax, Tmax and AUC values. As shown in Fig. 8, the maximum concentration level (Cmax = 3925 ng/ml) was observed upon 15 min,

Fig. 8. Flagellin concentration in serum following a single intramuscular administration of the vaccine. Maximum serum concentration 3925 ng/ml (Cmax) of agellin was observed after 15 min (Tmax). Half (T 12) of the total exposure quantity was obtained within 30 min post-dosing. The area under the serum concentrationtime curve of 15,027 ng/ml indicates the bodys total exposure over time to the vaccine.

No traces of protein in the serum could be detected at 12 h postinjection. 3.8. Safety 3.8.1. Allergy For the evaluation of potential allergic response to the vaccine, IgE levels were measured following immunization of mice

Fig. 6. Protection of transgenic mice from lethal dose (LD80 ) challenge of H5N1. Groups of 12 mice were immunized intranasally (left panel) or intramuscularly (right panel) with the recombinant vaccine (dark line) or with PBS (stripped line) The schedule of administration was 3 times at 3-week intervals, and the challenge infection with a lethal dose of avian inuenza given 3 weeks after the last boost. A signicant difference in survival was observed in both routes of administration of the vaccine (p = 0.039 between two survival curves of the IN administration and p = 0.018 between two survival curves of the IM administration).

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with different combination of epitopes or with native agellin at concentrations of 8240 g/mouse. Using either intranasal or intramuscular administration, after a single or multiple administrations, low titers (<20 ng/ml) of IgE were detected in the immunized animals. This level is normal and similar to the IgE level found in non-immunized mice as shown in our study and in [31]. 3.8.2. Safety in mice Male and female BALB/c mice were used in this toxicology study as this is the animal model in which this vaccine concept was established. The mice were administered intramuscularly with the epitopebased vaccine in PBS and the toxicology ndings were compared to those obtained in control group administered with PBS. The standard acute toxicology parameters were monitored, including in-life and necropsy analysis (both macroscopic and microscopic). No abnormalities were found in the in life phase. The macroscopic examination revealed a normal appearance of examined organs both in early- and late-study termination groups (Day 2 and Day 14, respectively) in both genders. Histologically, no treatment-related changes were found following a single IM injection of the vaccine in all major organs examined. Transient minimal to mild changes in the heart of both genders were seen within the early termination (Day 2) in the vaccine and PBS control treated groups. The hearts of all groups were normal with no changes observed at study termination (Day 14). The severity and characteristics of changes observed in early termination PBS control groups consisted of degeneration and brosis as well as sub-acute epicardium inammation in the region of the auricles in a minimal level. In the vaccine treated group, increased incidences of minimal to mild severity of these phenomena were detected. A thorough literature review revealed that BALB/c mice that were used for this study are characterized by spontaneous lesions and epicardial mineralization, resulting in increased susceptibility of heart inammatory reactions aiming to repair these lesions [32]. This phenomenon was manifested in our toxicology study by the minimal inammation observed in the PBS BALB/c treated mice. In a similar study conducted with ICR inbred mice, this observation was found at tens of micrograms doses only and the safe dose was determined. 4. Discussion Despite of the potential advantages associated with peptidebased vaccines, in reality, peptide vaccines against infective agents have not yet realized their initial promise; none of the peptide vaccines had entered advanced phases of clinical trials. This results from their limited immunogenicity, their structural instability and the complexity associated with cellular responses to them [33]. The purpose of this study was to further develop and evaluate a novel approach to vaccination, based on conserved inuenza epitopes within recombinant agellin that serves both as a carrier and as an adjuvant [11,34]. The efcacy of the vaccine was established in mice. Previous studies demonstrated the potential of a vaccine comprising four of the epitopes included in the current vaccine to protect human/mouse radiation chimera against H3N2, H2N2 and H1N1 viruses [14]. The present study reports on experiments with an improved vaccine, that includes two additional epitopes (HA 354372 and MI 212) within agellin and its ability to induce protection against the highly pathogenic avian inuenza strain. Each epitope was expressed individually within agellin and the vaccine consisted of a mixture of the six recombinant agellin proteins. The HA 354372 peptide resides at the highly conserved maturational cleavage site of the HA(0) precursor of the inuenza B

virus hemagglutinin. It can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive inuenza B virus lineages [35]. The MI 212 peptide includes a sequence that is dened as A2 restricted CTL epitope. These epitopes were added in view of their ability to broaden the scope of the vaccine to additional strains of the viruses. All the epitopes in the vaccine were presented in the form of chimeric protein of Salmonella agellin, and hence no external adjuvant was required. These properties of the agellin are ascribed to its association with TLR5 [20]. The toll-like receptor (TLR) system provides an inherent cellular recognition device that is of paramount importance for the development and function of innate as well as adaptive immunity [36,37]. Data on consequences of TLR5 activation for Th1 versus Th2 decisions in the murine system are not uniform [34,38,39]. In the current study, the effects of agellin (endotoxin-low), as well as the effect of the inuenza epitopes that are included in the vaccine on IFN-gamma secretion by human PBMC was investigated, as demonstrated (Table 3). We report that pre-incubation of PBMC from healthy volunteers with agellin signicantly up regulated secretion of IFN-gamma that is indicative of Th1 type of response. Similarly, incubation with some of the epitopes included in the vaccine and especially the T helper epitope HA 307319, resulted in signicant IFN-gamma secretion from them. These data are in accord with the study of Bachman et al. that specically characterized the agellin of Salmonella Menchen (the one employed in the current study) as an inducer of TH1 [40]. Virus-specic CTL are recruited to inuenza-infected lungs by a Th1 response, specically due to the production of IFN-gamma that has a clear anti-viral effect [41,42], and hence the signicance of inducing a Th1 response for protective immunity against inuenza. The contribution of agellin to the adjuvant effect stems also from the prolonged exposure of the immune system to the peptide when it is presented in the agellin. Degradation of peptides in the body occurs within few minutes [33] whereas the recombinant agellin was detected in the blood up to 12 h post-intramuscular administration. It should be noted that pre-existing anti-agellin antibodies had no signicant effect on the adjuvant activity of agellin [15]. Similarly, pre-exposure to the Salmonella administered by feeding did not lead to carrier suppression and hence did not affect the protective response of a recombinant agellin-based vaccine expressing a foreign antigen [43]. According to computerized algorithms, the epitopes NP 335350 and MI 212 included in the vaccine should induce a CTL response; indeed, binding assay by FACS revealed the specic binding of the peptides corresponding to the epitopes included in the vaccine to HLA A*0201 molecules (Fig. 1). Despite this, specic CTL lysis of target cells loaded with these peptides was not detected after immunization of HHD++2 /HLA A*0201 transgenic mice with recombinant agellin expressing these epitopes. For the induction of CTL lysis, the accepted procedure is to immunize with 100 g peptide in IFA at least once or three times with peptide loaded APCs at weekly intervals. However, in the agellin systemonly 3.5% of the recombinant agellin comprises the epitope hence, the actual peptide dose administered was only 5 g peptide, which might be too low for the induction of specic CTL response. NK cells express the TLR and therefore respond to the agellin. Lysis of YAC-1 cells that are sensitive to the cytotoxic activity of naturally occurring killer cells was shown after immunization with the agellin at levels of 1040%. This response was elevated and ranged from 30 to 70%, respectively, when the mice were immunized with the recombinant agellin.

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Lysis by NK contributes to the anti-viral response. It was shown that infected cells are more sensitive and are more susceptible to lysis than non-infected cells [44]. The general notion is that NK cells are non-specic lymphocytes that attack and kill cancer cells and cells infected by microorganisms without recognition of a specic antigen on it. However, Alter et al. [45] demonstrate that the sequence of the peptide presented by HLA class I molecules can inuence the ligation by inhibitory and activating killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells, and thereby modulate target cell recognition and lysis. Based on these structural and experimental data on the peptide-specicity of the KIR/HLA interaction, it is tempting to speculate that the recognition of target cells by NK cells is more specic than previously thought. A similarity in peptide motif between HLA A*0201 binders and HLAG binders was demonstrated by Diehl et al. [46], suggesting that A2 specic peptides activate NK activity. Hence, the NK cells induced by the epitopes employed in the present study may bear relevance to their efcacy. The antibodies produced following vaccination with the recombinant agellin vaccine that include the six epitopes, recognized the H5N1 strain but no hemagglutination inhibition was observed. Hence we conclude that the protective response against the H5 subtype was most likely mediated by T-cell and NK activity, and possibly by complement-mediated lysis. In rabbits immunized with the whole combination of epitopes, a 6-fold elevation was observed towards H5N1 virus in comparison to pre-immune serum. This recognition is signicantly higher than the recognition of H5N1 virus, observed in human sera (1.79fold) following immunization with a commercial seasonal vaccine, justifying the selection of epitopes included in the vaccine. This comparison also indicates the potential of this epitope-based vaccine to protect against the avian inuenza strain, by enhancing the immune response towards specic epitopes within the virus. Further indication on the protective effect of the vaccine stems from direct protection studies. The protection experiments show difference in the morbidity of the animals as well as a statistically signicant difference between the survival curves of the vaccinated groups and the control group that was administered with PBS, in both intranasal and intramuscular routes of administration. Under the experimental conditions, in which the challenge dose is orders of magnitude higher than that pertaining in natural infection, all the exposed mice are infected regardless of their immune state. But, whereas only 2030% of the control mice survived infection of this severity, 7080% of the immunized mice survived this challenge. These results demonstrate the efcacy of the vaccine against the highly pathogenic H5N1 avian inuenza in a transgenic mouse model, indicating that this vaccine could be used in humans against not only human inuenza strains but against avian strains as well. The intranasal route was employed in many of our previous studies using the epitope-based vaccine; it is based on the assumption that it will be effective in inducing local response in the lung and nasal airways, which are the primary targets in inuenza infection. The intramuscular route is the most common for commercial vaccines including inuenza and the current study demonstrate it is comparable in its protective effect to the intranasal route. Additional advantages of this route are its safety, the comparatively easy regulations associated with this route and the higher volume that can be administered as compared to the limited volume used in nasal vaccines. The regulations for intramuscular administration are simpler in view of the risk associated with intranasal vaccines that can affect the facial nerve [47]. The safety studies performed with a single administration of mice with the vaccine demonstrated its potential to induce transient effect in the heart at high doses, this effect is attributed to the

agellin included in the vaccine since in a control group administered with native agellin this phenomenon was enhanced. The vaccine is sterile and does not contain virus or bacterial particles (the agellin originates from non-virulent salmonella bacteria [48]), its endotoxin levels are controlled and are within the level approved for human use and in addition, it does not elicit allergic responses as shown in this study and by Lee et al. [49], nor autoimmune responses as deduced from histopathology analysis of major mouse organs following administration of the vaccine. The safety of this vaccine in human should be examined in clinical trials. Acknowledgements We wish to thank Dr. A. Meshorer, for his help in the analysis of the histological sections. We wish to acknowledge Mrs. Kate-Ilovitz and the laboratory team at BiondVax for their dedicated work on this research. References
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