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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
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A universal epitope-based influenza vaccine and its efficacy against H5N1
Y. Adara,1, Y. Singerb, R. Levib, E. Tzehovalc, S. Perkd, C. Banet-Noachd, S. Nagard,R. Arnonc, T. Ben-Yedidiab,∗
a Israel Institute for Biological Research, Ness Ziona, Israelb BiondVax Pharmaceuticals Ltd., 14 Einstein st., Ness Ziona, Israelc Department of Immunology, the Weizmann Institute of Science, Rehovot, Israeld Kimron Veterinary Institute, Beit Dagan, Israel
a r t i c l e i n f o
Article history:Received 29 July 2008Received in revised form 25 January 2009Accepted 2 February 2009Available online 13 February 2009
Keywords:PeptideFlagellinInfluenza H5N1Toxicology
a b s t r a c t
Previous studies have shown that a recombinant vaccine expressing four highly conserved influenza virusepitopes has a potential for a broad spectrum, cross-reactive vaccine; it induced protection against H1,H2 and H3 influenza strains. Here, we report on the evaluation of an epitope-based vaccine in which sixconserved epitopes, common to many influenza virus strains are expressed within a recombinant flagellinthat serves as both a carrier and adjuvant. In an HLA-A2.1 transgenic mice model, this vaccine inducedboth humoral and cellular responses and conferred some protection against lethal challenge with thehighly pathogenic H5N1 avian influenza strain. Hence, it is expected to protect against future strains aswell. The data presented, demonstrate the feasibility of using an array of peptides for vaccination, whichmight pave the way to an advantageous universal influenza virus vaccine that does not require frequentupdates and/or annual immunizations.
Influenza is a highly infectious disease caused by frequentlymutating influenza viruses. It spreads rapidly around the worldin seasonal epidemics, affecting 10–20% of the total population.According to the WHO, 250,000–500,000 people die annuallyworldwide of seasonal influenza epidemic outbreaks [1].
Influenza pandemic is one of the major concerns among healthauthorities due to recent bird flu outbreaks, the increasing inter-national travel, as well as over-population associated with poorsanitary conditions for humans and livestock living together insome developing countries. As a result there is a heightened riskfor emergence of new and more violent influenza virus strains forhuman as well as increased human infection by animal virus strains,as observed since 1997 with the appearance of avian influenzastrains (H5N1 and others) that infected humans.
The currently used influenza vaccines are comprised of threevirus strains that are selected on an annual basis. There arefour types of influenza vaccines available: (a) whole virusvaccines—inactivated or live-attenuated virus; (b) split virus vac-cines (virus fragments); (c) subunit vaccines or purified antigens(in which surface proteins hemagglutinin (HA) and neuraminidase
1 During sabbatical at the Weizmann Institute of Science.
(NA) are included); (d) virosomal vaccines: synthetic virus-likeparticles with embedded HA and NA virus surface proteins. Allthese vaccine types are strain-specific and their efficacy relies heav-ily on inclusion of antigens (viruses or their proteins) similar tothose that are likely to infect during the following influenza sea-son. Frequent changes in influenza viruses, as a result of antigenicdrift or shift, limit protection due to low correlation between thevaccines’ antigens and the current circulating wild-type influenzavirus. Commercially available strain-specific vaccines lead to a rel-atively poor clinical efficacy of approximately 40% when there isnot a match between vaccine and circulating strains [1,2]. Othershortcomings of influenza vaccines include their complicated andtime-consuming annual production cycles and the risk of allergy toegg products that they induce. These cumulative limitations are thedriving force for the development of novel vaccines.
One approach focuses on the identification of specific epitopesderived from infectious pathogens and has significantly advancedthe development of peptide-based vaccines. Improved understand-ing of the molecular basis of antigen recognition and humanleukocyte antigen (HLA) binding motifs has resulted in the develop-ment of rationally designed vaccines based on motifs predicted tobind to human class I or class II major histocompatibility molecules.Many studies showed the immunological efficacy of peptide-basedvaccines against infectious diseases in animal models, as reviewedin Ben-Yedidia and Arnon [3], as well as in clinical studies whichdemonstrated the responses to peptide vaccines candidates againstvarious infectious diseases including malaria [4,5], hepatitis B [6]
and HIV [7,8]. The use of synthetic peptides in vaccines offerspractical advantages such as the relative ease of construction andproduction, chemical stability, and the avoidance of any infectiouspotential hazard.
A CTL response to epitope-based vaccines is HLA dependent; thelarge degree of MHC polymorphism and the need for knowledge ofHLA restrictions in the population to be vaccinated make it difficultto design a vaccine that will be efficient for all. A vaccine intendedfor a broad population should include T cell epitope(s) that willinduce responses in the vast majority of the people; this can beachieved by selecting several T cell epitopes that are specific to theprevalent HLA genotypes in the population [9].
Peptides as such, are weak immunogens that are degraded fastwithin the body, and hence cannot serve as vaccines unless incor-porated into a carrier protein. The epitope-based vaccine againstinfluenza, where the epitopes are inserted into flagellin that servesboth as a carrier and as an adjuvant was developed by Arnon etal. [10–12]. The flagellin-based vaccine was found to be safe andprotective in several animal models [13,14].
The concept of utilizing flagellin as a carrier is well documentedin scientific literature [15–18].
The intensive response to flagellin is mediated by toll-like recep-tor 5, linking innate and adaptive immunity [19,20]. Consequently,the incorporation of TLR-ligands into vaccines could result in morepotent and efficacious vaccines.
We report herewith of pre-clinical studies with a vaccine con-taining a mixture of six recombinant flagellin constructs, eachexpressing multiple copies of a single influenza epitope. This vac-cine administered intranasally or intramuscularly, induces crossreactive antibodies response as well as cellular immune responses.These responses and the ability of the vaccine to protect miceagainst challenge infection with a highly pathogenic avian influenzastrain are discussed.
2. Materials and methods
All the experiments detailed in this paper were repeated at least3 times except of the protection study against H5N1 that was per-formed twice. One experiment with intranasal administration andone experiment with intra muscular administration due to techni-cal limitations of working under BSL3 conditions.
2.1. Animals
HHD++2 transgenic mice were kindly provided by Dr. F. Lemon-nier (Institute Pasteur, Paris, France). All animal procedures wereapproved by the Animal Research Committee at the WeizmannInstitute of science (Rehovot, Israel).
2.1.1. MiceDb−/−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 forchallenge experiments and for the evaluation of the cellular andhumoral immune response.
Three-month-old female and male BALB/c and ICR mice wereused throughout the study for evaluation systemic exposure (phar-macokinetic) and toxicology following a single intramuscular (IM)administration of the vaccine.
2.1.2. RabbitsThree-month-old female New Zealand white (NZW) rabbits
were immunized 3 times at 3 weeks intervals with the vaccine sus-pended in PBS. Blood collections were performed 14 days after thelast immunization.
A/PC/4/73 (H3N2), A/PR8/34, A/New Caledonia/20/99 (H1N1) andinfluenza B (B/Lee/40) were employed. Viruses were grown in theallantoic cavity of 9–11-day-old embryonated hen eggs (Bar OnHatchery, Israel). Using sucrose gradient, the viruses were furtherpurified and used for induction of cell proliferation responses andcoating ELISA plates. Virus growth and purification, as well as itstitration (by hemagglutination assay) were described by Barret etal. [22]. Titers were expressed as hemagglutination units (HAU).H5N1 virus used for coating plates was inactivated by a standardbeta-propiolactone treatment.
The highly pathogenic strain H5N1 used for infection was iso-lated in Israel as noted in Banet-Noach et al. [23]. This strainshares over 97% homology with the following H5N1 strains: A/duck/Novosibirsk/56/2005, A/grebe/Novosibirsk/29/2005, A/whooperswan/Mongolia/3/05, A/egret/Hong Kong/757.2/03, and A/Duck/Hong Kong/2986.1/2000.
2.2. Preparation of recombinant flagellin
Salmonella SL5928 bacteria is a flagellin deficient mutant, eachof the plasmids pBVX01–pBVX09 that code for flagellin müenchenwith the inserted influenza epitopes, are transformed into it, result-ing in motile flagellated bacteria. The flagellin is purified from thegrowth medium using Akta chromatography system (AmershamBioScience, Uppsala, Sweden) containing Q Sepharose column(Amersham BioScience, Uppsala, Sweden). Next, the recombinantflagellin is concentrated, dialyzed and concentrated. The resultantsamples are then filtered for sterilization and kept frozen at −70 ◦C.For vaccination, a mixture of the recombinant flagellin contain-ing equal quantities of the different recombinant flagellin, eachexpressing a single influenza epitope is prepared. Endotoxin con-centration in the vaccine preparation is lower than 100 EU/ml asrequired for administration to human in influenza vaccines.
2.3. Immunization procedures
For determination of cellular immune responses, transgenicmice were immunized once sub-cutaneously with 200 �g recom-binant flagellin emulsified in complete Freund’s adjuvant.
In protection experiments, the vaccine was administered to miceintranasally 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 afterthe last immunization. Infection with the H5N1 strain was per-formed in a bio safety level 3 (BSL3) facilities (Kimron VeterinaryInstitute, Beit Dagan, Israel).
Rabbits were immunized with a single epitope HA 91–108expressed in flagellin in 3 doses of 100, 300 and 600 �g admin-istered 3 times at 3 weeks intervals intramuscularly.
For the safety study, a single dose, intramuscular toxicologystudy was performed in BALB/c and ICR mice. Mice were admin-istered with a single intramuscular dose of 50 �g/50 �l/mouse onDay 0. One group of mice was sacrificed on Day 2 and another groupon 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 incu-bation with the NP 335 peptides was monitored as previouslydescribed [24]. Briefly, lymphocytes from the spleen and inguinallymph nodes were removed aseptically 10 days after a single immu-nization, and a single cell suspension was prepared. The cells werecultured in 0.2 ml medium in the presence of the peptide (5, 10 and
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Table 1Characteristics of epitopes included in the vaccine.
5/80;B/Shangdong/7/97;B/Memphis/13/03;B/LosAngeles/1/02;B/Nebraska/1/01;B/Hong Kong/548/2000 B/HongKong/156/99;B/Vienna/1/99;strain B/Lee/40 And many more (over 100)asfound by Blast search in NCBI data base
20 �g/well) or the virus (20, 100 and 500 HAU/well), and the prolif-erative response was evaluated 3 and 5 days later (when incubatedwith the virus or with the peptide, respectively) by pulsing thecells for 18 h with [3H] thymidine and monitoring the incorporatedradioactivity. Supernatant from proliferating cells was removedafter 48 h for determination of IFN-gamma concentration usingcommercially available enzyme-linked immunosorbent assay kits(ELISA; DuoSet® mouse IFN-gamma; R&D Systems, Minneapolis,USA) according to the manufacturer’s instructions.
2.5. IFN-gamma secretion from human peripheral bloodmononuclear cells (PBMCs)
Buffy coats from normal volunteers were layered onto Ficoll-Paque Plus solution (GE Healthcare Bio-Science AB, Uppsala)and spun at 2000 rpm for 20 min. The interlayer containingPBMC was collected, washed twice with PBS, and incubated at5 × 105 cells/well in the presence of 5 �g flagellin or individual pep-tides at concentrations of 10–40 �g/well. Incubation period withflagellin was for 75 h whereas incubation with the peptides was for120 h. Secreted IFN-gamma concentration was measured by ELISAkit (DuoSet® human IFN-gamma; R&D Systems, Minneapolis, USA)according to the manufacturer’s instructions.
2.6. Measurement of peptide binding by stabilization of cellsurface HLA
Peptide binding to HLA A*0201 was measured by stabilizationof HLA-A*0201 on T2 cells as determined by fluorescence-activatedcell sorting (FACS) analysis.
The affinity of peptides for HLA-A*0201 molecules was mea-sured by cell surface class I stabilization assay as described byTourdot et al. [25] with minor modifications. T2-A*0201 cells (106)were incubated in RPMI 1640 containing 100 ng/ml �-2 humanmicroglobulin (Sigma) and peptides (12.5, 25, 50 and 100 �M) at37 ◦C overnight. Samples were washed with PBS supplementedwith 0.5% BSA and 0.1% Azide and incubated with HLA-A2 spe-cific Monoclonal antibodies (BB7.2, ATCC) at 4 ◦C for 30 min, andthen with FITC-conjugated F(ab′)2 goat anti-mouse IgG Antibod-ies (Jackson, West Grove, PA) at 4 ◦C for 30 min. After furtherwashing, the cells were analyzed by FACScan with Cellquest soft-ware (BD Biosciences, Mountain View, CA). The affinity of eachpeptide for HLA-A*0201 molecule was determined by mean fluores-cence intensity (MFI) and calculated as: MFI of peptide incubatedcells—MFI of cells incubated in absence of peptide (background).
2.7. ELISA
Sera antibodies were detected in the enzyme-linkedimmunosorbent assay (ELISA). The virus (20 HAU/well) or thepeptides (0.25 �g/well) were used for overnight coating. Following
blocking of non-specific binding, sera in serial dilutions wereadded and incubated for 2 h at 37 ◦C. After washing, peroxidase-conjugated secondary antibodies were added. Next, TMB peroxidesubstrate solution (DakoCytomation, Glostrup, Denmark) wasadded and the optical density was measured (450 nm).
ELISA to measure IgE in sera following immunization with thevaccine 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 recombinantflagellin vaccine or with the native flagella (200 �g in PBS,sub-cutaneously) twice on Days 0, 21. A third vaccination (incomplete Freund’s adjuvant) was administered on Day 42. Twomice of each group were sacrificed 10 days after the secondor the third immunizations to follow the cytotoxic T cell (CTL)and natural killing (NK) responses in their spleens. Lymphocytesuspensions were prepared. One-third of the splenocytes werepulsed with 100 �M synthetic peptides for 2 h at 37 ◦C and thenadded to the rest of the splenocytes. Cultures were incubatedfor 5 days in lymphocyte medium [RPMI medium supplementedwith 10% FCS, 2 mM l-glutamine, 1 mM sodium pyruvate, 1%non-essential amino acids, 25 mM HEPES (pH 7.4), 50 �M 2-mercaptoethanol, and combined antibiotics]. CTL or NK assayswere carried out as described previously [26]. Briefly, viablecells (effectors cells) were separated by Lympholyte-M (Cedar-lane Laboratories Ltd., Hornby, British Columbia, Canada) gradientcentrifugation, washed, resuspended in lymphocyte medium, andadmixed at four effectors-to-target (E:T) ratios ranging from 100:1to 12.5:1 with 5000 [35S]methionine-labeled target cells. Resultsare expressed as fold specific lysis above the lysis of flagella alone.
2.9. Measurement of flagellin concentration
The recombinant flagellin was highly purified and hence a stan-dard Lowry assay [27] was used for evaluation of flagellin proteinconcentration. To assess the pharmacokinetic profile of flagellin inthe serum, sandwich ELISA assay was performed in which the plateswere coated with purified polyclonal rabbit antibodies againstflagellin and a secondary purified polyclonal guinea pig antibodyanti-flagella 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 prepa-ration, the levels found in the purified vaccine constructs was≤350 EU/dose using QCL-1000 kit (Lonza, Verviers, Belgium).
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Table 2In silico binding predictions of the CTL epitopes included in the vaccine.
T Cell Epitopesa HLA SYFPEITHIb BIMASc IC 50 (SMM)d
a The exact CTL epitope within NP 335–350 is NP 336–344, the epitope within NP 380–393 peptide is NP 380–388 and the exact CTL epitope within MI 2–12 is MI 3–11.b http://www.uni-tuebingen.de/uni/kxi/.c http://www-bimas.cit.nih.gov/molbio/hla bind/.d http://cagt.bu.edu/page/SMM submit.
2.11. Statistical analysis
The Lifetest procedure using Kaplan–Meyer method and log ranktest 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 wasconsidered statistically significant. The data was analyzed using theSAS Version 9.1 software (SAS Institute, Cary, NC).
3. Results
3.1. Selection of epitopes
Based on in silico examination of the influenza peptides and aliterature review, the epitopes listed in Table 1 were included inthe vaccine. Most of the epitopes confer a wide HLA coverage; theyare linear and common to many influenza strains. The name of eachepitope defines its origin in terms of protein name (HA for hemag-glutinin, NP for nucleoprotein) and the amino acids location withinit.
As shown in Table 2, all 3 peptides are classified as good binderswith similar values according to the SYFPEITHI algorithm, Whereasaccording to BIMAS, which classifies HLA-A*0201-restricted can-didate peptides according to a theoretical score reflecting theestimated dissociation half-life of the MHC/peptide complex [29],A high score of 2666 was calculated for the MI 2–12 peptide ascompared to lower values for the NP peptides.
The MI 3–11 epitope binds better than NP 380–388 and the NP336–344 dissociates easily from the HLA molecule. SMM methodis applied successfully to predict MHC binding, TAP transport andproteasomal cleavage [30]. Low values of IC 50 indicate of goodbinders and again, MI 3–11 seems to be better than the other twoepitopes.
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 incu-bated overnight with peptides (12.5–100 �M) MI 3–11, NP 336–344 and HA 91–108.Then stained with anti-HLA-A2 monoclonal antibodies and analyzed by FACS. Resultsare presented as MFI above background.
(deficient for TAP transporters that express low and unstableamounts of HLA A*0201 molecules). Upon binding of peptides, sta-ble and high levels of HLA A*0201 are expressed on the cell surfaceas demonstrated by flow cytometry. Fig. 1 shows a representativedata of 3 repeated FACS measures. A high and dose-dependent bind-ing of MI 3–11 peptide that is the specific CTL epitope within theMI 2–12 peptide was observed. The binding pattern for NP 336–344peptide is different: it is low at all doses. Both patterns are accept-able and may lead to efficient presentation of the peptide on humanantigen presenting cells. The superior binding of the MI peptide overthe Nucleoprotein NP 336–344 peptide is also shown by the in silicoanalysis of their binding.
The B cell epitope HA 91–108 peptide, which is longer than theHLA groove, showed binding capacity only at the higher (100 �M)concentration, probably due to the HLA motif in its N′-terminal. NP380–388 serves as a negative control.
3.3. Cross-reactive antibodies in rabbits
Sera from rabbits immunized 3 times with the recombinant flag-ellin expressing the single B cell epitope HA 91–108 were reacted
Fig. 2. Anti-influenza viruses’ antibody response in pooled sera of vaccinated rabbits. Total Ig was assayed in serum samples diluted 1:500, 2 weeks after the third immunizationwith recombinant flagellin expressing HA 91–108 peptide. ELISA plates were coated with live influenza 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 influenza B (B/Lee/40).
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Fig. 3. Lymphocytes proliferation following immunization with recombinant flag-ellin containing NP 335–350 epitope. Transgenic mice for HLA A*0201 wereimmunized once with flagellin expressing NP 335–350 epitopes. Proliferation oftheir splenocytes (SP) and lymph nodes lymphocytes (LN) was measured followingin vitro stimulation with NP 335–350 peptide (A) or with inactive influenza virus(A/Texas/1/77, H3N2) (B): (white squares) splenocytes from mice administered withPBS; (lined squares) splenocytes from mice immunized with flagellin expressing NP335–350 epitope; (black squares) lymph nodes lymphocytes from mice immunizedwith flagellin expressing NP 335–350 epitope. The proliferation of cells from immu-nized mice is significantly 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 theproliferation of splenocytes from each group when incubated without Ag.
in ELISA with several influenza viruses. The results in comparisonto 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 humansubjects, the Db−/−x�2 microglobulin (�2m) null mice, transgenicfor a recombinant HLA-A2.1/Db−/−x�2 microglobulin single chain(HHD++2 mice) were tested. These mice combine classical HLAtransgenesis with selective destruction of murine H-2 and showonly HLA A*0201-restricted responses [21].
Splenocytes from mice sacrificed 10 days after the 1st immu-nization were stimulated with 5, 10 and 20 �g the NP 335–350peptide or with 20, 100 and 500 HAU of H3N2 A/Texas/1/77virus per well. Representative data of three repetitive experi-ments of lymphocytes proliferation and IFN-gamma secretion thatwere evaluated are shown; significant proliferation response wasobserved in both lymph nodes and splenocytes following in vitroincubation with the peptide (Fig. 3A) and with influenza virusA/Texas/1/77 (Fig. 3B). In accordance, increasing IFN-gamma secre-tion was detected in the immunized group as compared to cellsfrom non immunized mice that were stimulated with the samepeptide antigen as shown in Fig. 4.
The potential of human cells to mount a cellular response tothe vaccine was examined in human PBMC (HLA A2; DR10 phe-notype). Cells from healthy volunteers were incubated with theindividual peptides that are included in the vaccine, at elevatedconcentrations of 10–40 �g/well. A significant IFN-gamma secre-tion was detected in response to incubation with the peptides
Fig. 4. IFN-gamma secretion from proliferating lymphocytes. HHD++2 transgenicmice (harboring the human A*0201 HLA) were immunized subcutaneously withrecombinant flagellin containing a single T cell epitope (NP 335–350) or with PBSin complete Freund’s adjuvant as a negative control. The lymphocytes cultures werestimulated with the NP 335 synthetic peptides and the secreted IFN-gamma inresponse to this stimulation was monitored. Only mice immunized with the recom-binant flagellin (F 335) responded to the NP 335 peptide by significantly higher(p < 0.05) secretion of IFN-gamma. These data represent 3 repetitive studies of pro-liferation and IFN-gamma measures in the medium of proliferating lymphocytes.
HA 307–319, HA 354–372, NP 335–350 and MI 2–12. Followingincubation with 0.2–5 �g of purified flagellin approximately 9-foldelevations in the secretion of IFN-gamma as compared to the con-trol (lymphocytes incubated with medium) was observed as shownin Table 3. Incubation of these cells with the recombinant flag-ellin containing influenza epitopes resulted in 9-fold elevation ofIFN-gamma secretion; it is not clear if this elevation results fromresponse to the specific peptides or to the flagellin. According to theresponse to each of them separately, it can be concluded that bothpeptides and flagellin has a potential to induce cellular immunityin human cells.
3.5. Activation of NK cells
The immunogenicity of the peptides, namely their potential toinduce specific CTLs, has been examined also in vivo. HHD++2 micewere immunized with recombinant flagellin vaccine or with thenative flagella. Following in vitro re-stimulation with the mixture ofpeptides, the CTL-dependent lysis of single peptide-pulsed targetswas evaluated. No CTL activity was observed following the secondand the third immunization (data not shown).
Natural killing (NK) lysis of sensitive YAC-1 cells was demon-strated after the second and third immunization of transgenic micewith a mixture of six recombinant flagellin peptides in Freund’sadjuvant. After the second immunization, the lysis of YAC-1 by NKcells from mice immunized with the recombinant flagellin vaccinewas higher than the lysis observed by NK cells from mice immu-nized with the native flagellin. The epitope-based vaccine induceda natural killing activity which was 2–3-fold higher than the activ-ity which was induced by the native flagella. The lytic activity wasaugmented following the third immunization (Fig. 5).
Table 3IFN-gamma secretion from human lymphocytes following incubation with the flag-ellin and the peptides included in the vaccine.
In vitro stimulator Stimulator concentration(�g/well)
Fig. 5. Induction of NK activity by the recombinant vaccine. Mice were immunizedwith recombinant flagellin vaccine or with the native flagella 2 or 3 times. Spleenswere removed on Day 10 after the last immunization (second or third) and stimu-lated by a mixture of 6 peptide included in the vaccine. NK activities as effectors cellswere assayed on Day 5 with YAC-1 as target cells. The percentages of specific lysis ateffector:target (E:T) ratios ranging from 100:1 to 12.5:1 was detected. Spontaneousrelease did not exceed 20% of maximal release. Results are shown as fold increaseabove NK activity induced by the native flagellin.
3.6. Protection against highly pathogenic avian influenza H5N1
To study the efficacy of the vaccine, the HHD++2 (HLA A*0201transgenic) mice model was used. The mice were immunizedintranasally (IN) or intramuscularly (IM) with 150 �g (IN) or 300 �g(IM) of the vaccine and challenged with a lethal dose (LD80) ofH5N1 avian influenza strain. Their survival was monitored for thefollowing 3 weeks. Fig. 6 shows the survival curve following theseimmunizations, where the final survival level of the intramuscularlyimmunized mice was 68% and the survival level of the PBS adminis-tered mice was 33%. Similar effect was found when the vaccine wasadministered via the intranasal route (81% survival of vaccinatedvs. 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 ELISAfor its ability to recognize this virus. The data demonstrate a doserelated response (Fig. 7), where only immunization with the highdose of the vaccine resulted in a significant elevation of H5N1 spe-cific response.
3.7. Pharmacokinetic and systemic exposure
To assess the pharmacokinetic profile of the vaccine following asingle administration, BALB/c mice were injected intramuscularlywith the vaccine and bled at 10 time points. Serum samples weremonitored by ELISA for their flagellin concentration, to calculateCmax, Tmax and AUC values. As shown in Fig. 8, the maximum con-centration level (Cmax = 3925 ng/ml) was observed upon 15 min,
Fig. 7. Antibodies against H5N1 virus. Pooled sera from 2 rabbits per antigen immu-nized 3 times with the vaccine containing the 6 epitopes reacted in a dose relatedpattern with H5N1 virus that was used as a coating antigen. The response is definedas the titer ratio of post/pre-immune serum. This ratio is higher in the rabbit immu-nized with 600 �g of the vaccine than the response of sera from rabbits immunizedwith native flagellin or with PBS.
Fig. 8. Flagellin concentration in serum following a single intramuscular administra-tion of the vaccine. Maximum serum concentration 3925 ng/ml (Cmax) of flagellinwas observed after 15 min (Tmax). Half (T1⁄2) of the total exposure quantity wasobtained within 30 min post-dosing. The area under the serum concentration–timecurve of 15,027 ng/ml indicates the body’s total exposure over time to the vaccine.
No traces of protein in the serum could be detected at 12 h post-injection.
3.8. Safety
3.8.1. AllergyFor the evaluation of potential allergic response to the vac-
cine, 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 lethaldose of avian influenza given 3 weeks after the last boost. A significant difference in survival was observed in both routes of administration of the vaccine (p = 0.039 betweentwo 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 flagellin atconcentrations of 8–240 �g/mouse. Using either intranasal or intra-muscular administration, after a single or multiple administrations,low titers (<20 ng/ml) of IgE were detected in the immunized ani-mals. This level is normal and similar to the IgE level found innon-immunized mice as shown in our study and in [31].
3.8.2. Safety in miceMale and female BALB/c mice were used in this toxicology study
as this is the animal model in which this vaccine concept was estab-lished.
The mice were administered intramuscularly with the epitope-based vaccine in PBS and the toxicology findings were compared tothose obtained in control group administered with PBS.
The standard acute toxicology parameters were monitored,including in-life and necropsy analysis (both macroscopic andmicroscopic).
No abnormalities were found in the “in life” phase. The macro-scopic examination revealed a normal appearance of examinedorgans both in early- and late-study termination groups (Day 2 andDay 14, respectively) in both genders.
Histologically, no treatment-related changes were found follow-ing a single IM injection of the vaccine in all major organs examined.Transient minimal to mild changes in the heart of both genders wereseen within the early termination (Day 2) in the vaccine and PBScontrol treated groups. The hearts of all groups were normal with nochanges observed at study termination (Day 14). The severity andcharacteristics of changes observed in early termination PBS controlgroups consisted of degeneration and fibrosis as well as sub-acuteepicardium inflammation in the region of the auricles in a minimallevel. In the vaccine treated group, increased incidences of minimalto mild severity of these phenomena were detected.
A thorough literature review revealed that BALB/c mice thatwere used for this study are characterized by spontaneous lesionsand epicardial mineralization, resulting in increased susceptibilityof heart inflammatory reactions aiming to repair these lesions [32].This phenomenon was manifested in our toxicology study by theminimal inflammation observed in the PBS BALB/c treated mice.
In a similar study conducted with ICR inbred mice, this observa-tion was found at tens of micrograms doses only and the safe dosewas determined.
4. Discussion
Despite of the potential advantages associated with peptide-based vaccines, in reality, peptide vaccines against infective agentshave not yet realized their initial promise; none of the peptide vac-cines had entered advanced phases of clinical trials. This resultsfrom their limited immunogenicity, their structural instability andthe complexity associated with cellular responses to them [33].
The purpose of this study was to further develop and evaluate anovel approach to vaccination, based on conserved influenza epi-topes within recombinant flagellin that serves both as a carrier andas an adjuvant [11,34]. The efficacy of the vaccine was establishedin mice.
Previous studies demonstrated the potential of a vaccine com-prising four of the epitopes included in the current vaccine toprotect human/mouse radiation chimera against H3N2, H2N2 andH1N1 viruses [14]. The present study reports on experiments withan improved vaccine, that includes two additional epitopes (HA354–372 and MI 2–12) within flagellin and its ability to induce pro-tection against the highly pathogenic avian influenza strain. Eachepitope was expressed individually within flagellin and the vaccineconsisted of a mixture of the six recombinant flagellin proteins.
The HA 354–372 peptide resides at the highly conserved mat-urational cleavage site of the HA(0) precursor of the influenza B
virus hemagglutinin. It can elicit a protective immune responseagainst lethal challenge with viruses belonging to either one of therepresentative, non-antigenically cross-reactive influenza B viruslineages [35]. The MI 2–12 peptide includes a sequence that isdefined as A2 restricted CTL epitope. These epitopes were added inview of their ability to broaden the scope of the vaccine to additionalstrains of the viruses.
All the epitopes in the vaccine were presented in the form ofchimeric protein of Salmonella flagellin, and hence no externaladjuvant was required. These properties of the flagellin are ascribedto its association with TLR5 [20].
The toll-like receptor (TLR) system provides an inherent cel-lular recognition device that is of paramount importance for thedevelopment and function of innate as well as adaptive immunity[36,37]. Data on consequences of TLR5 activation for Th1 versusTh2 decisions in the murine system are not uniform [34,38,39]. Inthe current study, the effects of flagellin (endotoxin-low), as wellas the effect of the influenza epitopes that are included in the vac-cine on IFN-gamma secretion by human PBMC was investigated, asdemonstrated (Table 3).
We report that pre-incubation of PBMC from healthy volunteerswith flagellin significantly up regulated secretion of IFN-gammathat is indicative of Th1 type of response. Similarly, incubation withsome of the epitopes included in the vaccine and especially theT helper epitope HA 307–319, resulted in significant IFN-gammasecretion from them.
These data are in accord with the study of Bachman et al. thatspecifically characterized the flagellin of Salmonella Müenchen (theone employed in the current study) as an inducer of TH1 [40].
Virus-specific CTL are recruited to influenza-infected lungs bya Th1 response, specifically due to the production of IFN-gammathat has a clear anti-viral effect [41,42], and hence the signifi-cance of inducing a Th1 response for protective immunity againstinfluenza.
The contribution of flagellin to the adjuvant effect stems alsofrom the prolonged exposure of the immune system to the peptidewhen it is presented in the flagellin. Degradation of peptides inthe body occurs within few minutes [33] whereas the recombinantflagellin was detected in the blood up to 12 h post-intramuscularadministration.
It should be noted that pre-existing anti-flagellin antibodies hadno significant effect on the adjuvant activity of flagellin [15]. Sim-ilarly, pre-exposure to the Salmonella administered by feeding didnot lead to carrier suppression and hence did not affect the protec-tive response of a recombinant flagellin-based vaccine expressinga foreign antigen [43].
According to computerized algorithms, the epitopes NP 335–350and MI 2–12 included in the vaccine should induce a CTL response;indeed, binding assay by FACS revealed the specific binding of thepeptides corresponding to the epitopes included in the vaccineto HLA A*0201 molecules (Fig. 1). Despite this, specific CTL lysisof target cells loaded with these peptides was not detected afterimmunization of HHD++2/HLA A*0201 transgenic mice with recom-binant flagellin expressing these epitopes. For the induction of CTLlysis, the accepted procedure is to immunize with 100 �g peptidein IFA at least once or three times with peptide loaded APCs atweekly intervals. However, in the flagellin system—only 3.5% of therecombinant flagellin comprises the epitope hence, the actual pep-tide dose administered was only 5 �g peptide, which might be toolow for the induction of specific CTL response. NK cells express theTLR and therefore respond to the flagellin. Lysis of YAC-1 cells thatare sensitive to the cytotoxic activity of naturally occurring killercells was shown after immunization with the flagellin at levels of10–40%. This response was elevated and ranged from 30 to 70%,respectively, when the mice were immunized with the recombinantflagellin.
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Lysis by NK contributes to the anti-viral response. It was shownthat infected cells are more sensitive and are more susceptibleto lysis than non-infected cells [44]. The general notion is thatNK cells are non-specific lymphocytes that attack and kill cancercells and cells infected by microorganisms without recognition ofa specific antigen on it. However, Alter et al. [45] demonstrate thatthe sequence of the peptide presented by HLA class I moleculescan influence the ligation by inhibitory and activating killer cellimmunoglobulin-like receptors (KIRs) expressed on NK cells, andthereby modulate target cell recognition and lysis. Based on thesestructural and experimental data on the peptide-specificity of theKIR/HLA interaction, it is tempting to speculate that the recognitionof target cells by NK cells is more ‘specific’ than previously thought.A similarity in peptide motif between HLA A*0201 binders and HLA-G binders was demonstrated by Diehl et al. [46], suggesting that A2specific peptides activate NK activity. Hence, the NK cells inducedby the epitopes employed in the present study may bear relevanceto their efficacy.
The antibodies produced following vaccination with the recom-binant flagellin vaccine that include the six epitopes, recognizedthe H5N1 strain but no hemagglutination inhibition was observed.Hence we conclude that the protective response against the H5subtype was most likely mediated by T-cell and NK activity, andpossibly by complement-mediated lysis.
In rabbits immunized with the whole combination of epitopes,a 6-fold elevation was observed towards H5N1 virus in compari-son to pre-immune serum. This recognition is significantly higherthan the recognition of H5N1 virus, observed in human sera (1.79-fold) following immunization with a commercial seasonal vaccine,justifying the selection of epitopes included in the vaccine. Thiscomparison also indicates the potential of this epitope-based vac-cine to protect against the avian influenza strain, by enhancing theimmune response towards specific epitopes within the virus.
Further indication on the protective effect of the vaccine stemsfrom direct protection studies. The protection experiments showdifference in the morbidity of the animals as well as a statisticallysignificant difference between the survival curves of the vaccinatedgroups and the control group that was administered with PBS, inboth intranasal and intramuscular routes of administration.
Under the experimental conditions, in which the challenge doseis orders of magnitude higher than that pertaining in natural infec-tion, all the exposed mice are infected regardless of their immunestate. But, whereas only 20–30% of the control mice survived infec-tion of this severity, 70–80% of the immunized mice survived thischallenge.
These results demonstrate the efficacy of the vaccine againstthe highly pathogenic H5N1 avian influenza in a transgenic mousemodel, indicating that this vaccine could be used in humans againstnot only human influenza strains but against avian strains as well.
The intranasal route was employed in many of our previous stud-ies using the epitope-based vaccine; it is based on the assumptionthat it will be effective in inducing local response in the lung andnasal airways, which are the primary targets in influenza infection.The intramuscular route is the most common for commercial vac-cines including influenza and the current study demonstrate it iscomparable in its protective effect to the intranasal route. Addi-tional advantages of this route are its safety, the comparatively easyregulations associated with this route and the higher volume thatcan be administered as compared to the limited volume used innasal vaccines.
The regulations for intramuscular administration are simpler inview of the risk associated with intranasal vaccines that can affectthe facial nerve [47].
The safety studies performed with a single administration ofmice with the vaccine demonstrated its potential to induce tran-sient effect in the heart at high doses, this effect is attributed to the
flagellin included in the vaccine since in a control group adminis-tered with native flagellin this phenomenon was enhanced.
The vaccine is sterile and does not contain virus or bacterial par-ticles (the flagellin originates from non-virulent salmonella bacteria[48]), its endotoxin levels are controlled and are within the levelapproved for human use and in addition, it does not elicit allergicresponses as shown in this study and by Lee et al. [49], nor autoim-mune responses as deduced from histopathology analysis of majormouse organs following administration of the vaccine. The safetyof this vaccine in human should be examined in clinical trials.
Acknowledgements
We wish to thank Dr. A. Meshorer, for his help in the analysis ofthe histological sections. We wish to acknowledge Mrs. Kate-Ilovitzand the laboratory team at BiondVax for their dedicated work onthis research.
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