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Vaccine 27 (2009) 3083–3089

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

valuation of conserved and variable influenza antigens for immunizationgainst different isolates of H5N1 viruses

mi Patelb,c, Kaylie Tranb, Michael Grayb, Yan Lia,b, Zhujun Aoc, Xiaojian Yaoc,d,arwyn Kobasaa,b,c,∗, Gary P. Kobingerb,c,∗

Respiratory Viruses, National Microbiology Laboratory, Public Health Agency of Canada, CanadaSpecial Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, CanadaDepartment of Medical Microbiology, University of Manitoba, Winnipeg, CanadaLaboratory of Molecular Human Retrovirology, University of Manitoba, Winnipeg, Canada

r t i c l e i n f o

rticle history:eceived 18 November 2008eceived in revised form 2 March 2009ccepted 9 March 2009vailable online 1 April 2009

eywords:

a b s t r a c t

The combination of rapid evolution and high mortality in human cases of infections has raised concernsthat the H5N1 avian influenza virus may become a new, possibly severe, pandemic virus. Vaccination islikely to be the most efficient strategy to mitigate the impact of the next influenza pandemic. The presentstudy evaluates B and T cell immune responses generated by the H5N1 viral antigens, hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), or the M2 ion channel in parallel, expressed from a DNA vaccinevehicle. Protection studies of immunized mice challenged with 100 LD50 of homologous or heterologous

vian influenza5N1NA vaccineell-mediated immunity

H5N1 viruses indicate that HA afforded better protection than the NA, NP or M2 DNA vaccines. Theantibody response was also higher in HA-vaccinated mice as determined by hemagglutination inhibition(HI) and neutralizing antibodies (NAB) assays. Interestingly, the T cell response was higher against HAthan against NA, NP or M2 and was detectable at low doses of the DNA–HA vaccine capable of inducingcomplete protection, despite the absence of a detectable B cell response. This study emphasizes the needto evaluate the relationship between both arms of the adaptive immune responses in regards to protective

virus

efficacy against influenza

. Introduction

Influenza A viruses infect a variety of hosts and cause a rangef respiratory and gastrointestinal complications. The recent emer-ence of the highly pathogenic avian H5N1 influenza A viruses inird populations and transmission to humans, with high mortality,as raised concerns that the next human influenza pandemic mayerive from this subtype. Avian H5N1 viruses infect wild aquaticird and domestic poultry and transmission within bird popula-ions in Asia, Europe, and Africa has been largely associated with

igratory routes [1–4]. Since 2003, numerous clinical cases haveeen reported in humans who live and/or work in close contactith infected birds and as of October 2008, there have been over

80 confirmed human cases and 243 deaths, the majority of whichave occurred in Southeast Asia (Indonesia and Vietnam) [5]. Thepanish Flu (H1N1-1918) was likely derived from an avian virusnd it has been demonstrated in animal models that multiple genes

∗ Corresponding author at: 1015 Arlington Street, Winnipeg, MB, R3E 3R2, Canada.el.: +1 204 784 5923; fax: +1 204 789 2140.

E-mail addresses: darywn kobasa@phac-aspc.gc.ca (D. Kobasa),ary kobinger@phac-aspc.gc.ca (G.P. Kobinger).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.03.023

.© 2009 Elsevier Ltd. All rights reserved.

contributed to its virulence [6,7]. This virus was responsible for thelargest human influenza A pandemic in recorded history, and wasresponsible for 40–50 million deaths [8]. It is evident that appro-priate treatment and prophylaxis is necessary to better prevent themortality associated with a future influenza A pandemic.

The most effective prophylaxis against influenza viruses hasbeen vaccination. Several vaccination strategies have been eval-uated for optimal prevention of influenza, with the conventionalformaldehyde-inactivated virus being the most widely used [9,10].More recently, the live-attenuated FluMistTM vaccine has beenapproved [11]. These vaccines provide reasonable protectionagainst yearly influenza infections because they are reformulatedannually to match the antigenicity of the dominant circulatingstrains. However, the effectiveness of conventional inactivated vac-cines may be limited in the event of a sudden appearance of anew pandemic virus. Vaccines against the H5N1 virus that havealready been produced and evaluated may provide limited pro-tection against an antigenic variant that ultimately emerges as a

pandemic strain and current production methods are not optimalto meet global demand in the event of a pandemic [12]. A single vac-cine which could protect against divergent H5N1 influenza isolateswould be advantageous and therefore alternative vaccination plat-forms are currently being evaluated. DNA-based subunit vaccines

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ffer several advantages including stability and ease of productionor mass distribution [2,13,14]. In addition, recent improvementsn the efficacy of DNA-based vaccines using optimized antigenicxpression cassettes were demonstrated by the induction of signif-cant immune responses in humans [2,13,15,16]. However, whetherNA-based vaccines will be suitable for protection against influenza

nfection in humans remains to be demonstrated.Robust immune stimulation and development of a compre-

ensive memory response is likely to be necessary for successfulrotection against antigenically divergent strains of H5N1 influenzairus. Humoral responses resulting in the production of neutralizingntibodies (NAB) are considered one of the most important corre-ates of protection against influenza virus infection [17]. However,he H5N1 virus has evolved into several antigenically distinguish-ble clades and neutralizing antibodies may not provide extendedrotection against all clades. Therefore, strategies to stimulate atrong T cell-mediated immune response against conserved anti-enic regions of influenza virus, concomitant to a robust antibodyesponse were explored by many [18–20]. In this study, the B andcell responses, developed after immunization with an H5N1 HA,A, NP or M2 antigen expressed from a unique DNA vaccine vehi-le, were evaluated in parallel. Immune responses were correlatedo survival against homologous or heterologous H5N1 lethal chal-enges in mice.

. Materials and methods

.1. Viruses and cells

Influenza virus strain A/Hanoi/30408/2005 (Hanoi05; H5N1)as generously provided by Q. Mai Le and T. Hien Nguyen, National

nstitute of Hygiene and Epidemiology, Hanoi. Madin Darby canineidney (MDCK) cells were maintained in minimal essential mediumMEM) supplemented with 10% fetal bovine serum and antibiotics./Hong Kong/483/97 (HK/97; H5N1) and other viruses were prop-gated, and quantitated by standard plaque assay, on MDCK cellsultured with virus diluent (MEM containing 0.3% bovine serumlbumin and antibiotics) containing 1.0 �g/ml TPCK-treated trypsinTPCK-trypsin).

.2. Construction of plasmid DNA

The cDNA of Hanoi05 HA, NA, M2, and NP genes was codon opti-ized for more efficient expression in mammalian systems [21]

nd genes were reconstructed from overlapping oligonucleotiderimers. Each gene was inserted into the pCAG� vector under theontrol of a chicken �-actin promoter resulting in the following con-tructs: H5N1: pCAG�-HA, pCAG�-NA, pCAG�-M2, and pCAG�-NP.CAG� was derived from a pCAGGS expression vector following aeletion of 829 bp between the Eco47III/XbaI sites. DNA for eachaccine was scalable using endotoxin-free mega or gigapreps (Qia-en).

.3. Animal models

Six to eight-week-old female BALB/c mice (Charles River Canada)ere used to evaluate protection as well as B and T cell immune

esponses against influenza antigens as previously described [22].he T cell response was analyzed by evaluating the number of spotsorming on, and the frequency of, CD8+ T cells positive for IFN�

roduction. Twelve male cynomologous macaques (Macaca fasci-ularis) ranging from 9 to 19 years of age were obtained (Healthanada, Non-Human Primate Colony, Ottawa, Canada) by the Publicealth Agency of Canada according to the guidelines of the Cana-ian Council on Animal Care.

(2009) 3083–3089

2.4. Transfection and detection of H5N1 proteins

Human embryonic kidney (HEK) 293 T cells were cultured in60 mm dishes to 80–90% confluency and transfected with 10 �g ofH5N1 pCAG�-HA, pCAG�-NA, pCAG�-M2, or pCAG�-NP by usingcalcium phosphate precipitation. Cells were incubated for 48–72 hat 37 ◦C, 5% CO2 and harvested using radioimmunoprecipitationassay (RIPA) buffer (Triton ×100, 1 M Tris–HCl pH 7.7, 5 M NaCl,2% sodium deoxycholate, 0.1% SDS, 0.5 M EDTA pH 8.0, water) toprevent protein degradation, while passing through a 21 1/2-guageneedle to lyse the cells. Standard Western blot techniques wereused to visualize all four proteins. Briefly, protein samples wereseparated by electrophoresis on a 10% SDS–PAGE (sodium dodecylsulfate–polyacrylamide gel electrophoresis) gel and wet transferredto a PVDF (GE Healthcare) membrane overnight. The HA blot wasincubated with sera obtained from mice infected with a sub-lethaldose of the corresponding influenza virus. The three other proteinswere incubated with a primary anti-mouse antibody against NA,M2 or NP. All samples were probed with goat anti-mouse anti-body conjugated to horseradish peroxidase secondary antibody.The protein band was visualized using an ECL Detection kit (Amer-sham).

2.5. Immunization of mice with DNA vaccines and viral challenge

BALB/c mice were immunized with a range of doses of eachDNA vaccine individually or in selected combinations by intramus-cular (I.M.) injection of 50 �l in each of the right and left hindlimb. Anesthetized mice were challenged by intranasal inoculationof 100 times the amount of virus required for 50% survival (50%lethal dose or LD50, equivalent to 100 infectious dose 50), in 50 �lvirus diluent (MEM, 0.3% BSA, penicillin/streptomycin) of differentisolates of H5N1 influenza viruses [14]. After challenge, animalswere weighed daily for 15–20 days and monitored for clinical signsusing an approved scoring sheet. All procedures and the scoringsheet were approved by the Institutional Animal Care Committeeat the National Microbiology Laboratory (NML) of the Public HealthAgency of Canada (PHAC) according to the guidelines of the Cana-dian Council on Animal Care. All infectious work was performed inbiocontainment level 4 at the NML, PHAC.

2.6. Detection of antibody

Sera collected from immunized mice were evaluated by neu-tralization and/or hemagglutination inhibition assays as previouslydescribed [17,23,24]. For neutralization assays, sera were treatedovernight at 37 ◦C with the receptor destroying enzyme (RDE) andthen inactivated at 56 ◦C for 45 min. Twofold serial dilutions of eachsample, starting with a 1:10 dilution, were prepared in virus dilu-ent and mixed with equal volume of the homologous influenzavirus isolate used for immunization (100 plaque forming units [PFU]per well) and incubated at 37 ◦C for 60 min. The mixture was thentransferred onto subconfluent MDCK cells in 96-well flat-bottomedplates and incubated for 5–10 min at room temperature. Controlwells were infected with equal amount of viral vector withoutthe addition of serum or with non-immune control serum. Onehundred microliters of virus diluent supplemented with 2.0 �g/mlTPCK-trypsin was then added to each well and plates were incu-bated at 37 ◦C, 5% CO2 for 48 h. Cells were subsequently scored forthe presence or the absence of cytopathic effects (CPE) under a

light microscope. The highest serum dilution not exhibiting CPE wasscored positive for neutralizing antibody and neutralization titerswere reported as the reciprocal of this dilution. All infectious invitro work was performed in the biocontainment level 4 laboratoryof the NML, PHAC.

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Table 1Efficacy of different influenza antigen-based DNA vaccines following homologouschallenge (Hanoi05).

Treatment Protection (%) Weight loss (%)a Signs of diseaseb

Vehicle 0 >25 DeathHA (100 �g) 100 0 NoneNA (100 �g) 100 12 MildNP (100 �g) 100 24 Severe

A. Patel et al. / Vacc

.7. Hemagglutination inhibition assays

RDE-treated sera serially diluted in 2-fold steps starting with a:10 dilution and 50 �l/well added to a V-bottom 96-well microtiterlate. Four agglutinating doses (AD) of Hanoi05 H5N1 virus weredded to each well and the plate was incubated at room temper-ture for 1 h. Following incubation, 50 �l of 0.5% guinea pig, 0.5%orse, or 0.5% turkey red blood cells were added to each well andhe assay was incubated at room temperature for 1 h, 1 h, or 45 min,espectively. The hemagglutination inhibition titer was scored ashe highest dilution where red blood cell agglutination did not occurnd data were reported as the reciprocal of this dilution. Each assayas performed in triplicate.

.7.1. Cellular immune responsesELISPOT assays were performed with the ELISPOT Mouse Set

BD Biosciences) according to manufacturer’s instructions. 96-Wellicrotiter plates (with a PVDF membrane) were coated overnight

t 4 ◦C using purified anti-mouse-IFN� antibody. The followingay, each plate was blocked for at least 3 h with RMPI 1640 sup-lemented with 10% FBS, 1% penicillin/streptomycin. Splenocytesere harvested from mice 10 days post-vaccination to screen for T

ell responses by ELISPOT or flow cytometry. Spleens were groundgainst a fine mesh filter in L-15 medium (Gibco) and mononuclearells were isolated by filtration and resuspended in L-15. 15 mereptides with 10 amino acid overlaps were obtained (Mimitopes,ustralia) for the entire H5N1 HA, NA, NP, M2 proteins (112, 87,7, and 17 peptides, respectively). Each peptide was resuspendedn dimethyl sulfoxide (DMSO), and pooled (10 peptides/pool) forase of assay. Each peptide pool was diluted in RMPI 1640 anddded to the microtiter plate wells to give a final concentrationf 2.5 �g/ml per well of peptide. Splenocytes were resuspended inPMI 1640 (supplemented with 10% FBS, penicillin/streptomycin,-glutamine, non-essential amino acids, sodium pyruvate, HEPESuffer, 5 × 10−3 2-ME) and 5 × 105 cells were added to each well. TheLISPOT plates were incubated overnight at 37 ◦C, 5% CO2. The fol-owing day, samples were washed and incubated with biotinylatednti-mouse-IFN� for 2 h at room temperature. Following the addi-ion of streptavidin conjugated-horseradish peroxidase (HRP) IFN�ositive cells were detected using the BD AEC Substrate Reagentet (BD Biosciences). IFN� positive cells were visualized as spotsn the ELISPOT membrane and counted using an ELISPOT Plateeader (AID ELISPOT reader, Cell Technology, Colombia, MD). Allxperiments were repeated independently in triplicate.

Flow cytometry was performed to further characterize theature of the cellular immune response. Splenocytes werearvested at day 10 post-vaccination and plated at 2 × 106

ells/well. Cells were restimulated for 5 h with 9 mer pep-ides corresponding to the immunodominant H5N1 HA epitoper control NP peptide in Dulbecco’s modified Eagle’s mediumDMEM, supplemented with 10% FBS, penicillin/streptomycin, l-lutamine, non-essential amino acids, sodium pyruvate, HEPESuffer, 5 × 10−3 2-ME) containing GolgiStop (Brefeldin A, BD Bio-ciences) and IL2. Following incubation, cells were stained withither anti-mouse CD8-FITC (fluorescein isothiocynate) or anti-ouse CD4-PerCPCy5.5 (peridinin–chlorophyll–protein complex)

t 4 ◦C for 30 min. Splenocytes were fixed and permeabilized usinghe BD Cytofix and permwash protocols (BD Biosciences). The nextay, cells were stained for interferon gamma using IFN�-PE (phy-oerythrin). All samples were read on the LSRII Flow cytometer (BDiosciences). Data was analyzed using BD FACSDiva 6.0.1 software

BD Biosciences).

.7.2. Cellular immune responses in non-human primatesA study designed to assess the pathogenesis of the H5N1 virus

n twelve cynomologous macaques (M. fascicularis) included two

M2 (100 �g) 90 >25 Severe

a Percentage difference from pre-challenge weight to the lowest weight observed.b Change in observed clinical signs. None: no change from pre-challenge signs.

animals for the evaluation of the cellular immune response byELISPOT assays. All animals were confirmed seronegative againstthe H5N1 (A/Vietnam/1203/2004) influenza virus and infectedwith 7 × 106 pfu of virus per animal by intranasal (0.5 ml per nos-tril), intratracheal (4 ml), oral (1 ml) and ocular (0.5 ml per eye)administrations. Animals were monitored daily for signs of dis-ease using an approved scoring sheet including pulse rate, bloodpressure, temperature, respiration rate, and blood O2 saturationlevels. Peripheral blood mononuclear cells (PBMCs) were isolatedat day 12 post-challenge and ficoll purified cells were stimulatedwith pools of peptides overlapping the HA, NA, NP or M2 pro-teins. IFN� production was evaluated by ELISPOT as a measureof the activated cellular response (ELISPOT Human Set; BD Bio-sciences).

2.8. Statistical analysis

Data were analyzed for statistical difference by performingunpaired t-test, one-way analysis of variance (ANOVA) when appro-priate. The differences in the mean or raw values among treatmentgroups were considered significant when p < 0.05.

3. Results

3.1. Evaluation of protection induced by DNA vaccines expressingthe Hanoi H5N1 influenza HA, NA, NP or M2 antigen against alethal challenge with the homologous virus

The HA, NA, and M2 antigens were selected based on surfaceexposure and potential for generating an antibody response. Addi-tionally, M2 and NP are both highly conserved among divergingH5N1 isolates and are attractive candidates for activating a cellu-lar immune response. The HA, NA, NP and M2 antigens derivedfrom the H5N1 Hanoi05 isolate were generated by gene syn-thesis with overlapping primers encoding for the correspondingcodon optimized sequence for improved protein synthesis in mam-malian cells. Each antigen was cloned under the control of theCMV enhancer/chicken �-actin promoter in pCAG� [24], a modifiedpCAGGS plasmid (see Section 2 for details). The bands correspond-ing to proteins with the predicted molecular weight calculatedfor HA, NA, M2, and NP were detected by Western blot analy-sis of plasmid transfected 293 T cells using polyclonal anti-H5N1serum, anti-NA, anti-M2, or anti-NP antibody. The transfer indi-cated successful expression from the engineered pCAG� plasmidDNA (Fig. 1). The plasmids pCAG�-HA, pCAG�-NA, pCAG�-NPor pCAG�-M2 were used as DNA vaccines and each adminis-tered in parallel to groups of 10 BALB/c mice by intramuscularinjection. Mice were challenged after 28 days with 100 LD50 ofthe homologous Hanoi05 virus by intranasal administration. Mice

were weighed and clinical signs were monitored daily. Vaccina-tion with the pCAG�-HA plasmid afforded the best protection with100% survival and no weight loss or detectable signs of disease(Table 1). Protection with any of the NA, NP or M2 vaccine wasincomplete and mice exhibited mild to severe clinical symptoms

3086 A. Patel et al. / Vaccine 27 (2009) 3083–3089

Fig. 1. Detection of H5N1 HA, NA, M2, and NP by Western blot. HEK 293T cells weretransfected with H5N1 pCAG�-HA, pCAG�-NA, pCAG�-M2, or pCAG�-NP. HA, NA,M2 and NP proteins were detected by Western blot and probed with polyclonalamw

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Table 2Heterologous challenge (HK/97).

Treatment Protection (%) Weight loss (%)a Signs of diseaseb

Vehicle 0 >25 DeathHA (50 �g) 70 >25 Mild to severeHA (100 �g) 70 21 Mild to severeNA (100 �g) 0 >25 DeathNP (100 �g) 0 >25 DeathM2 (100 �g) 0 >25 Death

nti-H5N1 serum, anti-NA, anti-M2 or anti-NP antibody followed by a goat anti-ouse-HRP. Control well (−) contained protein derived from 293T cells transfectedith the empty plasmid.

ncluding weight loss of 12, 24 or >25%, respectively. Decreasingoses of the pCAG�-HA vaccine were administered to identify theinimum dose sufficient to fully protect mice against a lethal

hallenge with the homologous H5N1 virus. A single injectionf 10 �g/mouse was sufficient to confer 100% survival with no

igns of disease whereas partial protection with 50% survival andeight loss was observed with 5 �g/mouse (Fig. 2 and data not

hown).

ig. 2. Dose titration of H5N1-HA DNA vaccine. Decreasing doses of the pCAG�-HAaccine (100 �g, 50 �g, 10 �g, 5 �g) were administered to groups of 10 BALB/c mice.urvival was monitored following homologous (Hanoi05) challenge.

HA/HA (100 �g/100 ug) 100 0 None

a Percentage difference from pre-challenge weight to the lowest weight observed.b Change in observed clinical signs. None: no change from pre-challenge signs.

3.2. Evaluation of the DNA vaccines against a lethal challengewith the heterologous HK/97 H5N1 virus

One hundred micrograms of the pCAG�-HA, pCAG�-NA, pCAG�-NP, or pCAG�-M2 vaccines were administered side-by-side I.M. in asingle dose. Additional mice vaccinated with pCAG�-HA were alsoadministered a boost 28 days after immunization. All groups of micewere challenged intranasally with 100 LD50 of the heterologousHK/97 H5N1 virus. The HA gene of the HK/97 virus has a 95.5% aminoacid identity to the Hanoi05 HA gene encoded by pCAG�-HA. Theprime/boost regimen was fully protective against the HK/97 viruswith no clinical signs of disease or weight loss observed (Table 2).Mice vaccinated with a single dose of pCAG�-HA at 50 �g or 100 �gexhibited significant weight loss with a survival rate of 70. ThepCAG�-NA, pCAG�-NP, or pCAG�-M2 vaccines at 100 �g did notafford any significant protection over the control mice against thelethal challenge.

3.3. Immune responses in immunized mice

T cell immune responses were assessed by ELISPOT 10 daysafter immunization with 50 �g of each DNA-based vaccine permouse. A library of overlapping peptides fully representing theHA, NA, NP or M2 proteins of the Hanoi05 isolate was used forrestimulating splenocytes isolated from groups of 4 vaccinatedmice. Stimulated splenocytes were evaluated for the productionof IFN� by ELISPOT. After restimulation, the number of activatedIFN� secreting cells varied for each peptide (Fig. 3a). Interestingly,samples obtained from mice immunized with the pCAG�-HA vac-cine contained the highest number of activated IFN� producingcells followed by mice vaccinated with pCAG�-NA, pCAG�-NP orpCAG�-M2 DNA with in average a total of 10,096 ± 179, 3551 ± 132,2775 ± 53 and 245 ± 3.42 spot-forming cells (SFC)/million mononu-clear cells (MNCs) for HA, NA, NP and M2, respectively. Individualimmunodominant T cell epitopes were identified for Hanoi05 HA.An alignment between Hanoi05 and HK/97 HA revealed high con-servation between shared immunodominant epitopes, differing byonly a single amino acid in two epitopes (inset, Fig. 3a). Flow cytom-etry was used to further analyze the cellular immune response andcharacterize CD8+ and CD4+ T cell responses through detection ofIFN� secretion. The CD8+ and CD4+ responses following restimula-tion with the H5N1 HA immunodominant epitope were 1.8 ± 0.3%and 0.3 ± 0.1%, respectively and 0.1% and 0.1%, respectively, withcontrol NP peptide (Fig. 3b).

The B cell response was analyzed by measuring hemaggluti-nation inhibition (HI) and neutralizing antibody levels against theHanoi05 isolate from sera of mice 25–28 days after a single immu-nization with variable doses of the pCAG�-HA. Increasing HI levels

were observed in mice vaccinated with pCAG�-HA at increasingdoses from 1 to 100 �g (Fig. 4a). Reciprocal titers detected withguinea pig, horse, and turkey red blood cells ranged from averages of10 ± 9 to 32 ± 20, 0 to 60 ± 35, and 0 to 66 ± 23, respectively, in mice.An average NAB titer of 20 was measured in mice immunized with

A. Patel et al. / Vaccine 27 (2009) 3083–3089 3087

Fig. 3. ELISPOT-IFN� T cell responses from BALB/c mice against H5N1 HA, NA, NP, M2 following immunization. BALB/c mice were vaccinated with the HA, NA, NP, or M2DNA vaccines. (A) Splenocytes were harvested at 10 day post-immunization and 5 × 105 cells were stimulated with pools of 15 mer peptides covering the entire H5N1 HA,N uctior lenocyH unodC

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A, NP and M2 proteins Responses were visualized as spots representing IFN� prodepeated three times. Error bars represent the standard deviation of the data. (B) SpA. 2 × 106 cells were stimulated with a 9 mers corresponding to the H5N1 HA immD4+ T cells was detected by flow cytometry.

00 �g of pCAG�-HA, the lowest dose able to generate detectableAB (Fig. 4b). Hemagglutination inhibition was not detected from

era of HA-immunized mice against the heterologous HK/97 H5N1irus with guinea pig or turkey erythrocytes (Fig. 4c). However, HIiters of 33 ± 10 were detected against HK/97 with horse red bloodells from mice vaccinated with 100 �g of pCAG�-HA. Although at

ig. 4. Hemagglutination inhibition (HI) and neutralizing antibody (NAB) assays for H5accinated BALB/c mice and four agglutinating doses of virus were added to each well. Thlood cells and hemagglutination inhibition (HI titer) was reported as the reciprocal of thA) HI titers against H5N1 Hanoi05 virus following vaccination by H5N1-HA DNA vaccineetermined after administration of each of the vaccines (1 �g, 5 �g, 50 �g, 100 �g). (C) HHA 1 �g, 5 �g, 50 �g, and 100 �g). Groups of 5–10 mice were analyzed. Error bars repres

n by cytotoxic T cells. Four mice were analyzed per group and the experiment wastes were harvested on day 10 post-immunization from mice vaccinated with H5N1ominant epitope or control NP peptide. The percentage of IFN� secreting CD8+ and

or below the limit of detection of the assay, an average HI titer of7 ± 6 was observed from mice vaccinated with 50 �g of pCAG�-HA.

The NAB response could not be detected against HK/97 H5N1. Thesame assays were also unable to detect antibody from NA-, NP- orM2-vaccinated mice against the homologous Hanoi05 virus (datanot shown).

N1 following vaccination. Serial dilutions were performed on sera obtained frome sera and virus were incubated with ( ) guinea pig, ( ) horse, or ( ) turkey rede highest dilution of serum which did not block the agglutination of erythrocytes.

s (HA 1 �g, 5 �g, 50 �g, and 100 �g). (B) The presence of NAB against Hanoi05 wasI titers against H5N1 HK/97 virus following vaccination by H5N1 HA DNA vaccinesent the standard deviation of the data.

3088 A. Patel et al. / Vaccine 27 (2009) 3083–3089

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ig. 5. ELISPOT-IFN� from one NHP infected with H5N1 influenza (Hanoi05). (A) ELIumber and intensity of spots on the ELISPOT-IFN�. (B) Number of spots/million ce

.4. Cellular immunogenicity to HA, NA, NP and M2 influenzantigens in infected non-human primates

Cellular immune responses to different influenza antigensere evaluated in the context of an infection with the H5N1

irus in cynomologous macaques (M. fascicularis). The non-humanrimates were infected with the H5N1 (A/Vietnam/1203/2004)

nfluenza virus at 7 × 106 pfu per animal by intranasal, intratracheal,ral and ocular administrations. For T cell response, PBMCs weresolated at day 12 post-challenge. Ficoll purified cells were stim-lated with pools of peptides overlapping the HA, NA, NP or M2roteins and IFN� production was evaluated by ELISPOTS as a mea-ure of the activated cellular response. Production of IFN� by PBMCsfter peptide pool restimulation of isolated cells was detectable forll individual H5N1 pools (Fig. 5a). Interestingly, the total numberf spots added from the pools of each antigen and adjusted per mil-ion cells showed that HA pools stimulated the highest response

ith 1125 spots followed by NA, NP and M2 with 517, 513, and 129pots, respectively (Fig. 5b). In contrast, isolated mononuclear cellstimulated with the same concentration of an unrelated peptidesed as a control resulted in 7 detectable spots per million cells.esults obtained from H1N1-1918 infected ferrets and non-humanrimates showed similar relative responses for each antigen withA stimulating more spots per million PBMCs followed by NA, NPnd M2 (data not shown).

. Discussion

The systematic evaluation of several individual influenza pro-eins as antigenic candidates for vaccination will facilitate theevelopment of an optimized subunit immunization strategy forrotecting against the constantly evolving H5N1 virus. Addition-lly, the assessment of humoral and cellular immune responses toifferent individual influenza antigens will contribute to a betternderstanding of immune parameters correlating with protection.xtensive work has previously been done to develop and evalu-te different HA-based and other subunit influenza vaccines ([25],eviewed in [26,27]). Individual antigens have been assessed in theontext of protection following challenge and neutralizing antibodyesponses, however the role of cell-mediated immunity remains

nclear. The current study evaluated for the first time survivalgainst homologous or heterologous lethal H5N1 challenge in con-unction with both T and B cell responses in mice vaccinated inarallel with a DNA vaccine vehicle expressing one of the followingour H5N1 antigens: HA, NA, NP or M2.

IFN� spots for HA, NA, NP, M2, and Ctrl. Positive T cell responses are indicated by theociated with respective peptide pools. Sections represent individual peptide pools.

Although the antibody response is well documented to be a pre-dictor of protection against influenza infection, the cellular arm ofthe immune response may also contribute to the establishmentof protective immunity. Full protection of different animal speciesincluding mice, chicken, and ferrets against H5N1 infection was fre-quently observed in the absence of detectable antibody before viralchallenge following vaccination with a DNA-based vaccine express-ing HA [23,25,28]. This phenomenon was also observed in thisstudy after vaccination with pCAG�-HA, NA or NP which were capa-ble of inducing variable levels of protection against homologousor heterologous lethal challenges despite the absence of signifi-cant antibody level by HI or NAB assays. Although assay sensitivitywas improved using horse erythrocytes, partial protection was stillobserved from vaccinated mice with antibody titers at or below thethreshold of detection. It was hypothesized that an internal antigensuch as NP can induce protection mainly through activation of thecellular immune response [18,19,29]. However, the real contribu-tion of the T cell response to protection following immunizationwith antigen, such as HA, is unknown. The present study indicatesthat HA was more protective against H5N1 challenge than NA fol-lowed by NP and finally M2. The HA antigen was also confirmedto be the main target of the antibody response as determined byHI and NAB. Interestingly, the T cell response was proportional toprotective efficacy with the highest signal observed from mice vac-cinated with HA followed by NA, NP and M2. A similar observationwas also obtained from an infected non-human primate. Althoughit is difficult with these simple experiments to conclude on the pre-cise contribution provided by each arm of the immune response, itis noteworthy to mention that the top immunodominant epitopesfound in HA are highly conserved among most H5N1 isolates. Inaddition to low levels of detectable antibody, this may partiallyexplain the better protection observed from HA-vaccinated miceagainst the HK97 heterologous virus. Additionally, the contributionof both CD4+ and CD8+ T cell responses may be important towardsheterologous protection. The evaluation of the T cell response byFACS provides evidence that effector CD8+ T cells are activated andproduce IFN� in response to peptide restimulation. Experiments inT or B cell response knockout mice and depletion studies are cur-rently underway to better define the role of each arm of the immuneresponse relative to protection against a lethal challenge with H5N1viruses.

Overall, an influenza vaccine with broad spectrum efficacy islikely to necessitate the induction of a significant T cell immuneresponse targeting conserved regions shared by several virus iso-lates. Together with higher antibody response, the dominance ofthe T cell response against HA may also contribute to the reported

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fficacy of many HA-based influenza vaccines such as DNA- ordenovirus-vector [30,31]. However, this does not exclude theossibility that the addition of other influenza antigen(s) may sig-ificantly improve cross-protection to a wide variety of H5N1 virus

solates. A repeated hypothesis in the field is that cross-protectionay be better achieved with the additional contribution of other

onserved influenza genes such as NP [20,32–34].Overall, the present study sheds additional light on the overall

mmunogenicity of HA, NA, NP or M2 influenza antigens evaluatedn parallel in mice in conjunction with protection against challenge

ith a homologous or a heterologous H5N1 virus. The observationseported here may also be of interest for the development of subunitaccines using different delivery strategies such as recombinantrotein or viral vectors. Better characterization of a more global

mmune response should provide valuable information for futureevelopment of influenza vaccines.

cknowledgements

This work was funded by The Public Health Agency of Canadand grants # 166339 and # 310641 from the Canadian Institute forealth Research (CIHR) awarded to DK and GK.

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