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Vaccine 27 (2009) 2506–2512

Contents lists available at ScienceDirect

Vaccine

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

NA vaccination protects against an influenza challenge in a double-blindandomised placebo-controlled phase 1b clinical trial

uzanne Jonesa, Kirsten Evansa, Hilary McElwaine-Johnna, Michaela Sharpeb, John Oxfordc,ob Lambkin-Williamsc, Tim Mantd, Andrew Noland, Maria Zambone, Joanna Ellise,

ohn Beadle f, Peter T. Loudonb,∗

PowderMed Ltd., 4 Robert Robinson Ave, Oxford Science Park, Oxford OX4 4GA, UKPfizer PGRD, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UKRetroscreen Virology Ltd., Medical Building, Queen Mary School of Medicine, University of London, 327 Mile End Road, London E1 4NS, UKGuy’s Drug Research Unit, Quintiles Ltd., 6 Newcomen Street, London SE1 1YR, UKHealth Protection Agency, 61 Colindale Avenue, London NW9 5HT, UKEggPharm LLP, Whitelilies Farm, Downham Road, Ramsden Heath, Essex CM11 1JS, UK

r t i c l e i n f o

rticle history:eceived 14 May 2008eceived in revised form 10 February 2009ccepted 18 February 2009vailable online 24 February 2009

eywords:nfluenzaNA vaccinehallenge

a b s t r a c t

Background: We have developed a Trivalent DNA vaccine for influenza consisting of three plasmidsexpressing haemagglutinin from different seasonal influenza virus strains delivered using PMEDTM (par-ticle mediated epidermal delivery). We set out to determine whether this vaccine (with and without amolecular adjuvant DNA Encoded Immunostimulator-Labile Toxin (DEI-LT)) could protect subjects froma controlled influenza virus challenge.Methods: Healthy adult subjects were screened for susceptibility to infection with influenza A/H3Panama/2007/99 then vaccinated with 4 �g Trivalent influenza DNA vaccine, 2 �g Trivalent influenzaDNA vaccine plus DEI-LT or placebo. Safety and serological responses to vaccination were assessed andon Day 56 subjects were challenged with A/H3 Panama/2007/99 virus.Results: Vaccination with 4 �g Trivalent or 2 �g Trivalent/DEI-LT was well tolerated and induced antibody

responses to two of the three influenza virus vaccine strains. Post challenge, subjects in the 4 �g Trivalentgroup (N = 27) showed reductions in disease symptoms and viral shedding compared to placebo (N = 27),with an overall vaccine efficacy of 41% (95% confidence interval (CI) = −1.5, 67.7) for ‘Any illness with orwithout fever’ and 53% for ‘Upper respiratory tract infection’ (95% CI = 8.0, 77.7).Conclusion: It was concluded that PMED vaccination with 4 �g Trivalent influenza DNA vaccine was safe

cal reman

and elicited immunologireport of protection of hu

. Introduction

Influenza infection is the cause of considerable worldwide mor-idity and mortality, especially in the elderly [1]. Conventionalaccines provide protection against influenza, but have some limi-ations especially because of the difficulties of manufacturing newirus strains in egg or mammalian cell culture to annually updatehe vaccine and keep track of antigenic drift [2,3].

DNA vaccines provide an alternative to conventional influenza

accines, and use plasmids to express antigens in vivo in order toenerate immune responses. We have developed DNA vaccineshat encode influenza haemagglutinin (HA) and are delivered usingarticle mediated epidermal delivery (PMEDTM). PMED provides

∗ Corresponding author. Tel.: +44 1304 640778; fax: +44 1304 651813.E-mail address: peter.t.loudon@pfizer.com (P.T. Loudon).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.02.061

sponses that protected human subjects from influenza; this is the firstsubjects from disease by DNA vaccination.

© 2009 Elsevier Ltd. All rights reserved.

a needle-free technology to deliver DNA vaccines on microscopicgold beads to the epidermis, a tissue rich in antigen presentingcells and capable of stimulating potent immune responses [4]. DNAvaccines are produced in bacteria and therefore bypass the needfor egg or mammalian cell culture methods, allowing manufactureof a new strain to start as soon as the relevant HA gene sequenceis available. A previous phase I clinical study demonstrated that amonovalent PMED DNA vaccine (pPJV1671) for a seasonal influenzaH3 strain could generate potent immune responses in man [5].The present report concerns the next stage in this programme, theformulation of a trivalent seasonal influenza PMED DNA vaccineand testing in a clinical influenza challenge study. The selected

influenza virus challenge strain was A/Panama/2007/99 (H3N2)since this was representative of circulating annual influenza strainsfrom recent years and was the strain used to derive the vaccinein the previous PMED influenza vaccine study in man [5]. In thepresent study Trivalent vaccine administered alone was compared

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ith Trivalent vaccine co-administered with a molecular adjuvant,he DNA Encoded Immunostimulator-Labile Toxin (DEI-LT) [6].EI-LT is a plasmid vector that expresses the A and B subunits ofscherichia coli heat labile enterotoxin and has been shown to be aotent enhancer of immune responses in preclinical studies [7]. Therimary objective was to assess the efficacy of the Trivalent DNAMED vaccine with and without DEI-LT against controlled influenzairus challenge in healthy adults. Secondary objectives of the studyere to assess immunogenicity, to correlate this to protection and

o evaluate the safety of the Trivalent and Trivalent/DEI-LT vaccines.

. Materials and methods

.1. Study design

This was a double-blind randomised, placebo-controlled studyo assess the efficacy of a PMED DNA vaccine against a controllednfluenza virus challenge in healthy adult volunteers.

Fig. 1 represents the trial design. Subjects were randomised tone of three treatment groups of 35 in an equal ratio: 4 �g Trivalentaccine (Group 1), 2 �g Trivalent/DEI-LT vaccine (Group 2), placeboaccine (Group 3) and were vaccinated on an outpatient basis onay 0. The blinding between groups was maintained using ND10evices that were identical in outward appearance and by admin-

stering each treatment using two ND10 devices concurrently withlacebo devices being used where necessary. All administrations ofaccine were made to the upper, inner arm.

Subjects then attended the study site for three further outpatientollow-up visits on Days 7, 21 and 35. On Day 54 post-vaccination0 subjects were admitted to a quarantine facility for an in-patienteriod of 10 days. They were initially monitored for 2 days to ensurehey were eligible for virus challenge and replaced from the remain-ng 15 vaccinated subjects if they were not. On Day 56, eligibleubjects received a controlled challenge with 106.0 Egg Infectiousose50/ml of influenza A/Panama/2007/99, administered as four

ntranasal doses of 0.25 ml; this dose had previously been showno achieve infection rates of 50–70% of unvaccinated subjects [12].hey were then assessed until Day 63 for signs and symptoms ofnfluenza prior to release from quarantine. Five days after chal-enge, all subjects were prescribed a short course of an antiviraloseltamivir or amantadine) to ensure cessation of virus shedding.ll vaccinated subjects attended the study site for two further out-atient follow-up visits on Days 84 ± 1 and 98 ± 3.

.2. Patient population and setting

Eligible subjects for this study were healthy males or females ofon-child-bearing potential, 18–50 years of age providing written

nformed consent. Subjects were required to have a documentedeciprocal haemagglutination inhibition (HAI) titre ≤10 against the

ig. 1. Scheme of the phase 1b clinical trial to assess the efficacy of a trivalent DNA influenznfluenza virus challenge.

(2009) 2506–2512 2507

influenza challenge virus strain (A/Panama/2007/99) to ensure sus-ceptibility to challenge, no contraindication to receiving antiviraltherapy, no history during adulthood of asthma, no immuno-suppression due to disease or treatment, no allergy to gold, notvaccinated for influenza in the current or previous season and notin the currently recommended groups to receive influenza vaccina-tion.

This study was performed according to Good Clinical Practiceat Guy’s Drug Research Unit, Quintiles Ltd., London in 2006/2007;the virus challenge phase of the study was performed at a desig-nated purposely modified quarantine unit managed by RetroscreenVirology Ltd., London. Ethical approval was received from the GeneTherapy Advisory Committee prior to the enrolment of subjects.

2.3. Vaccines and assays

The three influenza vaccine plasmids used in the trivalent vac-cine were all based on plasmid pPJV1671 [5] and encoded influenzastrains used in conventional influenza virus vaccines in the north-ern hemisphere in recent years: pPML7800 (expressing HA frominfluenza strain H1: A/New Caledonia/20/99), pPJV1671 (expressingHA from influenza strain H3: A/Panama/2007/99) and pPML7794(expressing HA from influenza strain B/Jiangsu/10/2003). Theseplasmids express the full HA sequences including the signal peptide.The HA sequence in pPML7800 was codon optimised for opti-mum expression in mammalian cells because the native sequenceplasmid did not produce well in E. coli. To generate the Trivalent for-mulation the three plasmids were co precipitated onto gold beads1–3 �m in diameter and filled into cassettes inside helium poweredsingle-use disposable delivery devices called ND10 [8] as describedpreviously [9] such that each device delivered a total of 2 �g DNA(0.67 �g of each plasmid) on 1 mg of gold. In the Trivalent/DEI-LTformulation the three HA expressing plasmids were co-precipitatedonto gold beads with DEI-LT pPJV2012, a plasmid that expressesthe A and B subunits of E. coli heat labile enterotoxin [6] such thateach ND10 device delivered 2 �g DNA consisting of 0.6 �g of eachHA expressing plasmid (ratio 1:1:1) in combination with 0.2 �g ofDEI-LT. Placebo ND10 devices contained gold beads without DNA.

Determinations of anti-influenza serological responses both atscreening and post vaccination were made by haemagglutinationinhibition (HAI) assay [10]. The screening assay performed at Retro-screen used whole Panama virus particles, and was selected sinceit had previously been shown to identify subjects with HAI titreto Panama of ≤10 as susceptible to infection with the selectedchallenge strain. The assay of the post vaccination samples was per-

formed at Focus Diagnostics (USA) due to their capacity to assaythe required numbers of samples and used Tween-ether treatedPanama virus to facilitate transfer of reagent between laboratories.Nasal washes were assayed by real-time polymerase chain reaction(RT-PCR) for the presence of H3 Panama. This assay is expected to

a vaccine administered by particle mediated epidermal delivery against a controlled

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etect genomic RNA present in infectious virus particles, but alsohat released from infected cells in non-infectious forms; it mayherefore lead to higher values of detection compared to an infec-ivity assay, and is used to provide a comparative value betweenhe different treatment groups. The methods for extraction of viralNA from samples, standards and controls have been previouslyescribed [11]. Quantitative RT-PCR was carried out in a 25 �l reac-ion mixture with 7.5 �l extracted viral RNA template, 12.5 �l 2×T-PCR Reaction mix (SuperScript III Platinum One-Step Quantita-ive RT-PCR kit, Invitrogen, Paisley, Scotland), 900 nM each primerAH3 forward: 5′-CCT TTT TGT TGA ACG CAG CAA-3′, AH3 reverse:′-CGG ATG AGG CAA CTA GTG ACC TA-3′), 200 nM TaqMan minorroove binder probe (AH3 Probe: 5′-VIC-CCT ACA GCA ACT GTT ACC-GBNFQ-3′), 0.5 �l SuperScript III RT/Platinum Taq mix, and 0.1 �l

OX Reference Dye. The PCR thermal profile consisted of an initialT step of 50 ◦C for 30 min, followed by 15 min at 95 ◦C, and 40 cyclesf 15 s at 94 ◦C and 60 s at 60 ◦C. Amplification, detection and datanalysis were performed on an ABI Prism 7500 real-time thermalycler, selecting ‘VIC No Quencher’ as the detector. Ct values <38.00ere considered as positive detection by amplification of the tar-

et template. The RT-PCR assay method was considered validated ifinear regression analysis of the standard curve dilutions producedslope of −3.1 to −3.6, and a correlation coefficient (R2) of >0.99.he level of detected RNA was related to pfu by use of a standardurve using H3 Panama titrated by infectivity assay.

.4. Outcome measurement

During the 10-day quarantine phase, efficacy was assessedsing a combination of twice daily subject questionnaires solicitingymptoms of upper respiratory infection, daily directed physicianxaminations of the respiratory tract, counts and weights of tissuessed in each 24-h period as a surrogate of symptom severity andral temperature monitoring six times daily. In addition nasal washnd throat swab samples were taken once per day for detection ofirus by RT-PCR assay. The timings of these assessments were basedn data from previous unpublished viral challenge studies.

Immunogenicity of each vaccine was determined by collectinglood samples pre-vaccination on Day 0 and on Days 21, 35, 55nd 84 ± 1 for determination of HAI titres against the three vac-ine strains. Adverse events (AEs) were recorded from the time ofaccination until the end of the study. Safety laboratory tests wereerformed at screening, pre-vaccination on Day 0 and on Days 21,5, 55 and 84 ± 1. A physical examination including vital signs waserformed at screening and on Days 54, 63 and 98 ± 3. Local reac-ogenicity was assessed at each actuation site pre-vaccination onay 0 and post-vaccination on Days 0, 7, 21, 56 and 98 ± 3. Subjectsere also given a diary card to record systemic reactions for 14 daysost-vaccination and to record oral temperature and local reactionsor 3 days post-vaccination.

.5. Statistical analyses

A sample size of 90 challenged subjects was selected based uponhe calculation that a group size of 30 (with 28 evaluable subjects)ould yield at least 80% power to detect at least a 70% reduction

n the rate of the primary efficacy variable ‘Any illness’. The pri-ary efficacy analysis was based upon prevention of ‘Any illnessith and without fever’; secondary analyses were ‘Upper respira-

ory infection’ (URI), with and without ‘Fever’, ‘Lower respiratorynfection’ (LRI), with and without ‘Fever’ and ‘Laboratory-confirmed

nfluenza illness’; these terms are defined in Table 2. The esti-

ate of vaccine efficacy was calculated as the proportion of casesrevented by the vaccine, and was presented with its two-sided5% confidence interval (CI) [12]. The Chi-square test was used toompare the two treatment groups, for each of the three compar-

(2009) 2506–2512

isons. No adjustment of the level of significance for multiplicity wasmade. These analyses were defined prior to unblinding of the study.No formal statistical analysis of immunogenicity and safety wasplanned. A post hoc analysis of the association between virus shed-ding post challenge and disease symptoms was carried out using aMann–Whitney non-parametric test to compare the median areaunder the curve (AUC) for shedding between the symptom-freegroup and the with-symptoms group. A post hoc exploratory analy-sis of the association between HAI titre before challenge and diseasesymptoms post challenge was conducted using logistic regressionincluding only those covariates and factors significant (at two-sided5% level) in the model. Factors and covariates considered were nat-ural logarithm of HAI Panama strain GMT at baseline and at Day55 and vaccination group. The odds ratio and its 95% confidenceinterval was used to describe the findings.

3. Results

3.1. Subject population

The flow of subjects through the study is shown in Fig. 2. Onehundred and five subjects were enrolled into the study and vac-cinated; these made up the safety population. Of these 104 werefollowed up and one subject withdrew. A total of 86 of the 90 sub-jects planned in the protocol were challenged with influenza virusand made up the efficacy population; four subjects did not meetthe criteria for challenge upon entry to the quarantine facility. Thecharacteristics of the efficacy population are shown in Table 1. Sub-jects ranged between 20 and 49 years of age, were predominantly ofCaucasian origin and there was a much higher percentage of malesthan females since all females had to be of non-child-bearing poten-tial. The three treatment groups were generally well-balanced, andthe small differences between proportions of vaccinated subjectsin the three groups deemed eligible for virus challenge were notconsidered to have biased the results.

3.2. Effect of influenza virus challenge

Challenge of the subjects in the Placebo group with H3 Panamaled to the array disease symptoms expected for this virus (Table 2).Sixty three percent of placebo subjects showed ‘Any illness with orwithout fever’ as defined by the protocol compared to 37% in Group1 (4 �g Trivalent) and 56.3% in Group 2 (2 �g Trivalent/DEI-LT). Asimilar pattern was seen in the constituent symptoms of ‘Any illnesswith or with fever’; for each of ‘Upper respiratory tract infection(URI)’, ‘Lower respiratory tract infection (LRI)’, ‘Systemic illness’ and‘Fever’ the highest scores were seen in Group 3 (Placebo) and thelowest in Group 1 (4 �g Trivalent). ‘Laboratory confirmed influenzaillness’ was recorded in 61.5% of Placebo, 50% of 2 �g Trivalent/DEI-LT and 33.3% of 4 �g Trivalent subjects. Similar results were alsoseen for severity of influenza as measured by the mean numberof tissues and mucus weight post virus challenge (Fig. 3); for bothmeasurements the highest scores were seen in Group 3 (Placebo)and the lowest in Group 1 (4 �g Trivalent).

Virus genomic material was detected in nasal wash samplespost challenge in each treatment group; the limit of detection of theassay was 2.25 pfu per assay which was considered fit for purpose.In the Placebo group virus shedding in nasal wash samples peakedover Days 58–60, whereas in the 4 �g Trivalent group the amountof virus detected dropped after Day 58 (Fig. 4), and this was

reflected in 75% reduction in total amount of virus shed comparedto the placebo (area under the curve [AUC] for the Placebo groupwas 13.1 plaque forming units (pfu) per subject per day, AUC for4 �g Trivalent was 3.3 pfu). As with other measures in this study,the pattern of virus shedding detected in the 2 �g Trivalent/DEI-LT

S. Jones et al. / Vaccine 27 (2009) 2506–2512 2509

Fig. 2. Participant flow diagram. *A total of 18 subjects ineligible for virus challenge (Reasons: Influenza-like or related illnesses, N = 6; raised liver function test (LFTs) results,N = 4; unable to attend or withdrew consent, N = 5; positive drugs of abuse test, N = 3).

Table 1Summary demographics of the Efficacy population.

Characteristic Group 1 4.0 �g Trivalent (N = 27) Group 2 2.0 �g Trivalent/DEI-LT (N = 32) Group 3 Placebo (N = 27) Total (N = 86)

Gender (N [%])Male 26 (96.3) 29 (90.6) 24 (88.9) 79 (91.9)Female 1 (3.7) 3 (9.4) 3 (11.1) 7 (8.1)

Race (N [%])Afro Caribbean 2 (7.4) 4 (12.5) 2 (7.4) 8 (9.3)Asian/Oriental 1 (3.7) 2 (6.3) 1 (3.7) 4 (4.7)Caucasian 22 (81.5) 21 (65.6) 22 (81.5) 65 (75.6)Other 2 (7.4) 5 (15.6) 2 (7.4) 9 (10.5)

Age (years)Mean (SD) 32.9 (9.08) 36.9 (8.44) 35.0 (8.11) 35.0 (8.61)Median 30.0 38.5 31.0 33.0Range 20–49 23–48 25–49 20–49

Height (cm)Mean (SD) 175.43 (9.409) 174.93 (7.791) 175.37 (7.762) 175.22 (8.228)Median 176.00 172.25 176.50 175.00Range 150.5–190.0 165.0–193.0 160.5–185.0 150.5–193.0

Weight (kg)Mean (SD) 75.74 (16.479) 84.07 (11.139) 79.91 (9.375) 80.15 (12.930)Median 78.00 83.95 78.60 79.35Range 47.1–114.3 63.3–105.6 62.8–103.7 47.1–114.3

2510 S. Jones et al. / Vaccine 27 (2009) 2506–2512

Table 2Number of subjects showing symptoms of influenza infection post challenge in each treatment group.

Influenza symptom Number (%) of subjects

Group 1 4.0 �g Trivalent (N = 27) Group 2 2.0 �g Trivalent/DEI-LT (N = 32) Group 3 Placebo (N = 27) Total (N = 86)

‘Any illness’ (with/without ‘Fever’) 10 (37.0) 18 (56.3)a 17 (63.0) 45 (52.3)URI (with/without ‘Fever’) 7 (25.9) 13 (40.6)a 15 (55.6) 35 (40.7)LRI (with/without ‘Fever’) 3 (11.1) 5 (15.6)a 7 (25.9) 15 (17.4)‘Fever’ 4 (14.8) 10 (31.3) 9 (33.3) 23 (26.7)‘Systemic illness’ 6 (22.2) 10 (31.3)a 10 (37.0) 26 (30.2)

‘Laboratory confirmed influenza illness’ 9 (33.3) 16 (50)a 16 (61.5) 41 (47.7)

‘Any illness’ = displaying at least one symptom of URI, LRI, ‘Fever’ or ‘systemic illness’. Table presents number of subjects with ‘Any illness’ and then broken down by eachcomponent category of ‘Any illness’, i.e. URI, LRI, ‘Fever’ and ‘Systemic illness’. URI = ‘Upper respiratory infection’; LRI = ‘Lower respiratory infection’. URI = Presence of at leastone of: rhinorrhea, nasal congestion, sore throat, nasal discharge, otitis, sinus tenderness, pharyngitis on two consecutive days when a Grade 2 severity was present on atleast one of those days, or if a Grade 3 was attained at any time. LRI = Presence of at least one of: cough, new wheezing, rhonchi, râles or signs of consolidation on twoconsecutive days when a Grade 2 severity was present on at least one of those days or if a Grade 3 was attained at any time. ‘Fever’ = Any oral temperature ≥37.9 ◦C. ‘Systemici tive d3 of bo

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llness’ = Presence of at least one of: headache, myalgia or arthralgia on two consecuwas attained at any time. Laboratory confirmed influenza = Meeting the definitiona A subject symptom questionnaire was not completed for Subject 1018. It was as

nd systemic illness rather than ‘missing’ therefore a ‘worst case’ approach has not

roup was intermediate between the 4 �g Trivalent and Placeboroups (AUC for Group 2 was 6.8 pfu per subject per day). A postoc analysis demonstrated a strong association between AUC andisease symptoms post challenge (p � 0.01).

Overall, a vaccine efficacy of 41.2% was achieved for the pri-ary efficacy outcome ‘Any illness with or without fever’ at the�g Trivalent dose which approached but did not achieve statisti-al significance (p = 0.057; 95% CI = −1.5, 67.7). Also at this dose theecondary efficacy outcome of ‘URI’ achieved statistically signifi-ant efficacy of 53.3% (p = 0.027; 95% CI = 8.0, 77.7); efficacy versusLaboratory-confirmed influenza illness’ was 43.8% (p = 0.056; 95%I = −1.5, 70.4). None of the comparisons for the 2 �g Trivalent/DEI-T dose were statistically significant (Fig. 5).

.3. Serological response to vaccination

The antibody response to vaccination was determined by HAIssay and is shown in Fig. 6. Modest increases in responses to H1ew Caledonia and H3 Panama were noted in the two active groupsompared to the placebo at Days 21, 35 and 55, but vaccination

id not generate responses to B Jiangsu in either active group. Theesponses to 4 �g Trivalent and 2 �g Trivalent/DEI-LT were similaror all three strains at each time point. Note that the assay used tocreen subjects for susceptibility to infection used a somewhat dif-

ig. 3. Mean number of tissues and mean mucus weight collected per subject forach of the treatment groups post challenge.

ays when a Grade 2 severity was present on at least one of those days or if a Gradeth illness (any) and influenza infection (detectable virus PFU > 2).d responses were ‘none’, in the calculations of upper respiratory, lower respiratoryollowed for these three parameters.

ferent protocol to that used to analyse responses to vaccination (seeSection 2). Hence although all subjects enrolled showed a responseto Panama of ≤10 at Day 0 in the screening assay, Fig. 6 shows thatsome subjects had a response greater than this when measuredby the second assay. Since the expected proportion of subjects inthe Placebo group showed influenza illness post challenge, the HAI

Fig. 4. Shed virus detected in nasal wash samples by PCR. Scatter plots showingamount of virus detected per subject per day and geometric means for the 4 �gTrivalent (A), 2 �g Trivalent/DEI-LT (B) and Placebo groups (C).

S. Jones et al. / Vaccine 27

Fig. 5. Efficacy of the 4 �g Trivalent and 2 �g Trivalent/DEI-LT vaccines, calculated asthe proportion of ‘URI’ (upper respiratory tract infection), ‘Any illness’ or ‘Laboratory-confirmed influenza illness’ cases prevented by vaccination (*p = 0.027). The othercomparisons were not significant (p < 0.05).

Fig. 6. Serological responses to vaccination. Graphs show reverse distribution curvesof HAI titres to each of the three vaccine strain components at Day 55 (prechallenge);A = H1 New Caledonia, B = H3 Panama, C = B Jiangsu.

(2009) 2506–2512 2511

anti-H3 Panama HAI titre on Day 55 (prior to challenge) and subse-quent development of influenza disease symptoms. In this analysis,data across the three treatment groups were pooled. Associationswere demonstrated between anti-Panama geometric mean titre(GMT) at Day 55 and ‘Any illness with or without fever’ (odds ratio0.48, 95% CI 0.31–0.75) and ‘URI’ (odds ratio 0.45, 95% CI 0.29–0.70).For these analyses, after including in the logistic regression modelDay 55 GMT, the factor of vaccination group and covariate pre-vaccination GMT did not have additional statistically significantinfluence on outcome, hence were not included in the model.

3.4. Adverse events (safety population)

All three formulations were generally well tolerated and therewere no serious adverse events (SAEs) or withdrawals due to AEs(Table 3). The most common AEs were application site burning(36.2% subjects), headache (29.5% subjects) and application sitestinging (18.1% subjects). Actuation site AEs were all were mild,consistent with those seen in previous PMED studies [5,13] and allresolved without treatment, most events resolving within severalminutes to hours. There were no marked differences between thetreatment groups in terms of the frequency of AEs.

4. Discussion

DNA vaccines have been proposed as an attractive alternative toprotein-based approaches because of their simplicity of manufac-ture and ability to generate balanced serological and cell mediatedimmune responses [4]. However, despite exciting results in preclini-cal models and the licensure of DNA vaccines for veterinary diseases[14], an effective DNA vaccine for human disease has remained elu-sive. One particular aspect of DNA vaccination that has been thesubject of intensive study is the optimal mode of delivery, sinceadministration by needle and syringe is known to be an inefficientprocess. We have developed a device to deliver DNA vaccines ongold particles to the epidermis, and have shown that small (micro-gram) quantities of DNA can elicit potent immune responses inpreclinical studies and in man [5,9,13,15]. We believe the potency ofPMED derives both from the delivery of DNA into cells (without theneed to rely on inefficient uptake mechanisms to cross the plasmamembrane) and from the targeting of the epidermis where antigenpresenting cells such as Langerhans cells can express the DNA, tran-sit to the local lymph node and stimulate immune responses [16].

In the present report we show that vaccination with a trivalentPMED influenza DNA vaccine can protect subjects from influenzadisease, and we believe that this is the first time a DNA vaccinehas been shown to protect human subjects from disease. Subjectsin the 4 �g Trivalent group showed a consistent pattern of reduceddisease symptoms, fever, mucus weight and number of tissues usedcompared to the Placebo group. Virus shedding was also markedlyreduced in the 4 �g Trivalent group, with the median area underthe curve being 25% of that in the Placebo group. Although the pri-mary efficacy outcome (’Any illness’) showed a favourable responsecompared to placebo, it did not reach statistical significance, nor did‘Laboratory confirmed influenza illness’. Statistical significance wasachieved for efficacy versus URI.

The response of the 2 �g Trivalent/DEI-LT group to challenge wasintermediate between the Placebo and 4 �g Trivalent groups. Sincethis study did not include groups receiving the same dose of Triva-lent vaccine with and without DEI-LT a direct comparison of theeffects of DEI-LT are not possible; however, it is clear that the inclu-

sion of DEI-LT in the 2 �g dose group was not able to compensate forthe lower total DNA dose compared to the 4 �g Trivalent treatment,and hence the effect of DEI-LT is marginal at best. A proportion ofsubjects in the Trivalent/DEI-LT group made antibody responsesto LT confirming expression in vivo, and there was no apparent

2512 S. Jones et al. / Vaccine 27 (2009) 2506–2512

Table 3Summary of common adverse events (≥10% any treatment group) in the safety population.

System organ class/preferred term Number (%) of subjects

Group 1 4.0 �g Trivalent (N = 35) Group 2 2.0 �g Trivalent/DEI-LT (N = 35) Group 3 Placebo (N = 35) Total (N = 105)

General disorders and administration site conditionsApplication site burning 13 (37.1) 11 (31.4) 14 (40.0) 38 (36.2)Application site stinging 4 (11.4) 6 (17.1) 9 (25.7) 19 (18.1)Application site paraesthesia 6 (17.1) 8 (22.9) 1 (2.9) 15 (14.3)Malaise 4 (11.4) 3 (8.6) 4 (11.4) 11 (10.5)Application site pruritus 4 (11.4) 3 (8.6) 3 (8.6) 10 (9.5)Lethargy 4 (11.4) 1 (2.9) 5 (14.3) 10 (9.5)

Nervous system disordersHeadache 10 (28.6) 10 (28.6) 11 (31.4) 31 (29.5)

Respiratory, thoracic and mediastinal disordersPharyngolaryngeal pain 4 (11.4) 3 (8.6) 5 (14.3) 12 (11.4)Nasal congestion 3 (8.6) 3 (8.6) 4 (11.4) 10 (9.5)Cough 0 (0.0) 5 (14.3) 0 (0.0) 5 (4.8)

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Diarrhoea 1 (2.9) 5 (

nfections and infestationsRhinitis 4 (11.4) 3 (

elationship between antibody responses to LT and influenza HAresults not shown). The lack of a clear effect of DEI-LT is some-hat surprising given the potent enhancement of immunity seenith DEI-LT in preclinical studies [6,7], and may indicate that DNAelivery of LT protein is ineffective in humans, or that the level ofxpression was insufficient in man compared to preclinical species.nly one dose level of DEI-LT could be included in the current studynd this was well tolerated, therefore there may now be justificationor testing a range of DEI-LT doses in man.

Vaccination with Trivalent and Trivalent/DEI-LT vaccines led toodest antibody responses to the H1 and H3 components as deter-ined by HAI assay; the H1 component was codon optimised butas similarly immunogenic to the native H3 vaccine. An associa-

ion was noted between HAI titre to H3 Panama prior to challengend protection, with increasing levels of HAI at Day 55 indicativef greater protection. Serological HAI titre is widely regarded as aorrelate of protection for conventional influenza vaccines, with aeciprocal titre of 40 or above proposed as a level that is likely toe protective [17]. Thus the present study did not find any evidencehat the immunological mechanism of protection of healthy adultss different for DNA and protein-based influenza vaccines, althoughell mediated immune responses to vaccination were not measurednd may be a useful inclusion in future studies, especially since DNAaccination is known to induce T cell responses [4] and protection ofhe elderly from influenza has been correlated with T cell responsesn man [18].

The level of 41–53% efficacy seen in the present study usingMED DNA vaccination was less than the 70% reported in adultsor trivalent inactivated influenza vaccines [1], and the serologi-al responses to PMED vaccination were below those required foricensing of annual influenza vaccines in the European Union, espe-ially for the B strain component. Hence this technology needsurther optimisation before it can compete against conventionalnfluenza vaccines, and will need to provide an assurance of safetyquivalent to that obtained with licensed products. However, theseesults provide proof of principle that DNA vaccination can protectuman subjects from influenza and, given the potential advantagesf this technology over traditional vaccines, provides a rationale forurther studies to improve potency.

cknowledgements

We thank Jim Fuller (Recombiworks) for construction of plas-ids, Helen Amine-Edine (KataCliniKa) and Peter Treasure for

[

[

3 (8.6) 9 (8.6)

7 (20.0) 14 (13.3)

statistical analysis. We are also grateful to John Treanor (Univer-sity of Rochester) for scientific advice and for chairing the SafetyReview Board.

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