safety, immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, icc-1132...

Vaccine 23 (2005) 857–864

Safety, immunogenicity and efficacy of a pre-erythrocytic malariacandidate vaccine, ICC-1132 formulated in Seppic ISA 720

Michael Walthera,∗, Susanna Dunachiea, Sheila Keatinga, Jenni M. Vuolaa, Tamara Berthouda,Annette Schmidtb, Carolin Maierb, Laura Andrewsa, Rikke F. Andersena, Sarah Gilberta,Ian Poultona, Daniel Webstera, Filip Dubovskyc, Eveline Tierneyc, Pramod Sarpotdarb,

Simon Correaa, Angela Huntcookea, Geoffrey Butcherd, Jack Williamse,Robert E. Sindend, George B. Thorntonb, Adrian V.S. Hilla

a Nuffield Department of Clinical Medicine, Centre for Clinical Vaccinology and Tropical Medicine, Oxford University,Old Road, Headington, Oxford OX3 7LJ, UK

b Apovia Inc., 11125 Flintkote Avenue Suite A, San Diego, CA 92121, USAc Malaria Vaccine Initiative, PATH 6290 Montrose Road, Rockville, MD 20852, USA

d Department of Biology, Imperial College Road, London SW7 2AZ, UKe Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA

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Received 26 March 2004; accepted 17 August 2004Available online 19 September 2004

bstract

ICC-1132, a recombinant virus-like particle comprising of a modified hepatitis B core protein with a B cell (NANP) and two T cell epitopef Plasmodium falciparumcircumsporozoite protein (CSP), was administered i.m. as a single 50�g dose in Seppic ISA 720 to 11 volunteeocal reactogenicity and systemic side effects were acceptable with the predominant finding being mild pain at the injection site. Th

nduced anti-NANP antibodies in 10/11 and modest T cell responses. There was no evidence of protection from experimental cha. falciparumsporozoites. Other formulations and/or multi-dose regimens will be required to enhance the immunogenicity and eCC-1132.

2004 Elsevier Ltd. All rights reserved.

eywords:Malaria vaccine; ICC-1132; Sporozoite challenge

. Introduction

With a global mortality of 2–3 millions per annum[1]lasmodium falciparummalaria remains a major health prob-

em. Immunization with irradiated sporozoites can elicit ster-le immunity against challenge withPlasmodiumspp. sporo-oites in mice and humans[2,3], suggesting the feasibility ofpre-erythrocytic vaccine. Much attention has been focusedn the circumsporozoite protein (CSP) which is a major com-onent of the sporozoite surface. Its central part consists of

∗ Corresponding author. Tel.: +44 1865 857419; fax: +44 1865 857471.E-mail address:[email protected] (M. Walther).

tandem repeat sequences (NANP) that may act as T cedependent B cell epitopes that have been identified aimmunodominant region for antibody responses[4]. There isalso evidence that the NANP antibody response may bedependent[5]. Apart from NANP repeats many CS peptvaccines include parasite-specific T cell epitopes, impoboth for anamnestic antibody responses and for cytokineduction, notably interferon gamma (IFN-�), to inhibit devel-opment of sporozoites within hepatocytes. In recent phclinical trials, the (NANP)3 B cell epitope has been testedcombination with one or twoP. falciparumCSP T cell epitopes that have been identified using CD4+ T cell clones obtained from human volunteers successfully immunized

264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2004.08.020

858 M. Walther et al. / Vaccine 23 (2005) 857–864

irradiatedP. falciparumsporozoites[6,7]. One of the epi-topes, termed T1, mapped to the minor repeat region of CSPof the NK54 strain and was contained in the NANPNVDP-NANP sequence[8]. The same T cell clones also recognizedoverlapping but distinct epitopes within a 20-mer peptide rep-resenting the amino acids (aa) 326–345 of the CSP[9]. Thissequence comprises parts of the highly conserved region IIas well as parts of a polymorphic domain and was denoteduniversal T cell epitope (UTC) as it could bind to a broadrange of HLA class II molecules in vitro[10]. A triepitopepolyoxime vaccine construct combining the (NANP)3 B cell-, the T1- and the UTC epitope, was able to induce immuneresponses in human volunteers of diverse HLA types[7].

Different approaches have been chosen to enhance im-munogenicity of malaria antigens used in subunit vaccines.The leading malaria vaccine candidate, RTS,S/AS02A, usesrecombinant hepatitis B surface antigen as a carrier for a trun-cated CSP[11], taking advantage of the high immunogenicityof the virus-like particle. In animal models, recombinant hep-atitis B core (Hep B core) particles have been shown to beeven more immunogenic than the surface antigen at both theB and T cell level[12,13], and are highly suitable as a car-rier for foreign epitopes[14,15]. Optimal humoral immuno-genicity was observed when B cell epitopes were engineeredinto an immunodominant loop region at the tip of the sur-f Sr i-Co rs,sm tionf orem ingt e im-m l endo l andc hosts[ -i fur-t 720[ ju-v osefid eda portt atedi e.

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adult volunteers. The vaccine and trial protocol were re-viewed by the UK Medicines and Healthcare RegulatoryAgency. The study was conducted according to GCP ICHguidelines and independently monitored by PPD Develop-ment, USA. Ethical approval was obtained by the OxfordResearch Ethics Committee and the Human Subjects Protec-tion Committee at the Program for Appropriate Technologyin Health, Seattle, USA. Twenty-six adults were screenedand 17 healthy malaria-naıve subjects, aged 19–39 (nine fe-males, eight males, median age 26 years) were subsequentlyenrolled after having given written, informed consent.

2.2. The vaccine

ICC-1132 is a virus-like particle produced inEscherichiacoli comprising a recombinant hepatitis B core protein intowhich B and T cell epitopes ofP. falciparumCSP derivedfrom the NF 54 strain have been engineered. It is formulatedwith Seppic ISA 720, that contains a natural, metabolizableoil, composed of squalene and squalane, and a highly-refinedemulsifier from the mannide monooleate family[21]. The ra-tio of adjuvant to antigen is fixed at 70% (w/w) Seppic ISA720 to 30% (w/w) aqueous solution containing ICC-1132.The product was manufactured by Apovia Inc., USA. Thevirus-like-particles have a size of roughly 30 nm in diame-t TheB epi-t NPt gion,w -r inoa n in-s and7 cids,F inale

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ace spikes[16]. Hybrid Hep B core particles carrying Cepeats ofP. bergheior P. yoeliielicited high levels of antSP antibodies and protection in mice[17,18], but insertionf P. falciparumCS repeats induced only low antibody titeuggesting a benefit from T helper cell epitopes[18]. Opti-ization in mice helped identifying the most suitable posi

or the T cell epitopes T1 and UTC within the Hep B colecule[19]. A compound denoted ICC-1132, contain

he T1 and B repeat epitopes inserted in the Hep B corunodominant loop and the UTC fused to the C-terminaf the truncated Hep B core elicited the strongest humoraell-mediated immune responses in rodent and simian20]. In Rhesus monkeys (macaca mulatta), immunogenicty of a single i.m. dose of ICC-1132 could be enhancedher when the compound was emulsified in Seppic ISADubovsky F., et al. Abstract from 5th Annual Novel Adant Meeting WHO/TDR Annecy France, June 2003]. A dnding phase I trial conducted at the University of Tubingenemonstrated a 50�g dose of ICC-1132 was well toleratnd immunogenic [Nardin et al., submitted]. Here, we re

he first study assessing the efficacy of ICC-1132 formuln Seppic ISA 720 in volunteers in a sporozoite challeng

. Material and methods

.1. Study design

An open labeled phase I/IIa study was carried out to eate safety, immunogenicity and efficacy of a single df ICC-1132 in Seppic ISA 720 administered to hea

er, containing 240 copies of the ICC-1132 monomers.cell epitope includes three NANP repeats. One T cell

ope, termed T1, consists of alternating NANP NVDP NAetramers, derived from the adjacent CS minor repeat rehile the UTC epitope, CSP326–345, is derived from a non

epeat region of the C-terminal end of the CSP. A 24-amcid peptide comprising the T1/B cell epitopes has beeerted in the immunodominant loop between position 789 of a truncated hepatitis B core protein (149 amino aig. 1). The 20-mer UTC epitope is fused to the C-termnd of the Hep B core protein.

.3. Study procedures

.3.1. VaccinationWithin 11 days, 11 volunteers (6 females, 5 males) w

iven 0.5 ml containing 50�g of the vaccine as an intramuular injection into the deltoid muscle of the non-dominrm. Subjects were followed up to 1 h after vaccine ad

stration and on days 2, 7, 14, and 28 and after 6 monthach visit vital parameters were measured, the injectionas inspected and the largest diameter of any swellingess or warmth, as well as the degree of pain reported fo

ime of the visit were documented. An assessment forible systemic adverse events (AE) as reported for thef the visit was carried out and solicited AE (local and s

emic) that may have occurred since the last visit wereocumented. The relationship of systemic adverse reacas assessed by the investigator. Subjects were asked

heir temperature and to document any symptoms on aasis in a diary card for 14 days post-vaccination.

M. Walther et al. / Vaccine 23 (2005) 857–864 859

Fig. 1. Schematic view of ICC-1132. The T1/B repeat epitopes are inserted between hepatitis B core amino acids 78 and 79, a region forming the immunodominantloop at the tip of the core particle surface spikes. The universal T cell epitope (UTC) is fused to the C terminus of the truncated hepatitis B core protein.

Routine parameters for biochemistry and haematologywere assessed on day of vaccination, at 7, 14, 28 and 168days post-vaccination and on days 7 and 90 post-challenge(days 49 and 133).

2.3.2. ChallengeUsing a method adapted from Chulay[22], all 11 vacci-

nees underwent sporozoite-challenge by five bites from in-fectiousAn. stephensimosquitoes between 5 and 6 weekspost-immunization alongside six non-vaccinated controls.The challenge was performed at Imperial College, London,using in this challenge mosquitoes reared at the Walter ReedArmy Institute of Research (Silver Spring, MD, USA) withdetailed follow up of volunteers as described elsewhere[23].

2.4. Elispots

Peripheral blood mononuclear cells (PBMC) of each vol-unteer were obtained on the day of vaccine administration, at7, 14, 28, 42 (day of challenge), 49, 133 and 168 days post-injection, to perform ex-vivo ELISPOTS for IFN-�. PBMCwere incubated with 50�l of the peptides listed inTable 1and assays performed as decribed previously[23].

2.5. P. falciparum PCR

ina ndedi ningt ) tod e re-s (Qi-

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agen, Crawley, UK), and PCR was performed using a Rotor-Gene machine (Corbett Research, Cambridge, UK), with theQuantitect SYBR Green I PCR kit (Qiagen). The multicopy18S (small subunit) ribosomal RNA genes ofP. falciparumwere amplified to give a 174 bp product as described else-where [Andrews et al., in preparation]

2.6. Antibodies

Serum from each volunteer, obtained at the time of injec-tion (day 0), at 14, 28, 42 (day of challenge), 56, 77, 133 and168 days post-injection was assayed for anti-NANP IgG andanti-hepatitis B core antibodies. The titers of IgG antibodiesspecific for the B cell epitope were determined by peptide-based ELISA using (NANP)5 (Multiple Peptide Systems,San Diego, CA) as the coating antigen. The NANP-peptide(100 ng/well) was coated onto microtiter plates overnightat 37◦C. Antibodies to hepatitis B core antigen were mea-sured using a recombinant hepatitis B core particle (rHBcAg)truncated at aa 149 and C-terminal stabilized. rHBcAg(200 ng/well) was coated onto microtiter plates overnight atroom temperature (RT). For both, detection of anti-NANPand anti HBcAg IgG, plates were blocked for at least 1 hat 37◦C with 200�l/well of PBS containing 1% heat in-activated porcine serum (blocking buffer). After removal oft luted1 test tem-p per-o outh-e atR n atR anD dingH ader( re-s ce oft timesS s aree tablel t oft

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tiona iate.T sing

Briefly, 2 ml of EDTA blood were centrifuged for 5 mt 2500 rpm and plasma removed. Cells were resuspe

n PBS and vacuum filtered using 24 well plates contaiwo layers of Whatman VFE (Whatman, Maidstone, UKeplete leucocytes. DNA was extracted from 0.5 ml of thuspended sample using the Qiagen DNA Blood Mini kit

able 1eptides used for the IFN-� ELISPOT assays

eptide Description

TL Pool of 22 known, non vaccine-related common CTepitopes from Influenza A, EBV and CMV; used aspositive control

CC-1132 Complete ICC-1132 construct without adjuvantTC Universal T cell epitope

(EYLNKIQNSLSTEWSPCSVT)1 T cell epitope (NANPNVDPNANP)ANP B cell epitope (three NANP repeats)TC 7G8 Pool of three peptides corresponding to the UTC

sequence of a heterologous strain 7G8. The peptidare: CS11G: NEEPSDKHIEQYLKK, CS12G:DKHIEQYLKKIKNSI and CS 13G:QYLKKIKNSISTEWS

SP Recombinant CSP from vaccine strain NF 54

he blocking buffer, the human serum samples were di/50–1/12,800 with blocking buffer and added in triplica

o the assay plates. Plates were kept for 1 h at ambienterature and subsequently incubated with horseradishxidase conjugated mouse anti-human IgG (1/8000, Srn Biotechnology Associates, Birmingham, AL) for 1 hT. After washing the plates were developed for 30 miT with tetramethlybenzidine (TMB, BD Pharmingen, Siego, CA). The enzymatic reaction was stopped by ad2SO4 and the plates were read at 450 nm in an ELISA re

Spectramax 340, Molecular Devices, Sunnyvale, CA). Ault was considered positive when the mean absorbanhe sample was higher than the mean absorbance + three.D. of the corresponding pre-immunization serum. Titerxpressed as geometric mean titers (GMT). For undetec

evels of antibodies a value of 50% of the detection limihe assay was substituted to calculate the GMT.

.7. Statistics

Quantitative data were assessed for normal distribund log-transformation was performed where approprhe difference between two means was compared u


Student’st-test (paired or unpaired) and for non-normal dis-tributed data the Wilcoxon rank sum test was performed us-ing Sigma Plot 2000 (Jandel Scientific Graphic Software,Erkrath, Germany). Kaplan–Meier analysis of time to para-sitaemia between groups used log rank tests, using SPSS forWindows Version 11.0 (Lead Tools). Ninety-five percent con-fidence intervals (95% CI) were calculated for proportions,arithmetic and geometric means as described by Altman et al.[24], using CIA 2.1 (Tevor Bryant, Southampton). ELISPOTresponses and antibody titers were assessed for correlationto time to parasitaemia and to each other, using Spearman’srank correlation performed with SPSS for Windows.

3. Results

Of the vaccinated group, two subjects dropped out be-tween days 133 and 168 after the vaccination. Two partic-ipants in the control group could not be seen for their lastfollow up on day 90 after the challenge. All four participantshad completed the chloroquine treatment.

3.1. Safety

The i.m. administration of 50�g of ICC-1132 in Sep-p cts.N ventsw (notv d thei lvedw

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3.4. Laboratory adverse events

For all subjects, electrolytes and routine parameters forliver and kidney function remained within the normal rangethroughout the study. One subject showed a leucocytosis onthe day of vaccine administration that was followed by symp-toms of a common cold.

For six subjects (three vaccinees, three controls), serumlevels of creatine kinase were found to be elevated. In allcases, this was most likely to be exercise induced, since otherpotential causes could be excluded.

3.5. Sporozoite challenge

On 2 subsequent days, all 11 vaccinees and 6 malaria naıvecontrols were challenged withP. falciparumsporozoites byfive infectious mosquito bites. Challenge took place 5- to6-weeks post-vaccination and all challenged individuals de-veloped malaria. After challenge, one subject in the controlgroup revealed repeated previous malaria infection and wastherefore excluded from further analysis. No significant dif-ference in time to parasitaemia was observed for vaccineesand controls. The mean time to onset of parasitaemia de-tected by slide reading was 11.2 days (95% CI: 10.7–11.7)for vaccinated subjects and 11.6 days (95% CI: 10.8–12.4)fm sitesw (95%C trols

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ablep on-t -N vol-u -I uslyp (95%Ca or-r slide( ue>

nti-b ldb hreeo 90p -I partf omt st).

ic ISA 720 was generally well tolerated in all 11 subjeo vaccine related serious AE occurred. Two adverse eere graded as “severe”: a fracture of the left scaphoidaccine related) and warmth of 60 mm in diameter arounnjection site 1 h after vaccine administration that resoithin 8 h.

.2. Local reactogenicity

Within the initial 4 weeks follow up, 10 of 11 volunteeeported the injection site to be painful on touch (gradeeast once, amongst them two experienced a partial restrf activities due to pain (grade 2) on day 0 which lasteday 1 in one case. Seven of 11 felt pain for 1–3 days, withubjects reporting a longer episode of pain (grade 1) for 61 days, respectively. In one case, the injection site beainful again on day 12 until day 18 without any other londings. Swelling at the injection side was not observedocal redness (grade 1) was reported for two subjects. Waas experienced by two volunteers on the day of vaccina

n one case with a diameter of 60 mm (grade 3).

.3. Systemic side effects

For 6 of 11 subjects, no systemic AE were observedng the initial 4 weeks follow-up period. Of the remainve subjects, two experienced systemic AE that were grpossibly related to the vaccine” (fatigue on day 0 analaise on day 0). The 6 months follow up (performedof 11) did not reveal any long term effects related to

accine.

or malaria naıve controls, respectively (Fig. 2a). Taking aeasure of PCR positivity as >1000 parasites/ml, paraere detected in vaccinees on average at 10.2 daysI: 9.5–10.8), and 10.8 days (95% CI: 10.5–11.1) in conubjects (Fig. 2b).

.6. Anti-NANP antibodies

Individual anti-NANP IgG titers for various time poinre shown inFig. 3a. A summary of the IgG-GMT for vainees and controls is given inFig. 4.

In all subjects, anti-NANP antibodies were undetectrior to vaccination (vaccinees) or prior to challenge (c

rols). Following vaccination with 50�g of ICC-1132 antiANP IgG antibodies could be detected in all vaccinatednteers at least for one time point (Fig. 3a). The anti-NANP

gG GMT for all vaccinated subjects increased continuoeaking at day 77, 35 days after the challenge at 483I: 288–804), and declined slightly afterwards (Fig. 4). Thenti-NANP IgG titer on the day of challenge did not celate with time to parasitaemia defined as a positiverho = 0.046;p= 0.893) or the time until the first PCR val1000 parasites/ml (rho =−0.12,p= 0.726).

Sporozoite challenge also induced anti-NANP-IgG aodies in previously malaria naıve controls. Antibodies coue detected in one of four (day 14 post-challenge), tf five (day 35 post-challenge), and two of three (dayost-challenge) control subjects (Fig. 4). The anti-NANP

gG titers after challenge were higher in vaccinees but—arom the day of challenge—not significantly different frhe titer determined in control subjects (Wilcoxon rank te


Fig. 2. Kaplan–Meier analysis of time to parasitaemia for vaccinees (continuous line) and controls (broken line), based on microscopic slide reading (a), andquantitative PCR (b).Y-axis: cumulative probability to remain slide negative or for a parasite count less than 1000 parasites/ml in the PCR, respectively.X-axis:days after challenge.p-values as calculated with the log rank test.

3.7. Anti-Hep B core antibodies

By day 42, anti-Hep B core IgG antibodies could bedetected in all 11 vaccinees at a GMT of 1216 (95% CI:332–4447). Hep B core antibodies persisted in all subjectsuntil day 77 (GMT 782, 95% CI: 218–2807) and were stilldetectable at a GMT of 355 (95% CI: 45–2591) in six ofeight vaccinees seen on day 168. None of the sera from non-vaccinated controls tested positive for Hep B core antibodies.

3.8. IFN-� ELISPOT

Fig. 5 summarizes the IFN-� ELISPOT responses to thevaccine epitopes and the complete vaccine construct ICC-

F oint (a d the T 1e listed o ific CD4T 7, and ide hasb the U exprst e to pa spondera lack HL and/ove

1132 without adjuvant for various time points for all vacci-nees.

The average background was 8 spot forming unit(SFU)/million cells (95% CI: 5–11). Therefore, the cutoff defined as twice the background level averaged at16 SFU/million cells (S.D.: 29.3).

Values clearly above the cut off could be observed in re-sponse to ICC-1132 and a pool of 22 known, non-vaccinerelated CTL epitopes used as a positive control (Table 1).However, the number of IFN-� secreting cells in responseto the peptides representing the individual vaccine epitopeswas modest, and hardly ever exceeded the cut off level. Whencomparing IFN-� ELISPOT responses to single peptidesfrom vaccinees and controls no significant difference was

ig. 3. Comparison of individual anti-NANP-IgG titers for each time ppitope (c) in vivo (6, 7, and 9) or in vitro (10). HLA class II moleculescell responses inP. falciparumsporozoite immunized volunteers (6,

een demonstrated in vitro (10). HLA molecules listed in italics boundhis HLA molecule. Anti NANP titers, IFN-� ELISPOT responses or timlleles expressed by a volunteer. Anti NANP titers for individuals who
ivo were not significantly different from those determined for the remaining vpitopes to specific HLA types in humans.
) and HLA class II molecules, known to bind the UTC epitope (b), ann grey background have been shown to function in T1- or UTC-spec+

9). For HLA molecules listed in bold high affinity binding to the peptTC peptide with low affinity (10). A cross indicates that the volunteeresserasitaemia did not correlate to the number of known HLA class II reA class II molecules known to bind the UTC peptide with high affinityr in
olunteers, suggesting that the vaccine overcomes genetic restriction of antigenic


Fig. 4. Anti-NANP-IgG geometric mean titers for different time points, cal-culated for all volunteers. Grey bars: vaccinees; white bars: controls; errorbars: 95% confidence intervals. For vaccinees, the denominator is 11, excepton day 168 where it is 8. For controls,n= 4 (day 56),n= 5 (day 77),n= 3(day 133).

observed, apart from a borderline significant result(p= 0.048) obtained for the response to ICC-1132 seven dayspost-challenge. No significant correlation was found betweenanti-NANP-IgG titer on the day of challenge and ELISPOTresponses to the various peptides for that day.

However, 14 days post-vaccination anti-NANP-IgG anti-body levels did correlate with the IFN-� response to ICC1132in the ELISPOT (rho = 0.682,p= 0.021) and antibody titersobtained for days 28, 42, 56, 77, 133 and 168 all showeda weak but significant correlation to the number of IFN-�secreting cells in response to ICC-1132 detected on day 28.

F d diffe per millionP ving th ent ouF see Ta

4. Discussion

The hepatitis B virus core platform has shown promiseas an efficient delivery system for foreign antigens[14,15].Using the CSP repeat epitopes ofP. bergheiandP. yoeliias antigens, it has been used to develop a pre-erythrocyticmalaria vaccine capable of inducing high titers of protectiveanti-repeat antibodies in these murine models[18]. In ICC-1132, when well-defined B and T cell epitopes ofP. falcipaumCSP were engineered into Hep B core particles anti-NANPantibodies exceeding 106 in mice with remarkable longevitywere observed[20]. Similar antibody titers remaining at ahigh level for up to a year could be induced in Rhesus mon-keys when ICC-1132 was formulated with Seppic ISA 720[Dubovsky F., et al. Abstract from 5th Annual Novel Adju-vant Meeting WHO/TDR Annecy France, June 2003]. Impor-tantly, a second dose given 8 weeks later did not significantlyincrease the antibody levels in these animals. Based on thisobservation and the finding that animals having received twoinjections of 90�g ICC-1132 in Seppic ISA 720 developeda sterile abscess at the injection site [Langermans et al., sub-mitted], a single dose regimen was chosen for this trial. Thedose of 50�g ICC-1132 in Seppic ISA 720 has previouslybeen shown to be safe and immunogenic in human volunteers[Nardin et al., submitted].

cy ofI anv ram-e thatt7 inlyc ase,l onta-n wasp ne of

ig. 5. Summarized IFN-� ELISPOT responses for various peptides anBMC with the individual background deducted in a box plot format gior more detailed information on the peptides used to stimulate cells,

rent time points. Results are expressed as spot forming units (SFU)e median, the 10th, 25th, 75th and 90th percentile. Single dots represtliers.ble 1.

This paper reports the first study assessing the efficaCC-1132 formulated in Seppic ISA 720 in healthy humolunteers in a sporozoite challenge. Review of safety paters collected in this study for up to 168 days suggests

he i.m. administration of 50�g of ICC-1132 in Seppic ISA20 is generally well tolerated. Local reactogenicity maonsisted of grade 1 pain at the injection site. In one cocal tenderness reoccurred on day 12 and resolved speously on day 18. At no point in time, a nodule or lumpalpable. Systemic side effects were negligible. Since no


the 11 vaccinated volunteers experienced a severe or seriousadverse event related to the vaccine, the level of confidencethat the true rate of severe or serious adverse events in thegeneral population would be less than 20% is 92%.

ICC-1132 aims to protect against the pre-erythrocyticstage ofP. falciparummalaria primarily by inducing highlevels of anti-sporozoite antibodies. A single dose of 50�gof ICC-1132 in Seppic ISA 720 induced anti-NANP-IgG an-tibodies in 10 of 11 volunteers prior to challenge. For thetotal cohort, the peak response of a GMT of 483 (95% CI:288–804) was seen on day 77, 35 days post-challenge (Fig. 4).However, the antibody levels induced here in humans areconsiderably lower than those described in Rhesus andM.fascicularismonkeys. Here, a GMT titer of 310 has beencalculated for day 42 post-vaccination. In contrast, inM. fas-cicularis monkeys vaccinated with a single dose of 20�gICC-1132 in Seppic ISA 720, anti-NANP repeat titers deter-mined using similar ELISA techniques had reached GMTsof 105 by this time point already and peaked at levels of 9×105 a month after a second dose was given[20].

A recent study in human volunteers assessed the immuno-genicity of a triepitope polyoxime malaria vaccine constructcomprising the same epitopes as were included into ICC1132[7]. A month after the first vaccination 9 of 10 volunteers hadanti-NANP-IgG antibodies at a GMT of 296. For this timep -b er,b

doser erec sm larian anti-N to1 ouldbt mia.C nti-bS then thept l-l im-m asw

eenT nti-b t theU romp ra-s at thei rec-oT se-q

CD8+ T cells from volunteers immunized three times with thepolyoxime construct[7] produced IFN-�, a likely inhibitorof intracellular hepatic stages of the parasite.

Here, a single dose of 50�g of ICC-1132 induced onlymodest numbers of IFN-� secreting cells in an ex vivoELISPOT in response to the peptides representing the in-dividual vaccine epitopes (Table 1). The highest values ofSFU/million cells were observed when cells were stimulatedwith the complete vaccine ICC-1132. For day 14, a significantcorrelation between IFN-� ELISPOT responses to ICC-1132and anti-NANP antibody titers could be observed. As de-scribed for the polyoxime construct[7] a multi-dose regimenmay induce much stronger cellular immune responses.

To further enhance immunogenicity and efficacy of ICC-1132 multi-dose regimens could be tested, and a higher anti-gen dose should be considered. Repeated injections of otherantigens at doses as used in this study, adjuvanted with Sep-pic ISA 720, had an acceptable side effect profile[27,28].However, vaccine formulations combining the adjuvant withother antigens at doses ranging from 200 to 1000�g showedconsiderable local reactogenicity, especially after the secondvaccination[29,30]. Work in rabbits suggests that local sideeffects can be reduced with a booster containing the sameantigen in aluminum hydroxide[31]. This may represent apotential strategy for ICC-1132 too since immune responsese bee lum[

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oint, the responder rate after 50�g of ICC-1132 is comparale [9 of 11], and the GMT for responders is slightly higheing 370 (95% CI: 188–728).

In the current study, to assess the efficacy of a singleegimen of 50�g ICC-1132 all vaccinated volunteers whallenged withP. falciparumsporozoites by five infectiouosquito bites 5–6 weeks post-vaccination alongside maaıve controls. Despite the presence of vaccine inducedANP antibodies in 10 of 11 volunteers with titers up600, no significant difference in time to parasitaemia ce observed for vaccinees and controls (p= 0.408,Fig. 2), and

he antibody titer did not correlate to time to parasitaelearly, the amount and/or quality of anti-sporozoite aodies induced by a single injection of 50�g ICC-1132 ineppic ISA 720 is not sufficient to substantially reduceumber of sporozoites that infect liver cells. However,olyoxime construct referred to earlier[7] elicited antibody

iters exceeding 103−104 in the majority of vaccinees foowing three immunizations, suggesting the potential of

unologic maturation with multiple doses for ICC-1132ell.Nardin et al.[7] also observed a strong correlation betw

cell proliferation in response to UTC and anti-NANP aody titers after the third immunization, suggesting thaTC epitope functioned as a T helper cell epitope. Apart froviding help for B cells this epitope may also elicit paite specific cell-mediated immune responses directedntracellular stages of the parasite, since CTL epitopesgnized by human CD4+ CTL [25] as well as human CD8+

cells [26] have been described within this amino aciduence. When restimulated with UTC peptide CD4+ but not

licited in mice with ICC-1132 in Seppic ISA 720 couldfficiently boosted by a second dose of ICC-1132 in a

20].

cknowledgements

We are grateful to all the subjects who volunteered toicipate in this study, to Martin Schletz and Stephan Schor help with the ELISAs and to Elizabeth Nardin for caully reading the manuscript. This work was supportedrants from the Malaria Vaccine Initiative, PATH andellcome Trust.

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safety, immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, icc-1132...

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