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A vaccine for hypertension based on virus-like particles:preclinical efficacy and phase I safePatrice M. Ambuhla,M, Alain C. Tissotb,M, AlmaJuerg efKatja fiHans uMarti

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relatively long-lasting effects and do not require daily

dosing. Here we describe the preclinical development and

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and improving patient compliance. J Hypertens 25:6372

Q 2007 Lippincott Williams & Wilkins.

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Original article 63

Part of thisAmerican H The first

0263-6352 2007 Lippincott Williams & Wilkinsfrom 1970 to 1990, subsequent US surveys haved a progressive increase in the prevalence of

nsion among adults [1]. Primary arterial hyper-is a multifactorial and probably polygenic diseaseever, the reninangiotensinaldosterone systemis probably the single most important regulator of

c blood pressure. The binding of angiotensin II to

turn cleaved to angiotensin II by angiotensin-convertingenzyme (ACE). The pharmacological treatment ofhypertension with both ACE inhibitors and AT1Rblockers has proved very successful. Nevertheless, onlyapproximately one-third of hypertensive patients in theUnited States are presumed to have adequate controlof blood pressure, i.e. blood pressure maintained below140/90 mmHg [3]. In addition to inadequate treatment,low compliance to the application of medication andadherence to prescribed treatment is a major reason for

work has been presented as an abstract at the scientific sessions of theeart Association 2005 (no. 2445, Circulation 2005; 112:II511).

three authors contributed equally to this work.ntihypertensive vaccine.

s and results An angiotensin II-derived peptide

jugated to the VLP Qb (AngQb). AngQb was highly

genic in mice and rats. To test for efficacy,

eously hypertensive rats (SHR) were immunized

mg AngQb or VLP alone. Group mean systolic blood

e (SBP) was reduced by up to 21 mmHg (159 W 280 W 5 mmHg, P < 0.001), and total angiotensin IIntibody-bound and free) were increased ninefold

versus 9 W 1 pmol/l, P U 0.002) compared with VLP. SHR treated with the angiotensin-converting

(ACE) inhibitor ramipril (1 mg/kg per day by mouth)

an SBP of 155 W 2 mmHg. Twelve healthyrs of a placebo-controlled randomized phase I trial

ected once with 100mg AngQb. Angiotensin

c antibodies were raised in all subjects (100%

er rate) and AngQb was well tolerated.

Journa

Keywosystem

aRenalZurichSwitzeDermaBerlin

CorresBiotecTel: +4e-mail:

ConflicfundingM.R., Pstock o

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uctionmately one billion adults worldwide suffer fromblood pressure. The highest prevalence is in the

States and eastern Europe, where approximatelyadults are hypertensive [1]. After two decades of

its releadsall cogenetensiht Lippincott Williams & Wilkins. Unauthorized: angiotensin, antibodies, blood pressure, hypertension, immune

ision, University Hospital, Zurich, bCytos Biotechnology AG,hlieren, cDivision of Angiology and Hypertension, CHUV, Lausanne,, dInterdisciplinary group of Molecular Immunopathology,y/Medical Immunology, eInstitute of Medical Immunology, Charite,

fCharite Research Organisation, Berlin, Germany

dence and requests for reprints to Martin F. Bachmann, Cytosogy AG, Wagistrasse 25, 8952 Schlieren, Switzerland4 7334747; fax: +41 44 7334740;[email protected]

f interest: P.M.A., J.N., R.S., H-D.V. and F.W. have received researchm Cytos Biotechnology AG. A.C.T., A.F., P.M., C.S., K.S., K.R., T.P.,G.T.J. and M.F.B. are employees of Cytos Biotechnology AG and havens or stock holdings in Cytos.

15 May 2006 Revised 17 August 200621 August 2006

torial commentary on page 41

tor, the angiotensin receptor subtype 1 (AT1R),o vasoconstriction and aldosterone secretion,ributing to its pressor effect. Angiotensin II ised in two steps, first through cleavage of angio-en by renin, yielding angiotensin I, which is ine I clinical trial testing of a virus-like particle (VLP)-l of Hypertension 2007, 25:6372Nussbergerc, Robert Sabatd, Vera NiSladkob, Kirsten Roubicekb, Thomas P-Dieter Volke, Frank Wagnerf, Philipp Mn F. Bachmannb

ound Despite the availability of efficacious drugs,

ess of treating hypertension is limited by patients

tent drug intake. Immunization against angiotensin

ffer a valuable alternative to conventional drugs for

tment of hypertension, because vaccines induce

Conc

levels

and w

angio

antihty and immunogenicityFulurijab,M, Patrik Maurerb,

a, Charlotte Schellekensb,sterb, Manfred Rettenbacherb,llerb, Gary T. Jenningsb and

ions AngQb reduces blood pressure in SHR to

btained with an ACE inhibitor, and is immunogenic

tolerated in humans. Therefore, vaccination against

sin II has the potential to become a useful

rtensive treatment providing long-lasting effects reproduction of this article is prohibited.

Copyrigh

the high percentage of patients with arterial hypertension[4]. Impis thus asive age

In ordean immbodies abiting ivaccinaattemptcompletin humrecentlypressureraised bvaccineimmunisurfaceparticleerating[1012]p1) or atrials animmunoover, thin the acompati

In thisagainstpeptideconjugarats. Thangiotenpressurethe phaimmunothe angdemonsand rapiantibodshow thpeptideapproac

MethoVaccineAngioteits N-teVLP (Fphase cUSA; oRNA bstudy. Tfrom 1monom

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dylce,gate was coated onto ELISA plates at 10mg/ml in

bonate buffer overnight at 48C. After blocking withovine serum albumin solution in phosphate-bufferede/Tween, sera were incubated for 2 h on the plate, andtion was performed with a goat anti-rat IgG horse-

h peroxidase conjugate (Jackson Immunoresearch,Grove, Pennsylvania, USA). Titres were expressed

e serum dilution giving half-maximal binding. Theficity of the signal was confirmed by assaying pre-une serum, which gave a signal minimally aboveground. Pre-immune titres were thus set as the lowestion measured in the assay. Inhibition ELISA were

64 Journal of Hypertension 2007, Vol 25 No 1

Fig. 1

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teneineTh

e oroving patient compliance by appropriate therapyprimary goal of newly developed antihyperten-

nts.

r to overcome these limitations, we have testedunological approach aimed at inducing auto-anti-gainst endogenous angiotensin II, thereby inhi-ts effect on blood pressure. Although active

tion against angiotensin peptides has beened in the past, it was either unsuccessful, or usede Freunds adjuvant, which is prohibited for useans [58]. An anti-angiotensin I vaccine hasbeen tested in humans, but did not reduce blood[9]. The anti-angiotensin I antibody response

y that vaccine was found to be reversible, and thedemonstrated good safety and tolerability. A newzation technology that conjugates antigens to theof the highly repetitive structure of virus-like

s (VLP) has been shown to be effective in gen-strong B-cell responses against self-antigens

. VLP conjugated with either a peptide (Derhapten (nicotine) have been tested in clinical

d have been shown to be well tolerated and highlygenic, with a 100% responder rate [13,14]. More-

ese antibody responses were induced in humansbsence of adjuvant or by using the mild human-ble adjuvant aluminum hydroxide.

study we have tested this vaccination strategya self-antigen and as a therapy for hypertension. A

derived from angiotensin II was covalentlyted to VLP, and used to immunize hypertensivee vaccine was shown to induce antibodies againstsin II, and consequently reduce arterial bloodin spontaneously hypertensive rats (SHR). In

se I arm of the trial, safety, tolerability andgenicity were assessed after one injection of

iotensin II vaccine. In 12 healthy volunteers wetrated that the vaccine was well tolerated, safedly induced high levels of angiotensin II-specificies, which declined over time. These resultsat immunotherapy using angiotensin II-deriveds coupled to VLP represents a promising newh for the treatment of hypertension.

dssynthesesnsin II was modified with amino acids CGG atrminus to permit directional conjugation to theig. 1). The peptide was synthesized by solid

hemistry (Eurogentec, Belgium; Sigma Genosys,r Bachem, Switzerland). VLP derived from theacteriophages Qb and AP205 were used in this

he VLP are macromolecular assemblies formed80 copies of their respective coat proteiner, which spontaneously assemble into a capsid

strucEschetargegatiododeperfogels;tratioford

The(an adataAngQvaccione scoupcounmodito thefollowto thguide

EnzyPeptthe Rnosoconjunimi(Pierconjua car2% bsalindetecradisWestas thspeciimmbackdilut

Structcompo(angioa cyst(VLP).surfact Lippincott Williams & Wilkins. Unauthorized e [15]. VLP were recombinantly expressed inhia coli, purified, and covalently coupled to thentigen, as described previously [16,17]. Conju-f the peptide to VLP was verified by lithiumsulphatepolyacrylamide gel electrophoresised under reducing conditions (12% Nu-Pagevitrogen, Switzerland). The protein concen-

of the vaccine was determined using the Brad-thod.

ptide was coupled at high density to the Qb VLPrage of two to three peptides per VLP subunit,t shown). The resulting vaccine is referred to aswhich for the sake of simplicity also refers to thebased on AP205 VLP. AP205 VLP was used forle injection in the telemetry experiment. Thevaccine had similar epitope density to its Qb

part. Vaccine for the clinical trial comprised thed angiotensin II peptide (Fig. 1) covalently linkedLP Qb, termed CYT006-AngQb. It was producedg current good manufacturing practice accordingternational Conference on Harmonization (ICH)es.

-linked immunosorbent assays-specific antibody titres were measured by coatingAse-peptide conjugate on enzyme-linked immu-nt assay (ELISA) plates. The RNAse-peptidete was prepared using the cross-linker sulfosucci-

6-(30-[2-pyridyldithio]-propionamido)hexanoateRockford, Illinois, USA). The RNAse-peptide

VLP DRVYIHPFCGG

Angiotensin IISpacer

ngQb

of the AngQb vaccine. The modified angiotensin II peptide isd of the amino acid sequence of angiotensin18 octapeptidesin II) fused at its N-terminus to a spacer sequence containing

to permit directional conjugation to the Qb virus-like particlee orientation of the N-terminally coupled peptides on thef the VLP is depicted.reproduction of this article is prohibited.

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performed to measure relative apparent affinities of anti-bodies f

EnzymeFor thea subjecdetermithe geomserum tQualification ofquantifi

InhibitioSera weangiotentensinogferred topeptidebationantibodwas furtaccordinapparen[18]. Thas in th

ImmunoImmuntitre andrats (nand 28400mgAlhydrocollecteAntiboddetermi

AngiotePlasmameasurebodiesdescribediamine450ml ppolyproclonal aaddedextracterected40 1%from 5samplescision3.0 pmoand onenot be a

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Antihypertensive angiotensin II virus-like particle vaccine Ambuhl et al. 65or antigen(s).

-linked immunosorbent assay of human seraELISA of human sera, a responder was defined ast whose titre was above a cutoff titre. This wasned individually for each patient as the sum of

etric mean titre of at least a duplicate analysis ofaken at week 0 plus three standard deviations.ation experiments showed a coefficient of vari-less than 13% for the titres and a lower limit of

cation of 1 : 30.

n enzyme-linked immunosorbent assaysre first incubated with serial dilutions of eithersin I, angiotensin II, angiotensin III or angio-en for 1 h at room temperature, and then trans-an ELISA plate coated with RNAse-conjugatedat a low concentration, to minimize the pertur-

of the equilibrium between free peptide andies in the serum. The coating concentrationher decreased in a second set of assays performedg to Friguet et al. to better estimate of thet affinity of the antibodies for angiotensin IIe detection of bound antibodies was performed

e standard ELISA described above.

genicity in ratsogenicity and the effect of adjuvant on antibody

reversibility were determined in rats. Groups of5) were injected subcutaneously on days 0, 14

with 400mg AngQb, or with either 25, 100, orAngQb formulated in aluminum hydroxide (2%gel; Brenntag Biosector, Denmark). Sera wered on days 0, 14, 28, 35, 49, 63, 91, 119 and 147.y titres against the immunizing peptide werened by ELISA.

nsin II measurementsimmunoreactive angiotensin II levels wered by radioimmunoassay using monoclonal anti-[19] after ethanol extraction, as previouslyd [20]. Briefly, proteins of 50ml cold ethylene-tetraacetic acid plasma were precipitated by

ure ethanol and the supernatants were dried inpylene tubes coated with buffer proteins; mono-ntibodies and iodinated tracer angiotensin II werefor the radioimmunological quantitation ofd plasma angiotensin II. Results were not cor-

for recoveries, which were low but constant atwhen radiolabelled angiotensin II was extracted

0ml ethylenediamine tetraacetic acid plasma. The coefficient of variation for intra-assay pre-was 0.095. The plasma detection limit wasl/l. Samples from two rats in the AngQb grouprat in the VLP group were haemolytic and couldnalysed.

moniSHRAngQsimilmeasA thitor, r(1 mgmeas(Prescelonin a rof 25weretensi

EfficamoniSHRtelemthe aBaugperitrats wto enAnimRats(n 8(n 7vacciAP20logicadmigavagTherthe e

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PrecPreclthe Ibiotecal pStudht Lippincott Williams & Wilkins. Unauthorizeded by tail-cuff blood pressure measurementsere immunized subcutaneously with 400mgor VLP in aluminum hydroxide. Animals were

y boosted on days 14 and 28. Antibody titres wered from sera collected on days 0, 7, 14, 21 and 28.

group of rats was treated with an active compara-ipril, administered daily via the drinking water

g bodyweight). Arterial blood pressure wasd on days 9, 16, 23, and 30 by the tail-cuff methode Meter LE-5000 Series; Letica, Cornella`, Bar-Spain). During measurements, animals were heldraining device. SBP was calculated as the mediandings for each animal at each timepoint. Animals

lled on day 35, and blood was sampled for angio-I determination.

experiment in spontaneously hypertensive ratsed by telemetry12 weeks of age were surgically implanted withry devices, and a pressure sensor was introduced inominal aorta (Centre de Recherches Biologiques,France). Sodium pentobarbital (45 mg/kg, intra-al route) was used as an anaesthetic. After surgery,e allowed to recover for 2 weeks and monitoredre the stabilization of blood pressure readings.were then randomly sorted into groups (day 0).

re vaccinated subcutaneously with 400mg AngQbor with 400mg VLP in aluminum hydroxide

on days 1, 15, 29 and 43. For the injection ofs on day 29, the VLP was switched from Qb toanother VLP with similar properties but sero-distinct. A third group of animals (n 7) was

tered an ACE inhibitor (enalapril) by daily oralt a dose of 10 mg/kg bodyweight from days 1 to 28.ter the dose was sequentially lowered throughouteriment.

amples were collected on days 0 (pre-immune),42, 56 and 70, and antibody titres against angio-I were measured. Blood pressure from individual

was measured every 15 min throughout thef the experiment. The median of the 48 readings

d during each of the 12-h day and night periodsculated for each 24-h period. For the sake ofdata for the day period only are presented,night-time results were equivalent. Blood pres-

ues were subtracted from their baseline value foral analysis, which was performed at the Centre dehes Biologiques.

cal toxicological safetycal toxicology studies were performed followingguidelines for the preclinical safety evaluation of

nology-derived pharmaceuticals and the preclini-macological and toxicological testing of vaccines.were performed according to the principles of reproduction of this article is prohibited.

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good laboratory practice by a contract research organiz-ation wiTranen

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66 Journal of Hypertension 2007, Vol 25 No 1th expertise in vaccine toxicology (Charles River,t, UK).

al analysestail-cuff experiment, data were analysed by two-lysis of variance (ANOVA) for repeated measure-and in case of significance a Bonferroni post-test.

telemetry experiment, blood pressure valuesnalysed using two-way ANOVA for repeatedments, and in case of significance a Dunnettst. P-values less than 0.05 were considered stat-significant. Data were analysed using the soft-

ckage Graphpad Prism (GraphPad Software, SanCalifornia, USA).

studysign

ective of the first-into-man, randomized, placebo-ed, double-blind, phase I study was to assess theolerability and pharmacodynamic effect (immu-ity) of the CYT006-AngQb vaccine. The phase Iorted here is part of an ongoing phase I/II study, ofnly the phase I part was unblinded. The studyd a single-dose regimen consisting of a subcu-injection of 100mg CYT006-AngQb or placebo

ted in aluminum hydroxide. Twelve subjectsn active drug and four on placebo. Subjectsd at the clinic 24 h after dosing, and were mon-

or safety and tolerability on weeks 1, 2, 3 and 4.armacodynamic effect (antibody responses) wasd on weeks 0 (pre-dosing), 1, 2, 3, 4, 8 and 16.ive renin concentration and angiotensin II levelsot measured in this study arm, but are beingned in the phase II arms of the study in hyper-patients. Proteinuria was monitored at screening,after injection, and on days 7, 14 and 21 (Combur

st; Roche Diagnostics GmbH, Mannheim,y). Study protocol and other relevant documents

viewed and approved (1 November 2004) by theommittee of the Landesarztekammer Branden-

ermany, before the initiation of the study, whichducted in accordance with ICH GCP GuidelinesDeclaration of Helsinki (1964) and subsequent

s. Written consent was obtained from all subjects.

ment of immune complexes

ncentrations of activated complement factors5a) and of immune complexes were quantifieds from BD Biosciences (Heidelberg, Germany)

TEOmedical (Bunde, Germany), respectively.

sand immunogenicity of anti-angiotensin IIsffective, a vaccine against hypertension interfer-

the RAAS pathway should ultimately prevent

curreangioresidwithinduTo aconjuantibsin IIgen,miceWe cwhethydraloneincrealumsibilititresdecli131indusiblenum

In aspeciwithwe fpredsin IIwasangioIII, athe sinhibagainfollowan oaverareduconstminacentrantibangio(averby e1.0 nFriguof lig

Antibtratioent aleastt Lippincott Williams & Wilkins. Unauthorized models predict that residues spanning the wholesin II molecule are involved in receptor contact,2, 4 and 8 of angiotensin II are known to interactAT1R [21], we designed a vaccine (AngQb) to

antibodies specifically recognizing angiotensin II.ieve this a modified angiotensin II peptide wasted via its N-terminus to the VLP. In this wayies specific for the carboxy terminus of angioten-hich differs from angiotensin I and angiotensino-uld be generated. The vaccine was first tested ind was found to be highly immunogenic (Fig. 2a).firmed the immunogenicity in rats and also testedr the inclusion of a mild adjuvant, aluminumde, further increased antibody titres. AngQbduced a strong antibody response. Titres wered twofold by the inclusion of the adjuvantm hydroxide (Fig. 2b). We also tested the rever-

of the antibody response by measuring antibody-m rats over a period of 147 days (Fig. 2c). Titres

exponentially with an average half-life ofdays, demonstrating that the immune response

against angiotensin II, a self-antigen, was rever-ter three immunizations with or without alumi-droxide (Table 1).

rther set of experiments, we characterized theity of the antibody response. Using ELISA assayset of angiotensin II peptides coupled to RNAse,d that the AngQb vaccine induced antibodies

inantly recognizing the C-terminus of angioten-ata not shown). The specificity of the antibodiesther assessed by testing their ability to bindsin I, angiotensin II, angiotensin28 (angiotensinctive cleavage product of angiotensin II bindinge receptors) and angiotensinogen in solution (i.e.n ELISA). As seen in Fig. 3, antibodies raisedAngQb bound most strongly to angiotensin II,

by angiotensin III. Binding to angiotensin I wasr of magnitude lower. In order to estimate anaffinity for angiotensin II, coating densities wereaccording to Friguet et al. [18]. A dissociation

t (Kd) of 11.5 2.5 nmol/l (average of two deter-ns) was obtained using the optimal coating con-on. We further measured the concentration ofies by equilibrium dialysis using radiolabelledsin II, and found a value of 300 86 nmol/lof two determinations). The affinity measured

ilibrium dialysis (dissociation constant of 2.6l/l) was higher than obtained when using theassay, but was measured over a smaller range

d concentrations (0.216 nmol/l).

ies did not bind angiotensinogen at the concen-used in the assay, demonstrating that the appar-

nity of the antibodies for angiotensinogen is atorder of magnitude lower than for angiotensin II.reproduction of this article is prohibited.

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In additensin150010gen tha

Antihypertensive angiotensin II virus-like particle vaccine Ambuhl et al. 67

Table 1 Half-life of angiotensin II-specific IgG antibody titres in ratsimmunized against AngQb

25mg Al. 100mg Al. 400mg Al. 400mg

(days) 19 16 17 13I (days) 1333 1420 1519 1114

ensin II-specific IgG titre half-lives from rats immunized with 25, 100 or

Fig. 2

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tion, inhibition ELISA performed using angio-

14 as a surrogate for angiotensinogen showed a00-fold lower affinity of the sera for angiotensino-n for angiotensin II (data not shown).

Vaccination reduces blood pressure in a rat model ofhypertensionThe effect of immunization on blood pressure was testedin the SHR model using two different methods ofmeasurement. In the first experiment, systolic bloodpressure (SBP) was monitored by the tail-cuff methodin all rats on days 9, 16, 23 and 30 of the regimen. SBP inthe AngQb-vaccinated animals was significantly lowercompared with the VLP control group on days 23 and30 (Fig. 4a). By day 30, a difference of 21 mmHg(P< 0.001) was observed in comparison with the VLPcontrol. As expected, treatment with ramipril alsodecreased SBP (25 mmHg, P< 0.001). We observedan inverse correlation between antibody titre and bloodpressure (r0.54, P 0.006 for ln titre versus SBP,correlation within animals [22]).

In order to monitor the effect of immunization on bloodpressureprecise

400mg of AngQb in aluminum hydroxide (Al.) or 400mg AngQb. The sera of fiveanimals of each group at each timepoint were pooled for the measurement. Half-lives were fitted to a single exponential decay. CI, confidence interval.

00

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400 g 400 g Alum

0 25 50 75 100 1250

00

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Days post d35

enicity in mice and rats immunized with AngQb andty of antibody response. (a) Balb/c mice (n5) wered on days 0 and 14 with 100mg AngQb. Anti-angiotensin IItitres were measured in the sera of days 14 and 21 with anked immunosorbent assay (ELISA) against the modifiedin II peptide coupled to RNAse. The titre is expressed as thef serum giving half-maximal binding in the assay (opticalD 50%). The pre-immune titre was 1 : 100. Error bars show

ard error of the mean. (b) Rats (n5 per group) wered with 400mg AngQb with or without aluminum hydroxide on4 and 28. The pre-immune titre was 1 : 100. Bars show themean titres of the groups against angiotensin II and error bars

he standard error of the mean. (c) Titre was measured byainst angiotensin II in the pooled sera of rats immunized withngQb in aluminum hydroxide collected on days 35, 49, 63, 91,147.

Fig. 3

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Inhibitor concentration [mol/l] reproduction of this article is prohibited.

y and crossreactivity of antibodies raised against AngQb.of immunized spontaneously hypertensive rats (day 42 of experiment) were pooled and assessed for binding toin II (&), angiotensin III (~), angiotensin I (!) andinogen (^) by inhibition enzyme-linked immunosorbent assay.urves were normalized for comparison between experiments.ensin II inhibition curve as reference was included in everyt. Assays were performed in duplicate, and error bars show

ard deviation.

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Fig. 4

140

150

160

170

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Angiotensin II level measurementsA decrease in blood pressure in SHR vaccinated againstangiotensin II was expected to enhance the secretion ofrenin in the kidney. This is caused by reduced AT1Rstimulation relieving feedback suppression of reninsecretion, the baroreceptor reflex and the tubuloglomer-ular feedback mechanism. Consequently, an increase inthe total angiotensin II concentration was expected,angiotensin II being either free or antibody-bound. Wemeasurecuff exthe totaAngQbt-test). Aand ramnot be dbecauseangioten

activity, which is assessed by a competition radio-immunoassay with radiolabelled angiotensin I and ananti-angiotensin I antibody.

Treatment of spontaneously hypertensive rats withvaccine for 177 daysAlthough the small size of angiotensin II virtually pre-cludes the binding of two antibodies at the same time,there exists a theoretical possibility that high levels ofantibodies and angiotensin II in the kidney could giverise to renal inflammation, for example in the event ofimmune complex deposition. To examine this possib-ility, SHR were immunized, and their kidneys and otherorgans examined by histopathology for signs of inflam-mation. A high antibody response was induced and main-tained until they were killed on day 177. There were nofindings in the kidneys of animals vaccinated withAngQb, whereas nephritis was reported in one animalimmunized with VLP (Table 3). Minor findings in theheart, lung or liver were also observed in the ramiprilcontrol group, and are consistent with the type of back-ground lesions previously reported for SHR (Table 3)[23,24].

Toxicolocolratityioninuwarescte

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Antihypertensive angiotensin II virus-like particle vaccine Ambuhl et al. 69

3d fo

Table 2 Changes in arterial blood pressure in the AngQb andenalapril groups compared with virus-like particle controls duringthe 12-h day and night period

SBP DBP MAP

AngQb Day 15M 5 10yNight 13M 6 9y

Enalapril Day 31M 22M 26MNight 25M 15M 20M

DBP, Diastolic blood pressure; MAP, mean arterial pressure; SBP, systolic bloodpressure. Mean SBP, MAP and DBP in mmHg were calculated for the periodbetween days 1 and 56 and days 5779 and adjusted for the baseline level.Values shown are differences in arterial blood pressure between the AngQb andvirus-like particle (VLP) control group for days 5779, or the enalapril and VLPgroups for days 156 (high-dose enalapril treatment). MP

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Phase I clinical trialAll subjects were male, had a median age of 33.5 years(range 2252), median body weight of 77.4 kg (range62.397.5), and a median height of 181 cm (range 172192). Vaccination with CYT006-AngQb was well toler-ated. Fourteen out of 16 subjects showed local adverseevents such as erythema, oedema, pain and induration atthe injection site, all of mild intensity. A mild headachereported by one subject (on active treatment) was alsoconsidered to be possibly related to treatment. All othersystemic adverse events (one report of blocked nose andone sore throat) were not considered to be drug related, aswell as the symptoms of one subject (on active treatment)who experienced spinal pain caused by a herniated disc,and who underwent surgery. As expected, no significantchanges occurred in blood pressure in these healthynormotensive volunteers. Heart rate and 12-lead electro-cardiogram data were unchanged, and laboratory para-meters showed no clinically significant deviations fromthe normal range.

All volunteers receiving CYT006-AngQb responded withhigh IgG titres against angiotensin II within 2 weeks ofimmunization (n 12). Titres peaked on week 3 anddeclined with an average half-life of 19 days (n 10,95% confidence intervals 1225, Fig. 7). Volunteersreceiving placebo showed no detectable antibodyrespons

The indtensin Idepositi

immune complexes containing C1, C3, IgM, IgA or IgGat baselchangesilarly, nserum fimmuniin humimmune

DiscusVLP aragainstshowedderivedantibodimmobistudy w100% intitres afour precVLP-coB-cellresponsand its

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Angiotenssingle immof the 16immunosothe subjeactive arm(n4, opwhich wae against angiotensin II (n 4).

uction of antibodies against endogenous angio-I could theoretically lead to immune complexon. We therefore measured the concentration of

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in II-specific IgG titres in healthy human volunteers after aunization with CYT006-AngQb. Anti-angiotensin II IgG titreshealthy volunteers were determined by enzyme-linkedrbent assay (ELISA). Bars denote geometric mean titres of

cts with 95% confidence intervals. Titres for subjects in theare shown as filled bars. All subjects receiving placebo

en bars) showed titres below the lower limit of quantification,s set to 1 : 30.t Lippincott Williams & Wilkins. Unauthorized ine and 7 and 14 days after immunization. Noin immune complex levels were observed. Sim-

o changes in the levels of C3a and C5a in therom baseline were detected 7 and 14 days afterzation. CYT006-AngQb was highly immunogenicans and induced no signs of inflammation or

complex formation.

sione a very potent means of inducing antibodiesself-antigens [10,25]. In preclinical studies wethat our vaccine comprising an angiotensin II-peptide coupled to Qb VLP induced a strong

y response against angiotensin II, both whenlized on a plate or in solution. A phase I clinicalith our VLP vaccine showed a response rate of

healthy human volunteers with high antibodyter only one immunization, a comparable result tolinical experiments in rats. This is the first time anjugate vaccine has been demonstrated to breakunresponsiveness in humans. Moreover, thee to the vaccine was shown to be reversible,use was safe and well tolerated.

, angiotensin-specific antibodies effectivelyblood pressure in two independent experiments

fferent methods for monitoring blood pressure. Ineriment in which blood pressure was measured bycuff method, the reduction was comparable to theeen with the ACE inhibitor, ramipril. In theexperiment involving telemetry, the blood pres-ucing effect was clear and statistically significant,

s demonstrated to be long lasting. The blood-reducing effect of AngQb was also observed

ond model of hypertension, the Dahl salt-sensi-(data not shown).

h affinity and concentration of antibodies raisedb (approximately 311 and 300 nmol/l, respect-

d to a significant blockade of the RAAS, whichin a decrease in blood pressure and a consequentin total angiotensin II (antibody-bound and

his was presumably caused by a rise in renin.ount of antibodies was, however, high enough tothe increase in angiotensin II, and thus reduceressure. By the law of mass action, a concentrationody 26115-fold (range of Kd values determined)han its dissociation constant is predicted to bind

of angiotensin II, fully consistent with thed efficacy. The level of antibodies raised is alsoough to bind 8597% of angiotensin II in tissues,

steady-state antibody levels in the interstitiale 3.5-fold lower than in plasma [26]. Previouss to immunize against angiotensin I have beenssful at convincingly lowering blood pressure ind humans [6,9]. This may have been the result ofreproduction of this article is prohibited.

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lower levels of antibodies raised in those studies, but asantiboddirect clocationrender ito intertensin ImuscleII antibexperimtensin I[27,28],tensin Iphysioloof antibconcentbetweenangioten

Consistvaccinebegan tspecificconsistethe devtitres. Inin bloodtelemereductioreductioof a mean analanimalsexperimblood p(predomdue to afindingssequestthe vacc

Two safangiotenimmuneresponsexposedneitherof immadditionkidneytolerateNo chadetectetensin Isible, wSimilarlteers, w

response, demonstrating that angiotensin II-specific anti-responses are indeed reversible and decline over

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Antihypertensive angiotensin II virus-like particle vaccine Ambuhl et al. 71ies were reported as titres and not concentrations,omparisons cannot be made. Alternatively, the

of ACE on the endothelial cell surface mayt more difficult for anti-angiotensin I antibodiescept angiotensin I before its cleavage to angio-I. In contrast, the AT1R is located on the smoothcell surface, making it easier for anti-angiotensinodies to sequester angiotensin II. Immunizationents looking at the pressure response upon angio-infusion showed a blood pressure-lowering effectbut were performed with concentrations of angio-one-to-two orders of magnitude higher than itsgical concentration. The kinetics of the bindingodies would be much reduced at physiologicalrations, and might explain the discrepancy

blood pressure measurements in SHR andsin I infusion data in normotensive rats.

ent with the proposed mechanism of action of a, the blood pressure of test and control groupso diverge with the development of angiotensin-antibody titres. A statistically significant and

nt reduction in blood pressure occurred withelopment of high angiotensin-specific antibodythe tail-cuff experiment, a sustained reductionpressure occurred from day 23 onwards. In the

try experiment, the first statistically significantn occurred on day 46, whereas a continuousn was achieved from day 57 onwards. Supportive

chanism of action relying on induced antibodies,ysis of titres and blood pressure for individualshowed an inverse correlation in the tail-cuffent. Furthermore, the antibody-mediated drop inressure led to increased total angiotensin IIinantly antibody bound) which was presumablyn increase in renin secretion. Collectively, thesesupport a mechanism of action based on the

ration of angiotensin II by antibodies induced byine.

ety prerequisites for a therapeutic vaccine againstsin II are the absence of inflammation-inducingcomplexes and the reversibility of the antibody

e. In a toxicological study, normotensive ratsto high antibody titres against angiotensin II

showed signs of systemic toxicity nor evidenceune complex deposition or inflammation. In, long-term treatment of SHR did not result ininflammation. Vaccination of humans was welld with no systemic vaccine-related side-reactions.nge in the level of immune complexes wasd upon the induction of antibodies against angio-I. In addition, the antibody response was rever-ith a half-life of approximately 23 weeks in rats.y, antibody titres dropped in all human volun-ith an average half-life of 19 days after the peak

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10 JeAfoht Lippincott Williams & Wilkins. Unauthorizedntage of vaccination over the daily administrationmolecular drugs is that antibodies circulate in the

or weeks to months, resulting in a continuouse of angiotensin II. Current medications, takenhibit peak-to-trough variations in activity that arel for the treatment of a chronic condition in whichilibrium of healthy processes is slightly but con-distorted; as is the case for hypertension.

lusion, we have demonstrated a sustained low-blood pressure in rats after therapeutic vaccina-

h a VLP-based vaccine targeting angiotensin II.wed that efficacy can be modelled based on theand specificity of the antibodies, and that bloodreduction presumably led to elevated renin. The

tration of only one dose of vaccine in aluminumde (the most used commercial human adjuvant)an volunteers resulted in a strong antibodye with a 100% response rate, similar to whaterved in the rat model. The fact that the sameformulation is used in both preclinical and

studies increases the likelihood that a therapeuti-levant reduction in blood pressure may also bed in humans. Towards this end the vaccine isy being evaluated in a phase II efficacy study innsive patients.

wledgementthors are grateful to Dr Hans Stocker for hisof statistical analysis procedures, and Dr Dacea for help with immunizations.

ncesey PM, Whelton M, Reynolds K, Whelton PK, He J. Worldwidelence of hypertension: a systematic review. J Hypertens 2004;19.EAD. The genetics of human hypertension. Semin Nephrol 2002;

34.WJ. The economic impact of hypertension. J Clin Hypertensnwich) 2003; 5:313.r M. Long-term compliance with antihypertensive therapy: another

of chronotherapeutics in hypertension. Blood Press Monit 2000; 5l 1):S31S34.

l JB, Guettier C, Reade R, Sayah S, Corvol P, Menard J. Immunologicaches to blockade of the reninangiotensin system: a review. AmJ 1989; 117:756767.l JB. Reninangiotensin vaccine: old story, new project efficacy

s safety. Clin Sci (Lond) 2004; 107:145147.ton CI, Hutchinson JS, Mendelsohn FA. Biological significance ofangiotensin immunization. Circ Res 1970; 27 (Suppl 2):215.tlieb AR, Biber TU, Hickler RB. Studies on the role of angiotensin inimental renovascular hypertension: an immunologic approach. J Clint 1969; 48:15061518.n MJ, Coltart J, Gunewardena K, Ritter JM, Auton TR, Glover JF.mized double-blind placebo-controlled study of an angiotensin

notherapeutic vaccine (PMD3117) in hypertensive subjects. Clin Sci) 2004; 107:167173.

lehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kundig T, et al.ecular assembly system that renders antigens of choice highly repetitiveuction of protective B cell responses. Vaccine 2002; 20:31043112. reproduction of this article is prohibited.

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11 Lechner F, Jegerlehner A, Tissot AC, Maurer P, Sebbel P, Renner WA, et al.Virus-like particles as a modular system for novel vaccines. Intervirology2002; 45:212217.

12 Spohn G, Bachmann MF. Therapeutic vaccination to block receptorligand interactions. Expert Opin Biol Ther 2003; 3:469476.

13 Maurer P, Jennings GT, Willers J, Rohner F, Lindman Y, Roubicek K, et al.A therapeutic vaccine for nicotine dependence: preclinical efficacy, andphase I safety and immunogenicity. Eur J Immunol 2005; 35:20312040.

14 Kundig TM, Senti G, Schnetzler G, Wolf C, Prinz Vavricka BM, Fulurija A,et al. Der p 1 peptide on virus-like particles is safe and highly immunogenicin healthy adults. J Allergy Clin Immunol 2006; 117:14701476.

15 Golmohammadi R, Fridborg K, Bundule M, Valegard K, Liljas L. The crystalstructure of bacteriophage Q beta at 35 A resolution. Structure 1996;4:543554.

16 Freer G, Giannecchini S, Tissot A, Bachmann MF, Rovero P, Serres PF,et al. Dissection of seroreactivity against the tryptophan-rich motif of thefeline immunodeficiency virus transmembrane glycoprotein. Virology 2004;322:360369.

17 Jegerlehner A, Storni T, Lipowsky G, Schmid M, Pumpens P, BachmannMF. Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation. Eur J Immunol 2002; 32:33053314.

18 Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME.Measurements of the true affinity constant in solution of antigenantibodycomp1985

19 Nussbwith h6:S42

20 NussbA simEndo

21 Miuraswitch1999

22 Blandobser

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24 Komadynamof ang213.

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72 Journal of Hypertension 2007, Vol 25 No 1lexes by enzyme-linked immunosorbent assay. J Immunol Methods; 77:305319.erger J, Keller I, Waeber B, Brunner HR. Angiotensin II measurementigh-affinity monoclonal antibodies. J Hypertens Suppl 1988;4S425.erger J, Beckerhoff R, Vetter W, Armbruster H, Siegenthaler W.

ple and sensitive radioimmunoassay for plasma angiotensin II. Actacrinol Suppl (Copenh) 1973; 173:157.S, Feng YH, Husain A, Karnik SS. Role of aromaticity of agonistes of angiotensin II in the activation of the AT1 receptor. J Biol Chem

; 274:71037110.JM, Altman DG. Calculating correlation coefficients with repeated

vations: Part 1-Correlation within subjects. BMJ 1995; 310:446.H, Behrend M, Park JK, Schaberg T, Luft FC, Distler A. Monocyte

tion and c-fms expression in hearts of spontaneously hypertensiveypertension 1995; 25:132138.

tsu K, Frohlich ED, Ono H, Ono Y, Numabe A, Willis GW. Glomerularics and morphology of aged spontaneously hypertensive rats Effectsiotensin-converting enzyme inhibition. Hypertension 1995; 25:207

kerian B, Lenz P, Lowy DR, Schiller JT. Determinants of autoantibodytion by conjugated papillomavirus virus-like particles. J Immunol; 169:61206126.H, Gyenge CC, Tenstad O. The interstitial distribution ofmolecules in rat tumours is influenced by the negatively chargedcomponents. J Physiol 2005; 567:557567.

ham MR, Auton TR, Rosul A, Sharp HL, Sjostrom L, Rushton A, et al.ation of two carrier protein-angiotensin I conjugate vaccines to assessuture potential to control high blood pressure (hypertension) in man.lin Pharmacol 2003; 56:505512.

ner SM, Auton TR, Downham MR, Sharp HL, Kemp PA, March JE,Active immunization with angiotensin I peptide analogue vaccinesively reduces the pressor effects of exogenous angiotensin I inious rats. Br J Pharmacol 2000; 129:11781182.t Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety andimmunogenicityIntroductionMethodsVaccine synthesesEnzyme-linked immunosorbent assaysEnzyme-linked immunosorbent assay of human seraInhibition enzyme-linked immunosorbent assaysImmunogenicity in ratsAngiotensin II measurementsEfficacy experiment in spontaneously hypertensive rats monitored by tail-cuff blood pressure measurementsEfficacy experiment in spontaneously hypertensive rats monitored by telemetryPreclinical toxicological safetyStatistical analysesHuman studyStudy designMeasurement of immune complexes

ResultsDesign and immunogenicity of anti-angiotensin II vaccinesVaccination reduces blood pressure in a rat model of hypertensionAngiotensin II level measurementsTreatment of spontaneously hypertensive rats with vaccine for 177 daysToxicologyPhase I clinical trial

DiscussionAcknowledgement

References

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