Original article 63

A vaccine for hypertension based on virus-like particles:

preclinical efficacy and phase I safety and immunogenicity
Patrice M. Ambuhla,M, Alain C. Tissotb,M, Alma Fulurijab,M, Patrik Maurerb,
Juerg Nussbergerc, Robert Sabatd, Vera Niefa, Charlotte Schellekensb,
Katja Sladkob, Kirsten Roubicekb, Thomas Pfisterb, Manfred Rettenbacherb,
Hans-Dieter Volke, Frank Wagnerf, Philipp Mullerb, Gary T. Jenningsb and
Martin F. Bachmannb
Background Despite the availability of efficacious drugs,
the success of treating hypertension is limited by patients
inconsistent drug intake. Immunization against angiotensin
II may offer a valuable alternative to conventional drugs for
the treatment of hypertension, because vaccines induce
relatively long-lasting effects and do not require daily
dosing. Here we describe the preclinical development and
the phase I clinical trial testing of a virus-like particle (VLP)based antihypertensive vaccine.
Methods and results An angiotensin II-derived peptide
was conjugated to the VLP Qb (AngQb). AngQb was highly
immunogenic in mice and rats. To test for efficacy,
spontaneously hypertensive rats (SHR) were immunized
with 400 mg AngQb or VLP alone. Group mean systolic blood
pressure (SBP) was reduced by up to 21 mmHg (159 W 2
versus 180 W 5 mmHg, P < 0.001), and total angiotensin II
levels (antibody-bound and free) were increased ninefold
(85 W 20 versus 9 W 1 pmol/l, P U 0.002) compared with VLP
controls. SHR treated with the angiotensin-converting
enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth)
reached an SBP of 155 W 2 mmHg. Twelve healthy
volunteers of a placebo-controlled randomized phase I trial
were injected once with 100 mg AngQb. Angiotensin
II-specific antibodies were raised in all subjects (100%
responder rate) and AngQb was well tolerated.

Introduction
Approximately one billion adults worldwide suffer from
elevated blood pressure. The highest prevalence is in the
United States and eastern Europe, where approximately
30% of adults are hypertensive [1]. After two decades of
decline, from 1970 to 1990, subsequent US surveys have
identified a progressive increase in the prevalence of
hypertension among adults [1]. Primary arterial hypertension is a multifactorial and probably polygenic disease
[2]. However, the reninangiotensinaldosterone system
(RAAS) is probably the single most important regulator of
systemic blood pressure. The binding of angiotensin II to
Part of this work has been presented as an abstract at the scientific sessions of the
American Heart Association 2005 (no. 2445, Circulation 2005; 112:II511).

The first three authors contributed equally to this work.

Conclusions AngQb reduces blood pressure in SHR to

levels obtained with an ACE inhibitor, and is immunogenic
and well tolerated in humans. Therefore, vaccination against
angiotensin II has the potential to become a useful
antihypertensive treatment providing long-lasting effects
and improving patient compliance. J Hypertens 25:6372
Q 2007 Lippincott Williams & Wilkins.
Journal of Hypertension 2007, 25:6372
Keywords: angiotensin, antibodies, blood pressure, hypertension, immune
system
a
Renal Division, University Hospital, Zurich, bCytos Biotechnology AG,
ZurichSchlieren, cDivision of Angiology and Hypertension, CHUV, Lausanne,
Switzerland, dInterdisciplinary group of Molecular Immunopathology,
Dermatology/Medical Immunology, eInstitute of Medical Immunology, Charite,
Berlin and fCharite Research Organisation, Berlin, Germany

Correspondence and requests for reprints to Martin F. Bachmann, Cytos

Biotechnology AG, Wagistrasse 25, 8952 Schlieren, Switzerland
Tel: +41 44 7334747; fax: +41 44 7334740;
e-mail: martin.bachmann@cytos.com
Conflicts of interest: P.M.A., J.N., R.S., H-D.V. and F.W. have received research
funding from Cytos Biotechnology AG. A.C.T., A.F., P.M., C.S., K.S., K.R., T.P.,
M.R., P.M., G.T.J. and M.F.B. are employees of Cytos Biotechnology AG and have
stock options or stock holdings in Cytos.
Received 15 May 2006 Revised 17 August 2006
Accepted 21 August 2006

See editorial commentary on page 41

its receptor, the angiotensin receptor subtype 1 (AT1R),

leads to vasoconstriction and aldosterone secretion,
all contributing to its pressor effect. Angiotensin II is
generated in two steps, first through cleavage of angiotensinogen by renin, yielding angiotensin I, which is in
turn cleaved to angiotensin II by angiotensin-converting
enzyme (ACE). The pharmacological treatment of
hypertension with both ACE inhibitors and AT1R
blockers has proved very successful. Nevertheless, only
approximately one-third of hypertensive patients in the
United States are presumed to have adequate control
of blood pressure, i.e. blood pressure maintained below
140/90 mmHg [3]. In addition to inadequate treatment,
low compliance to the application of medication and
adherence to prescribed treatment is a major reason for

0263-6352 2007 Lippincott Williams & Wilkins

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64 Journal of Hypertension 2007, Vol 25 No 1

the high percentage of patients with arterial hypertension

[4]. Improving patient compliance by appropriate therapy
is thus a primary goal of newly developed antihypertensive agents.
In order to overcome these limitations, we have tested
an immunological approach aimed at inducing auto-antibodies against endogenous angiotensin II, thereby inhibiting its effect on blood pressure. Although active
vaccination against angiotensin peptides has been
attempted in the past, it was either unsuccessful, or used
complete Freunds adjuvant, which is prohibited for use
in humans [58]. An anti-angiotensin I vaccine has
recently been tested in humans, but did not reduce blood
pressure [9]. The anti-angiotensin I antibody response
raised by that vaccine was found to be reversible, and the
vaccine demonstrated good safety and tolerability. A new
immunization technology that conjugates antigens to the
surface of the highly repetitive structure of virus-like
particles (VLP) has been shown to be effective in generating strong B-cell responses against self-antigens
[1012]. VLP conjugated with either a peptide (Der
p1) or a hapten (nicotine) have been tested in clinical
trials and have been shown to be well tolerated and highly
immunogenic, with a 100% responder rate [13,14]. Moreover, these antibody responses were induced in humans
in the absence of adjuvant or by using the mild humancompatible adjuvant aluminum hydroxide.
In this study we have tested this vaccination strategy
against a self-antigen and as a therapy for hypertension. A
peptide derived from angiotensin II was covalently
conjugated to VLP, and used to immunize hypertensive
rats. The vaccine was shown to induce antibodies against
angiotensin II, and consequently reduce arterial blood
pressure in spontaneously hypertensive rats (SHR). In
the phase I arm of the trial, safety, tolerability and
immunogenicity were assessed after one injection of
the angiotensin II vaccine. In 12 healthy volunteers we
demonstrated that the vaccine was well tolerated, safe
and rapidly induced high levels of angiotensin II-specific
antibodies, which declined over time. These results
show that immunotherapy using angiotensin II-derived
peptides coupled to VLP represents a promising new
approach for the treatment of hypertension.

Methods
Vaccine syntheses

Angiotensin II was modified with amino acids CGG at

its N-terminus to permit directional conjugation to the
VLP (Fig. 1). The peptide was synthesized by solid
phase chemistry (Eurogentec, Belgium; Sigma Genosys,
USA; or Bachem, Switzerland). VLP derived from the
RNA bacteriophages Qb and AP205 were used in this
study. The VLP are macromolecular assemblies formed
from 180 copies of their respective coat protein
monomer, which spontaneously assemble into a capsid

Fig. 1

AngQb

VLP

CGG

DRVYIHPF

Spacer

Angiotensin II

Structure of the AngQb vaccine. The modified angiotensin II peptide is

composed of the amino acid sequence of angiotensin1 8 octapeptide
(angiotensin II) fused at its N-terminus to a spacer sequence containing
a cysteine to permit directional conjugation to the Qb virus-like particle
(VLP). The orientation of the N-terminally coupled peptides on the
surface of the VLP is depicted.

structure [15]. VLP were recombinantly expressed in

Escherichia coli, purified, and covalently coupled to the
target antigen, as described previously [16,17]. Conjugation of the peptide to VLP was verified by lithium
dodecylsulphatepolyacrylamide gel electrophoresis
performed under reducing conditions (12% Nu-Page
gels; Invitrogen, Switzerland). The protein concentration of the vaccine was determined using the Bradford method.
The peptide was coupled at high density to the Qb VLP
(an average of two to three peptides per VLP subunit,
data not shown). The resulting vaccine is referred to as
AngQb, which for the sake of simplicity also refers to the
vaccine based on AP205 VLP. AP205 VLP was used for
one single injection in the telemetry experiment. The
coupled vaccine had similar epitope density to its Qb
counterpart. Vaccine for the clinical trial comprised the
modified angiotensin II peptide (Fig. 1) covalently linked
to the VLP Qb, termed CYT006-AngQb. It was produced
following current good manufacturing practice according
to the International Conference on Harmonization (ICH)
guidelines.
Enzyme-linked immunosorbent assays

Peptide-specific antibody titres were measured by coating

the RNAse-peptide conjugate on enzyme-linked immunosorbent assay (ELISA) plates. The RNAse-peptide
conjugate was prepared using the cross-linker sulfosuccinimidyl 6-(30 -[2-pyridyldithio]-propionamido)hexanoate
(Pierce, Rockford, Illinois, USA). The RNAse-peptide
conjugate was coated onto ELISA plates at 10 mg/ml in
a carbonate buffer overnight at 48C. After blocking with
2% bovine serum albumin solution in phosphate-buffered
saline/Tween, sera were incubated for 2 h on the plate, and
detection was performed with a goat anti-rat IgG horseradish peroxidase conjugate (Jackson Immunoresearch,
West Grove, Pennsylvania, USA). Titres were expressed
as the serum dilution giving half-maximal binding. The
specificity of the signal was confirmed by assaying preimmune serum, which gave a signal minimally above
background. Pre-immune titres were thus set as the lowest
dilution measured in the assay. Inhibition ELISA were

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Antihypertensive angiotensin II virus-like particle vaccine Ambu hl et al. 65

performed to measure relative apparent affinities of antibodies for antigen(s).

Enzyme-linked immunosorbent assay of human sera

For the ELISA of human sera, a responder was defined as

a subject whose titre was above a cutoff titre. This was
determined individually for each patient as the sum of
the geometric mean titre of at least a duplicate analysis of
serum taken at week 0 plus three standard deviations.
Qualification experiments showed a coefficient of variation of less than 13% for the titres and a lower limit of
quantification of 1 : 30.
Inhibition enzyme-linked immunosorbent assays

Sera were first incubated with serial dilutions of either

angiotensin I, angiotensin II, angiotensin III or angiotensinogen for 1 h at room temperature, and then transferred to an ELISA plate coated with RNAse-conjugated
peptide at a low concentration, to minimize the perturbation of the equilibrium between free peptide and
antibodies in the serum. The coating concentration
was further decreased in a second set of assays performed
according to Friguet et al. to better estimate of the
apparent affinity of the antibodies for angiotensin II
[18]. The detection of bound antibodies was performed
as in the standard ELISA described above.
Immunogenicity in rats

Immunogenicity and the effect of adjuvant on antibody

titre and reversibility were determined in rats. Groups of
rats (n 5) were injected subcutaneously on days 0, 14
and 28 with 400 mg AngQb, or with either 25, 100, or
400 mg AngQb formulated in aluminum hydroxide (2%
Alhydrogel; Brenntag Biosector, Denmark). Sera were
collected on days 0, 14, 28, 35, 49, 63, 91, 119 and 147.
Antibody titres against the immunizing peptide were
determined by ELISA.
Angiotensin II measurements

Plasma immunoreactive angiotensin II levels were

measured by radioimmunoassay using monoclonal antibodies [19] after ethanol extraction, as previously
described [20]. Briefly, proteins of 50 ml cold ethylenediamine tetraacetic acid plasma were precipitated by
450 ml pure ethanol and the supernatants were dried in
polypropylene tubes coated with buffer proteins; monoclonal antibodies and iodinated tracer angiotensin II were
added for the radioimmunological quantitation of
extracted plasma angiotensin II. Results were not corrected for recoveries, which were low but constant at
40  1% when radiolabelled angiotensin II was extracted
from 50 ml ethylenediamine tetraacetic acid plasma
samples. The coefficient of variation for intra-assay precision was 0.095. The plasma detection limit was
3.0 pmol/l. Samples from two rats in the AngQb group
and one rat in the VLP group were haemolytic and could
not be analysed.

Efficacy experiment in spontaneously hypertensive rats

monitored by tail-cuff blood pressure measurements

SHR were immunized subcutaneously with 400 mg

AngQb, or VLP in aluminum hydroxide. Animals were
similarly boosted on days 14 and 28. Antibody titres were
measured from sera collected on days 0, 7, 14, 21 and 28.
A third group of rats was treated with an active comparator, ramipril, administered daily via the drinking water
(1 mg/kg bodyweight). Arterial blood pressure was
measured on days 9, 16, 23, and 30 by the tail-cuff method
(Pressure Meter LE-5000 Series; Letica, Cornella`, Barcelona, Spain). During measurements, animals were held
in a restraining device. SBP was calculated as the median
of 25 readings for each animal at each timepoint. Animals
were killed on day 35, and blood was sampled for angiotensin II determination.
Efficacy experiment in spontaneously hypertensive rats
monitored by telemetry

SHR 1112 weeks of age were surgically implanted with

telemetry devices, and a pressure sensor was introduced in
the abdominal aorta (Centre de Recherches Biologiques,
Baugy, France). Sodium pentobarbital (45 mg/kg, intraperitoneal route) was used as an anaesthetic. After surgery,
rats were allowed to recover for 2 weeks and monitored
to ensure the stabilization of blood pressure readings.
Animals were then randomly sorted into groups (day 0).
Rats were vaccinated subcutaneously with 400 mg AngQb
(n 8), or with 400 mg VLP in aluminum hydroxide
(n 7) on days 1, 15, 29 and 43. For the injection of
vaccines on day 29, the VLP was switched from Qb to
AP205, another VLP with similar properties but serologically distinct. A third group of animals (n 7) was
administered an ACE inhibitor (enalapril) by daily oral
gavage at a dose of 10 mg/kg bodyweight from days 1 to 28.
Thereafter the dose was sequentially lowered throughout
the experiment.
Blood samples were collected on days 0 (pre-immune),
14, 28, 42, 56 and 70, and antibody titres against angiotensin II were measured. Blood pressure from individual
animals was measured every 15 min throughout the
course of the experiment. The median of the 48 readings
recorded during each of the 12-h day and night periods
was calculated for each 24-h period. For the sake of
clarity, data for the day period only are presented,
because night-time results were equivalent. Blood pressure values were subtracted from their baseline value for
statistical analysis, which was performed at the Centre de
Recherches Biologiques.
Preclinical toxicological safety

Preclinical toxicology studies were performed following

the ICH guidelines for the preclinical safety evaluation of
biotechnology-derived pharmaceuticals and the preclinical pharmacological and toxicological testing of vaccines.
Studies were performed according to the principles of

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66 Journal of Hypertension 2007, Vol 25 No 1

good laboratory practice by a contract research organization with expertise in vaccine toxicology (Charles River,
Tranent, UK).
Statistical analyses

For the tail-cuff experiment, data were analysed by twoway analysis of variance (ANOVA) for repeated measurements, and in case of significance a Bonferroni post-test.
For the telemetry experiment, blood pressure values
were analysed using two-way ANOVA for repeated
measurements, and in case of significance a Dunnetts
post test. P-values less than 0.05 were considered statistically significant. Data were analysed using the software package Graphpad Prism (GraphPad Software, San
Diego, California, USA).
Human study
Study design

The objective of the first-into-man, randomized, placebocontrolled, double-blind, phase I study was to assess the
safety, tolerability and pharmacodynamic effect (immunogenicity) of the CYT006-AngQb vaccine. The phase I
arm reported here is part of an ongoing phase I/II study, of
which only the phase I part was unblinded. The study
evaluated a single-dose regimen consisting of a subcutaneous injection of 100 mg CYT006-AngQb or placebo
formulated in aluminum hydroxide. Twelve subjects
were on active drug and four on placebo. Subjects
remained at the clinic 24 h after dosing, and were monitored for safety and tolerability on weeks 1, 2, 3 and 4.
The pharmacodynamic effect (antibody responses) was
measured on weeks 0 (pre-dosing), 1, 2, 3, 4, 8 and 16.
The active renin concentration and angiotensin II levels
were not measured in this study arm, but are being
determined in the phase II arms of the study in hypertensive patients. Proteinuria was monitored at screening,
and 24 h after injection, and on days 7, 14 and 21 (Combur
10 Test; Roche Diagnostics GmbH, Mannheim,
Germany). Study protocol and other relevant documents
were reviewed and approved (1 November 2004) by the
Ethics Committee of the Landesa rztekammer Brandenburg, Germany, before the initiation of the study, which
was conducted in accordance with ICH GCP Guidelines
and the Declaration of Helsinki (1964) and subsequent
revisions. Written consent was obtained from all subjects.
Measurement of immune complexes

The concentrations of activated complement factors

(C3a, C5a) and of immune complexes were quantified
by assays from BD Biosciences (Heidelberg, Germany)
and OSTEOmedical (Bu nde, Germany), respectively.

the binding of angiotensin II to its receptor. Whereas

current models predict that residues spanning the whole
angiotensin II molecule are involved in receptor contact,
residues 2, 4 and 8 of angiotensin II are known to interact
with the AT1R [21], we designed a vaccine (AngQb) to
induce antibodies specifically recognizing angiotensin II.
To achieve this a modified angiotensin II peptide was
conjugated via its N-terminus to the VLP. In this way
antibodies specific for the carboxy terminus of angiotensin II, which differs from angiotensin I and angiotensinogen, would be generated. The vaccine was first tested in
mice and was found to be highly immunogenic (Fig. 2a).
We confirmed the immunogenicity in rats and also tested
whether the inclusion of a mild adjuvant, aluminum
hydroxide, further increased antibody titres. AngQb
alone induced a strong antibody response. Titres were
increased twofold by the inclusion of the adjuvant
aluminum hydroxide (Fig. 2b). We also tested the reversibility of the antibody response by measuring antibodytitres from rats over a period of 147 days (Fig. 2c). Titres
declined exponentially with an average half-life of
1319 days, demonstrating that the immune response
induced against angiotensin II, a self-antigen, was reversible after three immunizations with or without aluminum hydroxide (Table 1).
In a further set of experiments, we characterized the
specificity of the antibody response. Using ELISA assays
with a set of angiotensin II peptides coupled to RNAse,
we found that the AngQb vaccine induced antibodies
predominantly recognizing the C-terminus of angiotensin II (data not shown). The specificity of the antibodies
was further assessed by testing their ability to bind
angiotensin I, angiotensin II, angiotensin28 (angiotensin
III, an active cleavage product of angiotensin II binding
the same receptors) and angiotensinogen in solution (i.e.
inhibition ELISA). As seen in Fig. 3, antibodies raised
against AngQb bound most strongly to angiotensin II,
followed by angiotensin III. Binding to angiotensin I was
an order of magnitude lower. In order to estimate an
average affinity for angiotensin II, coating densities were
reduced according to Friguet et al. [18]. A dissociation
constant (Kd) of 11.5  2.5 nmol/l (average of two determinations) was obtained using the optimal coating concentration. We further measured the concentration of
antibodies by equilibrium dialysis using radiolabelled
angiotensin II, and found a value of 300  86 nmol/l
(average of two determinations). The affinity measured
by equilibrium dialysis (dissociation constant of 2.6 
1.0 nmol/l) was higher than obtained when using the
Friguet assay, but was measured over a smaller range
of ligand concentrations (0.216 nmol/l).

Results
Design and immunogenicity of anti-angiotensin II
vaccines

To be effective, a vaccine against hypertension interfering with the RAAS pathway should ultimately prevent

Antibodies did not bind angiotensinogen at the concentrations used in the assay, demonstrating that the apparent affinity of the antibodies for angiotensinogen is at
least an order of magnitude lower than for angiotensin II.

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

Table 1 Half-life of angiotensin II-specific IgG antibody titres in rats

immunized against AngQb

Fig. 2

(a)

25 mg Al.

100 mg Al.

400 mg Al.

400 mg

19
1333

16
1420

17
1519

13
1114

Half-life (days)
95% CI (days)

100000
Angiotensin II-specific IgG titre half-lives from rats immunized with 25, 100 or
400 mg of AngQb in aluminum hydroxide (Al.) or 400 mg AngQb. The sera of five
animals of each group at each timepoint were pooled for the measurement. Halflives were fitted to a single exponential decay. CI, confidence interval.

10000
1000
100

d14

Vaccination reduces blood pressure in a rat model of

hypertension

d21

The effect of immunization on blood pressure was tested

in the SHR model using two different methods of
measurement. In the first experiment, systolic blood
pressure (SBP) was monitored by the tail-cuff method
in all rats on days 9, 16, 23 and 30 of the regimen. SBP in
the AngQb-vaccinated animals was significantly lower
compared with the VLP control group on days 23 and
30 (Fig. 4a). By day 30, a difference of 21 mmHg
(P < 0.001) was observed in comparison with the VLP
control. As expected, treatment with ramipril also
decreased SBP (25 mmHg, P < 0.001). We observed
an inverse correlation between antibody titre and blood
pressure (r 0.54, P 0.006 for ln titre versus SBP,
correlation within animals [22]).

(b)

10000

1000

100

400 g

400 g Alum

(c)

In order to monitor the effect of immunization on blood

pressure continuously over time in the most sensitive and
precise manner we chose the method of telemetry in the

20000

15000

Fig. 3

10000

125
5000

In addition, inhibition ELISA performed using angiotensin114 as a surrogate for angiotensinogen showed a
5001000-fold lower affinity of the sera for angiotensinogen than for angiotensin II (data not shown).

50

25

1.

0
1

0 0

05

0
10
1.

1.

0
10

06

0
0 0

Immunogenicity in mice and rats immunized with AngQb and

reversibility of antibody response. (a) Balb/c mice (n 5) were
immunized on days 0 and 14 with 100 mg AngQb. Anti-angiotensin II
antibody titres were measured in the sera of days 14 and 21 with an
enzyme-linked immunosorbent assay (ELISA) against the modified
angiotensin II peptide coupled to RNAse. The titre is expressed as the
dilution of serum giving half-maximal binding in the assay (optical
density; OD 50%). The pre-immune titre was 1 : 100. Error bars show
the standard error of the mean. (b) Rats (n 5 per group) were
immunized with 400 mg AngQb with or without aluminum hydroxide on
days 0, 14 and 28. The pre-immune titre was 1 : 100. Bars show the
geometric mean titres of the groups against angiotensin II and error bars
indicate the standard error of the mean. (c) Titre was measured by
ELISA against angiotensin II in the pooled sera of rats immunized with
400 mg AngQb in aluminum hydroxide collected on days 35, 49, 63, 91,
119 and 147.

75

0
1

125

1.

100

75

0 0

50

Days post d35

0
1

25

1.

100

09

0
10

ELISA titre (OD50%)

25000

1.

ELISA titre ( OD50%)

100000

Relative binding (%)

ELISA titre ( OD50%)

1000000

Inhibitor concentration [mol/l]

Specificity and crossreactivity of antibodies raised against AngQb.
The sera of immunized spontaneously hypertensive rats (day 42 of
telemetry experiment) were pooled and assessed for binding to
angiotensin II (&), angiotensin III (~), angiotensin I (!) and
angiotensinogen (^) by inhibition enzyme-linked immunosorbent assay.
Binding curves were normalized for comparison between experiments.
An angiotensin II inhibition curve as reference was included in every
experiment. Assays were performed in duplicate, and error bars show
the standard deviation.

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68 Journal of Hypertension 2007, Vol 25 No 1

Fig. 4

Fig. 5

(a)

(a)

190

SBP (mmHg)

190

Systolic BP (mmHg)

180

180
170
160

170
150
0

10 15 20 25 30 35 40 45 50 55 60 65 70 75

Days
160

(b)
20000

140
d9

d16

d23

d30

(b)

ELISA titre (OD50%)

50000

ELISA titre (OD50%)

150
15000

10000

5000

40000
0
d14

d28

d42

d56

d70

30000

20000

10000

0
d7

d14

d21

d28

Effect of vaccination on systolic blood pressure (BP) in spontaneously

hypertensive rats (SHR) monitored by the tail-cuff method. Groups of
SHR (n 8) were immunized on days 0, 14 and 28 with AngQb (^),
virus-like particles (~) or adminstered ramipril (&) as described.
Systolic BP was determined by tail-cuff measurement, antibody titres
were determined by enzyme-linked immunosorbent assay (ELISA). (a)
Effect of vaccination on systolic BP. Systolic BP was recorded for each
animal on days 9, 16, 23 and 30. Data are presented as the average of
each group, and error bars indicate the standard error of the mean.
Systolic BP for the AngQb group was reduced compared with the VLP
group by 17 mmHg on day 23 (P < 0.01) and 21 mmHg on day 30
(P < 0.001, two-way repeated measure analysis of variance with
Bonferroni post test). (b) Antibody titres in vaccinated rats. OD,
Optical density. IgG titres against angiotensin II were measured in the
sera of the AngQb group sampled on days 0, 7, 14, 21 and 28. The
pre-immune titre was 1 : 100. Titres are shown as box and whiskers plot
with median 25- and 75-percentile, whiskers show minimal and maximal
values.

second efficacy experiment. Reductions in SBP relative

to the VLP control group first achieved statistical significance on days 46 and 47 (Fig. 5a). Thereafter, SBP was
statistically significantly lower compared with the VLP
control group from days 57 to 79. The mean SBP for

Effect of vaccination on blood pressure in spontaneously hypertensive

rats (SHR) monitored by telemetry. Groups of SHR were immunized
on days 1, 15, 29 and 43 with AngQb (n 8), virus-like particles
(VLP; n 7), as described. Systolic blood pressure (SBP) was
measured by telemetry and antibody titres were determined by enzymelinked immunosorbent assay (ELISA). (a) Effect of vaccination on SBP.
SBP measurements are averages of the groups. Data are only shown
for the AngQb (&) and VLP (^) groups. Error bars show standard
error of the mean. Significant differences (P < 0.05) for the AngQb
versus the VLP group were found on days 46 and 47, and on days
5779 (marked with a bracket and a star), with the exception of days
62, 63 and 68 (two-way analysis of variance for repeated
measurements, with Dunnetts post test). (b) Antibody titres in
vaccinated rats. OD, Optical density. IgG antibody titres against
angiotensin II were measured in the sera of the AngQb group collected
on days 0, 14, 28, 42, 56 and 70. The pre-immune titre was 1 : 100.
Titres are shown as box and whiskers plot with median 25- and
75-percentile, whiskers show minimal and maximal values.

the period extending from days 57 to 79 was 15 mmHg

lower in the AngQb group compared with the VLP
controls (P < 0.01, Table 2). Mean arterial pressure was
also reduced during this period by 7% (P < 0.05) or
10 mmHg, (Table 2). Immunization against angiotensin
II thus yielded a consistent reduction in blood pressure
lasting more than 35 days after the last boost. Angiotensin II-specific antibody titres in the AngQb group peaked
on day 42, later than was observed in the tail-cuff
experiment, and remained high until completion of the
experiment (Fig. 5b). Finally, no significant differences
in heart rate were detected in any of the treatment
groups.

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

Changes in arterial blood pressure in the AngQb and

enalapril groups compared with virus-like particle controls during
the 12-h day and night period

Table 2

SBP
AngQb
Enalapril

Day
Night
Day
Night

15
13M
31M
25M

DBP

MAP

5
6
22M
15M

10y
9y
26M
20M

DBP, Diastolic blood pressure; MAP, mean arterial pressure; SBP, systolic blood
pressure. Mean SBP, MAP and DBP in mmHg were calculated for the period
between days 1 and 56 and days 5779 and adjusted for the baseline level.
Values shown are differences in arterial blood pressure between the AngQb and
virus-like particle (VLP) control group for days 5779, or the enalapril and VLP
groups for days 156 (high-dose enalapril treatment). M P < 0.01 versus VLP.
y
Data analysed in percentage: 7% reduction (P < 0.05). Data analysed in absolute
variation: threshold value for significance versus VLP at the 95% confidence limit:
11 mmHg for the day values, and 10 mmHg for the night values.

Angiotensin II level measurements

A decrease in blood pressure in SHR vaccinated against

angiotensin II was expected to enhance the secretion of
renin in the kidney. This is caused by reduced AT1R
stimulation relieving feedback suppression of renin
secretion, the baroreceptor reflex and the tubuloglomerular feedback mechanism. Consequently, an increase in
the total angiotensin II concentration was expected,
angiotensin II being either free or antibody-bound. We
measured total angiotensin II levels in rats from the tailcuff experiment (Fig. 6) on day 35. A ninefold increase in
the total angiotensin II concentration was observed in the
AngQb group (61 pmol/l, P < 0.01 versus VLP group,
t-test). Although plasma renin activity for the VLP control
and ramipril groups was in the expected range, it could
not be determined in AngQb-vaccinated animals. This is
because the antibodies generated by AngQb also bind
angiotensin I, interfering with the measurement of renin

activity, which is assessed by a competition radioimmunoassay with radiolabelled angiotensin I and an

anti-angiotensin I antibody.
Treatment of spontaneously hypertensive rats with
vaccine for 177 days

Although the small size of angiotensin II virtually precludes the binding of two antibodies at the same time,
there exists a theoretical possibility that high levels of
antibodies and angiotensin II in the kidney could give
rise to renal inflammation, for example in the event of
immune complex deposition. To examine this possibility, SHR were immunized, and their kidneys and other
organs examined by histopathology for signs of inflammation. A high antibody response was induced and maintained until they were killed on day 177. There were no
findings in the kidneys of animals vaccinated with
AngQb, whereas nephritis was reported in one animal
immunized with VLP (Table 3). Minor findings in the
heart, lung or liver were also observed in the ramipril
control group, and are consistent with the type of background lesions previously reported for SHR (Table 3)
[23,24].
Toxicology

Toxicology studies with CYT006-AngQb in normotensive rats demonstrated no evidence of local or systemic
toxicity resulting from either single or multiple administrations of the vaccine in the presence or absence of
aluminum hydroxide. A histiocytic response at injection
sites was noted for animals receiving CYT006-AngQb in
the presence of aluminum hydroxide, and was of the type
expected when this adjuvant is used. In particular, no
signs of inflammation were detected in the kidney,
indicating no inflammatory immune complex deposition.

Fig. 6

Histological findings in spontaneously hypertensive rats

treated for 177 days

Table 3

Ang II (pmol/l)

175
150

Organ

Histological finding

125

Kidney
Heart

100

Heart

75

Heart
Heart

Nephritis
Low level multifocal
histiocytic myocarditis
Low level focal
histiocytic myocarditis
Low level focal myocard fibrosis
Focal swelling of the
media in intramural arteries
Intima sclerosis in vein or artery
Necrosis, with histiocytic
inflammation
Myocytic proliferation in the
intima of a few arteries

50

Heart
Liver

25

Lung

0
AngQb

VLP

AngQb

Ramipril

1
4

0
3

0
0

0
0

1
2

1
0

0
1

0
0

1
1

VLP

Angiotensin II (Ang II) levels in the plasma of vaccinated rats

determined by radioimmunoassay. Angiotensin II was extracted from
plasma sampled at time of death (day 35) of vaccinated rats.
Angiotensin II levels were determined by radioimmunoassay, and are
expressed in pmol/l. Horizontal bars indicate the average of the groups,
symbols indicate individual values. VLP, Virus-like particles.

VLP, Virus-like particle. Number of animals in which the listed findings were
identified are indicated for each treatment group. Spontaneously hypertensive
rats (n 6) were immunized on day 0 with 400 mg AngQb or VLP in aluminum
hydroxide. They were boosted on days 14, 28, 42, 70 and 162 with 200 mg
vaccine in aluminum hydroxide. A control group (n 6) was administered ramipril
daily by mouth in the drinking water (days 097: 1 mg/kg per day; days 98125:
0.5 mg/kg per day; days 126161: 0.25 mg/kg per day; days 162177: 1 mg/kg
per day). Animals were killed and organs removed for histological analysis on
day 177.

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70 Journal of Hypertension 2007, Vol 25 No 1

Phase I clinical trial

All subjects were male, had a median age of 33.5 years

(range 2252), median body weight of 77.4 kg (range
62.397.5), and a median height of 181 cm (range 172
192). Vaccination with CYT006-AngQb was well tolerated. Fourteen out of 16 subjects showed local adverse
events such as erythema, oedema, pain and induration at
the injection site, all of mild intensity. A mild headache
reported by one subject (on active treatment) was also
considered to be possibly related to treatment. All other
systemic adverse events (one report of blocked nose and
one sore throat) were not considered to be drug related, as
well 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 significant
changes occurred in blood pressure in these healthy
normotensive volunteers. Heart rate and 12-lead electrocardiogram data were unchanged, and laboratory parameters showed no clinically significant deviations from
the normal range.
All volunteers receiving CYT006-AngQb responded with
high IgG titres against angiotensin II within 2 weeks of
immunization (n 12). Titres peaked on week 3 and
declined with an average half-life of 19 days (n 10,
95% confidence intervals 1225, Fig. 7). Volunteers
receiving placebo showed no detectable antibody
response against angiotensin II (n 4).
The induction of antibodies against endogenous angiotensin II could theoretically lead to immune complex
deposition. We therefore measured the concentration of
Fig. 7

ELISA titre

10000

1000

100

10
0

16

Week
Angiotensin II-specific IgG titres in healthy human volunteers after a
single immunization with CYT006-AngQb. Anti-angiotensin II IgG titres
of the 16 healthy volunteers were determined by enzyme-linked
immunosorbent assay (ELISA). Bars denote geometric mean titres of
the subjects with 95% confidence intervals. Titres for subjects in the
active arm are shown as filled bars. All subjects receiving placebo
(n 4, open bars) showed titres below the lower limit of quantification,
which was set to 1 : 30.

immune complexes containing C1, C3, IgM, IgA or IgG

at baseline and 7 and 14 days after immunization. No
changes in immune complex levels were observed. Similarly, no changes in the levels of C3a and C5a in the
serum from baseline were detected 7 and 14 days after
immunization. CYT006-AngQb was highly immunogenic
in humans and induced no signs of inflammation or
immune complex formation.

Discussion
VLP are a very potent means of inducing antibodies
against self-antigens [10,25]. In preclinical studies we
showed that our vaccine comprising an angiotensin IIderived peptide coupled to Qb VLP induced a strong
antibody response against angiotensin II, both when
immobilized on a plate or in solution. A phase I clinical
study with our VLP vaccine showed a response rate of
100% in healthy human volunteers with high antibody
titres after only one immunization, a comparable result to
our preclinical experiments in rats. This is the first time a
VLP-conjugate vaccine has been demonstrated to break
B-cell unresponsiveness in humans. Moreover, the
response to the vaccine was shown to be reversible,
and its use was safe and well tolerated.
In rats, angiotensin-specific antibodies effectively
reduced blood pressure in two independent experiments
using different methods for monitoring blood pressure. In
one experiment in which blood pressure was measured by
the tail-cuff method, the reduction was comparable to the
effect seen with the ACE inhibitor, ramipril. In the
second experiment involving telemetry, the blood pressure-reducing effect was clear and statistically significant,
and was demonstrated to be long lasting. The blood
pressure-reducing effect of AngQb was also observed
in a second model of hypertension, the Dahl salt-sensitive rat (data not shown).
The high affinity and concentration of antibodies raised
by AngQb (approximately 311 and 300 nmol/l, respectively) led to a significant blockade of the RAAS, which
resulted in a decrease in blood pressure and a consequent
increase in total angiotensin II (antibody-bound and
free). This was presumably caused by a rise in renin.
The amount of antibodies was, however, high enough to
absorb the increase in angiotensin II, and thus reduce
blood pressure. By the law of mass action, a concentration
of antibody 26115-fold (range of Kd values determined)
higher than its dissociation constant is predicted to bind
9699% of angiotensin II, fully consistent with the
observed efficacy. The level of antibodies raised is also
large enough to bind 8597% of angiotensin II in tissues,
because steady-state antibody levels in the interstitial
fluid are 3.5-fold lower than in plasma [26]. Previous
attempts to immunize against angiotensin I have been
unsuccessful at convincingly lowering blood pressure in
SHR and humans [6,9]. This may have been the result of

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Antihypertensive angiotensin II virus-like particle vaccine Ambu hl et al. 71

lower levels of antibodies raised in those studies, but as

antibodies were reported as titres and not concentrations,
direct comparisons cannot be made. Alternatively, the
location of ACE on the endothelial cell surface may
render it more difficult for anti-angiotensin I antibodies
to intercept angiotensin I before its cleavage to angiotensin II. In contrast, the AT1R is located on the smooth
muscle cell surface, making it easier for anti-angiotensin
II antibodies to sequester angiotensin II. Immunization
experiments looking at the pressure response upon angiotensin I infusion showed a blood pressure-lowering effect
[27,28], but were performed with concentrations of angiotensin I one-to-two orders of magnitude higher than its
physiological concentration. The kinetics of the binding
of antibodies would be much reduced at physiological
concentrations, and might explain the discrepancy
between blood pressure measurements in SHR and
angiotensin I infusion data in normotensive rats.
Consistent with the proposed mechanism of action of a
vaccine, the blood pressure of test and control groups
began to diverge with the development of angiotensinspecific antibody titres. A statistically significant and
consistent reduction in blood pressure occurred with
the development of high angiotensin-specific antibody
titres. In the tail-cuff experiment, a sustained reduction
in blood pressure occurred from day 23 onwards. In the
telemetry experiment, the first statistically significant
reduction occurred on day 46, whereas a continuous
reduction was achieved from day 57 onwards. Supportive
of a mechanism of action relying on induced antibodies,
an analysis of titres and blood pressure for individual
animals showed an inverse correlation in the tail-cuff
experiment. Furthermore, the antibody-mediated drop in
blood pressure led to increased total angiotensin II
(predominantly antibody bound) which was presumably
due to an increase in renin secretion. Collectively, these
findings support a mechanism of action based on the
sequestration of angiotensin II by antibodies induced by
the vaccine.
Two safety prerequisites for a therapeutic vaccine against
angiotensin II are the absence of inflammation-inducing
immune complexes and the reversibility of the antibody
response. In a toxicological study, normotensive rats
exposed to high antibody titres against angiotensin II
neither showed signs of systemic toxicity nor evidence
of immune complex deposition or inflammation. In
addition, long-term treatment of SHR did not result in
kidney inflammation. Vaccination of humans was well
tolerated with no systemic vaccine-related side-reactions.
No change in the level of immune complexes was
detected upon the induction of antibodies against angiotensin II. In addition, the antibody response was reversible, with a half-life of approximately 23 weeks in rats.
Similarly, antibody titres dropped in all human volunteers, with an average half-life of 19 days after the peak

response, demonstrating that angiotensin II-specific antibody responses are indeed reversible and decline over
time.
An advantage of vaccination over the daily administration
of small molecular drugs is that antibodies circulate in the
blood for weeks to months, resulting in a continuous
blockade of angiotensin II. Current medications, taken
daily, exhibit peak-to-trough variations in activity that are
not ideal for the treatment of a chronic condition in which
the equilibrium of healthy processes is slightly but constantly distorted; as is the case for hypertension.
In conclusion, we have demonstrated a sustained lowering of blood pressure in rats after therapeutic vaccination with a VLP-based vaccine targeting angiotensin II.
We showed that efficacy can be modelled based on the
affinity and specificity of the antibodies, and that blood
pressure reduction presumably led to elevated renin. The
administration of only one dose of vaccine in aluminum
hydroxide (the most used commercial human adjuvant)
to human volunteers resulted in a strong antibody
response with a 100% response rate, similar to what
was observed in the rat model. The fact that the same
vaccine formulation is used in both preclinical and
clinical studies increases the likelihood that a therapeutically relevant reduction in blood pressure may also be
observed in humans. Towards this end the vaccine is
currently being evaluated in a phase II efficacy study in
hypertensive patients.

Acknowledgement
The authors are grateful to Dr Hans Stocker for his
review of statistical analysis procedures, and Dr Dace
Skrastina for help with immunizations.

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