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Influenza virosomes as a vaccine adjuvant and

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Influenza virosomes as a vaccine

adjuvant and carrier system
Expert Rev. Vaccines 10(4), 437446 (2011)

Christian Moser1
Mario Amacker1 and
Rinaldo Zurbriggen2
Pevion Biotech AG,
Worblentalstrasse32, CH-3063 Ittigen,
Switzerland
2
Lonza AG, CH-3930 Visp, Switzerland

Author for correspondence:

Tel.: +41 315 504 424
Fax: +41 315 504 445
christian.moser@pevion.com
1

Influenza virosomes have been used for more than 10 years in commercial vaccines. The
technology has been further developed as a carrier and adjuvant system for subunit vaccines,
in particular for synthetic peptides. The extensive amount of preclinical and clinical data supports
the notion that influenza virosomes represent a platform technology that ensures robust and
long-lasting immune responses against subunit antigens with an excellent safety profile.
Structurally and functionally, virosomes are enveloped virus-like particles, although they are
assembled invitro. This unique feature ensures a tight control of their composition and at the
same time provides the flexibility to adapt the particle to various types of antigens. The mode
of action of virosomes is complex and includes carrier as well as immune-stimulatory functions.
Keywords : adjuvant antigen delivery influenza vaccines virosomes

The need for safe and efficacious vaccines has

promoted the use of subunit antigens in order
to remove antigenic components that are unrelated to protection, potentially harmful or even
immune suppressive. Subunit antigens can be
purified from the respective pathogens, as illustrated by influenza vaccines, but recombinant proteins or synthetic peptides are increasingly being
used. However, the reduction in antigen complexity in combination with a high purity frequently
impacts negatively on the immunogenicity.
The induction of an adaptive immune
response via vaccination is a highly complex process, which requires several distinct cell types to
interact, differentiate and migrate between the
administration site and lymphatic tissue within
a time course of several weeks [1,2] . The goal of
any vaccination is to generate populations of
mature, antigen-specific effector and accessory
cells, including memory cells, for sustained,
long-lasting immunity. Vaccine adjuvants can
come to action at any stage of this process in
order to amplify the desired type of immune
response [1] .
Historically, vaccine adjuvants were identified
empirically by evaluation of their potential to
enhance immune responses against coadministered antigens. Novel insights into the interplay
between innate and adaptive immunity have
not only led to more rational selection of novel
adjuvant candidates, such as specific ligands of
innate danger and tissue damage sensors [35] ,
but have also provided explanations for the
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10.1586/ERV.11.15

mode of action of established adjuvants [38] .

Antigen-presenting cells (APCs), in particular
dendritic cells (DCs), play a central role in this
process. DCs express a wide range of innate sensor molecules and strongly influence the activation of antigen-specific B and T lymphocytes in
reaction to innate stimuli [1,6,9,10] .
In-depth knowledge of the mode of action
of adjuvants provides a rational basis for risk
assessments, and for the design of conclusive
toxicology studies and clinical trials. It should
be emphasized that safety concerns are more difficult to address in clinical trials than immuno
genicity because severe side effects are not
acceptable even at very low frequencies [11,12] .
Therefore, a thorough safety assessment is also
the key issue in regulatory guidelines for clinical testing and market approval of novel adjuvants [1316] . A wide variety of adjuvants are
capable of enhancing the immunogenicity of
weak antigens but, aside from aluminum salts,
only very few are actually applied in licensed
human vaccines, namely MF59 (Novartis, Basel,
Switzerland), AS03 and AS04 (GSK, London,
UK), and influenza virosomes (Crucell, Leiden,
The Netherlands; Solvay, Brussels, Belgium).
Virus-like particles (VLPs) represent a distinct class of adjuvants. Aside from their individual components, their particle characteristics
contribute to their function as adjuvants, and
thus, they have a complex mode of action [3,17] .
Originally, VLPs were defined as nonreplicating, recombinant virus capsids [18,19] , but it

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ISSN 1476-0584

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Moser, Amacker & Zurbriggen

seems appropriate to expand the definition and to include viruslike structures derived from enveloped viruses [20,21] . Common
to all VLPs is their particulate structure that mimics a virus
combined with their complete inability to replicate. These features ensure a high safety level combined with the advantages
of a VLP structure enhancing the immune stimulation and the
stability of the antigen.
Virus-like particles can be derived from a variety of nonenveloped and enveloped viruses, and are generally manufactured
in recombinant expression systems. VLPs are applied in several licensed vaccines (hepatitis B virus, human papillomavirus,
influenza and hepatitis A virus) and in a considerable number
of prophylactic and therapeutic vaccine candidates in clinical
development in the field of infectious diseases, cancer and allergy.
Originally, VLPs represented just another form of vaccine
antigen, but their unique versatility soon promoted their use as
carrier and adjuvant systems suitable for heterologous antigens,
where they provide a higher, immune-potentiating particle structure [3,2226] . Most VLP-based vaccines are combined with other
adjuvant types such as alum salts and Toll-like receptor (TLR)
agonists, while influenza-derived VLPs, including virosomes, are
generally used as stand-alone products.
Virosome technology

The concept of virosomes and the idea to use them as vaccines

dates back to the 1970s [27,28] . Virosomes are spherical, unilamellar vesicles composed of membrane lipids and integrated viral
envelope proteins. Unlike most VLPs, virosome particles are not
produced by host cells but assembled invitro in a three-step process from the parental virus. First, the purified and inactivated
virus is dissolved in detergent. Second, the envelope fraction
comprising the membrane-associated viral proteins and lipids
is separated from the core complex containing the genetic material and internal proteins. Third, detergent removal triggers the
formation of empty membrane vesicles.
This simple principle of assembling empty envelope particles
invitro is applicable to all enveloped viruses, for the purpose of
functional studies or as vaccine candidates, most recently for HIV
and respiratory syncytial virus [25,29,30] .
Influenza virus was used in the early studies on virosomes[27,28] .
In the following years, the production of influenza virosomes was
further optimized [22,31] and established as an industrial scale
process compliant with good manufacturing practice (GMP)
guidelines [32] . To date, influenza virosomes remain the only
virosome type that is applied in licensed human vaccines[33,34] .
In the past few years, several clinical trials have been performed,
using influenza virosomes as a carrier and adjuvant system for
heterologous subunit antigens [3537] . The dominance of the
influenza virus as the substrate for virosome-based products may
be explained by two aspects, namely, by the biological properties of the viral envelope proteins, in particular of influenza
hemagglutinin (HA), and by the availability of virus material.
GMP-grade influenza virus is produced in embryonated chicken
eggs every year for several hundred million doses of influenza
vaccines. The same material has proven suitable and cost-efficient
438

for the generation of influenza virosomes. Notably, influenza

virus grown in cell culture is equally suited for the production
of virosomes [201] .
The envelope of influenza virus contains predominantly HA
trimers, approximately fivefold less neuraminidase tetramers,
and traces of M2e tetramers [38] . All these proteins are also present in influenza virosomes in similar ratios as in the parental
virus. Retaining the authentic conformation and functionality
of the viral proteins is a key aspect of the virosome formulation
process, as exemplified by the pH-dependent fusion activity of
HA[39,40] . As a consequence, a thorough quality control of virosomal vaccines goes far beyond content analysis and includes
particle characterization, as well as a verification of the fusion
activity [40] .
The addition of phospholipids to the solubilized viral envelope
fraction has proven essential for a robust, scalable particle assembly step. The addition of lipids minimizes the loss of influenza
components during detergent removal and ensures the assembly
of a homogenous particle population that renders further purification steps obsolete [32] . However, it should be noted that the
density of the envelope proteins on the virosome surface is reduced
in comparison to the parental virus particle, since the density is a
direct function of the protein:lipid ratio. The physico-chemical
and biological properties of the resulting virosome particles can
be modulated not only by the amount, but also by the type of
lipids added [4143] . The addition of charged lipids has proven
useful to associate and deliver negatively charged molecules such
as nucleic acids [39,44,45] .
Conventional influenza virosomes have a limited stability
unless stored at 28C, and are particularly sensitive to freezing. The addition of sugars, with or without cationic lipids,
was shown to protect the virosomes from the negative effects
of freezing and to generate temperature-insensitive virosomes
that can be lyophilized. These formulations are stable and retain
their fusion activity in a broad temperature range [46,47] . In
addition, lyophilization can stabilize labile subunit antigens,
and enables a novel approach to efficiently encapsulate small
antigens like peptides, by reconstituting lyophilized empty
virosomes with a peptide solution [46] . Influenza virosomes are
protected by a considerable number of patents, covering multiple aspects of use as a vaccine platform and drug delivery
system, the formulation process including lyophilization and
combination with other adjuvants.
A wide range of antigen types, including peptides, proteins
and carbohydrates, have been combined successfully with influenza virosomes in order to obtain immunogenic vaccine candidates [4856] . In addition, virosomes were shown to function as
delivery system for nucleic acids and drugs [39,44,45,57] .
The physical association of the payload, the antigen of interest (AoI), with the virosome particle is a prerequisite for the full
immune-enhancing function [49,58] . Depending on the preferred
type of immunity, antibody or cytotoxic T lymphocytes (CTL),
the antigen is either formulated to be displayed on the surface or
encapsulated in the lumen, respectively [40,59] . For surface display, the AoI needs to be anchored in the lipid bilayer, either
Expert Rev. Vaccines 10(4), (2011)

Influenza virosomes as a vaccine adjuvant & carrier system

via a hydrophobic protein domain in the AoI, or via a synthetic

phospholipid conjuguated to the AoI. By contrast, efficient encapsulation is achieved by a specifically designed formulation process [46,48] , but does not require integration of the AoI into the
lipid membrane.
Antigens of interest displayed on the outer surface of the virosomes in a repetitive array are optimal to prime B lymphocytes for
antibody production. After uptake by APCs, AoI on the virosome
surface are processed within the late endosome and presented in
the context of MHC class II to generate T-helper cells. For AoI
encapsulated in the lumen, the pH-dependent fusion activity of
HA enables virosomes to act as cytoplasmatic delivery vehicles.
After uptake by APCs, the acidification within the late endosome
triggers HA-mediated fusion between the virosomal and endosomal membrane. As a result, the encapsulated AoI is released into
the cytoplasm, thereby providing access to MHC class I presentation and CTL induction [46,48,6062] . CTL induction critically
depends on the fusion activity of HA [63] .
Mode of action

Virus-like particles including virosomes exert their effects on

APCs and on antigen-specific lymphocytes via their overall particle structure and their individual components [3,17,23,64] . In
addition, pre-existing immunity significantly impacts on the
adjuvant function of virosomes [40,49] . Therefore, virosomes represent an excellent example for a multifunctional carrier and
adjuvant system (Figure1).

Carrier

Review

Adjuvant

Delivery
Antigen protection

Repetitive
antigen display

PAMP signals
Costimulation

VLP
structure
APC targeting

CD4+ help

Pre-existing immunity
Antibodies

T-cell memory

Figure1. Multifunctional mode of action of virosomes.

Schematic representation of the overlapping functions of
virosomes to highlight the central role of the VLP structure. The
carrier functions result predominantly from the particle structure.
The adjuvant functions relate to both the particle structure and
the individual virosome components with stimulatory effects and
synergistic potential. Pre-existing immunity against influenza
affects the carrier as well as the adjuvant functions. Antibodies
interact directly with the particle structure, while cellular immunity
expands the pool of T-helper cells.
APC: Antigen-presenting cell; PAMP: Pathogen-associated
molecular pattern; VLP: Virus-like particle.

Carrier functions

The carrier function is related to the particle structure and plays

a key role in the early events after vaccine administration. The
VLP structure facilitates a rapid uptake by APCs. The embedding of the AoI in the virosome particle can also protect it from
premature degradation by extracellular proteases. Virosomes
are rapidly taken up by cells, both invitro and invivo [58,62,65] .
When administered via intramuscular injection, virosomes can
access the draining lymph node via free lymph drainage, or in
association with migrating cells. The free draining hypothesis
is supported by a recent report, demonstrating that nanoparticles up to 200nm in size and nonenveloped VLPs are detectable in APCs resident in the lymph node, but larger particles
were found only in macrophages originating from the injection
site[66] . Within the lymph node, virosomes are thought to interact with B cells as intact particles, as it has been shown for other
viruses and particulate antigens [67,68] . Therefore, the repetitive display of the AoI on the virosome surface acts as a strong
activation signal for antigen-specific B cells via cross-linking of
immunoglobulin receptors [69,70] .

invitro stimulation with virosomes [62] , although this cell line

is highly responsive to both DNA and RNA. Nucleic acids
are degraded by treatment with b-propiolactone, which is performed to inactivate the influenza virus prior to virosome assembly. Accordingly, no nucleic acids are detectable in virosomes.
However, recombinant influenza HA was reported to activate
both murine and human DCs invitro [7173] . Further investigations are needed to clarify whether virosomes can directly
activate DCs, and if so, by which mechanisms. Virosomes have
been shown to stimulate human peripheral blood mononuclear
cells (PBMCs) invitro, resulting in cytokine secretion (TNFa, GM-CSF, IFN-g and IL-2 but not IL-4) consistent with a
Th1 profile, and proliferation of influenza-specific memory T
cells in PBMCs obtained from adult donors but not from cord
blood [61,74] . Notably, TNF-a and GM-CSF were secreted at
high levels rapidly after exposure to virosomes, suggesting that
these cytokines at least do not originate from the proliferating
memory cells but from a more abundant, so far unidentified,
cell type present in PBMC preparations.

Adjuvant functions

Role of pre-existing immunity

The adjuvant effect of virosomes relates to their ability to

enhance antigen processing and presentation by APCs.
Direct activation of DCs by virosomes was demonstrated in
the murine system [63] , while a human plasmacytoid DC cell
line did not increase expression of activation markers upon

Pre-existing immunity against the carrier proteins can impair

the function of viral vectors and VLPs, and quench the immune
response against the AoI [7577] . By contrast, the immune potentiating effect of influenza virosomes is enhanced by pre-existing immunity against influenza. This feature is of particular

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Moser, Amacker & Zurbriggen

Reactivation of influenza-specific memory cells, as demonstrated in human PBMCs [74] , is considered the main cause
for cell-mediated immune enhancement. B cells specific for
the AoI are activated upon contact with the antigen displayed
on the surface of virosomes. Subsequently, these cells process
and present all virosome components in the context of MHC
classII, including the influenza proteins. Therefore, these cells
can obtain their signal 2 not only from antigen-specific but also
from influenza-specific CD4 + T-helper cells. In a situation of
pre-existing immunity, influenza-specific CD4 + T cells are more
abundant and faster activated than naive CD4 + T cells reactive
to the AoI. Therefore, influenza-specific CD4 + memory cells can
significantly contribute to the differentiation and proliferation
of AoI-specific effector cells.
Multiple infections by constantly drifting virus strains result
in a complex immunity against influenza in the human population. For that reason, no conclusive clinical data are available
on the positive effect of pre-existing immunity on the response
against a heterologous AoI applied in the context of virosomes.
The hemagglutination inhibition (HI) titer against the virus
strain used in virosomes A/Singapore/6/86 (H1N1) was measured in several clinical trials. Consistently, no significant correlation was found between the response against hepatitis A virus
and the baseline HI titer against influenza, thereby excluding at
least a negative effect [33,80] . However, the value of these data is
limited because HI titers account for neither humoral immunity
against heterologous influenza strains nor
for any cellular immunity. No correlation
Anti-virosome A d21
100,000
was detectable between either pre-existing
Anti-malaria d42
cellular immunity against influenza and
Anti-malaria d63
the response against the malaria-derived
10,000
peptides formulated on virosomes [79] .
Without truly naive subjects available as
negative controls, it appears extremely difficult to clinically verify the positive cor1000
relation between baseline anti-influenza
immunity and response to the AoI, as it
has been clearly demonstrated in animal
100
models [49] .
Further preclinical and clinical studies
are necessary to dissect the contributions
10
of the different elements and to determine
None
Influenza A
Influenza B
Virosome A
whether the positive effect directly correlates
Pre-immunization
with the magnitude of pre-existing immunity or rather requires threshold levels of
Figure2. Positive effect of pre-existing immunity against influenza is strain
pre-existing humoral and cellular immunity.
ELISA titer

importance since pre-existing immunity against influenza is

widespread among the human population and can be detected
in nearly every individual [74,78,79] .
Preclinical studies in mice have clearly demonstrated that
pre-existing immunity against influenza enhances the antibody
response against unrelated antigens administered subsequently
in the context of virosomes [49] . The enhancer effect is strain
independent, since immunity against influenza B had the same
positive effect as the homologous influenza A/H1N1 strain used
to assemble the virosomes (Figure2) . By contrast, the magnitude
of CTL responses against virosome-encapsulated peptides was
not significantly increased by pre-existing immunity against
influenza [46,48] .
Both humoral and cellular elements of pre-existing immunity
against influenza are thought to contribute to the immuneenhancing effects. Anti-influenza antibodies are thought to target virosomes to Fc receptors of APCs, thereby accelerating and
enhancing uptake by APCs. For this purpose, the antibodies
need neither virus neutralizing nor hemagglutination inhibiting
capacity. Mere binding to any of the influenza proteins present
on virosomes is sufficient. This hypothesis is consistent with the
observed strain-type independence. A speculative explanation for
the lack of enhanced CTL responses is interference of antibodies
with the HA function necessary for the cytoplasmatic delivery,
which might be sufficient to compensate for the positive effects
of improved APC targeting.

independent. BALB/c mice were pre-immunized at day 0 with either influenza A/H1N1
virus, influenza B virus or virosomes made from homologous influenza A. At day 21 and
day 42, the animals were immunized with a virosomal vaccine composed of a
malariaderived peptide and virosomes assembled from influenza A. Antibody titers were
determined by ELISA: first, against influenza A virosomes at day 21, before the first
immunization with the virosomal peptide vaccine (anti-Virosome A d21) and, second,
against the malaria peptide 3weeks after the first and second immunization with the
virosomal peptide vaccine, respectively (anti-malaria d42 and anti-malaria d43). In the
presence of pre-existing immunity, a single immunization induces antipeptide titers
comparable to those achieved with two immunizations in naive animals.
Data provided by Pevion Biotech AG (Ittigen, Switzerland).

440

Clinical experience with virosomes

Over the last two decades, a considerable

amount of clinical data has been generated with influenza virosomes. Four vaccines based on the virosome technology
are licensed for commercial use, while over
70million doses of Crucells influenza and
hepatitis A vaccines have been distributed
Expert Rev. Vaccines 10(4), (2011)

Influenza virosomes as a vaccine adjuvant & carrier system

so far (Table1) . Since 2005, five novel product candidates have been tested in clinical
trials or are part of ongoing studies, all
of them based on subunit antigens in the
form of synthetic peptides or recombinant
proteins (Table2) .
Hepatitis A vaccines

Review

Table 1. Licensed vaccines based on virosomes.

Product

Vaccine composition

Launched
(year)

Ref.

Epaxal (Crucell,
Leiden,
TheNetherlands)

A(H1N1) virosomes +
inactivated hepatitis A virus

1994

[33,81]

Inflexal V (Crucell)

Virosomes from three influenza strains 1997

A(H1N1), A(H3N2) and B

[34]

The hepatitis A vaccine, Epaxal (Crucell),

was approved for commercial use in 1994 Nasalflu (Berna
[89,90]
Virosomes from three influenza strains 2000,
as the first vaccine on the basis of the influ- Biotech, Bern,
A(H1N1), A(H3N2) and B + heat labile withdrawn 2001
toxin adjuvant
enza virosome technology. The virosomes Switzerland)
in this product are generated from the Invivac (Solvay,
[91,92]
Virosomes from three influenza strains 2004,
influenza strain A/Singapore/6/86 (H1N1) Brussels, Belgium)
A(H1N1), A(H3N2) and B
not on the market
since 2005
and coated after assembly with the inactivated hepatitis A virus as the AoI. The Epaxal Junior (Crucell) A(H1N1) virosomes + inactivated
[33,80]
2008
same virus strain has been used for all subhepatitis A virus
sequent applications of influenza virosomes
as carrier and adjuvant system for heterologous antigens.
influenza strain [32] . Nasalflu (Berna Biotech, Bern, Switzerland)
Epaxal and the follow-up product Epaxal Junior were character- was a virosomal influenza vaccine for intranasal application, which
ized in numerous clinical studies with regard to safety, immunoge- contained heat labile toxin from Escherichia coli as an additional
nicity and efficacy [33,81] . Notably, when compared with alum-adju- mucosal adjuvant [89] . The product was launched in 2000, but
vanted competitor products, the virosomal products demonstrated withdrawn from the market due to an association between heat
in several independent studies a significantly better tolerability[8285] labile toxin adjuvanted vaccination and Bells palsy [90] . In 2004,
and a clear trend towards improved immunogenicity [80,82] .
Solvay launched Invivac, a trivalent seasonal influenza vaccine
for intramuscular injection based on the virosome concept [91,92] .
Influenza vaccines
However, the product was only commercially available during the
When the virosome concept is applied to influenza vaccines, season 2004/2005.
the basic building blocks of virosomes, namely the influenza
proteins themselves, represent the AoI. Compared to conven- Vaccines in clinical development
tional, nonparticulate subunit influenza vaccines, the assembly Malaria
of virosomes provides the antigens a VLP structure and native Based on two synthetic peptides, a bivalent malaria virosomal vacpresentation. Both aspects are expected to improve the immuno cine was developed to induce protective antibodies against sporozological properties [59,86] . By contrast to the application of viro- ite and blood-stage parasites [93,94] . The vaccine has been tested in
somes for heterologous antigens, the positive effects related to the three clinical trials, including a Phase I safety trial, an experimental
presence of influenza proteins, such as stimulatory signals and challenge study and a PhaseI trial in an endemic area (Tanzania).
expanded CD4 + T-cell help, do not come into play in the con- The vaccine proved to be safe and immunogenic, and induced
text of influenza vaccines. Virosomes and
conventional subunit vaccines essentially Table2. Virosome-based vaccines in clinical development.
contain the same components. Therefore,
Ref.
when applied to influenza vaccines, the Target Vaccine composition Status
[36,37,79,95,96]
role of virosome is limited to providing Malaria Two peptides
Three Phase I/II trials with bivalent
+ A(H1N1) virosomes
vaccine completed:
the antigen a particle structure.
safe and immunogenic
Comparative clinical studies with conventional subunit vaccines demonstrated Hepatitis Three peptides
PhaseI completed:
[46,100]
[Pevion Biotech AG,
+ A(H1N1) virosomes
safe but insufficiently
that the virosome formulation did not sig- C virus
Unpublished Data]
immunogenic
nificantly increase immunogenicity, but did
[35]
improve tolerability [34,87] . Whether the lat- Breast
Three peptides
PhaseI completed:
+ A(H1N1) virosomes
safe and immunogenic
ter results from the particle structure or from cancer
the higher purity of the product remains HIV
One peptide
PhaseI initiated 2010
[301]
[Pevion Biotech AG,
unclear [88] . InflexalV (Crucell) is a triva+ A(H1N1) virosomes
Unpublished Data]
lent seasonal influenza vaccine successfully
[99,101,302]
Candida
One
recombinant
PhaseI
initiated
2010
marketed since 1997 [34] . The product is
albicans
protein
+
A(H1N1)
a blend of three different virosome parvirosomes
ticles, each assembled separately from one

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Moser, Amacker & Zurbriggen

long-lasting functional antibody titers [36,37,95] . The challenge study

showed evidence of blood-stage efficacy [37] , illustrated by lower
rates of parasite growth and by appearance of morphologically
abnormal parasites in vaccinated volunteers. The third study demonstrates the safety and immunogenicity results for the lyophilized
vaccine formulation, comparable to the previous studies performed
with the liquid, temperature sensitive form of the vaccine [96] .
Hepatitis C virus

The virosomal hepatitis C virus vaccine was designed as a therapeutic T-cell vaccine to clear chronic hepatitis C virus infection.
The vaccine included two peptides representing CTL epitopes,
encapsulated in virosomes, and a peptide acting as a CD4 epitope
displayed on the virosome surface. Preclinical models demonstrated induction of both peptide-specific CTL and CD4 + lymphocytes[46] . In the Phase I clinical trial the vaccine was well tolerated
and safe but failed to induce satisfactory levels of T-cell responses
[Pevion Biotech, Unpublished Data] . It remains unclear whether this
shortcoming is due to the peptide design, or indicates a limitation
of the virosome technology.
Breast cancer

The B-cell vaccine was designed to induce antibodies against Her2,

a target validated by the established passive immunotherapy with
Trastuzumab [97] . Encouraging preclinical results with a virosome
formulation containing three synthetic peptides warranted a Phase
I clinical trial. Beyond meeting the safety end points, the vaccine
induced antibodies in eight out of ten breast cancer patients [35] ,
recognizing not only the peptides in the vaccine but also the fulllength Her2/neu protein. Notably, this response was achieved with
only 10g of each peptide.
HIV

The concept of the prophylactic HIV vaccine is to induce a protective mucosal immunity that has been shown to efficiently prevent
infection [98] . The candidate vaccine is composed of subunit antigens derived from HIV gp41 displayed by virosomes. The liquid
formulation was designed for intramuscular as well as mucosal
administration, and both routes are currently being tested with a
monovalent peptide vaccine in a Phase I clinical trial for safety and
immunogenicity [301] .
Candida

The Candida vaccine aims to prevent recurrent vulvovaginal candidiasis. The vaccine is based on a recombinant, truncated form
of the Sap2 protein, a virulence factor of Candida albicans [99] .
Lyophilized virosome formulations for intramuscular and intravaginal application were developed with the intention to induce
mucosal immunity via a systemic prime/mucosal boost regime. The
vaccine presentation in a capsule for intravaginal application represents a novelty with advantages regarding delivery and patient compliance. The capsule is designed to dissolve upon administration,
resulting in the reconstitution of the lyophilized vaccine invivo,
directly on the mucosal surface. The vaccine is currently being
tested in a Phase I clinical trial for safety and immunogenicity [302] .
442

Expert commentary

The regulatory hurdles for clinical testing and market licensure of

novel vaccines have become increasingly stringent, with a strong
focus on safety and product definition. Subunit vaccines meet
these requirements, in particular when based on synthetic peptides and recombinant proteins. However, owing to their generally
poor immunogenicity, subunit vaccines frequently need to be
combined with adjuvants, which in turn raise safety issues on their
own. Thus, the mode of action of adjuvants has become a central
theme in vaccinology, driven by increasingly detailed insights into
the underlying biological mechanisms.
The knowledge of the mode of action of influenza virosomes
remains fragmented, but there is sufficient data to declare them
multifunctional. Although difficult to investigate, this complexity might be the key to a balanced, efficient, but well-tolerated
immune stimulation, as it mimics the broad range of stimulatory
signals mediated by a natural infection.
With three products on the market, 15years of clinical experience and 70million doses distributed to date, a solid clinical track
record for virosomes has been established, which emphasizes the
safety and efficacy of the technology [33,34] . Over the past 5years,
early stage clinical studies have demonstrated that the same virosome technology is a safe and potent adjuvant system for synthetic
peptides designed as B-cell vaccines against malaria and breast
cancer [35,36,79,95] . Antibodies were induced even in elderly breast
cancer patients, notably against low dosages of peptides derived
from self-antigen [35] . However, the T-cell vaccine approach for
hepatitis C virus failed to induce robust responses in humans
despite encouraging preclinical data [46] .
Unlike most VLPs, virosomes are assembled invitro in a tightly
controlled process. Modifications of the particle structure or
inclusion of additional compounds can be achieved independently
of a host cell system, which saves time and increases reproducibility. The successful development of a temperature-insensitive,
lyophilized form of virosomes illustrates the flexible application
of the system [46] .
Five-year view

A further validation of the virosome technology can be expected

from ongoing clinical studies with new targets (e.g., Candida
and HIV), thereby expanding the antigen spectrum from synthetic peptides to larger recombinant proteins. In addition, both
approaches include a mucosal route of application and may thus
provide further information on the potential of virosomes as a
mucosal adjuvant system. The clinical proof of concept for virosomal subunit vaccines represents the next level of validation,
specifically the demonstration of protective efficacy in humans.
Novel antigens and targets for the virosome technology are
continuously being evaluated at the preclinical stage and are likely
to enter the clinical development phase in the near future.
The mode of action of adjuvants will remain a key topic in vaccinology, and novel insights can be expected on a regular basis.
Regarding virosomes, further investigations on the mode of action
are needed to better define the carrier and adjuvant functions, and
the impact of pre-existing immunity.
Expert Rev. Vaccines 10(4), (2011)

Influenza virosomes as a vaccine adjuvant & carrier system

Financial & competing interests disclosure

Christian Moser and Mario Amacker are employees of Pevion Biotech AG.
All three authors are shareholders of Pevion Biotech AG. The authors have
no other relevant affiliations or financial involvement with any organization

Review

or entity with a financial interest in or financial conflict with the subject

matter or materials discussed in the manuscript apart from those
disclosed.
No writing assistance was utilized in the production of this manuscript.

Key issues
Virosomes are enveloped virus-like particles assembled invitro, composed of purified or synthetic phospholipids and the envelope
fraction of influenza virus.
Virosomes are a commercially validated vaccine platform with an excellent safety and efficacy track record.
Synthetic peptides displayed on virosomes are safe and immunogenic in humans, even at low doses.
Virosomes are an adjuvant and carrier system for subunit vaccines; owing to their structure and composition they feature a
multifunctional mode of action.
Pre-existing immunity against influenza has no negative effect on the functionality of virosomes as adjuvant and carrier system.
The invitro assembly process is current good manufacturing practice compatible and established at commercial scale. It allows for
flexible particle design and integration of various antigen types. The lyophilized form of virosomes gives access to numerous novel
applications of the technology.

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