International Journal of PharmTech Research
CODEN (USA): IJPRIF
ISSN: 0974-4304
Vol.2, No.4, pp 2327-2339,
Oct-Dec 2010
Virosomes: A Novel Carrier for Drug
Delivery
Rajat Sharma1, Mohd Yasir*1
1ITS
College of Pharmacy, Muradnagar,Ghaziabad, UP,201204 (India)
*Corres. Author: mohdyasir31@gmail.com
Tel.: +91-9761131206
Abstract: Live, replicating, vaccines have the advantage that they closely mimic the actual infection and therefore
induce a broad and physiologically relevant immune response, involving both a humoral immune response (antibody
production) and cell-mediated immunity (cytotoxic T lymphocytes). However, there is an increasing concern about the
adverse side effects that may occur as a result of vaccination with replicating pathogen preparations. Therefore, in
general killed whole pathogens or (recombinant) subunit vaccines are used for vaccination. These preparations induce
satisfying antibody responses although less efficient than live, replicating, vaccines. This is due to the way in which the
antigens are processed and presented to the immune system. The development of antigen delivery systems to introduce
nonreplicating antigens into presentation pathways that result in activation of the humoral arm of the immune response,
but also the cytotoxic T-cell arm is therefore of major interest. Virosomes are virus-like particles consisting reconstituted
viral envelope lacking the viral genetic material. Virosomes are virus-like particles consisting reconstituted viral
envelope lacking the viral genetic material. Virosomes represent such a unique system for presentation of antigens to the
immune system. In this review, we will focus on structure of virosomes, preparation of virosomes as carrier vehicles for
the intracellular delivery of drugs, protein antigens and DNA for the induction of a cellular immune response against
encapsulated protein antigens etc, interaction of virosomes with immune cells, pharmacokinetics of virosomes,
applications, advances & future prospects of virosomes.
Keywords: Antigen delivery, Antibody, DNA delivery, Drug delivery Vaccines, Virosomes, etc.
INTRODUCTION
As a tool in the evaluation vesicles of drug efficacy in
living cells and in the treatment of human diseases.
The drug delivery system (DDS) is attractive as a
therapeutic method. To enhance the efficiency of gene
delivery by the introduction of molecules directly into
cells, virosomes have been developed by combining
various agents like drug, antigen and DNA with
fusiogenic viral envelope proteins.1, 2 Therefore;
enhancement of delivery efficiency in vivo is a major
objective in the field of virosomes research. Despite
improvements in viral and non-viral vector systems, a
major hurdle in the delivery of drugs and other
macromolecules into the desired cell types is crossing
the permeability barrier imposed by the plasma
membrane followed by the controlled release inside
the cytoplasm 1. On the other hand, it is known that
controlled release of various agents can be
significantly improved by using virosomes carriers 2, 3
First, virosomes closely resemble the envelope of the
virus they are derived from and therefore constitute
antigen-presentation form superior to isolated surface
antigens.3 In addition, properly assembled virosomes
retain the membrane fusion activity of the native virus
and, therefore, virosomes may be used to deliver
encapsulated, unrelated, antigens to the cytosol of
antigen-presenting cells. In this respect, virosomes
differ from conventional liposomes which will target
enclosed antigens primarily to the phagolysosomal
system of macrophages. We have recently exploited
both aspects of virosomes, derived from influenza
virus, to induce CTL activity against a virosomeencapsulated antigenic peptide and whole protein.4
Virosomes are spherical, unilamellar vesicles with a
mean diameter of 150 nm. Essentially, (fig. 1)
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
virosomes represent reconstituted empty influenza
virus envelopes, devoid of the nucleocapsid including
the genetic material of the source virus.4, 5 Virosomes
are not able to replicate but are pure fusion-active
vesicles. In contrast to liposomes, virosomes contain
functional viral envelope glycoproteins: influenza
virus hemagglutinin (HA) and neuraminidase (NA)
intercalated in the phospholipid bilayer membrane.5
The unique properties of virosomes partially relate to
the presence of biologically active influenza HA in
their membrane. This viral protein not only confers
structural stability and homogeneity to virosome-based
formulations, but it significantly contributes to the
immunological properties of virosomes, which are
clearly distinct from other liposomal and
proteoliposomal carrier systems.6 It has been shown
that a physical association between the virosome and
the antigen of interest is necessary for the full adjuvant
effect of virosomes. Such physical association can be
achieved by a variety of methods, depending on the
properties of the antigen. Antigens can be incorporated
into virosomes, adsorbed to the virosome surface, or
integrated into the lipid membrane; either via
hydrophobic domains or lipid moieties cross-linked to
the antigen.7 Virosomes therefore represents an
innovative, broadly applicable adjuvant and carrier
system with prospective applications in areas beyond
conventional vaccines. They are one of only five
adjuvant systems widely approved by regulatory
authorities and the only one that has carrier
capabilities.8
Fig. 1: Structure of influenza virosome
Key Strengths 3, 9
Virosome technology provides a broadly
applicable delivery system for antigens or
2328
DNA/RNA encoding specific immune stimulatory
proteins.
 Virosome technology enables target-specific
delivery of antigens and amplification of the
immune response.
 Virosomes stimulate both arms of the immune
system – eliciting antibody and cellular
immune responses - against inserted immune
stimulatory proteins derived from human
pathogens.
 Virosomes are completely biodegradable and
can exert an immune response via different
routes of administration
KEY ADVANTAGES OF VIROSOMES 10, 11,
12
High quality, effective and long-lasting antibody
responses
 Efficient delivery and presentation of antigens
to the immune system
 Conformational stabilization of protein and
peptide antigens, even of barely soluble
antigens
 Antigen
protected
from
extracellular
degradation
 Presentation of antigen to B cells in repetitive
array on virosome surface
 Natural uptake and processing of the antigen
by professional APC
 Strong immunostimulation through efficient
recruitment of T cell help
 Excellent safety and tolerability profile
 Suited for vulnerable populations such as
elderly, infants, and immnunosuppressed
 Mainly
synthetic
and
biodegradable
components
VIROSOME- BASED TECHNOLOGY
PLATFORM
Virosomes are a market approved carrier and
adjuvant system for the delivery of immunologically
active substances. This predominantely synthetic
carrier is broadly applicable with almost
any antigen. Peptides, (recombinated) proteins or
carbohydrates of interest are combined with a lipid
anchor, which enables the attachment on the surface of
virosomes: 13, 14
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
2329
Fig. 2: Modular virosome-based vaccine design
PREPARATION OF VIROSOMES
Virosomes made from influenza virus retain the cell
entry and membrane Fusion capacity of this virus 15, 16.
Functionally reconstituted influenza virosomes will
bind to sialic acid residues on the surface of cells and
enter the cell via receptor mediated endocytosis 17, 18.
Upon endocytosis, the low pH in the endosomes
induces fusion of the virosomal membrane with the
endosomal membrane, causing the release of the
contents of the virosome into the cytoplasm of the cell.
The fusion process is mediated by hemagglutinin, the
major envelope glycoprotein of influenza virus 19, 20.
These procedures involved in the insertion of influenza
HA and NA into immunostimulating complexes
(ISCOMS) 21, 22, plain liposomes, liposomes containing
immunomodulators such as muramyldipeptide 23, 24, or
liposomes containing cationic lipids 3, 25. These
structures are called immunopotentiating reconstituted
influenza
virosomes
(IRIVs),
(fig.
1)
Immunopotentiating reconstituted influenza virosomes
(IRIVs) are prepared by the detergent removal of
influenza surface glycoproteins, hemagglutinin (HA)
and neuraminidase (NA), which are subsequently
combined with natural and synthetic phospholipids.
The phospholipids consist of 70% lecithin, a structural
component, 20% cephalin, a structural component that
also stimulates B cells independently of T-cell
determinants and can bind hepatitis A antigen, and
10% envelope phospholipids, originating from the
selected influenza virus. The resulting IRIVs are
spherical, unilamellar vesicles with a mean diameter of
approximately 150 nm and, due to the low viral and
avian protein content, are a virtually non-immunogenic
delivery system. Sendai virus virosomes have been
generated by reconstitution of the Sendai fusion
protein (F-protein), with or without the hemagglutinin–
neuraminidase protein (HN-protein) in viral lipids 26.
Rubella virus virosomes were prepared by
incorporating E1 and E2 envelope glycoproteins into
liposomes 27 and vesicular stomatitis virus (VSV)
virosomes were generated by adding the G-protein of
VSV to preformed liposomes 28, 29. In addition,
virosomes have been generated based on Epstein–Barr
virus 30, human immunodeficiency virus 31, Semliki
Forest virus 32, Friend murine leukemia virus 33, herpes
simplex virus 34 and Newcastle disease virus
Preparation of Virosomes for Drug Delivery
The concept of conferring viral functions to liposomes
will lead to a new field in drug delivery new virosome
vectors will possess the properties of efficient delivery
derived from viral molecules and of lessened toxicity
derived from liposomes. Recently, a new approach,
constructing influenza virosomes with a cationic lipid
was reported. Influenza virus A was solubilized with
detergent and mixed with a cationic lipid,
dioleoyldimethylamonium chloride (DODAC), at 30%
to form a cationic virosomes. Plasmid DNA was
complexed with these cationic virosomes and was
successfully transferred to cultured cells by low pHdependent membrane fusion.35. The main feature is
also attractive for cytoplasmic drug delivery if efficient
encapsulation of the respective compound and specific
targeting of the vehicle can be achieved. A successful
application of the concept has been described by
Waelti et al.36, showing the inhibition of tumour
progression in a mouse model after treatment with a
virosomal formulation. The formulation included
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
phosphatidylethanolamine-PEG-an-chored antibodies
for targeting and HA for cytoplasmic delivery of the
encapsulated doxorubicin. After receptor mediated
endocytosis and endosomal fusion, virosomes are able
to deliver their content (e.g. drugs) into the cytosol.
Using monoclonal antibodies to tumor associated
antigens, virosomes can be targeted to cancer cells and
therefore allow specific immunotherapy 36.
Preparation of Virosomes for Antigen Delivery
The potentialfor virosomes as delivery systems for
peptide and nucleic acid based vaccines has been
investigated for several diseases including malaria,
melanoma, hepatitis C virus and Alzheimer’s disease
37
. When virosomes are to be used for the delivery of
antigen into the cytosol of cells, fusion activity of the
virosomes is of major importance. Studies have shown
that virosomes as a delivery system are suitable for the
efficient induction of antibody responses against
conformational epitopes by use of cyclic-template
bound peptidomimetics. Virosomes as antigen carriers
both protect the incorporated peptide and adjuvants
and provide additional immunogenicity. By virtue of
the fact that reconstituted viral envelopes closely
mimic the outer surface of the virus they are derived
from, virosomes also represent a very useful system
for the induction of antibody responses against the
native virus 38. Furthermore, virosomes may also be
used to incorporate other unrelated antigens in the
virosomal membrane. For example, Glqck et al. have
incorporated hepatitis A virions (HAV) in influenzaderived virosomes and observed a strong stimulation
of the HAV-specific antibody response 23. A
virosomal HAV vaccine (Epaxal) is currently on the
market. Cytoplasmic delivery of influenza virosomeencapsulated material has been conclusively
demonstrated using fragment A of diphtheria toxin
(DTA) 39. Fusion-active influenza virosomes have the
ability to efficiently deliver encapsulated DTA to the
cytosol of cells, as evidenced by complete inhibition of
cellular protein synthesis. The capacity of virosomes to
deliver antigen to the MHC class I presentation route
in vivo and to activate antigen-specific CTLs was first
assessed in immunization experiments in mice, using
influenza virosomes containing a synthetic
Peptide epitope from influenza NP 40.Antigen doses as
low as 0.7 μg per injection sufficed to induce CTL
activity (Bungener et al., Vaccine, in press).
Intramuscular, intraperitoneal and subcutaneous
immunisations effectively induced CTLs. Although the
booster immunisation potentiated the CTL response, a
single immunisation of as little as 1.5 μg of OVA in
fusion-active virosomes sufficed to induce CTL
activity. Fusion-inactive OVA virosomes were also
able to induce CTL responses, yet the percentages of
specific lysis were lower than for mice immunised
2330
with fusion-active virosomes. This indicates that the
fusion activity of the virosomes potentiates, but is not
essential for CTL induction in vivo. Immunisation
with OVA in virosomes also resulted in a humoral
immune response as demonstrated by the presence of
anti-OVA
IgG in the sera of immunised mice.
Preparation of Virosomes for DNA/RNA Delivery
Virosomes are also suitable as a DNA/RNA carrier
system, since these fusogenic particles mimic a virus.
The genetic material is enclosed in the virosome and is
therefore protected from DNAase and RNAase 3. Gene
therapy requires vectors that are efficient and safe, and
simple to prepare. Although viral vectors, in general,
very efficiently transfect a variety of cell types, safety
issues for most viral vector systems is still a major
concern. Accordingly, nonviral techniques for gene
transfer that is as efficient as viral vectors are to be
developed. DNA vaccines have been developed and
numerous clinical studies have been performed.
Preclinical studies demonstrated that expression of an
antigen or antigens from plasmid DNA may elicit both
humoral and cellular immune responses. DODACcontaining DNA-virosomes based on influenza virus
lipids and proteins, demonstrated the potential of the
influenza virus fusion protein HA to promote uptake
and intracellular delivery of virosome-associated
plasmid DNA41. DOTAP-influenza virosomes
demonstrated DNA or oligonucleotide delivery into
human tumour cells and human DCs 42. In vitro as well
as in vivo studies demonstrated the potential of DNAvirosomes based on Sendai virus for gene transfection
and expression 43. In an elegant study by Ramani et
al.43, it was shown that upon intravenous injection of
Sendai Fvirosomes containing a CAT-encoding
plasmid, CAT gene expression, both mRNA and
protein, was observed predominantly in the liver. Up
to now, only three studies have been published on the
immune responses elicited by the administration of
DNA-virosomes. The influenza DOTAP virosomes
used in the first study were exogenously loaded with a
plasmid DNA encoding the mumps virus
hemagglutinin or the mumps virus fusion protein 44.
DNA expressing the parathyroid hormone related
peptide (PTH-rP), a protein secreted by prostate and
lung carcinoma cells was coupled to virosomes or
IRIVs DNA immunization has emerged as a strikingly
novel approach to immunoprophylaxis. Immunization
with naked DNA encoding variousproteins, promises
to be a valuable vaccine approach especially if its
immunogenicity can be optimized. In order to avoid
the injection of high amounts of DNA for vaccination,
efficient gene transfer techniques must be employed
for an acceptable vaccine in humans. Virus mediated
gene transfer generally presents an
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
underned risk of infection or Inflammation.45
KEY PROBLEMS ASSOCIATED WITH
VIROSOMES PREPARATION 44, 45
Problems
encountered
in
development
of
pharmaceutical virosomes:
 Shelf-life is too short
 Scale up related problems
 Poor quality of raw material
 Pay-load is too slow
 Absence on any data on safety of these carrier
systems on chronic use
But in recent years several solutions have been worked
upon to remove above mentioned problems: 46
 High quality products with improved purification
protocols and validated analytical techniques are
available
 Quality control assay can be performed using
sophisticated instruments and batch to batch
variability is can be checked
2331
 Payload problems can be sorted out using either
lipophillic drug/lipophillic prodrug of hydrophilic
drugs or using active (remote loading) techniques.
 Shelf life can be improved using appropriate
cryoprotectant and lyoprotectant and product can
be successfully freeze dried.
 Scaling up can be improved by carefully selecting
method of prepration, sterilization by autoclaving
or membrane filtration (0.2 micrometer) coupled
with aseptic and pyrogen removal using properly
validated Lal test
 By choosing candidate potent drugs with narrow
therapeutic window (eg. Cytotoxic drugs, and
fungicides) the drug related safety problems can be
alleviated
INTERACTIONS OF VIROSOMES WITH
CELLS OF THE IMMUNE SYSTEM
By virtue of the repetitive arrangement of
haemagglutinin on the virosome surface, virosomes
interact efficiently with immunoglobulin receptors on
B lymphocytes.3, 25
Fig. 3: Interactions of Virosomes with Cells of the Immune System
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
2332
Fig. 4: Mechanism of Virosomes to Stimulate the Immune Systems
Virosomes are also taken up avidly by antigenpresenting cells, particularly dendritic cells. Antigens
on the virosome surface, as well as antigens derived
from degraded virosomes, enter the MHC class II
pathway, activating T helper cells. Antigens inside the
virosomes, through fusion of the virosomes, access the
cytosolic MHC class I presentation pathway, activating
cytotoxic T lymphocytes (CTL) (fig. 3)
Virosomes can be used for cell-and organ or tissue
specific drug delievery of pharmaceutically active
substances in the body.. 5, 9, 47 The unique properties of
virosomes partially relate to presence of HA in their
membrane. This viral protein not only confers
structural stability and homogeneity to virosomal
formulations, but also significantly contributes to the
virosomal formulations, but also significantly
contributes to the fusion activity of virosomes which
induces endolysomal pathway.48 On the virosomal
surface, ligands are attached. This function is crucially
for the targeted drug delievery of drugs. Therefore,
virosomes selectively bind to their ligands to the target
cell. Likewise, the virosomes HA PROMOTES
promotes BINDING TO THE TARGET CELL and
receptor mediated endocytosis. In the endosome, the
virosomal HA- triggered by an acidic e enviourmentmediates membrane fusion, and therapeutically active
substances escape from endosomes into cytoplasm of
target cell. This concept has been validated in in-vivo.
The cytotoxic drugs doxorubicin was encapsulated into
the virosomes. On their surface, monoclonal antibodies
were cross-linked, mediating specific targeting carrier
to the cancer cells. 48(fig. 4)
CHARACTERISTICS OF VIROSOME
Virosome technology is competitive
Virosome technology represents a new frontier in
vaccinology, simultaneously improving the delivery of
specific antigens to target immune cells, while
bypassing the problems of conventional adjuvantbased vaccines. Thus virosomes stimulate antibody
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
and cellular immune responses against the inserted
antigens of a human pathogen. Virosomes are
completely biodegradable and virosome-based
vaccines do not exert the adverse reactions usually
associated with adjuvant activity.49 Virosome
formulations are compatible with intramuscular,
intradermal and intranasal administration and are
broadly applicable with almost any given antigen
Virosome technology for highly immunogenic
vaccines
Two marketed products validate virosome technology:
vaccines against hepatitis A and influenza. These
products are registered for human use in more than 40
countries and have been administered to more than 30
million individuals. Virosomal vaccines have been
shown to elicit highly protective immune responses
with excellent safety profiles. Side effects are much
less frequent after the application of virosomal
vaccines compared to alum-precipitated antigens
Virosome–based products from bench to clinic
Today many companies in world has fully operational
GMP laboratories dedicated to virosomal products
with integrated upstream and downstream capacities
up to a scale of several 100,000 doses. These units are
used to develop and produce any new virosomal
formulation under strict cGMP conditions.
VIROSOME PHARMACOKINETICS
Pharmacokinetics information can be used to interpret
the differences in the pharmacological effect of
liposomal-entrapped drug and free drug, and
subsequently
can
be
exploited
for
dose
designing.virosome pharmacokinetics deal with time
course of the absorption, distribution and degradation
of the virosomal carriers in vivo.The pharmacokinetics
of virosomes requires the knowledge of possible
accessible sites after intravenous administration as this
is the most accepted route for various virosomal
formulations exploited for clinical therapeutics except
topical formulations.Virosomes alter both the tissue
distribution and the rate of clearance of a drug as they
are affected by the pharmacokinetics parameters of
carrier. Under optimal conditions, the drug is carried
within the virosomal aqueous phase during circulation,
but leaks at sufficient rate to become bioavailable on
arrival at tissue or other sites. Bioavailability in case of
virosomal carriers can be defined as the amount of free
drug that is able to escape the confines of the carrier
and thus become available for redistribution to
neighboring tissue.
Final outcome of virosomal delivery of drugs on
biopharmaceutical and pharmacokinetic parameters
are: 21, 50
 Higher therapeutic index
2333
 Decrease in the amount and type of
nonspecific toxicity.
 Protection of drug from metabolism and
inactivation in plasma.
 Reduced volume of distribution and hence
decrease in non-specific localization.
 Increased in concentration of drug at target
site.
RATIONALE FOR USING AND DEVELOPING
VIROSOMES 51
 Potential for sustained release dosage forms –
dexamethasone palmitate, barbiturates,
physostigmine salicylate.
 Site specific delivery to various organs:
cytotoxic agents.
 Solubilization of poorly water soluble drugs:
diazepam, vitamin a, propofol,dexamethasone
palmitate.
 Prevention of drug uptake by infusion sets:
diazepam,perilla ketone.
KEY POINTS FOR EVALUATION OF
VIROSOME 52, 53
 Vesicle shape and surface morphology:
Transmission electron microscopy, freeze
fracture electron microscopy
 Vesicle size and size distribution: Dynamic
light
scattering,
transmission
electron
microscopy, zetasizer, photon correlation
spectroscopy, laser light scattering, gel
permeation and gel exclusion
 Surface charge: free flow electrophoresis
 Electrical surface potential and surface ph:
zeta potential measurements and ph sensitive
probes
 lamellarity: small angle x-ray scattering,
freeze fracture electron microscopy, 13p- nmr
 Phase behavior: freeze fracture electron
microscopy, differential scanning colorimetry
 Percent of free drug: mini column
centrifugation, gel exclusion, ion exchange
chromatography,
protamine
aggregation,
radiolabel ling
 Drug release: diffusion cell/ dialysis
 Pyrogenicity: rabbit fever response test or
limulus ambeocyte lysate (lal) test
 Animal toxicity: monitoring survival rates,
histology and pathology
 Chemical analysis of surface: static
secondary
ion
mass
spectrometry,
spectrometer
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
3, 6, 10, 36
APPLICATIONS OF VIROSOMES
(A) General Application
1. Genetic(dna) vaccination
2. Blood substitutes for hemoglobin
3. Immunoadjuvat, immunomodulator,
immunodiagnosis
4. Artificial blood surrogates
5. Pharmaceutical pigments or dyes
6. In gene delivery
7. In tumor therapy a carrier of small cytotoxic
molecules
8. Vehicle for macromolecules as cytokines or
genes
9. As biological response modifiers
10. Virosomal drugs
11. Virosomes as drug/protein drug delivery
vehicles
12. As radiopharmaceutical and radio diagnosis
carriers
13. As gene therapy genetic targeted intracellular
material delivery
14. Enzyme replacement therapy and lysosomal
storage disorders.
15. In cosmetics and dermatology
16. Enhanced drug solubilization
17. In antifungal (lung therapy), antiviral and anti
microbial therapy
18. Altered pharmacokinetics and biodistribution
19. Masking action
20. Drug overdose treatment
21. Separation and extraction technique
22. In fabrication of microcapsulated dosage form
23. Enzyme immobilization
(B) Biomedical Applications
1. Virosomal loaded antibiotics used potentially
for treatment of – Protozoan- leshminiasis
(antimonials,
amphotericinB)
Bacterialtuberclosis,
leprosis,
salmonellosis,
brucellosis,
Fungalhistoplasmosis
(amphotericinB), cryptococcoses(amphotericin
b),Viral- herpes simplex virus, aids
2. Antigens and allergens extract given by im, sc,
oral used in desensitization desiring high yield
allergen entrapment, stability.
3. Virosomes associated with hemoglobin,
synthetic oxygen transporters given by i.v route
used as blood surrogates desiring high yield hb
entrapment, stability and long circulating .
2334
4. Virosomes associated with plasmid dna,
antisense oligonucleotides given by
iv,im,ia,it
topical and oral used in gene and antisense therapy
desiring high yield
DNA incorporation,
fusogenic(cationic,virosome,phsensitive),and
targeted
5. As endogenous cytokines (IL, IFN, TNF, GMCSF)
given
by
iv,im,sc,it
used
as
immunomodulators
desiring
high
yield
incorporation, stability, controlled Clearance rates
and targeted
6. Used in macrophage activation for protection
against klebesiella pneumniae
7. Used in macrophage activation for tumoricidal
properties against spontaneous metastases
(C) List of Antigens Where Virosomes Have
Been Used as Immuno Adjuvants
1. Rabies glycoprotein- antigen specific
interleukin-2-enhancement
2. Herpes simplex virus- mlv with lipid A,
enhanced ab level
3.
Bacterial
polysacchrides-superirr
immunoadjuvants.
4. Influenza subunit antigen-intranasal, protects
animal from virus
5. Streptococcus mutant carbohydrate antigenincreased antibody titre in salivary glands
6. Poliovirus peptides-enhanced antibody level
7. Cholera toxin-superior immunoadjuvants
8. Hepatitis virus surface antigan-PC/chol/DCP,
higher antibody response
9. Tetanus toxoid- Mannose mediated targeting,
increased antibody titre
10. Filamentous haemagglutinis and detoxified
pertussis toxin of bortella pertussis-very effective
vaccine.
ADVANCES AND FUTURE PROSPECTS
Until other carrier systems of age, virosomal
technology will remain a hotspot active area for future
research.54 They represent the one of the simplest yet a
novel drug carrier based on the fundamental principle
of self assembly.Pevion Biotech Company is currently
developing products for human use and is currently
testing the commercial medical applications of
virosomes. The coming years represent a critical time
in this field as commercial applications has been
explored. In near future virosomes based delivery
system with their ability to provide controlled and site
specific drug delivery revolutionize disease
management.55
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
2335
Table 1: Marketed Products of Virosomal drug delivery 57
S. No. Virosomal preparations
Application
A
Virosomes antigen based products
1 Hepatitis A virus envelope proteins
1
Hepatitis a
(EpaxalW)
2
Influenza virus (InflexalW V)
Influenza
Virosomal antigen preparations
B
under clinical trials
1
Diphtheria/tetanus toxoid virus envelope
Diphtheria, Tetanus
proteins
2
Peptidomimetic of loop I from domain III Malaria
of Plasmodium falciparum AMA-1
3
PEV6
Breast cancer
C
Virosomal antigen preparations under
pre clinical trials
1
Doxorubicin
Cancer
2
Doxorubicin
Ovarian carcinoma
3
L-myc antisense ODNs
Cancer
DNA-encoded TAA Prostate
4
Carcinoma
5
DNA-encoded mumps antigen
Mumps
6
Melan A/Mart-1 peptides
Melanoma
7
RSV F protein
RSV
8
Hepatitis C peptides
Hepatitis C
9
VSV G protein
Vesicular stomatitis
10
NDV envelope proteins
Newcastle disease
11
HIV proteins
AIDs
12
PEV4
RSV
13
PEV7
Candida
14
PEV 8
Universal influenza
Table 2: Recent US Patents of Virosomal Drug Delivery 58
S.No.
US Patent No.
Title
1
7576066
Nucleic acid compositions for stimulating immune responses
2
7615227
Use of CpG oligodeoxynucleotides to induce angiogenesis
3
7615377
Fluorescein-based metal sensors
4
7615539
Nucleic acid-lipophilic conjugates
5
7618641
Functionally reconstituted viral membranes containing adjuvant
Recently berna a crucell company has been granted a
patent from us patent office for F-VIROSOMES. (US
Patent No.5, 683, 866 dated November 4, 1997).
1) This delivery vehicle (F-virosome) consisting
of the envelope of Sendai virus containing
only the fusion protein (F) and devoid of its
own genetic material and other undesirable
proteins, holds minimum risk of any side
effects.
2) The F-virosomes containing the genes of
interest (e.g. CAT and luciferase) specifically
bind and fuse with liver parenchymal cells
(both in vitro and in vivo) resulting in high
efficiency gene transfer to the nucleus of these
cells, bypassing the degradative endocytotic
route.
3) A single intravenous administration of DNA
loaded F-virosomes into Balb/c mice resulted
in expression of CAT mRNA and the
corresponding protein till four months post
injection. The delivered DNA was found in the
mouse chromosomes and persisted till two
months.
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
4) No adverse immune side effects (antibodies
against F protein) were observed in Fvirosome injected mice.
5) Preliminary experiments have been done in
delivering a therapeutic gene (bilirubin-UDPglucuronosyl transferase, BUGT) into Gunn
rats (lacking the BUGT gene showing high
levels of serum bilirubin) through F-virosomes
and found to be encouraging in terms of
reduction of serum bilirubin levels.
Fig 5: Safety Profile of Virosomes
Novelty of F-Virosome Mediated Gene Delivery 56,
1. Specific cytosolic delivery of genes and their
expression in cells using Sendai viral envelope devoid
of hemagglutinin-neuraminidase and its own genetic
material thereby totally eliminating the possibility of
any side effects. Sendai virus, which is the starting
material for developing this delivery system, is known
to be non pathogenic to humans.
2 Dual roles (in binding and membrane fusion) of F
protein of Sendai virus in gene delivery
3. Targeted delivery of genes into liver cells in culture
using modified Sendai viral envelopes.
4. Considerably higher efficiency of gene delivery as
compared to existing delivery systems such as
liposome and commercially available systems like
lipofectin. The genes delivered by this system remain
intact and are able to express biological by active
protein products.
2336
Uses: some of the Potential Diseases for which
therapeutics can develop using this delivery system
such as: Hemophilia, HBV, Diabetes, Cancer.
CONCLUSION
Vaccines have been well accepted and used effectively
for more than 100 years. Traditional vaccines are
generally composed of whole inactivated or attenuated
microorganisms that have lost their disease-producing
properties, or they are composed of subunits of
organisms such as an outer coat protein. These
classical prophylactic vaccines evoke a humoral
immune response and are therefore immunogenic, but
they often have been associated with an unfavorable
safety profile. For the evaluation of vaccines different
key factors have to be considered. One key factor is its
immunogenicity or effectiveness in immunizing an
individual. A second key factor in evaluating a vaccine
is its tolerability with respect to the vaccination
recipient. A vaccine’s tolerability is largely linked to
the type of adjuvant it uses to enhance its
immunogenicity. Aluminum-based compounds are the
oldest adjuvants and have been used in vaccines for the
past 70 years. Most commercial adjuvanted vaccines
today use aluminum salts, such as aluminum
hydroxide, as an adjuvant. However, as aluminum has
been linked to localized side effects such as rashes,
swelling and pain at the injection site of the vaccine,
efforts have been made to develop other potent
adjuvants that are better tolerated by vaccine
recipients.
A new generation of prophylactic and therapeutic
vaccines is needed: vaccines capable of inducing a
strong specific immune response and possessing a
more favorable safety profile than the classical
preparations. These issues can be addressed by
developing vaccines which are not based on isolated
biological material but which are produced
synthetically. An increasing number of antigens (e.g.
proteins, peptides or carbohydrates) capable of
inducing humoral and cellular immune responses have
been identified. However, these new epitopes are often
weak immunogens. It is therefore important to present
them to the immune system in such a way that a
specific and strong immune response is induced.
Virosomes fulfill all the prerequisites necessary to
serve as new gold standard adjuvant and carrier system
for next generation prophylactic vaccines.
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
REFERENCES
1. Targeting HER-2/neu with Antirat Neu Virosomes
for Cancer Therapy, Cancer Res 62, 437-444
2. Huckriede A, Bungener L, Stegmann T, Daemen
T, Medema J, Palache AM, Wilschut J. The
virosome concept for influenza vaccines
University Medical Center Groningen, Department
of Medical Microbiology, Molecular Virology
Section, University of Groningen, Ant.
Deusinglaan 1, 9713 AV Groningen, The
Netherlands
3.
Schumacher R, Adjuvance of influenza virosomes
in CTL induction in vitro, PhD Thesis, University
of Basel, Faculty of Science, 2005
4. Ernst W, Nina W, Ruth S, Antionette W, Carsten
W, Barbara RR, Claude DG, Targeting HER-2/neu
with Antirat Neu Virosomes for Cancer Therapy,
Cancer Res ry 15 (1), 437,2002
5. Peter H,
Richard G, Fusogenic Virosomes
Prepared by Partitioning of Vesicular Stomatitis
Virus G Protein into Preformed Vesicles,From the
Department of Molecular Genetics, Biochemistry,
and Microbiology, University of Cincinnati
College of Medicine,269(6), 4050-4056, 1994
6. Trudel M, Marchessault F. Payment P, Rubella
virosomes: Preparation and ultrastructure, Journal
of Virological Methods, 3, (4), 187-192, 1981
7. Toos D, Laura B, Anke H, Jan W, Virosomes as
an Antigen Delivery System, Journal of Liposome
Research, 10, (4 ) 2000 , 329 – 338
8. Karin M, Karin M, Evgeni G., Michael R,
Michael FG. Schmidt, Andreas H..Lipid
Composition of Virosomes Modulates Their
Fusion Efficiency with Cryopreserved Bull Sperm
Cells, Cloning, 3(1), 2004, 135-148
9. Xinyu L, Sibylle S, Mario A, Rinaldo Z, Gerd P
and PH. Seeberger, Enhancement of the
Immunogenicity of Synthetic Carbohydrates by
Conjugation to Virosomes: A Leishmaniasis
Vaccine Candidate, ACS Chem. Biol., 1 (3),
2006,161–164
10. Komal R, Roop S ,Mukesh K, Sandeep T, Feeb L,
Novel gene delivery to liver cells using engineered
Virosomes, Current Medicinal Chemistry, 404, (
2), 1997, 164-168
11. Michel A, Walter P. Weberb, Rachel R, Reto S,
Paul Z, Ulrich G, Daniel MF, Daniel O, Markus Z,
Michael H, Giulio C S, Heterologous prime-boost
immunotherapy of melanoma patients with
Influenza virosomes, and recombinant Vaccinia
virus encoding 5 melanoma epitopes and 3 costimulatory molecules. A multi-centre phase I/II
open labeled clinical trial, Contemporary clinical
trials , 29 (2), 2008, 165-181
2337
12. . Elena A, Conference Report - Adjuvants and
Delivery:
Improving
on
Vaccine
Immunogenicity,Highlights From the Viral
Vaccine Meeting; October 25-28, 2003;, Spain
13. Khoshnejad, M., Young, PR., Toth I. Minchin RF
Modified Influenza Virosomes: Recent Advances
and Potential in Gene Delivery. Current Medicinal
Chemistry, 14 29, 2007 3152-3156.
14. Shoji J, tanihara Y, uchiyama T, kawai A,
Preparation of Virosomes Coated with the
Vesicular Stomatitis Virus Glycoprotein as
Efficient Gene Transfer Vehicles for Animal Cells,
Microbiol Immunol, 48 (3), 2004, 163-174
15. Bron R, Ortiz A, Dijkstra J, Stegmann T,
Wilschut J. Preparation, Properties and
applications of reconstituted influenza virus
envelopes (virosomes). Methods Enzymol, 220,
1993, 313–331.
16. . Stegmann T, Morselt HW, Booy FP, van
Breemen JF, Scherphof G, Wilschut J. Functional
reconstitution of influenza virus envelopes, EMBO
J, 6, 1987, 2651–2659.
17. Matlin KS, Reggio H, Helenius A, Simons K.,
Infectious entry pathway of influenza virus in a
canine kidney cell line. J Cell Biol, 91, 1981, 601–
613.
18. Lanzrein M, Schlegel A, Kempf C., Entry and
uncoating of enveloped viruses, Biochem
J.,302(Part 2), 1994, 313–20.
19. Wilschut J, Bron R. The influenza virus
hemagglutinin: membrane fusion activity in intact
virions and reconstituted virosomes. In: BentzJ,
editor. Viral fusion mechanisms. Boca Raton:
CRC Press, 1993, 133–61.
20. Skehel JJ and Wiley DC., Receptor binding and
membrane fusion in virus entry: the influenza
hemagglutinin, Annu Rev Biochem, 69, 2000,
531–69.
21. Morein B., Barz D., Koszinowski U.,.
Schirrmacher V, Integration of a virus membrane
protein into the lipid bilayer of target cells as a
prerequisite for immune cytolysis. Specific
cytolysis after virosome target cell fusion, J. Exp.
Med. 150, 1979, 1383– 1398.
22. Coulter A., Harris R., Davis R., Drane D., Cox J.,
Ryan D., Sutton P., Rockman S., Pearse M.,
Intranasal vaccination with ISCOMATRIX
adjuvanted influenza vaccine, Vaccine 21,2003,
946– 949.
23. Nerome K., Yoshioka Y., Ishida M., Okuma K.,
Oka T., Kataoka T., Inoue A., Oya A.,
Development of a new type of influenza subunit
vaccine made by muramyldipeptideliposome:
enhancement of humoral and cellular immune
responses, Vaccine, 8, 1990, 503–509.
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
24. Iinuma H., Nerome K., Yoshioka Y., Okinaga K,
Characteristics of cytotoxic T lymphocytesdirected
to influenza virus haemagglutinin elicited by
immunization with muramyldipeptide- influenza
liposome vaccine, Scand. J. Immunol, 41, 1995, 1–
10.
25. Glqck R, Mischler R., Brantschen S., Just M.,
Althaus B.,. Cryz SJ Immunopotentiating
reconstituted influenza virus virosome vaccine
delivery system for immunization against hepatitis
A, J. Clin. Invest. 90, 1992, 2491–2495.
26. Bagai S, Sarkar DP, Effect of substitution of
hemagglutinin– neuraminidase with influenza
hemagglutinin on Sendai virus F protein mediated
membrane fusion, FEBS Lett.,353, 1994, 332– 336
27. Orellana A., Mottershead D., van der Linden I.,
Keinanen K., Okerblom C., Mimicking rubella
virus particles by using recombinant envelope
glycoproteins and liposomes, J.Biotechnol., 75,
1999, 209–219.
28. Loh D. Ross AH, Hale AH, Baltimore, Eisen HN,
Synthetic phospholipid vesicles containing a
purified viral antigen and cell membrane proteins
stimulate the development of cytotoxic T
lymphocytes, J. Exp. Med., 150, 1979, 1067–
1074.
29. Shoji J, Tanihara Y, Uchiyama T.,. Kawai A,
Preparation of virosomes coated with the vesicular
stomatitis virus glycoprotein as efficient gene
transfer vehicles for animal cells, Microbiol.
Immunol, 48, 2004, 163– 174.
30. Grimaldi S.,. Giuliani A, Giuliani A., Ferroni L.,
Lisi A., Santoro N., Pozzi D., Engineered
liposomes and virosomes for delivery of
macromolecules, Res. Virol. 146, 1995, 289– 293.
31. Cornet B., Vandenbranden M., Cogniaux J.,
Giurgea L., Dekegel D.,. Ruysschaert JM,
Virosomes
reconstituted
from
human
immunodeficiency virus proteins and lipids,
Biochem. Biophys. Res. Commun. 167, 1990,
222– 231.
32. Helenius A., Fries E, Kartenbeck J, Reconstitution
of Semliki Forest virus membrane, J.Cell Biol., 75,
1977,866– 880.
33. Schneider J., Falk H., Hunsmann G., Virosomes
constructed from lipid and purified Friend
leukaemia virus glycoprotein, J. Gen. Virol., 64,
1983, 559– 565.
34. Johnson DC, Wittels M, Spear PG, Binding to
cells of virosomes containing herpes simplex virus
type 1 glycoproteins and evidence for fusion, J.
Virol., 52, 1984, 238– 247.
35. Kapczynski DR, Tumpey TM, Development of a
virosomes vaccine for Newcastle disease virus,
Avian Dis., 47, 2003, 578– 587.
2338
36. Waelti E, Wegmann N, Schwaninger R,
Wetterwald A, Wingenfeld C, Rothen-Rutishauser
B, Gimmi CD: Targeting her-2/neu with antirat
Neu virosomes for cancer therapy. Cancer Res.,
62, 2002, 437-444.
37. Yasufumi K, Virosomes: evolution of the
liposome as a targeted drug delivery
system,Advanced Drug Delivery, 43, 2000, 197–
205.
38. Huckriede A., Bungener L., ter Veer W.,. Holtrop
M, Daemen T., Palache A.M., Wilschut
J,Influenza virosomes: combining optimal
presentation of hemagglutinin immunopotentiating
activity, Vaccine, 21, 2003, 925– 931.
39. Bron R., Ortiz A., Wilschut J., Cellular
cytoplasmic delivery of a polypeptide toxin by
reconstituted
influenza
virus
envelopes
(virosomes), Biochemistry, 33,1994, 9110 – 9117.
40. Arkema A., Huckriede A, Schoen P, Wilschut J.
Daemen T, Induction of cytotoxic T lymphocyte
activity by fusion-active peptide-containing
virosomes, Vaccine, 18, 2000, 1327– 1333.
41. Schoen P, Chonn A, Cullis PR, Wilschut J.,
Scherrer P., Gene transfer mediated by fusion
protein hemagglutinin reconstituted in cationic
lipid vesicles, Gene Ther., 6, 1999, 823– 832.
42. . Kim HS, Park YS, Gene transfection by
quantitatively reconstituted Sendai envelope
proteins into liposomes, Cancer Gene Ther., 9,
2002, 173– 177.
43. Ramani K, Hassan Q, Venkaiah B, Hasnain SE,
Sarkar DP, Site-specific gene delivery in vivo
through engineered Sendai viral envelopes, Proc.
Natl. Acad. Sci. U. S. A., 95,1998, 11886– 11890.
44. Cusi MG, Zurbriggen R, Valassina M., Bianchi S,
Durrer P, Valensin P.E, Donati M,.Guck R,
Intranasal immunization with mumps virus DNA
vaccine delivered by influenza virosomes elicits
mucosal and systemic immunity, Virology, 277,
2000, 111– 118.
45. Andreas RK, Mario A , Silvia R, Nicole
Westerfeld, Christel Gremion, Danielle N, Rinaldo
Z, A new and versatile virosomal antigen delivery
system to induce cellular and humoral immune
responses, Vaccine 25 (2007) 7065–7074.
46. Cusi MG, Gluck R. Potential of DNA vaccines
delivered by influenza virosomes. Vaccine,18,
2000, 1435.
47. Hu KF, Ekstrom J, Merza M, Lovgren-Bengtsson
K, Morein B., Induction of antibody responses in
the common mucosal immune system by
respiratory syncytial virus immunostimulating
complexes, Med Microbiol Immunol, 187, 1999,
191–198.
48. Gluck R, Mischler M, Brantschen S, Just M,
Althaus B, Cryz SJ., Immunopotentiating
Mohd Yasir et al /Int.J. PharmTech Res.2010,2(4)
49.
50.
51.
52.
53.
54.
reconstituted influenza virosome (IRIV) vaccine
delivery system for immunization against hepatitis
A, J. Clin Invest, 90, 1992, 2491-2495.
Gluck R. Adjuvant activity of immunopotentiating
reconstituted influenza Virosomes (IRIVs).
Vaccine, 17, 1999, 1782-1787.
Toos D, Arjan M, Laura B, Jbrgen J, Anke H, Jan
W, Virosomes for antigen and DNA delivery,
Advanced Drug Delivery, 57, 2005,451– 463.
Christine L and Ralf W, Virus-like particles—
universal molecular toolboxes, Current Opinion in
Biotechnology, 18, 2007, 537–545.
3. Zurbriggen R., Immunostimulating reconstituted
influenza virosomes, Vaccine, 21, 2003, 921– 924.
. Matlin, KS, Reggio, H., Helenius, A. and
Simons, K., J. Cell Biol. 91, 1981, 601-613.
Yoshimura, A. and Ohnishi, S., J. Virol. 51, 1984,
497-5046. Norrby, E., Viral vaccines: The use of
currently available products and future devel
opments, Arch. Viral, 73, 1983, 163-165.
*****
2339
55. Vadolas J, Davies JK, Wright P. J., Strugnell RA.,
Intranasal immunization with liposomes induces
strong mucosal immune responses in mice, Eur J.
Immunol , 25, 1995, 969–975.
56. Cusi MG, Zurbriggen R, Bianchi S, Valassina M,
Valensin PE, Donati M, et al, Intranasal
immunization with mumps virus DNA vaccine
delivered by influenza virosomes elicits mucosal
and systemic immunity, Virology, 277, 2000, 111–
118.
57. Saravanan. R.,Senthilkumar. K., Dhachinamoorthi.
D., Chellaram.S., Mallikarjuna Reddy, Virosomes
: As a Versatile Carrier for Delivery System,
journal of pharmacy research, Virosomes : As a
Versatile Carrier for Delivery System, , 3(6),
20101283-1287
58. www.uspto.gov