Korean Journal of Microbiology (2019) Vol. 55, No. 4, pp. 327-343 pISSN 0440-2413DOI https://doi.org/10.7845/kjm.2019.9089 eISSN 2383-9902Copyright ⓒ 2019, The Microbiological Society of Korea
Review
Virus like particles- A recent advancement in vaccine development
Bishajit Sarkar* , Syed Sajidul Islam, Umme Salma Zohora, and Md. Asad Ullah
Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka, Bangladesh
(Received August 12, 2019; Accepted September 23, 2019)
*For correspondence. E-mail: [email protected];
Tel.: +8801627614976
The unintentional reversion of various attenuated and inactivated
vaccines has led the way of discovering and development of the
virus like particles (VLPs) in the 1970s and 80s. The VLPs
contain only the antigenic determinant portion of the structure
of a target virus that is able to produce the same immune
responses like the original viruses, however, since the particles
do not contain any genetic material, the particles can’t infect
like the actual viruses. They are able to induce both the humoral
and cell mediated immune responses. Since infection doesn’t
occur, this technology is a safe and reliable way to combat
infectious diseases. Currently, VLPs are produced for a good
number of infectious diseases and researches are going on to
develop VLPs against many more diseases. Based on the
structure, VLPs can be divided into two types: enveloped and
non-enveloped VLPs. Enveloped VLPs have greater flexi-
bility. To produce VLPs, various expression systems can be
used: yeast, bacterial, mammalian, insect, and plant cells can be
used. Insect cell culture system is mainly used in VLP pro-
duction. The appropriate system should be determined based on
the VLP type in question. The production and purification of all
the VLPs are quite similar: the cloning of the gene for the
antigenic portion, cell harvesting, disruption (if necessary),
clarification, concentration and characterization, these steps are
done using various means. This review summarizes the types of
VLPs, their immunogenic properties, various expression
systems that can be used for production and the production
procedures of various VLPs.
Keywords: chromatography, immune systems, insect cell culture
system, vaccines, virus like particles
Vaccination is being practised from the late 18th Century.
Over time, more and more advanced vaccines have been
developed. Vaccines for viral diseases are sometimes based on
attenuated or inactivated form of live viruses. There is a risk
that the attenuated or inactivated viruses may revert to virulent
form that can lead to the development of various terrible
diseases (Noad and Roy, 2003). For this reason, many of those
early vaccines, sometimes, caused unexpected viral outbreaks,
for example, the outbreak of foot-and-mouth disease virus
(FMDV). To avoid such type of outbreaks, scientists discovered
in the 1970s that only a single protein from the virus can be used
to produce a vaccine (Roldão et al., 2010). In 1981, world’s
first genetically engineered vaccine for using in veterinary
animals, was prepared by cloning the antigenic segment of
FMDV called ‘VP3’ within Escherichia coli (Kleid et al., 1981).
Later, by using recombinant DNA technology, the first recom-
binant vaccine for using in human, was produced, which was
the hepatitis B virus surface antigen (HBsAg) (Michel and
Tiollais, 2010). Although the recombinant HBV vaccine was
the first vaccine derived from virus like particles (VLPs), it is
not a true VLP because it is not a direct mimic to the intact
virion. VLPs are actually subunit vaccines that can mimic the
overall structure of virus particles and they do not require
the infectious genetic material to do so. In fact, many VLPs
completely lack the genetic material of the target virus. For this
reason, there is no risk of reversion and re-assortment to the
virulent infectious form. VLP preparations work based on the
fact that expression of the capsid proteins of viruses leads to the
formation of particles that are structurally similar to the real
viruses. Therefore, VLPs can stimulate the B-cell-mediated
immune responses as well as cytotoxic T lymphocyte (CTL)
responses. This feature of VLPs plays the major role for selecting
them as vaccines (Noad and Roy, 2003). Using various tech-
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Table 1. Examples of some VLPs with description
Name of the
virus
Expression
systemCells
Recombinant
proteins
Yields
(bulk)
Yields
(purified)References
Adeno Associated
Virus (AAV)
Insect cell culture
system &
mammalian cell
culture system
Sf-9 & HEK 293
VP1, VP2 & VP3
(for both the cell
culture systems)
18 µg/ml
(for insect cell
system)
500 µg/ml
(for mammalian
cell culture system)
Aucoin et al. (2007)
Human
Papillomavirus-11
(HPV 11)
Transgenic plantsTransgenic potato
and tobacco plantsL1 protein
About 20 ng/g of
fresh tuberNA
Warzecha et al.
(2003)
HIV-1
Baculovirus-insect
cell expression
system
Sf-21
HIV gag proteins
like GagTN and
GagRT VLPs and
other proteins
460.5 ng/ml
(for GagTN)
& 406.1 ng/ml
(for GagRT)
112.6 ng/ml
(for GagTN) &
74.2 ng/ml
(for GagRT)
Pillay et al. (2009)
Infectious bursal
disease virus (IBDV)
Insect cell culture
systemSf-9 VP2 & VP3
2.87 × 107
molecules/cell/h
for VP2 protein
NAHu and Bentley
(2001)
Indian peanut clump
virus (IPCV)
Transgenic plants
systemTobacco Coat protein
50~100 pg/g
fresh weighNA Bragard et al. (2000)
Marburg & Ebola
virus
Mammalian cell
culture system293T cells
VP40 and
glycoproteinsNA NA
Swenson et al.
(2005)
Newcastle disease
virus (NDV)Avian cells HN, F, NP, and MP NA
387 µg/ml
(average)
McGinnes et al.
(2010)
Polyomavirus
(human)
Yeast cell culture
system
Saccharomyces
cerevisiaeVP1 protein NA 38 µg/ml approx.
Sasnauskas et al.
(2002)
Polyomavirus
(hamster)
Yeast cell culture
system
Saccharomyces
cerevisiaeVP1 protein NA 40 µg/ml approx.
Sasnauskas et al.
(2002)
Polyomavirus
(avian)
Yeast cell culture
system
Saccharomyces
cerevisiaeVP1 protein NA 5 µg/ml approx.
Sasnauskas et al.
(2002)
Polyomavirus
(murine)
Yeast cell culture
system
Saccharomyces
cerevisiaeVP1 protein NA 30 µg/mlapprox.
Sasnauskas et al.
(2002)
SARSInsect cell culture
systemSf-9
S protein, E protein
& M protein
200 µg VLP per
1 × 109 infected cells
NAMortola and Roy
(2004)
Influenza ABaculovirus
expression systemSf-9
H1N1 protein,
M1 protein etc.0.4 µg/ml approx. NA
Krammer et al.
(2010)
MS2Bacterial cell
culture systemE. coli Coat protein NA 442 µg/ml Bundy et al. (2008)
Swine Vesicular
Disease Virus (SVDV)
Insect cell culture
systemSf-9 P1 & 3CD NA 2 µg/ml Ko et al. (2005)
niques of molecular biology, it is now possible to express one
or more specific antigens on the multimeric protein structures
of VLPs so that they will be much more potent, efficient and
broadly applicable. Moreover, VLPs can induce a protective
response at doses lower than the conventional vaccines, which
is also cost-effective. Until now, VLPs are produced for a lot of
different, structurally diverse viruses that infects human and
other animals. Some of the examples are listed in Table 1 with
description. However, VLP based vaccines cannot be generated
for all types of viruses which may be a potential drawback.
Many VLPs are generated for fundamental researches, for
example: assembly and structural studies, protein-protein
interaction studies etc. (Roldão et al., 2010). Recently, VLPs
are being applied in nanoparticle biotechnology (Ludwig and
Wagner, 2007).
Types of VLPs on the Basis of Structure
VLPs can be divided into two major categories based on
their structure. They are: non-enveloped VLPs and enveloped
VLPs (Kushnir et al., 2012).
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Korean Journal of Microbiology, Vol. 55, No. 4
Fig. 2. Examples of structural diversity of enveloped VLPs (Lua et al.,
2014).
Fig. 1. Examples of structural diversity of non-enveloped VLPs (Lua et al.,
2014).
Non-enveloped VLPs
Non-enveloped VLPs are the VLPs that are comprised of
one, two or more components of a target or specific micro-
organisms and those components can self-assemble into a
complete particle (Steven et al., 1997; Phelps et al., 2000;
Zlotnick and Mukhopadhyay, 2011) (Fig. 1). These newly
formed whole particles do not include any host component.
Non-enveloped VLPs are used for the development of vaccines
against various types of parvovirus, for example, porcine,
mink, canine parvoviruses etc. (López de Turiso et al., 1992;
Christensen et al., 1994; Antonis et al., 2006). VLPs for
hepatitis E virus (HEV) are produced by two types of insect cell
culture system: Sf-9 and Tn-5 and it is made of the 50 kDa
truncated capsid protein of the virus (Li et al., 1997). HBsAg
can be successfully produced in other expression systems by
exploiting recombinant DNA technology. Hepatitis B surface
antigen particles can be generated in yeast (Valenzuela et al.,
1982).
Hepatitis B surface antigen particles that carries a specific
receptor for human serum albumin can be synthesized in animal
cell expression systems (Michel et al., 1984). These recombinant
HBsAg VLPs varies from one another due to genetic engineering
and type of the host cell that is used (Diminsky et al., 1997).
Moreover, various chimeric non-enveloped VLPs are recently
developed where the target protein for the vaccine is fused to
the particles, on their surface. The particles consist of a com-
ponent of animal or plant pathogens and these particles have
self-assembling capability. For example, an anti-malaria vaccine,
RTS, S, that contains the malarial antigen circumsporozoite
protein fused with HBsAg (on the surface of VLP), showed a
good response against malaria (Stoute et al., 1997).
Recently, target antigens for various mammalian pathogens
are being created in to plant expression systems, where the coat
proteins (CPs) of several plant viruses are engineered and fused
with antigens of mammalian viruses. For example, the coat
proteins of alfalfa mosaic virus (AlMV) are engineered to act as
carrier molecule for rabies virus and HIV-1 (Yusibov et al.,
1997). Some non-enveloped VLPs show more complexity.
These viral particles contain multiple capsid proteins. For this
reason, a number of discrete mRNAs are expressed. The members
of reoviridae family contain such kind of complex structures
that contain many complicated concentric layers of different
capsid protein (Roy and Noad, 2008).
Enveloped VLPs
Enveloped VLPs are relatively more complicated than the
non-enveloped VLPs since their structures consist of the cell
membrane of the host cell called envelope and this envelope
contains integrated target antigens that are displayed on the
surface (Fig. 2). Enveloped VLPs show higher degrees of
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Fig. 3. Activation of immune responses by VLPs. Here, G, Golgi; ER, endoplasmic reticulum; NC, nucleus; P, proteasome; TV, transport vesicle; LE, late
endosome; R, receptor; MyD88, Myeloid Differentiation primary response 88 protein (Ludwig and Wagner, 2007).
flexibility than the non-enveloped VLPs. The common examples
of enveloped VLPs are: VLPs containing vaccine target antigens
from influenza viruses and retroviruses. The steps for the
assembly of enveloped VLPs are: expression of the desired
structural infectious agent proteins, assembly of the viral proteins
into particles, incorporation of the particles into host membranes
and the release of mature and fully formed particles from the
cell membrane by ‘budding’ process (Kushnir et al., 2012).
The enveloped influenza A/Udorn/72 (H3N2) VLPs can be
produced in insect cell culture system Sf-9. The assembly
and budding of the VLPs have been attained successfully by
expressing four structural proteins of the virus simultaneously:
hemagglutinin (HA), neuraminidase (NA), matrix protein (M1),
and ion channel protein (M2). The VLPs produced by this system
resembled the native influenza virus by size, morphology and
fine surface spikes (Latham and Galarza, 2001).
Retroviruses for example, HIV virus and simian immuno-
deficiency virus (SIV) also showed the correct assembly and
release of VLPs when they are produced in baculovirus expression
system. Moreover, scientists have also produced enveloped
VLPs containing target antigens from heterologous viruses,
such as SIV gag protein and HIV env protein (Gheysen et al.,
1989; Yamshchikov et al., 1995).
Immunogenic Mechanisms
It has already been demonstrated that VLP preparations
are more immunogenic than several subunit and recombinant
immunogenic particles and can stimulate both the humoral
and cellular immune systems. Utilizing VLPs to stimulate the
immune system is proved to be an effective and safe approach.
Using VLP preparations, antibodies against the surface antigens
of varied sorts of pathogens can be generated. Some VLPs show
the ability to introduce or present more than one immunogenic
proteins to the cells of the immune systems. VLPs are sometimes
conjugated with adjuvants to achieve the immune stimulation.
The VLPs are designed in such a way that is appropriate for uptake
by dendritic cells (DCs) and then the particles are processed
and presented by MHC class I and class II molecules. The
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Korean Journal of Microbiology, Vol. 55, No. 4
Fig. 4. Activation of DC cells by VLPs (Ludwig and Wagner, 2007).
uptake of the particles can be mediated by normal receptors
present on the cell surface (Grgacic and Anderson, 2006). VLPs
are highly effective for inducing immune responses because
they can precisely target DCs, delivery to both the MHC class
I and class II molecules occurs efficiently and they can directly
activate DC cells (Chackerian, 2007).
VLPs activate the immune responses by some complicated
mechanisms (Fig. 3), where baculo-derived VLP preparations
activate the DCs and baculovirus is used as adjuvant. The VLPs
and baculovirus adjuvant are regarded as “danger signals” by
the immune system. The particles are taken up by both
receptors or by endocytosis. When the particles are taken up by
endocytosis (Fig. 3-1), they are first processed by late
endosome (LE) and after that presented by the MHC class-II
pathway. Moreover, when the particles are taken up by receptor
(gp64) mediated fusion (2a in Fig. 3), then the particles are at
first processed by the proteasomes and later presented by either
MHC class II and MHC class I molecules. The baculovirus
adjuvant is also taken up the gp64 receptor (2b in Fig. 3) and
toll like receptors-9 (TLR-9) in the endosome recognise the
CpG rich baculovirus DNA and then processed and transmitted
via various MyD88 (Myeloid Differentiation primary response
88)-dependent or independent signalling pathways, which
ultimately results in the production of various types of inter-
ferons and cytokines.
VLPs can also activate the DC cells (Fig. 4). In response to
the VLPs and their adjuvants (baculovirus in this case), various
maturation markers like CD40, CD80, CD86 etc. are expressed
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in the DC cells, which are responsible for the activation of DC
cells. The activated DC cells later present the antigen particles
via MHC class II and MHC class I molecules to CD4+ T-cells
and CD8+ T-cells, respectively (Ludwig and Wagner, 2007).
Expression Systems for VLP Production
Bacteria
Bacterial expression system is not desirable because of the
absence of proper post translational modifications. However,
bacteria can be used to generate non-enveloped VLPs. These
VLPs are created from the infectious elements of a pathogen
that have the self-assembling ability within the bacterial host or
as fused proteins with the partnership of vaccine target antigens
(Zhang et al., 1998; Nardin et al., 2004; Tissot et al., 2010).
Human papilloma virus (HPV) infection is a common sexually
transmitted infection in the world. Of them, types: 6, 11, 40, 42
etc. are in the lower risk group and 16, 18, 31, 33 etc. are in the
highest-risk group that cause cervical cancer (Paavonen, 2007).
HPV VLPs can be produced by expressing the major capsid
protein of the HPV type 16, L1, in Lactobacillus casei, under
the control of a lactose promoter which is a lactose inducible
expression system (Aires et al., 2006).
Vaccine candidates against hepatitis B virus (HBV) and
hepatitis C virus (HCV) infections were created by using HBV
core (HBc) virus-like particles as carriers in E. coli. Chimeric
VLPs carriyng the virus-neutralizing protein molecule HBV
pre-S1 epitope for HBV and extremely conserved N-terminal core
epitope for HCV, were gernerated in the E. coli (Sominskaya et
al., 2010). Malaria vaccine is another example of chimeric
non-enveloped VLPs produced in E. coli. These VLPs contain
HBcAg as carrier that carries genetically fused P. falciparum
circumsporozoite protein (CSP) containing B- and T-cell epitopes
from the repeat region and also a universal T-cell epitope from
the C terminal end of the CS protein (Milich et al., 2001; Birkett et
al., 2002). Moreover, cowpea chlorotic mottle virus like particles
are shown to be properly expressed in bacteria Pseudomonas
fluorescens (Phelps et al., 2007).
Yeast
Yeast cells are eukaryotic cells but they grow in a similar
fashion to bacterial cells and require very cheap and simple
media for growth. Since they are eukaryotes, they can perform
the proper post translational modifications of complex recom-
binant proteins which is much more efficient than bacteria.
However, they have some drawbacks. Although they can carry
out N-glycosylation, the glycan structures sometimes differ
from the mammalian counterparts and their post translational
modifications could be completely different from those found
in mammalian cells. Hypermannosylation, plasmid loss and
lower yields of protein can be other issues. Yeast cells can be
used to produce various types of products as well as VLPs, for
example, the production of hepatitis B surface antigen-based
vaccines and VLPs. (Engerix-B created by GlaxoSmithKline
and Recombivax HB created by Merck) (Mett et al., 2008;
Yusibov and Rabindran, 2008).
Yeast cell culture system can be utilized in producing human
immunodeficiency virus (HIV) type 1 p55 gag virus-like particles
(VLPs). These yeast VLPs can efficiently incorporate into
dendritic cells (DCs) via macropinocytosis or endocytosis. This
uptake of yeast VLPs can induce the maturation of DCs and
enhance the production of cytokines, like interleukin-12 p70.
Thus, the gag particles encapsulated by yeast membrane can
stimulate immune responses. Moreover, it has been found that,
yeast VLPs have the potential to activate both CD4+ and CD8+
T cells of HIV-infected individuals (Tsunetsugu-Yokota et al.,
2003).
Yeast expression system is also utilized in generation of
VLPs to act as vaccines against dengue virus. The major
structural protein on the surface of mature flavivirus is the
envelope glycoprotein E. The complexes of glycoprotein E and
premembrane (prM), play important roles in virus assembly
and maturation and also inducing potential immunity. Therefore,
the vaccine can be produced by inserting the cDNA encoding prM
and E proteins of dengue virus type 2 (DENV-2) in the genome
of Pichia pastoris, under the control of the glyceraldehyde-
3-phosphate dehydrogenase (GAP) which is a constitutive
promoter (Liu et al., 2010).
Insect cells
For a past few decades, insect cell culture system has been
used for the production of foreign proteins. Insect cells have
post-translational protein modification machinery quite similar
Virus like particles- a recent advancement in vaccine development ∙ 333
Korean Journal of Microbiology, Vol. 55, No. 4
to that of mammalian cells which is appropriate for making
numerous complex proteins. Baculovirus, that is the most
commonly used in insect cell culture system, can’t infect
vertebrates because their promoters remain in mammalian
cells, this provides insect cell culture system a bonus over other
systems for the expression of different types of proteins (Mett
et al., 2008).
VLPs against HIV can be generated in insect cell culture
system. Recombinant baculovirus infected Sf-9 cell lines are
capable of producing and the correct processing of gag gene
products that can self-assemble into large enveloped (Gheysen
et al., 1989; Overton et al., 1989). These HIV-VLPs can induce
the immune reaction by activating monocyte-derived dendritic
cells (MDDCs) and human CD4+ T cells (Buonaguro et al.,
2006).
Human papilloma virus (HPV)-VLPs can be generated into
insect cell culture. The HPV types 16-L1 and L1-L2 efficiently
self-assemble into VLPs, when they are expressed by using a
baculovirus double-expression vector (Kirnbauer et al., 1993).
VLPs against norwalk virus can also be produced in Sf-9 cell
culture system using recombinant baculovirus system. The NV
capsid protein can be generated in such a system which
self-assembles into VLPs that mimics the native capsid protein
by appearance and size and the VLPs are able to induce
immune response (Jiang et al., 1992).
Another vital VLP generated from insect cell culture is the
RV-VLPs. Once VP2 and VP6 from heterologous RV strains
(bovine and simian) are produced in Sf-9, the particles self-
assembled and form VLPs (Labbe et al., 1991). Insect cell
culture systems are also utilized in production of VLPs against
herpes simplex virus type-1 (HSV-1) (Tatman et al., 1994).
Mammalian and avian cells
Mammalian cells are used in expression of various proteins
because of their ability to carry out the post translational
modifications (PTM). An additional advantage of this system
is that the proteins are secreted in their native, mature form.
Bacterial and other expression systems are sometimes unable
to carry out these functions (Grillberger et al., 2009).
An important example of the production of VLPs in mam-
malian cell line is the production of HBsAg VLPs. The VLPs
have been successfully created in chinese hamster ovary (CHO)
cell line (Michel et al., 1984; Patzer et al., 1986).
DENV VLPs against dengue virus has already been generated
in chinese hamster ovary cell line. DNA plasmids that can
express flavivirus premembrane (prM/M) and envelope (E)
proteins in the form of virus like particles, were inserted into the
CHO cell line and significant amount of VLPs were produced
(Purdy and Chang, 2005).
In avian cell culture system, VLPs for vaccination against
the human respiratory syncytial virus (RSV) has been produced.
ELL-0 cells (avian fibroblasts) were used for this purpose. The
VLPs were created by combining newcastle disease virus
(NDV) membrane proteins and nucleocapsid with the RSV
proteins F and G. The VLPs showed predominant TH1 response
(McGinnes et al., 2010).
Plant cells
Plant cells are eukaryotes, therefore, they can produce com-
plex, properly folded proteins with proper post translational
modifications. Moreover, plant based systems have some other
benefits, for example lower cost, more safety and easy processes.
Moreover, plants do not harbor pathogens that attack the humans
(Yusibov and Rabindran, 2008).
In tobacco plants, HIV-1 VLPs have been produced by
inserting the gag polyprotein of the virus, Pr55 gag, into the
genome of the plant. Both stable and transient transfection can
be used. The gag proteins produced in such transgenic plants
were able to assemble into VLPs and they were similar to the
VLPs that are produced in insect cells and E. coli systems
(Scotti et al., 2009).
In transgenic tomato, bivalent HBV-HIV vaccine candidates
have been prepared. These bivalent vaccines contain the HBsAg
VLPs displaying immunogenic epitopes from HIV-1, env and
gag proteins (Shchelkunov et al., 2006).
VLPs against human papillomavirus (HPV) type 16 has
been generated into Nicotiana tabacum cv. Xanthi plants. The
genes for HPV type 16 L1 major capsid protein were integrated
into the N. tabacum genome and the expressed proteins showed
VLPs like assembly (Varsani et al., 2003).
Transgenic plants are also used to produce VLPs against the
norwalk virus infection. The norwalk virus capsid proteins
were generated in transgenic plants (transgenic tomato) and
they showed VLP like assembly and act as excellent immune
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inducers when tested on mice (Huang et al., 2005).
Production and Purification
of Some VLP Vaccines
Hepatitis B virus VLP vaccine production
At present, almost 2 billion people (approximately one third
of the world’s population) have serological evidence that
infection with the HBV has occurred for once in their life time.
The infection can cause acute and chronic liver diseases.
According to a study of WHO, every year, 1 million deaths
occur due to the HBV-related liver diseases. Hepatitis B is most
common in Asia, Africa, southern Europe and Latin America
and in these regions, almost 2 to 20% of the total population
carries HBsAg (Kao and Chen, 2002).
The HBV is an enveloped DNA virus that belongs to the
hepadnaviridae family. The DNA of HBV is an unusual
double-stranded circular DNA. The genome is only 3.2 kb and
HBV is one of the smallest DNA viruses. The double-shelled
virions (42–47 nm) contain an outer envelope made of lipid and
three envelope proteins. A nucleocapsid core (27-nm) resides
inside the envelope, which contains the structural C protein and
this protein is called hepatitis B virus core antigen (HBcAg).
The three envelope proteins of HBV are known as small (S),
middle (M), and large (L) envelope proteins. They can self-
assemble into the subviral HBsAg particles (Roldão et al.,
2010).
VLPs against HBsAg can be cloned and expressed in a
variety of expression systems. The proteins can be generated in
Escherichia coli (Burrell et al., 1979). Moreover, eukaryotic
expression hosts such as yeast and mammalian cells, for
examples, Pichia pastoris, Hansenula polymorpha, human
serum albumin etc. can be used (Michel et al., 1984; Janowicz
et al., 1991; Vassileva et al., 2001).
The choice of the host cell depends on the desired type of
VLP and of the production process. For example, the molecular
weight, size and monomer numbers are different for two
recombinant HBsAg molecules produced by two different
expression systems: chinese hamster ovary (CHO cells) and H.
polymorpha (Zhou et al., 2006).
The production and purification of HBsAg was done in
Pichia pastoris. At first, recombinant P. pastoris was created to
express the HBsAg. Then the recombinant P. pastoris strain
was kept as a master seed lot at -70°C. After that, under
carefully controlled conditions, the HBsAg gene-containing
yeast cells were passed from shake flasks into bioreactors and
finally into large scale bioreaction unit. The medium used for
this culture in the bioreactors contained some common inorganic
salts like (NH4)4SO4, KH4PO4 etc., glycerol, vitamins like biotin,
riboflavin etc. For the optimal growth, 30°C temperature, 5 pH,
proper agitation and aeration etc. were maintained. Later,
methanol was added to promote the intracellular synthesis of
HBsAg. The culture reached cell densities of 75–88 gm dry-cell
weight/L at the end of the bioreaction. Next ther yeast cells
were harvested. And after harvesting, the yeast cells were dis-
rupted. After that, to recover and purify HBsAg, a series of
steps were done, including acid precipitation, adsorption/
desorption and successive purification through immunoa-
ffinity, ion-exchange and gel-filtration chromatography. In
these sequence of steps, HBsAg can be prepared from P.
pastoris cell culture (Hardy et al., 2000).
Human papillomavirus VLP vaccine production
Human papillomavirus (HPV) infection is one of the most
common sexually transmitted diseases in the world. The virus
can be transmitted through skin-to-skin contact (Villa, 2006).
In human, more than 100 types of HPV have been dis-
covered so far. 30 of them are genital and mucosal HPVs. They
can be divided into low-risk (e.g. HPV 6, 11, 72, 81 etc.) and
high-risk (e.g. HPV16, 18, 31, 33, 35, etc.) groups (Munoz et
al., 2003). Many HPV types are responsible for cervical cancer.
The most frequently found types in cervical cancer are HPV-
16, -18, -31, -33, -35, etc. Moreover, HPV types alpha-5, 6, 7
etc. are also found in cervical cancer. These types belong to the
high-risk group of HPV (Bouvard et al., 2009). Furthermore,
some other types of cancers like oropharyngeal cancer is caused
by HPV type 16 (D'souza et al., 2007). Cervical cancer is the
common type of cancer in the developing world and it is the
leading cause of mortality due to cancer in most countries.
Majority of cervical cancers are caused by HPV type 16 and 18
(Kane et al., 2006).
HPV particle contains two virally encoded proteins. They are
designated as L1 and L2. These proteins form an icosahedral
Virus like particles- a recent advancement in vaccine development ∙ 335
Korean Journal of Microbiology, Vol. 55, No. 4
capsid which is approximately 60 nm in diameter. L2 protein is
less abundant and is mainly found in the interior of the viral
particle and L1 is the main protein found in the capsid that
forms the outer shell of the virion (Baker et al., 1991; Chen et
al., 2000). The production and purification steps of many of the
HPV type VLPs are quite similar. This includes: HPV type -11,
16, 18, 33, and many more.
A vector was first constructed that contains the gene(s) for
L1 or L1 and L2 proteins, or L1 and L2: fusion protein. These
proteins can self-assemble into VLPs. The vector might contain
some other genes like transcription and translation controlling
signals, marker genes etc. Now, the vector was inserted into a
host cell. The host cell can be a yeast cell (Saccharomyces
cerevisiae), insect cell, bacterial cell or mammalian cell. Yeast
cell is the most preferred cell used in HPV VLP production.
The host cells (in this case S. cerevisae) were allowed to grow
and divide. After proper cell concentration was reached, the
cells were harvested and can be frozen for storage at -70°C
temperature. The frozen cells were thawed 3 h at room tem-
perature followed by approximately 18 h at 4°C. And after that
the cells were stirred for 15 min and disrupted by homogenizer.
After that, cross flow microfiltration is used for clarification.
After clarification, diafiltration was done. Finally, ion-
exchange chromatography was done to get the desired purified
product. The fractions were collected in sterile bottles, after the
chromatography and stored at 4°C (Cook, 2003).
Enterovirus 71 VLP vaccine production
Enterovirus 71 (EV71) causes major outbreaks of hand, foot,
and mouth disease (HFMD). This virus most frequently affects
children. Humans are the only known host of enterovirus
(Solomon et al., 2010).
Enterovirus 71 contains a core of single stranded RNA. The
viral capsid is icosahedral in shape and composed of 60
identical protomers and each of these protomers is made of four
structural proteins, VP1-VP4. The virus contains a single
open reading frame (ORF) that codes for a large, single
polyprotein and this protein is cleaved into three smaller,
functional proteins: P1, P2, and P3. The P1 region encodes the
four structural proteins: VP1, VP2, VP3, and VP4. The P2 and
P3 regions encode seven non-structural proteins, including 2A,
3-CD, 2AB, 3AD etc. (McMinn, 2002).
VLPs against enterovirus 71 can be generated using the P1
and 3-CD genes. For this purpose, Spodoptera frugiperda
(Sf-9) insect cells were used. At first, the genes coding for P1
and 3CD were amplified by polymerase chain reaction and then
the genes were cloned into the multiple cloning site (MCS) I
and II of a plasmid vector (pFastBacTM
DUAL plasmids). The
P1 gene fragment was inserted into MCS I under the polyhedrin
promoter and the 3CD gene fragment was integrated into MCS
II control of p10 promoter. Then the recombinant baculo-
viruses were produced using Bac-to-Bac® system and they
were designated Bac-P1, Bac-3CD and Bac-P1-3CD according
to the genes they encoded. In this way the recombinant ba-
culovirus was generated.
After the confirmation of the protein expression, the Sf-9
cells were infected by the recombinant baculoviruses and
allowed to grow. When the satisfied cell concentration had
been reached, the cells are harvested (after 2 days). The cell
suspension was supplemented with required amount of 10X
HO buffer and cell lysis was performed using sonication method
for 3 min. Now, centrifugation was done at 13,000 × g and the
supernatant was collected and filtered by a 0.22 µm filter. After
filtration, the filtrate was collected and ultracentrifugation was
done at 27,000 × g for 4 h. After that the pellets were collected
and resuspended in TE buffer. Again, sonication for 30 sec was
performed and after sonication, the remaining substances were
loaded onto discontinuous sucrose gradient. After another
round of ultracentrifugation at 27,000 × g for 3 h, the milky
white band between the interfaces of sucrose was collected and
was carefully taken on top of a 1.33 g/cm3 cesium chloride
(CsCl) solution. Ultracentrifugation at 34 000 × g for 24 h was
carried out and the bands collected from CsCl gradient were
collected. Now, dialysis was performed against phosphate-
buffered saline and a final ultracentrifugation was done at
34,000 g for 5 h. At last, the pellets containing the viral particles
were collected and resuspended in pure water (Chung et al.,
2006).
Norwalk virus VLP vaccine production
Norwalk virus (NV) and norwalk-like viruses (NLVs) are
human caliciviruses, that are the main cause of epidemic acute,
non-bacterial gastroenteritis in the USA. NLVs are transmitted
through feces-contaminated food and water, direct contact to
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미생물학회지 제55권 제4호
contaminated surfaces and also person-to-person contact
(Lindesmith et al., 2003).
Norwalk virus contains a single-stranded RNA genome which
is positive-sense and the genome is about 7.7 kb. The genome
is incorporated within a shell and the shell consists of multiple
copies of a single capsid protein. The capsid is an icosahedral
38 nm structure and it is assembled from 90 dimers of the
capsid protein (CP) (Prasad et al., 1999). The capsid protein of
NV is used to produce the VLPs against the NV infection. The
capsid protein is highly immunogenic and capable of inducing
the B-cell and T-cell immune responses. The NV VLPs were
generated in Nicotiana benthamiana plant, with the help of
Agrobacterium tumefaciens bacteria.
At first, MagnICON vectors were created for NVCP and
GFP gene was inserted as the reporter gene. Then the vector
was inserted into the A. tumefaciens strain GV3101 cells. The
cells were transformed by electroporation. Then the cells
containing the vectors were then multiplied. Next, the seeds of
N. benthamiana plant were collected and grown to a full plant
in a greenhouse system under proper conditions like 25 ±
0.5°C, 16 h Light / 8 h dark cycle etc. When the plantlets
reached at a satisfied growth stage, the agroinfiltration was
performed and then incubation was done for the plantlets to
grow. Agroinfiltrated N. benthamiana leaves were harvested
on days 6, 7, and 8 days after the day when infiltration was
performed (days post infiltration or ‘dpi’). Total leaf protein
was extracted by homogenization with extraction buffer. The
homogenates were then centrifuged at 6,000 × g for 30 min at
4°C to remove leaf debris and the plant enzyme RuBisco and
the supernatant was collected. Next, after an additional incubation
step that lasted for 24 h at 4oC temperature, centrifugation was
done at for 30 min at 6,000 × g at 4°C to further remove
RuBisco and this step was continued for three times to remove
any RuBisco. The supernatant was collected and filtered using
a 0.22 µM filter. Next, the concentration of the clarified
supernatant was performed using ultrafiltration/diafiltration.
After that, anion exchange chromatography was performed to
further purify the retentate of the ultrafiltration/diafiltration.
Finally, a final ultrafiltration/diafiltration was done to concentrate
the purified product, which were the desired virus like particles
(Lai and Chen, 2012).
Human parvovirus VLP vaccine production
Among many human parvoviruses, the human parvovirus
B19 is considered to be pathogenic to humans. Human parvovirus
is responsible for the diseases like transient aplastic crisis,
erythema infectiosum, chronic anemia, arthritis etc. (Anderson,
1990).
Parvovirus B19 is a non-enveloped virus and it is icosahedral
in symmetry. It consists of two types of capsid proteins, one is
the major capsid protein known as VP2 protein and its MW is
58 kDa. The other one is minor capsid protein known as VP1
and its MW is 83 kDa. The parvovirus VLPs can be produced
in Sf-9 cell line, human bone marrow culture, fetal liver culture
etc. (Tsao et al., 1996).
The production and purification processes using Sf-9 cells
involved the inoculation of Sf-9 cells in a bioreactor and then
co-infection with bacVP1 and bacVP2 viruses that encode VP1
and VP2 protein, respectively. After that the cell viability was
measured and centrifuged at 800 × g for 30 min. The pellet is
collected and resuspended in Tris buffer with leupeptin. Now,
2-3 ion-exchange steps were carried out with different types of
chromatographic column. After that, the proteins purified by
chromatographic steps were formulated in sucrose and tween
and in the last step, sterile filtration was done using 0.22 µm
filter to get the final product (Roldão et al., 2010).
HIV VLP vaccine production
The HIV-1 gag and env particles are used for the production
of VLPs and CLPs respectively, against the HIV-1. The insect
cell culture system Sf-9 is mainly used for the production of
HIV-1 VLPs. Since the insect cells are eukaryotic, for this
reason they can give the proper post translational modifications
to the VLPs. For this reason, the VLPs can mimic the actual
viral particles. Pr55 gag from the gag protein is the protein used
for generating VLPs. Naturally, the Pr55 gag is a 55 kDa
polyprotein precursor that is processed by the virion protease
and gives rise to the matrix protein (p17), nucleocapsid protein
(p7), and the capsid protein (p24). Moreover, the natural
envelope glycoprotein gp160 is also processed and gives rise
to two subunits: the membrane protein gp41 and the large
external glycoprotein gp120. In the insect cell culture system,
the Pr55gag and the gp160 are targeted to the plasma mem-
Virus like particles- a recent advancement in vaccine development ∙ 337
Korean Journal of Microbiology, Vol. 55, No. 4
brane of the cells and assemble in 100 to 120 nm particles that
bud from the cell surface, leading to the production of CLP or
VLP (Cruz et al., 2000).
Using VLPs as a HIV vaccine has several advantages. First,
the immune system responds better to the VLPs that mimic
the actual viral particles. Second, more than one antigen or
immunostimulatory molecule can be displayed on the VLPs.
Third, these particles don’t replicate and contain the HIV
genome, for this reason, they cannot produce more viruses.
Thus, the VLPs eliminate the safety issues with inactivated and
attenuated vaccines. Fourth, less amount of VLPs need to be
administered than the conventional vaccines to get the same
result (Doan et al., 2005).
The production of HIV-1 VLPs was done using the Sf-9 cell
culture system. At first, the generation of recombinant baculo-
virus was done. To carry out this process, the cDNA encoding
Pr55gag and gp160 were inserted in to a baculo transfer vector
under the appropriate promoters and then the vectors were
inserted into the Sf-9 cells by co-transfection with Autographa
californica nuclear polyhedrosis (AcNPV) linear viral DNA
and viral infection took place. The Sf-9 cells were grown into in
a cell free medium in a controlled stirred bioreactor. During this
process, the cell concentration was measured by haemacytometer
and cell counts and cell viability were detected by trypan blue
exclusion dye. During the production of Pr55 gag, the concent-
ration was measured by immunological techniques, the density
was measured by sucrose gradient centrifugation and the
characterization was done by gel filtration chromatography.
When the appropriate concentration and density was reached,
the downstream processing was performed. The centrifugation
was done and the supernatant was collected and ELISA was
done to determine the product titre. To remove the debris,
dead-end microfiltration was done. Then an ultracentrifugation
was carried out for partial purification and finally gel filtration
chromatography was used to produce the purified product VLPs
(Cruz et al., 2000).
Influenza VLP vaccine production
Using VLPs as vaccines against influenza is a safe approach
to prophylaxis. This technology can also be used to eliminate
the problem associated with the pandemics that are caused by
various newly emerging strains of influenza. The influenza
VLPs are created by expressing the viral structural proteins:
hemagglutinin (HA), neuraminidase (NA), matrix (M1), in
Sf-9 cell culture system (Galarza et al., 2005).
The glycoprotein spikes of HA and NA are embedded in the
influenza virion envelope and the ratio is approximately four to
one. A smaller number of matrix (M2) ion channels are also
present. The envelope and its integral membrane proteins (HA,
NA, and M2) overlay the M1 protein that encloses the virion
core (Bouvier and Palese, 2008).
The production of influenza VLPs was done successfully in
the Sf-9 cells. First, the infection of Sf-9 cells were done with
recombinant insect baculovirus that express HA, NA, and M1
genes, in a 100L bioreactor. After 72 h, VLPs were harvested
using tangential flow filtration (TFF). The TFF was used for
concentration, clarification, and diafiltration of the product.
Next, the concentrated VLPs were separated from debris and
contaminants using ion exchange chromatography, sucrose
gradient ultracentrifugation and diafiltration. In the last step, a
0.22 µm filtration step was done to get the purified VLP
product. The sterile 2009 VLPs were then stored at 2~8°C
(Milián and Kamen, 2015).
Rotavirus VLP vaccine production
Infection by rotavirus causes diarrhea in infants and kills
more than half million children each year. Virus like particles
(VLPs) as vaccines against rotavirus are excellent vaccine can-
didates. The rotavirus genome contains 11 segments of double-
stranded RNA. The dsRNA is enclosed in the viral capsid, which
is composed of three layers. The major rotavirus structural
proteins are VP2, VP4, VP6, and VP7. The inner layer contains
60 dimers of VP2. The middle layer is formed by 260 trimers of
VP6 and the outer layer contains 780 monomers of the VP7
carrying spikes formed by the 60 trimers of VP4. The rotavirus
VLPs are formed in baculovirus expression system where triple
layered multiprotein VLP complexes are formed. The triple
layers contains VP2, VP6, and VP7 proteins (Vieira et al.,
2005).
The successful production process of rotavirus VLPs included
the construction of tricistronic baculovirus that express the
VP2, VP6, and VP7 genes. Sf-9 cell line was obtained and the
cells were infected with baculovirus, in a bioreactor. After 120
h of infection, the bioreactor was harvested. The density and
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미생물학회지 제55권 제4호
Table 2. Examples of the tools used in characterization of VLPs (Lua et al., 2014)
Name of tools Type Applications
Mass Spectrometry (MS) BiochemicalDetermination of molecular weight (MW) and amino
acid composition
Reverse Phase- High Performance Liquid Chromatography (RP-HPLC) Biochemical Determination of the purity of the sample
Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis Biochemical Determination of MW and purity
Transmission Electron Microscopy (TEM) Biophysical Visualization and size determination
Cryo- Electron Microscopy Biophysical Visualization and size determination
Disc centrifugation Biophysical Determination of size and polydispersity
Negative agarose gel electrophoresis Biophysical Determination of surface charge and polydispersity
Analytical ultracentrifugation Biophysical Determination of velocity
Atomic Force Microscopy (AFM) Biophysical Visualization and size determination
ElectroSpray Differential-Mobility Analysis (ES-DMA) Biophysical Determination of size
Enzyme linked immune sorbent assay (ELISA) Biological Detection of specific antibody
Surface Plasmon Resonance (SPR) Biological Detection of specific antibody
viability of infected cells were determined by haemocytometer
cell counts and trypan blue exclusion dye, respectively. ELISA
was used for quantification of the VLPs and SDS-PAGE and
western blotting were done to evaluate the VLPs protein content.
After that, the purification steps were started. In the purification
processes, first ultracentrifugation using CsCl gradients was
done. Now, to promote cell lysis and removal of lipid content,
the collected bulk was incubated with Triton X-100 for 30 min
at 37°C. The clarification was performed by using a depth filter
with pores of 3 µm. After that, cross-flow ultrafiltration/
diafiltration was done for concentration of the product. Next, a
polishing step using size exclusion chromatography was done
to get the purified product. In the final concentration step,
ultracentifugation at 110,000 × g for 90 min was done (Peixoto
et al., 2007).
Quantification and Characterization of VLPs
Conventionally, the quantification of VLPs is performed by
electrophoresis, western blot, ELISA, flow cytometry (Meghrous
et al., 2005; Vieira et al., 2005). These methods are relatively
time-consuming, require high sample volumes for operation,
have low specificity and sensitivity and sometimes give faulty
results (Roldao et al., 2010). Recently more developed tech-
nologies are used, for example, gel permeation chromatography,
SDS-capillary gel electrophoresis method, MALDI-TOF mass
spectrometry and RT-PCR (Mena et al., 2005; Mellado et al.,
2008; Franco et al., 2010; Debbink et al., 2013).
For characterization, various modern tools are used, a list of
such tools along with their applications are shown in Table 2.
The tools can be divided into three categories: biochemical,
biophysical and biological tools. Biochemical tools include
mass spectrometry (MS), RP-HPLC, SDS-PAGE; biophysical
tools include transmission electron microscopy (TEM), negative
agarose gel electrophoresis, cryo-electron microscopy, analytical
ultracentrifugation etc.; the biological tools include ELISA and
Surface Plasmon Resonance (SPR) (Lua et al., 2014).
Summary and Future Prospects
The vaccines that are produced to combat various infectious
diseases are mostly attenuated or chemically inactive vaccines.
However, the possibility of such vaccines to revert to their
virulent form which may lead to unexpected outbreaks, has
been a major issue for a long time. That’s where, the virus like
particles (VLPs) come to play (Noad and Roy, 2003). Since,
VLPs can mimic the original antigenic proteins of the pathogen,
then can activate and stimulate the immune cells that take part
in both the innate and adaptive immune responses (Grgacic and
Anderson, 2006). As VLPs do not contain the genetic materials
of the original pathogen, they are safe and can be used for
prophylactic and therapeutic purposes (Galarza et al., 2005).
Virus like particles- a recent advancement in vaccine development ∙ 339
Korean Journal of Microbiology, Vol. 55, No. 4
Fig. 5. Common flowchart of production and purification steps of VLPs
(Zeltins, 2013).
Table 3. A list of VLP based vaccines along with their respective current stages of development
VLP based vaccine Vaccine target Stage of development References
Hepatitis B Virus (HBV)-VLP Hepatitis B Virus (HBV) Clinical trials (Human trials) Plummer and Manchester (2011)
Rotavirus-VLP Rotavirus Pre-clinical trials (Animal trials) Plummer and Manchester (2011)
Alfalfa mosaic virus-VLP Respiratory syncytial virus (RSV) Trials in non-human primates Plummer and Manchester (2011)
Ebola virus-VLP Ebola virus Pre-clinical trials Vicente et al. (2011)
Norwalk virus-VLP Norwalk virus Phase I Vicente et al. (2011)
Human Papilloma Virus (HPV)-VLP Human Papilloma Virus (HPV) Clinical trials Roldão et al. (2010)
Influenza A virus-VLP Influenza A virus Preclinical trials Roldão et al. (2010)
Norovirus-VLP Norovirus Clinical trials Mohsen et al. (2017)
Hepatitis E Virus (HEV)-VLP Hepatitis E Virus Approved in China in 2011 Mohsen et al. (2017)
Chikungunya Virus -VLP Chikungunya Virus Clinical trial phage-I Mohsen et al. (2017)
Based on whether the VLPs are encapsulated or not, they can be
divided into two type: enveloped and non-enveloped VLPs
(Kushnir et al., 2012). For the production of VLPs, various
expression systems can be used. Bacterial expression system,
yeast expression system, insect cell system, mammalian cell
culture system, plant expression system can be used effectively.
The choice of expression systems depends on the right balance
between yield and VLP composition (Roldão et al., 2010).
Among all the expression systems, the insect cell culture system
is widely used (Noad and Roy, 2003).
The basic steps in production of the VLPs are quite similar.
The first stage of production is the cloning of the necessary
antigenic genes and expressing the antigenic genes into an
optimum cell culture system. The cell culture system can be
any one of the systems mentioned above. Later, the purification
and characterization of VLPs are done. The choice of the
purification and characterization processes can vary. If the
VLPs are produced intracellularly, in that case, cell disruption
is done. However, if the VLPs are produced extracellularly,
then cell disruption should not be done. After that, clarification,
concentration and polishing steps are done to get the purified
VLPs (Fig. 5). In these steps, various centrifugation and ultra-
centrifugation techniques, various chromatographic techniques
etc. are applied. Then the characterization of VLPs are per-
formed by using various tools (Zeltins, 2013).
Currently, scientists are working on the production of VLP
based vaccines against many infectious viruses, for example,
human papillomavirus, norwalk and norwalk like virus, hepatitis
C and E virus, HIV, polio virus etc. Scientists are also trying to
develop VLP based vaccines against many viruses that infect
animals, for example, chicken anemia virus, canine parvovirus,
porcine parvovirus etc. Although most of the VLP based vaccines
are still in the developmental stages and only few VLP based
vaccines have gained license (for example, papillomavirus VLP)
(Roy and Noad, 2008). A list of VLP based vaccines along with
their respective current stages of development has been shown
in Table 3. Since most of these VLP based vaccines are in
clinical and pre-clinical stages of development, in the near future
they will gain commercial access to the market, which then can
lead the way to combat various lethal infectious diseases.
340 ∙ Sarkar et al.
미생물학회지 제55권 제4호
Conclusion
VLP technology is a rapidly growing field and has great
potential in the field of vaccination and gene therapy. It is a
multidisciplinary field that combines topics from vaccinology,
microbiology, biotechnology, genetic engineering and immu-
nology. The conventional vaccines may cause toxicity in the
body of the recipients however, VLPs are safer, more flexible,
more stable, and easier to produce and also have the ability to
induce better immune responses than most of the vaccines.
Although, at present, only a few types of VLPs are currently
available in the market, however, in the near future, it will be
possible to generate VLPs against most of the infectious
diseases. VLPs can be seen as an incredible achievement in the
quest for the rationale recombinant vaccine design. VLP
technology will lead the way in combating various diseases in
the “Biotechnological era”.
Conflict of Interest
The authors declare that there is no conflict of interest
regarding the publication of the manuscript.
Acknowledgements
This study was supported by the Department of Biotech-
nology and Genetic Engineering, Jahangirnagar University,
Savar, Dhaka, Bangladesh.
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