review virus like particles- a recent advancement in vaccine …327-343]kjm19-089.pdf ·...

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-

https://orcid.org/0000-0001-8439-6994

328 ∙ Sarkar et al.

미생물학회지 제55권 제4호

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


Sasnauskas et al.

(2002)

Polyomavirus

(avian)

Yeast cell culture

system

Saccharomyces


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).

Virus like particles- a recent advancement in vaccine development ∙ 329

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



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



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



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



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



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



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



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-



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



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).



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.



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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