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 recombinant 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 techniques 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).

Table 1

Examples of some VLPs with description

Name of the virus	Expression system	Cells	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 plants	Transgenic potato and tobacco plants	L1 protein	About 20 ng/g of fresh tuber	NA	Warzecha et al. (2003)
HIV-1	Baculovirus-insect cell expression system	Sf-21	HIV gag proteins like GagTN and GagRT VLPs and other roteins	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 system	Sf-9	VP2 & VP3	2.87 × 107 molecules/cell/h for VP2 protein	NA	Hu and Bentley (2001)
Indian peanut clump virus (IPCV)	Transgenic plants system	Tobacco	Coat protein	50~100 pg/g fresh weigh	NA	Bragard et al. (2000)
Marburg & Ebola virus	Mammalian cell culture system	293T cells	VP40 and glycoproteins	NA	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 cerevisiae	VP1 protein	NA	38 μg/ml approx.	Sasnauskas et al. (2002)
Polyomavirus (hamster)	Yeast cell culture system	Saccharomyces cerevisiae	VP1 protein	NA	40 μg/ml approx.	Sasnauskas et al. (2002)
Polyomavirus (avian)	Yeast cell culture system	Saccharomyces cerevisiae	VP1 protein	NA	5 μg/ml approx.	Sasnauskas et al. (2002)
Polyomavirus (murine)	Yeast cell culture system	Saccharomyces cerevisiae	VP1 protein	NA	30 μg/mlapprox.	Sasnauskas et al. (2002)
SARS	Insect cell culture system	Sf-9	S protein, E protein & M protein	200 μg VLP per 1 × 109 infected cells	NA	Mortola and Roy (2004)
Influenza A	Baculovirus expression system	Sf-9	H1N1 protein, M1 protein etc.	0.4 μg/ml approx.	NA	Krammer et al. (2010)
MS2	Bacterial cell culture system	E. coli	Coat protein	NA	442 μg/ml	Bundy et al. (2008)
Swine Vesicular Disease Virus (SVDV)	Insect cell culture system	Sf-9	P1 & 3CD	NA	2 μg/ml	Ko et al. (2005)

VLPs can be divided into two major categories based on their structure. They are: non-enveloped VLPs and enveloped VLPs (Kushnir et al., 2012).

Non-enveloped VLPs

Non-enveloped VLPs are the VLPs that are comprised of one, two or more components of a target or specific microorganisms 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).

Fig. 1.

Examples of structural diversity of non-enveloped VLPs (Lua et al., 2014).

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 component 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 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).

Fig. 2.

Examples of structural diversity of enveloped VLPs (Lua et al., 2014).

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 immunodeficiency 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).

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 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 interferons and cytokines.

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).

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).

Fig. 4.

Activation of DC cells by VLPs (Ludwig and Wagner, 2007).

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 recombinant 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 mammalian 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 T_H1 response (McGinnes et al., 2010).

Plant cells

Plant cells are eukaryotes, therefore, they can produce complex, 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).

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 disrupted. After that, to recover and purify HBsAg, a series of steps were done, including acid precipitation, adsorption/ desorption and successive purification through immunoaffinity, 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 discovered 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 temperature 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 (pFastBac^TM 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 baculoviruses 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 baculovirus 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 4^oC 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 membrane 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 baculovirus 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 concentration 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 candidates. 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 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).

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 technologies 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).

Table 2

Examples of the tools used in characterization of VLPs (Lua et al., 2014)

Name of tools	Type	Applications
Mass Spectrometry (MS)	Biochemical	Determination 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

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).

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 ultracentrifugation techniques, various chromatographic techniques etc. are applied. Then the characterization of VLPs are performed by using various tools (Zeltins, 2013).

Fig. 5.

Common flowchart of production and purification steps of VLPs (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.

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)

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 immunology. 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”.

This study was supported by the Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka, Bangladesh.

The authors declare that there is no conflict of interest regarding the publication of the manuscript.