Subviral particle as vaccine and vaccine platform.

Recombinant subvirual particles retain similar antigenic features of their authentic viral capsids and thus have been applied as nonreplicating subunit vaccines against viral infection and illness. Additionally, the self-assembled, polyvalent subviral particles are excellent platforms to display foreign antigens for immune enhancement for vaccine development. These subviral particle-based vaccines are noninfectious and thus safer than the conventional live attenuated and inactivated vaccines. While several VLP vaccines are available in the markets, numerous others, including dual vaccines against more than one pathogen, are under clinical or preclinical development. This article provides an update of these efforts.

Current Opinion in Virology 2014, 6:24–33

This review comes from a themed issue on Vaccines

Edited by Shan Lu

For a complete overview see the and the

Available online 21st March 2014

1879-6257/$ – see front matter, © 2014 Elsevier B.V. All rights reserved.

Introduction

Most viruses share common spherical or rod-shaped capsids built by multiple subunits of capsid proteins that encapsulate the viral genome. Through bioengineering technology viral capsid proteins can be produced in vitro, resulting in self-assembled, empty virus-like particles (VLPs) (reviewed in [1, 2••]). In addition, smaller particles with less subunits can be produced for some viruses by expression of portions of the major viral capsid proteins [3, 4, 5, 6, 7]. These artificial subviral particles retain the structures and antigenic properties of their native viruses, including the virus-specific molecular patterns and high density of B-cell and T-cell epitopes to induce potent innate, humoral, and cellular immune responses, respectively, in animals and humans [1, 2••]. Thus, these subviral particles are excellent source of materials for vaccine development against many viruses and their associated diseases. VLPs are usually made by an eukaryotic expression system, including the baculovirus/insect cells, yeast, and mammalian cells, while the smaller subviral particles and hepatitis B virus (HBV) VLPs can be produced through the E. coli expression system (Table 1 ), which is more cost-effective. Sevral subviral particle-based vaccines are currently available in the market, while many others are under clinical or preclinical development.

Table 1

Some known subviral particles that have been studied as vaccines or immunogens

Virus family	Virus species	Subviral particle	Production system	Immune responses in lab animals (mice)	Neutralization/protection against virus and diseases (mice)	Clinical trial/commercial use	Reference
Arteriviridae	PRRSV	VLP	Baculovirus	Ab, T cell	Neutralization		[46]
Birnaviridae	IBDV	VLP	Baculovirus	Ab (chicken),	Neutralization, protection (chicken)		[47, 48]
Bunyaviridae	RVFV	VLP	Baculovirus	Ab, T cell (rat)	Neutralization/protection (rat)		[49]
Caliciviridae	NoV	VLP	Baculovirus	Ab, T cell	Block NoV-receptor interaction, protection (human)	Phase I and II	[19••, 50]
	RHDV	VLP	Baculovirus	Ab (rabbit)	Protection (rabbit)		[51, 52]
	NoV	P particle	E. coli	Ab, T cell	Block NoV-receptor interaction		[9•, 38•]
	NoV	Polyvalent complex	E. coli	Ab, T cell	Block NoV-receptor interaction		[53]
Circoviridae	PCV	VLP	E. coli	Ab (pig)	Protection (pig)	Commercial use	[17, 18]
Coronaviridae	SARS-CoV	VLP	Baculovirus	T cell			[54, 55]
	IBV	VLP	Baculovirus	Ab, T cell (chicken)	Neutralization (chicken)		[56]
Filoviridae	EBOV	VLP	Mammalian cells	Ab (guinea pig)	Protection (guinea pig)		[57, 58]
Flaviviridae	HCV	VLP	Baculovirus	Ab, T cell (primate)	Protection		[59, 60, 61, 62]
Hepadnaviridae	HBV	VLP	Yeast	Ab (monkey, chimpanzee)	Protection (chimpanzee, human)	Commercial use	[14, 15]
	HBV	VLP	E. coli	Ab, T cell (human)		Phase I	[26, 63]
	HBV	VLP	E. coli	Ab	Protection against B. burgdorferi		[64, 65]
Hepeviridae	HEV	VLP	Baculovirus	Ab	Protection (monkey, human)	Phase I and II	[20, 21, 66, 67]
	HEV	E2 particle	E. coli	Ab (monkey)	Protection (monkey, human)	Commercial use	[3, 68]
Herpesviridae	EVB	VLP	HEK293 cell line	Ab, T cell			[69]
Nodaviridae	BV	VLP	Baculovirus		Protection (European Sea Bass)		[70, 71]
	FHV	VLP	Baculovirus	Ab (rat)			[72]
Orthomyxoviridae	Flu virus	VLP	Baculovirus	Ab, T cell (ferret)	Protection (ferret)		[73, 74, 75]
Paramyxoviridae	NDV	VLP	Baculovirus	Ab (chicken)	Protection (chicken)		[76]
	RSV	VLP	Baculovirus	Ab	Neutralization/protection		[77]
Parvoviridae	PPV	VLP	Baculovirus	Ab (guinea pig, pig)	Protection (pig)		[78]
	CPV	VLP	Baculovirus, E. coli	Ab (dog), T cell	Protection (dog)		[79, 80, 81]
	GPV	VLP	Baculovirus	Ab (goose)	Neutralization		[82]
	PV B19	VLP	Baculovirus, yeast	Ab (human)	Neutralization (human)	Phase I	[83, 84]
Papillomaviridae	HPV	VLP	Baculovirus, yeast	Ab (rabbit)	Neutralization, protection (human)	Commercial use	[10, 11, 12, 13]
	HPV	Capsomere	E. coli	Ab (dog)	Protection (dog)		[7, 11, 85, 86]
Picornaviridae	EMCV	VLP	Baculovirus	Ab (pig)	Neutralization		[87]
	CVB3	VLP	Baculovirus	Ab	Protection		[88]
	CVA16	VLP	Baculovirus	Ab	Protection		[89]
	EV71	VLP	Baculovirus, yeast	Ab, T-cell (monkey)	Neutralization (monkey), protection		[90, 91, 92]
	FMDV	VLP	Baculovirus, E. coli	Ab, T cell (dog, cattle)	Protection (guinea pig, dog, cattle)		[93, 94]
	PyV	VLP	Yeast	Ab, T cell			[95, 96, 97]
	PyV	VLP	Yeast	T cell	Protection		[98]
	PyV	VLP	E. coli	Ab	Protection		[99]
	PyV	VLP	Baculovirus	Ab, T cell	Against tumor growth, protection		[100, 101]
	PyV	Pentamer/capsoid	E. coli	Ab (piglet)			[102]
Polyomaviridae	SV40	VLP	Baculovirus	Ab, T cell			[103]
Reoviridae	RV	VLP	Baculovirus, E. coli	Ab, T cell	Protection (mouse, pig)		[104, 105, 106]
	BTV	VLP	Baculovirus	Ab	Neutralization, protection (sheep)		[107, 108, 109]
Retroviridae	HIV	VLP	Baculovirus	Ab, CTL	Neutralization		[110]
Togaviridae	CHIKV	VLP	Baculovirus	Ab (monkey)	Protection (monkey)		[111]

Ab, antibody; B. burgdorferi, Borrelia burgdorferi; BV, betanodavirus; BTV, bluetongue virus; CHIKV, chikungunya virus; CoV, coronavirus; CPV, conine parvovirus; CTL, cytotoxic-T-lymphocyte; CVA16, coxsackievirus A-16; CVB3, coxsackievirus B3; E. coli, Escherichia coli; EBOV, ebolavirus; EMCV, encephalomyocarditis virus; EV71, Enterovirus 71; EBV, epstein–barr virus; FHV, flock house virus; Flu virus, influenza virus; FMDV, foot-and-mouth disease virus; GPV, Goose parvovirus; HBV, hepatitis B virus; HCV, hepatitis C virus; HEV, hepatitis E virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; IBDV, Infectious bursal disease virus; IBV, infectious bronchitis virus; NDV, Newcastle disease virus; NoV, norovirus; PCV, porcine circovirus; PPV, porcine parvovirus; PRRSV, porcine reproductive and respiratory syndrome virus; PV, parvovirus; PyV, polyomavirus; RV, rotavirus; RHDV, Rabbit haemorrhagic disease virus; RSV, respiratory syncytial virus; RVFV, Rift Valley fever virus; SARS, severe acute respiratory syndrome; SV40, simian vacuolating virus 40 or simian virus 40; VLP, virus-like particle.

The self-assembled, polyvalent subviral particles are also excellent platforms for antigen presentation to enhance immunogenicity. Through genetic engineering or chemical conjugation heterologous antigens or peptide epitopes can be inserted or conjugated onto the surface of the subviral particles. The polyvalent presentation of the foreign antigens or epitopes on the subviral particles leads to enhanced immunogenicity, providing an effective approach for novel vaccine development. On the other hand, the immunogenicity of the subviral particle is generally maintained without disrupption by the foreign insertion, and thus the chimeric particles can be used as dual or even multivalant vaccines against two or more pathogens. A number of such chimeric particles have been under preclinical development, pointing to a new direction of highly efficient, low cost vaccines against major infectious diseases.

Subviral particles as vaccines

Over 30 different subviral particles (Table 1), representing at least 21 viral families, have been generated so far through recombinant baculovirus, yeast, mammalian cells and E. coli expression systems. Most of them are VLPs comprising one or more full-length viral structural proteins, while others are smaller subviral particles formed by truncated capsid proteins [3, 4, 5, 6, 7]. The most complex subviral particles are VLPs of the rotavirus, influenza virus and coronavirus that contain up to four structure proteins. The smaller subviral particles include the E2 particles (∼23 nm) of the hepatitis E virus (HEV) that are composed of the truncated protruding (P) P1 and P2 domains (∼30 kDa) of HEV VP1 [2••, 3, 8] and the P particles (∼20 nm) of norovirus (NoV) that are formed by 24 copies of the P domain (∼34 kDa) of the NoV capsid protein VP1 [4, 6, 9•].

Most subviral particles can be easily produced in the laboratory (Table 1) and several of them have reached the markets as effective vaccines after successfully scaled-up production through Good Manufacturing Practices (GMP). These subviral particles are excellent immunogens inducing strong humoral and cellular immune responses as shown by numerous studies (Table 1). Immunization of subviral particle vaccines in different animal species and humans, through various routes, such as intranasal, intramuscular, and intraperitoneal administrations, stimulated high antibody as well as high CD4+ proliferative and cytotoxic T lymphocyte (CTL) responses (Table 1). These features support the subviral partciels to be highly efficient vaccines against many infectous diseases.

To date five subviral particle-based vaccines are commercially available for human use. The two VLP vaccines against human papillomavirus (HPV) are made by L1, the major capsid protein of HPV16 [10], through recombinant yeasts (Gardasil®, Merck & Co., NJ, USA) or baculoviruses in insect cells (Cervarix®, GlaxoSmithKline, London, UK) [10, 11, 12, 13]. Both vaccines have been proven for the prevention of cervical and anogenital infection and diseases associated with HPVs. The other two commercial VLP vaccines against hepatitis B viruses (HBVs), Recombivax HB® (Merck & Co., NJ, USA) and Engerix-B® (GlaxoSmithKline, London, UK), are made by the small surface antigen of HBV (HBsAg) through recombinant yeasts (Saccharomyces cerevisiae) [14, 15]. These vaccines have been proven effective worldwide against HBV infection. Most recently, a further subviral particle vaccine against HEVs, the HEV 239/Hecolin® (Xiamen Innovax Biotech, Xiamen, China) that is made through the E. coli system, has been proven by the Chinese health authorities for human use in China [16]. In addition, there are two other subviral particle vaccines, the Ingelvac CircoFLEX® (Boehringer Ingelheim, Germany) and Porcilis PCV® (Intervet International, The Netherlands), that are commcercially available for use in domestic pigs against porcine circovirus infection and diseases [17, 18]. Furthermore, the NoV VLP vaccine has shown significant protection against NoV diarrhea in phase II clinical trials [19••, 20, 21], while many other subviral particle vaccines are under intensive preclinical development (Table 1).

Subviral particles as vaccine platforms

In addition to being vaccines, the subviral particles can also be used as vaccine platforms to present foreign antigens and small peptide epitopes of heterologous pathogens for novel vaccine development. The highly stable structures of most subviral particles tolerate an exogenous insertion, which can be achieved through either recombinant DNA technology or chemical conjugation. The native antigenic properties of the inserted antigens or epitopes usually are preserved on the surface of the chimeric particles, while the immunogenicity of the antigen/epitope is significantly enhanced by the polyvalent nature of the subviral particles functioning as an adjuvant. In addition, the major antigenic determinants of the subviral particle carriers are generally preserved, and thus the resulting chimeric particles can be used as a dual vaccine against the pathogens of the insertion and the carrier.

Numerous chimeric particles with antigen or epitope insertions on the surface have been produced (Table 2 ), in which the foreign antigen is usually inserted into a surface loop of the subviral particles. The capacity of a foreign insertion is subviral particle-dependent, with a maximal insertion of 238 residues (green fluorescence protein, GFP) for the HBV VLP [22] and 159 residues (VP8* antigen of rotavirus) for the P particle of NoV [9] being reported. A selection of proper sites of a subviral particle for insertion of exogenous antigens and/or epitopes is important for the generation of stable chimeric particles, the distal end of a flexible surface loops is generally a good choice.

Table 2

Some subviral particle platforms for display of heterologous antigens and epitopes for vaccine development

Virus species	Subviral particle	Displayed epitope or antigen	Production system	Immune response in animal (mouse)	Neutralization/protection against pathogens and diseases (mouse)	Clinical trial	Reference
Vaccine candidates that are in clinical trials
HBV	VLP	CSP antigen of P. falciparum	Yeast	Ab, T cell (human)	Protection against malaria (human)	Phase I, II, and III	[23•, 24••, 112, 113]
	VLP	CSP epitopes of P. falciparum	E. coli	Ab (monkey, human)	Protection against malaria (monkey)	Phase I	[25, 26]
	VLP	M2e epitope (influenza virus)	E. coli	Ab	Protection	Phase I	[27, 29, 33]
Bacteriophage	VLP	Nicotine	E. coli	Ab (human)	Increase smoking cessation (human)	Phase I, II	[34, 35]
Qβ	VLP	Angiotensin II epitopes	E. coli	Ab (rat, human)	Reduces blood pressure (rat)	Phase I	[36]
	VLP	allergen Der p 1 epitope	E. coli	Ab (human)		Phase I	[114]
Some vaccine candidates that are in preclinical development
CPMV	Virion	VP2 epitope of MEV	Cowpea leaf	Ab	Protection (minks)		[115]
	Virion	Protein F epitope of P. aeruginosa	Cowpea leaf	Ab	protection		[116]
	Virion	FnBP epitope of T. aureus	Cowpea leaf	Ab	protection against endocarditis (rat)		[117]
FHV	VLP	Toxin of Bacillus anthracis	Baculovirus	Ab	Neutralization, protection (rat)		[72]
Influenza virus	VLP	IBV S1 protein	Baculovirus	Ab, T cell (chicken)	Neutralization, protection (chicken)		[118]
	VLP	HA/NA Epitope of NDV	Baculovirus	Ab (chicken)	Protection (chicken)		[119]
	VLP	F or G antigen of RSV	Baculovirus	Ab	Neutralization/protection		[77]
HAV	VLP	Angiotensin II epitopes	Baculovirus	Ab (rat)	Reduced blood pressure (rat)		[120]
HBV	VLP	SP55/SP70 epitopes of EV71	E. coli	Ab	Neutralization/protection		[121]
	VLP	epitopes of HCV	E. coli	Ab, T cell, CTL			[122]
	VLP	HVR1 epitope of E2 of HCV	E. coli	Ab	Neutralization		[123]
	VLP	EDIII antigen of DENV-2	Yeast	Ab	Neutralization		[124, 125]
	VLP	E1 epitope of rubella virus	E. coli	Ab			[126]
	VLP	CSP epitopes of P. falciparum	E. coli	Ab, T cell (human)		Phase I	[26, 63]
	VLP	OspA antigen of B. burgdorferi	E. coli	Ab	Protection		[64, 65]
	VLP	VP2 five-mimotope of IBDV	E. coli	Ab (chicken)	Protection (chicken)		[127]
	VLP	CFP-10 antigen of MTB	E. coli	Ab, T cell			[128]
HIV	VLP	Domain III of DENV1 or WNV	Baculovirus	Ab	Neutralization		[129]
	VLP	F/G surface antigens of HMPV	Baculovirus	Ab	Neutralization/protection		[130]
NoV	P particle	VP8* antigen of RV	E. coli	Ab	Neutralization/protection		[9•]
	P particle	M2e epitope of influenza virus	E. coli	Ab	Protection		[41]
	Polyvalent complex	VP8* antigen of RV	E. coli	Ab, T cell	Neutralization/protection		[53]
	Polyvalent complex	M2e epitope of influenza virus	E. coli	Ab	protection		[53]
	Polyvalent complex	P domain antigen of HEV	E. coli	Ab	Neutralization		[131]
PyV	VLP	Pre-S1 epitope of HBV	Yeast	Ab			[95]
	VLP	N-termini of NP of PUUV	Yeast	Ab			[96]
	VLP	CTL epitope of mucin 1	Yeast	Ab, T cell			[97]
	VLP	GP33 CTL epitope of LCMV	Yeast	T cell	Protection		[98]
	VLP	J8i antigen of GAS	E. coli	Ab	Protection		[99]
	VLP	Her2 antigens of tumors	Baculovirus	T cell	Protection against tumor growth		[100]
	VLP	PSA antigens of D2F2 tumors	Baculovirus	Ab, T cell	Protection against tumor growth		[101]
	VLP	H190 epitope of influenza virus	E. coli	Ab			[132]
	Pentamer capsoid	B cell epitopes	E. coli	Ab (pig)			[102]
RHDV	VLP	3A protein epitope of FMDV	Baculovirus	Ab, T cell (pig)			[51]
SV40	VLP	HLA-CTL epitope of flu virus	Baculovirus	Ab, T cell	Protection		[133]

Ab, antibody; B. burgdorferi, Borrelia burgdorferi; CFP-10, antigen of culture filtrate protein 10; CPMV, cowpea mosaic virus; CSP, circumsporozoite protein; CTL, cytotoxic-T-lymphocyte; DENV-1/2, dengue virus type-1/2; EDIII, envelope domain III; EV71, Enterovirus 71; FHV, Flock House virus; Flu virus, influenza virus; FMDV, foot-and-mouth disease virus; FnBP, fibronectin-binding protein; GAS, Group A streptococcus; GP, glycoprotein; HA/NA, hemagglutinin/neuraminidase; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HEV, hepatitis E virus; HMPV, human metapneumovirus; HVR1, hypervariable region 1; IBDV, infectious bursal disease virus; IBV, infectious bronchitis virus; INF, interferon; LCMV, lymphocytic choriomeningitis virus; M2e, ectodomain of influenza A virus M2 protein; MEV, Mink enteritis virus; MTB, mycobacterium tuberculosis; NDV, Newcastle disease virus; NP, nucleocapsid protein; NoV, norovirus; P. falciparum, Plasmodium falciparum; P. aeruginosa, Pseudomonas aeruginosa; PyV, polyomavirus; PUUV, Puumala hantavirus; RV, rotavirus; RHDV, rabbit haemorrhagic disease virus; RSV, respiratory syncytial virus; SV40, simian vacuolating virus 40 or simian virus 40; T. aureus, Taphylococcus aureus; VLP, virus-like particle; WNV, West Nile virus.

The HBV VLP has been extensively studied as a vaccine platform for presentation of heterologous antigens and epitopes, with a chimeric VLP vaccines reaching to phase III and other two to phase I human trials. One is the RTS,S/AS01 malaria vaccine (GlaxoSmithKline) that comprises of the C-terminal half (189 residues) of the circumsporozoite protein (CSP) of Plasmodium falciparum on the surface of the HBV VLP (HBsAg) with adjuvant AS01 [23]. This chimeric vaccine is currently under phase III evaluations with high protective efficacy [24] and thus will most likely be the first malaria vaccine ever licensed and the first vaccine with a VLP-displayed antigen. Another VLP-based malaria vaccine is ICC-1132 (Malarivax) that is composed of a HBV VLP (HBcAg) displaying multiple epitopes of the P. falciparum CSP [25, 26]. After testing in rodents and nonhuman primates [25], this vaccine candidate was assessed for safety and immunogenicity by a phase I human trial, which showed malaria- and HBV-specific immune responses [26], supportingg ICC-1132 as a potential dual vaccine. The other HBV VLP-based dual vaccine is the M2e-HBcAg chimera, in which the conserved M2e epitope of influenza A virus M2 protein is linked to the HBV VLP through either recombinant DNA technology [27] or chemical conjugation [28]. After a number of animal experiments showing specific immune reponses against the M2e epitope and HBV, as well as protective immunity against influenza virus infection [28, 29, 30, 31], the the first phase I trial was performed in 2008, demonstrating its safety and immunogenicity in humans [32, 33]. These data prove the concept that subviral particle can be a practical strategy of novel vaccine development.

Another well studied subviral partcile platform is the bacteriaphage Qβ VLPs that have been used to develop vaccines to control smoking addiction, hypertention and allergy. Nicotine was cross-linked to Qβ VLPs, forming nicotine-Qβ chimeric particle vaccine. Both phase I and II human trials of smokers showed high nicotine-specific immune responses in vaccinated subjects and revealed significantly increased abstinence rates of smoking [34, 35]. The Qβ VLP was also used to display the epitopes of angiotensin II (Ang-Qβ) and the chimeric vaccine induced high level of angiotensin II-specific IgG and reduced systolic blood pressure in vaccinated rats [36]. A phase I human trial confirmed the high immunogenicity and safety of the chimeric vaccine [36]. In a separate study, an epitope of allergen Der p1 was covalently coupled to the Qβ VLPs (Der-P1-Qβ). This vaccine induced high immune response and has been shown to be safe in humans [37].

There are many other chimeric subviral particle-based vaccines that are in the preclinical evaluation, including those derived from VLPs of polyomaviruses, cowpea mosaic viruses, flock house virus, and NoVs (Table 2). The P particle of NoV that is formed by 24 copies or 12 dimers of the protruding (P) domain of NoV capsid protein (VP1) is highly stable and immunogenic [38•, 39]. Three surface loops are identified on each of the P monomer that tolerate a heterologous insertion of at least 159 residues [9•, 40]. Two chimeric P particles, each with the rotavirus surface spike protein VP8* [9] and the conserved M2e epitope of influenza A viruses [41], have been successfully constructed. Both chimeric vaccines revealed strong humoral and cellular immune responses, neutralization and protective efficacies against these viruses in mouse models [9•, 38•, 41], supporting the two chimeric particles as dual vaccines against rotavirus and NoV, and influenza virus and NoV, respectively.

Challenges and future directions

The non-replicating subunit vaccine is an important option against many viral pathogens, particularly those that an in vitro cultivation system remains lacking such as human NoV, and that are too dangerous to culture, such as variola virus and Ebola virus. It is also a choice for future vaccines to avoid the safety concerns of conventional live attenuated or inactivated vaccines, such as a safe vaccine for eradication of poliovirus. The recent reports on the increased risk of intussusception of the two live attenuated rotavirus vaccines to vaccinated children [42•, 43, 44] is a new example of such concerns that could be prevented by a non-replicating subunit vaccine. However, based on current technology, it seems not possible to produce subviral particles of all known viral pathogens. Thus, the technology of subviral particle-based antigen presentation provides an important strategy for vaccine development against those viral pathogens. As shown in the two tables, many subviral particles are capable antigen carriers. Since the major antigenic determinants of many viral pathogens are known (Table 2), it would be straightforward for design and producing a new vaccine by taking advantage of this technology.

The past experience suggests that success of a chimeric vaccine may rely on certain levels of structural and/or chemical compatibility between the carriers and the inserted antigens. There is no simple solution to this technical challenge. If such a problem occurs, attempts of other carrier-antigen combinations are encouraged. In addition, a modification of the carrier vectors by including short flexible peptide adaptors to the two arms of the surface loops is an option. Furthermore, the maximal size of an inserted antigen may vary among different carriers, and therefore, selection of proper carriers for larger antigens is also recommended. Finally, selection of appropriate carrier–antigen combinations should be considered based on the target pathogens and host populations. For example, both NoVs and rotaviruses cause acute gastroenteritis in children, the selection of NoV P particle as carrier to present the rotavirus surface antigens is an ideal combination for a highly effective dual vaccine against the two most important causes of acute gastroenteritis in children.

Subviral particle-based vaccines may not be as immunogenic as those replicating viruses following a natural infection. Thus, development of strategies for a maximal efficacy of the subviral vaccines is important, for which optimization of the vaccine formulations and vaccination regimes may be the key, including increase of vaccine doses and dosages, identification of the best administration routes, and use of appropriate adjuvants. In the case that the antigen-presentation approach is used, rational designs of the vaccines by increasing the copy numbers of the inserted antigens/epitopes on each subviral particle carrier should be considered. In addition, insertion of a universal immunue stimulate elements, such as the T cell epitope, may be considered.

Currently, production of most subviral particles relies on a eukaryotic expression system, such as baculovirus/insect cells, yeasts or mammalian cells. Since bacteria can produce subviral particles at lower cost, attempt to improve the prokaryotic expression system for production of more subviral particles would help to reduce the cost of vaccine delivery in the developing countries. It is worth to point out that several smaller or simpler subviral particles, including the VLP of HBV (HBcAg) [22], the P particles of NoV [4, 6], the E2 particles of HEV [3, 8], and the small VLP [7] and the L1 capsomeres [45] of HPV, can be readily produced in E. coli with excellent quality and yields. Since all these viral pathogens are prevalent in the developing countries, further study to develop their subviral particles into cost-effective vaccines and vaccine platform for broad application in the developing world is highly significant. Finally, new concept of vaccine delivery, such edible vaccines produced by transgenic vegetbles containing related subviral particles should be explored.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest