The Virosome as a Novel Concept for High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV) Vaccines

Journal of Integrative Agriculture

2013, 12(7): 1215-1224

July 2013

REVIEW

The Virosome as a Novel Concept for High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (HP-PRRSV) Vaccines CAO Zhengand LÜ Feng-lin College of Bioengineering and Key Lab of Biorheological Science and Technology, Chongqing University, Chongqing 400044, P.R.China

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an envelope, positive, single-strand RNA virus and is a member of the Arteriviridae family, Nidovirales order. PRRSV is the viral pathogen responsible for porcine reproductive and respiratory syndrome (PRRS) and caused reproductive failure and high rate of late abortion and early farrowing in sows and respiratory disease in all age. In 2006, a large scale outbreak of atypical PRRS occurred in China is characterized by high fever (41-42°C), high morbidity (50-100%) and high mortality (20-100%). The disease was caused by a highly pathogenic PRRSV with a 30 amino acid deletions in its Nsp2 coding region. Because the PRRSV strains are genetically heterogeneous, and elicit delayed and weak cell-mediated immune (CMI) and antibody responses after vaccination the current vaccines are failed to provide sustainable disease control. Virosomes are virus-like particles, consisting of reconstituted virus envelopes without genetic material of the native virus. Since the virosomes has being similar to the original virus in terms of morphology and cell entry characteristics. Virosomes provide a vaccine platform that has the capacity to combine the antigen and an adjuvant within a single particle that could activate both the humoral and the cellular arm of the immune system. Furthermore, the virosomes are also providing a novel promising approach for the development of an efficacious vaccine against HP-PRRSV. Key words: PRRSV, virosome, vaccines

THE VIRUS AND THE ANTIGENICITY OF THE VIRUS PROTEINS Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failure in sow and respiratory distress in all ages (Done and Paton 1995; Bøtner et al. 1997) and is one of the most economically important diseases of swine industry worldwide. The etiologic agent for this disease, porcine reproductive and respiratory syndrome virus (PRRSV), is an envelope, positive, single-strand RNA virus belonging to the

Arteriviridae family (Meulenberg et al. 1995; Dea et al. 2000). PRRSV, based on their antigenic and genetic characteristics, is divided into two distinct genotypes. The virus was initially isolated in the Netherlands in 1991 (Wensvoort et al. 1991; Evans et al. 2008), known as Lelystad virus, the representative of European type. A similar virus strain, ATCC VR-2332, was isolated in the United States in 1992 (Collins et al. 1992), and considered to be representative of the North American type. The PRRSV genome is approximately 15.4 kb in length and contains nine open reading frames (ORFs), which in order from the 5´ end of the genome. ORF1a

Received 24 April, 2012 Accepted 4 September, 2012 Correspondence LÜ Feng-lin, E-mail: [email protected]

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and ORF1b comprise about 80% of the genome and encode proteins with replicase and polymerase activities and the ORF1a and ORF1b are translated into two large polyproteins and processed into 13 nonstructural proteins (NSPs) (Boon et al. 1995; Snijder and Meulenberg 1998), which are mostly involved in genome replication and transcription. The seven smaller ORF2a, ORF2b, and ORF3 to ORF7 are located at the 3´ end of the genome and code for N-glycosylated structure protein GP2a, GP3, GP4, GP5, and nonglycosylated envelope small membrane protein (E), membrane protein (M), and nucleocapsid protein (N) (Meulenberg et al. 1995, 1996; Wu et al. 2001; Spilman et al. 2009). GP5, M, and N are the three major structural proteins. The 15 kD non-glycosylated N protein encapsidates the viral RNA genome and is highly basic, which facilitates its interaction with the genome RNA in the assembly of the nucleocapsid (Meulenberg et al. 1996). The antibodies directed to N, generated by PRRSV-infected pigs, are mostly abundant. By date, the immunodominant antigenic domain was mapped in the central region of the protein, 56-66 and 80-90 aa. In addition, the N-terminal is important of antibodies binging (Wootton et al. 1998). The GP5 protein is a 25 kD N-glycosylated protein, which spans the membrane three times and contains a 50-72 residues plasmic domain and a 30 residues ectodomain. The GP5 protein is containing major neutralization epitopes and associated with the neutralizing antibosies and host protection. The GP5 has formed disulphide-linked heterodimers with the 18-kD nonglycosylated M protein. M protein is the most conserved structural protein and its N-terminal spans the membrane three times, only leaving a 10-18 residues exposed at the surface (Mardassi et al. 1996; Ostrowski et al. 2002; Snijder et al. 2003). The heterodimers formed by GP5 and M is essential for virus infectivity and serves as a ligand for CD163, which is the PRRSV internalization receptor on alveolar macrophages (de Vries et al. 1995; van Breedam et al. 2010). The 29-30 kD GP2a, 45-50 kD GP3 and the 31-35 kD GP4 are minor envelope glycoprotein of the virus particle and are typical class I membrane proteins (van Nieuwstadt et al. 1996; Spilman et al. 2009). The GP2a protein forms a heterotrimer complex with the GP3

and GP4 protein by disulphide linkage (Wissink et al. 2005) and, detected by co-immunoprecipitation, the heterotimers were interacted with the GP5 protein, but only GP2 and GP4 were found to interact with CD163 receptor (Das et al. 2010).

THE PATHOGENESIS History of HP-PRRSV PRRS is characterized by reproductive failure in sows and respiratory disease in pigs and is one of the most economically important diseases of swine industry worldwide (Neumann et al. 2005). The known history of PRRS is relatively short. This disease was first observed in the North America in 1987 and in Europe in 1990 and spread throughout much of the rest of the world in the last 20 years (Wensvoort et al. 1991; Baron et al. 1992; Kuwahara et al. 1994; PU et al. 2009). In China, the first PRRSV isolated was in North China at the end of 1995 and the disease spread to the pig farms in the rest of China within the succeeding years (Chang et al. 2008; Chen et al. 2010). In May 2006, a disease with high fever, called as swine high fever disease (SHFD) outbroke in the central region of China and millions of pigs were killed during this outbreak (Tian et al. 2007). This severe infection was initially suspected to be co-infections caused by classical swine fever virus, porcine circovirus type 2, or peudorabies virus, etc. However, several laboratories in China have isolated the same one virus in the epidemic pig farms of the outbreak. By pathogenic and genomic analysis, the virus was considered to be a highly pathogenic PRRSV (HP-PRRSV) and was the main causative agent of the epidemic (Tian et al. 2007; Zhou et al. 2009a). Therefore, this outbreak should be called atypical PRRS instead of SHFD. By the results of genomic analysis of the HP-PRRSV, these HP-PRRSVs belong to the North American group and with a remarkable 30 amino acid (aa) discontinuous deletions in their non-structure protein 2 (NSP2) coding region. A single one Aa deletion was at position 482 of NSP2, and a 29 aa deletion was located at position 533-561 of NSP2 (Li et al. 2007; Zhou et al. 2009a, b). However, this 30 aa deletion in HP-PRRSV is not

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The Virosome as a Novel Concept for High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

related to its virulence and cannot be used as a marker to characterize the high virulence of the virus but can be used as a molecular marker for distinguishing the high pathogenic PRRSV from other PRRSV strains (Zhou et al. 2009a). The complete genomic sequence and the evolution analysis of BJ0706 (isolated in 2007 in China) indicate that the newly emerging HP-PRRSV displays higher homology with previous strains, in particular the CH1a (the first PRRSV isolated in 1996 in China) (Zhou et al. 2009b). Interestingly, the BJ0706 only have 1 aa deletion at position 481 of NSP2. Thus, it could be postulated that the HP-PRRSV emerging in China experiences a gradual variation and accumulation progress of genome change from previous PRRSV in China and the BJ0706 might be an intermediate virus during this evolution (Zhou et al. 2009b; An et al. 2010).

Clinical sign Before 2006, the PRRSV was the major pathogen causing dead fetuses, irregular abortions, early farrowing, and stillborn and other productive failure in sows and respiratory disorder in all age. The following co-infections of PRRSV with other pathogens, such as classical swine fever virus (CSFV), porcine parvovirus (PPV), and mycoplasma hyopneumonide, can cause more complicated situations. In particular, the co-infections of PRRSV with porcine circovirus type 2 (PCV-2) were easy to be detected after the postweaning multisystemic wasting syndrome outbreak in the pig farms (Zhou et al. 2006; Shen et al. 2010; Karniychuk et al. 2011). In comparison with typical PRRS, the atypical PRRS exhibited high morbidity (50-100%), high mortality (20100%) and other unique characteristics. The clinical symptoms exhibited by infected pig include high fever, depression, rube-faction on skin and ears, constipation or diarrhea and most pigs showed sneezing, coughing, eye secretion increasing, conjunctivitis and other respiratory distress. The atypical PRRS has obvious epidemic and transmission features, once the outbreak occurred in one pig farm and quickly spread the whole pig herd within 3-5 d and transmitted to nearby pig farms in the following 1-2 wk (Zhou et al. 2008; Zhou and Yang 2010). In most pig farms, the disease emerged initially in pregnant sows or finishing pigs and then transmitted to piglets. Affected cases were observed, the

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body temperature of the infected pigs reached 41-42°C and disease course was usually 1-3 wk. The prevalence rate ranged from 20% in finishing pigs to 100% in piglets. The death case could be found within 5-7 d and the morbidity was usually 50-100%. The survived pigs showed pale, rough hair, and emaciation, and some of them could recover gradually (An et al. 2010; Zhou and Yang 2010). The common lesions showed by infected pigs are unique for atypical PRRS, which include pulmonary edema, congestion and consolidation; lymph node enlarged and edema; larynx and trachea congestion; intestinal hydropsia and ulcers; brain edema and congestions; kidney hemorrhages; spleen infarct. Because the virus can induce a multi-systemic infection in piglet, the hallmark lesions in piglet are pulmonary consolidation and lymph node edema, especially the mesenteric and superficial inguinal lymph nodes (Feng et al. 2008; Zhou et al. 2008; Wei et al. 2011). With co-infection and secondary infections, the hemorrhagic pneumonia in lung, pericarditis, fibrinous pleuropneumonia and peritonitis could be detected (Zhou and Yang 2010).

CURRENT VACCINES Problems with current PRRSV vaccines Several PRRS vaccines are currently available; however, they are not effective in protecting against infections with the diverse field strains of PRRSV (Huang and Meng 2010). The first PRRSV vaccine (RespPRRS) was an attenuated moderately virulent PRRSV strain and introduced in North America in 1994 (Done and Paton 1995). It was only recommended for use in 318-wk-old pigs and in non-pregnant sows for preventing the respiratory facet of PRRS (Danneskiold-Samsøe et al. 2013). Another modified live-attenuated vaccine (MLV), Prime Pac PRRS, could reduce the severity of disease following challenge (Scortti et al. 2006). However, it cannot prevent the pigs by virulent heterologous strain (Meng 2000). Scortti et al. (2006) compared three commercial vaccines in their ability to induce protection against high virulence PRRSV; they found that vaccines could induce protection against clini-

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cal disease but not against infection. The attenuated PRRSV vaccines have shown some efficacy in reducing disease occurrence, but protection is always more pronounced in a homologous situation (Labarque et al. 2003). Some countries, such as UK, only permit the use of inactivated PRRSV vaccines. However, the inactivated vaccines were considered ineffective, even against homologous challenge. In order to control the PRRS in China, an inactivated vaccine derived from the first isolate CH1a was developed in 2000, and an attenuated vaccine derived from the same virus was registered in 2007. However, the clinical case associated with productive failure in pregnant sows and respiratory disorder in piglet occurred frequently in the vaccinated pig farms (Zhou and Yang 2010). After the outbreak of the atypical PRRS in China, an inactivated vaccine derived from the isolated HP-PRRSV (JXA1) was developed. However, the vaccine could not induce protection as expected. Jiang et al. (2006, 2007) has developed a recombinant live pseudorabies virus expressing GP5, M proteins of PRRSV and a recombinant plasmid DNA expressing GP5 of PRRSV, which could induce effective protection to PRRSV infection in pigs and antibody response in mouse model, but none of them was practically applied in field.

Challenge of PRRSV vaccine development In China, vaccines for PRRS have been available for more than 10 years. However, the atypical PRRS remains difficult to control. A number of small size pig farms still suffer the atypical PRRS. The inactivated vaccines were considered ineffective and not very promising. Although the attenuated vaccines were generally effective against homologous strains, but none of them is able to against heterologous strains (Cano et al. 2007b). Cano et al. (2007a, b) examined an attenuated vaccines of pigs previously infected. This vaccines significantly reduced the number of pigs persistently infected with a homologous strains, but not of pigs persistently infected with a heterologous strains. The attenuated vaccines also associated with some problems such as persistent infection, incomplete protection, and reversion to virulence (Storgaard et al. 1999). Key et al. (2003) have isolated a vaccine-derived PRRSV

strains and that virus strains could cause disease in pigs. The effectiveness of against hetreologous strains will depend on the antigenic relatedness of the virus strains. Qiu et al. (2005) used swine pseudorabies virus (PRV) as vector for expressing the GP5 protein of PRRSV which could induce protection against clinical disease, and reduce pathogenic upon PRRSV challenge, but failed to neutralize antibodies. Jiang et al. (2008) used replication-defective adenovirus recombinants as vector to examine the immunogenicity of GP3, GP4, and GP5 protein. Vector expressing the hybrid GP3-GP4 or GP3GP4-GP5 protein appeared more immunogenic than a single protein. Currently, safety and immunogenicity issues challenge the development of PRRSV vaccines. All data available so far suggest that all structural protein of PRRSV are essential for inducing cell-mediated immune (CMI) and antibody responses after vaccination. Therefore, the design of future vaccines must take the antigenic diversity and all structure protein of PRRSV into consideration.

VIROSOMES The background of virosomes Virosomes or “reconstituted viral envelopes” are viruslike particle, consisting of reconstituted viral envelopes, lacking the viral genetic material. Almeida et al. first introduced virosomes by insertion of purified spike proteins of the influenza virus into pre-formed liposome in 1975 (Almeida et al. 1975; de Jonge et al. 2006). Thereafter, virosomes derived from different virus have been described. Since the virosomes are closely mimicking the intact virus and they display the viral envelope glycoprotein and the most important viral antigens for immune responses in a native configuration, they are highly suitable for use as vaccines (Huckriede et al. 2005; de Vries et al. 2009). A virosomal vaccine has been commercially available for more than ten years (Inflexal® V), which is derived from influenza H1N1, H3N2, and B strains and consists of a mixture of this three monovalent virosomes. Inflexal® V is used as a vaccine for immunization against influenza in 38 countries, and is the only adjuvanted influenza vaccine

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The Virosome as a Novel Concept for High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

licensed for all age groups (Mischler and Metcalfe 2002; Herzog et al. 2009). Clinical studies have demonstrated that intramuscular administration of this vaccine to humans inducing haemagglutination-inhibition titers is similar to those induced by conventional whole virus. Chrstian et al. reported that, during its eleven years on the market, Inflexal® V has shown an excellent tolerability profile due to its biocompatibility (Herzog et al. 2009). Since the virosomes preserve the receptor-binding and membrane fusion activity of the viral enveloped protein, the virosomes are similar to the original virus in terms of morphology and cell entry characteristics (de Jonge et al. 2006). Moreover, the receptor-binding and membrane fusion properties allow the use of virosome as transport vehicles for cellular delivery of biologically active macromolecules, and does not result in infection of the cells (Cusi et al. 2002; Homhuan et al. 2009).

Interactions of virosomes with the immune system Membrane-associated immunoglobulin receptor molecules can recognize the enveloped protein on the surface of virosomes, as well as the foreign antigens carried by virosomes, on B lymphocytes (Bungener et al. 2002; Bungener et al. 2005; Wilschut et al. 2009). Since the internalization and intracellular trafficking of virosomes are similar to their native virus, the antigen delivered by virosome is mimicked by the natural pathway of antigen processing and presentation. The virosomes enter the antigen-presenting cells (APCs), in particular the dendritic cell, via receptor mediated endocytosis, and acidic pH triggers fusion events between virosomes and endosomal membranes resulting in encapsulated antigen release into cytosol of the ACPs. The antigen can thus into the conventional MHC class I presentation pathway and interact with T cell receptor on CD8+ lymphocytes, that offers the possibility to induce cytotoxic T lymphocyte (CTL) responses (Bungener et al. 2005; Homhuan et al. 2009). However, not all of the virosomes are likely to fuse with the endosomal membrane after uptake by ACPs; a fraction of the particles will continue their fate in the endosomal/ lysosomal pathway, resulting in the generation of peptides derived from the viral spike proteins as well as

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from virosome encapsulated antigen. The peptides generated would then be presented in the context of MHC class II molecules. The presentation of MHC class II/ peptide complexes on the APCs surface to CD4+ T cells will induce strong T helper responses that are of crucial importance for the stimulation of CTLs, and for the support of antibody-forming B cells (Nakanishi et al. 2000; Bungener et al. 2002; Homhuan et al. 2009; Wilschut et al. 2009). T helper cell responses after immunization of mice with virosomal vaccine have also been examined. The T lymphocytes obtained from the spleens of mice produced IFNγ as well as IL-4 indicating that virosome induced a balanced Th1/Th2 response. Furthermore, the numbers of IFNγ-producing cells induced by virosomes are higher than those induced by subunit vaccines, and the T helper responses are more closely resembles that observed upon infection with live virus (Nakanishi et al. 2000; Bungener et al. 2002). Inactivated virus do not have access to the cytosol and cannot enter into the MHC class I presentation pathway, thus they are inefficient in activating CTLs. The CTL activity is importance for the destruction of virus-infected cells, and the clearance of virus infections. Virosomes, like their native virus, are thus having the capacity to deliver antigen for presentation in both MHC class I, and II, activated both humoral and the cellular arm of the adaptive immune system. This opens the possibility to introduce antigens into the cytosolic pathway of antigen presentation, and the virosomes interact efficiently with the immune system, leading to activation of B cells, dendritic cells and T cells as found during native virus infection. Therefore, to develop the next generation of HP-PRRS vaccines may thus be tailored to induce CTL activity not only against the GP5 protein, but the GP2-GP3-GP4 heterotrimer complex and other major antigens.

Virosomal delivery system The escape potential and the ability of virosomes to induce a strong immunogenic response have been proven by many studies. Zurbriggen et al. (2000) and Homhuan et al. (2009) have demonstrated that a mixture of streptavidin with influenza virosomes induced a four-fold higher antibody immune response than the antigen given alone. In addition, delivery of the antigen

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via virosomes induced approximately 4 500-fold higher antibody titer than the antigen given alone, that elucidating the capability of virosomes as delivery systems. The subunit A of diphtheria toxin (DTA) is a membraneimpermeable toxin that is non-toxin as it can’t enter the cell by itself. Bron et al. (1994) demonstrated that the DTA could be delivered to the cytosol and toxic to the cell when it was encapsulated into virosomes. Angel et al. (2007) have reported that virosomes containing encapsulated Melan-A peptide have the capacity to bind human plasmacytoid DC and virosomes were endocytosed within vesicles in the cytosol. Bungener et al. (2002) have used ovalbumin (OVA) as a model antigen to examine the ability of virosomes to deliver antigen to the cytosol. The study shown that influenza virosomes are highly effective in the delivery of OVA for MHC class I presentation by mouse DC. In recent years, some studies have investigated whether virosomes encapsulating tumor-associated antigens (TAA) are able to induce specific CTL activity and to inhibit the tumor progression in mice. Bungener et al. (2006) have demonstrated that influenza virosomes containing early protein 7 (E7) of human papillomavirus 16 (HPV-16) have the ability to induce strong E7-specific CTL responses and protected 70% of mice against tumor challenge with TC-1 cells (E7-expressing tumor cells). Schumacher et al. (2005) have reported that influenza virosomes containing Mart-1/Melan-A peptide derive from melanoma epitope could induce pacific Mart-1/Melan-A CTL responses and the proliferation of CD4+ T cells. Virosomes retain the functional capacity of the original virus to enter antigen-presenting cells and shown their potential to induce a strong immune response. This capacity opens the possibility to deliver foreign antigen to the cytosol of ACP, and shown the potential as novel selective antigen deliver systems. Adjuvants are substances that enhance the immune response. Some commonly used adjuvants, like aluminum salts or mineral oil serve to form a physical depot retaining the antigen for presentation to cells of the immune system. However, these adjuvants do not effectively augment CTL responses. Virosomes containing adjuvants incorporated into their membrane offer the unique advantage of combining of ATCs and could enhance the immune response. Spohn et al. (2004) have

reported that lipopeptide adjuvant N-palmitoyl-S-2,3 (bispalmitoyloxy)-propyl-cysteinyl-seryl-(lysil)3-lysine (P3CSK4) incorporated that in influenza, virosomes could be recognized by Toll-like receptor 2 of B lymphocytes and dendritic cells. As compared to virosome alone, injection of the adjuvanted virosomes in mice resulted in a 150-fold increased IgG response. Stegmanna et al. (2010) have demonstrated that mice injected with respiratory syncytial virosomes (RS virosome) with P3CSK4 induced five-fold neutralizing antibodies titers than that with the adjuvanted F protein of RSV. After vaccinations the amounts of IgG1 and IgG2a isotopes were determined to assess the Th1/Th2 skewing of the response. As compared to virosome alone, injection of virosomes with P3CSK4 induced more IgG2a which can thus lead to a more balanced Th1/Th2 response. de Vries et al. (2009) reported that LpxL1 (a detoxifie lipopolysaccharide adjuvant) could be co-reconstituted in influenza H5N1 virosomal membranes. To compare non-adjuvanted virosomes, LpxL1-virosomes significantly increased HI titers and total IgG titers to virosomes. Moreover, immunization with LpxL1-virosomes resulted in increased Th1-type antibody (IgG2a) levels, higher IgG2a/IgG1 ratios, suppression of IL-4-producing T cells and higher IFNγ/ IL-4 ratios. Several studies have demonstrated that virosomes have the capacity to induce a strong immune response, and also to provide the unique opportunity to combine an antigen and an adjuvant within a single particle.

POTENTIAL APPROACHES FOR DEVELOPING THE VIROSOME-BASED PRRSV VACCINES The development of safe and effective vaccines against HP-PRRSV infection remains a challenge and a significant priority of the swain industry. Several vaccines suffer from poor immunogenicity, insufficient seroconversion rate, lack of hetero-subtypic protection and inability to induce cell-mediated immune response and antibody response (Jiang et al. 2008; Prieto et al. 2008). Cinta et al. reported that immunization of growing pigs with a recombinant fusion GP5 protein not only failed to provide protection from subsequent

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The Virosome as a Novel Concept for High Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

viral challenge, but also appeared to exacerbate disease (Prieto et al. 2011). The lack of protection afforded by vaccines against HP-PRRSV has been attributed to their inability to elicit a robust immune response and to the significant antigen diversity of the virus. The plasmacytoid dendritic cells (pDCs) are now being considered as the target in the design of new vaccines by enhancing the immunogenicity of vaccines (McCullough and Summerfield 2009). Developments of delivery systems capable of targeting the vaccine antigen to pCDs are recognized as the research priority. This avenue could be applied to pigs by using pCD169 (sialoadhesin receptor) as the target on porcine monocyte-derived DCs (MDDCs) (Revilla et al. 2009). Revilla et al. (2009) have compared the T cell proliferative response induced by an anti-pCD169 Mab with that of a non-targeting Mab. The anti-pCD169 Mab could rapidly internalize and induce recall T cell response at concentrations of 100-fold lower than the non-targeting Mab. Since the pCD169 has been identified as the entry receptor for PRRSV, targeting the PRRSV antigens directly to pCD169 will increase the vaccine efficacy (de Vries et al. 1995; Revilla et al. 2009). The challenge of novel vaccine development remains to design vaccine formulations that closely mimic the HP-PRRSV without bedding HP-PRRSV themselves. The generation of HP-PRRSV virosomes is a promising step in this direction. A key feature of virosomes is that they closely resemble the native virion in terms of surface organization and functional characteristics. The HP-PRRSV virosome will interact with the immune system in various ways eventually leading to activation of B cells, dendritic cells and T cells as found during the native virus infection. The type of antigen is an important factor in vaccines design. Virosomes retain the functional capacity of the native virus penetrate cells and fuse with the limiting membranes of intracellular compartments. That opens the possibility of virosomes as a delivery system to introduce different antigens into the cytosolic pathway of antigen presentation. These antigens are presented in the context of MHC class I to prime CTL activity. Novel generations of HP-PRRSV vaccines may thus be tailored to induce CTL activity not only against the viral enveloped protein, but also against the other antigens such as nucleoprotein and matrix protein, through encapsulation of these antigens

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in the virosomal lumen. Another factor is becoming increasingly apparent that specific stimulation of dendritic cells is of key important to the quality and quantity of the immune response to vaccine antigens. Novel development in this regard involves the use of adjuvants, by which virosomes represent an excellent platform for inclusion of lipophilic or amphipathic adjuvants targeting the specific signaling receptors on dendritic cells to improve the quality and quantity of the induced immune response. Virosomes provide a vaccine platform that has the capacity to combine the antigen and an adjuvant within a single particle that could activate both the humeral and the cellular arm of the immune system. The virosomes also provide a novel promising approach for the development of an efficacious vaccine against HPPRRSV.

Ackownledgements This work was supported by the National Natural Science Foundation of China (11032012), the Natural Science Foundation Project of Chongqing CSTC, China (2009BA5068) and the “111 Project” for Biomechanics and Tissue Repair Engineering, China.

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