Innovation of a New Virus-Like Nanoparticle Vaccine System for the Specific Aerosol Relative Disease

Human spend a lot of time indoors and indoor air quality (IAQ) has a great effect on quality of life in humans. Traditionally, bioaerosol in IAQs is focused on bacteria and fungus. Airborne viruses, particularly for the nanoparticletype virus, on the bioaerosol particulates are associated with the quality of human health in indoor environment. We establish the human like animal model, like pig, for testing the efficacy of the nano virus like particle (VLP) vaccine for aerosol relative disease. The porcine reproductive and respiratory syndrome virus (PRRSV), which is an airborne and a droplet infection virus coated on the small size of bioaerosol, carries a major infectious disease in the pig raising industry worldwide. There are commercial vaccines including attenuated and inactivated vaccines available, but their efficacy remains to be improved. VLP represents safe and effective vaccine platforms based on nano-technology. The fusion protein of capsid protein (Cap) from the porcine circovirus 2 (PCV2) and the glycoprotein 5 (GP5) from PRRSV were expressed by E. coli system. The fusion protein self-assembled with the GP5 to become a VLP nanoparticle. For testing, BALB/c mice were immunized with 10 μg of chimeric VLP nanoparticle once, which induced neutralization by antibody responses to PRRSV. The VLP nanoparticle vaccine induced high T cell proliferation, not evident in commercial vaccines tested on mice. Our results suggest that the cheaper VLP–based nanoparticle is a possibly viable approach model for specific airborne-relative-diseases human vaccine in the future.


INTRODUCTION
A microenvironment or indoor environment is a main source of human exposure to several types of contaminants like inorganic chemicals (e.g., lead, arsenic, and chromium), allergens, organic contaminants (e.g., polycyclic aromatic hydrocarbons, polybrominateddiphenyl ethers, and phthalates), allergenic or mutagenic substances, bacteria, fungus, and airborne virus probably due to human spending more than 90% in the indoor environment (Robert et al., 2009;Lee et al., 2011;Chang et al., 2015;Cheruiyot et al., 2015;Shy et al., 2015).Currently, the IAQ parameters including total bacteria counts, total fungi counts, carbon monoxide, carbon dioxide, particulate matter, ozone, and total volatile organic compounds (TVOCs) in workplaces, hospitals, transportation stations, department stores, supermarkets, and public buildings shall be met to the national standards in Taiwan (Hsu et al., 2012;Hsu et al., 2015), but airborne virus is not regulated yet.Airborne viruses and their vaccine are of particular interest due to their potentially adverse impact on human or animal health in the indoor environment.
Many chemical and biological aerosol contaminants influence indoor air quality as well as the quality of health (Hsu et al., 2012;Lee et al., 2012;Kram and Dmowska, 2013;Chen et al., 2015;Sidra et al., 2015).Bioaerosol contain many human pathogens including virus which is one of the biggest threats for human health.Vaccination with different delivery system is the best way for aerosol relative virus diseases.The vaccination delivery system plays the crucial role for inducing good protection efficacy of vaccine.In this research, we evaluate the new virus like particle nanoparticle delivery system for vaccine development.Indoor air viruses including porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are bioaerosol nanoparticles that infect hosts in indoor environment.The Taiwanese IAQ standard, only specifies airborne bacteria and fungi, but nano-size virus particles are not included (Hsu et al., 2012;Wang et al., 2014).Porcine reproductive and respiratory syndrome (PRRS) is one of the dangerous virus diseases of pig raising industry (Albina, 1997;Ellis et al., 1999).The clinical symptoms of PRRSV include cyanosis of the ear, severe reproductive failure and respiratory distress.PRRSV was first identified in North America in 1987 and in Europe in 1990.PRRS drew significant attention due to large-scale outbreaks resulting in annual losses of one billion US dollars in the North American pig industry, alone.There are two distinct genotypes: European (type 1) and North American (type 2).In Taiwan, type 2 PRRSV is widely present and has continuously evolved since the infection was discovered (Lee et al., 2015).Recently, the aerosol spreading pathway of the PRRSV transmission has been completely documented (Brockmeier and Lager, 2002;Otake et al., 2002).The best way of prevention today is vaccination of various forms.There are many commercially attenuated or inactivated vaccines for the prevention of PRRS.Research suggests that the attenuated vaccines still might have the ability to mutate to virulent types, and cause vaccine-associated outbreaks (Opriessnig et al., 2002).On the other hand, the immunity provided by these inactivated vaccines is not enough for the animal stock (Nilubol et al., 2004).
Viruses like particles (VLPs) are capable of self-assembling along virus protein, if similar size and morphology exist (Garcia Durán et al., 2016).Most importantly, they are non-infectious, since they lack genetic contents (Schiller et al., 2012;Rodriguez-Limas et al., 2013).On the field of immunization, their safety is higher than that of attenuated vaccines, and also induces better humoral and cellular immune responses (Grgacic and Anderson, 2006;Roy and Noad, 2009).The reason of efficiency in immune response is that the conformational epitope of VLPs is the same as that found in the virion (Grgacic and Anderson, 2006;Roy and Noad, 2009;Zhao et al., 2013).For example, the prokaryotic and eukaryotic expressions of viral capsid proteins can self-assemble to VLPs (Yin et al., 2010;Wu et al., 2012;Zhao et al., 2013).Some of these viruses include the human papilloma virus (Bryan, 2007;Kim et al., 2014), hepatitis C virus (Mihailova et al., 2006) and the porcine circovirus 2 (Liu et al., 2008;Wu et al., 2012).
The PCV2 is a small, non-enveloped, icosahedral particle with diameter of 15-20 nm containing a circular, single strand DNA (Faurez et al., 2009).There are two major open reading frames (ORF) of the PCV2 genome.The capsid protein (Cap) of PCV2 is the major structural protein.Research shows that the expressed capsid protein of baculovirus or Escherichia coli, spontaneously assembles to virus like particles (VLPs) (Liu et al., 2008;Wu et al., 2012).In our current study, PCV2-based nano VLP with PRRSV protein is produced by the prokaryotic system.Most of the VLP antigens were produced by baculovirus or yeast expression systems, and their production cost was about 10 to 100 times higher than ours (Liu et al., 2001;Wang et al., 2008;Yin et al., 2010;Wu et al., 2012).In this research, we expressed the PCV2-PRRSV antigen by a lower-cost bacterial system.We also evaluated the efficiency of the new vaccine.This is the first study reporting the development of a PCV2based chimeric VLP vaccine for PRRSV.

Genetic Construction
For cloning of the GP5 gene, the TY-1 PRRSV strain, isolated from a field outbreak in Taiwan in 2004, was used.The in-framed fusion genes of PCV2, cap (JQ390467) and PRRSV gp5 (EU273667.1),were synthesized into pET-28a with Himd III and Nco I restriction enzymes (Fig. 1).For more detail, see the site of Taiwan BioMed (Taiwan BioMed, Taipei, Taiwan).

PEG Precipitation of Virus Like Particles
The PCV2-PRRSV chimeric VLP were produced by E. coli system with 1 mM IPTG induction for 6 h.The culture medium was centrifuged for 20 min at 15,000 rpm with initial volume of 1L, and the bacterial pellet was resuspended in 10 mL PBS.The resuspension was disrupted by sonication with 20% pulsing cycles for 3 times (MISONIX Sonicator ® 300).After sonication, the cell debris was removed by centrifuging for another 20 min at 15,000 rpm.The VLP was precipitated from the supernatant by first adding 7% PEG 6000 and 2% sodium chloride, then and agitating at 4°C for 4 h before centrifuging at 9500 × g at 4°C for 30 min.Eventually, the supernatant was discarded, and the VLPcontaining pellet was resuspended in 4-5 mL PBS.The VLP solution was stored at -20°C.

LAL Assay
For the determination of endotoxin activity, the LAL assay (Pyrotell Associates, Cape Cod, MA) was used, according to the manufacturer's instructions.The LPS concentrations in the purified VLP were less than 0.1 EU mL -1 .Complete clotting of the lysate was indicative of a positive reaction.

Electron Microscopy
The slotted carbon grid was coated with purified VLPs, and stained with phosphotungstic acid for 60 s.The morphology of stained VLPs was observed by transmission electron microscope (H-7500, Hitachi, Japan).

Animal Trial and Sample Collection
Six-week-old inbred female SPF BALB/c mice were housed in cages (8 mice per cage) and had ad libitum access to water and food.The mice were vaccinated intramuscularly (i.m.) with 10 µg VLP with Freund's adjuvant on day 0. The commercial vaccine group was vaccinated with 10 4 50% tissue culture infectious dose (TCID50) modified live vaccines (MLV) according to the manufacturer's recommendations.The negative control group was injected with PBS.The blood samples for antibody detection were collected from mice tail vein, on day 0, day 14, and day 21, after vaccination.Blood was collected and centrifuged at 2,000 rpm for 30 minutes at room temperature.After centrifugation, the supernatant serum was collected for antibody and neutralizing tests.Lymphocytes were collected from mice spleen samples and were used for T cell proliferation analysis.The experiments were carried out in accordance with the ethical guidelines of The Institutional Animal Care and Use Committee (IACUC) in NPUST.

Evaluation of Antibody Responses
After immunization, the mice produced antibodies to PRRSV, which we were able to detect from the serum.The serum was evaluated by enzyme-linked immunosorbent assay (ELISA).The ELISA plate was coated with 1 × 10 4 TCID 50 PRRSV and used as target for antibodies.The serum was diluted 1,000-fold, and we used 10,000-fold diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Southern Biotech, AL, USA) as secondary antibody.The ELISA was carried out with ABTS substrate for 15 minutes.We read out the optical density (O.D.) with an ELISA plate reader at 450 nm.

T cell Proliferation Analysis
To determine the T cell proliferation activity, the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used.This colorimetric assay determines cell activity by measuring the breakdown of mitochondria of a viable cell, which correlates with the proliferation of the cells.The lymphocytes were isolated from spleen and seeded into 96well plates, with 1 × 10 5 cells per well in 100 µL medium with or without 1 × 10 4 TCID 50 PRRSV.After 72 h of incubation, 10 µL of MTT (0.5 mg mL -1 ) was added into the wells and was incubated for 4 h at room temperature.The plate was centrifuged for 15 min at 1,500 rpm.The supernatant was discarded and the cells were lysed in the buffer (0.01 M HCl + 10% SDS).The extent of the color change was 570 nm, measured by ELISA reader (Bio-TEK).The T cell proliferation activity was presented as stimulation index (S.I.).The S.I. is defined as S.I. = (O.D. of PRRSV stimulated -O.D. of PRRSV non-stimulated)/ (O.D. of PRRSV non-stimulated).Experimental data from three experiments showed eight replicates in the samples, each time.

Virus Neutralization Assay
The 100 TCID 50 of PRRSV was incubated with 100-fold diluted heat inactivated serum samples at 37°C for 2 h.The 1 × 10 4 MARC-145 cells were co-incubated with the mixture for another 2 h.The un-infected viruses were removed and fresh medium was added and incubated.After 72 h of incubation, the supernatant were collected and the virus genome was determined by Q-PCR.
The MARC-145 cell culture supernatant was used for RNA extraction with the help of RNeasy Plus Mini Kit (Qiagen, Mississauga, ON, Canada). 100 ng of RNA was added for RT-PCR using the Super Script™ III Platinum ® Two-Step RT-qPCR Kit (Invitrogen).The cDNA, primer and IQ SYBR Green Supermix (Bio-Rad, Hercules, CA) were mixed together.The qPCR conditions were: 95°C for 3 min, followed by 45 cycles at 95°C each lasting 15 s (denaturation), then 3 s at 55°C (annealing), and finally 30 s at 72°C (elongation).The normalized amounts of PRRSV genome relative to the VLP vaccination group were analyzed by the 2 -Ct method (Schmittgen and Livak, 2008).

Statistical Analysis
The GraphPad Prism 5 software (Graphpad, San Diego, PCV2-ORF2 PRRSV-ORF5 pET28a NcoⅠ HimdⅢ pET28a-PCV2-PRRSV CA, USA) was used for statistical analysis.Statistical comparison between the VLP and other groups was performed using the two-tailed Student t test.The level of significance was set to p < 0.05 for all analyses.Data are presented as mean ± standard division (S.D.).

Production and Morphology Analysis of Virus Like Particles
The Cap-GP5 VLPs were expressed by a prokaryotic expression system.The expressed protein was purified and characterized by SDS-PAGE and Western blot (Figs.2(a) and 2(b)).To find the real molecular weight of the protein, the VLPs purified by PEG, were disrupted by 6 M urea, and characterized by Western blot with anti-His antibodies (Fig. 2(c)).The structure of the purified VLPs was confirmed by electron microscopic examination, revealing the morphology of VLPs with a 20 nm diameter (Fig. 2(d)).The antigenicity of the VLPs with PRRSV, was also determined with PRRSV (Fig. 3(a)), PCV2 (Fig. 3(b)) infected or SPF pig serum (Fig. 3(c)).

Antibody Responses to Immunization in Mice
The antibody response of the chimeric nano VLPs was evaluated in a mouse immunization trial in the presence of Freund's adjuvants.GP5 protein was used as an antigen for PRRSV antibody detection.Mice vaccinated with either the VLPs or commercially modified live vaccine formulations, increased the anti-GP5 antibody responses after the first vaccination (Fig. 4).The VLP and the positive control groups all showed IgG antibody levels that were significantly higher than that of the negative control group (Fig. 4).The differences in IgG antibody levels between the VLP group and the commercial vaccine group were statistically significant in favor of our VLPs (p < 0.05).Namely, the chimeric VLPs induced significantly higher IgG antibody levels in response to the PRRSV GP5 protein than the negative control and the MLV groups (Fig. 4).The production of an immune response to the GP5 protein indicated that intramuscular chimeric VLP vaccination effectively induces a similar response to antigens to what we saw in the case of commercial live vaccines.

The Cytokine expression Level of Chimeric Virus Like Particles Vaccine in Mice
The cellular immune response was evaluated by a T cell proliferation assay.Splenocytes were harvested from vaccinated mice.The cells were stimulated with 1 × 10 4 TCID50 PRRSV.The T cell proliferation was determined by the MTT colorimetric assay system.The data indicated that cell proliferation was induced in the chimeric VLP group, but was not in the MLV or the negative control group (Fig. 5, P < 0.05).It is notable that the cellular immune response of the VLP vaccinated mice was higher than that of the other groups.This observation might be helpful in suppressing viral replication and reducing the infectiousness of PRRSV.

The PRRSV Neutralizing Antibodies Induced by Virus Like Particles
Neutralization plays a crucial role against PRRSV infection.Our neutralization assays were based on detecting the number of replicated PRRSV RNA copies.As shown in Fig. 6, the PRRSV replication was inhibited by PRRSVspecific antibodies, starting on the 21st day after primary vaccination.Our chimeric VLPs significantly decreased the RNA genome of the virus, compared with the effects of the negative control and the MLV groups.We also found significantly elevated neutralizing antibody levels in the

Groups
Relative PRRSV RNA (fold) Fig. 6.Anti-PRRSV neutralization antibodies in mice.Mice were vaccinated with MLV, VLP and PBS.The vaccinated serum were collected and used for neutralization antibody assay.The 200 fold diluted serum were mixed with PRRSV for 2 hrs and co-culture with MARC-145 cells for 3 days.The relative virus RNA was evaluated by real time PCR method.The data represent the mean ± standard errors for eight mice.The differences in the relative PRRSV RNA between the vaccinated groups were statistically significant (**p < 0.05).chimeric nano VLP group compared with the PRRSV MLV group (P < 0.05).Splenocytes proliferated vigorously in response to stimulation with PRRSV.This data demonstrates that our chimeric nano VLPs vaccine system is an effective candidate for PRRSV vaccine development.Intramuscular vaccination of mice with this vaccine induces both humoral and cytokine immune responses.
Our VLPs form multimeric nanostructures assembled by structural viral proteins.Their structures increase cell entry efficiency, and thus, tissue-specific targeting by the antigens.The uptake of VLPs by the macrophage and dendritic cells increases immune responses, resulting in IFN-γ release and the expressions of CD80 and CD40 (Chroboczek et al., 2014;Rosenthal et al., 2014).According to these studies, our VLP-based vaccine can induce better APC responses and stimulate humoral and cellular immunity.The Recombivax HB was the first VLP-based vaccine approved by FDA against hepatitis B (Bryan, 2007;Rodriguez-Limas et al., 2013).There are many VLP-based vaccines licensed for human and veterinary use (Landry et al., 2010;Kim et al., 2014;Landry et al., 2014).In recent years, baculovirusbased expression system-type VLP vaccines have been licensed against porcine diseases in the USA and Europe (Patterson et al., 2011).Most of the VLP antigens were produced by baculovirus or yeast expression systems and the production cost is high.Results show that bacterially produced PCV2 VLPs system can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine for porcine in the future.

CONCLUSIONS
Compared with the traditional vaccine method for airborne virus, the chimeric VLP nanoparticle can induce more immunological effects in an in-vivo model.The production of chimeric VLP nanoparticle is also faster, cheaper and presents a lower risk than the traditional method.Our research recommends this nano-technology approach may have great potential for some aerosol transmitted human or animal diseases vaccine development in the future.

Fig. 2 .
Fig. 2. Analysis of PEG-purified chimeric VLPs.(a) The transformed BL21 cell was induced by 0.6 mM IPTG for 6 hour (lane 1) and purified by PEG (lane 2).The protein was analysis by 7.5% SDS-PAGE (a) or treat with (c) or without (b) 6M urea and analysis by western blotting with anti-his antibody.The forms of the VLPs (d) were observed by TEM after negative staining of the samples.

Fig. 3 Fig. 4 .
Fig. 3 Analysis the antigenicity of chimeric VLPs.The PEG purified VLPs were analysis by western with serum from artificial PRRSV challenged SPF porcine (a) serum from artificial PCV2 challenged SPF porcine (b) or SPF porcine serum.

Fig. 5 .
Fig. 5. Analysis the T cell proliferation response in mice.The splenocytes were isolated and used for T cell proliferation assay from mice on the 21th days after the initial vaccination.The isolated splenocytes were incubated with PRRSV for 72 hrs and T cell proliferation were examined by MTT assays (*p < 0.05).