1. Introduction
Newcastle disease (ND), an acute and highly contagious avian disease caused by avian paramyxovirus, remains endemic throughout Africa, Asia, and Europe, with frequent epidemics occurring despite the availability of global commercial vaccinations since the 1930s [
1,
2]. When the virus is introduced into a susceptible flock, including domestic poultry, virtually all the birds will be infected within two to six days (World Organisation for Animal Health, OIE, 2019). According to the World Animal Health Information Database (WAHIS), an average of 60 countries reported NDV outbreaks yearly from 2013 to 2018, and the number of viral genotypes is increasing (OIE, 2019), which might be related to the mutation and reconstitution of live vaccines [
3]. Therefore, ND continues to be a threat and to cause considerable economic losses to the poultry industry worldwide. ND in its highly pathogenic form is listed in the World Organization for Animal Health (OIE) Terrestrial Animal Health Code and must be reported to the OIE.
Due to the constant threat of introduction of the virus from wild birds, vaccination and the establishment of poultry biosecurity are essential to the prevention of ND. At present, ND vaccines are available in live, inactivated forms and are licensed for use in many countries worldwide. Where vaccination is carried out, serological surveillance is of limited value because it cannot discriminate between NDV infection and vaccination. Positive NDV antibody test results can have four possible causes: (i) natural infection with NDV; (ii) vaccination against ND; (iii) exposure to vaccine virus; and (iv) maternal antibodies. This is a challenge for ND eradication plans in many countries. Chicken-producing countries that are vaccinated with whole virus are adversely affected by restrictions on international trade designed to avoid the importation of chickens that may be infected with NDV into ND-free zones [
4]. Therefore, the development of the next generation of NDV vaccines is essential to distinguish between infected and vaccinated animals while ensuring disease control. As an alternative to live and inactivated vaccines, subunit vaccines require only an immunogenic portion of the target virus, without the risk of virus spread and recombination, and can be designed to develop a differentiating infected from vaccinated animals (DIVA) strategy and disease eradication plan for ND control. The greatest dilemma in avian vaccine research is the balance between product prices and immunogenicity. Currently, the prices of NDV inactivated vaccines and live vaccines used in China are much lower than 0.1 RMB per dose. Traditional expression systems such as insect cells are not easily accepted by the market, and low-cost
E. coli expression systems cannot readily obtain highly immunogenic antigens; thus, there are currently no NDV subunit vaccines on the market.
The envelope of NDV contains two transmembrane glycoproteins, the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), which form spike-like protrusions on the outer surface of the virion. The HN protein is responsible for the attachment of the virus to the sialic acid receptor on the host cell and for releasing progeny virions from the surface of infected cells [
5,
6]. Fusion protein (F) glycoprotein is an important protective protein of NDV that promotes the merger of viral and cellular bilayers and the opening of a pore to deliver the viral genome into the cytoplasm of the host [
7]. These proteins are the two major vaccine antigen candidates. F glycoprotein has been shown to be the major contributor to the induction of neutralizing antibodies and protective immunity, followed by the HN protein, which conferred partial protection against an intravenous challenge [
8,
9]. Therefore, compared with the HN protein, the F protein is more ideal as a subunit vaccine antigen.
The “transgenic plant vaccine” was proposed in 1992 [
10], and transgenic plants are promising vehicles for recombinant proteins [
11,
12,
13]. Compared to traditional systems, plant hosts exhibit easy scalable production, very low production costs, high production quality, lack of pollution, and the process of eukaryotic protein modification [
14,
15]. Moreover, vaccines produced by plants avoid the culture of viruses and bacteria, eliminating the risk of infection [
16]. A number of pharmaceutical proteins produced by transgenic plants are currently in clinical development. ZMapp antibodies against the Ebola virus and influenza vaccines have shown the great potential of the plant system.
Rice seeds are a cost-effective bioreactor for the large-scale production of pharmaceuticals [
14]. To obtain inexpensive and effective subunit ND vaccines to meet market needs, in this study, we expressed an
Oryza sativa recombinant F protein from transgenic rice seeds. The expression level of the F protein was increased by hybridizing F-transgenic rice with low-gluten rice. Immunoassays and protective assays have demonstrated that recombinant proteins do not trigger a stress response, and 4.5 μg immunization doses protect chickens from lethal viruses. This was the first time that the immunogenicity of the plant-made F vaccine was comprehensively characterized in vivo. Moreover, plant-produced F vaccine enables the differential diagnosis of vaccination and natural infection by detecting HN-specific antibodies. Our results demonstrate that transgenic rice engineering is a promising approach for the future production of an affordable ND vaccine.
2. Materials and Methods
2.1. Construction of Plant Vector and Rice Genetic Transformation
The DNA sequence coding for the F gene (GenBank accession No. JN618348.1) was synthesized by GenScript Corporation using rice codon preferences. The F gene was subcloned into the
MlyⅠ-Xho Ⅰ site of intermediate vector pMP3 containing the Gt13a promoter, a signal peptide and the
t-nos terminator (Healthgen Biotechnology Co., Ltd., Wuhan, China). The recombinant plasmid pMP3-F was digested by
EcoR Ⅰ and
Hind Ⅲ and cloned into the plant vector pCAMBIA1300 (Healthgen Biotechnology Co., Ltd., Wuhan, China), which includes the hpt Ⅱ (hygromycin resistance) gene as a selective marker and the right and left borders necessary for T-DNA transmission. The plasmid pCAMBIA1300-F was transformed into the callus regenerated from rice cultivar TP309 by
Agrobacterium-mediated transformation as described previously [
14]. Positive callus was obtained after hygromycin-resistance screening and transplanted into the greenhouse.
2.2. PCR Analysis to Select Positive Transgenic Plants
Total genomic DNA was extracted from young leaves of transgenic rice by the CTAB method. Positive transgenic plants were doubly identified by the hygromycin gene and the F gene. The F gene was amplified with forward primer (5’-CACATCCATCATTATCCATCCACC-3’) and reverse primer (5’-GAGGAGGGTGGTGAGGGT-3’). The hyg gene was amplified with forward primer (5’-CGATTCCGGAAGTGCTTGAC-3’) and reverse primer (5’-CGTCTGCTGCTCCATACAAG-3’).
2.3. SDS-PAGE and Western Blotting
The transgenic rice extract and the pure F protein were separated on a 12% polyacrylamide gel and then transferred to a PVDF membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 5% skim milk in phosphate-buffered saline Tween 20 (PBST) buffer to prevent non-specific reactions. After 2 h, the Western blot membrane was washed 3 times with PBST buffer and incubated with a monoclonal antibody 13A5 to the F protein (Li et al., 2016) at a dilution of 1:1000. Subsequently, a goat anti-mouse antibody (1:8000) conjugated with HRP (Abbkine, Beijing, China) was reacted with a monoclonal antibody. Finally, the hybridization reaction was detected by chemiluminescence detection.
2.4. F Protein Purification
The rice seeds were ground into a powder and extracted with phosphate buffer (25 mM PB with 50 mM NaCl, pH 6.3) in a 1:6 (wt/vol) ratio at room temperature for 2 h with constant stirring. The seed residue and precipitate were removed by centrifugation at 10,000 rpm for 30 min at 4 ℃. The supernatant was aspirated, and the conductance value and pH were measured. The pH of the extract supernatant was adjusted to 5.0 with 1 M hydrochloric acid to precipitate some of the impurities. The sediment was discarded by filtration with a 0.8 to 0.22 μm filter. Then, the clarified extract was loaded into an CaptoTM MMC purification column (GE Healthcare, Boston, MA, USA) with 25 mM PB buffer (25 mM PB with 50 mM NaCl, pH 5.0) and eluted with 1 M sodium phosphate dibasic (pH 9.0). The resulting collected fractions were purified with Q Bestarose Fast Flow (Bestchrom, Shanghai, China). At this time, the F protein flowed out of the Q column to be separated from the impurities bound to the column. The F fractions from Q Bestarose were finally purified through a Superdex 75 pg column (GE Healthcare, Boston, MA, USA). The purity of F was determined by SDS/PAGE.
2.5. Enzyme-Linked Immunosorbent Assay (ELISA)
The F protein concentration in the extract was measured by ELISA quantitative assay. Briefly, chicken anti-NDV IgG was diluted 1:500 in carbonate buffer (pH 9.6) and then applied to a 96-well microwell overnight at 4 °C. After washing 3 times with PBST, the plate was blocked with 5% skim milk at 37 °C for 2 h. The rice extract and the pure F protein standard were each subjected to gradient dilution and incubated at 37 °C for 1 h. The nontransgenic plant TP309 was set as a negative control. After washing 5 times, an anti-F protein mouse monoclonal antibody 13A5 was added to the well and incubated for 1 h at a dilution of 1:1000. Subsequently, a goat anti-mouse IgG/HRP antibody (Abbkine, Beijing, China) was added, and the reaction was carried out at 37 °C for 1 h. After washing five times, tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Darmstadt, Germany) was added to each well. After 10 min, the OD450 value was read with a microplate reader. A standard curve was drawn based on the OD value and the concentration of the standard F protein, and then the total soluble protein (TSP) content was determined from the OD value of the sample.
2.6. Animal and Vaccine
All chickens in the experiment were four-week-old SPF chickens (n = 80) purchased from Beijing Boehringer Biotechnology Co., Ltd., (Beijing, China) The chickens were acclimated for one week prior to the beginning of the vaccine study. The chickens were numbered individually and randomly assigned in isolators into nine treatment groups. To verify that the four-week-old SPF chickens had no prior exposure to NDV, twenty chickens randomly selected prior to the start of the trial were bled, and the sera were tested using the Newcastle disease virus Antibody Test Kit (BioChek, Reeuwijk, The Netherlands) according to the manufacturer’s instructions. The samples to positive control ratio (S/P) values (0.08 to 0.21) were negative (S/P < 0.3) in all cases.
The plant-derived F vaccine was prepared by mixing the purified F protein and an adjuvant. The purity of the F protein after three purification steps reached 95%. According to the vaccine dose in each group, the pure F protein produced by plants was diluted in PBS and mixed with Montanide™ ISA 71 VG adjuvant (Seppic, Paris, France) at a ratio of 3:7. The commercial live vaccine LaSota was purchased from the manufacturer. According to the label, the EID50 of the commercial vaccine (HARVAC, Harbin, China) is ≥ 106 per recommended dose (0.5 mL) for initial immunization or booster injections.
2.7. Efficacy Study in Chickens
In the nine experimental groups, six groups were intramuscularly (IM) inoculated with different doses of plants to produce F protein (0.5 μg, 1.5 μg, 4.5 μg, 9 μg, 18 μg, and 36 μg) of 100 µL each. The seventh and eighth groups were intramuscularly inoculated with the same volume of non-transgenic rice TP309 and PBS as two negative controls. The ninth group was vaccinated with 50 µL of commercial live vaccine (LaSota strain) by eye drop as a positive control. At day 0 before the vaccination, 1 mL of blood was sampled from the wing vein of each of ten randomly selected chickens to confirm that the SPF chickens had no prior exposure to NDV. All treatment groups were boosted with the respective vaccine 28 days after the initial vaccination. Sera were collected on days 7, 14, 21, and 28 post inoculation after the first and booster immunizations to determine the level of antibody response to treatment. Chickens were routinely monitored daily for the first 7 days after immunization to assess safety. Lesions at the injection site, animal behavior, and body weight were also recorded. F-specific antibodies were detected with a commercial Newcastle Disease Virus Antibody Test Kit (BioChek, Reeuwijk, The Netherlands) according to the manufacturer’s instructions. The sample to positive control ratio (S/P) was calculated from the optical density at OD450 for each sample. S/P > 0.3 is considered positive. The virus challenge experiment was performed 28 days after booster immunization. The chickens were challenged via the oculo-nasal route with a 50% egg infectious dose (EID50) of 106.5 of the highly virulent NDV gene type Ⅶ strain XX-08. Chickens were observed daily throughout the trial for clinical signs of disease until 15 days post challenge.
2.8. Virus Neutralization Assay
To verify the ability of the vaccine to resist NDV viruses of different strains, we selected an F48E8 strain homologous to the plant-produced vaccine and an XX-08 strain heterologous to the vaccine produced by the plant for neutralization experiments. First, BHK21 cells showing good growth were plated into 96-well cell plates and incubated at 37 °C in a CO2 incubator for 16 h. Subsequently, 50 μL of 100 TCID50 NDV F48E8 strain and XX-08 strain were mixed with an equal volume of 2-fold serially diluted chicken sera in a 96-well plate. Both virus and serum were diluted in serum-free DMEM. After incubation at 37 °C for 1 h, virus and serum mixtures were added to 96-well cell plates in sequence. The 96-well plate was incubated for 2 h to allow the virus to fully infect the cells, and then the solution in the well was discarded, and the cell plate was gently washed twice with sterile PBS. Then, 100 μL of Dulbecco’s modified Eagle Medium (DMEM) containing 2% fetal bovine serum (FBS) was added to each well and cultured at 37 °C in a CO2 incubator. The cell plate was removed at 48 h. The cells were fixed by adding 100 μL of pre-cooled absolute ethanol to each well. Immunocytochemical staining of NDV in cells was performed as with anti-HN protein monoclonal antibody 5F2 and goat anti-mouse IgG/HRP antibody (Abbkine, Beijing, China). The virus staining results were observed under a microscope. When the antibodies in the serum completely neutralize the virus, there is no cell staining in the wells. Conversely, when neutralizing antibodies do not completely neutralize the virus, red-stained cells appear in the wells. The neutralizing antibody titer in the serum sample is expressed as the reciprocal of the highest dilution that causes 100% neutralization.
2.9. Immunochromatographic Strip
We previously developed two immunochromatographic strips, which are used to detect antibodies that recognize HN protein and F protein of NDV, respectively. The recombinant HN or F proteins were coated in an antigen pad. The anti-HN mAb or anti-F mAb was labeled with colloidal gold as the detector. A chicken anti-NDV polyclonal antibody and staphylococcal protein A (SPA) were blotted on the nitrocellulose membrane for the test and control lines, respectively. Chicken serum under different immunization conditions was added into the sample well of the strip. In the absence of a specific antibody in the serum, when the serum flowed through the antigen pad, the recombinant antigen was released and combined with the AuNPs-labeled mAb to form a complex. The complex was then captured by the polyclonal antibody immobilized on the test-zone and appeared as a red band. If specific antibody existed in the serum, the antibody in the serum would compete against the captured antibody on the test zone to specifically bind to the limited recombinant antigen. The test zone did not appear as a red bond. The results of the test-line (T) and the control-line (C) were recorded. Two red lines indicate a negative result, and only the C-line indicates a positive result.
2.10. Ethics Statement
All animal experiments involved in this study were carried out under the approval of the Animal Experimental Committee of Henan Academy of Agricultural Sciences, with ethic approval number LLSC4102019058. According to Chinese animal ethics procedures and guidelines, all animals received humane care.
4. Discussion
NDV is a major viral disease that severely restricts the development of the global poultry industry [
17]. The control of ND must include strict biosecurity that prevents virulent NDV from contacting poultry, as well as the stringent and appropriate administration of vaccines. Currently, the effective containment of ND outbreaks is normally achieved with the utilization of a combination of vaccinations, rapid diagnostic assays, and culling of infected flocks. From the early 1950s, live and inactivated ND vaccines were the only vaccine platforms available for most developing countries and were used to decrease economic losses resulting from morbidity and mortality. Due to the severe production cost constraints of avian vaccines, genetically engineered vaccines such as subunit vaccines are currently only at the research stage and are not recognized by poultry farmers. Plant molecular farming offers a cost-effective and scalable approach to the expression of recombinant proteins, providing an alternative to conventional production platforms for developing countries [
4]. Therefore, this study was conducted in a rice expression system to obtain low-cost and efficient recombinant antigens in rice endosperm.
The strains of ND can be divided into two classes. Viruses from class I mainly infect water-fowl and captured wild birds. Class II viruses are present in both wild birds and domestic poultry and are further divided into 16 genotypes based on the sequence and phylogenetic analysis of the F protein gene. Genotype VII of class II NDV is the main strain that has caused outbreaks in Europe, south America, the Middle East, and Africa in recent years [
1,
18]. Therefore, the F protein gene design in this study was based on genotype VII as the backbone [
19]. By comparing the 32 strains of ND in GenBank, we replaced the highly mutant sequence of genotype VII to increase the resistance of the plant vaccine to heterologous ND. As the main storage protein of rice seeds, glutelin accounts for approximately 60% to 80% of total endosperm protein. Low-storage protein mutants provide more space for the accumulation of foreign gene products than the normal host plant [
20], allowing higher accumulation of foreign gene products. To enhance the expression of the F protein, we hybridized TP309 rice expressing the F protein (TP-F) with low-glutelin rice to obtain the hybrid rice H-F. After hybridization, the expression of F protein can be increased 3.6-fold.
Recently, rice seeds have been demonstrated to be an effective bioreactor for molecular pharming [
14,
21]. The rice endosperm provides the ideal environment, as the seed not only provides a stable site for the deposition of recombinant proteins to prevent degradation by proteases but also can be stored at ambient temperatures for several years [
22]. The N-glycosylation of α-1,3-fucose in seed cells was as low as 10%, and the N-glycosylation patterns of the proteins in endosperm cells were much simpler than those in leaf cells [
21]. Lower α-1,3-fucose contents might make rice seed a much more favorable platform for the production of recombinant proteins. In this study, we compared the weight change and clinical symptoms of the live virus ND vaccine group and the plant-produced F vaccine group after immunization. Chickens immunized with plant-produced F vaccine did not show adverse clinical reactions. The weight of chickens after inoculation were similar to those of PBS-immunized groups, but the live virus vaccine group gained weight slowly in the seven days after vaccination (
Figure 3b).
DIVA is important for international trade and disease control. The country is obliged to report to the OIE when the poultry is infected with virulent ND. Trading partners may suspend imports of poultry or poultry products from that country. Whole virus vaccines elicit immune responses against the full complement of viral proteins, making it difficult to distinguish between immunized animals and vaccinated animals by serological methods. Among them, HN and F, as the main protective antigens, can elicit a strong antibody response in chickens. Plant-produced F vaccine does not contain HN protein, and no genetic material exists. Therefore, vaccinated chickens can be distinguished by detecting HN-specific antibodies. For example, the presence of F- but absence of HN-specific antibodies indicates a vaccine response, while the presence of HN antibodies indicates exposure to wild viruses. HN-specific antibodies can be detected using traditional hemagglutination inhibition (HI) methods. As a traditional serological method, the protocol of the HI test is simple and presents no special requirements for the skills of operators. In addition, we have previously established an immunochromatographic strip to detect antibodies that recognize the HN and F protein of Newcastle disease virus. The strip can quickly distinguish F-vaccinated animals from natural-infected animals within 10 minutes.
With the growing demand for animal protein, coupled with increasing concerns about animal welfare, microbial resistance to antibiotics, and food safety, the focus of poultry health has shifted from treatment to prevention. The plant F vaccine developed in this study is safer, and there is no need to worry about poultry infections caused by improper vaccine administration regimens. Moreover, the subunit vaccines would also achieve a DIVA strategy to allow the differentiation of infected birds from vaccinated birds. The use of 4.5 μg/dose provided immune protection for chickens in this study, showing that the low cost of the F vaccine can meet the needs of the market. In addition, rice as a bioreactor can be stored for long periods of time providing good control of production scale. In conclusion, our study provides evidence that transgenic rice can achieve high accumulation of recombinant protein with correct modifications and folding. Plant-produced F vaccine is safe, efficient, and inexpensive. The plant-derived F vaccine along with the immunochromatographic strips could be useful in the implementation of an NDV eradication program.
5. Conclusions
In this study, we provided a new transgenic rice-based ND subunit vaccine and a rapid differential diagnostic platform. Compared to traditional live vaccines, plant produced F vaccine is safer and has no adverse effects on chickens. Two doses of 4.5 μg fully protected chickens from a lethal ND challenge without any clinical symptoms. In addition, the rapid detection platform in this study can distinguish vaccine-immunized animals from naturally infected animals within ten minutes. Our study provides evidence that transgenic rice is a promising bioreactor. The plant-produced F vaccine along with the immunochromatographic strips could be useful for ND eradication.6. Patents
In this study, we have applied to the State Intellectual Property Office of the People’s Republic of China for a patent of “an immunochromatographic strip to detect antibodies targeting HN protein of NDV”. The publication Patent Number is CN 110018304 A.