Adjuvanticity of sliver nanoparticles with Infectious bursal disease virus VP2 protein and its effect on humoral immune response in chickens

The present study was conducted to decipher the effect of silver nanoparticles conjugated marker recombinant VP2 immunogen of Infectious bursal disease virus on humoral immune response in chickens. The hypervariable VP2 gene segment of eld Infectious bursal disease virus, consisting of major and minor hydrophilic loops, was amplied using reverse transcription-polymerase chain reaction. The gene segment of size 664 bp was cloned into pGEM-T Easy plasmid followed by subcloning into pET32a plasmid vector. Truncated recombinant VP2 protein (rVP2, 19 kDa) was expressed in a prokaryotic expression system using Escherichia coli BL32DE3 cells. The rVP2 protein showed reactivity with specic anti VP2 chicken antibodies. The results of Western blot revealed its utility in serological diagnosis. The recombinant antigen was tested for immunogenic potential by vaccinating the chickens with and without silver nanoparticles. The rVP2 protein blended with adjuvant grade montanide oil showed a highly signicant rise in serum IgY titers. The titers induced by rVP2 protein mixed with montanide oil were non-signicant when compared with titers induced by the conventional vaccines. The IgY response was highly signicant in chickens vaccinated with rVP2 protein blended with montanide oil and silver nanoparticles than in chickens vaccinated with conventional vaccines or rVP2 protein. The results represent Infectious bursal disease virus rVP2 protein as a promising candidate for the DIVA vaccine and sero-diagnostic tool. For the rst time, the current study elucidated the adjuvanticity effect of silver nanoparticles on avian Infectious bursal disease virus rVP2 vaccine potency. isolation groups. However, from the 14th-day onwards, an exponential and signicant increase (p < 0.01) in serum IgY level was recorded till the 28th-day post-vaccination. Similar trends were observed for all the treatment groups. The birds from groups vaccinated with rVP2-montanide oil showed signicantly increased but non-immune titers on the 7th-day post-vaccination. On the 14th-day, a signicant increase in the serum antibody titers was recorded in the group vaccinated with rVP2- montanide oil, commercial vaccine, and rVP2-montanide oil-AgNP compared with the placebo group. On the 21st-day, serum antibody titers were more signicant in the group vaccinated with commercial vaccine booster followed by rVP2-montanide oil-AgNP booster, rVP2-montanide oil-AgNP, commercial vaccine, rVP2-montanide oil booster, and rVP2-montanide oil. The non-signicant increase was recorded in serum IgY titers in groups vaccinated with rVP2-Page This study compares the adjuvanticity of AgNP, montanide oil, and commercial live poultry vaccines. The results indicated a signicant rise of serum IgY titers in birds who received rVP2 vaccine blended with montanide oil and AgNP compared with commercial vaccines and rVP2 vaccine mixed with montanide oil alone. The AgNP can be used in veterinary vaccine preparations for a more promising and long-lasting immune response with the additional advantage of less animal handling. Moreover, booster vaccination can be withdrawn because the primary vaccination could produce comparable IgY titers, as evident in the present investigation. The mechanism of AgNP in potentiating antigenicity is not well elucidated in the literature. However, the accumulation property of the AgNP in water is believed to be the primary mechanism involved in antigen trapping and its slow release. Other suggested mechanisms involve cytokine release, leukocyte recruitment, and up-regulation of major histocompatibility complex (MHC II) expression of peripheral macrophages [24]. Abd reported follicular hyperplasia due to increased B cell number in rabbit spleen and increased humoral response after AgNP immunization [44].


Introduction
The Infectious bursal disease (IBD) is a highly contagious disease of chickens causing heavy morbidity and mortality [1]. Transient immuno-suppression and decreased productivity due to the infection result in substantial direct and indirect economic losses to the poultry industry worldwide [2,3].
The conventional live vaccines derived from the "Intermediate" and "intermediate plus" vaccine strains of Infectious bursal disease virus (IBDV) have better e cacy and breakthrough higher levels of antibodies [4]. However, these vaccines induce moderate to severe bursal lesions leading to mild immunosuppression with low mortality [5]. Increased morbidity and mortality were documented in vaccinated ocks indicating that these vaccines may not fully protect chickens from vvIBDVs [6]. The recent reports suggested that conventional vaccines do not offer complete protection against new IBDV variants [7,8]. Thus, the safety and e cacy of these vaccines remain a signi cant concern [9].
The structural VP2 protein is considered the best candidate for developing diagnostics and subunit vaccines as it induces a protective immune response in chickens [10][11][12][13][14]. The virus neutralizing humoral immune response is generated against this protein [15]. Therefore, it is considered for the development of recombinant vaccine candidates. Very limited antigenic determinants can trigger the B and T cell responses, and the epitopes present in VP2 protein produce virus-neutralizing antibodies [15,16]. Two conformational virus-neutralizing epitopes were suggested and mapped in the 204-359 amino acids on VP2 protein [17,18].
The recombinant vaccine candidates are considered to be weak immunogens in producing antiviral responses. Therefore, adjuvants are used to enhance their immunogenicity. An ideal adjuvant should stimulate a long-term immune response with minimum side effects.
Various adjuvants are used in veterinary vaccines. The newer developments in metal nanotechnology exhibited a variety of different applications. The nanoparticles can trap proteins. Therefore, they can be used for antigen delivery and immune system stimulation by targeting antigen-presenting cells (APC). Antigen presentation to the APCs is an essential issue in the improvement of vaccine potency. The adjuvanted nanoparticle vaccines open the avenues for controlled antigen delivery to immune cells [19]. Recent studies showed the adjuvanticity of silver, gold, polylactide-co-glycolide, and calcium phosphate nanoparticles in enhancing the immunogenicity of antigens [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Despite other applications of nanoparticles, they are used as immune potentiators. Moreover, few recent studies elucidated the immunomodulatory effects of silver nanoparticles (AgNPs) on the immune response to weaker antigens like albumin and Rabies veterinary vaccine [24,25].
In the present study, the potential effect of the AgNPs was evaluated to enhance the humoral immune response against recombinant VP2 protein (rVP2) of IBDV, and the results were compared with the existing commercially available live vaccines.

Materials And Methods
Extraction of viral dsRNA The samples were aseptically collected from broiler birds exhibiting Infectious bursal disease (IBD) from central India. The bursa of Fabricius was frozen at -80 o C and minced in ne powder to obtain 10% suspension. The clari ed suspension was subjected to total RNA extraction using TRI reagent (Sigma Aldrich, USA) following the manufacturer's instructions. The resultant RNA pellet was resuspended in 200 μl DEPC treated water. Lithium chloride and 3M Sodium acetate (Sigma Aldrich, USA) precipitation was followed to extract viral dsRNA as per the method described by Green and Sambrook [26]. The RNA pellet was resuspended in 25 μl of 1X TE buffer and quanti ed using nanodrop 1000 (Eppendorf, Germany).

Ampli cation of hypervariable segment of VP2 gene
The cDNA was synthesized using a Superscript TM III First-Strand Synthesis kit (Invitrogen USA). The polymerase chain reaction was set up in 50 μl volume (5.0 μl of 10X PCR buffer, 1.5 μl of 50 mM MgCl 2 , 1.0 μl of 10 mM dNTPs, 2.0 μl of each 10 pmol primers and 1.0 unit of Taq DNA polymerase (Promega, USA). The F 5'-ACT GTC CTC AGC TTA CCC ACA T-3' and R 5'-TCT GTG ACC AGG TTC TTT GCT A-3' primers speci c to a 678 bp VP2 gene segment, consisting of the hypervariable region, were used for ampli cation. The ampli cation was carried out in 35 cycles at an annealing temperature of 49 o C with a nal extension at 72 o C for 10 minutes. The amplicon was sequenced using commercial sequencing services (Euro ns, India). The sequence was deposited in GenBank (Accession No. MK172062).

Sub-cloning of VP2 gene segment
The agar gel-puri ed VP2 gene insert (12 ng/μl) from recombinant pGEM-T Easy plasmid was isolated after EcoRI digestion. The insert was ligated into EcoRI restriction sites of pET32a (40 ng/μl, Novagen, USA) plasmid. The recombinant plasmid DNA was transformed into BL32DE3 Escherichia coli cells. The insert orientation in recombinant clones was determined by restriction endonuclease digestion using BamHI (Fermentas, USA).

Expression and puri cation of rVP2
The rVP2 protein was expressed into a prokaryotic expression system using BL21DE3 Escherichia coli cells [23]. The overnight grown culture (100 μl) of a single transformed colony was transferred to 10 ml LBAmp broth in a 100 ml conical ask. The ask was incubated in an orbital shaker incubator. The expression was induced at the mid-exponential phase with one mM IPTG for three hours at 37 o C. Bacterial pellets were resuspended and boiled in Laemmli buffer for ve minutes. The expression of rVP2 protein was con rmed by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) following the method described by Moore and Burke [27]. Large-scale protein expression was carried out at an optimum concentration of 0.5 mM IPTG for 6 hrs. The bacterial pellet was resuspended in a binding buffer and subjected to three cycles of sonication at ve μm amplitude for 10 seconds pulse each in an ice bath with 30 seconds gap between each cycle. The slurry was centrifuged at 10000 g for 30 min at 4 o C. The supernatant was subjected to the puri cation of rVP2 protein using charged Ni-NTA resin (Thermo, USA). The Ni-NTA resin column was equilibrated by adding a ve-column volume of equilibration buffer with one ml per minute ow rate. The supernatant from the above step was loaded onto the column, and ow-through was collected. The washing was done with a 10 column volume of washing buffer at one ml per minute ow rate. The protein was eluted with a ve-column volume of elution buffer. The rVP2 protein was quanti ed by the Bradford method [28].

Western blot
The puri ed rVP2 protein (3 μl) was dotted onto nitrocellulose membrane and allowed to bind for an hour at room temperature. The membrane was blocked in 5% skimmed milk for half an hour. The membrane was immersed in primary chicken antibodies (non-immune, 1:400) and anti IBDV antibodies (1:400) for half an hour. Anti-chicken horse reddish peroxidase conjugate (Sigma, USA) was allowed to react for half an hour. The membrane was subjected for spot development in 3'3' Diaminobenzidine (0.3%, Sigma, USA) and hydrogen peroxide (0.6%) for 10 minutes.
Day-old chicken birds were randomly divided into seven groups consisting of 20 birds per group. Sera were randomly collected from 12 birds/group before vaccination. The birds were vaccinated with the above blends keeping placebo as negative control and commercial vaccines as a positive control. Each preparation was evaluated with and without a booster dose. Post-vaccination sera were collected on days 7, 14, 21, and 28 (Table 1). The sera were subjected to IBDV rVP2 ELISA (IDVet, France) following the manufacturer's instructions. The OD was taken at 450 nm in an ELISA reader (BioTech, India). The results were interpreted by calculating the S/P ratio and antibody titer be using the following formulas. The ELISA antibody titer greater than 1324 was taken as positive as indicated by the manufacturer.

Ampli cation VP2 gene and construction of recombinant plasmid
The RT-PCR ampli ed 664 bp nucleotide sequence speci c to the VP2 gene hypervariable region from position 548 to 1248 on segment A of the IBDV genome. The homology of deduced amino acid sequence with other IBDV sequences is shown in gure 1.
The codon-optimized VP2 gene segment (aa 178-385) was ligated into EcoRI restriction sites of the pGEM-T Easy vector. The recombinant plasmid was transformed into JM109 Escherichia coli cells. The plasmid DNA was extracted and digested with EcoRI. The excised VP2 gene insert was subcloned into EcoRI restriction sites of dephosphorylated pET32a expression vector. BL21DE3 Escherichia coli cells were transfected with this recombinant pET32a plasmid. The recombinant pET32a plasmid yielded two fragments of 6336 bp and 64 bp on endonuclease digestion using BamHI. The digestion patterns showed correct insert orientation in the expression vector ( Figure 2).

Expression, puri cation, and characterization truncated rVP2
The truncated rVP2 protein of IBDV was expressed into a prokaryotic expression system under the control of the T7 promoter. The induced clones were lysed, and rVP2 was puri ed from cell-free supernatant using His tag puri cation. The Ni-NTA puri cation yielded rVP2 protein with a concentration of 1600 μg/ml. The induction and puri cation of rVP2 protein were monitored by SDS-PAGE ( Figure 3). The recombinant clones expressing a speci c 19 kDa protein were selected for further evaluation. The western blot identi ed the expressed protein as IBDV rVP2 with no speci c reaction in negative controls (Figure 4), indicating that the protein is expressed correctly and has a reaction with chicken anti-IBDV antibodies.
Humoral response rVP2 protein blends The mean serum antibody titers are shown in table 2 and gure 5. The results showed that the commercial vaccines and rVP2 protein blend preparations induced signi cant (p < 0.01) and protective titers after the 14th day of vaccination. The titers of birds from the placebo group were non-signi cant and non-protective throughout the experiment. The birds vaccinated with the commercial vaccine showed a signi cant decrease in the serum IgY titers on the 7th-day post-vaccination. The titers on the 7th-day post-vaccination were non-immune in all the treatment groups. However, from the 14th-day onwards, an exponential and signi cant increase (p < 0.01) in serum IgY level was recorded till the 28th-day post-vaccination. Similar trends were observed for all the treatment groups. The birds from groups vaccinated with rVP2montanide oil showed signi cantly increased but non-immune titers on the 7th-day post-vaccination. On the 14th-day, a signi cant increase in the serum antibody titers was recorded in the group vaccinated with rVP2-montanide oil, commercial vaccine, and rVP2-montanide oil-AgNP compared with the placebo group. On the 21st-day, serum antibody titers were more signi cant in the group vaccinated with commercial vaccine booster followed by rVP2-montanide oil-AgNP booster, rVP2-montanide oil-AgNP, commercial vaccine, rVP2-montanide oil booster, and rVP2-montanide oil. The non-signi cant increase was recorded in serum IgY titers in groups vaccinated with rVP2-montanide oil (both primary and booster) on the 21st-day compared with the respective titers on the 14th day. On the 28th-day, a signi cant increase in the serum antibody titers was recorded in the group vaccinated with rVP2-montanide oil-AgNP booster followed by rVP2montanide oil-AgNP, commercial vaccine booster, rVP2-montanide oil booster, commercial vaccine, and rVP2-montanide oil. The results indicated that rVP2 expressed in the prokaryotic expression system has immunogenic potential.

Discussion
The VP2 protein is the major structural protein of IBDV and is routinely used in diagnosis and epidemiology [29,30]. It forms the outer surface of the viral capsid and possesses neutralizing epitope. The neutralizing antibodies and protective immune response have been demonstrated against this protein [10,11,31,32]. The VP2 protein comprises the major conformational epitopes. These epitopes are responsible for the induction of virus-neutralizing antibodies. Therefore, the IBDV major capsid protein VP2 is utilized for developing diagnostics and novel subunit vaccines [12,14]. Recent studies used Escherichia coli [7,33], Lactococcus lactis [34], and plant [14] expression systems to express IBDV rVP2 based virus-like particles and had a molecular mass of more than 40 kDa. This study elaborated the diagnostic and immunogenic potential of a truncated 19 kDa rVP2 protein.
Puri ed rVP2 protein concentration of 50 μg/dose induced signi cant serum IgY titers. These titers were comparable with the titers induced against commercial vaccines. In the placebo group, the titers remained non-immune throughout the experiment. Recently, a 40 kDa IBDV rVP2 expressed in Nicotiana benthamiana demonstrated IBDV speci c neutralizing antibody titers in chicken comparable to those induced by the commercial vaccine [14].
The signi cant (2.31 to 2.67 fold) increase in the serum IgY level was recorded on the 14th-day post-vaccination in the birds vaccinated with rVP2 protein and commercial vaccines. On the 14 th -day, the titers against rVP2-Montanide oil (2.48 fold) were comparable but signi cantly higher than the titers produced by the commercial vaccine (2.31 fold). The birds vaccinated with rVP2-Montanide oil-AgNP showed equivalent but signi cantly higher serum IgY titers (2.55 to 2.67 fold) than the commercial vaccine.
On the 21 st -day, a signi cant rise in serum IgY level (4.95 to 5.11 fold) was recorded in birds vaccinated with booster dose compared to birds that did not receive booster dose (2.92 to 3.94 fold). Comparable but non-signi cant titers were observed in the birds with (2.97 fold) and without (2.92 fold) booster vaccination with rVP2-Montanide oil blends. However, the group receiving booster rVP2-Montanide oil-AgNP booster showed signi cantly higher titers (4.95 fold).
On the 28 th -day, titers were signi cantly higher in all the vaccinated groups except in placebo (0.06 fold) compared with the respective titers on the 21 st -day. All the groups receiving booster doses viz. commercial vaccine, rVP2-Montanide oil, and rVP2-Montanide oil-AgNP showed signi cantly higher levels of serum IgY titer (14.55, 14.07, and 17.84 fold, respectively) as compared to the corresponding groups which did not receive booster vaccination (12.00, 11.91, and 16.37 fold, respectively). The titers in the groups receiving rVP2-Montanide oil-AgNP were signi cantly higher than those reported in the commercial vaccine booster group. At the same time, the titers in the group receiving rVP2-Montanide oil booster were comparable with the titers recorded in the commercial booster vaccine group. The titers in groups not receiving booster doses of rVP2-Montanide oil were comparable with the titers observed in the group receiving only a primary dose of commercial vaccine. Exceptionally, the titers in the group receiving only primary rVP2-Montanide oil-AgNP were signi cantly higher throughout the experiment. Earlier reports documented signi cantly higher anti-IBDV antibody titer against rVP2 after two weeks of immunization [7,34].
Wang et al. reported that the IBDV SH619-VLP vaccine induced comparatively lower antibody titers than the commercial vaccine [33]. Our ndings also demonstrated signi cantly high IgY titers after two weeks of vaccination. However, the titers produced against rVP2-Montanide oil were comparatively lower than those induced by commercial vaccines.
The results indicated that the immunogenic potential of rVP2 is comparable to commercial vaccines. Moreover, serum IgY response in birds receiving rVP2-Montanide oil-AgNP was superior when compared with the commercial vaccines. Secondly, the results are suggestive of potentiating immune response of rVP2 when blended with AgNP. Similarly, developing a recombinant DIVA vaccine could be possible as antibody response to VP3 structural protein will be absent in marker vaccine preparations. The commercial live vaccines could not differentiate the infected versus vaccinated birds due to the induction of similar immune responses. Recombinant VP2 vaccine may discriminate vaccinated versus naturally infected birds as naturally infected birds show antibodies to VP3 viral protein [35]. Earlier studies reported induction of protective immune response in chickens immunized with VP2 antigen [10,11]. The recombinant VP2 protein expressed in the prokaryotic expression system was utilized in diagnostic assays [13]. Recombinant VP2 subunit vaccines have been experimentally demonstrated [36,37]. The available commercial vaccines are prepared from "Intermediate" and "intermediate plus" or "hot" strains, which lead to the bursal changes attributing the immunosuppression, which was being altered by the use of the recombinant VP2 protein-based vaccines. The bene ts of using recombinant VP2 immunogen are that it harbors most of the neutralizing epitopes with a crystalline structure and will not compromise sero-surveillance of IBD.
The mineral oils are often used in a vaccine to form stable water in oil emulsions, ensuring depot formation and steady antigen release [38][39][40]. The montanide oil used in this study yielded stable emulsions after blending with rVP2 and rVP2-AgNP. It is a proven adjuvant and is safer than Freund's adjuvant, aluminum hydroxide, and aluminum phosphate and can be used in veterinary vaccines [41]. A recent report indicated that the chickens vaccinated with rVP2 neutralizing epitope antigen blended in oil emulsion adjuvant-induced more robust humoral immune response with no side effects [7,42]. This investigation reported the adjuvanticity of montanide oil in signi cant induction of anti VP2 antibodies when blended with rVP2.
In the present study, we reported adjuvanticity of AgNP in poultry vaccine. Availability of a novel adjuvant is the current need of veterinary vaccines with more safety, adjuvanticity, and targeted antigen delivery. Many adjuvants have been used in the development of veterinary vaccines. But issues like antigen-dependent adjuvanticity, their physicochemical properties, and toxicity limited their applications. Mineral oil like montanide oil and other derivatives are routinely used in vaccine formulations. However, nanoparticles showed more promising results in potentiating the immune response [23].
Moreover, AgNPs were experimentally used in vaccine preparations in laboratory animals like dogs, rabbits, and mice for viral antigens like Rabies [25,43,44]. There are proven reports of silver toxicity in vitro and in vivo; however, AgNP is considered non-toxic due to the negligible release of Ag ions from AgNP in water [24]. Asgary et al. documented no toxicity of AgNP at the triple dose rate (60 mg/Kg body weight) in laboratory animals [25,43]. The present study utilized the AgNP at the dose rate of 20 mg/Kg body weight in poultry, which is considered safe [25,43].
This study compares the adjuvanticity of AgNP, montanide oil, and commercial live poultry vaccines. The results indicated a signi cant rise of serum IgY titers in birds who received rVP2 vaccine blended with montanide oil and AgNP compared with commercial vaccines and rVP2 vaccine mixed with montanide oil alone. The AgNP can be used in veterinary vaccine preparations for a more promising and long-lasting immune response with the additional advantage of less animal handling. Moreover, booster vaccination can be withdrawn because the primary vaccination could produce comparable IgY titers, as evident in the present investigation. The mechanism of AgNP in potentiating antigenicity is not well elucidated in the literature. However, the accumulation property of the AgNP in water is believed to be the primary mechanism involved in antigen trapping and its slow release. Other suggested mechanisms involve cytokine release, leukocyte recruitment, and up-regulation of major histocompatibility complex (MHC II) expression of peripheral macrophages [24]. Abd reported follicular hyperplasia due to increased B cell number in rabbit spleen and increased humoral response after AgNP immunization [44].

Conclusions
The rVP2 protein has diagnostic potential as evident by dot blot assay and immunogenic potential as it induced serum IgY titers signi cantly at the protective level. The AgNPs had boosting effect on humoral response and produced IgY at par with the commercial vaccines.