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.
Purified rVP2 protein concentration of 50 μg/dose induced significant 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 specific neutralizing antibody titers in chicken comparable to those induced by the commercial vaccine [14].
The significant (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 14th-day, the titers against rVP2-Montanide oil (2.48 fold) were comparable but significantly higher than the titers produced by the commercial vaccine (2.31 fold). The birds vaccinated with rVP2-Montanide oil-AgNP showed equivalent but significantly higher serum IgY titers (2.55 to 2.67 fold) than the commercial vaccine.
On the 21st-day, a significant 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-significant 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 significantly higher titers (4.95 fold).
On the 28th-day, titers were significantly higher in all the vaccinated groups except in placebo (0.06 fold) compared with the respective titers on the 21st-day. All the groups receiving booster doses viz. commercial vaccine, rVP2-Montanide oil, and rVP2-Montanide oil-AgNP showed significantly 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 significantly 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 significantly higher throughout the experiment. Earlier reports documented significantly 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 findings also demonstrated significantly 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 benefits 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 [38-40]. 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 significant 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 significant 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].