مروری آینده نگر بر روش‌های تولید واکسن علیه بیماری برونشیت عفونی

Introduction
Infectious bronchitis is a contagious viral disease with a high economic impact on the poultry industry. Schalk and Hawn first detected the disease in North Dakota in 1931. Since then, the disease has been distributed to different continents worldwide (Hajijafari Anaraki et al., 2022). The virus mainly affects the respiratory, kidney, and reproductive systems. Economic losses result from the high morbidity of the virus: Weight loss, condemnation at slaughterhouses, decreased egg production, and lower inner and outer egg quality. Nowadays, the strategies for disease control comprise a combination of good biosecurity and, most importantly, vast vaccination in broiler and breeder flocks (De Wit et al., 2010; Cavanagh, 2005).
Infectious bronchitis (IB) is caused by a single-stranded enveloped RNA virus belonging to the Igacovirus subgenus of the Gammacoronavirus genus and the Coronaviridae family confined to the avian population. The IB virus genome, the largest avian RNA virus, is 27 to 32 kb long. It encodes 4 structural proteins: Spike (S), matrix (M), nucleocapsid (N), and envelope (E). They are interspaced by 3 and 5 accessory genes and at least 15 nonstructural proteins encoded by the first two-thirds of the genome. S and N proteins were proposed to be immunomodulatory proteins in the virus. 
The most important virus protein is the glycoprotein spike projecting from the virus’s surface. It is responsible for bird immune system reactions and neutralizing antibody production in chickens. Infectious bursal disease (IBV) S-protein comprises 3400 nucleotides (Beheshtian et al., 2020). Furin or its related enzymes cleave S protein into S1 520 at the amino-terminal and S2 625 amino acids at the carboxyl-terminal in the Golgi complex of the host cell. Typically, S1-glycoprotein plays a significant role in host cell receptor binding and membrane fusion via cell-to-cell and virus-to-cell interactions, and S2 contributes to the fusion mechanism (Koch et al., 1990). A complication arises when the protein shows variability. It has at least 3 hypervariable regions at positions 24–61, 132–149, and 291–398 amino-acid residues. They continuously evolve into new variants different from classic strains, so natural or vaccinal protection against them cannot confront variant viruses (Lee et al., 2003). N protein attaches to the genome and causes the helical capsid complex.

Essential proteins in immunomodulation following virus infection or vaccination
As previously said, S1 is hypervariable. Unfortunately, sequence comparisons of variants with vaccines fail to identify the site of the gene responsible for protection induction. The highly conformational nature of S1 complicates the antigenic characterization of S1 and whether cell-mediated immune or humoral responses are the main protection mechanisms. Researchers have tried to map antibody-inducing sites and cell-mediated immune  (CMI) response factors in S1 protein (Cavanagh et al., 1992; Wei et al., 2014; Ignjatovic et al., 2006; Ignjatovic & Galli 1995; Ignjatovic & Galli 1994). To date, 5 antigenic sites, which all were conformation dependent and responsible for virus neutralizing antibody production, have been mapped within the hypervariable region (HVR) of S1 at positions 24–61 and 132–149, as well as outside the hypervariable region at 291–398 (Kant et al., 1992; Kusters et al., 1989; Moore et al., 1997). Because of this, monoclonal antibodies are the primary tool used in antigenic epitope recognition on S1. 
The S2 also induces antibodies to cross-react in ELISA and cell-mediated responses but not protective responses. The two antigenic sites in the N terminus of the S2 between 546-577 amino acids are reported to be immunodominant and cause cross-reactive antibodies (Kusters et al., 1989).
The N protein induces cross-reactive ELISA antibodies and CMI (Ignjatovic & Galli 1994). However, it is not involved in disease protection. The carboxy-terminal portions of N protein has B cells epitopes; a region between 78–94 amino acid residues was mapped, which induces a T cell response in addition to protection (Jayaram et al., 2005). N protein functionally binds with the genomic RNA to form a helical ribonucleoprotein complex, thus aiding transcription, replication, translation, and packaging of the viral genome during replication (Jayaram et al., 2005).
RNA virus is dangerous because of its fast and continuous evolutions. Two critical phenomena in shaping the genome of coronaviruses are mutation and recombination (Domingo & Holland, 1997). It is difficult to define how the IBV genome evolves; however, three major theories have been hypothesized. First, the lack of proofreading in RNA polymerase may lead to mistakes in the genome, which, in turn, causes mutations (deletions, insertions, or point mutations of nucleotides). Second, multiple serotype infections or using different live attenuated vaccines leads to recombination and favors the emergence of new variants (Kusters et al., 1989). Mutations in the hypervariable region of S1 affect its subpopulation and make new viruses of different pathogenicity and virulence. In this regard, quasi-species viruses are generated with a mixture of multiple genetic mutants of the same strains (Nix et al., 2000). The most common regions in the IBV genome for recombination are encoding regions of nsp 2, 3, and 16, plus the glycoprotein spike (Thor et al., 2011). Third, other suggested factors are the presence of immunosuppressive agents like Marek’s disease, infectious bursal disease, and chicken anemia virus, which affect the evolutionary dynamic of the IBV (Bande et al., 2015).

Control and vaccination strategies
Several IBV strains and variants have been detected and classified by serotype and genotype. Some have limited geographical distribution (Italy 02; variant 2), and some are globally distributed, including M41, QX, and so on (Valastro et al., 2016). As mentioned, IBV is currently controlled by vaccination with live-attenuated and inactivated vaccines (Motamed & Bashashati, 2022, Motamed et al., 2021). Vaccination programs in one geographical area differ from other sites because vaccines typically induce restricted cross-protection between strains (De Wit et al., 2010). Live-attenuated vaccines are administered to young chicks through sprays or drinking water. In older ages, breeders and or laying may then be boosted with either live or inactivated vaccines (Sjaak de Wit et al., 2011). The ease of application favors live-attenuated vaccines; however, the risk of reversion to virulence and breakdown limits their application. Although the molecular mechanism of attenuation has not been discovered, it happens only with few consecutive level mutations. Hence, over passaging of the vaccine in the field provides reversion to virulence possibility (Oade et al., 2019). 

Live-attenuated vaccines
The infectious bronchitis virus is a pathogen of economic and welfare concern for the poultry industry. Today, the most effective controlling tool is vaccination (Motamed & Bashashati, 2022). Live-attenuated vaccines are generated by serial passage of a virulent isolate in embryonated chicken eggs for at least 80 consecutive passages until attenuation is achieved. The mechanism of the attenuation is unknown (Motamed, 2022). This kind is the most widely-used vaccine with an old production history. In addition to high benefits, it has some disadvantages, such as reversion to virulence. Even with efforts to decrease virulence after more than 80-100 passages, severe outbreaks in the farm may occur. Also, tissue damage results in secondary bacterial infections or pathological disorders, especially in day-old chicks.
Another problem is the interference by maternal-derived antibodies. Finally, co-circulation of the vaccine virus and field viruses may lead to recombination events and the emergence of new variants. To reduce problems associated with vaccine reversion, researchers explored the options of using reverse genetic technology to create a vaccine virus that is potentially pathogenic in the host but capable of replication and inducing an immune response (Zhou et al., 2013). Exploring various virus clones showed that some vaccine strains, such as Ark, could not induce enough protection because of multiple virus subpopulations in a vaccine seed. So, protection did not reach the optimum, and chicks were still susceptible to the disease (Sjaak de Wit et al., 2011). The ability of some ArkDPI-derived vaccine viruses to persist and be selected in vaccinated chick may lead to a high frequency of Ark field viruses (Gholami et al., 2018). So, the persistence of the vaccine virus in the field offers the required materials for recombination with other circulating viruses or mutation, resulting in increased pathogenesis (Ndegwa et al., 2014).
Mutations and recombination contribute to the emergence of new variants following the intensive use of live, often multiple vaccines. Therefore, attempts have been made over recent years to develop new generation vaccines, such as subunit vaccines, to allow the use of a part of protective antigen but to vaccinate against different genotypes in the same vaccine (Ignjatovic et al., 2006).

Inactivated virus vaccines
Inactivated vaccines are used alone or in combination with live ones. Usually, they are administered to laying and breeder flocks at 12-18 weeks of age, and they cannot replicate. So, they do not revert to virulence; however, limitations are present. For example, their administration is difficult and impractical, especially in large settings; they have shorter immune duration with just antibody response and do not produce T cells. Thus, a prime boost with the live-attenuated vaccine is usually needed. Also, the elimination of the whole or parts of the carcass in slaughter because of the reactions in the injection site can happen. Slow release of antigen and long-lasting immunity throughout the laying period are achieved by intramuscular or subcutaneous injection of vaccines that incorporate an adjuvant and IBV that has been inactivated.
Researchers have taken many approaches to designing new vaccines against pathogenic microorganisms. These methods are dictated by the nature of the infection mechanism and pathogen and practical factors about its use (Motamed, 2022). 
In light of COVID-19, which is classified in the Coronaviridae family with a fast-evolving nature, researchers have considered new-generation vaccine approaches for confronting viral diseases. We can no longer trust live or inactivated vaccines. We need to think about new methods with more flexibility in manipulation or substitution. We should reach a new vaccine with new emerging strains in a shorter time. The continuous fast pace of vaccine development and quality controls represents a substantial achievement that was impossible previously, strongly demonstrating the utility of modern technology vaccines in new emerging diseases.

Pathogen outbreaks 
Considering new outbreaks, a live-attenuated vaccine usually takes 3 to 5 years to get official administration and distribution permission. However, according to studies, an emerged variant may persist not more than 4-5 years in an area. So it could be expected that neither a licensed variant vaccine, which is now a bit different from the predominant variant after 5 years, nor classic vaccines can fight effectively against field viruses. The same thing is expected over protectotype vaccination, which takes a long time and costs to be studied against variants. However, nowadays, protectotypes are considered as most effective vaccination method to make an optimum immune status in a flock (Sjaak de Wit et al., 2011). Over the last years, many reports declared that 793/B-like- and mass-like-based vaccine combination seems to provide broad protection against heterologous (Awad et al., 2015b; Awad et al., 2015a; Habibi et al., 2017) strains, such as Variant 2 and QX (Franzo et al., 2017; Lisowska et al., 2017). So, it is commonly adopted in most regions worldwide (Jordan, 2017). The vaccine industry has to develop rationally live vaccines. Past research has evaluated vaccines including the S glycoprotein as DNA or by viral vectors (Song et al., 1998; Yan et al., 2016; Toro et al., 2014; Zhao et al., 2017a; Shirvani et al., 2018; Ellis et al., 2018). Spike is the primary attachment factor and virus entry, making it a main target in the development of vaccines. Studies have shown it induces virus-neutralizing antibodies (Koch et al., 1990; Kant et al., 1992). Vaccines currently used against SARS-CoV-2 aim to generate neutralizing antibodies by following this method to deliver S protein (Folegatti et al., 2020; Jackson et al., 2020). The vaccine offers a reasonable degree of protection after multiple doses. They cannot stimulate complete protection in mild to moderate clinical disease or infection and replication of the SARS-CoV-2 (Ellis et al., 2018; De Wit et al., 2010). If the industry is going to be ready to respond to the newly-emerged viruses, it will have to develop and set up alternative techniques to ensure efficient reactions in a proper and fast manner

mRNA vaccines
mRNA vaccines have attracted considerable attention recently because they can accelerate vaccine development, improve safety and efficacy, and combat diseases that cannot be prevented by other methods. mRNA is a non-infectious agent. It is non-integrated and degraded by normal cellular processes shortly after injection, reducing the risk of toxicity and long-term side effects. Intracellular antigen expression by mRNA may produce strong T cell responses typically seen with viral vector-based or replication-defective virus-based vaccines (Gebre et al., 2022). Also, mRNA vaccines do not induce vector-specific immunity and counteract preexisting or newly developed vector immunity that can interfere with subsequent vaccinations (Gebre et al., 2022). In late 2020, Moderna and Pfizer launched their new mRNA vaccine against COVID-19. mRNA gene delivery system has numerous applications in cancer, vaccines, and disease. The concept of this method was initially developed in 1990. mRNA is a delivery system that encodes antigens of a specific pathogen in the host. Infected cells translate it, and the immune system recognizes the antigen. To increase stability and ease of entrance, mRNA is usually delivered in a lipid nanoparticle (LNP) without an immune response, allowing multiple LNP-mRNA use. The simple and fast nature of assembling new sequences in mRNA systems in vaccine formulas will promise mRNA vaccine pioneers in the near future (Folegatti et al., 2020). 
Two main limiting reasons that hindered the research of novel IBV vaccines are considered. The first factor is cost. Poultry plants have a minimal profit margin, so any extra cost seems weighty. Classic live-attenuated and inactivated vaccines have a few costs per dose. Other standard recombinant vaccines commonly used against poultry pathogens (other than IBV) are still frequently used due to their ease of application and good efficaciousness, even though they are expensive. If a new IBV vaccine can match those specifications, its cost would not be an impediment. The second limiting reason is the method of application. Each vaccine should be applied in mass and compatible on a large scale. 

Vaccine development in viral vectors 
The recombinant viral-vectored vaccines hold promises for the commercial poultry industry. Adenovirus vector vaccines have also evolved to become promising vaccine platforms. Optimal adenovirus vaccine vector design involves the selection of uncommon vector serotypes. The structural components of Adenovirus (Ad) vectors can be harnessed and modified to enhance tropism, efficient delivery, and optimal antigen expression. Ad vectors can be rapidly developed and produced commercially, and the potency and stability characteristics of the vector support single-shot vaccines that do not require cryopreservation. However, this technology does have limitations. The issue of preexisting immunity or maternally derived immunity interferes with the live vector itself. It reduces the antigen uptake by the antigen-presenting cells and, consequently, the transgene expression and specific immune response.
Developing promoter vectors against multiple pathogens shows their flexibility and promises for current and future vaccine applications. 

Vectors replicating or non-replicating viruses 
At present, Fowlpox virus, Marek’s disease virus, turkeys Herpes virus, Newcastle disease virus, and members of the Retroviridae family have been most extensively used as expression vectors. Genes of antigenic proteins of Newcastle virus, infectious bursal disease virus (IBDV) (Darteil et al., 1995), and infectious laryngotracheitis virus (Vagnozzi et al.; 2012) are inserted into these systems. They induced the appropriate immune reaction in chickens. Someone attempted to insert S1 in these viruses (Toro et al., 2014). They all had a different immune response to the homologous challenge. Still, they did not meet the vaccination requirements against IBV, especially mass application, which is not feasible commercially. Adenoviral vectors have efficient transduction in various cell types, are widely used in COVID-19 vaccine production, and reach into global consumption admissions (Mendonca et al., 2021). Hence, they can easily express inserted antigens accompanied by economic advantages of mass production of Ad-vectored viruses by cell lines in serum-free media and large bioreactors with simple chromatography separation techniques.
The potency of human Ad-vectored vaccines for immunizing chickens was reported by stimulating immune responses in chickens following intramuscular injection (Zeshan et al., 2010). Those adenoviruses are non-replicating, without causing tissue damage and tropism to different dividing and non-dividing cells, allow sustained antigen release with effective immune response, and reduce problems associated with reversion to virulence or mutation. However, a lack of posttranslational modifications, such as protein folding and glycosylation, can alter epitope arrangement and affect immunogenicity or vaccine efficacy. A recombinant adenovirus vaccine containing infectious bronchitis S1-glycoprotein showed a prominent antibody response, conferring 90%–100% protection. Zeshan et al., (2010) conducted a study on developing a human adenovirus-vector IB vaccine for use in ovo; they constructed a recombinant adenovirus expressing the S1 gene of nephropathogenic IBV (rAd-S1) and reported that in ovo vaccination and then an intramuscular inoculation by rAd-S1 led to dramatic augmentation of cellular and humoral responses against homologous challenge, decreasing lesion and clinical signs. In a study, expressing the N gene of IBV by a replication-defective human adenovirus was evaluated in SPF chickens using immune response and protective efficiency against challenge. Despite the recognized immunogenic response, the N protein had no proliferative response to IBV in vaccinated chickens without significant protection. Some researchers have designed oral adenovirus vector vaccines to avoid neutralization of vectors by maternally derived or preexisting antibodies. Other advantages are the ease of application and lower handling associated stress, but they had no T cell responses, so complementary studies are needed to stimulate CMI.
A modified baculovirus, the BacMam virus, that mediates expression under the control of the mammalian promoter has emerged as a versatile and safe vector in the development of vaccines. In a study, an improved BacMam virus expressing S1 of IBV was generated. S1 glycoprotein was displayed on the baculovirus envelope expressed in mammalian cells. The vaccine BV-Dual-S1 has elicited significant humoral and cell-mediated immunity in specific pathogen-free chickens. Protection rates of immunized chickens with BV-Dual-S1 reached 83% compared to 89% inactivated vaccine following the challenge with IBV-M41 (Zhang et al., 2014). Other vectors may replicate in bird bodies and have their specific effects. Shi et al. (2011) showed a recombinant fowlpox virus vaccine expressing S1-IBV gene and chicken INF-y gene [rFPV-IFN-S1] that enhanced humoral and CMI and protected birds against heterologous and homologous IBV challenge (Shi et al., 2011). In another study, the expression of S1 gene with interleukin (IL)-18 in a fowlpox virus vector caused a significant increase in humoral immunity, CD4+, and CD8+ activity (Chen et al., 2017). Metapneumoviruses, Newcastle disease virus, and duck enteritis viruses are other viral backbones for inserting S1 or S2, especially metapneumoviruses which can be massively used and replicate in the respiratory system and may stimulate the head-associated lymphoid tissue to a higher extent and produce high immune response (Falchieri et al., 2013; Li et al., 2016; Toro et al., 2014; Zhao et al., 2017b).

Subunit and peptide-based vaccines
Peptide subunit vaccines contain an antigenic portion of the pathogen, amino acid short segments presented to the immune system. Often, these peptides are synthetic and represent the immunostimulant antigen. S1- and N-gene epitopes have been used for neutralizing antibodies and cytotoxic T lymphocytes, respectively. Some vaccines showed promising results in protection challenges (Wei et al., 2014; Yang et al., 2009). A novel chimeric infectious bronchitis-Newcastle disease (IB-ND) virus-like particles (VLPs) vaccine constructed from the rF, rS, and M IBV proteins has been designed. It stimulates humoral and cell-mediated immunity, causes 100% protection against clinical signs, and reduces virus shedding (Wu et al., 2019). The immune response following administration of these vaccines must be enhanced by combining with factors such as cytokines adjuvants. 
The second limitation is the method of administration, commonly by injection, which is not favored in large rearing settings and usually costs much more than other production methods. They may be expressed in E. coli, yeast, insects, etc. (heterologous expression system). Today, most under-investigation vaccines are based on purified subunit antigens or recombinant proteins.
 The hepatitis B virus (HBV) vaccine is among the best recombinant protein vaccines. A study has reported that synthetic peptide epitopes from S20 to S255 react well with polyclonal antibodies, demonstrating the potential use for poly-topic IB vaccines (Wang et al., 1995). Some researchers have focused on developing multi-epitope peptide vaccines against various IBV genotypes. Yang et al., (2009) demonstrated that an IBV multi-epitope vaccine based on S1- and N-protein genes can cause remarkable cell-mediated and humoral immune responses. The Lactococcus lactis bacterial system could deliver peptide IBV vaccines orally and with mucosal immune response induction (Cao et al., 2012; Cao et al., 2013).

Plasmid DNA vaccines
DNA vaccine does not involve a live vector and uses plasmids to express the DNA of the immunogenic portion of the pathogen genome. No licensed DNA vaccine for poultry use is available yet, primarily because of the administration route, which is injection. This problem can be overcome by oral or in ovo administration. The nanoparticle delivery system may protect the DNA from enzyme degradation and enhance mucosal responses. As DNA vaccines can be administrated in the presence of maternally derived antibodies, they could overcome poultry challenges related to vaccination at an early age. Other advantages are the ability to induce humoral and cell-mediated immunity, possible expression of multiple epitopes, safety, and lower cost production, especially compared with peptide vaccines. Short production time is a valuable tool for confronting emergencies. They can be combined with adjuvants and cytokines (Zuo et al., 2021; Yang et al., 2009).
Zuo et al. (2021) designed and developed a DNA vaccine based on Spike protein with consecutive nucleotide sequences. After two times intramuscular injections of the vaccine, 85% protection was observed against challenges with the M41 infectious bronchitis virus. The vaccine could stimulate humoral and cell-mediated immunity.
A designated S1-DNA vaccine, pDKArkS1-DP, based on Arkansas IBV, was developed. In ovo vaccination, followed by a live attenuated IB vaccine 2 weeks later, caused a high immune response and 100% protection against challenge. The DNA vaccine’s efficacy and protection improve whenever a cationic liposome carrier is used. Alternatives to improve the effectiveness of that kind of vaccine are combination with specific adjuvants such as cytokines, whether mono- or polyvalent, DNA vaccines encoding nucleocapsid or S1 or even membrane protein with IL-2 chicken (GM-CSF). A liposome-encapsulated multi-epitope DNA vaccine from S1, S2, and N regions in intramuscular injection resulted in increased numbers of CD4+ and CD8+ cells and an 80% protective immune response in immunized birds (Leyson et al., 2017; (Jordan, 2017).

Reverse genetic vaccines
A reverse genetic vaccine is a new technology for manipulating viral genes and producing a new virus from a classic one. For example, a recombinant Beau-R-IBV vaccine was constructed by replacing the antigenic sites of S1 of the Beau-IBV strain with S1 from a pathogenic strain M41 and 4/91 strains (Armesto et al., 2011; Hodgson et al., 2004). These changes lead to protective responses without making the manipulated Beau-R strain to be pathogenic. Reversion to virulence, which is a weak point for live attenuated vaccines, is abrogated and can be used in the presence of maternal immunity. Whether this vaccine generation will increase or reduce the mutation rate or selection pressure is not clearly understood (Zhou et al., 2013).
Previous research has focused on developing an attenuated recombinant IBV (rIBV), a molecular clone of the highly attenuated Beaudette-CK strain, for S1 expressing of other heterologous genotypes. The rIBV, as a vaccine backbone, mimics a natural infection to induce both local and systemic immune responses. Beau-R with the S gene of 4/91 strain could achieve 65% protection after homologue challenge (Keep et al., 2020; Keep et al., 2022b). The replication of rIBV Beau-R in the host is minimal and, in cell culture, is completely inhibited at a natural chicken body temperature of 41°C. There was a hypothesis that the temperature sensitivity of the recombinant backbone is a desired character for the production of live-attenuated vaccines that are temperature sensitive and so restricted to causing virulent infection in chickens but have limited replication to induce immunity. Temperature sensitivity is not a new issue in virology and has been used to develop viral vaccines such as influenza (Keep et al., 2022a; Martinez-Sobrido et al., 2018).
Posttranslational modifications in different expression systems, including yeast, bacteria, plant, or baculovirus, influence the vaccine production outcome. For example, an attempt with the IBV vaccine on vaccinia could not produce enough antigen and then enough antibody responses, which is probably because of poor replication of the designed vaccine in avian cells and maybe the immune response of the mice to the vaccine that hindered the antigen-presenting for IBV gene (Bande et al., 2015). Another attempt to develop a baculovirus-based vaccine to produce the Korean KM91 strain S1-glycoprotein generated just 50% immunity because of post-translation modifications (Jung et al., 2022). The potato was chosen to express S1 of IBV using the cauliflower mosaic virus (35S) promotor gene (Zhou et al., 2004). The vaccine had promising results suggesting the method for food-based IB vaccines. Application of infectious bronchitis Beaudette virus as a background was a technology for a new generation of vaccines. The method was successful because it mimicked the virus replication cycle in the host without virulence but provided a possibility for vaccine production in cell culture, especially in the Vero cell, which is a licensed cell media to produce viral vaccines (Keep et al., 2022b). The delivery method and route of administration in vaccination can affect immune responses and significant histocompatibility complex responses. Live vaccines are administrated through water or spray. Inactivated vaccines are used via injection. The question is when you can use a vaccine in poultry as a mass application. It is beneficial and easy to use. Some vaccines have been designed to be administrated via feed, which is a very applicable route (Jordan, 2017). A DNA vaccine expressing S1-protein was administered orally with an attenuated Salmonella enterica strain (Jiao et al., 2011)—mucosal immune responses increased following oral or intranasal immunization. As a system to deliver the vaccine, Lactococcus lactis bacteria causes an effective immune response in mucosa (Peng et al., 2013). 

Conclusion
The future vision is that an IBV vaccine should provide broad protection against variable genotypes or induce strong protection in response to challenges posed by emerging viruses. Novel-designed vaccines should escape maternal antibody effects, be user-friendly, and have a rapid and cost-effective development process, considering the constant evolution of the viruses and the emergence of new variants that might lack cross-protection with the classic vaccine’s immunity. In light of novel technology, it is possible to develop vaccines that can reduce reversion to virulence. However, inactivation by maternal antibodies, especially following viral vectors and live-attenuated vaccines, is still a significant concern.
The recombinant vaccines, such as plasmid and multi-epitope vaccines, can deliver multiple antigens, inducing both humoral and cell-mediated immunity. New genotypes can also be manipulated and inserted into an established mechanism. Their preferences include oral or oculonasal routes for administration by developing these vaccines in novel delivery techniques such as nanoparticles or virus-like particles. Whether DNA or protein vaccines are produced in the future, it should be remembered that the protection is not 100%, and boosting with other kinds of vaccines must be contributed. These novel vaccines may be delivered by oral or mucosal route, but gaining the best protection requires repetition of vaccination. 

Ethical Considerations

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Conflict of interest
The author declared no conflict of interest.

Acknowledgments
The author would like to express her gratitude to her colleagues in the Poultry Vaccine Research and Production Department, Razi Vaccine and Serum Research Institute for their sincere efforts through vaccines production and developments.

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