3.5 Clinical signs
In all groups, no adversary vaccine reaction was observed at administration site. Also, there were no clinical symptoms, apparent phenotype alteration or mortalities were recorded in any bird after challenge exposure that also includes all non-vaccinated controls. The birds were also monitored for its fertility and did not cause any fertility issue in all birds we studied.
Discussion
In chickens, Mycoplasma gallisepticum is the primary etiological agent of CRD. MG as major respiratory pathogen of poultry that causes severe inflammatory response in the trachea of its host [23]. The pathogen leads to develop clinical symptoms such as ocular, sneezing and coughing etc. and in severe cases causes pneumonia, bronchitis and airsacculitis in chickens and turkeys [24]. After infection, MG resides on mucosal surface, and causes epithelial cell degeneration and also result in inflammatory cell infiltration into the mucosa. The infectious disease affecting poultry industry that accounts for huge financial loses by drastically reducing productivity. Besides, lower production, commercially available vaccines are administered to control the severity also add to additional costs. The MG infection control is achieved by maintaining disease free breeding flock [25] and also by both in inactive and live attenuated vaccines. The commercially available vaccine such as strain 6/85 [26] F strain [25] and ts-11are applied for disease control, however they are expensive and require cold chain for storage and distribution.
The availability of vaccines for chronic respiratory disease in chickens are limited and expensive. Producing vaccine using conventional production system such as microbial or animal cell culture-based system are prohibitively expressive because of associated maintenance, storage, distribution and safety that accounts for 80% higher production cost in comparison to plant-based system. Plants have natural ability to store numerous proteins in seed tissues are excellent destination for production of recombinant proteins [27]. Numerous proteins have been targeted into seed tissue, where the target proteins showed stability as well as functionality several months after storage at ambient temperature [10]. The vaccine candidates as expressed in plant tissues induced the immunogenic responses has encouraged this study to express vaccine candidate against CRD in chickens in cost effective manner.
In addition, in poultry industry, in developing world, the more threatening is that chickens are supplied with numerous antibiotics to control different infectious diseases. In addition, the residue of such antibodies has been found in chickens which are directly consumed by the humans could be threatening for their health. Therefore, the promotion of alternative production systems for cost effective vaccine development is required to ensure access to modern medicine and improve health particularly in developing world.
In this study, the construct seed specific promoter was incorporated to induce TM-1 expression in seed tissues to achieve goal of oral delivery. The integration of an expression cassette carrying transgene into wheat developing callus was achieved by stable transformation biolistic. The TM-1 protein was governed by a rice endosperm-specific promoterGluB-4 with focus on seed tissues. Although, the fusion protein was also detected in vegetative tissues, however, such data was not included in this manuscript, and achieving goal of oral delivery of seed-based system is reported and discussed. The seed-based system is favored because such tissues are natural production and accumulation tissues for numerous proteins. Taking advantage of such ability we targeted our candidate vaccine antigen to achieve higher expression and its bioencapsulation. Seeds are relatively lower in water (10-40%) in comparison to other plant tissues where water contents are up to 90% of their wet weight [28]. Thus, plant seeds are ideal vehicle with lower water content with denser proteins concentration which favors oral delivery of vaccines. Taking advantage of seed tissues, several vaccine antigens and antibodies showed prolonged stability for years without loss of activity at normal room conditions [28, 29].
The oral delivery for PDs is novel method for developing edible vaccine and has been reported earlier in several studies [14-16, 30-34]. The oral delivery of PDs does not necessitate the downstream processing which accounts for are more than 80% of total production cost. The cold chain requirement for vaccine storage and delivery are the major concerns to equitably vaccinate the resource poor population in developing countries where the average daily income is less than $2. In the developing world, children die because of the unavailability of vaccines against life threatening infectious diseases. In this scenario, availability of vaccine to against veterinary disease is very unlikely. This is because maintaining cold chain without continuous electricity supply for its storage and distribution is near to impossible. Likewise, poultry industry is heavily affected by zoonotic diseases where high cost and unavailability of vaccines are major concerns, and those if available are costly and require cold temperature.
In this study, we demonstrated the oral route for vaccine delivery which is promising alternative to drastically reduce the cost of production, where vaccine candidate does not require further purification steps. Due to plant cell wall natural ability of bioencapsulation of the vaccine antigen to eliminate the probability of its degradation by providing protection from enzymatic conditions and preserved their antigen immunogenicity and functionality in the gastrointestinal tract (The unique characteristic of plant cells is their ability to withstand the digestive enzymes as present in humans and animals. The presence of certain bacteria in small intestine has the ability to break the plant cell wall to release the vaccine and are absorbed. The successful oral delivery of recombinant TM-1 antigen produced in wheat seeds was evidenced by results showing dramatically increased anti-MG antibody titer in chicken sera after vaccination compared to those vaccinated with WT and PBS buffer (Fig. 4). This was further supported by the observation that oral vaccination of recombinant TM-1 antigen efficiently protected chickens from MG infection later, while chickens in control groups were greatly infected and developed corresponding clinical symptoms (Fig. 5, Table 2).
To date, there are fewer plant made vaccine and monoclonal antibody candidates received regulatory authorization. Plant made products, i.e. poultry vaccine against NDV and antibody against HBsAg are the front runners in 30 years long down the road journey of molecular farming. The NDV vaccine is produced in suspension culture of tobacco cell, however, there is no oral product so far in market for orally delivered plant made vaccine. The oral delivery of plant made vaccine antigen received publicity and attention for their ease of delivery and cost effectiveness. This study is indeed can be as precedent for oral delivery of vaccine antigens against animal diseases where seed-based tissue showed decent expression efficacy, immunogenicity, and stability.
Concluding remarks
This study presents a proof of concept of producing oral vaccine in wheat seeds against Chronic Respiratory Disease (CRD) in poultry. The recombinant CTB-TM-1 protein of Mycoplasma gallisepticum (MG) expressed in transgenic wheat seeds remained stable and functional after long-term storage at ambient temperature. In addition, oral delivery of CTB-TM-1 vaccine bioencapsulated in wheat seeds successfully induced immune responses and protective effect against MG challenge in chicken without sacrifice of healthy development. Thus, this study demonstrates the feasibility, simplicity, and efficiency of seed-bioencapsulated oral vaccine in treating zoonotic infectious diseases.