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.