\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Seed-based System for Cost-Effective Production of Vaccine Against
Chronic Respiratory Disease in Chickens}



\author[1]{Imran Khan}%
\author[2]{Peyman Habibi}%
\author[3]{Madiha Saeed}%
\author[3]{Ayesha Haq}%
\author[1]{Yao Shi}%
\affil[1]{University of Pennsylvania}%
\affil[2]{University of Pennsylvania Perelman School of Medicine}%
\affil[3]{International Islamic University}%


\vspace{-1em}



  
  \date{June 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The production of vaccines in plant cells, termed as plant-made
pharmaceuticals or molecular farming, is a promising technology.
Compared to mammalian cell lines, like HEK293 and CHO as established
platform, plants can be grown cost effectively on a large-scale without
necessitating any sophisticated technologies. An innovative application
of this alternative system is the production of vaccines in edible
tissues that can be consumed orally to deliver protein antigen without
any further processing. In this project we reported stable expression of
TM-1 protein of MG as vaccine candidate antigen against Chronic
Respiratory Disease (CRD) in wheat seeds tissues. The molecular and
Immunoblotting analysis confirmed the integration of recombinant protein
of 41.8 kDa with expression level of 1.03mg/g DW in endosperm tissues.
When orally delivered, the plant made vaccine were highly effective in
terms of developing antibodies in chicken without any detectable weight
loss. Two doses of orally delivered plant-made TM-1 vaccine candidate
elicited an immune response and protective effect against MG virus
challenge at the level comparable to commercially available inactivated
vaccine against CRD. Our study demonstrated that plant-made vaccines are
not only safe but also similarly effective to commercially available
vaccines.%
\end{abstract}%



\sloppy


\textbf{Seed-based System for Cost-Effective Production of Vaccine
Against} \textbf{Chronic Respiratory Disease in Chickens}

Imran Khan\textsuperscript{1}, Peyman Habibi\textsuperscript{2}, Ayesha
Naveed Ul Haq\textsuperscript{3}, Madiha Saeed\textsuperscript{3}, Yao
Shi\textsuperscript{1}*

\textsuperscript{1}Department of Basic and Translational Sciences,
School of Dental Medicine, University of Pennsylvania, Philadelphia, PA,
USA.

\textsuperscript{2}Department of Pathology and Laboratory Medicine and
Center for Cellular Immunotherapies, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

\textsuperscript{3}SA-Center for Interdisciplinary Research in Basic
Sciences, International Islamic University, Islamabad, Pakistan

*Corresponding Author:

Yao Shi

School of Dental Medicine,

University of Pennsylvania,

240 South 40th Street,

547 Levy Building

Philadelphia PA 19104-6030

Email: yaoshi@upenn.edu

Phone: 414-520-6920

\textbf{Running title} : Seed-based oral vaccine against Chronic
Respiratory Disease in chickens

\textbf{Key words} : Molecular farming, oral vaccine, Chronic
Respiratory Disease, immunization, seed-based expression

\textbf{Abbreviations} : CTB, cholera toxin B subunit;
MG,\emph{Mycoplasma gallisepticum} ; CRD, Chronic Respiratory Disease;
PDs, Protein drugs.

\textbf{Abstract}

The production of vaccines in plant cells, termed as plant-made
pharmaceuticals or molecular farming, is a promising technology.
Compared to mammalian cell lines, like HEK293 and CHO as established
platform, plants can be grown cost effectively on a large-scale without
necessitating any sophisticated technologies. An innovative application
of this alternative system is the production of vaccines in edible
tissues that can be consumed orally to deliver protein antigen without
any further processing. In this project we reported stable expression of
TM-1 protein of MG as vaccine candidate antigen against Chronic
Respiratory Disease (CRD) in wheat seeds tissues. The molecular and
Immunoblotting analysis confirmed the integration of recombinant protein
of 41.8 kDa with expression level of 1.03mg/g DW in endosperm tissues.
When orally delivered, the plant made vaccine were highly effective in
terms of developing antibodies in chicken without any detectable weight
loss. Two doses of orally delivered plant-made TM-1 vaccine candidate
elicited an immune response and protective effect against MG virus
challenge at the level comparable to commercially available inactivated
vaccine against CRD. Our study demonstrated that plant-made vaccines are
not only safe but also similarly effective to commercially available
vaccines.

\textbf{Introduction}

Plants can be an attractive system to produce immunogens to control
different human and veterinary diseases. The idea of utilizing plants as
bioreactor for the expression of veterinary vaccines has been studied
extensively in the last decade. Progress in plant genetic engineering
has opened novel opportunities to use plants tissues as bioreactors for
safe and cost-effective production of vaccine antigens. Plants provide
an alternative expression system to microbes or mammalian cells grown in
fermenters, which have limited capacity, higher production costs and in
case of bacteria, lack of post translational modifications. Moreover,
molecular farming has an edge over other production systems for
recombinant proteins in terms of risk of the contamination with human
and animal pathogen, microbial toxins, or oncogenic sequences. In the
last two decades, plants have been used as viable and efficient
bioreactor for the large-scale production of recombinant proteins
including pharmaceutical proteins, antibodies {[}1{]}, hormones,
replacement enzymes {[}2{]}, industrial enzymes {[}3{]} as well as
vaccine antigens against infectious diseases {[}4{]}. Till now, variety
of vaccines against veterinary diseases have been expressed using plant
tissues as bioreactor {[}5{]}. The proof-of-concept for such studies has
already been accomplished in previous studies and plants have been found
suitable host for expression of edible vaccines for Newcastle disease
{[}6{]}, influenza, Ebola, infectious bronchitis virus (IBV), infectious
bursal disease virus (IBDV) in chickens {[}7{]}. The plant made ZMapp
antibodies against Ebola and influenza were not only encouraging but
also paved a way for plants to be further utilized as bioreactor to
combat infectious diseases in cost effective manner. Plant expressing
vaccines for poultry against Newcastle disease virus has already been
approved by USDA in 2006 {[}8{]} where vaccine development for animals
was intensified, therefore, regulatory approval of plant made vaccines
against veterinary disease can be achieved following same regulatory
pathway.

Worldwide, poultry is the largest livestock group, and according to
estimates with market value of \$309 billion in 2019 and expected to
grow nearly 6\% annually till 2023 (Poultry world, 2019). The most
prevalent problem in poultry especially at large scale setup includes
zoonotic diseases which not only is threat to poultry producers but also
is a great risk for poultry genetic resources {[}6{]}. Thus, suitable
veterinary strategies against such diseases are in crucial need of
poultry setups. At present, there are occurrences of Chronic Respiratory
Disease (CRD) worldwide and possess implacably drastic effects on
small-scale poultry producers in underdeveloped countries.
\emph{Mycoplasma gallisepticum} ~(MG) is a bacteria-like organism
that~causes respiratory disease primarily in chickens and turkeys, that
can spread both vertically and horizontally, that and can also infect
gamebirds, pigeons, ducks, geese, peafowl, and wild birds.~Symptoms of
CRD includes stress caused by moving the birds, by de-beaking,
operations handlings or unfavorable conditions (bad ventilation or
cold). At present, poultry management activities such as feeding and
watering, treatment with herbs, antibiotics, vaccines, and cleaning
poultry houses are mainly carried out against diseases {[}9{]}. The use
of antibiotics is one of many important tools in the treatment of
poultry diseases. Use of antibiotics has brought unintended impacts on
poultry production industries, such as the increase of infections in
animals and the decrease of animal production. Antibiotics residues can
remain in body for long time and may also greatly reduce scavenger
wildlife populations. The potential relationship between circulating
antibiotic residues and the presence of bacterial and fungal pathogens
causing severe diseases. To overcome these problems, an innovative
strategy is to develop cost effective vaccines against poultry diseases,
mainly formed through molecular farming is urgently required because
vaccination offers more long-term protection and prevention against
diseases in poultry. Vaccination also enhance the immune system, without
affecting the normal bacteria in body and can easily be decompose. Plant
based vaccines should also be encouraged as they can easily be
manufactured and less expensive as compared to other vaccines and
antibiotics.

In this study, in view of mounting production cost for veterinary
vaccines, we expressed candidate antigen vaccine against CRD in wheat
seeds as promising model for cost effective vaccine development in
plants that can be delivered orally without requiring any purification
or downstream processing. The plant's seeds have naturally evolved
system to express and accumulate storage proteins in large amount up to
40\% of their weight {[}10{]}. The accumulation of such large amount of
proteins distinguish seed over green tissues and tubers, where lower
amount of proteins expression can be found {[}11{]}. Protein stability
in seeds tissues for longer duration at ambient temperature is huge
advantage where antibodies and vaccine antigens expressed in higher
amount remain functional and stable for several years at room
temperature {[}10,12,13{]}.

We also demonstrate the feasibility of developing plant based orally
delivered vaccines as cost effective alternative. The oral delivery of
vaccine candidate has been reported earlier in numerous studies
{[}14-16{]}. The downstream processing that accounts for more than 80\%
of total production cost can be eliminated if the vaccine can be
delivered orally without necessitating further processing. The oral
delivery of protein drugs (PDs) is facilitated by plant cell wall that
contains \selectlanguage{greek}β-\selectlanguage{english}1,4 and \selectlanguage{greek}β-\selectlanguage{english}1,6 linkages resistant to hydrolysis and allows PDs
to be functionally active without any degradation during passing through
enzymatic conditions in stomach. Further, certain gut bacteria that have
the ability to hydrolyze plant cell wall contributes to release of PDs
and its absorption into blood stream.

In this study, we stably engineered TM-1 protein as promising candidate
antigen in wheat seeds tissues. The vaccine candidate was orally
administered in chickens produced antibodies those able to inhibit
infections after second drug dosage. Our findings demonstrate that oral
delivery of vaccine is a promising approach that can revolutionize
current production and delivery system for safe and affordable vaccine
development.

\textbf{Materials and methods}

\textbf{2.1 Construct development and wheat transformation}

The binary vector pPZP211 vector was purchased commercially (Creative
Biogene). A rice endosperm-specific promoter \emph{GluB-4} {[}17{]} and
a Nos terminator (GenBank: \textbf{AJ007623} ) were inserted upstream
and downstream of the multiple cloning site (MCS) at\emph{EcoR}
I/\emph{Kpn} I and \emph{Xba} I/\emph{Hind} III sites, respectively. The
codon-optimized complete CDS of TM-1 protein of\emph{Mycoplasma
gallisepticum} (MG; GenBank: \textbf{E09896} ) was followed by the
endoplasmic reticulum-retention signal KDEL (Lys-Asp-Glu-Leu) linked to
C-terminal of TM-1. The CTB (cholera toxin B subunit) coding sequence
followed by a furin enzymatic cleavage site (RRKRSV) were inserted
immediately upstream of TM-1. The entire
PGluB-4-CTB-furin-TM-1-KDEL-Tnos cassette was synthesized (GenScript,
NJ, USA) and subcloned in pPZP211 at \emph{Kpn} I/\emph{Xba} I sites
(Figure 1A).

Wildtype wheat (\emph{Triticum aestivum} L. cv Bobwhite) was used to
generate transgenic plants in this study. PDS-1000/He particle gun or
biolistics (Biorad, Germany) was utilized for direct bombardment. The
20ug plasmid DNA was coated with gold particles of size (0.6 \selectlanguage{greek}µ\selectlanguage{english}m)
following procedure as described by Sanford et al., 1993, and with 950
psi rapture disks for bombardment. The immature embryos were isolated 14
days after anthesis (size 0.5-1.5mm) under aseptic conditions and were
placed on induction media for 5-6 days in dark at 26C before bombardment
for callus induction.

The total DNA was extracted from wheat seeds for confirmation of genetic
integration of PGluB-4-TM-1-Tnos construct in wheat seed genome by
Polymerase Chain Reaction (PCR) and sequencing with primers targeting
transgene TM-1 (Seq Forward: 5'-attgcaaagctaccttttttctatta-3'; Seq
Reverse: 5'-atcgcgcgcggtgtcatctatgtt-3'; Fig. 1A).

\textbf{2.2 Regeneration and Selection on Media}

The transgenic plants were grown in soil to obtained T1 wheat seeds. In
order to confirm the transgenic nature of the plants, the T1 seeds were
soaked overnight in distil water and the embryos were removed
aseptically. The excised embryos were placed on MS half media with 50
mg/L of kanamycin for selection.

\textbf{2.3 Nucleic acid isolation and PCR confirmation}

Plant genomic DNA will be extracted from 100mg in vitro plant material
i.e. seeds using the CTAB (cetyltrimethylammonium bromide) method using
DNA extraction buffer 1\% (w/v) SDS, 100mM NaCl, 100mM Tris base, 100mM
Na\textsubscript{2}EDTA, pH=8.5 by HCl), as described earlier by Murray
et al., (1980). PCR Primer (PGluB-4 Forward:
5'-gcacgccagaaaatataatgata-3'; TM-1 Reverse: 5'- cagtacgccgagcatact-3')
were utilized to confirm the transgene integration. The Polymerase Chain
Reactions (PCR) analysis was conducted for the presence of DNA encoding
vaccine antigen. Specific primers to promoter region and TM-1 gene
amplified the sequences in transgenic wheat seeds tissues. The DNA
extracted from transgenic wheat seeds was used as a template for PCR
analysis. The amplification for TM-1 was carried out at 94\selectlanguage{ngerman}°C for 5 min,
94°C for 1 min, 61°C for 1.5 min and 72°C for 1 min, with total of 35
cycles, and final extension of 72°C for 10 min. The amplified PCR
product was resolved at 1\% SDS gel electrophoresis and was finally
analyzed under UV light to confirm the presence of estimated band size.
After confirmation, all the positive plants were self-pollinated to
obtain T1 generation and were utilized for all analysis.

\textbf{2.4 Recombinant Protein extraction}

To find out the accumulation of recombinant fusion proteins, 50 mg of
seed powder was used to mix with 500 \selectlanguage{greek}µ\selectlanguage{english}l of \emph{N} -hexane followed by
centrifugation for 5 min at 5000\selectlanguage{ngerman}×g, and supernatant was discarded. The
process is repeated 3 times and fine seed powder was dried in vacuum.
Double dH\textsubscript{2}O was added to powder with 1/10 (w/v) seed
weight/water ratio and were incubated for 1 hour with agitation,
followed by centrifugation of 13000 rpm at 4°C. The samples was
homogenized with 3 volume of protein extraction buffer (3\% (w/v) CTAB,
1,4 M NaCl, 0,2 \% (v/v) \selectlanguage{greek}β-\selectlanguage{english}Mercaptoethanol, 20 mM EDTA, pH 8.0, 100 mM
Tris-HCl, pH 8.0, 1 \% (w/v) PVP40). Centrifuge for 15sec and the 100ul
of supernatant was added to new 1.5 Eppendorf tube. Centrifuge again for
40 min at 13,000 rpm at 4\selectlanguage{ngerman}°C. The supernatant will be discarded, and the
pellet will be re-suspended in 5X loading buffer for further analysis.
The total soluble protein (TSP) concentration of the extracts was
determined by Bradford assay.

\textbf{2.5 SDS-PAGE and western blot analysis}

The total protein extracted from transgene positive wheat seeds tissues
was then separated by SDS-PAGE on their molecular masses. The expression
of vaccine antigen in seeds was verified by western blot analysis. The
band were separated using 8\% gel and were incubated with PBST
containing 5\% skimmed milk for one hour. The blot was then incubated
with primary with 1:10000 ration with polyclonal rabbit anti-CTB
antibody (Sigma-Aldrich, Inc,) over night at 4°C with continuous
shaking. The blot was then washed three times with PBST and then was
incubated with secondary goat anti\selectlanguage{english}-rabbit IgG\selectlanguage{english}-HRP antibody (Southern
Biotechnology, Birmingham, AL) with ratio 1:5000 in PBST containing 5\%
skimmed milk. The expected bands corresponding to TM-1 protein was
detected by substrate NBT/ BCIP and developed with X-Ray for 5 seconds
exposure. The quantitation of specific band was performed using software
ImageJ.

\textbf{2.6 ELISA quantification} Enzyme-linked immunosorbent assay
(ELISA) was performed using Pierce\selectlanguage{english} Nickel Coated Plates (Thermo Fisher
Inc.) to detect the expression of TM-1 recombinant vaccine in wheat
seeds by following manufacture's instruction. All the plant samples were
analyzed in duplicate. For detection of antibody titer in immunized
chickens, chickens were bled, and the serum was analyzed by the ELISA
against GM antibody (Idexx Laboratories) following manufacture's
instruction.

\textbf{2.7 Immunization and challenge procedure}

We included 100 two-days-old chicks separated randomly into four groups
of 25 chicks each. Three groups were vaccinated orally with seeds powder
containing 15 \selectlanguage{greek}µ\selectlanguage{english}g recombinant TM-1 protein, same amount of WT seeds
powder, and equal amount of PBS (Phosphate-buffered saline) buffer twice
at day 7 and 21. The fourth group was inoculated with MG attenuated
vaccine (8 x 10\textsuperscript{6} CFU) via ocular method. MG virus S6
strain (5 x 10\textsuperscript{6} CFU) challenge was conducted through
eye route for the all groups at week 4 and 5. Clinical parameters were
recorded daily after challenge with MG virus strain as described
previously for two weeks {[}18{]}. Dead chickens before termination of
the study were necropsied. All others were euthanized and examined two
weeks after MG challenge. Briefly, the main clinical scores consisted of
four levels in terms of symptomatic severity as follows: 0 - no symptom;
1 - clear nasal exudate; 2 - cloudy nasal exudate; 3 - turbid nasal
exudate with foamy eyes and/or swollen infra-orbital sinuses. For air
sac lesion, scores were observed and examined at 14 days after challenge
as follows {[}19{]}: 0 - no lesions; 1 - mild lesions; 2 - moderate
lesions; and 3 - severe lesions. Individual bodyweight was also recorded
every week. All experiment groups were challenged with MG virulent
strain (5 x 10\textsuperscript{6} CFU) one week after second
immunization by ocular delivery, followed by recording clinical
parameters for additional two weeks.

\textbf{2.8 Statistical analysis}The statistical analysis of this study
was carried out using the one-way ANOVA analysis and indicated by
\emph{P} value.

\textbf{Results}

\textbf{3.1 Creation and characterization of transgenic wheat seeds
expressing recombinant TM-1.}

Lower expression of plant made biopharmaceutical is a great concern and
hurdle for its commercialization. Several efforts including enhanced
promoters, regulatory elements, protein targeting, utilization of
various plants and their tissues and codon optimizing improve overall
yield. It has been known that mRNA stability and translational
efficiency are enhanced by codon optimization due to idealized tRNA
usage preference of recombinant TM-1 biosynthesis in host cells
{[}20{]}. Therefore, coding sequence of TM-1 was optimized during which
88 out of 262 codons in native sequence were replaced, resulting in an
elevated GC\% from 31.93\% to 46.44\% (supplementary Fig. S1). The rice
seed-specific promoter \emph{GluB-4} was included in construct as it can
drive and target recombinant protein in rice endosperm with the highest
translational activity among other 14 rice seed-specific promoters
{[}17{]} (Fig. 1A). The ER retention signal peptide KDEL linked to 3'
end of TM-1 coding sequence has been shown to dramatically increase
recombinant protein expression in rice endosperm {[}21{]}. CTB fused
immediately upstream of TM-1 allows transport of TM-1 through
gastrointestinal epithelia to circulation system by binding to a mucosal
membrane receptor ganglioside GM1 {[}20{]}. Furin cleavage site can be
digested by furin, a ubiquitous protease in human cells, enabling
release of TM-1 after transmucosal transfer carried by CTB {[}22{]}.

Three independent transgenic wheat lines were generated with particle
bombardment and its subsequent regeneration and selection, and the line
with highest expression was selected for further analysis. To verify the
insertion of CTB-TM-1 construct in wheat genome, PCR with
construct-specific primers was performed using genomic DNA from wheat T1
transgenic endosperm (Fig. 1B). The presence of CTB-TM-1 construct was
detected in all three lines (Fig. 1B). The Western blot further
confirmed the presence of recombinant TM-1 in wheat seeds from all lines
with mean expression level of 0.84\selectlanguage{ngerman}±0.046, 1.03±0.068, and 0.92±0.096
mg/g dry weight (DW) respectively and correct size as expected
(\textasciitilde{}41 kDa) (Fig. 1C and 1D). To evaluate the proper
formation of CTB pentamer that binds GM1 receptor, a GM1 ELISA assay was
carried out with crude extract of wheat seeds expressing recombinant
CTB-TM-1. The results showed CTB-TM-1 correctly bond to GM1 \emph{in
vitro} when compared to CTB standard (Fig. 1E). In addition, unless
otherwise indicated, the T1 seeds of line TM-1-2 was used for all
downstream analysis and processes due to its highest TM-1 expression
level and prominent affinity to GM1 receptor.

The transgenic nature of wheat plants transformed with CTB-TM-1 cassette
by biolistics were tested on MS ½ media containing antibiotic as
selection marker. All the embryo excised from PCR confirmed transgenic
T0 plants showed efficient regeneration as compared to wild type embryos
as negative control. Total of 25 embryos excised from transgenic wheat
line were confirmed by transgene integration and showed regeneration
(Fig. 2).

\textbf{3.2 Stability, functionality, and immune response is preserved
after storage for 6 months}

Plant seeds has natural ability to protect the vaccine antigen by
bioencapsulation in the plant tissues, and therefore allow its storage
at ambient temperature, moisture, and light conditions, and thus
simplifies its production and distribution. We have tested our plant
made vaccine antigen for confirmation of immunogenicity after long-term
storage. The immunoblot (Fig. 3) confirmed TM-1 vaccine antigen can be
stored at ambient temperature without degradation for at least six
months. The confirmation of immune response is evident of such
bioencapsulation where vaccine antigen was intact during passing through
digestive system. Cell wall mediated bioencapsulation allow proteins to
withstand enzymatic condition and can reach the small intestine, where
the TM-1 vaccine antigen was incorporated into M cells in the
follicle-associated epithelium (FAE), and finally to systemic immune
responses. The CTB is FDA approved as fusion protein for delivery of
biopharmaceuticals into immune system. The furin sites are incorporated
between CTB and TM1 antigen for its delivery upon its cleavage. In
addition, such results are encouraging because this protection to
withstand biodegradation elements and stability at normal room
conditions would allow plant made vaccine to eliminate the moderate to
ultra-cold conditions required for vaccine storage and distribution.
Also, we confirmed the feasibility of oral delivery of plant made
vaccine production and its efficacy for animals with efficient delivery
vehicle such as CTB and cleavage sites

\textbf{3.3 Immune response to vaccination of recombinant TM-1}

To evaluate the humoral immune response to vaccination of recombinant
TM-1, serum from vaccinated chickens were analyzed with ELISA against
anti-MG antibody weekly starting from the first week after the initial
vaccination. The titer of anti-MG antibody started to increase in TM-1
and MG attenuated vaccine groups one week after initial immunization,
although not significantly higher compared to control groups, until two
weeks later (Fig. 4). Moreover, while MG attenuated vaccine caused
higher anti-MG antibody titer, it is not significantly different from
recombinant TM-1 group (Fig. 4). Notably, amount of anti-MG antibody was
improved even more one week after second vaccination, leading to a
greater significance compared to control group vaccinated with WT and
PBS buffer (Fig. 4).

\textbf{3.4 Immunization of recombinant TM-1 protected chicken against
MG infection.}

We next tested if twice vaccination could adequately protect chickens
from MG infection by monitoring change of clinical sings and bodyweight
after MG virulent strain inoculation. After two weeks of challenge, the
mean severity-based clinical scores in control and WT group was
2.73±0.28 and 2.65±0.26, which were significantly higher than groups
vaccinated with recombinant TM-1 and MG attenuated vaccine with a score
of 0.72±0.13 and 0.58±0.19 respectively (Fig.5), indicating an efficient
systemic protection against MG virus infection. Additionally, Bodyweight
remained similar among all groups up to week 5, suggesting vaccination
of recombinant TM-1 antigen and attenuated MG vaccine did not affect
gaining weight (Table 1). Nevertheless, chickens in TM-1 and attenuated
MG vaccine groups gained significantly more weight than those in control
group one week after MG virus challenge (Table 1), confirming the
protective effect of recombinant TM-1 antigen prior to MG infection.
Scores of air sac lesion showed similar pattern. After two weeks of MG
virus challenge, presence of lesions was found significantly more in
control groups compared to other two groups, suggesting the recombinant
TM-1 vaccine specifically protects air sac in avian respiratory system
(Table 2).

\textbf{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.

\textbf{Discussion}

In chickens, \emph{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
promoter\emph{GluB-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.

\textbf{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 \emph{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.

\textbf{Acknowledgments}

We acknowledged International Islamic University for providing wildtype
wheat plants for creation of transgenic lines, as well as animals and
facilities necessary for immunization experiments used in this study.

\textbf{Conflict of interest statement}

The authors declare no commercial or financial conflict of interest

\textbf{Funding} : This work was supported by the Higher Education
Commission of Pakistan (HEC). Project number: NRPU10635.

\textbf{Table 1.}

\textbf{Weekly bodyweight change}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Group & Bodyweight (kg) & Bodyweight (kg) & Bodyweight (kg) & Bodyweight
(kg) & Bodyweight (kg) & Bodyweight (kg) & Bodyweight (kg)
&\tabularnewline
\midrule
\endhead
& Week 0 & Week 1 & Week 2 & Week 3 & Week 4 & Week 5 & Week 6 & Week
6\tabularnewline
PBS & 0.09\selectlanguage{ngerman}±0.01 \textsuperscript{a} & 0.15\selectlanguage{ngerman}±0.01 \textsuperscript{a} &
0.34\selectlanguage{ngerman}±0.03 \textsuperscript{a} & 0.64\selectlanguage{ngerman}±0.03 \textsuperscript{a} &
0.96\selectlanguage{ngerman}±0.06 \textsuperscript{a} & 1.55\selectlanguage{ngerman}±0.12 \textsuperscript{a} &
2.00\selectlanguage{ngerman}±0.11 \textsuperscript{a} & 2.00\selectlanguage{ngerman}±0.11
\textsuperscript{a}\tabularnewline
WT & 0.09\selectlanguage{ngerman}±0.01 \textsuperscript{a} & 0.16\selectlanguage{ngerman}±0.01 \textsuperscript{a} &
0.35\selectlanguage{ngerman}±0.03 \textsuperscript{a} & 0.66\selectlanguage{ngerman}±0.03 \textsuperscript{a} &
0.96\selectlanguage{ngerman}±0.07 \textsuperscript{a} & 1.54\selectlanguage{ngerman}±0.09 \textsuperscript{a} &
2.01\selectlanguage{ngerman}±0.13 \textsuperscript{a} & 2.01\selectlanguage{ngerman}±0.13
\textsuperscript{a}\tabularnewline
TM-1 & 0.09\selectlanguage{ngerman}±0,01 \textsuperscript{a} & 0.16\selectlanguage{ngerman}±0.01 \textsuperscript{a} &
0.35\selectlanguage{ngerman}±0.02 \textsuperscript{a} & 0.64\selectlanguage{ngerman}±0.04 \textsuperscript{a} &
0.94\selectlanguage{ngerman}±0.06 \textsuperscript{a} & 1.57\selectlanguage{ngerman}±0.10 \textsuperscript{a} &
2.18\selectlanguage{ngerman}±0.14 \textsuperscript{b} & 2.18\selectlanguage{ngerman}±0.14
\textsuperscript{b}\tabularnewline
MG attenuated vaccine & 0.09\selectlanguage{ngerman}±0,01 \textsuperscript{a} & 0.15\selectlanguage{ngerman}±0.01
\textsuperscript{a} & 0.36\selectlanguage{ngerman}±0.02 \textsuperscript{a} & 0.65\selectlanguage{ngerman}±0.04
\textsuperscript{a} & 0.97\selectlanguage{ngerman}±0.08 \textsuperscript{a} & 1.59\selectlanguage{ngerman}±0.10
\textsuperscript{a} & 2.21\selectlanguage{ngerman}±0.15 \textsuperscript{b} & 2.21\selectlanguage{ngerman}±0.15
\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Note: The superscript letters (a, b) represent date from groups that
were statistically significant (\emph{P} \textless{}0.05) scales when
compared to each other.

\textbf{Table 2.}

\textbf{Air sac lesion scores.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Group & Chicken number (N) & Air sac lesion scores\tabularnewline
\midrule
\endhead
PBS & 25 & 2.6 (0,3) \textsuperscript{a}\tabularnewline
WT & 25 & 2.5 (0,3) \textsuperscript{a}\tabularnewline
TM-1 & 25 & 0.7 (0,2) \textsuperscript{b}\tabularnewline
MG attenuated vaccine & 25 & 0.5 (0,1)
\textsuperscript{b}\tabularnewline
\bottomrule
\end{longtable}

Note: The superscript letters (a, b) represent date from groups that
were statistically significant (\emph{P} \textless{}0.05) scales when
compared to each other.

\textbf{References}

{[}1{]}. Kn\selectlanguage{ngerman}ödler M., Buyel J.F., Plant-made immunotoxin building blocks:
A roadmap for producing therapeutic antibody-toxin
fusions.\emph{Biotechnol Adv} . 2021 \emph{47} :107683. doi:
10.1016/j.biotechadv.2020.107683.

{[}2{]}. Stoger E., Ma J. K., Fischer R., Christou P., Sowing the seeds
of success: pharmaceutical proteins from plants. \emph{Curr Opin
Biotechnol} . 2005;\emph{16(2):} 167-173.
doi:10.1016/j.copbio.2005.01.005

{[}3{]}. Fischer, R., Twyman, R. M., and Schillberg, S. Production of
antibodies in plants and their use for global health. \emph{Vaccine} ,
2003, \emph{21} , 820-825. doi: 10.1016/s0264-410x(02)00607-2.

{[}4{]}. Mikschofsky, H., König, P., Keil, M., Hammer, M., et al.
Choleratoxin B (CTB) is functional as an adjuvant for cytoplasmatic
proteins if directed to the endoplasmatic reticulum (ER), but not to the
cytoplasm of plants. \emph{Plant Sci} , 2009, \emph{177} , 35--42.

{[}5{]}. Ceballo Y., Tiel K., López A., Cabrera G., et al. High
accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1)
influenza. \emph{Transgenic Res} . 2017, \emph{26(6)} :775-789. doi:
10.1007/s11248-017-0047-9.

{[}6{]}. Shahid, N., Samiullah, T. R., Shakoor, S., Latif, A., et al.
Early Stage Development of a Newcastle Disease Vaccine Candidate in
Corn.~\emph{Front Vet Sci} ,~2020\emph{, 7} , 499. doi:
10.3389/fvets.2020.00499

{[}7{]}. Salem R., Assem S. K., Omar O. A., Khalil A. A., et al.
Expressing the immunodominant projection domain of infectious bursal
disease virus fused to the fragment crystallizable of chicken IgY in
yellow maize for a prospective edible vaccine. \emph{Mol Immunol} .
2020,\emph{118} :132-141. doi: 10.1016/j.molimm.2019.12.015.

{[}8{]}. Vermij P. USDA approves the first plant-based vaccine.\emph{Nat
Biotechnol} , 2006, \emph{24} , 233--234.

{[}9{]}. Kanci A., Wijesurendra D. S., Wawegama N. K., Underwood G. J.,
et al. Evaluation of Mycoplasma gallisepticum (MG) ts-304 vaccine as a
live attenuated vaccine in turkeys. \emph{Vaccine} . 2018,\emph{36(18}
):2487-2493. doi: 10.1016/j.vaccine.2018.02.117.

{[}10{]}. Khan I., Twyman R. M., Arcalis E., Stoger E., Using storage
organelles for the accumulation and encapsulation of recombinant
proteins. \emph{Biotechnol J} . 2012, \emph{7(9):} 1099-108. doi:
10.1002/biot.201100089.

{[}11{]}. Arcalis E., Ibl V., Peters J., Melnik S., et al. The dynamic
behavior of storage organelles in developing cereal seeds and its impact
on the production of recombinant proteins. \emph{Front Plant Sci} .
2014,\emph{3;5} :439. doi: 10.3389/fpls.2014.00439.

{[}12{]}. Hudson L. C., Garg R., Bost K. L., Piller K. J., Soybean
seeds: a practical host for the production of functional subunit
vaccines. \emph{Biomed Res Int} . 2014: 340804. doi:
10.1155/2014/340804.

{[}13{]}. Vamvaka E., Arcalis E., Ramessar K., Evans A., et al. Rice
endosperm is cost-effective for the production of recombinant
griffithsin with potent activity against HIV. \emph{Plant Biotechnol J}
. 2016, \emph{14(6):} 1427-37. doi: 10.1111/pbi.12507.

{[}14{]}. Park Y., An D-J., Choe S., Lee Y., et al. Development of
recombinant protein-based vaccine against classical swine fever virus in
pigs using transgenic Nicotiana benthamiana. \emph{Front Plant Sci} .
2019, \emph{10} , 624. doi: 10.3389/fpls.2019.00624

{[}15{]}. Reddy P. H., Johnson A. A., Kumar J. K., Naveen T., et al.
Heterologous expression of Infectious bursal disease virus VP2 gene in
Chlorella pyrenoidosa as a model system for molecular farming. Plant
Cell Tissue Organ Culture, 2017, \emph{131} , 119--26. doi:
10.1007/s11240-017-1268-6

{[}16{]}. Gunter CJ., Regnard GL., Rybicki EP., Hitzeroth II.
Immunogenicity of plant produced porcine circovirus-like particles in
mice. \emph{Plant Biotechnol J} , 2019, \emph{17} , 1751--9. doi:
10.1111/pbi.13097

{[}17{]}. Qu L. Q., Takaiwa F., Evaluation of tissue specificity and
expression strength of rice seed component gene promoters in transgenic
rice. \emph{Plant Biotechnol J} . 2004, \emph{2(2):} 113-25. doi:
10.1111/j.1467-7652.2004.00055.x.

{[}18{]}. Naylor, C.J., Al-Ankari A.R., Al-Afaleq, A.I., Bradbury, J.M.
et al. Exacerbation of Mycoplasma gallisepticum infection in turkeys by
rhinotracheitis virus. \emph{Avian Pathol} , 1992, \emph{21} , 295--305.
doi: 10.1080/03079459208418844.

{[}19{]}. Ganapathy, K. \& Bradbury, J.M. Pathogenicity of mycoplasma
imitans in mixed infection with infectious bronchitis virus in
chickens.\emph{Avian Pathol} , 1999, \emph{28} , 229--237. doi:
10.1080/03079459994713.

{[}20{]}. Takaiwa F., Wakasa Y., Takagi H., Hiroi T. Rice seed for
delivery of vaccines to gut mucosal immune tissues. \emph{Plant
Biotechnol J} . 2015, \emph{13(8):} 1041-55. doi: 10.1111/pbi.12423.

{[}21{]}. Takaiwa F., Yang L., Wakasa Y., Ozawa K. Compensatory
rebalancing of rice prolamins by production of recombinant
prolamin/bioactive peptide fusion proteins within ER-derived protein
bodies. \emph{Plant Cell Rep} . 2018 \emph{37(2} ):209-223. doi:
10.1007/s00299-017-2220-2.

{[}22{]}. Thomas G. Furin at the cutting edge: from protein traffic to
embryogenesis and disease. \emph{Nat Rev Mol Cell Biol} .
2002,\emph{3(10):} 753-66. doi: 10.1038/nrm934.

{[}23{]}. Haycraft, C. J., Schafer, J. C., Zhang, Q., Taulman, P. D., et
al. Identification of CHE-13, a novel intraflagellar transport protein
required for cilia formation. \emph{Exp Cell Res.} 2003;\emph{284(2):}
251-263. doi:10.1016/S0014-4827(02)00089-7

{[}24{]}. Nunoya T., Yagihashi T., Tajima M., and Nagasawa Y.,
Occurrence of keratoconjunctivitis apparentl,y caused by Mycoplasma
gallisepticum in layer chickens. \emph{Vet Pathol.} ,1995, \emph{32} ,
11--18. doi:10.1177/030098589503200102.

{[}25{]}. Levisohn S., and Kleven S. Avian mycoplasmosis (Mycoplasma
gallisepticum). \emph{Rev Sci Tech} . 2000;\emph{19(2} ):425-442.
doi:10.20506/rst.19.2.1232.

{[}26{]}. Evans R., and Hafez Y., Evaluation of a Mycoplasma
gallisepticum strain exhibiting reduced virulence for prevention and
control of poultry mycoplasmosis. \emph{Avian Dis.} 1992, \emph{36} ,
197--201. doi:10.2307/1591490

{[}27{]}. Ramessar, K., Capell, T., and Christou, P.. Molecular pharming
in cereal crops.\emph{Phytochemical Reviews} 2008, 7, 579-592.

{[}28{]}. Boothe J., Nykiforuk C., Shen Y., et al. Seed-based expression
systems for plant molecular farming \emph{Plant Biotechnol J} .
2010,\emph{8(5):} 588--606. doi: 10.1111/j.1467-7652.2010.00511.x.

{[}29{]}. Stoger E., Vaquero C., Torres E., Sack M., et al. Cereal crops
as viable production and storage systems for pharmaceutical scFv
antibodies. \emph{Plant Mol Biol} . 2000, \emph{42(4):} 583-90. doi:
10.1023/a:1006301519427.

{[}30{]}. Shim B. S., Hong K. J., Maharjan P M., and Choe S. Plant
factory: new resource for the productivity and diversity of human and
veterinary vaccines. \emph{Clin Exp Vaccine Res} , 2019, \emph{8} ,
136--9. doi: 10.7774/cevr.2019.8.2.136

{[}31{]}. Firsov A., Tarasenko I., Mitiouchkina T., Ismailova N., et al.
High-yield expression of M2e peptide of avian influenza virus H5N1 in
transgenic duckweed plants. \emph{Mol Biotechnol} , 2015, \emph{57} ,
653--61. doi: 10.1007/s12033-015-9855-4

{[}32{]}. Rizwan H. M., Khan M. K., Iqbal Z., and Deeb F. Immunological
and therapeutic evaluation of wheat (Triticum aestivum) derived
betaglucans against coccidiosis in chicken. International Journal of
Agricultural Biology, 2016, 18, 895--902. doi: 10.17957/IJAB/15.0182

{[}33{]}. Berinstein A., Vazquez-Rovere C., Asurmendi S., Gómez E., et
al. Mucosal and systemic immunization elicited by Newcastle disease
virus (NDV) transgenic plants as antigens. \emph{Vaccine} ,
2005,\emph{23} , 5583--9. doi: 10.1016/j.

{[}34{]}. Yang M., Sun H., Lai H., Hurtado J., et al. Plant-produced
Zika virus envelope protein elicits neutralizing immune responses that
correlate with protective immunity against Zika virus in
mice.\emph{Plant Biotechnol J} . 2018;\emph{16(2} ):572-580. doi:
10.1111/pbi.12796\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figures:}

\textbf{Figure 1. Development and characterization of CTB-TM-1
expression cassette in transgenic wheat seeds. A.} The schematic diagram
of expression cassette of pPZP211: CTB-TM-1. Genetic components included
are: PGluB-4, promoter of glutelin gene GluB-4; CTB, Cholera non-Toxin B
subunit; Furin, Furin cleavage site; TM-1, TM-1 protein
of\emph{Mycoplasma gallisepticum} (MG); KDEL, Endoplasmic reticulum
retention signal; Tnos, termination of the nopaline synthase gene
from\emph{Agrobacterium tumefaciens;} CaMV 35S, promoter of 35S RNA
from\emph{Cauliflower Mosaic Virus} ; Kan\textsuperscript{R}, Kanamycin
resistant gene; CaMV Poly(A), \emph{Cauliflower Mosaic
Virus}polyadenylation signal; LB and RB, left and right border of T-DNA
repeat respectively. Arrows indicate PCR and sequencing primers.
\textbf{B.}characterization of genomic integration of pPZP211: CTB-TM-1
cassette in different transgenic lines by PCR using primers (CTB-F and
TM-1-R).\textbf{C.} Validation of three independent lines for
recombinant CTB-TM-1 expression by Western blot with anti-CTB antibody.
\textbf{D.}quantification of recombinant CTB-TM-1 expression in C.
\textbf{E.} GM1 ELISA detecting the folding of CTB-TM-1 and its binding
with GM1 receptor. *, \emph{P} \textless{}0.05, **, \emph{P}
\textless{}0.01, ***,\emph{P} \textless{}0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2. Process of developing transgenic plant using Biolistics:
Segregation of transgenic wheat lines} : A1. Immature embryos after
bombardment with plasmid carying TM-1. A2. Callus development under dark
with antibiotic selection. A3. Plant regeneration in media containing
selection marker. A4. Plant regeneration and rooting in growth chamber.
A5. Wheat plant expressing TM-1 vaccine candidate in rooting media,
before moving to soil for growth. B. Embryo rescue analysis. Wheat
embryo excised from WT and positive plants and their germination on
selection media. B. Embryo excision for transgene selection with
kanamycine supplemented germinating media. B1. Wild type BobWhite as
negative control. B2. Growth TM-1 positive embryo excised from seeds
after 10 days on MS half media as control for the experiment.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Stability of recombinant CTB-TM-1 expressed in wheat
seeds after long-term storage at ambient temperature (transgenic line:
TM-1-2)} . The recombinant CTB-TM-1 was extracted from seeds powder
stored immediately after harvest and six months later as described in
method and the expression level was quantified by western blot with
anti-CTB antibody. NS: non-significant (\emph{P} \textgreater{}0.05)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Immune responses to recombinant TM-1 vaccine} .
Anti-MG antibodies in chicken serum were quantified by ELISA after oral
delivery of recombinant TM-1 vaccine, WT, and PBS except for group
innoculated with attanuated MG vaccine {[}MG(av){]} by injection. (V)
and (C) represent the week when vaccination or challenge occured
respectively. *, \emph{P} \textless{}0.05, **, \emph{P} \textless{}0.01,
***,\emph{P} \textless{}0.001.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Protective effect of recombinant TM-1 immunization
against MG challenge} . Clinical severity was observed and recorded for
two weeks after MG challenge. Score categories were assigned according
to the description in materials and methods section in text. *,\emph{P}
\textless{}0.05, **, \emph{P} \textless{}0.01, ***,\emph{P}
\textless{}0.001.

\selectlanguage{english}
\FloatBarrier
\end{document}

