Seed-based System for Cost-Effective Production of Vaccine
Against Chronic
Respiratory Disease in Chickens
Imran Khan1, Peyman Habibi2, Ayesha
Naveed Ul Haq3, Madiha Saeed3, Yao
Shi1*
1Department of Basic and Translational Sciences,
School of Dental Medicine, University of Pennsylvania, Philadelphia, PA,
USA.
2Department of Pathology and Laboratory Medicine and
Center for Cellular Immunotherapies, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
3SA-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
Running title : Seed-based oral vaccine against Chronic
Respiratory Disease in chickens
Key words : Molecular farming, oral vaccine, Chronic Respiratory
Disease, immunization, seed-based expression
Abbreviations : CTB, cholera toxin B subunit; MG,Mycoplasma gallisepticum ; CRD, Chronic Respiratory Disease; PDs,
Protein drugs.
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.
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. 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 β-1,4 and β-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.
Materials and methods