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