Recent advances in expression and purification strategies for plant made vaccines.

Plants have been explored as a platform to produce pharmaceutical proteins for over 20 years. Important features such as the cost-effectiveness of production, the ease of scaling up to manufacturing capacity, the lack of cold chain requirements and the ability to produce complex therapeutic proteins which are biologically and functionally identical to their mammalian counterparts, make plants a strong alternative for vaccine production. This review article focuses on both the expression as well as the downstream purification processes for plant made vaccines. Expression strategies including transgenic, transient and cell suspension cultures are outlined, and various plant tissues targeted such as leaves and seeds are described. The principal components used for downstream processing of plant made vaccines are examined. The review concludes with a reflection of the future benefits of plant production platforms for vaccine production.

Seed-Based System for Cost-Effective Production of Vaccine Against Chronic Respiratory Disease in Chickens.

The production of vaccines in plant cells, termed plant-made pharmaceuticals or molecular farming, is a promising technology for scalable production. Compared to mammalian cell lines, like Chinese Hamster Ovary (CHO) or bacterial cells, plants can be grown with less cost on a large scale to make vaccines antigens and therapeutics affordable and accessible worldwide. 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 report stable expression of amino acid sequences corresponding to the TM-1 gene of Mycoplasma gallisepticum as a candidate vaccine antigen against Chronic Respiratory Disease (CRD) in chickens using wheat seed's tissues as a production host. Molecular and immunoblotting analysis confirmed the ubiquitous expression of a recombinant 41.8-kDa protein with an expression level of 1.03 mg/g dry weight in the endosperm tissues. When orally delivered, the plant-made vaccine was effective in terms of developing antibody response in animal model i.e., chicken without any detectable weight loss. Two doses of orally delivered plant-made TM-1 vaccine candidate elicited the immune response and protective effect against MG virus challenge at the level comparable to commercially available inactivated vaccine against CRD. Our study demonstrates that plant-made vaccines are not only safe but also scalable and cost-effective with prolonged stability at room temperature.

Plants as Sources of Natural and Recombinant Antimalaria Agents.

Malaria is one of the severe infectious diseases that has victimized about half a civilization billion people each year worldwide. The application of long-lasting insecticides is the main strategy to control malaria; however, a surge in antimalarial drug development is also taking a leading role to break off the infections. Although, recurring drug resistance can compromise the efficiency of both conventional and novel antimalarial medicines. The eradication of malaria is significantly contingent on discovering novel potent agents that are low cost and easy to administer. In this context, plant metabolites inhibit malaria infection progression and might potentially be utilized as an alternative treatment for malaria, such as artemisinin. Advances in genetic engineering technology, especially the advent of molecular farming, have made plants more versatile in producing protein drugs (PDs) to treat infectious diseases, including malaria. These recent developments in genetic modifications have enabled the production of native pharmaceutically active compounds and the accumulation of diverse heterologous proteins such as human antibodies, booster vaccines, and many PDs to treat infectious diseases and genetic disorders. This review will discuss the pivotal role of a plant-based production system that expresses natural antimalarial agents or host protein drugs to cure malaria infections. The potential of these natural and induced compounds will support modern healthcare systems in treating malaria infections, especially in developing countries to mitigate human fatalities.

Health-risk behaviors among Iranian university students, 2019: a web-based survey.

BACKGROUND: High-risk behaviors are among the most serious threats for the physical and mental health of adolescents and young adults. Our aims in this study were to investigate the subgroups of students based on risky behaviors and to identify the prevalence rate of these subgroups. METHODS: This cross-sectional web-based survey was conducted from July to August 2019 in Tabriz, Iran. We performed proportional sampling in all nine universities of the city, according to the number of students in each university. Applying an online survey questionnaire, the data were collected from 3649 students and analyzed using Latent Class Analysis. RESULTS: For total sample, standardized prevalence rates of cigarette smoking, hookah use, alcohol consumption, substance abuse and unsafe sex were 18.5 (Confidence Interval (CI) 95%: 17.3-19.8), 9.1 (CI 95%: 8.2-10.1), 9.2 (CI 95%: 8.3-10.2), 8.3 (CI 95%: 7.4-9.3) and 14.5 (CI 95%: 13.3-15.7), respectively. Three latent classes of risky behaviors were determined among students: a) low risk b) smoking and c) high risk. About 18% of boys and 1.5% of girls were in the high risk class. Cigarette smoking (18.5%, CI 95%: 17.3-19.8) and substance abuse (8.3%, CI 95%: 7.4-9.3) were the most and the least common risky behaviors among the students. CONCLUSION: In this we-based survey, a considerable number of students, particularly boys (18%), was at high-risk class, stressing the need for preventive interventions for this group of youth. Our findings are beneficial for planning and development of risky-behavior preventive strategies to prevent high-risk behaviors among college students.

Corrigendum: Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris.

[This corrects the article DOI: 10.3389/fpls.2019.00414.].

Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a deadly and uncurable disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. Angiotensin Converting Enzyme 2 (ACE2) and its product, angiotensin-(1-7) [ANG-(1-7)] were expressed in lettuce chloroplasts to facilitate affordable oral drug delivery. Lyophilized lettuce cells were stable up to 28 months at ambient temperature with proper folding, assembly of CTB-ACE2/ANG-(1-7) and functionality. When the antibiotic resistance gene was removed, Ang1-7 expression was stable in subsequent generations in marker-free transplastomic lines. Oral gavage of monocrotaline-induced PAH rats resulted in dose-dependent delivery of ANG-(1-7) and ACE2 in plasma/tissues and PAH development was attenuated with decreases in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance and pulmonary artery remodeling. Such attenuation correlated well with alterations in the transcription of Ang-(1-7) receptor MAS and angiotensin II receptor AGTRI as well as IL-1ß and TGF-ß1. Toxicology studies showed that both male and female rats tolerated ~10-fold ACE2/ANG-(1-7) higher than efficacy dose. Plant cell wall degrading enzymes enhanced plasma levels of orally delivered protein drug bioencapsulated within plant cells. Efficient attenuation of PAH with no toxicity augurs well for clinical advancement of the first oral protein therapy to prevent/treat underlying pathology for this disease.

Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris.

Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance.

The potential of plant systems to break the HIV-TB link.

Tuberculosis (TB) and human immunodeficiency virus (HIV) can place a major burden on healthcare systems and constitute the main challenges of diagnostic and therapeutic programmes. Infection with HIV is the most common cause of Mycobacterium tuberculosis (Mtb), which can accelerate the risk of latent TB reactivation by 20-fold. Similarly, TB is considered the most relevant factor predisposing individuals to HIV infection. Thus, both pathogens can augment one another in a synergetic manner, accelerating the failure of immunological functions and resulting in subsequent death in the absence of treatment. Synergistic approaches involving the treatment of HIV as a tool to combat TB and vice versa are thus required in regions with a high burden of HIV and TB infection. In this context, plant systems are considered a promising approach for combatting HIV and TB in a resource-limited setting because plant-made drugs can be produced efficiently and inexpensively in developing countries and could be shared by the available agricultural infrastructure without the expensive requirement needed for cold chain storage and transportation. Moreover, the use of natural products from medicinal plants can eliminate the concerns associated with antiretroviral therapy (ART) and anti-TB therapy (ATT), including drug interactions, drug-related toxicity and multidrug resistance. In this review, we highlight the potential of plant system as a promising approach for the production of relevant pharmaceuticals for HIV and TB treatment. However, in the cases of HIV and TB, none of the plant-made pharmaceuticals have been approved for clinical use. Limitations in reaching these goals are discussed.

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana.

The exploration of emerging host organisms for the economic and efficient production of protein microbicides against HIV is urgently needed in resource-poor areas worldwide. In this study, the production of the novel HIV entry inhibitor candidate, griffithsin (GRFT), was investigated using Nicotiana benthamiana as the expression platform based on a non-viral vector. To increase the yield of recombinant GRFT, the RNA silencing defense mechanism of N. benthamiana was abolished by using three gene silencing suppressors. A transient expression system was used by transferring the GRFT gene, which encodes 122 amino acids, under the control of the enhanced CaMV 35S promoter. The presence of correctly assembled GRFT in transgenic leaves was confirmed using immunoglobulin-specific sandwich ELISA. The data demonstrated that the use of three gene silencing suppressors allowed the highest accumulation of GRFT, with a yield of 400 µg g-1 fresh weight, and this amount was reduced to 287 µg g-1 after purification, representing a recovery of 71.75%. The analysis also showed that the ability of GRFT expressed in N. benthamiana to bind to glycoprotein 120 is close to that of the GRFT protein purified from E. coli. Whole-cell assays using purified GRFT showed that our purified GRFT was potently active against HIV. This study provides the first high-level production of the HIV-1 entry inhibitor griffithsin with a non-viral expression system and illustrates the robustness of the co-agroinfiltration expression system improved through the use of three gene silencing suppressors.

Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L(-1) 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L(-1)). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L(-1) of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days.

Increasing Scopolamine content in Hairy Roots of Atropa belladonna using Bioreactor

The aim of this study was to use the, hairy root system for increasing the scopolamine content in Atropa belladonna. Agrobacterium rhizogenes ATCC15834 was utilized to produce hairy roots. The culture was carried out in a 1.5-l bioreactor using the inoculum size of 0.5 g fr. wt of 10-day-old hairy roots and various parameters, including agitation, aeration, conductivity and the consumption of sucrose were evaluated. Results revealed that the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per volume per minute) and 70 rpm, respectively. Study of conductivity and the consumption of sucrose showed that the highest amount of sucrose consumption and the highest amount of minerals consumption also was at 1.25 vvm and 70 rpm. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR).