Improved virus inhibition by microencapsulated monoclonal antibody against porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has been affecting the swine industry, especially in suckling pigs in with a high mortality rate. Among all the strategies to overcome PEDV, boosting mucosal immunity in pig intestine via oral administration appears to be more efficient than other routes. However, there are biological obstacles such as acidic environment that could damage biologics, a product from organisms often used for PEDV treatment. The plant-derived 2C10 monoclonal antibody (mAb) from Nicotiana benthamiana produced by transient expression was revealed as one of the potential candidates against PEDV through oral delivery. Herein, we demonstrated the calcium-alginate microencapsulation system to protect the 2C10 mAb from the harsh condition in the stomach and to be released the 2C10 mAb when arriving in the intestine. The pH-responsive encapsulated 2C10 mAb microbeads were constructed from the calcium-alginate system. The microbeads were well-tolerated under the acidic environment of simulated gastric fluid (SGF) and were digested under the alkaline condition of simulated intestinal fluid (SIF). The encapsulated 2C10 mAb in the SPF-treated microbeads exhibited high virus neutralization efficiency in Vero cells when compared to the unencapsulated 2C10 mAb treated by SPF that cannot neutralize the virus. For these reasons, calcium-alginate microencapsulation system is an attractive platform to be considered as a candidate for the next generation of oral vaccine development.

Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana).

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM'M-UD, coupled with an artificial terminator, 3PRt. FM'M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis-acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM'M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4-6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM'M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM'M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.

Preclinical evaluation of immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052-Alum adjuvant.

Cost-effective, and accessible vaccines are needed for mass immunization to control the ongoing coronavirus disease 2019 (COVID-19), especially in low- and middle-income countries (LMIC).A plant-based vaccine is an attractive technology platform since the recombinant proteins can be easily produced at large scale and low cost. For the recombinant subunit-based vaccines, effective adjuvants are crucial to enhance the magnitude and breadth of immune responses elicited by the vaccine. In this study, we report a preclinical evaluation of the immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052 (TLR7/8 agonist)-Alum adjuvant. This vaccine formulation, named Baiya SARS-CoV-2 Vax 2, induced significant levels of RBD-specific IgG and neutralizing antibody responses in mice. A viral challenge study using humanized K18-hACE2 mice has shown that animals vaccinated with two doses of Baiya SARS-CoV-2 Vax 2 established immune protection against SARS-CoV-2. A study in nonhuman primates (cynomolgus monkeys) indicated that immunization with two doses of Baiya SARS-CoV-2 Vax 2 was safe, well tolerated, and induced neutralizing antibodies against the prototype virus and other viral variants (Alpha, Beta, Gamma, Delta, and Omicron subvariants). The toxicity of Baiya SARS-CoV-2 Vax 2 was further investigated in Jcl:SD rats, which demonstrated that a single dose and repeated doses of Baiya SARS-CoV-2 Vax 2 were well tolerated and no mortality or unanticipated findings were observed. Overall, these preclinical findings support further clinical development of Baiya SARS-CoV-2 Vax 2.

The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking ß-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of ß-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

Impact Of Plant Based Vaccines On Covid-19

Human life has always been under a constant threat to emerging deadly viruses, Covid-19 is the newest. This deadliest virus become pandemic within a short span of time and brought great amount of concern to fight against it and overcome dynamic challenges. It demands the speedy manufacture of vaccines and drugs at the industrial level. A conventional vaccine is effective but has risk of being infected with foreign agents;to overcome this problem plant based vaccine is superior alternative. The VLPs are generated by recombinant technology and consumed orally and functionally plant cell distributes the antigen. The process consumes time, cost effective, easily conveyed and mucosal immunity induction. Benefit of plant counteract, they are free from any corruption and has minute risk of anomalous responses. VLPs are more stable than conventional vaccines and have immense potential to treat diseases. It contains few bioethical issues, such as transferring of allergens to humans. It requires the safe sites and skilled staff for the smooth administration of operations. Copyright © 2022 Wolters Kluwer Medknow Publications. All rights reserved.

Plant-produced recombinant SARS-CoV-2 receptor-binding domain; an economical, scalable biomaterial source for COVID-19 diagnosis.

The outbreak of the novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread rapidly causing a severe global health burden. The standard COVID-19 diagnosis relies heavily on molecular tests to detect viral RNA in patient samples; however, this method is costly, requires highly-equipped laboratories, multiple reagents, skilled laboratory technicians, and takes 3-6 hours to complete. To overcome these limitations, we developed a plant-based production platform for the SARS-CoV-2 receptor-binding domain as an economical source of detection reagents for a lateral-flow immunoassay strip (LFIA) which is suitable for detection of IgM/IgG antibodies in human samples. Further, we validated the plant-produced SARS-CoV-2 receptor-binding domain-based LFIA as a useful diagnostic tool for COVID-19. A total of 51 confirmed COVID-19 serum samples were tested using the LFIA, and the obtained results were consistent with those from polymerase chain reaction assays, while providing sensitivity and specificity of 94.1% and 98%, respectively. The developed LFIA is rapid, scalable, user-friendly, and relatively inexpensive with a simple test procedure, making it useful for the routine monitoring of COVID-19 in clinical settings. This study was approved on March 19, 2020 by the Ethics Committee of the Faculty of Medicine, Chulalongkorn University (COA No. 354/2020 and IRB No. 236/63).

Recombinant proteins of spike protein of SARS-CoV-2 with the Omicron receptor-binding domain induce production of highly Omicron-specific neutralizing antibodies.

Various vaccines have been developed to fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 pandemic. However, new variants of SARS-CoV-2 undermine the effort to fight SARS-CoV-2. Here, we produced S proteins harboring the receptor-binding domain (RBD) of the Omicron variant in plants. Plant-produced S proteins together with adjuvant CIA09A triggered strong immune responses in mice. Antibodies in serum inhibited interaction of recombinant human angiotensin-converting enzyme 2 with RBD of the Omicron variant, but not RBD of other variants. These results suggest that antibodies induced by RBD of the Omicron variant are highly specific for the Omicron RBD, but not for that of other variants.

SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

Edible vaccines: a nutritional substitute for traditional immunization

Edible vaccines are created from transgenic plants and animals and contain immunostimulant. Edible vaccines, to put it simply, are medications generated from plants or animals. In underdeveloped countries, oral vaccines are less expensive and more widely available. Researchers came up with the idea of edible vaccines, in which edible plant pieces are employed as a vaccine factory. To make edible vaccinations, scientists put desired genes into plants and then force the plants to generate the proteins expressed in the genes. Transgenic plants are the result of transformation, whereas transformation is the act of converting plants. The edible vaccination promotes mucosal immunity. Dendritic cells in the gut can assist native T cells activate and differentiate into follicular T-helpers (Tfh). T and B cells will respond precisely to a reliable, digestible immunization. Potato, tomato, banana, carrots, tobacco, papaya, algae, and a variety of other plants are utilised as alternative agents for standard vaccinations. Malaria, cholera, hepatitis, rabies, measles, rotavirus, diarrhoea cancer treatments and treatment of covid-19 are among the illnesses for which plant-based vaccines have been created. It takes time and dedication to develop and sell edible vaccinations. Many edible vaccines for animal and human ailments have been developed and have gone through various levels of clinical testing. The importance of plant-based vaccinations is emphasized in this article.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Effects of the COVID-19 pandemic and the preventive and compulsory social isolation on the workers of two circulatory territories in Argentina's agriculture

In this paper, we analyze the effects of the COVID-19 pandemic and the preventive and compulsory social isolation, called ASPO in Argentina (Decree 297/2020), on the temporary and cyclical mobility of agricultural workers towards different productive areas of the country. On the one hand, we observe the effects on workers of Salta province who were involved in harvesting and packing tasks in the viniculture sector in Mendoza and the fruit growing sector in Rio Negro when the ASPO was declared by the government. On the other hand, we inquiry about the impact on Bolivian workers that were doing different works in tobacco and horticulture farms in Salta province at the beginning of the pandemic and the isolation measures. Moreover, we observe some effects on Bolivian people that were permanently (residents) or temporarily in Salta at the time when the ASPO and the closing of the border between Argentina and Bolivia began. The findings are based on interviews to agricultural workers, leaders of social organizations both from Bolivia and Salta origin, and civil servants of the Bolivian consulate in Salta carried out during 2020 and 2021. We incorporate in our analysis the collection of national and provincial news articles, web portals, decrees and official resolutions. We also include the findings of our previous research conducted before the pandemic about Bolivian mobility associated to tobacco and horticulture labor market in Salta province.

COVID-19 and the political economy of tobacco and maize commodity circuits: Makoronyera, the 'connected' and agrarian accumulation in Zimbabwe

This paper analyses the global commodity circuits (value chains) for maize and tobacco in Zimbabwe, in the context of a reconfigured agrarian economy and COVID-19 induced shocks. The study focuses on the political economy dynamics of agricultural commodity circuits to reveal how they can contribute to understanding the drivers and constraints of agricultural commercialisation in Zimbabwe. This paper traces the circuits of maize and tobacco, the two major crops for food security and foreign currency earnings in Zimbabwe.

Plant-based expression and characterization of SARS-CoV-2 virus-like particles presenting a native spike protein.

We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

COVID-19 and smoking: a high-risk association. (Special Section: COVID-19 - public health contributions.)

The objective of the article was to examine the relationship of tobacco smoking with the risk of acquiring COVID-19. Gas exchange, lung function, and blood circulation, processes directly affected in COVID-19, improve quickly after smoking cessation. Quitting smoking and avoiding exposure to tobacco smoke and vapors can have a positive impact, reducing the risk involved in COVID-19 and smoking.

Tobacco: an invisible and immediate threat for COVID 19. (Special Issue: The epidemiologist and the pandemic.)

One of the major steps to prevent damage due to any pandemic is to focus on risk factors related to the disease. Tobacco consumption is emerging as a major factor out of all for COVID 19. There is hardly any country who has warned the public about this or has made the Tobacco control measures stringent in view of COVID 19. Factors making tobacco consumers more vulnerable to COVID 19 infections are low immunity, damaged cilia in smaller air ways leading to decreased lung capacity and previous history of respiratory illness. Smokers have more expression of ACE2 receptor gene which is also responsible for SARS-COV-2 virus replication in host. More viral load makes smokers potent "carrier". COVID 19 can be transmitted via tobacco consumers by sharing of smoked tobacco and release of vapour droplets. Spitting of smokeless tobacco in public places puts community at risk. Cardiovascular diseases and cancers caused by tobacco consumption act as comorbidities aggravating the symptoms in COVID 19 infection. Scope of FCTC framework can be extended to address prevention and control of COVID 19. Recommendations: tobacco products should be banned immediately to control the spread. MPOWER strategy of FCTC can be utilized in this pandemic to prevent transmission.

Cloning and functional characterization of a terpene synthase gene AlTPS1 from Atractylodes lancea

Atractylodes lancea (Thunb.) DC has been used widely as a medicinal herb for centuries and is now being used to treat COVID-19 pneumonia. Terpenoids are thought to be its main pharmacologically active constituents. However, their biosynthesis remains uncharacterized in this species. In this study, the terpene synthase gene AlTPS1 was cloned and functionally characterized. We found that AlTPS1 was a bifunctional enzyme that catalyzed the conversion of farnesyl diphosphate to nerolidol and geranyl diphosphate to linalool in vitro. However, it functioned only in the nerolidol production in vivo by transient expression of the AlTPS1 gene in Nicotiana benthamiana leaves maybe due to subcellular compartmentalization of the AlTPS1 in the cytosol. Furthermore, AlTPS1 was highly expressed in leaves, considered to be the sites of nerolidol synthesis. This study is the first in which the cloning and expression of the AlTPS1 gene from A. lancea were analyzed, and it has provided new insights into terpene biosynthesis in A. lancea.

Soluble Human Angiotensin- Converting Enzyme 2 as a Potential Therapeutic Tool for COVID-19 is Produced at High Levels In Nicotiana benthamiana Plant With Potent Anti-SARS-CoV-2 Activity.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs a safe, cost-effective SARS-CoV-2 vaccine as well as therapeutic and antiviral drugs to combat COVID-19. Angiotensin-converting enzyme 2 (ACE2), as a key receptor for SARS-CoV-2 infections, has been proposed as a potential therapeutic tool in patients with COVID-19. In this study, we report a high-level production (about ∼0.75 g/kg leaf biomass) of human soluble (truncated) ACE2 in the Nicotiana benthamiana plant. After the Ni-NTA single-step, the purification yields of recombinant plant produced ACE2 protein in glycosylated and deglycosylated forms calculated as ∼0.4 and 0.5 g/kg leaf biomass, respectively. The plant produced recombinant human soluble ACE2s successfully bind to the SARS-CoV-2 spike protein. Importantly, both deglycosylated and glycosylated forms of ACE2 are stable at increased temperatures for extended periods of time and demonstrated strong anti-SARS-CoV-2 activities in vitro. The half maximal inhibitory concentration (IC50) values of glycosylated ACE2 (gACE2) and deglycosylated ACE2 (dACE2) were ∼1.0 and 8.48 µg/ml, respectively, for the pre-entry infection, when incubated with 100TCID50 of SARS-CoV-2. Therefore, plant produced soluble ACE2s are promising cost-effective and safe candidates as a potential therapeutic tool in the treatment of patients with COVID-19.

Vaccine mimics virus particle

The article talks about production of a new vaccine which mimics Covid-19 virus particle with spike proteins from a genetically engineered plant, a tobacco cousin called Nicotiana benthamiana produced by Medicago.