The use of food-grade organisms as recombinant vaccine expression hosts and delivery vehicles has been explored during the past 25 years, opening new avenues for vaccinology. Considering that oral ...immunization is a beneficial approach in terms of costs, patient comfort, and protection of mucosal tissues, the use of food-grade organisms can lead to highly advantageous vaccines in terms of costs, easy administration, and safety. The organisms currently used for this purpose are bacteria ( Lactobacillus and Bacillus) , yeasts, algae, plants, and insect species. Herein, a comparative and updated scenario on the production of oral vaccines in food-grade organisms is provided and placed in perspective. The status of clinical evaluations and the adoption of this technology by the industry are highlighted.
MINCLE is a C‐lectin receptor mainly expressed in macrophages to recognize carbohydrates enhanced by the innate immune response in mammals and fish. Toxin A (ToxA) from Vibrio parahaemolyticus N16 ...was evaluated as an antigenic protein; it was observed to induce IgM production, increase cytokine gene expression, and offer immunoprotection against bacterial challenges in fish. This study was aimed at analysing the MINCLE receptor from the Pacific red snapper (Lutjanus peru) through bioinformatics tools, and the stimulating effects of β‐glucans from Sterygmatomyces halophilus 4 N16 (β‐Glu/Sh) combined with an antigenic protein on head–kidney leukocytes (HKLs) before and on challenge with Vibrio parahaemolyticus N16. LpMincle was mainly expressed in the intestine, eyes and head–kidney tissues. Before and after V. parahaemolyticus challenge, an up‐regulation of the receptors (Mincle, Marco and tlr2) and pro‐inflammatory cytokines (il‐1β and il‐6) gene expression was observed in HKLs incubated with β‐Glu/Sh. Respiratory burst activity was mainly enhanced in cells incubated with ToxA before and after V. parahaemolyticus infection. Nitric oxide (NO) production increased in leukocytes incubated with β‐Glu/Sh or ToxA, whereas myeloperoxidase (MPO) activity augmented only after ToxA stimulation before the bacterial challenge. In conclusion, MINCLE from L. peru was characterized in silico and its effects on HKLs was identified. Receptors and pro‐inflammatory cytokine genes up‐regulated by β‐Glu/Sh alone or in combination with ToxA can be elicitors of innate immune parameters. These findings evidence the function and recognition of novel β‐glucans from yeast either alone or in combination with antigenic proteins by β‐glucan receptors and their cytokines response.
Given the dramatic impact of the COVID-19 pandemic, it is imperative to divulge all the available technologies with the potential to fight against this virus. Plant biotechnology offers potential ...solutions to this pandemic through the development of low-cost vaccines and antibodies useful for therapy, prophylaxis, and diagnosis. The technology to produce plant-made biopharmaceuticals is already established; two examples of these are: a therapeutic enzyme that has entered the market and the influenza vaccines that are currently under clinical trials with encouraging results. Thus far, some companies have started developing anti-COVID-19 antibodies and vaccines. In particular, plant-made antibodies might be timely produced and approved for human use in the short term, while the development of vaccines will take longer time (clinical evaluations could be concluded by the end of 2021); nonetheless, the candidates obtained will be valuable tools for future outbreaks. The key aspects that will define the exploitation of this technology in the fight against COVID-19 are discussed.
The aggregation and spread of alpha‐synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the ...establishment of a safe and effective disease‐modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB‐Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat‐labile Escherichia coli enterotoxin LTB as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB‐Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB‐Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 μg/mL. Furthermore, the purified soluble rLTB‐Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB‐Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies.
Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a low‐grade fever, watery stools, and ...minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon. Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant‐based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB‐CdeM), carrying the CdeM antigen, fused to the carboxi‐terminus of the B subunit of the Escherichia coli heat‐labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB‐CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB‐CdeM antigen in the range of 52–90 μg/g dry weight leaf tissues. The antigenicity of the plant‐made LTB‐CdeM antigen was evidenced by GM1‐ELISA and immunogenicity assessment performed in test mice revealed that the LTB‐CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant‐based vaccines against C. difficile infection.
The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology ...and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species
and
using
mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in
and
. The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 μg/g RBD and 1.61 ng/g FGF in
and 1.61 μg/g RBD and 1.025 ng/g FGF in
. Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.
The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as ...well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.
Although oral subunit vaccines are highly relevant in the fight against widespread diseases, their high cost, safety and proper immunogenicity are attributes that have yet to be addressed in many ...cases and thus these limitations should be considered in the development of new oral vaccines. Prominent examples of new platforms proposed to address these limitations are plant cells and microalgae.
. constitutes an attractive expression host for vaccine production because of its high biosynthetic capacity, fast growth in low cost culture media, and the availability of processes for industrial scale production. In addition, whole
. cells may serve as delivery vectors; especially for oral vaccines since
. is safe for oral consumption, produces immunomodulatory compounds, and may provide bioencapsulation to the antigen, thus increasing its bioavailability. Remarkably,
. was recently used for the production of a highly immunoprotective influenza vaccine. Moreover, an efficient method for transient expression of antigens based on viral vectors and
. as host has been recently developed. In this review, the potential of
. in vaccinology is placed in perspective, with emphasis on its use as an attractive oral vaccination vehicle.
The emergence of new pathogenic viral strains is a constant threat to global health, with the new coronavirus strain COVID-19 as the latest example. COVID-19, caused by the SARS-CoV-2 virus has ...quickly spread around the globe. This pandemic demands rapid development of drugs and vaccines. Plant-based vaccines are a technology with proven viability, which have led to promising results for candidates evaluated at the clinical level, meaning this technology could contribute towards the fight against COVID-19. Herein, a perspective in how plant-based vaccines can be developed against COVID-19 is presented. Injectable vaccines could be generated by using transient expression systems, which offer the highest protein yields and are already adopted at the industrial level to produce VLPs-vaccines and other biopharmaceuticals under GMPC-processes. Stably-transformed plants are another option, but this approach requires more time for the development of antigen-producing lines. Nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. Therefore, this is the most attractive approach in terms of cost, easy delivery, and mucosal immunity induction. The development of multiepitope, rationally-designed vaccines is also discussed regarding the experience gained in expression of chimeric immunogenic proteins in plant systems.
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