Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants.

Severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) can infect various human organs, including the respiratory, circulatory, nervous, and gastrointestinal ones. The virus is internalized into human cells by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor through its spike protein (S-glycoprotein). As S-glycoprotein is required for the attachment and entry into the human target cells, it is the primary mediator of SARS-CoV-2 infectivity. Currently, this glycoprotein has received considerable attention as a key component for the development of antiviral vaccines or biologics against SARS-CoV-2. Moreover, since the ACE2 receptor constitutes the main entry route for the SARS-CoV-2 virus, its soluble form could be considered as a promising approach for the treatment of coronavirus disease 2019 infection (COVID-19). Both S-glycoprotein and ACE2 are highly glycosylated molecules containing 22 and 7 consensus N-glycosylation sites, respectively. The N-glycan structures attached to these specific sites are required for the folding, conformation, recycling, and biological activity of both glycoproteins. Thus far, recombinant S-glycoprotein and ACE2 have been produced primarily in mammalian cells, which is an expensive process. Therefore, benefiting from a cheaper cell-based biofactory would be a good value added to the development of cost-effective recombinant vaccines and biopharmaceuticals directed against COVID-19. To this end, efficient protein synthesis machinery and the ability to properly impose post-translational modifications make microalgae an eco-friendly platform for the production of pharmaceutical glycoproteins. Notably, several microalgae (e.g., Chlamydomonas reinhardtii, Dunaliella bardawil, and Chlorella species) are already approved by the U.S. Food and Drug Administration (FDA) as safe human food. Because microalgal cells contain a rigid cell wall that could act as a natural encapsulation to protect the recombinant proteins from the aggressive environment of the stomach, this feature could be used for the rapid production and edible targeted delivery of S-glycoprotein and soluble ACE2 for the treatment/inhibition of SARS-CoV-2. Herein, we have reviewed the pathogenesis mechanism of SARS-CoV-2 and then highlighted the potential of microalgae for the treatment/inhibition of COVID-19 infection.

Pathogenicity of Helicobacter pylori in cancer development and impacts of vaccination.

Helicobacter pylori affect around 50% of the population worldwide. More importantly, the gastric infection induced by this bacterium is deemed to be associated with the progression of distal gastric carcinoma and gastric mucosal lymphoma in the human. H. pylori infection and its prevalent genotype significantly differ across various geographical regions. Based on numerous virulence factors, H. pylori can target different cellular proteins to modulate the variety of inflammatory responses and initiate numerous "hits" on the gastric mucosa. Such reactions lead to serious complications, including gastritis and peptic ulceration, gastric cancer and gastric mucosa-associated lymphoid structure lymphoma. Therefore, H. pylori have been considered as the type I carcinogen by the Global Firm for Research on Cancer. During the two past decades, different reports revealed that H. pylori possess oncogenic potentials in the gastric mucosa through a complicated interplay between the bacterial factors, various facets, and the environmental factors. Accordingly, numerous signaling pathways could be triggered in the development of gastrointestinal diseases (e.g., gastric cancer). Therefore, the main strategy for the treatment of gastric cancer is controlling the disease far before its onset using preventive/curative vaccination. Increasing the efficiency of vaccines may be achieved by new trials of vaccine modalities, which is used to optimize the cellular immunity. Taken all, H. pylori infection may impose severe complications, for resolving of which extensive researches are essential in terms of immune responses to H. pylori. We envision that H. pylori-mediated diseases can be controlled by advanced vaccines and immunotherapies.

Stable transformation of Spirulina (Arthrospira) platensis: a promising microalga for production of edible vaccines.

The planktonic blue-green microalga Spirulina (Arthrospira) platensis possesses important features (e.g., high protein and vital lipids contents as well as essential vitamins) and can be consumed by humans and animals. Accordingly, this microalga gained growing attention as a new platform for producing edible-based pharmaceutical proteins. However, there are limited successful strategies for the transformation of S. platensis, in part because of an efficient expression of strong endonucleases in its cytoplasm. In the current work, as a pilot step for the expression of therapeutic proteins, an Agrobacterium-based system was established to transfer gfp:gus and hygromycin resistance (hygr) genes into the genome of S. platensis. The presence of acetosyringone in the transfection medium significantly reduced the transformation efficiency. The PCR and real-time RT-PCR data confirmed the successful integration and transcription of the genes. Flow cytometry and ß-glucuronidase (GUS) activity experiments confirmed the successful production of GFP and the enzyme. Moreover, the western blot analysis showed a ~ 90 kDa band in the transformed cells, indicating the successful production of the GFP:GUS protein. Three months after the transformation, the gene expression stability was validated by histochemical, flow cytometry, and hygromycin B resistance analyses.

Current status and future prospective of vaccine development against Echinococcus granulosus.

Cystic echinococcosis (CE) is one of the most important zoonotic parasite diseases in human, livestock, and wildlife worldwide. Development of effective vaccines against CE appears to be the most promising strategy to control this infectious disease. Use of potential livestock and canine vaccines against the larval and adult stage of E. granulosus life cycle may be the key to the production of powerful vaccines. Some progress has been accomplished in the development of vaccines against hydatidosis using empirical approaches, while such immunotherapies often fail to induce adequate immune responses. Therefore, it is of great interest to identify antigens (Ags) with high immunogenicity and develop effective vaccines and adjuvant constructs against CE. To this end, various tools can be applied, including immune-based genomics and proteomics, immunoinformatics, systems vaccinology and mathematical/computational modeling. In this review, we aimed to provide comprehensive insights upon the current status of vaccination trials against E. granulosus, and also articulate some perspectives on the production of novel anti-CE vaccines. Use of novel prospective technologies is also discussed to highlight the importance of development and advancement of the next generation vaccines against E. granulosus.

A domain-based vaccine construct against SARS-CoV-2, the causative agent of COVID-19 pandemic: development of self-amplifying mRNA and peptide vaccines.

Introduction: Coronavirus disease 2019 (COVID-19) is undoubtedly the most challenging pandemic in the current century with more than 293,241 deaths worldwide since its emergence in late 2019 (updated May 13, 2020). COVID-19 is caused by a novel emerged coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Today, the world needs crucially to develop a prophylactic vaccine scheme for such emerged and emerging infectious pathogens. Methods: In this study, we have targeted spike (S) glycoprotein, as an important surface antigen to identify its B- and T-cell immunodominant regions. We have conducted a multi-method B-cell epitope (BCE) prediction approach using different predictor algorithms to discover the most potential BCEs. Besides, we sought among a pool of MHC class I and II-associated peptide binders provided by the IEDB server through the strict cut-off values. To design a broad-coverage vaccine, we carried out a population coverage analysis for a set of candidate T-cell epitopes and based on the HLA allele frequency in the top most-affected countries by COVID-19 (update 02 April 2020). Results: The final determined B- and T-cell epitopes were mapped on the S glycoprotein sequence, and three potential hub regions covering the largest number of overlapping epitopes were identified for the vaccine designing (I531-N711; T717-C877; and V883-E973). Here, we have designed two domain-based constructs to be produced and delivered through the recombinant protein- and gene-based approaches, including (i) an adjuvanted domain-based protein vaccine construct (DPVC), and (ii) a self-amplifying mRNA vaccine (SAMV) construct. The safety, stability, and immunogenicity of the DPVC were validated using the integrated sequential (i.e. allergenicity, autoimmunity, and physicochemical features) and structural (i.e. molecular docking between the vaccine and human Toll-like receptors (TLRs) 4 and 5) analysis. The stability of the docked complexes was evaluated using the molecular dynamics (MD) simulations. Conclusion: These rigorous in silico validations supported the potential of the DPVC and SAMV to promote both innate and specific immune responses in preclinical studies.

Vaccination with rEGVac elicits immunoprotection against different stages of Echinococcus granulosus life cycle: A pilot study.

Vaccination against dog-sheep transmission cycle is necessary to control cystic echinococcosis (CE) infection. A multi-epitope multi-antigenic recombinant vaccine was developed-comprising the three putative vaccine antigens EG95, Eg14-3-3 and EgEnolase-was cloned and expressed. In a pilot experiment, the multi-antigen vaccine was assessed in 15 dogs and 15 sheep (five experimental groups and three animals in each group) by two subcutaneous doses 28 days apart. To evaluate the efficacy of the vaccine candidate first immunological analysis were done comprising IgG and IgE antibodies and the cytokine IL-4 in sera of the immunized dogs and sheep. Serum IgG, IgE, and IL-4, in particular in the dogs, were increased after the two rounds of vaccine candidate injection, while the total number of hydatid cysts was reduced (~85.43%). This pilot trial indicated significant immune protection efficacy against E. granulosus especially in dogs, while its efficacy in sheep was not as high as dogs. The multi-antigenic candidate vaccine is proposed as a protective vaccine modality in both dogs and sheep.

Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2.

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium -2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris , and Dunaliella salina . The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines.

Towards a new avenue for producing therapeutic proteins: Microalgae as a tempting green biofactory.

Most of the recent approved therapeutic proteins are multi-subunit biologics, which need glycosylation and disulfide bridges for their correct conformation and biological functions. Currently, there exist many protein-based drugs that are mostly produced in the Chinese hamster ovary (CHO) cells. However, this expression system appears to associate with some limitations both in upstream and downstream processing steps, including low growth rate, sensitivity to different stresses and pathogens, and time-consuming purification processes. Some microalgae species offer a suitable expression system for the production of a wide range of recombinant proteins due to their key features such as fast-growing rate, having no common pathogens with the human, being used as the human food, and providing the possibility for the large-scale production in the closed/controlled bioreactors. More importantly, the protein biosynthesis machinery of some microalgae seems to be relatively similar to those of the human and animal cells. In fact, microalgae can assemble fully functional complex proteins that can be safely used in humans. In this review, we provide comprehensive insights into the currently used expression systems for the production of therapeutic proteins and discuss the essential features of the microalgae as a novel protein expression platform.

Lactobacillus plantarum induces apoptosis in gastric cancer cells via modulation of signaling pathways in Helicobacter pylori.

Introduction: Gastric cancer is considered the second prevalent cause of death around the world. This type of cancer is generally induced by Helicobacter pylori which could colonize within the gastric mucosa of the infected cases. To date, triple antibiotic therapy has routinely been utilized for controlling the H. pylori- induced infection. However, this strategy has been unsuccessful, in large part because of issues such as occurring point mutations in the H. pylori genome that can induce resistance to the antibiotics administered. Recently, it has been shown that different probiotics may have strong anti-cancer effects, in which they are capable of inhibiting H. pylori by both immunological and non-immunological mechanisms. Here, we aimed at finding possible anti-cancer impacts of the probiotic bacterium Lactobacillus plantarum on gastric cancer, AGS cells. Methods: The anti-cancer effects of the conditioned media of the locally isolated L. plantarum on the AGS cells were evaluated by different analyses such as flow cytometry, DNA ladder assay, DAPI staining, and RT-PCR. Results: Our findings showed that the conditioned media of L. plantarum can inhibit both H. pylori and AGS cells through up-/down-regulation of PTEN, Bax, TLR4, and AKT genes. The exudates of the probiotic L. plantarum bacteria can increase the expression of PTEN, Bax, and TLR4, and also decrease the expression of AKT gene. Conclusion: In agreement with different reports, our results proved the anti-cancer effects of the locally isolated L. plantarum through some immunological cell signaling pathways. Accordingly, it seems the probiotics could be considered as at least a complementary treatment for different types of malignancies.

Efficient and stable transformation of Dunaliella pseudosalina by 3 strains of Agrobacterium tumefaciens.

Introduction: Several platforms including mammalian, plant and insect cells as well as bacteria, yeasts, and microalgae are available for the production of recombinant proteins. Low efficiency of delivery systems, extracellular and intracellular degradation of foreign genes during transformation, difficulties in targeting and importing into the nucleus, and finally problems in integration into nuclear genome are the most bottlenecks of classical plasmids for producing recombinant proteins. Owing to high growth rate, no common pathogen with humans, being utilized as humans' food, and capability to perform N-glycosylation, microalgae are proposed as an ideal system for such biotechnological approaches. Here, Agrobacterium tumefaciens is introduced as an alternative tool for transformation of the microalga Dunaliella pseudosalina. Methods: The transformation of gfp gene into the D. pseudosalina was evaluated by three strains including EHA101, GV3301 and GV3850 of A. tumefaciens. The integrating and expression of gfp gene were determined by PCR, RT-PCR, Q-PCR and SDS-PAGE analyses. Results: The T-DNA of pCAMBIA1304 plasmid was successfully integrated into the genome of the microalgal cells. Although all of the strains were able to transform the algal cells, GV3301 possessed higher potential to transform the microalgal cells in comparison to EHA101 and GV3850 strains. Moreover, the stability of gfp gene was successfully established during a course of two months period in the microalgal genome. Conclusion : Agrobacterium is introduced as a competent system for stable transformation of Dunaliella strains in order to produce eukaryotic recombinant proteins.