Protective efficacy of the oral vaccine Tc24:Co1 produced in Schizochytrium sp. against Trypanosoma cruzi infection in a mouse model.

Trypanosoma cruzi parasite - causal Chagas disease agent - affects about 7 million people; no vaccine is available, and current medications have not been entirely effective. Multidisciplinary efforts are necessary for developing clinical vaccine prototypes. Thus, this research study aims to assess the expressed and whole-cell administration protection of the oral vaccine prototype Tc24:Co1 using Schizochytrium sp. microalga. High recombinant protein expression yields (675 µg/L) of algal culture were obtained. Additionally, Schizochytrium sp.-Tc24:Co1 resulted stable at 4 °C for up to six months and at 25 °C for three months. After receiving four oral doses of the vaccine, the mice showed a significant humoral immune response and a parasitemia reduction associated with a lack of heart inflammatory damage compared with the unvaccinated controls. The Schizochytrium sp.-Tc24:Co1 vaccine demonstrates to be promising as a prototype for further development showing protective effects against a T. cruzi challenge in a mouse model.

Trypanosoma cruzi Tc24 Antigen Expressed and Orally Delivered by Schizochytrium sp. Microalga is Immunogenic in Mice.

Chagas disease-caused by the parasite Trypanosoma cruzi-is a neglected tropical disease for which available drugs are not fully effective in the chronic stage and a vaccine is not available yet. Microalgae represent a promising platform for the production and oral delivery of low-cost vaccines. Herein, we report a vaccine prototype against T. cruzi produced in a microalgae platform, based on the candidate antigen Tc24 with a C terminus fusion with the Co1 peptide (Tc24:Co1 vaccine prototype). After modeling the tertiary structure, in silico studies suggested that the chimeric protein is antigenic, not allergenic, and molecular docking indicated binding with Toll-like receptors 2 and 4. Thus, Tc24:Co1 was expressed in the marine microalga Schizochytrium sp., and Western blot confirmed the expression at 48 h after induction, with a yield of 632 µg/L of algal culture (300 µg/g of lyophilized algal cells) as measured by the enzyme-linked immunosorbent assay (ELISA). Upon oral administration of whole-cell Schizochytrium sp. expressing Tc24:Co1 (7.5 µg or 15 µg of Tc24:Co1 doses) in mice, specific serum IgG and intestinal mucosa IgA responses were detected in addition to an increase in serum Th1/Th2 cytokines. In conclusion, Schizochytrium sp.-expressing Tc24:Co1 is a promising oral vaccine prototype to be evaluated in an animal model of Trypanosoma cruzi infection.

Sida chlorotic leaf virus: a new recombinant begomovirus found in non-cultivated plants and Cucumis sativus L.

Background: Begomoviruses are circular single-stranded DNA plant viruses that cause economic losses worldwide. Weeds have been pointed out as reservoirs for many begomoviruses species, especially from members of the Sida and Malvastrum genera. These weeds have the ability to host multiple begomoviruses species simultaneously, which can lead to the emergence of new viral species that can spread to commercial crops. Additionally, begomoviruses have a natural tendency to recombine, resulting in the emergence of new variants and species. Methods: To explore the begomoviruses biodiversity in weeds from genera Sida and Malvastrum in Colima, México, we collected symptomatic plants from these genera throughout the state. To identify BGVs infecting weeds, we performed circular DNA genomics (circomics) using the Illumina platform. Contig annotation was conducted with the BLASTn tool using the GenBank nucleotide "nr" database. We corroborated by PCR the presence of begomoviruses in weeds samples and isolated and sequenced the complete genome of a probable new species of begomovirus using the Sanger method. The demarcation process for new species determination followed the International Committee on Taxonomy of Viruses criteria. Phylogenetic and recombination analyses were implemented to infer the evolutionary relationship of the new virus. Results: We identified a new begomovirus species from sida and malvastrum plants that has the ability to infect Cucumis sativus L. According to our findings, the novel species Sida chlorotic leaf virus is the result of a recombination event between one member of the group known as the Squash leaf curl virus (SLCV) clade and another from the Abutilon mosaic virus (AbMV) clade. Additionally, we isolated three previously identified begomoviruses species, two of which infected commercial crops: okra (Okra yellow mosaic Mexico virus) and cucumber (Cucumber chlorotic leaf virus). Conclusion: These findings support the idea that weeds act as begomovirus reservoirs and play essential roles in begomovirus biodiversity. Therefore, controlling their populations near commercial crops must be considered in order to avoid the harmful effects of these phytopathogens and thus increase agricultural efficiency, ensuring food and nutritional security.

Immune Evaluation of Avian Influenza Virus HAr Protein Expressed in Dunaliella salina in the Mucosa of Chicken.

Avian influenza (AI) is a serious threat to the poultry industry worldwide. Currently, vaccination efforts are based on inactivated, live attenuated, and recombinant vaccines, where the principal focus is on the type of virus hemagglutinin (HA), and the proposed use of recombinant proteins of AI virus (AIV). The use of antigens produced in microalgae is a novel strategy for the induction of an immune response in the mucosal tissue. The capacity of the immune system in poultry, particularly in mucosa, plays an important role in the defense against pathogens. This system depends on a complex relationship between specialized cells and soluble factors, which confer protection against pathogens. Primary lymphoid organs (PLO), as well as lymphocytic aggregates (LA) such as the Harderian gland (HG) and mucosa-associated lymphoid tissue (MALT), actively participate in a local immune response which is mainly secretory IgA (S-IgA). This study demonstrates the usefulness of subunit antigens for the induction of a local and systemic immune response in poultry via ocular application. These findings suggest that a complex protein such as HAr from AIV (H5N2) can successfully induce increased local production of S-IgA and a specific systemic immune response in chickens.

Transformation of Dunaliella salina by Agrobacterium tumefaciens for the Expression of the Hemagglutinin of Avian Influenza Virus H5.

Avian influenza (AI) is one of the main threats to the poultry industry worldwide. Vaccination efforts are based on inactivated, live attenuated, and recombinant vaccines, where the virus hemagglutinin (HA) is the main component of any vaccine formulation. This study uses Dunaliella salina to express the AIV HA protein of an H5 virus. D. salina offers a system of feasible culture properties, generally recognized as safe for humans (GRAS), with N-glycosylation and nuclear transformation by Agrobacterium tumefaciens. The cloning and transformation of D. salina cells with the H5HA gene was confirmed by polymerase chain reaction (PCR). SDS-PAGE and Western blot confirmed HA5r protein expression, and the correct expression and biological activity of the HA5r protein were confirmed by a hemagglutination assay (HA). This study proves the feasibility of using a different biological system for expressing complex antigens from viruses. These findings suggest that a complex protein such as HA5r from AIV (H5N2) can be successfully expressed in D. salina.

Plant-Based Vaccines: Antigen Design, Diversity, and Strategies for High Level Production.

Vaccines for human use have conventionally been developed by the production of (1) microbial pathogens in eggs or mammalian cells that are then inactivated, or (2) by the production of pathogen proteins in mammalian and insect cells that are purified for vaccine formulation, as well as, more recently, (3) by using RNA or DNA fragments from pathogens. Another approach for recombinant antigen production in the last three decades has been the use of plants as biofactories. Only have few plant-produced vaccines been evaluated in clinical trials to fight against diseases, of which COVID-19 vaccines are the most recent to be FDA approved. In silico tools have accelerated vaccine design, which, combined with transitory antigen expression in plants, has led to the testing of promising prototypes in pre-clinical and clinical trials. Therefore, this review deals with a description of immunoinformatic tools and plant genetic engineering technologies used for antigen design (virus-like particles (VLP), subunit vaccines, VLP chimeras) and the main strategies for high antigen production levels. These key topics for plant-made vaccine development are discussed and perspectives are provided.

Two strains of a novel begomovirus encoding Rep proteins with identical ß1 strands but different ß5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Plant-made vaccines against parasites: bioinspired perspectives to fight against Chagas disease.

Introduction: Three decades of evidence have demonstrated that plants are an affordable platform for biopharmaceutical production and delivery. For instance, several plant-made recombinant proteins have been approved for commercialization under good manufacturing practice (GMP). Thus far, plant-based vaccine prototypes have been evaluated at pre- and clinical levels. Particularly, plant-made vaccines against parasitic diseases, such as malaria, cysticercosis, and toxoplasmosis have been successfully produced and orally delivered with promising outcomes in terms of immunogenicity and protection. The experience on several approaches and technical strategies over 30 years accounts for their potential low-cost, high scalability, and easy administration.Areas covered: This platform is an open technology to fight against Chagas disease, one of the most important neglected tropical diseases worldwide.Expert opinion: This review provides a perspective for the potential use of plants as a production platform and delivery system of Trypanosoma cruzi recombinant antigens, analyzing the advantages and limitations with respect to plant-made vaccines produced for other parasitic diseases. Plant-made vaccines are envisioned to fight against Chagas disease and other neglected tropical diseases in those countries suffering endemic prevalence.

Evaluation of acute and chronic exposure to aflatoxin B1 in indigenous women of the Huasteca Potosina, Mexico.

Aflatoxin B1 (AFB1) is one of the most studied mycotoxins due to its high occurrence in food and its hepatotoxic, immunosuppressive, carcinogenic, childhood growth, genotoxic, mutagenic, and teratogenic effects in humans and animals. Exposure to AFB1 is reported to be both, acute and chronic; the main exposure pathway to AFB1 is through the intake of contaminated food. In Mexico, although the reports of several studies addressing the problem of aflatoxins in maize and other foods, the evidence has been centered on exposure to AFB1 and to the quantification of the Aflatoxins themselves, but there is null evidence about genotoxic effects of aflatoxins in vulnerable populations. Therefore, this study focused on assessing chronic AFB1 exposure through the AFB1-lys biomarker adduct and acute exposure through total AFB1-DNA adducts in women from a rural indigenous community in the Huasteca Potosina (Mexico). A hundred percent of the studied population presented total AFB1-DNA and AFB1-lys adducts in concentrations of 1.08 (0.48-1.34) µmol of adduct/mol of DNA and 2.33 (1.08-102.6) pg/mg of albumin respectively (median (min-max)). Thus, continuous monitoring and important changes in regulations are desired and recommended. The results in this study provide enough evidence to support the toxic effects that the exposure to AFB1 represents, as well as the population risk that it poses, and in the same sense, the current need to create an intervention program that directly influences the control of the sources of exposure in order to reduce it.

Inducible expression of antigens in plants: a study focused on peptides related to multiple sclerosis immunotherapy.

Multiple sclerosis (MS) affects 2.3 million patients worldwide with no effective treatments available thus far. Depletion of autoreactive T-cells is considered the basis for immunotherapeutic approaches. For this purpose the peptides BV5S2, BV6S5, and BV13S1 have been identified as candidates for the development of a MS vaccine. Herein, the plant-based simultaneous production of these peptides is described as an effort to generate a new model of MS immunotherapy. A polyprotein comprising the sequence of the target peptides was designed having the picornaviral 2A sequence in between to mediate the release of the individual peptides upon translation. A codon optimized gene was cloned in vectors mediating constitutive (CaMV35S promoter) or inducible (AlcA promoter) expression. No transgenic tobacco plants were recovered from the constitutive vector suggesting toxicity of the target peptides. In contrast, several transformed lines were obtained with the inducible vector. The individual BV5S2, BV6S5, and BV13S1 peptides were detected in transformed lines upon ethanol-mediated induction and a quantitative analysis based on a OVA conjugate carrying the three peptides revealed accumulation levels up to 0.5 µg g-1 FW leaves. The plant-made peptides were able to induce humoral responses in orally immunized mice. This platform will be useful in the development of alternative immunotherapies against MS having low cost and safety as main attributes. Moreover the platform represents an attractive alternative for the expression of antigens having detrimental effects in plants.

Increased removal of cadmium by Chlamydomonas reinhardtii modified with a synthetic gene for γ-glutamylcysteine synthetase.

Bioremediation with genetically modified microalgae is becoming an alternative to remove metalloids and metals such as cadmium, a contaminant produced in industrial processes and found in domestic waste. Its removal is important in several countries including Mexico, where the San Luis Potosi region has elevated levels of it. We generated a construct with a synthetic gene for γ-glutamylcysteine synthetase and employed it in the chloroplast transformation of Chlamydomonas reinhardtii. In dose-response kinetics with media containing from 1 to 20 mg/L of cadmium, both the transplastomic clone and the wild-type strain grew similarly, but the former removed up to 32% more cadmium. While the growth of both decreased with higher concentrations of cadmium, the transplastomic clone removed 20 ± 9% more than the wild-type strain. Compared to the wild-type strain, in the transplastomic clone the activity of glutathione S-transferase and the intracellular glutathione increased up to 2.1 and 1.9 times, respectively, in media with 2.5 and 10 mg/mL of cadmium. While 20 mg/L of cadmium inhibited the growth of both, the transplastomic clone gradually duplicated. These results confirm the expression of the synthetic gene gshA in the transformed strain as revealed in its increased removal uptake and metabolic response.

Current advances in the algae-made biopharmaceuticals field.

INTRODUCTION: The biopharmaceuticals industry demands new production platforms to address several challenges; such as cost reduction to make biologics accessible in low-income countries, safety enhancement of the product, development of products administered by noninvasive routes, and expansion of potential biosimilars and biobetters. Microalgae are emerging hosts for biopharmaceuticals production with the potential to meet such requirements. AREAS COVERED: Nowadays successful cases on the production of vaccines, antibodies, antimicrobial peptides, growth factors/cytokines, and hormones in algae have been reported. This review comprises an updated outlook covering protein expression strategies, a compilation of functional biopharmaceuticals produced in algae, and companies investing in this technology. EXPERT OPINION: Key perspectives for the field include optimizing yields, scaling up production and completing preclinical trials. The experience from the field of plant-made biopharmaceuticals is commented as a key reference that will aid in the development of the algae-made biopharmaceuticals field.

Arsenic removal using Chlamydomonas reinhardtii modified with the gene acr3 and enhancement of its performance by decreasing phosphate in the growing media.

Arsenic contamination of groundwater is a significant problem in countries like Mexico, where San Luis Potosi is among the regions registering severe levels of it. Bioremediation with microalgae capable to absorb and metabolize metals or metalloids like arsenic reduces their toxicity and is a cost-effective approach compared to physical-chemical processes. We evaluated the capability of Chlamydomonas reinhardtii to remove arsenate and compared it with an acr3-modified recombinant strain, which we produced by transforming the wild-type strain with Agrobacterium tumefaciens using the construct pARR1 including a synthetic, optimized acr3 gene from Pteris vittata, a hyper-accumulator of arsenic. We monitored the growth of both strains in media with arsenate, containing a standard or a 10-fold decreased amount of phosphate. Comparing both strains in media initially with 0.5, 1, and 1.5 mg/L of arsenate, the acr3-modified strain removed 1.5 to 3 times more arsenic than the wild-type strain. Moreover, the arsenic uptake rate increased 1.2 to 2.3 times when growing the acr3-modified strain in media with decreased phosphate, while the uptake rate for the wild-type strain scarcely changed under the same conditions. These results confirm the expression of the acr3 gene in C. reinhardtii and its potential application to remove arsenic.

Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: Clues to comprehend the complex regulation of late genes.

A novel bipartite begomovirus, Blechum interveinal chlorosis virus (BleICV), was characterized at the genome level. Comparative analyses revealed that BleICV coat protein (CP) gene promoter is highly divergent from the equivalent region of other begomoviruses (BGVs), with the single exception of Tomato chino La Paz virus (ToChLPV) with which it shares a 23-bp phylogenetic footprint exhibiting dyad symmetry. Systematic examination of the homologous CP promoter segment of 132 New World BGVs revealed the existence of a quasi-palindromic DNA segment displaying a strongly conserved ACTT-(N7)-AAGT core. The spacer sequence between the palindromic motifs is constant in length, but its sequence is highly variable among viral species, presenting a relaxed consensus (TT)GGKCCCY, which is similar to the Conserved Late Element or CLE (GTGGTCCC), a putative TrAP-responsive element. The homologous CP promoter region of Old World BGVs exhibited a distinct organization, with the putative TATA-box overlapping the left half of the ACTT-N7 composite element. Similar CP promoter sequences, dubbed "TATA-associated composite element" or TACE, were found in viruses belonging to different Geminiviridae genera, hence hinting unsuspected evolutionary relationships among those lineages. To get cues about the TACE function, the regulatory function of the CLE was explored in distinct experimental systems. Transgenic tobacco plants harboring a GUS reporter gene driven by a promoter composed by CLE multimers expressed high beta-glucuronidase activity in absence of viral factors, and that expression was increased by begomovirus infection. On the other hand, the TrAP-responsiveness of a truncated CP promoter of Tomato golden mosaic virus (TGMV) was abolished by site-directed mutation of the only CLE present in it, whereas the artificial addition of one CLE to the -125 truncated promoter strongly enhanced the transactivation level in tobacco protoplasts. These results indicate that the CLE is a TrAP-responsive element, hence providing valuable clues to interpret the recurrent association of the CLE with the TACE. On the basis of the aforesaid direct evidences and the insights afforded by the extensive comparative analysis of BleICV CP promoter, we propose that the TACE might be involved in the TrAP-mediated derepression of CP gene in vascular tissues.

Prospects on the Use of Schizochytrium sp. to Develop Oral Vaccines.

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. Schizochytrium sp. 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 Schizochytrium sp. cells may serve as delivery vectors; especially for oral vaccines since Schizochytrium sp. is safe for oral consumption, produces immunomodulatory compounds, and may provide bioencapsulation to the antigen, thus increasing its bioavailability. Remarkably, Schizochytrium sp. 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 Schizochytrium sp. as host has been recently developed. In this review, the potential of Schizochytrium sp. in vaccinology is placed in perspective, with emphasis on its use as an attractive oral vaccination vehicle.

Expression of a Zika virus antigen in microalgae: Towards mucosal vaccine development.

Zika virus (ZIKV) infection has extended rapidly all over the world in the last decades affecting humans of all ages, inducing severe illness such as the autoimmune Guillain-Barré syndrome as well as fetal neurodevelopmental defects. Despite the epidemiological importance of ZIKV, today there are no commercially available drugs or vaccines to combat or prevent this infection. Microalgae are attractive hosts to produce and deliver vaccines, with some candidates under preclinical evaluation. Herein, algae-based expression was assessed for the production of a new vaccine candidate against ZIKV called ZK. The Algevir technology was applied to express an antigenic protein called ZK comprising the B subunit of the heat labile Escherichia coli enterotoxin along with 3 epitopes from the ZIKV envelope glycoprotein. Efficient expression of the ZK antigen was achieved in Schizochytrium sp. with yields of up to 365 µg g-1 microalgae fresh weight. Upon oral administration in mice, the microalgae-made ZK protein elicited significant humoral responses at a higher magnitude to those induced upon subcutaneous immunization. The algae-made ZK vaccine represents a promising candidate to formulate attractive vaccines against ZIKV.

Efficient Expression of an Alzheimer's Disease Vaccine Candidate in the Microalga Schizochytrium sp. Using the Algevir System.

Alzheimer's disease (AD) is the most common neurodegenerative disease, where ß-amyloid (Aß) plays a key role in forming conglomerated senile plaques. The receptor of advanced glycation end products (RAGE) is considered a therapeutic target since it transports Aß into the central nervous system, favoring the pathology progression. Due to the lack of effective therapies for AD, several therapeutic approaches are under development, being vaccines considered a promising alternative. Herein, the use of the Algevir system was explored to produce in the Schizochytrium sp. microalga the LTB:RAGE vaccine candidate. Algevir relies in an inducible geminiviral vector and led to yields of up to 380 µg LTB:RAGE/g fresh weight biomass at 48-h post-induction. The Schizochytrium-produced LTB:RAGE vaccine retained its antigenic activity and was highly stable up to temperatures of 60 °C. These data demonstrate the potential of Schizochytrium sp. as a platform for high production of thermostable recombinant antigens useful for vaccination against AD.

Assessment of Carrot Callus as Biofactories of an Atherosclerosis Oral Vaccine Prototype.

Atherosclerosis is a pathology leading to cardiovascular diseases with high epidemiologic impact; thus, new therapies are required to fight this global health issue. Immunotherapy is a feasible approach to treat atherosclerosis and given that genetically engineered plants are attractive hosts for vaccine development; we previously proved that the plant cell is able to synthesize a chimeric protein called CTB:p210:CETPe, which is composed of the cholera toxin B subunit (CTB) as immunogenic carrier and target epitopes from the cholesteryl ester transfer protein (CETP461-476) and apolipoprotein B100 (p210). Since CTB:p210:CETPe was expressed in tobacco at sufficient levels to evoke humoral responses in mice, its expression in carrot was explored in the present study looking to develop a vaccine in a safe host amenable for oral delivery; avoiding the purification requirement. Carrot cell lines expressing CTB:p210:CETPe were developed, showing accumulation levels up to 6.1 µg/g dry weight. An immunoblot analysis revealed that the carrot-made protein is antigenic and an oral mice immunization scheme led to evidence on the immunogenic activity of this protein; revealing its capability of inducing serum IgG responses against p210 and CETP epitopes. This study represents a step forward in the development of an attractive oral low-cost vaccine to treat atherosclerosis.

Oxidative Stress Modifies the Levels and Phosphorylation State of Tau Protein in Human Fibroblasts.

Since the tau protein is closely involved in the physiopathology of Alzheimer's disease (AD), studying its behavior in cellular models might lead to new insights on understanding this devastating disease at molecular levels. In the present study, primary cultures of human fibroblasts were established and used to determine the expression and localization of the tau protein in distinct phosphorylation states in both untransfected and tau gene-transfected cells subjected to oxidative stress. Higher immunopositivity to phospho-tau was observed in cell nuclei in response to oxidative stress, while the levels of total tau in the cytosol remained unchanged. These findings were observed in both untransfected cells and those transfected with the tau gene. The present work represents a useful model for studying the physiopathology of AD at the cellular level in terms of tau protein implications.