[Show abstract][Hide abstract] ABSTRACT: Key message The first report on the recombinant production of a candidate vaccine in the moss system. Abstract The need for economical and efficient platforms for vaccine production demands the exploration of emerging host organisms. In this study, the production of an antigenic protein is reported employing the moss Physcomitrella patens as an expression host. A multi-epi-tope protein from the Human Immunodeficiency Virus (HIV) based on epitopes from gp120 and gp41 was designed as a candidate subunit vaccine and named poly-HIV. Transgenic moss plants were generated carrying the corresponding poly-HIV transgene under a novel moss promoter and subsequently seven positive lines were confirmed by PCR. The poly-HIV protein accumulated up to 3.7 lg g -1 fresh weight in protonema cultures. Anti-genic and immunogenic properties of the moss-produced recombinant poly-HIV are evidenced by Western blots and by mice immunization assays. The elicitation of specific antibodies in mice was observed, reflecting the immuno-genic potential of this moss-derived HIV antigen. This is the first report on the production of a potential vaccine in the moss system and opens the avenue for glycoengineer-ing approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.
[Show abstract][Hide abstract] ABSTRACT: Implications of bioinformatics on the development of epitope-based vaccines are described.•A compilation of useful bioinformatics tools for vaccine development is presented.•Implications on developing vaccines against hypervariable viruses are described.•Perspectives for the field are identified.
[Show abstract][Hide abstract] ABSTRACT: The progressive interest in transgenic plants as advantageous platforms for the production and oral delivery of vaccines has led to extensive research and improvements in this technology over recent years. In this paper, the authors examine the most significant advances in this area, including novel approaches for higher yields and better containment, and the continued evaluation of new vaccine prototypes against several infectious diseases. The use of plants to deliver vaccine candidates against viruses, bacteria, and eukaryotic parasites within the last 5 years is discussed, focusing on innovative expression strategies and the immunogenic potential of new vaccines. A brief section on the state of the art in mucosal immunity is also included.
Expert Review of Vaccines 08/2014; · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an effort to initiate the development of a plant-based vaccination model against atherosclerosis, a cholera toxin B subunit (CTB)-based chimeric protein was designed to target both ApoB100 and CETP epitopes associated with immunotherapeutic effects in atherosclerosis. Epitopes were fused at the C-terminus of CTB to yield a protein called CTB:p210:CETPe. A synthetic gene coding for CTB:p210:CETPe was successfully transferred to tobacco plants with no phenotypic alterations. Plant-derived CTB:p210:CETPe was expressed and assembled in the pentameric form. This protein retained the target antigenic determinants, as revealed by GM1-ELISA and Western blot analyses. Higher expresser lines reached recombinant protein accumulation levels up to 10 µg/g fresh weight in leaf tissues and these lines carry a single insertion of the transgene as determined by qPCR. Moreover, when subcutaneously administered, the biomass from these CTB:p210:CETPe-producing plants was able to elicit humoral responses in mice against both ApoB100 and CETP epitopes and human serum proteins. These findings evidenced for the first time that atherosclerosis-related epitopes can be expressed in plants retaining immunogenicity, which opens a new path in the molecular farming field for the development of vaccines against atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: The high demand for less polluting, newer, and cheaper fuel resources has increased the search of the most innovative options for the production of the so-called biofuels. Chlamydomonas reinhardtii is a photosynthetic unicellular algae with multiple biotechnological advantages such as easy handling in the laboratory, a simple scale-up to industrial levels, as well as a feasible genetic modification at nuclear and chloroplast levels. Besides, its fatty acids can be used to produce biofuels. Previous studies in plants have found that the over expression of DOF-type transcription factor genes increases the synthesis and the accumulation of total lipids in seeds. In this context, the over-expression of a DOF-type transcription factor in C.reinhardtii was applied as approach to increase the amount of lipids. The results indicate higher amounts (around 2-fold) of total lipids, which are mainly fatty acids, in the genetically C. reinhardtii modified strains when compared with the non-genetically modified strain. In order to elucidate the possible function of the introduced Dof-type transcription factor, we performed a transcription profile of 8 genes involved in fatty acid biosynthesis and 6 genes involved in glycerolipid biosynthesis, by quantitative real time (qRT-PCR). Differential expression profile was observed, which can explain the increase in lipid accumulation. However, these strains did not show notable changes in the fatty acid profile. This work represents an early effort in generating a strategy to increase fatty acids production in C.reinhardtii and their use in biofuel synthesis.
[Show abstract][Hide abstract] ABSTRACT: Genetically engineered plants can be used for the biomanufacture and delivery of oral vaccines. Although a myriad of antigens have been produced using this approach, improving our knowledge of their oral immunogenic properties is a priority as this aspect has not been well researched. Some studies have provided evidence of a higher immunogenic activity for antigens that were orally administered in the form of plant-based vaccines in comparison with conventional pure antigens. The characteristics of the plant-derived vaccines that may influence oral immunogenicity are identified and discussed in this review. Among the hypotheses explaining these immunogenic properties are the following: bioencapsulation favors antigen uptake and displays a resistance to degradation; plant metabolites exert adjuvant activity; plant compounds, such as polysaccharides, exert mucoadhesive properties; differential glycosylation conferred by the plant cell machinery enhances immunogenicity. Perspectives on how these hypotheses may be assessed are examined.
Expert Review of Vaccines 04/2014; · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plants are considered advantageous platforms for biomanufacturing recombinant vaccines. This constitutes a field of intensive research and some plant-derived vaccines are expected to be marketed in the near future. In particular, plant-based production of immunogens targeting molecules with implications on the pathology of Alzheimer's has been explored over the last decade. These efforts involve targeting amyloid beta and β-secretase with several immunogen configurations that have been evaluated in test animals. The results of these developments are analyzed in this review. Perspectives on the topic are identified, such as exploring additional antigen configurations and adjuvants in order to improve immunization schemes, characterizing in detail the elicited immune responses, and immunological considerations in the achievement of therapeutic humoral responses via mucosal immunization. Safety concerns related to these therapies will also be discussed.
Expert Review of Vaccines 01/2014; · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The moss Physcomitrella patens has a number of advantages for the production of biopharmaceuticals, including: i) availability of standardized conditions for cultivation in bioreactors; ii) not being part of the food chain; iii) high biosafety; iv) availability of highly efficient transformation methods; v) a haploid, fully sequenced genome providing genetic stability and uniform expression; vi) efficient gene targeting at the nuclear level allows for the generation of mutants with specific post-translational modifications (e.g., glycosylation patterns); and vii) oral formulations are a viable approach as no toxic effects are attributed to ingestion of this moss. In the light of this panorama, this opinion paper analyzes the possibilities of using P. patens for the production of oral vaccines and presents some specific cases where its use may represent significant progress in the field of plant-based vaccine development. The advantages represented by putative adjuvant effects of endogenous secondary metabolites and producing specific glycosylation patterns are highlighted.
Expert Review of Vaccines 01/2014; · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of a vaccine is still a priority in the fight against human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Since conventional vaccine strategies have failed to provide a highly immunoprotective effect, approaches based on the rational design of vaccines composed of multiple HIV neutralizing epitopes have been proposed as potential vaccines. The aim of this study is to design a multiepitopic protein (Multi-HIV) carrying several neutralizing epitopes from both gp120 and gp41 as an effort to develop a new broad immunization scheme against HIV. This Multi-HIV was initially produced in a recombinant Escherichia coli strain either as a single protein or fused to glutathione-S-transferase. These proteins were purified by immobilized metal ion affinity chromatography and shown to be antigenic by positive reactivity in Western blot analyses using sera from HIV-positive patients for labeling. Since global immunization strategies are often limited by costs, platforms that require minimal processing are the priority in this field. Therefore, we explored the possibility of using transplastomic tobacco plants as an experimental model of a low cost plant-based vaccine against HIV. Transplastomic tobacco plants carrying the multi-HIV gene were developed and verified by PCR analyses. The expected Multi-HIV recombinant protein was localized in the chloroplast as proven first by confocal microscopy and subsequently by Western blot analysis. Tobacco-derived Multi-HIV protein was clearly able to evoke humoral responses in mice when orally administered without adjuvants. This report constitutes an effort to explore a new low-cost candidate that could have future implications on the development of affordable HIV vaccines.
Plant Cell Tissue and Organ Culture 01/2014; · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In contrast to traditional pharmacological treatments for hypertension, immunotherapies serve as promising alternatives as they are low-cost and afford better patient compliance. In this study, a chimeric protein targeting Angiotensin II via genetic fusion to a nucleocapsid antigen from Hepatitis B virus (HBcAg), serving as a carrier, is designed. This candidate immunogen designated as HBcAgII has been expressed in the alga specie Chlamydomonas reinhardtii, serving as an attractive vaccine expression system and delivery host. This alga can be grown on minimal media under controlled environmental conditions, and can serve as a safe oral delivery vehicle. Transgenic C.
reinhardtii lines have been developed, and the expected recombinant protein has been detected by Western blot and ELISA analyses. Levels of expression of this recombinant protein in some transgenic lines have reached 0.05 % of total soluble protein. The immunogenic properties of the HBcAgII algae-derived antigen will be assessed.
Plant Cell Tissue and Organ Culture 01/2014; · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Besides serving as a valuable model in biological sciences, Chamydomonas reinhardtii has been used during the last decade in the biotechnology arena to establish models for the low cost production of vaccines. Antigens from various pathogens including Plasmodium falciparum, foot and mouth disease virus, Staphylococcus aureus, classical swine fever virus (CSFV) as well as some auto-antigens, have been produced in C. reinhardtii. Although some of them have been functionally characterized with promising results, this review identifies future directions for the advancement in the exploitation of this robust and safe vaccine production platform. The present analysis reflects that important immunological implications exist for this system and remain unexplored, including the possible adjuvant effects of algae biomolecules, the effect of bioencapsulation on immunogenicity and the possible development of whole-cell vaccines as an approach to trigger cytotoxic immune responses. Recently described molecular strategies that aim to optimize the expression of nuclear-encoded target antigens are also discussed.
Expert Review of Vaccines 09/2013; 12(9):1011-9. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.
[Show abstract][Hide abstract] ABSTRACT: Alternatives to pharmacological treatments for atherosclerosis are highly desirable in terms of cost and compliance. During the last two decades several vaccination strategies have been reported as an effort to develop immunotherapeutic treatments. This approach consists on eliciting immune responses able to modulate either the atherosclerosis-associated inflammatory processes or the activity of some physiological mechanisms that are up-regulated under this pathologic condition. In particular, the apolipoprotein B100 (ApoB100) and the cholesterilester transferase protein (CETP) have been targeted in these strategies. It is considered that recent progress in the development of experimental models of oral vaccines against atherosclerosis has opened a new avenue in the field: as plant-based vaccines are considered a viable platform for vaccine production and delivery at low costs, they could serve as an oral-delivered therapeutic approach for atherosclerosis in an economical and patient-friendly manner. The rationale of the design, development and evaluation of possible plant-based vaccines against atherosclerosis is discussed in this review. We identify within this approach a significant trend that will positively impact the field of atherosclerosis vaccination.
[Show abstract][Hide abstract] ABSTRACT: Neglected tropical diseases (NTDs) impair the lives of 1 billion people worldwide, and threaten the health of millions more. Although vaccine candidates have been proposed to prevent some NTDs, no vaccine is available at the market yet. Vaccines against NTDs should be low-cost and needle-free to reduce the logistic cost of their administration. Plant-based vaccines meet both requirements: plant systems allow antigen production at low cost, and also yield an optimal delivery vehicle that prevents or delays digestive hydrolysis of vaccine antigens. This review covers recent reports on the development of plant-based vaccines against NTDs. Efforts conducted by a number of research groups to develop vaccines as a mean to fight rabies, cysticercosis, dengue, and helminthiasis are emphasized. Future perspectives are identified, such as the need to develop vaccination models for more than ten pathologies through a plant-based biotechnological approach. Current limitations on the method are also noted, and molecular approaches that might allow us to address such limitations are discussed.
[Show abstract][Hide abstract] ABSTRACT: Vaccines against hypertension constitute a viable approach to decrease blood pressure. In particular, two vaccines against hypertension (HP) targeting angiotensin II (AgII) have showed promising results and these are currently on evaluation in clinical trials. In parallel, plant-based vaccines have become a biotechnological application that has been assessed in clinical trials for some cases. This report proposes a hypothesis that involves developing a plant-based vaccine against HP. It is hypothesized that a plant-based vaccine having AgII or its AT1 receptor (ATR1) as targets, constitutes a safe, suitable and efficient therapeutic approach for HP. It is known that a number of carrier proteins can be produced in plants retaining its adjuvanticity. Therefore the production in plants of chimeric proteins where either AgII or ATR1 domains are fused to these carriers would be a promising approach to be investigated. Mucosal immunization using plant-derived AgII/ATR1 chimeric proteins would imply several advantages such as low cost and friendly delivery. However due to the lack of a detailed knowledge on the physiological role of AgII at the gastrointestinal tract, the effects of partially blocking the AgII action must be extensively evaluated. An alternative related to this aspect would be the use of transient expression systems where productivity is sufficiently high to allow the purification of the antigen of interest at convenient yields, so that it can constitute a parenteral vaccine. Proving the concept for a plant-based vaccine against HP may have profound implications on the development of a new HP therapy which offers convenient features such as low cost and easier compliance in comparison to pharmacological treatment.
Medical Hypotheses 08/2012; 79(5):555-9. · 1.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bioactive peptides are considered high value-added ingredients in functional foods, and the main sources of these are milk, egg, plants, among others. A major limitation in their commercial use is the cost of production. This study deals with the design and production of a chimeric protein in chloroplasts of the microalgae Chlamydomonas reinhardtii to generate bioactive peptides of antihypertensive, opioid, antimicrobial, and hypocholesterolemic activities. A synthetic gene, designated as NCQ, coding for the selected chimeric protein, is transferred to C. reinhardtii using biolistic bombardment. Transplastomic transformants have been identified by PCR and Western blots following selection on a spectinomycin-containing medium. An ELISA quantification assay has revealed that the expressed NCQ protein accumulated at levels ranging between 0.16 and 2.4 % of total soluble protein. These findings demonstrate that chloroplasts of C. reinhardtii could serve as a robust expression platform for production of bioactive peptides.
Plant Cell Tissue and Organ Culture 05/2012; 113(2). · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a serious disease of swine and contributes to severe worldwide economic losses in swine production. Current vaccines against PRRS rely on the use of an attenuated-live virus; however, these are unreliable. Thus, alternative effective vaccines against PRRS are needed. Plant-based subunit vaccines offer viable, safe, and environmentally friendly alternatives to conventional vaccines. In this study, efforts have been undertaken to develop a soybean-based vaccine against PRRSV. A construct carrying a synthesized PRRSV-ORF7 antigen, nucleocapsid N protein of PRRSV, has been introduced into soybean, Glycine max (L.) Merrill. cvs. Jack and Kunitz, using Agrobacterium-mediated transformation. Transgenic plants carrying the sORF7 transgene have been successfully generated. Molecular analyses of T(0) plants confirmed integration of the transgene and transcription of the PRRSV-ORF7. Presence of a 15-kDa protein in seeds of T(1) transgenic lines was confirmed by Western blot analysis using PRRSV-ORF7 antisera. The amount of the antigenic protein accumulating in seeds of these transgenic lines was up to 0.65% of the total soluble protein (TSP). A significant induction of a specific immune response, both humoral and mucosal, against PRRSV-ORF7 was observed following intragastric immunization of BALB/c female mice with transgenic soybean seeds. These findings provide a 'proof of concept', and serve as a critical step in the development of a subunit plant-based vaccine against PRRS.
[Show abstract][Hide abstract] ABSTRACT: To date, a number of antihypertensive peptides (AHPs) have been identified. Most of these are derived from proteins present in common edible consumables, including milk, egg, and plant foods. Consumption of these foods serves as means of AHP delivery and thus contributing favorable health benefits. It is hypothesized that food crops, either over-expressing AHP precursor proteins or producing particular peptides as heterologous components, may serve as viable vehicles for production and delivery of functional foods as alternative hypertension therapies. In recent years, genetic engineering efforts have been undertaken to add value to functional foods. Pioneering approaches have been pursued in several crop plants, such as rice and soybean. In this review, a summary of current tools used for discovery of new AHPs, as well as strategies and perspectives of capitalizing on these AHPs in genetic engineering efforts will be presented and discussed. The implications of these efforts on the development of functional foods for preventing and treating hypertension are also presented.
Plant Cell Tissue and Organ Culture 02/2012; 112(2). · 2.61 Impact Factor