Immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic HIV protein

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.     

Engineering production of antihypertensive peptides in plants

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.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

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.     

LTB-Syn: a recombinant immunogen for the development of plant-made vaccines against synucleinopathies

Planta - A recombinant antigen targeting α-synuclein was produced in the plant cell rendering an immunogenic protein capable to induce humoral responses in mice upon oral administration....     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Current status and perspectives of plant-based candidate vaccines against the human immunodeficiency virus (HIV)

Genetically engineered plants are economical platforms for the large-scale production of recombinant proteins and have been used over the last 21 years as models for oral vaccines against a wide variety of human infectious and autoimmune diseases with promising results. The main inherent advantages of this approach consist in the absence of purification needs and easy production and administration. One relevant infectious agent is the human immunodeficiency virus (HIV), since AIDS evolved as an alarming public health problem implicating very high costs for government agencies in most African and developing countries. The design of an effective and inexpensive vaccine able to limit viral spread and neutralizing the viral entry is urgently needed. Due to the limited efficacy of the vaccines assessed in clinical trials, new HIV vaccines able to generate broad immune profiles are a priority in the field. This review discusses the current advances on the topic of using plants as alternative expression systems to produce functional vaccine components against HIV, including antigens from Env, Gag and early proteins such as Tat and Nef. Ongoing projects of our group based on the expression of chimeric proteins comprising C4 and V3 domains from gp120, as an approach to elicit broadly neutralizing antibodies are mentioned. The perspectives of the revised approaches, such as the great need of assessing the oral immunogenicity and a detailed immunological characterization of the elicited immune responses, are also discussed.     

Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies

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.     

Production of an antigenic C4(V3)6 multiepitopic HIV protein in bacterial and plant systems

A C4(V3)6 multiepitopic protein was designed in an effort to pursue broad immunization against the human immunodeficiency virus (HIV). This C4(V3)6 chimeric protein is based on sequences of gp120, including epitopes from the fourth conserved domain (C4) and six tandem repeats of the third variable domain (V3), which represent different HIV isolates. The histidine-tagged C4(V3)6 was subsequently over-expressed in a recombinant Escherichia coli strain, and purified by immobilized metal ion affinity chromatography. Expression of the C4(V3)6 in both tobacco and lettuce plants was also achieved with no toxic effects on plant growth as transgenic plants were phenotypically normal. Moreover, the functional C4(V3)6 protein showed HIV antigenic determinants. The implications of these findings on the development of a new low-cost HIV vaccine are discussed.     

Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine

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.