The first report on the recombinant production of a candidate vaccine in the moss system. 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-epitope 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 µg g(-1) fresh weight in protonema cultures. Antigenic 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 immunogenic 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 glycoengineering approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.

Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.

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.

An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse. Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60 µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80 days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.

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