Tomasz Pniewski

A plant-derived edible vaccine against hepatitis B virus

The infectious hepatitis B virus represents 42 nm spherical double‐shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus‐like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus‐specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum‐IgG response to plant produced protein.—Kapusta, J., Modelska, A., Figlerowicz, M., Pniewski, T., Letellier, M., Lisowa, O., Yusibov, V., Koprowski, H., Plucienniczak, A., Legocki, A. B. A plant‐derived edible vaccine against hepatitis B virus. FASEB J. 13, 1796–1799 (1999)

Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

Oral immunization of human with transgenic lettuce expressing hepatitis B surface antigen.

A number of reports for the past few years have shown that higher plants can be important and useful tools for production and delivery of viral antigens. Oral route represents one of the most efficient and safe ways for administration of therapeutic proteins [1]. Among protein antigens expressed in transgenic plants is hepatitis B surface antigen as reported by several laboratories.

Oral administration of low doses of plant-based HBsAg induced antigen-specific IgAs and IgGs in mice, without increasing levels of regulatory T cells

Plant-based oral vaccines run the risk of activating regulatory T cells (Tregs) and suppressing the antigen-specific immune response via oral tolerance. Mice humanized for two HLA alleles (HLA-A2.1 and HLA-DR1) were used to measure changes in Tregs and antigen-specific immune responses induced by the oral administration of tobacco (Nicotiana tabacum), expressing the hepatitis B surface antigen (HBsAg). Antigen-specific CD8+ T cell activation was not detected, but the plant-based oral immunization, without adjuvant, resulted in humoral responses comparable to those obtained by adjuvanted DNA immunization. Treg titers did not increase with DNA immunization. In contrast, with plant immunization, Tregs increased linearly to reach a plateau at high antigen doses. The highest humoral IgA and IgG responses correlated with the lowest plant antigen dose (0.5 ng), while for DNA immunization the best antibody responses were obtained at higher antigen doses. These experiments suggest that plant-based oral vaccines could be adjusted to minimize tolerance, while still inducing an immune response. Oral tolerance and adjuvant engineering in plants are discussed.

Plant expression, lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation.

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B.

Freeze-Drying of Plant Tissue Containing HBV Surface Antigen for the Oral Vaccine against Hepatitis B

The aim of this study was to develop a freeze-drying protocol facilitating successful processing of plant material containing the small surface antigen of hepatitis B virus (S-HBsAg) while preserving its VLP structure and immunogenicity. Freeze-drying of the antigen in lettuce leaf tissue, without any isolation or purification step, was investigated. Each process step was consecutively evaluated and the best parameters were applied. Several drying profiles and excipients were tested. The profile of 20°C for 20 h for primary and 22°C for 2 h for secondary drying as well as sucrose expressed efficient stabilisation of S-HBsAg during freeze-drying. Freezing rate and postprocess residual moisture were also analysed as important factors affecting S-HBsAg preservation. The process was reproducible and provided a product with VLP content up to 200 µg/g DW. Assays for VLPs and total antigen together with animal immunisation trials confirmed preservation of antigenicity and immunogenicity of S-HBsAg in freeze-dried powder. Long-term stability tests revealed that the stored freeze-dried product was stable at 4°C for one year, but degraded at elevated temperatures. As a result, a basis for an efficient freeze-drying process has been established and a suitable semiproduct for oral plant-derived vaccine against HBV was obtained.

Agrobacterium-mediated transformation of yellow lupin to generate callus tissue producing HBV surface antigen in a long-term culture

The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using thenptII marker gene and theuidA reporter gene as well as variousAgrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 μg per g of fresh tissue.

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by usingAgrobacterium tumefaciens

Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.

HIV-1 derived peptides fused to HBsAg affect its immunogenicity

The hepatitis B virus (HBV) surface small antigen (HBsAg) self-assembles into virus-like particles (VLPs). HBsAg-based VLPs constitute a powerful vector for heterologous immunogenic peptides to develop a safe vaccine delivery system. HBV and the human immunodeficiency virus type 1 (HIV-1) are frequently associated in infection. An HIV-1 class I polyepitope was designed for an HIV-1/HBV vaccine prototype based on HBsAg VLPs. Invariable peptides from the original HIV-1 polyepitope were here permutated to study the influence of epitope order on HIV-1/HBV VLP immunogenicity. Anti-HIV-1 cellular responses were statistically comparable among polyepitope variants. Nevertheless, delivered HIV-1 polyepitopes impacted anti-HBsAg carrier immunogenicity in a polyepitope-specific manner. For a given set of epitopes, the choice of epitope order in polyepitopes is strategic to control immune responses towards HBsAg VLPs used as carrier of foreign immunogenic peptides.

Immunogenicity of parenterally delivered plant-derived small and medium surface antigens of hepatitis B virus

Key messageIntramuscularly delivered plant-derived M-HBsAg was compared to S-HBsAg, and as a result elicited specific anti-preS2 antibodies and significantly higher titre of anti-HBs antibodies, together with IgG isotype profile indicating some Th1 polarisation, apart from the main Th2 response.AbstractHBV prevalence is still threatening, regardless of prevention programmes using vaccines containing S-HBsAg, supplemented by third-generation vaccines, comprising also M- and L-HBsAg. Plant expression systems offer a cost-effective production option of the antigens. Plant-derived S- and M-HBsAg, intramuscularly delivered to mice, elicited anti-HBs antibodies several times higher than high responsiveness threshold titre. M-HBsAg induced stronger response of anti-HBs and also specific anti-preS2 antibodies. IgG isotype profiles indicated mainly Th2 response, yet Th1 polarisation was also pointed out, in some larger extent for M-HBsAg. These results correspond to research on CHO-derived M-HBsAg vs. commercial vaccines based on S-HBsAg and support potency of plant-derived antigens as alternative injection vaccines.