The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos.

Abiotic stresses occur more often in combination than alone under regular field conditions limiting in more severe way crop production. Stress recognition in plants primarily occurs in the plasma membrane, modification of which is necessary to maintain homeostasis in response to it. It is known that lipid transport proteins (ns-LTPs) participate in modification of the lipidome of cell membranes. Representative of this group, ns-LTP2.8, may be involved in the reaction to abiotic stress of germinating barley plants by mediating the intracellular transport of hydrophobic particles, such as lipids, helping to maintain homeostasis. The ns-LTP2.8 protein was selected for analysis due to its ability to transport not only linear hydrophobic molecules but also compounds with a more complex spatial structure. Moreover, ns-LTP2.8 has been qualified as a member of pathogenesis-related proteins, which makes it particularly important in relation to its high allergenic potential. This paper demonstrates for the first time the influence of various abiotic stresses acting separately as well as in their combinations on the change in the ns-LTP2.8 transcript, ns-LTP2.8 protein and total soluble protein content in the embryonal axes of germinating spring barley genotypes with different ns-LTP2.8 allelic forms and stress tolerance. Tissue localization of ns-LTP2.8 transcript as well as ns-LTP2.8 protein were also examined. Although the impact of abiotic stresses on the regulation of gene transcription and translation processes remains not fully recognized, in this work we managed to demonstrate different impact on applied stresses on the fundamental cellular processes in very little studied tissue of the embryonal axis of barley.

Plant-Based Vaccines in Combat against Coronavirus Diseases.

Coronavirus (CoV) diseases, including Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) have gained in importance worldwide, especially with the current COVID-19 pandemic caused by SARS-CoV-2. Due to the huge global demand, various types of vaccines have been developed, such as more traditional attenuated or inactivated viruses, subunit and VLP-based vaccines, as well as novel DNA and RNA vaccines. Nonetheless, emerging new COVID-19 variants are necessitating continuous research on vaccines, including these produced in plants, either via stable expression in transgenic or transplastomic plants or transient expression using viral vectors or agroinfection. Plant systems provide low cost, high scalability, safety and capacity to produce multimeric or glycosylated proteins. To date, from among CoVs antigens, spike and capsid proteins have been produced in plants, mostly using transient expression systems, at the additional advantage of rapid production. Immunogenicity of plant-produced CoVs proteins was positively evaluated after injection of purified antigens. However, this review indicates that plant-produced CoVs proteins or their carrier-fused immunodominant epitopes can be potentially applied also as mucosal vaccines, either after purification to be administered to particular membranes (nasal, bronchus mucosa) associated with the respiratory system, or as oral vaccines obtained from partly processed plant tissue.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II.

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.

Characterization of Chemically Activated Carbons Prepared from Miscanthus and Switchgrass Biomass.

Lignocellulosic biomass, including that of energy crops, can be an alternative source to produce activated carbons (ACs). Miscanthus and switchgrass straw were used to produce ACs in a two-step process. Crushed plant material was carbonized at 600 °C and then obtained carbon was activated using NaOH or KOH at 750 °C. The content of surface oxygen groups was determined using Boehm's method. The porosity of ACs was assayed using the nitrogen adsorption/desorption technique, while their thermal resistance using the thermogravimetric method. The ACs derived from miscanthus and switchgrass were characterized by surfaces rich in chemical groups and a highly developed porous structure. The highest specific surface areas, over 1600 m2/g, were obtained after carbon treatment with NaOH. High values of iodine number, 1200-1240 mg/g, indicate an extensive system of micropores and their good adsorption properties. The type of activator affected the contents of oxygen functional groups and some porosity parameters as well as thermal stability ranges of the ACs. Among obtained carbons, the highest quality was found for these derived from M. sacchariflorus followed by switchgrass, after activation with NaOH. Hence, while these crop species are not as effective biomass sources as other energy grasses, they can become valuable feedstocks for ACs.

Parenteral-Oral Immunization with Plant-Derived HBcAg as a Potential Therapeutic Vaccine against Chronic Hepatitis B.

Chronic hepatitis B (CHB) is the cause of severe liver damage, cirrhosis, and hepatocellular carcinoma for over 240 million people worldwide. Nowadays, several types of treatment are being investigated, including immunotherapy using hepatitis B core antigen (HBcAg) assembled into highly immunogenic capsid-like particles (CLPs). Immunogenicity of plant-produced and purified HBcAg, administered parenterally or intranasally, was previously reported. In this study, a novel parenteral-oral vaccination scheme is proposed using plant-derived HBcAg preparations. The antigen for injection was obtained via transient expression in Nicotiana benthamiana. HBcAg-producing transgenic lettuce was lyophilized and used as an orally delivered booster. The intracellular location of plant-produced HBcAg CLPs implies additional protection in the digestive tract during oral immunization. BALB/c mice were intramuscularly primed with 10 µg of the purified antigen and orally boosted twice with 5 or 200 ng of HBcAg. A long-lasting and significant systemic response after boosting with 200 ng HBcAg was induced, with anti-HBc titer of 25,000. Concomitantly, an insignificant mucosal response was observed, with an S-IgA titer of only 500. The profile of IgG isotypes indicates a predominant Th1 type of immune response, supplemented by Th2, after injection-oral vaccination. The results demonstrate that a low dose of parenteral-oral immunization with plant-derived HBcAg can elicit a specific and efficient response. This study presents a potential new pathway of CHB treatment.

Assembly and Characterization of HBc Derived Virus-like Particles with Magnetic Core.

Core-virus like particles (VLPs) assembly is a kinetically complex cascade of interactions between viral proteins, nanoparticle's surface and an ionic environment. Despite many in silico simulations regarding this process, there is still a lack of experimental data. The main goal of this study was to investigate the capsid protein of hepatitis B virus (HBc) assembly into virus-like particles with superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic core in relation to their characteristics. The native form of HBc was obtained via agroinfection of Nicotiana benthamiana with pEAQ-HBc plasmid. SPIONs of diameter of 15 nm were synthesized and functionalized with two ligands, providing variety in ζ-potential and hydrodynamic diameter. The antigenic potential of the assembled core-VLPs was assessed with enzyme-linked immunosorbent assay (ELISA). Morphology of SPIONs and core-VLPs was evaluated via transmission electron microscopy (TEM). The most successful core-VLPs assembly was obtained for SPIONs functionalized with dihexadecyl phosphate (DHP) at SPIONs/HBc ratio of 0.2/0.05 mg/mL. ELISA results indicate significant decrease of antigenicity concomitant with core-VLPs assembly. In summary, this study provides an experimental assessment of the crucial parameters guiding SPION-HBc VLPs assembly and evaluates the antigenicity of the obtained structures.

Plant lyophilisate carrying S-HBsAg as an oral booster vaccine against HBV.

A formulation of a defined antigen dosage together with an appropriate and convenient immunisation regime are the main problems of a plant-derived oral vaccine against HBV. Both factors have to be mutually adjusted to ensure efficacious vaccination and to minimise the risk of oral tolerance acquisition. As based on previous research, solely oral immunisation appears to be of limited effectiveness, but a combined immunisation scheme via injection priming and oral boosting can be proposed. Thus, the previously optimised plant lyophilisate with accumulated S-HBsAg was orally administered in a series of mouse immunisation trials. The impact of various S-HBsAg oral dosages and concentrations on the induction of an active immune response was studied. Immunisation via i.m. priming followed by double oral boosting in 6-week intervals was comparably efficient as standard i.m. vaccination. Among the tested dosages, 2× 5 or 200 ng triggered effective and exclusively systemic responses. Mucosal adjuvants CTB or quillaja bark saponins as well as alhydrogel either had no or a negative effect on immune response, which indicates that a low-dosed plant lyophilisate as an oral booster vaccine may not require exogenous adjuvants. Additionally, a positive effect of S-HBsAg encapsulation in lyophilised cells on immune response stimulation was confirmed by comparison with the antigen released in the plant extract. The low-dosed plant lyophilisate with concentrated S-HBsAg was proven as an effective and convenient oral booster vaccine. Results of immunisation via a mixed injection-oral route provide the foundation for research on further standardisation of a plant-derived oral vaccine against HBV and details of immune response together with effects for health condition.

HBcAg produced in transgenic tobacco triggers Th1 and Th2 response when intramuscularly delivered.

Hepatitis B core Antigen (HBcAg) assembled into Capsid-Like Particles (CLPs) is investigated as a therapeutic vaccine in treatment of chronic hepatitis B (CHB) and in diagnostic tests or as a carrier for various epitopes. While the expression of HBcAg has been thoroughly clarified in E. coli and yeast, it has also been investigated in other expression systems. Stably transformed tobacco expressed HBcAg at a level of 110-250µg/g fresh weight, therefore in view of its large leaf biomass it offers a production platform comparable with transient expression systems regarding the final yield of HBcAg. Several extraction and purification methods were tested and finally the antigen was purified up to 43% using sucrose density gradient centrifugation. The purified HBcAg retained its antigenicity, as confirmed by ELISA and western blot, while maintaining its CLP-structure as observed in TEM. In mice HBcAg intramuscularly delivered at 2×10µg triggered a significant response (serum anti-HBc titre around 150,000), being statistically equivalent to that induced by the reference antigen. Among anti-HBc IgG isotypes, IgG2a and then IgG1 were increasing during immune response. However IgG2b and IgG3 were also induced, especially in mice immunised with the plant-derived antigen. Analysis of the isotype profile indicates mainly Th1 polarisation, but completed with Th2 response. Obtained results indicate a considerable potential of plant-derived HBcAg as a therapeutic vaccine, since a mixed immune response with a stronger Th1 component is particularly required for treatment of CHB.

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants.

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.

Micropropagation of transgenic lettuce containing HBsAg as a method of mass-scale production of standardised plant material for biofarming purposes.

KEY MESSAGE: Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. Effective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5-5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40-85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

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

KEY MESSAGE: Intramuscularly 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. HBV 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.

Stability of S-HBsAg in long-term stored lyophilised plant tissue.

A potent plant-derived oral vaccine against Hepatitis B Virus (HBV) requires a durable and compact form for efficacious and convenient distribution and delivery. In the previous study we have devised a successful freeze-drying process of plant material containing the HBV small surface antigen (S-HBsAg) for the purpose of an oral vaccine against the virus, but product storage stability was limited to 4 °C. The aim of this study was to upgrade a freeze-dried product formula to facilitate successful long-term storage of S-HBsAg assembled into Virus-Like Particles (VLPs) at elevated temperatures. Series of additional excipients and storage conditions were tested. Atmosphere of nitrogen proved to preserve S-HBsAg VLPs most efficiently, with only minor degradation at the highest temperature of 37 °C. As a result, a semi-product for the oral plant-derived vaccine against HBV with good storage capabilities was obtained.

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.

The twenty-year story of a plant-based vaccine against hepatitis B: stagnation or promising prospects?

Hepatitis B persists as a common human disease despite effective vaccines having been employed for almost 30 years. Plants were considered as alternative sources of vaccines, to be mainly orally administered. Despite 20-year attempts, no real anti-HBV plant-based vaccine has been developed. Immunization trials, based on ingestion of raw plant tissue and conjugated with injection or exclusively oral administration of lyophilized tissue, were either impractical or insufficient due to oral tolerance acquisition. Plant-produced purified HBV antigens were highly immunogenic when injected, but their yields were initially insufficient for practical purposes. However, knowledge and technology have progressed, hence new plant-derived anti-HBV vaccines can be proposed today. All HBV antigens can be efficiently produced in stable or transient expression systems. Processing of injection vaccines has been developed and needs only to be successfully completed. Purified antigens can be used for injection in an equivalent manner to the present commercial vaccines. Although oral vaccines require improvement, plant tissue, lyophilized or extracted and converted into tablets, etc., may serve as a boosting vaccine. Preliminary data indicate also that both vaccines can be combined in an effective parenteral-oral immunization procedure. A partial substitution of injection vaccines with oral formulations still offers good prospects for economically viable and efficacious anti-HBV plant-based vaccines.

Is an oral plant-based vaccine against hepatitis B virus possible?

Prevention of hepatitis B, one of the most prevalent human diseases, still requires cheap and commonly available vaccines. Oral vaccines, including plant-based formulations, have been considered as alternatives or supplements for standard injection vaccines, due to the assumed low-cost production and simplified vaccination. Although plant production of HBV antigens is sufficiently efficient, despite almost 20 years of research still no anti-HBV plant-based vaccine has been developed. The basic difficulty has been to elaborate an effective immunisation procedure. Immunisation by parenteral priming and oral boosting with raw plant tissue adjuvanted with the cholera toxin, although effective, seemed to be unfeasible and controversial. Exclusively oral immunisation using lyophilised tissue, despite its appropriate form, appeared also impractical because of too low efficiency. Oral tolerance turned out to be the main barrier for anti-HBV plant-based vaccines. Based on previous results and knowledge on the mucosal immune system, a possible vaccine may consist of two components, parenteral for priming and oral for boosting. Probably the oral constituent could independently serve for further booster vaccinations. Both vaccine components can be produced in plants and used after some processing - purification for the injection constituent and lyophilisation for the oral one. Lyophilised tissue can be converted into tablets, capsules, etc. Previous and recent data show that the injection-oral immunisation regime may be efficient. A combination of parenteral and oral vaccination offers good prospects for a truly efficacious plant-derived anti-HBs vaccine and even a partial substitution of parenteral vaccines by an oral formula may prove to be economically reasonable.

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.

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.

Nanogram doses of alum-adjuvanted HBs antigen induce humoral immune response in mice when orally administered.

Mucosal immunity elicited by plant-based and other orally administered vaccines can serve as the first line of defense against most pathogens infecting through mucosal surfaces, but it is also considered for systemic immunity against blood-borne diseases such as hepatitis B (HB). Previous oral immunization trials based on multiple administration of high doses of HBs antigen elicited an immune response; however, a reproducible and long-lasting immunization protocol was difficult to design. The objective of this study was to evaluate the effect of dose and timing of orally delivered alum-adsorbed antigen on the magnitude of the anti-HBs humoral response. Mice were immunized orally by gavage intubation or parenterally by intramuscular injection three times, once every 2 weeks, with doses of 5, 50, or 500 ng alum-adjuvanted HBsAg. A low dose (10 ng) of HBsAg was orally administered three times in different time intervals: 2, 4, 6, and 8 weeks. The three consecutive 5-ng oral doses of the antigen induced immune response at the protective level (>or=10 mIU/ml), significantly higher than the reaction elicited by three 50 or 500 ng doses. In contrast, intramuscular delivery of these doses did not differ significantly; however, they induced a five to six times higher immune response than oral immunization. The 8-week period between each of the three oral immunizations appeared to be favorable to the anti-HBs humoral responses compared with the shorter schedules. The results presented here clearly identify the importance of low doses of antigen administered orally in extended intervals for a significantly higher anti-HBs response. This finding provides some indications concerning the strategy of orally administered vaccines, including plant-based ones.

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.