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

Abstract

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.

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

“…This, together with the reported optimisation of plant tissue conversion to lyophilisate and its long-term stability (Czyż et al 2014, 2016), makes it a successive essential step in manufacturing of a standardised oral vaccine against HBV of reliable efficacy. As lettuce becomes more and more attractive as a platform for expression of various heterologous proteins (Dong et al 2014), our results show that micropropagation can serve as a biofarming tool, enabling a considerable increase in the yield of production of biopharmaceuticals.…”

“…In the case of S-HBsAg, the expression yield reached around 60 or 70 µg/g FW in lettuce or maize, respectively, or 16–17 µg/g FW for M- and L-HBsAg in lettuce (Hayden et al 2012a; Pniewski et al 2011, 2012). Maize grains were treated with organic solvents to remove fats and the obtained pulp was pelleted or wafered (Hayden et al 2012b, 2014), while lettuce leaves were processed into lyophilisate (Czyż et al 2014, 2016). Lyophilised lettuce tissue was also used as a vehicle of an oral vaccine against tuberculosis and to induce tolerance in haemophilia therapy (Lakshmi et al 2013; Su et al 2015).…”

Plant‐made oral vaccines against human infectious diseases—Are we there yet?

“…Long‐term stability tests showed that the stored freeze‐dried product was stable at 4 °C for 1 year but degraded at room temperature. Animal oral immunization trials induced systemic IgG in mice (293 mIU/mL), confirming the preservation of antigenicity and immunogenicity (Czyż et al ., ).…”

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B

“…As with leafy vegetables, a variety of expression technologies have not yet been applied. In lettuce, the maximum expression level was 60 µg/g FW, which is the maximum anti-HBsAg antibody titer of 300 mIU in immunized mice serum [188,189,201]. Upon transformation into a soybean cell culture using a construct of HBsAg fused with the 3′ region from the soybean vegetative storage protein gene and as controlled by a chimeric ocs-mas promoter, the maximum expression level was 74 µg/g FW [174].…”

“…In the previous study, the storage stability of lyophilized powder form was limited at 4 °C [189]. In a recent study, successful long-term storage at 37 °C was achieved though improvements in the process [229].…”