Biomed Res Int. 2014;2014:485689. doi: 10.1155/2014/485689.

Freeze-drying of plant tissue containing HBV surface antigen for the oral vaccine against hepatitis B.

Czyż M1, Dembczyński R2, Marecik R2, Wojas-Turek J3, Milczarek M3, Pajtasz-Piasecka E3, Wietrzyk J3, Pniewski T1.
  • 1Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland.
  • 2Poznań University of Life Sciences, Wojska Polskiego 28, 60-995 Poznań, Poland.
  • 3Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland.



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.

PMID: 25371900



Although vaccines against hepatitis B are available for about 30 years, still approx. 350 – 400 million people worldwide are chronically infected and 1/3 of human population were HBV (Hepatitis B Virus) infected. Necessary mass prophylaxis could be provided by inexpensive and convenient in application oral vaccines of plant origin. A potent plant-derived anti-HBV oral vaccine requires a durable and compact form for efficacious and simple distribution and delivery. In the previous studies we have applied freeze-drying (lyophilisation) process of plant material containing the HBV small surface antigen (S-HBsAg). We managed to obtain a semi-product inducing an immune response and enabling tablet preparation, yet the antigen content was low [1].

Preservation of native complex structure of immunogenic proteins, especially formed in structures resembling viruses (i.e. Virus-Like Particles – VLPs) as S-HBsAg, is the main goal during developing vaccine formula. Loss of this characteristic quality poses significant issue when implementation of freeze-drying technique is needed to allow a vaccine distributed independently from cold chain. Precise mechanisms of protein preservation in the solid state have still not been completely understood, therefore development of a pharmaceutical formulation based on freeze-dried proteins still remains empirical efforts to some extent. In order to achieve a product, series of formulations and storage conditions usually must be tested to select the most potent variant.

During previous study we have optimised preparation of S-HBsAg incorporated in plant tissue, based on freeze-drying process [2]. We were able to establish drying conditions and addition of sucrose as an excipient that proved successful in inhibiting lyophilisation-induced degradation of S-HBsAg native VLPs structure and immunogenicity, as confirmed in animal trials. The scope of our next work included similar tests on M- and L-HBsAg, as components of potential multiantigenic vaccines. However, good preservation was obtained only for M-HBsAg, while L-HBsAg was generally unstable, whether with or without excipients.

Nevertheless storage stability of all antigens was tested. Decisive parameters for the selection of the most effectively lyophilised tissue formula and storage conditions were efficient retention of VLPs together with a minimal change in the total antigen pool. The latter is intrinsically associated with release of S-HBsAg dimers from VLPs and/or their denaturation, as described earlier [2]. Small antigen turn out to be stable in presence of sucrose for initial three month period, but only at 4°C. Remaining antigens, M- and L-HBsAg, expressed very erratic behaviour during storage – significant raise in VLPs content was often noted. This most probably represented severe disintegration of particulated structure of antigens resulting in formation of defect subparticles or aggregates of dimers. Used excipients showed only minor protective effect on analysed antigens, yet gave indications for further research. Combined results pointed toward focusing more intense work on the S-HBsAg, because it is the basic component of anti-HBV vaccines. We concentrated our work on improving its stability at elevated temperatures for extended, one-year storage.


Figure. Lyophilised plant tissue powder stored in sealed vial under nitrogen atmosphere (left) and stability of contained S-HBsAg – VLP-formed and total pool during one-year storage (right).


As the observed stability of S-HBsAg was lost with increasing storage temperature, we focused on countering destabilisation factors that are promoted by higher temperature, as molecular mobility, water and oxidative activities and consequently protein aggregation. In order to select and prevent most significant S-HBsAg degradation pathways we investigated several agents and their combinations: i/ improving mechanical properties of the ‘cake’ of lyophilised tissue – glycine; ii/ potentially stabilising protein structure – zinc ions; iii/ reducing residual moisture – silica gel beads; iv/ and antioxidants – sodium sulphite, ascorbic acid and nitrogen. After extensive tests, only storage under neutral atmosphere of nitrogen enabled for VLP-assembled S-HBsAg to remain significantly stable at all analysed temperatures. Only small (14%) decrease in VLPs content was observed at 37ºC, but this was triggered most likely by oxygen traces, penetrated the material before the vial silage under nitrogen and then accelerated by higher temperature. However, this degradation was soon – before the 3rd month – arrested. For the total antigen, only moderate instability was noted. This pool remained relatively stable when stored in cold, but at 22°C and 37°C its level dropped (to approx. 80% and 60%, respectively) in the second storage interval. However, it should be stressed that ‘free’ antigen forms are intrinsically more prone to degradation. Yet distinctly, those were preserved more effectively than in other variants (see above) and previous results [1, 2]. Moreover, the achieved stability of S-HBsAg stored at cold or room temperatures was comparable, while at 37°C several times more effective, than formulations based on the purified antigen [3].

This results pointed toward conclusion that neutralisation of oxidative-reductive activities during storage is crucial for stability of S-HBsAg, both VLPs and ‘free’ molecules. This may also suggest that storage stability of other HBV antigens – M- and L-HBsAg, may be significantly increased, when neutral atmosphere would be applied during their storage. Although this needs to be investigated during further tests, yet in the future it may allow to create a complex multiantigenic vaccine formulation administered orally.

The research results showed significant practical as well as cognitive value, expanding knowledge about the factors determining the preservation of immunogenicity of HBV antigens during the processing of plant material to a lyophilised form of the vaccine preparation. Thus, they can greatly contribute to the work on obtaining an anti-HBV plant-derived oral vaccine as well as the basis and starting point for preclinical studies, clinical trials and implementation process of a new generation vaccine against hepatitis B.



  1. Pniewski T, Kapusta J, Bociag P, Wojciechowicz J, Kostrzak A, Gdula M, Fedorowicz-Strońska O, Wójcik P, Otta H, Samardakiewicz S, Wolko B, Płucienniczak A. Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation. Journal of Applied Genetics, 2011, 52: 125-136.
  2. Czyż M, Dembczyński R, Marecik R, Wojas-Turek J, Milczarek M, Pajtasz-Piasecka E, Wietrzyk J, Pniewski T. Freeze-drying of plant tissue containing HBV surface antigen for the oral vaccine against hepatitis B. BioMed Research International, 2014, 485689.
  3. Diminsky D, Moav N, Gorecki M, Barenholz Y. Physical, chemical and immunological stability of CHO-derived hepatitis B surface antigen (HBsAg) particles. Vaccine, 1999, 18: 3 – 17.



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