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.
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland.
Marcin
0000-0002-5182-8022
M
Czyż
Poznań University of Life Sciences, Wojska Polskiego 28, 60-995 Poznań, Poland.
Radosław
0000-0001-7575-995X
R
Dembczyński
Poznań University of Life Sciences, Wojska Polskiego 28, 60-995 Poznań, Poland.
Roman
0000-0001-5387-990X
R
Marecik
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland.
Justyna
0000-0002-1349-3890
J
Wojas-Turek
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland.
Magdalena
M
Milczarek
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland.
Elżbieta
E
Pajtasz-Piasecka
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland.
Joanna
J
Wietrzyk
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland.
Tomasz
0000-0002-5754-483X
T
Pniewski
D000284:Administration, Oral; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D015153:Blotting, Western; D005612:Freeze Drying; D006509:Hepatitis B / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000008:administration & dosage* / Q000276:immunology*; D018545:Lactuca / Q000235:genetics / Q000502:physiology*; D008807:Mice, Inbred BALB C; D030821:Plants, Genetically Modified; D015203:Reproducibility of Results; D013395:Sucrose / Q000032:analysis; D013696:Temperature; D014771:Virion / Q000378:metabolism
D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D013395:Sucrose