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

Czyż M, Dembczyński R, Marecik R, Wojas-Turek J, Milczarek M, Pajtasz-Piasecka E, Wietrzyk J, Pniewski T - Biomed Res Int (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland.

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.

No MeSH data available.


Related in: MedlinePlus

Anti-HBs antibody response in mouse serum after oral boosting with S-HBsAg in powdered lyophilised tissue. Material for immunisation was freeze-dried under the 20°C/22 h–22°C/2 h profile with 500 mM sucrose as protective excipient. Symbols: ○ individual mouse response, – group mean value. Letter indexes mark statistically homogenous groups.
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fig6: Anti-HBs antibody response in mouse serum after oral boosting with S-HBsAg in powdered lyophilised tissue. Material for immunisation was freeze-dried under the 20°C/22 h–22°C/2 h profile with 500 mM sucrose as protective excipient. Symbols: ○ individual mouse response, – group mean value. Letter indexes mark statistically homogenous groups.

Mentions: To confirm the retained immunogenicity of S-HBsAg in freeze-dried plant tissue, animal immunisation trials were performed. Mice after i.m. priming with the Engerix B vaccine received an oral booster with powdered lyophilised tissue (see Section 2) based on the low-dose protocol reported previously [4]. Used tissue came from freeze-drying cycle number IV with 109% preservation of S-HBsAg VLPs, corresponding to 29 μg/g DW and 199% or 538 μg/g DW for the total antigen. Hence, the dose of 50 ng S-HBsAg was delivered in 1.72 mg of freeze-dried tissue per individual subject. Results of mouse vaccination are summarised in Figure 6. Titre of anti-HBs antibodies in mice boosted orally with lyophilised powder reached a mean value of 293 mIU/mL, while in the group boosted by injection with Engerix B they reached 397 mIU/mL. Although two of the orally boosted mice were not able to respond to preparation, the response pattern was similar to that observed for reference group boosted with Engerix B. When mice were orally delivered with the control tissue, no boosting effect was observed. Even though the used freeze-dried preparation exhibited some build-up of total S-HBsAg, apparently it did not hinder immune response development. This result might be obtained due to the considerably lower absolute level of total antigen in lyophilised tissue than that used previously [4], yet an exclusively oral immunisation pattern was then applied. The observed response, was lower but comparable to other reports on injection-oral vaccination trials. However, in those experiments the administered plant-associated S-HBsAg was CTB- or LTB-adjuvanted [52, 53]. Presented in our study induction of systemic immune response, confirmed that selected parameters of plant material processing ensure successful preservation of S-HBsAg antigenicity and immunogenicity.


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

Czyż M, Dembczyński R, Marecik R, Wojas-Turek J, Milczarek M, Pajtasz-Piasecka E, Wietrzyk J, Pniewski T - Biomed Res Int (2014)

Anti-HBs antibody response in mouse serum after oral boosting with S-HBsAg in powdered lyophilised tissue. Material for immunisation was freeze-dried under the 20°C/22 h–22°C/2 h profile with 500 mM sucrose as protective excipient. Symbols: ○ individual mouse response, – group mean value. Letter indexes mark statistically homogenous groups.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4209752&req=5

fig6: Anti-HBs antibody response in mouse serum after oral boosting with S-HBsAg in powdered lyophilised tissue. Material for immunisation was freeze-dried under the 20°C/22 h–22°C/2 h profile with 500 mM sucrose as protective excipient. Symbols: ○ individual mouse response, – group mean value. Letter indexes mark statistically homogenous groups.
Mentions: To confirm the retained immunogenicity of S-HBsAg in freeze-dried plant tissue, animal immunisation trials were performed. Mice after i.m. priming with the Engerix B vaccine received an oral booster with powdered lyophilised tissue (see Section 2) based on the low-dose protocol reported previously [4]. Used tissue came from freeze-drying cycle number IV with 109% preservation of S-HBsAg VLPs, corresponding to 29 μg/g DW and 199% or 538 μg/g DW for the total antigen. Hence, the dose of 50 ng S-HBsAg was delivered in 1.72 mg of freeze-dried tissue per individual subject. Results of mouse vaccination are summarised in Figure 6. Titre of anti-HBs antibodies in mice boosted orally with lyophilised powder reached a mean value of 293 mIU/mL, while in the group boosted by injection with Engerix B they reached 397 mIU/mL. Although two of the orally boosted mice were not able to respond to preparation, the response pattern was similar to that observed for reference group boosted with Engerix B. When mice were orally delivered with the control tissue, no boosting effect was observed. Even though the used freeze-dried preparation exhibited some build-up of total S-HBsAg, apparently it did not hinder immune response development. This result might be obtained due to the considerably lower absolute level of total antigen in lyophilised tissue than that used previously [4], yet an exclusively oral immunisation pattern was then applied. The observed response, was lower but comparable to other reports on injection-oral vaccination trials. However, in those experiments the administered plant-associated S-HBsAg was CTB- or LTB-adjuvanted [52, 53]. Presented in our study induction of systemic immune response, confirmed that selected parameters of plant material processing ensure successful preservation of S-HBsAg antigenicity and immunogenicity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland.

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.

No MeSH data available.


Related in: MedlinePlus