Factors Important in the Extraction, Stability and in Vitro Assembly of the Hepatitis B Surface Antigen Derived from Recombinant Plant Systems

Abstract

The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally processed oral vaccine, may prove valuable in increasing the immunogenicity of the plant-derived antigen.

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control

“…As well, fruits, tubers, and taproots can be stored for a limited time, especially in a chilled environment. Vaccine antigens have also been expressed in undifferentiated plant cell cultures such as, cell suspensions (Smith et al 2002), somatic embryos , and callus cultures (Kapusta et al 1999). High overall protein content in the target plant tissue is beneficial for high-level accumulation of recombinant proteins.…”

“…The most commonly used promoter to control antigen expression in dicotyledonous plant species is the strong and constitutive cauliflower mosaic virus 35S promoter (CaMV 35S). A version of CaMV 35S with a duplicated transcriptional enhancer region is also used to increase gene expression (Smith et al 2002;Warzecha et al 2003;Dong et al 2005). Another strong candidate promoter is the synthetic Super promoter (Ni et al 1995), which has been successfully used to drive vaccine antigen expression in plants (Tuboly et al 2000;Pogrebnyak et al 2005).…”

Production of Hepatitis B Surface Antigen in Recombinant Plant Systems: An Update

“…Protocols for the extraction of recombinant HBsAg from transgenic plant materials were studied. The effects of reducing agent 2-mercaptoethanol (BME), protease inhibitors, extraction buffers, temperature, storage at 4°C, detergent, antioxidant concentrations, and stabilizers (skimmed milk powder) were analyzed to optimize the extraction protocols to recover and obtain the required form of HBsAg from recombinant potato tubers (31,32).…”

“…This increase was influenced by the presence of sodium ascorbate. Although the initial mAb-HBsAg increased with increasing levels of antioxidants, it was retarded by the reducing environments later (31,32). The formation of multimeric particle is essential for the immunogenicity of HBsAg.…”

Plant cell factories and mucosal vaccines

“…Smith et al [43 ] performed a more comprehensive stability study. The quantification of antigenically reactive HBsAg was found to be strongly dependent on the ratio of detergent:cell concentration.…”