The use of transgenic plants in the production of recombinant proteins for human therapy, including subunit vaccines, is being investigated to evaluate the efficacy and safety of these emerging biopharmaceutical products. We have previously shown that synthesis of recombinant glycoprotein B (gB) of human cytomegalovirus can be targeted to seeds of transgenic tobacco when directed by the rice glutelin 3 promoter, with gB retaining critical features of immunological reactivity (E.S. Tackaberry et al. 1999. Vaccine, 17: 30203029). Here, we report development of second generation transgenic plant lines (T1) homozygous for the transgene. Twenty progeny plants from two lines (A23T1-2 and A24T1-3) were grown underground in an environmentally contained mine shaft. Based on yields of gB in their seeds, the A23T1-2 line was then selected for scale-up in the same facility. Analyses of mature seeds by ELISA showed that gB specific activity in A23T1-2 seeds was over 30-fold greater than the best T0plants from the same transformation series, representing 1.07% total seed protein. These data demonstrate stable inheritance, an absence of transgene inactivation, and enhanced levels of gB expression in a homozygous second generation plant line. They also provide evidence for the suitability of using this environmentally secure facility to grow transgenic plants producing therapeutic biopharmaceuticals.Key words: transgenic tobacco seeds, homozygous second generation, glycoprotein B, human cytomegalovirus, vaccine, underground mine.
Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems