| Human cytomegalovirus (HCMV) infection is usually asymptomatic in healthy individuals. However, severe disease and/or death can occur in immunocompromised patients. The overall objective of this project was to develop a plant-derived CMV subunit vaccine and determine its effectiveness. Since CMV is species-specific, guinea pig CMV (GPCMV) was used as a model for HCMV. Four gene constructs were engineered to contain either the rice glutelin-1 promoter (Gt1) or the maize ubiquitin promoter, and the gene for either glycoprotein B (gB) or phosphoprotein 65 (pp65) of GPCMV. Agrobacterium tumefaciens were transformed with the Gt1-gB construct and subsequently used to transform Oryza sativa (rice) or Arabidopsis thalina . Due to technical difficulties associated with rice transformations, experimental work was continued with only A. thaliana. PCR analysis of plant tissue confirmed maintenance of the gB transgene for seven generations, and Western blots of selected seed extracts revealed a gB-specific band not found in non-transformed A. thaliana extracts. Guinea pigs (4 per group) were subcutaneously immunized with 20 mg of seed protein extract from GPCMV gB A. thaliana plants (estimated to contain approximately 50 mug of gB), an equal amount of total protein from non-transformed A. thaliana seeds (negative control), or baculovirus-derived GPCMV gB (positive control). Two animals immunized with seed-derived gB demonstrated GPCMV-specific antibody responses when analysed by ELISA. Western blot analysis confirmed that this response was specific for GPCMV gB. Neutralization assays were performed using serum obtained 14 days after the last immunization. Three of the 4 animals immunized with the gB seed extract showed viral neutralizing responses, with titres ranging from 40 to 3240. These results indicate that GPCMV gB expressed in the seeds of A. thaliana can generate a GPCMV-specific immune response. Based on data from GPCMV-specific ELlSAs, Western blots, and viral neutralization assays using serum from immunized animals, I concluded that gB was authentically expressed in A. thaliana seeds with at least some epitopes similar to those found on native GPCMV gB that were able to neutralize viral infectivity. |
