Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine

Abstract

Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV.

Keywords: ELISA, enzyme-linked immunosorbent assay; GST; GST, glutathione-s-transferase; HPV, human papillomavirus; HPV-16 L1; L1_2xCysM, modified L1 gene; PCR, polymerase chain reaction; Plant-made vaccines; VLPs, virus-like particles; aadA, aminoglycoside 3'-adenylyltransferase; capsomeres; cervical cancer; plastid transformation.

Figure 1.

Schematic diagram of transformation vector construction (map not drawn to scale). (A) Precursor vector pPNG1014_MCS120 was used to clone transgenes. (B) Plasmid pPNGST-L1 obtained after the insertion of transgenes GST and L1_2xCysM in the precursor vector. (C) Final transformation vector pPNGST-L1-T for the transformation of plants, showing transgenes along with plastome flanks inserted within the tobacco plastid genome. PrrnPEP, plastid encoded polymerase promoter from rrn16 gene; Prrn-62NEP, nuclear encoded polymerase promoter; G10L RBS, ribosomal binding site from gene 10 leader sequence; GFP, first 14 amino acids of the green fluorescent protein; MCS, multiple cloning site; 5´ UTR, 5´ untranslated region; aadA, aminoglycoside 3'-adenylyltransferase; PNG10, cassette containing PrrnPEP, Prrn-62NEP and G10L RBS; CP, chloroplast DNA; GST, glutathione S-transferase gene; L1_2xCysM, modified L1 gene; TrbcL, terminator from large subunit of ribulose-bisphosphate carboxylase gene; INSR, right insertion site (trnR); INSL, left insertion site (trnN).

Figure 2.

PCR and Southern analysis of the transgenic lines. (A) PCR analysis of the transgenes correctly inserted within the targeting site of the plastid genome, Amplification of GST-L1_2xCysM gene (2582 bp) with the primers oli248 in the L1_2xCysM gene and oli252 located within the plastome, (B); Amplification of the aadA gene (1981 bp) with primers oli251 in the aadA gene and oli253 present within the plastid genome. Lanes 1-7: Seven independently generated transplastomic lines (C) Southern blot analysis of GST-L1_2xCysM transplastomic plants. Total plant DNA was digested with BglII. The DNA sequence P (773 bp) located within left insertion site (INSL) of plastid genome was amplified by PCR and served for probing in the Southern analysis. Lanes 1-7: Seven independently generated transplastomic lines analyzed for the transgene integration, M: marker, WT: wild type.

Figure 3.

Morphology of transplastomic tobacco plants carrying transgene GST-L1_2xCysM showing healthy phenotype with fertile flowers.

Figure 4.

Antigen capture Enzyme-linked immunosorbent assay (ELISA) of the 7 transplastomic lines showing the L1 protein accumulation in the leaf extracts. Baculovirus-derived VLPs served as positive control for the assay. Detection of conformational epitopes was done by monoclonal antibody Ritti01. WT: Wild type, as negative control.