Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures

Abstract

Human papillomavirus (HPV) tumor disease is a critical public health problem worldwide, especially in the developing countries. The recognized pathogenic function of E5, E6, and E7 oncoproteins offers the opportunity to devise therapeutic vaccines based on engineered recombinant proteins. The potential of plants to manufacture engineered compounds for pharmaceutical purposes, from small to complex protein molecules, allows the expression of HPV antigens and, possibly, the regulation of immune functions to develop very specific therapies as a reinforcement to available nonspecific therapies and preventive vaccination also in developed countries. Among plant-based expression formats, hairy root cultures are a robust platform combining the benefits of eukaryotic plant-based bioreactors, with those typical of cell cultures. In this work, to devise an experimental therapeutic vaccine against HPV, hairy root cultures were used to express a harmless form of the HPV type 16 E7 protein (E7*) fused to SAPKQ, a noncytotoxic form of the saporin protein from Saponaria officinalis, that we had shown to improve E7-specific cell-mediated responses as a fusion E7*-SAPKQ DNA vaccine. Hairy root clones expressing the E7*-SAPKQ candidate vaccine were obtained upon infection of leaf explants of Solanum lycopersicum using a recombinant plant expression vector. Yield was approximately 35.5 μg/g of fresh weight. Mouse immunization with vaccine-containing crude extracts was performed together with immunological and biological tests to investigate immune responses and anticancer activity, respectively. Animals were primed with either E7*-SAPKQ DNA-based vaccine or E7*-SAPKQ root extract-based vaccine and boosted with the same (homologous schedule) or with the other vaccine preparation (heterologous schedule) in the context of TC-1 experimental mouse model of HPV-associated tumor. All the formulations exhibited an immunological response associated to anticancer activity. In particular, DNA as prime and hairy root extract as boost demonstrated the highest efficacy. This work, based on the development of low-cost technologies, highlights the suitability of hairy root cultures as possible biofactories of therapeutic HPV vaccines and underlines the importance of the synergic combination of treatment modalities for future developments in this field.

Keywords: HPV – human papillomavirus; cancer; hairy root cultures; heterologous prime – boost; plant molecular farming; plant-produced antigens; therapeutic vaccines.

Figure 1

E7GGG (E7*), SAP-KQ, and E7*-SAPKQ genes were inserted in pEAQ-HT for both transient and stable plant expression. The expression cassette consisted of the cauliflower mosaic virus (CaMV) 35S promoter, the Cowpea Mosaic Virus (CPMV) 5′ and 3′ UTRs with the hyper-translatable mutation, the tomato bushy stunt virus posttranscriptional silencing inhibitor (p19), N- and C-terminal His₆-tag for affinity purification (His₆), and the nopaline synthase terminator sequence from A. tumefaciens (NPTII). Genes were cloned between the indicated sites to gain either a N-terminal, C-terminal, or no His₆-tag after expression.

Figure 2

Immunoblotting analysis of accumulation of recombinant E7*-SAPKQ, E7*, and SAPKQ. (A) Accumulation of recombinant E7*-SAPKQ in total extracts of N. benthamiana infiltrated leaves (anti-SAP polyclonal antibody). (+), His₆-E7*-SAPKQ from E. coli (25, 50, and 100 ng); (−), extract from leaves infiltrated with pEAQ-HT harboring an irrelevant gene; d.p.i., days post infiltration. (B) Soluble (S.) and insoluble (I.) fractions from leaves infiltrated with A. tumefaciens harboring His₆-E7*-SAPKQ and E7*-SAPKQ. (+), His₆-SAPKQ from E. coli (50 ng). (C) Accumulation of recombinant E7* in total extracts of N. benthamiana infiltrated leaves. (+), His₆-E7* purified from E. coli (25, 50, and 100 ng). (D) Accumulation of recombinant SAPKQ in total extracts of N. benthamiana infiltrated leaves. (+), His₆-SAPKQ purified from E. coli (1, 2.5, and 5 ng). M, SDS Molecular Weight Standard Mixture (Sigma) used during SDS-PAGE separation.

Figure 3

Micro-Tom (S. lycopersicum) clonal hairy root lines obtained after (A) transformation with empty A. rhizogenes A4, (B) expressing the recombinant His₆-E7*-SAPKQ, (C) His₆-SAPKQ, or (D) His₆-E7*. (E) Growth rate of one representative clone for each transformation was measured as root fresh weight at different time points after subculture over a 28-day period. Data represent average values ± SD of triplicate assays from two biological replicates.

Figure 4

Immunoblotting of extracts (15 μg TSP) from Micro-Tom hairy root clones obtained after transformation with His₆-E7*-SAPKQ, His₆-SAPKQ or His₆-E7* constructs. (A) Accumulation of recombinant His₆-E7*-SAPKQ and His₆-SAPKQ in extracts from different clonal hairy root lines (anti-SAP polyclonal antibody). Lanes 1–4, four His₆-E7*-SAPKQ best expressing clones; lane 5, hairy root clone expressing an irrelevant gene; M, Marker Color burst™, Sigma; lanes 6–8, three His₆-SAPKQ best expressing clones; lane 9, reference standard (His₆-SAPKQ from E. coli; 25 μg); hairy root clone expressing an irrelevant gene. (B) Immunoblotting on soluble (S.) and insoluble (I.) fractions from extract (5 μg TSP) of three representative His₆-E7*-SAPKQ-expressing clones (anti-E7 polyclonal antibody). (C) 5 μg TSP from the two best clones expressing His₆-E7*-SAPKQ were assayed at different time points after subculture (day 7, 14, 28, 35, and 42; anti-E7 polyclonal antibody was used). Day 7, lane 1 (clone 1); day 14, lane 2 (clone 1), lane 6 (clone 2); day 28, lane 3 (clone 1), lane 7 (clone 2); day 35, lane 4 (clone 1), lane 8 (clone 2); day 42, lane 5 (clone 1), lane 9 (clone 2); lane 10, reference standard (His₆-E7*-SAPKQ purified from E. coli, 100 ng; anti-E7 polyclonal antibody was used). (D) Assay of extracts from hairy root clones obtained after transformation with pEAQ-HT/His₆-E7*. M, Marker Color burst™; lanes 1–8, kanamycin-resistant clones obtained after transformation and not expressing His₆-E7*; lanes 9–11, reference standard (50, 75, 100 μg TSP from concentrated extracts of N. benthamiana expressing His₆-E7*-SAPKQ); lanes 12-13, 1.5 and 5 μg TSP of one representative hairy root clone expressing His₆-E7*-SAPKQ (anti-E7 polyclonal antibody).

Figure 5

Immunofluorescence microscopic analysis showing expression of His₆-E7*-SAPKQ in the root cap/apical meristem and along the region of elongation/maturation of a transformed hairy root clone from Micro-Tom. Original magnification 20×. Scale bar = 100 μm.

Figure 6

Immunization schedules. (A) Prime/boost regimens. A, empty pVax prime/mock hairy root extract boost; B, mock hairy root extract prime/mock hairy root extract boost; C, pVax-E7*-SAPKQ prime/pVax-E7*-SAPKQ boost; D, pVax-E7*-SAPKQ prime/E7*-SAPKQ extract boost; E, SAPKQ extract prime/SAPKQ extract boost. (B) Immunization of mice in the absence of tumor challenge or (C) in the presence of tumor challenge to evaluate immune response and tumor rejection, respectively.

Figure 7

Cell-mediated immune responses in vaccinated mice. C57BL/6 mice were immunized according to schedule and procedures as in Materials and Methods. IFN-gamma production was measured in an ELISPOT assay after specific antigenic stimulation with the E7 peptide RAHYNIVTF (aa 49–57). A, empty pVax prime/mock hairy root extract boost; B, mock hairy root extract prime/mock hairy root extract boost; C, pVax-E7*-SAPKQ prime/pVax-E7*-SAPKQ boost; D, pVax-E7*-SAPKQ prime/E7*-SAPKQ extract boost; E, SAPKQ extract prime/SAPKQ extract boost. Data are presented as fold-increase responses to the E7 peptide in comparison with mice vaccinated with empty vector/irrelevant root extract, and they represent the means of all of the mice in the groups ± SD.

Figure 8

Anticancer activity His₆-E7*-SAPKQ in TC-1 mouse model. C57BL/6 mice were challenged with 5 × 10⁴ TC-1 cells and thereafter treated with the different vaccine preparations as in Materials and Methods. Schedules of vaccination were as follows: A, empty pVax prime/mock hairy root extract boost; B, mock hairy root extract prime/mock hairy root extract boost; C, pVax-E7*-SAPKQ prime/pVax-E7*-SAPKQ boost; D, pVax-E7*-SAPKQ prime/E7*-SAPKQ extract boost; E, SAPKQ extract prime/SAPKQ extract boost. (A) Tumor volumes were recorded at different intervals after boost as in Materials and methods. Data represent means of five animals ± SEM. (B) Tumor weights were recorded when tumor controls reached >3 cm³ and all animals were euthanatized for ethical reasons. Data are means of five animals ± SD. ****p < 0.0001; ***p < 0.001; **p < 0.02.