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

Silvia Massa,Aldo Venuti,Carmela Marino,Rosella Franconi,Francesca Paolini

doi:10.3389/fpls.2019.00452

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.

Highlights

Over the past four decades, a wealth of literature has demonstrated the production of exogenous proteins in plants for health applications
To express a recombinant therapeutic Human papillomavirus (HPV) vaccine in plantbased systems, the E7*-SAPKQ fusion protein was cloned into the cauliflower mosaic virus (CaMV) 35 promoter/Nos terminator cassette of the pEAQ-HT plant expression vector, between appropriate restriction sites, in order to add an affinity purification His6-tag for affinity purification (His6)-tag either at the N- or C-terminus of the final product synthesized by plant cells
Constructs were introduced into plant cells either by agroinfiltration mediated by A. tumefaciens C58C1 in N. benthamiana plants for transient expression, or by transformation mediated by A. rhizogenes A4 to obtain stably expressing hairy root cultures from Micro-Tom leaf explants

Summary

Introduction

Over the past four decades, a wealth of literature has demonstrated the production of exogenous proteins in plants for health applications. Plant-based expression systems have great potential to produce different types of biologics at reasonable costs and with reduced risks of contamination by threatening pathogens. This approach is especially advantageous in the field of prevention and treatment of infections and cancer (Rybicki, 2014; Streatfield et al, 2015; Loh et al, 2017). Hairy roots are attractive for the industrialscale production of secondary metabolites (Miralpeix et al, 2013), but are considered for the expression of pharmaceutical proteins, due to better performances over plant cell suspension cultures in terms of genetic and biochemical stability, reduced presence or absence of toxic compounds, such as alkaloids, with respect to leaves. Recombinant proteins produced in engineered hairy root cultures can be secreted in the culture medium simplifying downstream purification processes (Guillon et al, 2006; Häkkinen et al, 2014)

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Conclusion