Although long-term protection is a key-point in evaluating HPV-vaccine over time, there is currently inadequate information on the duration of HPV vaccine-induced immunity and on the mechanisms ...related to the activation of immune-memory. Longer-term surveillance in a vaccinated population is needed to identify waning immunity, evaluating any requirements for booster immunizations to assess vaccine efficacy against HPV-diseases. Current prophylactic vaccines have the primary end-points to protect against HPV-16 and 18, the genotypes more associated to cervical cancer worldwide. Nevertheless, data from many countries demonstrate the presence, at significant levels, of HPVs that are not included in the currently available vaccine preparations, indicating that these vaccines could be less effective in a particular area of the world. The development of vaccines covering a larger number of HPVs presents the most complex challenge for the future. Therefore, long term immunization and cross-protection of HPV vaccines will be discussed in light of new approaches for the future.
Papillomaviruses (PVs) are established agents of human and animal cancers. They infect cutaneous and mucous epithelia. High Risk (HR) Human PVs (HPVs) are consistently associated with cancer of the ...uterine cervix, but are also involved in the etiopathogenesis of other cancer types. The early oncoproteins of PVs: E5, E6 and E7 are known to contribute to tumour progression. While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems. The Bovine PV (BPV) system has been the most useful animal model in understanding the oncogenic potential of PVs due to the pivotal role of its E5 oncoprotein in cell transformation. This review will highlight the differences between HPV-16 E5 (16E5) and E5 from other PVs, primarily from BPV. It will discuss the targeting of E5 as a possible therapeutic agent.
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
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
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.
Antigen-specific immunotherapy and, in particular, DNA vaccination provides an established approach for tackling human papillomavirus (HPV) cancers at different stages. DNA vaccines are stable and ...have a cost-effective production. Their intrinsic low immunogenicity has been improved by several strategies with some success, including fusion of HPV antigens with plant gene sequences. Another approach for the control of HPV cancers is the use of natural immunomodulatory agents like those derived from plants, that are able to interfere in carcinogenesis by modulating many different cellular pathways and, in some instances, to reduce chemo- and radiotherapy resistance of tumors. Indeed, plant-derived compounds represent, in many cases, an abundantly available, cost-effective source of molecules that can be either harvested directly in nature or obtained from plant cell cultures. In this review, an overview of the most relevant data reported in literature on the use of plant natural compounds and genetic vaccines that include plant-derived sequences against HPV tumors is provided. The purpose is also to highlight the still under-explored potential of multimodal treatments implying DNA vaccination along with plant-derived agents.
The E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions ...and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.
An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast psbD 5' UTR and the psbA 3' UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.
The C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.
Cervical cancer (CC) is the fourth most common pathology in women worldwide and presents a high impact in developing countries due to limited financial resources as well as difficulties in monitoring ...and access to health services. Human papillomavirus (HPV) is the leading cause of CC, and despite the approval of prophylactic vaccines, there is no effective treatment for patients with pre-existing infections or HPV-induced carcinomas. High-risk (HR) HPV E6 and E7 oncoproteins are considered biomarkers in CC progression. Since the E6 structure was resolved, it has been one of the most studied targets to develop novel and specific therapeutics to treat/manage CC. Therefore, several small molecules (plant-derived or synthetic compounds) have been reported as blockers/inhibitors of E6 oncoprotein action, and computational-aided methods have been of high relevance in their discovery and development. In silico approaches have become a powerful tool for reducing the time and cost of the drug development process. Thus, this review will depict small molecules that are already being explored as HR HPV E6 protein blockers and in silico approaches to the design of novel therapeutics for managing CC. Besides, future perspectives in CC therapy will be briefly discussed.
Human papillomavirus (HPV) still represents an important threat to health worldwide. Better therapy in terms of further improvement of outcomes and attenuation of related side-effects is desirable. ...The pharmaceutical industry has always targeted natural substances-phytochemicals in particular-to identify lead compounds to be clinically validated and industrially produced as antiviral and anticancer drugs. In the field of HPV, numerous naturally occurring bioactives and dietary phytochemicals have been investigated as potentially valuable in vitro and in vivo. Interference with several pathways and improvement of the efficacy of chemotherapeutic agents have been demonstrated. Notably, some clinical trials have been conducted. Despite being endowed with general safety, these natural substances are in urgent need of further assessment to foresee their clinical exploitation. This review summarizes the basic research efforts conducted so far in the study of anti-HPV properties of bio-actives with insights into their mechanisms of action and highlights the variety of their natural origin in order to provide comprehensive mapping throughout the different sources. The clinical studies available are reported, as well, to highlight the need of uniformity and consistency of studies in the future to select those natural compounds that may be suited to clinical application.
The immune response is a key factor in the fight against HPV infection and related cancers, and thus, HPV is able to promote immune evasion through the expression of oncogenes. In particular, the E5 ...oncogene is responsible for modulation of several immune mechanisms, including antigen presentation and inflammatory pathways. Moreover, E5 was suggested as a promising therapeutic target, since there is still no effective medical therapy for the treatment of HPV-related pre-neoplasia and cancer. Indeed, several studies have shown good prospective for E5 immunotherapy, suggesting that it could be applied for the treatment of pre-cancerous lesions. Thus, insofar as the majority of cervical, oropharyngeal and anal cancers are caused by high-risk HPV (hrHPV), mainly by HPV16, the aim of this review is to discuss the immune pathways interfered by E5 oncoprotein of hrHPV highlighting the various aspects of the potential immunotherapeutic approaches.
The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with ...cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells.
Envisioning clinical application of the best characterized anti-HPV16 E6 and -HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed.
We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway.
Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.
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