FIGURE 7 | Reduction of a S. enterica ssp. enterica contamination on different food matrices by salmocins. Fresh raw food matrices as whole egg (A), beef trims (B) and tuna filet trims (C) as shown in images were contaminated with a bacterial strain mix in equal cell number proportions [nalidixic acid resistant mutants of Salmonella strains of two serovars: Enteritidis (ATCC R 13076TM∗ ) and Typhimurium (ATCC R 14028TM∗ )]. The graphs show bacterial populations recovered from foods shown in the pictures above upon storage for various periods of time at 10◦C upon salmocin treatment (dark gray bars, initial contamination level; white bars, carrier treatment; light gray bars, salmocin treatment with SalE1b in concentration of 0.5 mg/kg food of contaminated food by intermixing. Error bars indicate standard deviation of biological replicates, N = 4. Statistically significant reductions (p < 0.005) in bacterial contamination were found by assessment of viable bacterial counts obtained from salmocin-treated in relation to carrier-treated food samples by analysis by unpaired parametric t-test with GraphPad Prism v. 6.01 at all timepoints showing efficacy of salmocin treatment. Experiments were performed using semi-purified salmocin SalE1b protein.

We already reported on the development of ethanol-inducible transgenic N. benthamiana lines for the expression of colicin M (Schulz et al., 2015) and salmocin E1b (Schneider et al., 2018). Currently, we are developing transgenic N. benthamiana lines for production of other colicins and salmocins. Alternative approaches for large-scale protein expression in plants, such as agroinfiltration or agrospray, require a fermentation facility to generate inoculum, plus containerization, plant transport and vacuum infiltration equipment in infiltration-based processes (Chen et al., 2013; Gleba et al., 2014; Tusé et al., 2014).

Such process requirements introduce complexity and ultimately drive up manufacturing capital and operating costs. Although higher cost of goods sold (cogs) might be tolerated for pharmaceutical or other high-value products produced through transient expression, they are undesirable in cost-constrained applications such as food safety.

Here we analyzed the performance of colM-producing N. benthamiana line (T4 generation plants homozygous for single-copy T-DNA insertion) depending on season; we also compared transient and transgenic colicin M expression (**Figure 9**). In our semi-controlled glass-facade greenhouse conditions, seasonal differences in plant biomass yield depend mostly on light intensity and the lowest amount of plant biomass was found in the winter season due to slower plant growth as this is usually observed for all plant species grown in the greenhouse (**Figure 9A**). The lower biomass yield of seasons with unfavorable speed of plant growth can be compensated by prolonged incubation of plants before treatment and harvest, as seen in comparison of summer and autumn plants (**Figure 9A**). There was no prominent difference between transgenic and transient production host (**Figure 9A**). Expression of recombinant proteins was more equally distributed between leaves and stems for transgenics compared to vacuum-infiltrated plants with predominant leaf expression (**Figure 9B**). Despite experiment-to-experiment variability, transgenic and transient expression hosts provided comparable levels of recombinant protein accumulation (**Figure 9B**). Phenotypically, transgenic plants were indistinguishable from wild type plants (**Figure 9C**).

#### Colicins/Salmocins: Regulatory Marketing Allowance as Food Antimicrobials

Development and regulatory approval of any product to be added to food or used as medicine is a complex, lengthy and usually costly process. Regulatory approvals for food additives vary significantly from country to country. We discuss here regulatory approval pathways for food antibacterials in the United States because this country represents by far the largest potential market for these products and because its regulatory review process can be relatively simple and fast (and relatively inexpensive), compared to the regulations in most other countries. In the United States, any substance to be intentionally added to food is a food additive and must be subjected to premarket review and approval by the FDA under the Federal Food, Drug and Cosmetic Act (FFDCA; the "Act"), unless the substance meets a listed exemption in the Act, or is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use6 .

GRAS' (Generally Recognized As Safe) is an FDA designation that a chemical or substance added to food is considered safe by experts, and so is exempted from the conventional premarket approval process by FDA. The developer of the new substance (the Notifier) conducts an analysis of safety and utility of its product using scientific procedures including corroboration from

6www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/

publically available information, and determines and documents that the substance is GRAS as specified by the FDA's Final Rule for GRAS Notices7 . The Notifier voluntarily submits its GRAS conclusion to FDA for review and comment. The FDA can either reject Notifier's conclusion of safety, cease to evaluate the submission upon request by the Notifier, or, ideally, issue a "No Questions" letter to Notifier. The latter verifies that the FDA agrees with Notifier on its conclusion that the substance is GRAS and equates to marketing allowance by FDA for the substance. The FDA may conduct the GRAS review on its own for certain types of food treatments, or solicit input from the United States Department of Agriculture (USDA) if the substance is to be applied to USDA-regulated products such as meat and egg products. The GRAS pathway can be used for substances added to human food or animal food, as well as for animal feed ingredients.

In addition to the FDA, an alternative body capable of conducting GRAS reviews is the Flavor and Extract Manufacturers Association (FEMA), which is the national association of the United States flavor industry. FEMA works with legislators and regulators to assure that the needs of members and consumers are addressed and can provide GRAS guidance, although their function is restricted to flavor substances.

The GRAS designations fall into several categories, the most relevant of which for food antibacterials is "Food Processing Aid." Substances added to food are classified as Food Processing Aids if they provide a rapid yet temporary effect, degrade and become part of the food matrix and thus have no functional effect on the food. If FDA accepts such a designation based on the evidence provided, the designation allows the manufacturer to avoid listing the substance on the treated food's product label; thus, there is no labeling requirement for the substance. Food additives or food ingredients, on the other hand, are typically persistent, are essential to or can modify the food's functionality, and need to be listed on the product label.

Facilitated regulatory pathways similar to GRAS exist also in a few other countries, for example Canada, Mexico, Australia, and New Zealand. In yet other territories, including countries of the European Union and Japan, approval of a new food additive involves a process similar to the USA's pre-market review of a new non-GRAS substance, requiring extensive toxicity/safety studies.

Nomad Bioscience is the first company to successfully obtain FDA concurrence for GRAS designation of its plant-made bacteriocins, such as colicins, as food antimicrobials. In its first GRAS notice (GRN 5938 ), the following arguments were used to support safety and suitability of colicins made in food species hosts as food antimicrobials.

Safety:

– Colicins are naturally occurring antibacterial proteins produced endogenously by commensal enteric bacteria in the human gut;

7www.federalregister.gov/documents/2016/08/17/2016-19164/substancesgenerally-recognized-as-safe

8https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices &id=593

FIGURE 9 | Transgenic EtOH-inducible hosts for colicin expression. Independent experiments performed in different seasons, summer, autumn, and winter, comparing transient and transgenic colicin M expression are shown. Methods of plant cultivation, transient expression of colicin M using N. benthamiana WT plants and vacuum infiltration of 1:100 dilutions of agrobacterial cultures or ethanol-induction of transgenic plants using 4% (v/v) EtOH solutions were described in Werner et al. (2011) or Schulz et al. (2015), respectively. The TMV-based constructs for transient colM expression (pNMD10221) and for EtOH-inducible colM expression (pNMD18381) were described in Schulz et al. (2015), Supplementary Figures S1, S3, respectively. Transgenic plants for EtOH-inducible colM expression used were T4 generation plants homozygous for single copy T-DNA insertion with characterized T-DNA ends and genomic insertion point of N. benthamiana plant line Nb18381T0#29 initiated as described in Schulz et al. (2015) (Supplementary Figure S2). (A) shows the yield of plant biomass in g fresh weight (FW) as average and standard deviation of 6 or 3 plants (also true for B) and sample description giving plant age at timepoint of treatment (vacuum infiltration or EtOH-induction) in days post sowing (dps), harvesting timepoint for biomass and recombinant protein analysis in days post treatment (dpt) and antimicrobial activity of colicin-containing TSP extracts in AU/mg FW plant biomass. (B) inspection of plant TSP extracts prepared with 2 vol. 20 mM citrate, 20 mM Na2HPO4, 30 mM NaCl, pH 4.0 using Angel Juicer 7500 and feeding buffer using peristaltic pump iPump2S by SDS-PAGE and Coomassie-staining; recombinant colM is marked with asterisks, loading corresponds to 2.5 mg FW (summer and winter) or 3.75 mg FW (autumn). (C) Plant phenotypes before treatment or at harvest.

#### Suitability:

In 2015, based on Nomad's submission, FDA accepted colicins as first-in-class GRAS antimicrobials for controlling pathogenic E. coli in fruits and vegetables (GRN 593), and in 2017, FDA and USDA accepted colicins as antimicrobials for controlling E. coli in meat products (GRN 6769 ). In both cases, the Agencies agreed with the "Food Processing Aid" definition and USDA has added colicins to its FSIS Directive 7120, which is a list of safe and suitable ingredients allowed for use in the production of meat, poultry and egg products (FSIS Directive 7120.1, Revision 42). Subsequently, Nomad also filed GRAS notices for Salmonella salmocins (GRN number pending) and C. perfringens bacteriophage endolysins (GRN 80210). Independently, Nomad also submitted a GRAS notice for the use of N. benthamiana (non-edible) production host for the manufacture of colicins (GRN 77511), which led to a "No Questions" letter from FDA. A list of allowed GRAS notices, notices currently under review by FDA, and notices in preparation is provided in **Supplementary Table S1**.

Regulatory experience to date suggests that additional plantmade colicins, phage-derived endolysins, other bacteriocins, defensins, etc., could also gain rapid marketing allowance. In particular, in 2018 Nomad received 'No Questions' letter from FDA for a product candidate in another category of food treatments, the natural sweeteners/taste modifiers thaumatins (GRN 73812). As long as the GRAS notice describes a natural or nature-identical substance, GRAS designation is a relatively simple, fast and inexpensive way for obtaining regulatory review and product marketing allowance, as evidenced by the success of Nomad's GRAS submissions to date. Based on this experience, we see a potential GRAS allowance 'space' for multiple classes of natural proteins such as:

Natural products that are used to treat food but that have no functional effect on food (food processing aids) are obviously the easiest cases to take through the GRAS process. For colicins, the inherent safety of these proteins was supported in part by the fact that colicins and colicin-like bacteriocins are very sensitive to proteases, and any traces of these proteins remaining in the treated food would be rapidly degraded in the stomach and duodenum; Nomad provided FDA with extensive data on gastroduodenal degradation of colicins in its dossier. Future uses of bacteriocins as food treatments to control bacteria in the human or animal gastrointestinal tract would likely require additional data on bacteriocin safety, bioavailability, and their functionality in the intestinal lumen.

#### Potential Markets for Bacteriocins as Food Antibacterials

Bacteriocins such as colicins and salmocins are promising alternatives to antibiotics for many markets, perhaps most importantly for food and medicinal uses. At present, antibiotics still constitute our main therapeutic toolbox for controlling pathogens; the situation is, however, rapidly changing with increasing number of pathogenic bacteria becoming resistant to most antibiotics. In the health care market, bacteriocins could probably be effectively used today in specific market niches in which the pathogen species are known and a topical application (direct surface delivery to skin, surface of lungs, surface of urogenital tract, and intestinal tract) is possible. Such indications could include treatment of cystic fibrosis patients by inhalation-based delivery of pyocins to treat Pseudomonas, or treatment of uropathogenic Escherichia by catheter delivery of colicins (**Tables 1**, **2**).

9https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices&id=676&sort= GRN\_No&order=DESC&startrow=1&type=basic&search=676

10https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices&id=802 11https://www.accessdata.fda.gov/scripts/fdcc/?set=GRASNotices&id=775

12https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices& id=738

TABLE 2 | Potential uses of bacteriocins in food and medicines/medical devices.

TABLE 3 | Potential United States food/feed safety and animal health markets for bacteriocins.

∗GRAS status agreement by FDA obtained; ∗∗GRAS status agreement by FDA expected Q1 2019.

Potential markets for food antibacterials are large and even more immediate because of the facile marketing channels in countries such as United States and Canada. **Table 3** lists potential market segments that are in need of better food safety through bacterial control, and those markets are sizeable and numerous. Given the current antibacterial intervention costs accepted or acceptable by the industry (\$0.025–0.1 per kg of food product), the estimated markets for antibacterials could potentially be very attractive. Some of the segments, such as processing of fresh and ground beef or processing of poultry and pigs, are oligopolistic with only a few companies controlling the majority of the United States market (i.e., Tyson Foods, JBS, Cargill, National Beef, and Pilgrim's Chicken). Therefore, a commercial alliance with just one such partner would provide access to 20% or more of the market segment.

#### DISCUSSION

Food safety market needs are shaped by two major trends, both of which revolve around real and perceived food safety issues. The first trend is the rapid increase of multidrug resistant forms among common bacterial pathogens present in food. The second is the desire by consumers to have a 'natural' food that is devoid of chemical additives or genetically modified ingredients ('organic food'). This second trend is actively exploited by food companies because it allows them to charge a premium for food classified as 'natural,' 'GMO-free,' 'antibiotic-free,' 'organic,' 'bio,' etc. Unfortunately, the modification of agricultural and husbandry practices and subsequent food processing so as to exclude previously accepted chemical interventions drastically increases the likelihood of food contamination by bacteria. Two examples illustrate this point. Contamination of food with pathogenic E. coli was originally dubbed a 'hamburger disease' because such contamination was initially traced to contaminated beef. The statistics from the United States Centers of Disease Control and Prevention (CDC) that tracks outbreaks in United States demonstrate that during 2006–2010 only one out of ten outbreaks was traced to contaminated vegetables. However, during 2011– 2016, nine out of thirteen E. coli foodborne outbreaks was due to vegetables/vegetable products, including five due to organically grown vegetables and sprouts. Another sad illustration is the case with Chipotle, a United States restaurant chain that in 2013 declared itself as the one intending to provide its customers with natural, organic and GMO-free foods. Within approximately the next 18 months, there were four outbreaks due to contamination involving three different pathogens (E. coli, Salmonella, and norovirus). As a result, the company stock took a serious hit and its market value had fallen by almost 65% by the end of 2017, somewhat recovering during the first half of 2018. However, in August 2018 the company experienced its largest outbreak to date, this one due to C. perfringens. In the minds of the general public, there still appears to be no correlation between 'organic' farming/food and higher risk of bacterial contamination. Nevertheless, since bacteriocins are natural proteins identical to the ones made by our intestinal bacterial flora, we believe that bacteriocins are more likely to be accepted as novel food safety interventions by the industry, non-governmental and governmental organizations and ultimately by consumers.

There is a concern about increase in resistance to colicins and salmocins upon their use as food antimicrobials. Bacterial resistance to bacteriocins is well-known, it was described in numerous publications (e.g., Riley and Gordon, 1996; Feldgarden and Riley, 1998; Kirkup and Riley, 2004, etc.). We believe that the application of colicins/salmocins

to food is unlikely to allow for selection of bacteriocinresistant bacteria in the intestinal tract of humans, for three main reasons:

There are other serious challenges for the developers of bacteriocins as food antibacterials. The food industry has traditionally been reluctant to make significant investments to develop new products unless market or other dynamics mandate it. In particular, antibiotics have been developed by pharmaceutical companies and afterwards adopted by the food industry. Development of the most recent antibacterial products, bacteriophages, has been pioneered and conducted almost entirely by small companies and academia. Most of our discussions with large companies active in food production and processing indicated that they would consider adopting a bacteriocin product if it were available on the market already, but not if it was a product candidate in late development, even if it had already been accepted by regulatory agencies. In other words, a bacteriocin developer may be expected to shoulder the majority of the costs and risks of not only development and regulatory review of the product but also of building manufacturing capacity and marketing resources. This is in stark contrast to the pharmaceutical industry, where there is a "division of labor" in which the R&D pipeline is serviced by multiple small and medium size companies each specializing in certain steps of product development, such as discovery, preclinical studies, or Phases I–II clinical studies. The risk is compounded by the fact that in case of eventual acquisition of the whole product pipeline and accompanying infrastructure by a food industry company (the preferred exit for a small developer), the developer is unlikely to receive multiples on the investment made that are comparable to the multiples enjoyed by developers in the pharmaceutical business (i.e., depending on the development phase at trade sale, average 3.7–4.8 multiples on investment can be realized).

Additional challenges stem from the regulatory requirements imposed on the food industry. Whereas there are strict 'zero tolerance' rules concerning contamination of food with E. coli, there are, for practical reasons, no such limits on contamination with Salmonella or other food pathogens. Correspondingly, large food producers' only "incentives" are the costs of food recalls due to contamination and the damage to their product brands, and sometimes, to their share price.

Some optimism is offered by recent successes of companies developing plant-based meat analogs, such as Beyond Meat and Impossible Foods. The latter company includes recombinant (yeast) leghemoglobin in its ingredients as a flavor enhancer, with the argument that it is less environmentally impactful to produce the heme protein recombinantly by fermentation than to obtain it by natural extraction of legumes (soybeans) grown in vast acreages. This concept appeals to the overall consumer base targeted by the company, whose business model is to offer meat-replacing foods that taste like meat but do not lead to deforestation to raise grains for animal feed, thereby combating global warming. The recombinant heme has achieved GRAS status (GRN 73713), and the plant-based meats are gaining acceptance with consumers in spite of GM ingredients. There is a lesson to be learned in this example that could apply to consumer acceptance of natural proteins such as colicins, salmocins and others that are known to be safe and effective, can address a major worldwide safety issue, and can be manufactured at scale in plant-based systems that are sustainable and environmentally compatible. The future will be interesting indeed.

# AUTHOR CONTRIBUTIONS

SH-L, ASt, AG, and YG designed the research. SH-L, ASt, SS, TS, and ASh performed the research. SH-L, ASt, SS, TS, DT, AG, and YG analyzed the data. SH-L, DT, AG, and YG wrote the manuscript.

# FUNDING

This work was partially financed by Investitionsbank Sachsen-Anhalt, Magdeburg, Germany (Grants 1204/00033, 1704/00088, and 1704/00087).

# ACKNOWLEDGMENTS

We thank Dr. Mirko Buchholz (Fraunhofer Institute for Cell Therapy and Immunology, Department of Drug Design and Target Validation, Halle, Germany) for protein and alkaloid analytical services, Dr. Antje Breitenstein, Lia Bluhm, and Anja Banke (BioSolutions Halle GmbH, Halle, Germany) for their support in evaluating colicins' and salmocins' antimicrobial activity. We thank Dr. Kristi Smedley (Center for Regulatory Services, Woodbridge, VA, United States) and Prof. Chad Stahl (University of Maryland, College Park, MD, United States) for valuable advice and guidance on regulatory strategy. We also thank Dr. Aušra Ražanskienë (Nomads UAB, Vilnius, Lithuania) for fruitful discussions.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00437/ full#supplementary-material

13 https://www.accessdata.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices& id=737

#### REFERENCES

fpls-10-00437 April 8, 2019 Time: 7:56 # 16

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Hahn-Löbmann, Stephan, Schulz, Schneider, Shaverskyi, Tusé, Giritch and Gleba. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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

*Silvia Massa1 \*, Francesca Paolini2 , Carmela Marino3 , Rosella Franconi3 and Aldo Venuti 2 \**

*1 Biotechnology Laboratory, Biotechnology and Agroindustry Division, Department of Sustainability, ENEA (Italian National Agency for New Technologies, Energy and Sustainable Economic Development), Rome, Italy, 2Virology Laboratory, HPV-UNIT, Department of Research, Advanced Diagnostic and Technological Innovation (RIDAIT), Translational Research Functional Departmental Area, IRCSS Regina Elena National Cancer Institute, Rome, Italy, 3Biomedical Technologies Laboratory, Health Technologies Division, Department of Sustainability, ENEA, Rome, Italy*

#### *Edited by:*

*Suvi Tuulikki Häkkinen, VTT Technical Research Centre of Finland Ltd, Finland*

#### *Reviewed by:*

*Carlo De Giuli Morghen, University of Milan, Italy Hugh S. Mason, Arizona State University, United States*

#### *\*Correspondence:*

*Silvia Massa<EMAIL_ADDRESS>Aldo Venuti<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science*

*Received: 12 January 2019 Accepted: 26 March 2019 Published: 11 April 2019*

#### *Citation:*

*Massa S, Paolini F, Marino C, Franconi R and Venuti A (2019) Bioproduction of a Therapeutic Vaccine Against Human Papillomavirus in Tomato Hairy Root Cultures. Front. Plant Sci. 10:452. doi: 10.3389/fpls.2019.00452*

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

# INTRODUCTION

Over the past four decades, a wealth of literature has demonstrated the production of exogenous proteins in plants for health applications. Indeed, 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).

Transient expression of target proteins achieved by plant viruses or by agroinfiltration often allows higher protein yield with respect to stable transformation due to the absence of chromosomal integration (Komarova et al., 2010) and represents also a means for evaluation of expression before starting the generation of transgenic plant-based expression platforms. Nevertheless, expression of therapeutic proteins using *in vitro* plant systems under contained conditions represents a profitable manufacturing approach in terms of uniform cultivation conditions, product quality, and downstream purification process (Rischer et al., 2013; Santos et al., 2016; Massa et al., 2018). Together with cell suspensions, organ cultures such as hairy root cultures (HRCs) offer advantages including containment, established cultivation conditions in hormone-free media, product homogeneity (Franconi et al., 2010; Schillberg et al., 2013). Hairy roots are particularly attractive for the industrialscale production of secondary metabolites (Miralpeix et al., 2013), but are also 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. Among plants used for generating hairy roots, crop plants such as tomato and potato were also used. Indeed, hairy roots of many different plant species have been utilized to produce various both secondary metabolites and recombinant proteins of pharmaceutical value at varying yields, such as enzymes (Woods et al., 2008), vaccines and hormones (Woffenden et al., 2008; Skarjinskaia et al., 2013), antibodies in different formats (Wongsamuth and Doran, 1997; Lonoce et al., 2016, 2018). Production of enzymes for replacement therapy of rare diseases was also reported (Rodriguez-Hernandez et al., submitted; Naphatsamon et al., 2018). Recombinant proteins produced in engineered hairy root cultures can be also secreted in the culture medium simplifying downstream purification processes (Guillon et al., 2006; Häkkinen et al., 2014).

Cervical cancer and cervical intraepithelial neoplasia (CIN) are known consequences of human papillomavirus (HPV) infection. Cervical cancer is the fourth most common cancer in female population, with about 569,847 new cases per year (of which 88% in developing countries) and over 311,365 deaths (GLOBOCAN 2018, https://gco.iarc.fr/today). HPV is also the agent behind the development of other tumors and of oropharyngeal carcinogenesis, now in significant rise, and has a causal role in 13% of all female cancers (i.e., 5% of all cancers). Expression of viral oncogenes such as E6 and E7, and, as it was more recently demonstrated, E5, leads to correlated malignant disease.

Although HPV infection is preventable through very efficient recombinant vaccines developed against variously incident oncogenic genotypes in yeast and insect cells, and despite cervical cytology and DNA testing, HPV-related preinvasive and invasive diseases remain critical public health problems. Furthermore, currently available treatments against HPV-related disease are only moderately successful, with radiotherapy, chemotherapy, and surgery very poorly efficient against highgrade lesions (Vici et al., 2014; Cordeiro et al., 2018). This highlights the need for specific treatment strategies. Among the most promising, there are therapeutic vaccines and novel therapeutics that may target the ability of HPV to influence host immune tolerance. If available, these tools may also imply milder side effects than conventional approaches such as radiotherapy and/or chemotherapy.

Immunological therapeutic approaches against HPV have been investigated in the last decades, facilitated by the availability of E5, E6, and E7 tumor-associated antigens, optimal targets for cancer immunotherapy. First examples of experimental HPV therapeutic vaccines were able to block tumor growth in animal models, with some being able to evoke specific cell-mediated immune responses (Cordeiro et al., 2018). However, poor presentation of viral antigens that are expressed at low levels and poor trafficking of effector T-cell populations to non-inflamed mucosal/skin sites were common limitations. The use of adjuvants was, indeed, demonstrated to be crucial for therapeutic efficacy (Gerard et al., 2001).

Studies have focused on enhanced E6 and E7 HPV peptidefusions in combination with bacterial toxins and/or adjuvants. Nevertheless, these approaches showed negligible correlation to good clinical outcomes and tumor regression (Skeate et al., 2016). An advantage of whole protein-based vaccines compared to peptides is that they theoretically cover all available cytotoxic T lymphocytes (CTLs) and T-helper epitopes. Therefore, the use of whole HPV E6 and E7 proteins or fusion proteins as the antigenic source has been widely employed in preclinical therapeutic vaccines tested in animal models and advanced into phase II and III clinical trials (Vici et al., 2016; Barros et al., 2018; Roden and Stern, 2018).

Among protein-based formulations, production of candidate HPV therapeutic/prophylactic vaccines using plant-derived expression platforms was also proven. Different plant-based expression systems were considered, from whole plant approaches for transient expression to stably transformed green microalgae, using single HPV antigens or fusion to peptides to improve accumulation yield, to intracellular targeting strategies. In many cases, evidence of immunogenicity and efficacy in animal models were reported (reviewed in Chabeda et al., 2018). In our previous experience, crude plant extracts containing HPV16 E7 antigen were shown to provide protection against challenge with E7-expressing TC-1 cells (Franconi et al., 2002, 2006). These responses were improved when E7 was produced in plants as fusion to a bacterial carrier (LicKM, *Clostridium thermocellum* beta-glucanase) (Massa et al., 2007; Venuti et al., 2009).

Besides protein-based formulations, genetic vaccination is also a promising immunotherapeutic tool due to amenability to engineer sequences (e.g., addition of sequences of immunological value), stability and ease of manufacturing, cost-effectiveness, safety, and general tolerability (Gurunathan et al., 2000). Genetic immunization is able to induce adaptive cell-mediated immune responses, including activation of CD4+ helper T cells and CD8+ cytotoxic T cells, crucial to the resolution of cancer (Stevenson et al., 2004). Indeed, the most remarkable result in the field of therapeutic HPV vaccines is the clinical activity showed by a phase IIb randomized trial performed with a DNA-based vaccine (VGX-3100) in either HPV16- or HPV18-positive CIN2/3 patients (Trimble et al., 2015). In this trial, DNA vaccine delivery included the use of intramuscular injection coupled with electroporation (i.e., administration of short electrical pulses at the site of the DNA vaccine injection to increase plasmid uptake and correlated immune response) and showed, for the first time, significant regression of CIN2 lesions.

In our search for innovative immune-stimulatory tools for the rational design of therapeutic DNA-based vaccines against HPV (Massa et al., 2008), we focused on the sequence encoding the saporin protein (SAP) from *Saponaria officinalis*, a member of the "Ribosome-Inactivating Proteins" (RIPs) family (Hartley and Lord, 2004; Stirpe, 2004; Zarovni et al., 2009). We demonstrated that the combination of a mutagenized SAP sequence (SAPKQ) with an attenuated, synthetic HPV16 E7 gene (E7GGG, thereafter indicated as E7\*), in the context of DNA-based vaccination, determined a modulation of E7-specific humoral and cell-mediated immune responses affecting the growth of E7-expressing tumors (Massa et al., 2011).

Heterologous DNA prime-protein boost regimen (i.e., administration of an immunogen as a DNA-based preparation in the first dose, followed by the same immunogen as a proteinbased preparation in the booster dose) is emerging as a tool for envisaging new therapeutic options in HPV-associated infection and cancer (Peng et al., 2016).

In the present study, hairy root cultures derived from tomato (*Solanum lycopersicum*) were used to stably express E7\*-SAPKQ in order to devise a protein-based experimental HPV therapeutic vaccine. HRC turned out to be a better platform to express E7\*-SAPKQ than *E. coli*, due to its ability to accumulate the recombinant vaccine in the soluble fraction of root extracts. The administration of the DNA-based E7\*-SAPKQ vaccine (prime dose) followed by E7\*-SAPKQ protein-containing hairy root extract (boost dose) was considered, together with homologous prime-boost regimens. 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 (i.e., DNA-based vaccination and plant-based expression systems), 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.

#### MATERIALS AND METHODS

#### Cells

*Agrobacterium tumefaciens* C58C1 strain was used for plantbased transient expression experiments. *Agrobacterium rhizogenes* A4 (*Rhizobium rhizogenes* ATCC 43057; American Type Culture Collection, Manassas, VA, USA) was used to generate hairy root clones. Bacteria were grown in YEB medium (5 g/l beef extract, 1 g/l yeast extract, 5 g/l peptone, 5 g/l sucrose, 2 mM MgSO4) at 28°C with shaking at 220 rpm. When necessary, kanamycin (50 μg/ml) was added to the culture medium.

E7-expressing TC-1 tumor cells were kindly gifted by T.C. Wu (Johns Hopkins Medical Institutions, Baltimore, MD) and were cultivated in RPMI (Invitrogen, Paisley, UK) containing 400 μg/ml G418 and 10% fetal calf serum.

#### Animals

Six- to eight-week-old female C57BL/6 mice were supplied by Charles River Laboratories. Animal handling and sacrifice were performed under specific pathogen-free conditions at the Animal House of the Regina Elena National Cancer Institute. All experimental procedures were approved by the Government Committee of National Minister of Health (85/2016-PR) and were carried out in accordance with EU Directive 2010/63/ EU for animal experiments.

#### Plant Material, Genes, and Construction of the Plant Expression Vector

Whole plant-based transient expression of E7\*-SAPKQ was assessed in 3-week-old *Nicotiana benthamiana* plants upon agroinfiltration. Plants were grown in soil in the Bio-Safety Level-2 green-house available at ENEA, under LED lighting (650 LED Lumigrow lamp; spectroradiometric data: lux 3106.5; total PAR 138.83; Watts 0.0011) with daylight integration and dark condition (16/8 h) until use. Nutrients (Idrofill base, K Adriatica, Italy) were added to water every 3 weeks.

Tomato (*S. lycopersicum* L.) cv. Micro-Tom leaf explants were used to generate hairy root clones upon infection with recombinant *A. rhizogenes* A4. Micro-Tom plants were grown in greenhouse under hydroponic conditions and LED lighting, as described for *N. benthamiana*.

The attenuated E7GGG gene (in this work indicated as E7\*) was obtained from the E7 gene of HPV16 (HPV16 genome NCBI Reference Sequence: K02718), as previously described (Massa et al., 2008). The gene encoding the leaf apoplastic saporin isoform (SAP, Genbank Acc. No. DQ105520) had been previously mutagenized to abolish toxicity (SAPKQ, IQMTAE176AAR179FRY > IQMTA**K**176AA**Q**179FRY) and to obtain the pVax-E7\*-SAPKQ fusion construct by fusing the E7\* sequence to the 3′ end of the SAPKQ gene (Massa et al., 2011).

In order to obtain the plant-expression constructs, genes were PCR-amplified from the abovementioned DNA construct. Cloning into the binary Ti plasmid pEAQ-HT (PBL Technologies) (Sainsbury et al., 2009) was performed with primers designed to add either a N- or C-terminal His6-tag, or, on the contrary, no His6-tag to final products (**Figure 1**).

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.

Genes were inserted under the CaMV 35 promoter/Nos terminator cassette downstream of the 5′-UTR of the Cowpea Mosaic Virus (CPMV) RNA-2 harboring the U162C mutation ("hypertranslatable," HT) and upstream of the 3′-UTR of the CPMV RNA-2. In this vector, the tomato bushy stunt virus (TBSV) p19 sequence serves as a posttranscriptional silencing suppressor.

#### Agroinfiltration of Whole *N. benthamiana* Plants for Evaluation of Expression of the E7\*, SAPKQ, and E7\*-SAPKQ Proteins in Plant Cells

The resulting pEAQ-HT constructs were transferred into *A. tumefaciens* strain C58C1 by electroporation and, then, introduced by vacuum infiltration into *N. benthamiana* for transient expression. Briefly, 2-ml starter culture harboring the pEAQ-HT-based constructs started from a fresh colony was grown overnight in YEB containing 50 mg/l kanamycin. Then, the culture was diluted 1:500 into 500 ml of YEB, 50 mg/l kanamycin, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 5.6, 2 mM MgSO4 and 20 μM acetosyringone, and grown overnight at 28°C, 220 rpm to O.D600nm = 1.7.

Bacteria were pelleted by centrifugation at 3000 × *g* for 15 min and resuspended to a final OD600nm = 2.4 with the addition of 200 μM acetosyringone in MMA medium (4.4 g/l Murashige and Skoog salts, 10 mM MES pH 5.6, 20 g/l sucrose). After 1–3 h of incubation at RT, the suspension was applied to 3-week-old *N. benthamiana* plants by vacuum infiltration cabinet (O.M.EC. Impiantistica, Grassobbio, Italy), and plants were returned to the growth module for observation. Leaves were harvested and stored at −80°C until use.

#### Assessment of E7\*, SAPKQ, and E7\*-SAPKQ Expression in *N. benthamiana* Plants

Leaf tissues of infiltrated plants were harvested 1–7 days post infiltration. Leaf biomass samples were finely ground in liquid N2 with mortar and pestle, resuspended and homogenized in extraction buffer (1:3 w/v; phosphate-buffered saline "PBS": 21 mM Na2HPO4, 2.1 mM NaH2PO4, 150 mM NaCl, pH 7.2; alternatively, GB buffer was used: 100 mM Tris-HCl pH 8.1; 10% glycerol; 400 mM saccharose; 5 mM MgCl2; 10 mM KCl; 10 mM 2-β-mercaptoethanol) containing a protease inhibitor cocktail (Complete™; Roche, Mannheim, Germany). Samples were incubated on ice for 30 min with gentle rocking and extracts were clarified by centrifugation at 11,000 g for 20 min. Supernatants were transferred to a fresh tube and kept on ice until use and total soluble protein (TSP) content was estimated by the Bradford assay (Bio-Rad Inc., Segrate, Italy). Pellets were resuspended in appropriate volumes of SDS-PAGE sample buffer (10% glycerol, 60 mM Tris-HCl pH 6.8, 0.025% bromophenol blue, 2% SDS, 3% 2-mercaptoethanol), constituting the insoluble fraction of leaf extracts. Samples containing 15 μg of TSP and the corresponding insoluble fraction in SDS-PAGE sample buffer were denatured at 95°C for 5 min and subsequently electrophoresed on a 12% SDS-polyacrylamide gel. Known amounts of proteins purified from *E. coli* were used as reference standards. Extract from leaves infiltrated with pEAQ-HT harboring an irrelevant gene was used as negative control. SDS Molecular Weight Standard Mixture (Sigma) was used during SDS-PAGE separation. Separated protein samples were transferred to a PVDF membrane (Millipore, Bedford, MA) by electro-transfer at 100 V with a Trans-Blot apparatus (BioRad). Filters were probed with either mouse anti-E7 or rabbit anti-SAP polyclonal antibodies (Massa et al., 2011) used at a dilution of 1:2,000 and, then, with 1:10,000 dilution of either a horseradish peroxidase-conjugated goat anti-mouse IgG antibody or a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (GE-Healthcare) for 1 h. The immune complexes were detected by developing chemiluminescence with the Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). The ImageQuant™ LAS 500 (GE Healthcare) was used for chemiluminescence signal detection and densitometric quantification of bands was performed by ImageJ software.

#### Hairy Roots Generation

*S. lycopersicum* (cultivar Micro-Tom) clonal root lines were obtained from wild-type leaf explants co-cultured with *A. rhizogenes* A4 (ATCC, 43057™) harboring the constructs. Bacteria were grown in YEB medium containing 50 mg/l rifampicin and 50 mg/l kanamycin to OD600nm = 0.6, at 28°C and 220 rpm. Bacteria were, then, pelleted by centrifugation at 3000 × *g* for 15 min and resuspended at OD600nm = 1 in Murashige and Skoog medium (MS, Duchefa) with 30 g/l sucrose and 200 μM acetosyringone, pH 5.8.

Leaves from 3-week-old Micro-Tom plants were harvested, sterilized in 0.1% (v/v) sodium hypochlorite solution (NaClO) for 15 min, and aseptically cut into explants of 1 cm × 1 cm. Explants were subsequently inoculated by immersion in the recombinant *A. rhizogenes* suspension for 15 min, in a rotary shaker at the minimum speed, in the dark. Explants were dried onto sterilized tissue paper and transferred on their adaxial side, onto co-culture plates containing MS agar medium and 100 μM acetosyringone and incubated under dark conditions for 3 days. The co-cultured explants were, then, blotted and transferred to a hormone-free MS medium supplemented with 200 μg/l cefotaxime (Cef; Sandoz, Varese, Italy) at 25°C. Fresh growing hairy roots were obtained after 8–10 days.

Emerging roots were excised and transferred to new plates. *A. rhizogenes* was eradicated by transferring roots every 15 days onto MS agar plates using decreasing Cef concentrations (0.25, 0.125, and 0.05 g/l) until no antibiotic was added. Transformation and *A. rhizogenes* eradication were confirmed by PCR using specific oligonucleotides for the exogenous sequences and for *rol B*/*rol C* genes, and *vir C* specific primers, respectively. The selected, kanamycin-resistant hairy root clones were considered for subsequent growth and analysis. The growth rate of one representative clone for each transformation was measured as root fresh weight at different time points after liquid subculture in Erlenmeyer flasks over a 28-day culture period and this was performed on two biological replicates for each hairy root clone. Hairy root biomass harvested for subsequent analysis was carefully handled, pulverized in liquid nitrogen, and immediately stored in −80°C.

#### Screening of Clonal Hairy Root Lines Expressing the Antigens by Immunoblotting Analysis

The selection of antigen-expressing hairy root clones was performed by immunoblotting. Root tissues were finely ground in liquid N2 with mortar and pestle and resuspended and homogenized in phosphate-buffered saline pH 7.2 (PBS, 1:3 w/v) containing a protease inhibitor cocktail (Complete™; Roche, Mannheim, Germany). Sample preparation for 12% SDS-PAGE acrylamide gels, immunoblotting, and detection of bands were performed as described for *N. benthamiana* samples. Protein molecular mass marker Color burst™ (Sigma) was used as reference for bands upon immunoblotting.

#### Immunofluorescence Detection of Antigens in Hairy Roots

Recombinant His6-E7\*-SAPKQ expressed from Micro-Tom hairy tissue samples located on glass slides was detected by immunofluorescence after softening tissues with 2% driselase in PBS for 40 min at 37°C following fixation with paraformaldehyde 4% in PBS for 1 h. Thereafter, samples were washed in PBS (pH 7.2) three times for 5 min each time with gentle shaking. Then the target was covered with 10% DMSO and 0.5% NP40 in PBS for 1 h at room temperature. Following removal of permeabilization solution, 5% BSA was applied for 1 h at RT and then, samples were dipped with primary rabbit anti-SAP polyclonal antibody diluted 1:300 and incubated at RT in wet box overnight. Then, the samples were washed three times. After a gentle drying, samples were incubated with antirabbit polyclonal antibody conjugated with phycoerythrin (sc-3739 goat anti-rabbit IgG-PE, Santa Cruz) diluted 1:1,000, in the dark for 1 h, washed, added with DAPI staining solution, and incubated in the dark at RT for 10 min. Subsequently, slides were washed three times in PBS. Samples were sealed with glycerol:PBS (1:1) for image collection under Nikon Eclipse TE2000-S epifluorescence microscope equipped with a Hg 100 lamp and filter sets appropriate for DAPI and Cy3 fluorescence.

#### Vaccination Schedules in Mouse Model

Six- to eight-week-old female C57BL/6 mice were immunized according to two immunization protocols (**Figure 6**). In the first protocol (i.e., immunization protocol), mice were subjected to vaccination and, thereafter, analyzed for immune response. In the second one (i.e., therapeutic protocol), mice were subcutaneously (s.c.) injected with TC-1 tumor cells before administration of the vaccines. Immunization protocol implied a priming with the E7\*-SAPKQ DNA vaccine (50 μg/mouse, intramuscularly, i.m.) into the tibia muscle, followed by electroporation according to standardized procedures (Cordeiro et al., 2015). To perform immunizations, root tissues were finely ground in liquid N2 with mortar and pestle, resuspended and homogenized in phosphate-buffered saline pH 7.2 (PBS, 1:3 w/v), and administered to mice. Antigen doses were quantified in the extract by immunoblotting using *E. coli*-purified fusion protein as standard, as previously described (Franconi et al., 2002). Animals were boosted after 1 week either with the same DNA- or with the E7\*-SAPKQ root extract-based vaccine (1 μg/mouse, s.c. in the trunk). Alternatively, mice were primed with the E7\*-SAPKQ root extract-based vaccine and boosted with the same E7\*-SAPKQ root extract-based vaccine.

The therapeutic protocol implied that mice were injected with 5 × 104 TC-1 tumor cells in 200-μl saline solution and, 3 days post tumor challenge, primed and boosted with the same preparations by the same time intervals used in the immunization protocol. Tumor growth was monitored by visual inspection and palpation two times a week. Animals were scored as tumor bearing when tumors reached a size of approximately 1–2 mm in diameter. For ethical reasons, the experiment was ended and all animals euthanized when tumor growth reached 3 cm3 in the control animals. Finally, all tumors were carefully removed from euthanized animals and tumor weight recorded.

#### IFN-Gamma Enzyme-Linked Immuno-Spot Assay

HPV16 E7-specific T-cell precursors were detected by enzymelinked immunospot assay (ELISPOT) 1 week after the boost, according to previous reported protocols (Massa et al., 2007). Briefly, single-cell suspension of splenocytes (1 × 106 cells per well) from each group of vaccinated mice was added to microtiter wells coated with anti-mouse IFN-γ antibody (5 μg/ml, BD Biosciences PharMingen, San Diego, CA), along with interleukin-2 (50 units/ml, Sigma-Aldrich Italia, Milan, Italy). Triplicate samples were incubated at 37°C for 48 h with the E7-specific H-2Db (10 μg/ml) cytotoxic T-lymphocyte (CTL) MHC class I epitope (amino acids 49–57, RAHYNIVTF). After peptide incubation, a biotinylated anti-mouse IFN-γ antibody (2 μg/ml) was added for 4 h at room temperature. Cell spots were detected by streptavidin-HRP incubation for 1 h at room temperature and staining with filtered 3-amino-9-ethylcarbazole substrate (BD Biosciences PharMingen, San Diego, CA), for 5 min. Spots were counted using a dissecting microscope.

#### Statistical Analysis

Comparisons between individual data points were analyzed by two-tailed Student's *t*-test using the GraphPad Prism 8 software.1 Data were expressed as means ± standard deviations (SD) or ± standard error of mean (SEM). A *p* < 0.05 was considered statistically significant.

#### RESULTS

#### E7\*-SAPKQ Protein Expression in *N. benthamiana* Plants

To express a recombinant therapeutic HPV vaccine in plantbased systems, the E7\*-SAPKQ fusion protein was cloned into the 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 either at the N- or C-terminus of the final product synthesized by plant cells. Also the single E7\* and SAPKQ sequences were cloned in the same manner in order to verify the accumulation behavior of the single components of the fusion protein E7\*-SAPKQ (**Figure 1**).

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.

The engineered proteins were firstly introduced in *N. benthamiana* plants by transient methodology. Protein extracts from leaves were analyzed by immunoblotting over a 7-day period (from day 1 to day 7 post infiltration) to assess the accumulation of the recombinant antigens. Extractions, initially performed using PBS-based buffer, turned out to be insufficient to satisfactorily detect expression of the recombinant proteins. Then, a stronger extraction using GB buffer was used, revealing that both E7\*-SAPKQ and the single antigens E7\* and SAPKQ were expressed preferentially as N-terminal His6-tagged and non-tagged form in extracts of infiltrated leaves (**Figure 2**).

Among all the constructs, mainly His6-E7\*-SAPKQ and E7\*-SAPKQ were produced upon agroinfiltration (**Figure 2A**), and accumulated as soluble proteins in leaf biomass (**Figure 2B**). The theoretical weight of plant-expressed His6-E7\*-SAPKQ and E7\*-SAPKQ was confirmed by the separation of the *E. coli* standard His6-E7\*-SAPKQ (Massa et al., 2011), showing a most distinct, specific band of the apparent molecular weight ≤ 45 kDa, that indicated the accumulation of the monomeric form of the fusion protein (**Figures 2A,B**). Additional faint bands at lower molecular weight are present as probable degradation by-products. Both His6-E7\*-SAPKQ and E7\*-SAPKQ accumulation show a peak 5 d.p.i. with a maximum expression level estimated of 3.20 and 3.90 μg/g fresh weight corresponding to 0.15 and 0.17% TSP, respectively. E7\*-SAPKQ-His6 was calculated as 0.40 μg/g fresh weight.

E7\* is produced upon agroinfiltration, given that a specific band is recognized by the anti-E7 antibody in plant extracts especially for the non-tagged protein 5 d.p.i. (**Figure 2C**). The band separates at a higher molecular weight than the *E. coli* reference standard (recombinant His6-E7\*, about 17 kDa) (Massa et al., 2008). E7\*-His6 seemed to accumulate at low levels 5 d.p.i., and as a dimer (about 34 kDa) 7 d.p.i. The maximum concentration calculated for His6-E7\* and for the non-tagged E7\* was 9.4 μg/g fresh weight (0.41% TSP), and 14.6 μg/g fresh weight (0.64% TSP), respectively. The dimeric E7\*-His6 was estimated to be 8.2 μg/g fresh weight (0.36% TSP).

His6-SAPKQ accumulated with a peak 7 d.p.i., accounting for at least 14.52 μg/g fresh weight (0.64% TSP). Non-tagged product was found to accumulate 10.27 μg/g fresh weight (0.45% TSP), mainly 7 d.p.i. Lower expression was found for SAPKQ-His6 that mainly accumulated 3 d.p.i. (3.20 μg/g fresh weight; 0.14% TSP). The theoretical weight of plant-expressed SAPKQ in the three forms was confirmed by the separation of the *E. coli* standard His6-SAPKQ, showing a specific band of the apparent molecular weight of 30 kDa (**Figure 2D**).

#### E7\*-SAPKQ Expression in Hairy Root Cultures

After confirming the possibility to express the antigens in plant cells, the N-terminal His6-tagged constructs were chosen to engineer clonal hairy root lines for *in vitro*, stable, plant-based

1 www.graphpad.com

bioproduction of the experimental therapeutic vaccine. Fortythree clonal hairy root lines were isolated from *S. lycopersicum* cv. Micro-Tom after leaf explants infection with recombinant *A. rhizogenes* A4 strain transformed with the expression vector pEAQ-HT/His6-E7\*-SAPKQ for subsequent analysis. Thirty-four hairy root clones were isolated after transformation with pEAQ-HT/His6-E7\*. Forty-five hairy root clones were isolated after transformation with pEAQ-HT/His6-SAPKQ.

Hairy root clones were isolated from Micro-Tom for the different constructs and were subcultured. Recombinant hairy root clones showed slower growth rate with respect to clones obtained after transformation with non-transformed *A. rhizogenes* A4 (**Figure 3A**), and different growth rate depending on the construct. The best growing clones were those expressing His6- E7\*-SAPKQ (**Figure 3B**), followed by those expressing His6- SAPKQ (**Figure 3C**) and by those transformed with pEAQ-HT/ His6-E7\* that had very low growth rates (**Figures 3D,E**).

The selection of hairy roots expressing the different products was performed by immunoblotting using either anti-E7\* or anti-SAP polyclonal antibodies (**Figure 4**). In the case of His6- E7\*-SAPKQ, 34 clones (about 79% of the isolated clones) showed the expected band at about 45 kDa indicating the presence of the full-size fusion protein. Four clones exhibited the most intense bands for the given quantity of total protein transferred (**Figure 4A**). Also in the case of the hairy rootproduced His6-E7\*-SAPKQ, the fusion protein accumulated mainly in the soluble fraction (**Figure 4B**). TSP from two out of the four best clones expressing His6-E7\*-SAPKQ was assayed by immunoblotting analysis at different time points after subculture (day 7, 14, 28, 35, 42). As shown in **Figure 4C**, accumulation of His6-E7\*-SAPKQ is stable until day 15 (i.e., along almost the whole cultivation period since clones were normally subcultured for maintenance every 21 days), to decrease thereafter. The lower weight band revealed by the anti-SAP polyclonal antibody (**Figure 4A**) but not by the anti-E7 polyclonal antibody (**Figures 4B,C**) might represent a proteolysis product containing SAPKQ, or an alternative splicing activity.

Fourteen clones resulted positive for expression of His6- SAPKQ (about 31% of the clones), with three clones showing the most intense bands at the expected molecular weight of about 30 kDa (**Figure 4A**). Among those transformed with His6-E7\*, only eight clones survived on selective medium and none of them showed detectable expression of His6-E7\* (**Figure 4D**).

All these clones were kept for subsequent subculture and measurements. His6-E7\*-SAPKQ best clone accounted for 35.49 ± 2.69 μg/g of fresh weight (1.25% TSP) and His6-SAPKQ accounted for 31.06 ± 4.79 μg/g of fresh weight (0.96% TSP).

Immunofluorescence analysis performed on samples of Micro-Tom hairy root clones expressing His6-E7\*-SAPKQ confirmed the accumulation of the recombinant protein by the detection of an intracellular specific signal. His6-E7\*-SAPKQ (red labeling) appeared to be localized both in the root cap/apical meristem and along the region of elongation/maturation of transformed hairy roots. This showed vaccine expression both in the root tips where cell proliferation occurs and also that it was stably accumulated in mature tissues. Tissue sampling in view of preparation of extracts for administration upon preclinical studies was performed taking into account this observation. No expression of His6-E7\*-SAPKQ in hairy roots obtained after transformation with an irrelevant gene was observed (**Figure 5**).

period. Data represent average values ± SD of triplicate assays from two

#### Mouse Immunological Responses to Vaccination

The immunological effects of vaccines were studied in C57BL/6 mice with the prime-boost schedule described in **Figure 6**. Cell-mediated immune responses were analyzed in ELISPOT assay for INF-γ secreting cells. One week after the boost, spleens were collected from immunized mice. Significantly positive scores were obtained only in the animal immunized with the root extract boost (**Figure 7**). In particular, heterologous prime (i.e., E7\*-SAPKQ DNA)/boost (i.e., E7\*-SAPKQ-containing root extract) exerted a dramatic increase in cell-mediated immune response specifically directed against E7 with respect to the other immunization combinations. The specific activity of vaccine preparation in root extracts was further confirmed by the absence of any activity of the root extracts without E7 (**Figure 7**).

The immunological responses evoked by E7\*-SAPKQcontaining root extract vaccines induced us to determine if a therapeutic potential of the heterologous schedule may still be envisaged in the TC-1 preclinical model. Indeed, TC-1 cell expressing the E7 oncogene is a well-known and validated animal model for the preclinical evaluation of therapeutic candidate HPV vaccines. The homologous schedule (i.e., administration of E7\*-SAPKQ DNA both as a prime and as a boost) was utilized for comparison. After injecting TC-1 cells into naïve C57BL/6 mice, prime and boost immunizations were performed on days 3 and 10, respectively (**Figure 6**). Tumor volume recorded 24 days after boost was mostly affected by the vaccine treatments implying the administration of the E7\*-SAPKQ-containing root extracts, showing higher, statistically significant differences with respect to controls (empty pVax and mock extract, respectively) (**Figure 8A**). Although not statistically significant, the heterologous schedule scored a slightly higher reduction in tumor volume than the homologous administration of E7\*-SAPKQ DNA both as prime and as boost.

Since tumor volume calculation *in vivo* cannot take into account all the tumor dimensions, tumors were excised from immunized mice and their weight recorded when tumor controls reached >3 cm3 and all animals were euthanatized for ethical reasons. Data obtained from tumor weight substantially confirmed those related to tumor volume: the heterologous E7\*-SAPKQ DNA/E7\*-SAPKQ-containing root extract schedule showed 4-fold reduction in tumor growth with respect to controls. In addition, the heterologous E7\*-SAPKQ DNA/E7\*-SAPKQcontaining root extract schedule showed statistically significant differences with respect to homologous E7\*-SAPKQ DNA (both as prime and boost) or E7\*-SAPKQ-containing root extracts (both as prime and boost) schedules, with *p* = 0.0009 or *p* = 0.0056, respectively (**Figure 8B**).

 HPV infection can also cause cancer in subsets of oropharyngeal tumors, including the base of the tongue and tonsils. Although HPV prevention is feasible, the effects of preventive vaccination on the incidence of HPV tumors

biological replicates.

will only be visible in the long term with a prediction of a 7.5% reduction in cases, if vaccination policies remain primarily "female-only" (Hartwig et al., 2017). In addition, current treatments are distant from the ideal ones and less effective in high-grade lesions. Expression of viral oncogenes E6, E7, and E5 leads to HPV-related malignant progression. Due to their peculiarities, HPV oncogenes represent an excellent target for cancer immunotherapy (de Freitas et al., 2017).

HPV peptide- and protein-based experimental vaccines have reached phase II trial phases, the whole protein-based having the advantage to more probably evoke efficient cell-mediated responses in patients. Nevertheless, it has been suggested that adjuvants and tools able to improve immunogenicity and endowed with less safety concerns for human health than those that are currently tested (e.g., calreticulin, lysosome-associated proteins, heat shock proteins, tetanus toxoid) are necessary to minimize autoimmune reactions, or preexisting immunity (Wolkers et al., 2002; Millar et al., 2003; Savelyeva et al., 2003). Therefore, there is still need for further improvements in terms of safety, efficacy and, possibly, of decreased costs of vaccine preparation.

Plant molecular farming (PMF) may be the approach to achieve these results. Indeed, PMF is devoted to produce active and secure cost-effective pharmaceutical proteins, and plants can be a source of immune-stimulating tools in terms of both primary and secondary metabolites. So far, production of valuable pharmaceutical proteins by PMF has been demonstrated, which can help the treatment of patients particularly in developing countries, where production and preservation costs of medicines cannot be afforded. Platforms for PMF are different and may involve the use of whole plants or plant cell/organ cultures subjected to a transient or stable expression. PMF may be intended for purification, or administration as a crude extract or whole plant tissues. All these aspects may emphasize the advantages of the plant-based systems for expression of pharmaceutical proteins. Indeed, it has been shown that genes encoding tumor-associated antigens and viral coat proteins of HPV can beexpressed in plants not only retaining their native immunological activity but also receiving adjuvant activity from plant extract itself (Franconi et al., 2002, 2006; Chabeda et al., 2018).

On the other hand, safety, efficacy, and potential immunogenicity are also features of DNA vaccines targeting HPV (Gurunathan et al., 2000; Stevenson et al., 2004; Massa et al., 2008). Literature data indicate that genetic immunotherapy is becoming a pharmacological tool and therapeutic option against cervical disease, with HPV DNA vaccines reaching encouraging results in phase II clinical trials phases (Vici et al., 2016).

We have already reported that the genetic fusion of E7\* with SAPKQ modulates E7-specific humoral and cell-mediated immune responses resulting in antitumor effects against E7-expressing tumors when administered as a DNA vaccine. These findings opened the way to a new application of this plant protein, known in medicine mainly as the cytotoxic component of immuno-toxins, and gave us further impulse to the development of candidate HPV therapeutic vaccine, expanding the nature of the possible immune-enhancers of HPV E7.

This manuscript describes the use of hairy root culture expression technology for the bioproduction of a candidate therapeutic vaccine endowed with specific cell-mediated response associated to anticancer activity against HPV in a mouse model. This is the first time, to our knowledge, that such plant-based expression platform is preclinically proven to produce HPV antigens with antitumor activity.

We report expression of E7\*-SAPKQ in both whole *N. benthamiana* plants by transient expression technology and hairy roots from tomato by stable transformation with the idea to develop a safe and affordable therapeutic vaccine for HPV malignancies. Our purpose was to accumulate the E7\* protein in a plant-based expression system maintaining/improving its antigenicity.

Expression of the SAPKQ protein had been already tried in *E. coli* (Massa et al., 2011). Indeed, bacterial expression of

SAPKQ was obtained with no bacterial growth block, demonstrating that the mutagenesis that had been introduced into the saporin leaf apoplastic isoform coding sequence of *S. officinalis* was able to highly reduce its cytotoxicity, otherwise leading to procaryotic/eucaryotic cell death (Stirpe, 2004). Nevertheless, when expression of different versions of E7\*-SAPKQ fusion protein was attempted, the majority of the recombinant fusion proteins was found in the insoluble fraction of the bacterial lysates, as a possible consequence of residual toxicity of the SAPKQ proteins and of its E7GGG fused derivatives in the bacterial host (usually able to neutralize "disturbing" exogenous proteins in inclusion bodies found in the insoluble fraction of lysates upon extraction procedures). Therefore, alternative expression systems were tried, and, in particular, eukaryotic plant-based expression chassis.

The engineered proteins E7\*, SAPKQ, and E7\*-SAPKQ were firstly introduced into *N. benthamiana* plants by transient methodology, primarily to assess if the recombinant protein expression was generally tolerated in plant cells. The single components of the fusion were utilized to investigate if protein bioaccumulation by plant cells was in function of the type of protein itself. It is noteworthy that while E7\* alone was difficult to accumulate (at least in the presence of the His6-tag), the different forms of SAPKQ were quite easily accumulated. This gave a clue about a possible positive influence of the SAPKQ in the accumulation of the fusion product E7\*-SAPKQ. Indeed, even though at low levels, His6-E7\*-SAPKQ was expressed in plants upon agroinfiltration in both un-tagged and His6-tagged forms, and turned out to be accumulated mainly as a soluble protein. This result also suggested the possibility to devise a different plant-based platform for stable transformation to possibly achieve better protein yield and purification in native conditions. However, the low and different accumulation levels of all the forms of E7\* prompted us not to purify these antigens from the infiltrated leaf biomass.

We demonstrated that infection of Micro-Tom leaf explants with recombinant *A. rhizogenes* can be used for rapid establishment (approximately 6 weeks) of hairy root clones stably expressing His6-E7\*-SAPKQ. Expression yield accounted for at least 35.5 μg/g of fresh weight. This value was tenfold higher than that obtained in whole plants upon agroinfiltration and it is comparable to or higher than those reported for

rejection, respectively.

other exogenous proteins accumulated in recombinant hairy roots. His6-E7\*-SAPKQ in hairy roots yielded approximately fivefold higher than a similar fusion protein intended for immunization, the rabies glycoprotein-ricin toxin B chain (i.e., another plant toxin adapted for therapeutic use like naturally found saporin) chimera, in optimized air-lift tomato hairy root-based bioreactors (6–8 μg/g) (Singh et al., 2015). Fusion of ricin toxin B chain with F1:V pneumonic plague vaccine antigen in tobacco hairy roots yielded 140-fold less than His6- E7\*-SAPKQ (0.25 μg/g) (Woffenden et al., 2008). De Guzman et al. (2011) reported production of the *E. coli* B-subunit heat labile toxin antigen in tomato hairy root cultures (~10 μg/g; the same protein was expressed in tobacco and petunia hairy roots ~100 μg/g).

His6-E7\*-SAPKQ expression in tomato clonal hairy root lines was maintained along subculture intervals, and lowering of expression was observed when hairy root clones were forced to age in culture vessels. All these results demonstrate the suitability of the hairy root system to our purposes also in absence of optimization of culture conditions. It is noteworthy that a mild extraction (i.e., performed in PBS, the same solution that was subsequently used for immunizations) was sufficient to obtain the recombinant proteins, while a stronger buffer was necessary to extract the same products from leaf tissues.

No hairy root clones expressing His6-E7\* alone were selected. This is not surprising since, in contrast to transient expression, that was demonstrated several times for E7 in plant-based expression systems (reviewed in Chabeda et al., 2018), constitutive expression could imply some toxicity affecting the onset and subsequent survival of hairy root clones upon stable transformation. Actively growing clones were, on the contrary, obtained for His6-SAPKQ, as for His6-E7\*-SAPKQ. This suggests also that the SAPKQ carrier may be helpful in determining a growth-compatible bioaccumulation of the candidate vaccine in the root tissues.

It is well known that heterologous DNA prime followed by recombinant protein boost regimens can be used to enhance HPV therapeutic vaccine potency (Smyth et al., 2004; Fiander et al., 2006; Peng et al., 2016; Chabeda et al., 2018).

Indeed, in this work, the boosting strategy with the E7\*-SAPKQ-containing extract was effective in strongly improving E7-specific T-cell responses and related anticancer activity after E7\*-SAPKQ DNA-based priming with respect to homologous prime-boost regimens, as proven by ELISPOT assay and biological tests. The model chosen to test the efficacy of these experimental therapeutic vaccines was the well-known and widely used TC-1 model. TC-1 cells are immortalized mouse lung epithelial cells transduced with HPV E6 and E7 genes, able to express these viral oncoproteins that are continuously processed through the proteasome pathway. This processing allows their epitopes to be exposed in the context of the MHC class I complex located on the cell membrane, mimicking the mechanism leading to epitope-specific killing by CTLs upon integration of the HPV genome in an infected target cell (Feltkamp et al., 1995).

E7-specific immunological and anticancer responses were also obtained with the homologous prime-boost regimen based on E7\*-SAPKQ-containing hairy root extracts, suggesting that hairy root expression system could be a useful tool in devising a therapeutic vaccine strategy against HPV. The good elicitation of specific immunity and anticancer activity was statistically significantly higher than that of the homologous prime-boost regimen based on E7\*-SAPKQ DNA, already demonstrated to be effective in a previous study (Massa et al., 2011). Data from tumor weight measurement confirmed efficacy of the treatment with statistically significant differences between experimental vaccines (either homologous or heterologous

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.

prime-boost regimens with E7\*-SAPKQ-based preparations) and controls.

Accumulation of E7 in hairy root tissues was demonstrated as a fusion to the LicKM carrier (Massa et al., 2009); however, in the present work, for the first time, the therapeutic potential of an E7\* produced in clonal hairy root lines is determined in animal studies.

Hairy root technology offers an easy handling alternative that can be conveniently biocontained in a controlled *in vitro* environment and scaled up depending on the demand for the target protein by the use of improved bioreactors. As mentioned, important experimental pharmaceutical proteins, among which vaccine antigens and their fusions with carriers, together with enzymes, hormones, and antibodies in different formats, have been reported. The hairy root system has also the advantage to theoretically allow to regenerate transgenic plants and to retain germplasm (De Guzman et al., 2011). In particular, generation of hairy roots from *S. lycopersicum* for production of foreign proteins has the advantage that there is less hazardous accumulation of alkaloids like in the case of tobacco (Singh et al., 2015), reducing the presence of unwanted compounds in extract or purified protein administration. These characteristics are essential to acquire regulatory approval for clinical administration of hairy rootproduced proteins in the future.

In addition, in the case of raw and partially purified preparations from candidate vaccine-expressing tomato hairy root clones, the presence of functional elements can be exploited. As an example, a molecular complex formulation (Tomatine) based on the natural adjuvant α-tomatine (i.e., nontoxic to humans when assumed in the amounts present in green tomatoes), was reported to stimulate antigenspecific humoral and cellular immune responses that determined protection against malaria, *Francisella tularensis* and regression of experimental tumors (Morrow et al., 2004; Zhang et al., 2006; Granell et al., 2010). Indeed, preliminary analysis of our candidate vaccine-expressing hairy roots showed the presence of α-tomatine (manuscript in preparation) which may have an additive role to that of SAPKQ in the immunological and anticancer responses observed after immunization with the E7\*SAPKQ-containing hairy root extracts, possibly enhancing the overall effect. Partially purified recombinant proteins have already proven to be able to induce an immune response in animal models (reviewed in Rigano et al., 2013) and may be considered in "rationally designed" formulates containing one or more active principles (e.g., vaccine antigen, bioactive secondary metabolites, and adjuvants such as α-tomatine).

Our data show that it is possible to obtain a recombinant E7\*-SAPKQ from clonal hairy root lines with immunological and anticancer activities against HPV experimental tumors, especially in combination with a DNA vaccine based on the same sequences. These results pave the way to undergo more studies on production of vaccines in plant-based expression systems and their combination with other treatment modalities for the development of effective and more specific therapeutic intervention against HPV infection and related cancer.

#### ETHICS STATEMENT

Animal handling and sacrifice were performed under specific pathogen-free conditions at the Animal House of the Regina Elena National Cancer Institute. All experimental procedures were approved by the Government Committee of National Minister of Health (85/2016-PR) and were carried out in accordance with EU Directive 2010/63/EU for animal experiments.

#### AUTHOR CONTRIBUTIONS

SM is the recipient of the special Grant from MIUR (Italian Ministry of University and Research) "ENEA 5 × Mille" (Young investigator Project: New therapeutic strategies for the treatment of cancer). She planned and designed the project, assembled the plant-expression constructs, undertook *N. benthamiana* agroinfiltration and generation of hairy root clones, analyzed plant material and hairy root recombinant clones, prepared extracts for immunization, planned and wrote the paper. FP undertook large-scale preparation of DNA plasmids, immunization experiments with mice, collected and analyzed data on immune responses and biological activity of the different vaccine preparations, wrote and revised the manuscript. AV planned the immunization protocols; supervised immunization experiments; analyzed and interpreted data on immune responses and biological activity of the different vaccine preparations; planned, wrote, and revised the manuscript. RF contributed to planning of the immunization experiments and in revising the manuscript. CM (Head of the Health Technologies Division) was committed in the active search of funding and revised the manuscript.

#### REFERENCES

#### FUNDING

This work was partially supported by the Special Grant 2015 from MIUR (Italian Ministry of University and Research) to ENEA (Young Investigator Progect 'New therapeutic strategies for the treatment of cancer) and by the Research Project "Regione Lazio-Lazio-Innova," funded under the L.R. 13/08.

#### ACKNOWLEDGMENTS

The authors thank PBL Technologies and Prof. George Lomonosoff for the license to use the pEAQ-HT vector; Dr. Eugenio Benvenuto (ENEA) who contributed important reagents and for the availability of the BSL2 Containment Greenhouse; Mrs Elisabetta Bennici (ENEA) for plant material growth setting and maintenance and for general technical assistance; and Dr. Debora Giorgi (ENEA) for help in fluorescence microscopy. SM is deeply grateful to Dr. Vidadi Yusibov and Dr. Marina Skarjinskajia (Fraunhofer Center for Molecular Biotechnology, DE, USA) for teaching and sharing advice and protocols concerning hairy root culture technology.

activity of a plant-derived HPV16 E7 vaccine. *Int. J. Immunopathol. Pharmacol.* 19, 785–795.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Massa, Paolini, Marino, Franconi and Venuti. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana

Ee Leen Pang1 , Hadrien Peyret2 , Alex Ramirez3 , Hwei-San Loh1 \*, Kok-Song Lai4 , Chee-Mun Fang5 , William M. Rosenberg3 and George P. Lomonossoff2 \*

#### Edited by:

Suvi Tuulikki Häkkinen, VTT Technical Research Centre of Finland Ltd., Finland

#### Reviewed by:

Markus Sack, RWTH Aachen University, Germany Hugh S. Mason, Arizona State University, United States Johannes Felix Buyel, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Germany

#### \*Correspondence:

Hwei-San Loh<EMAIL_ADDRESS>George P. Lomonossoff<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

Received: 01 December 2018 Accepted: 26 March 2019 Published: 16 April 2019

#### Citation:

Pang EL, Peyret H, Ramirez A, Loh H-S, Lai K-S, Fang C-M, Rosenberg WM and Lomonossoff GP (2019) Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana. Front. Plant Sci. 10:455. doi: 10.3389/fpls.2019.00455 1 School of Biosciences, University of Nottingham Malaysia, Semenyih, Malaysia, 2 Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom, 3 iQur Limited, London, United Kingdom, 4 Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Malaysia, 5 Division of Biomedical Sciences, School of Pharmacy, University of Nottingham Malaysia, Semenyih, Malaysia

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium-mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAgcEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Keywords: virus-like particles, epitope display, hepatitis B core antigen, dengue envelope glycoprotein, envelope glycoprotein domain III, dengue vaccine, tandem core technology

# INTRODUCTION

fpls-10-00455 April 13, 2019 Time: 8:57 # 2

The alarming rise of dengue epidemics has been highlighted to affect over 40% of the world population (Brady et al., 2012). The disease can be manifested as undifferentiated dengue fever or life-threatening conditions such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Murrell et al., 2011). Classified under the Flaviviridae virus family, dengue virus (DENV) is a single-stranded, positive-sense nonsegmented RNA virus with 40–50 nm enveloped particles (Guzman et al., 2010). The 10.6 kbp viral genome encodes a polypeptide that is processed into structural proteins [capsid (C); envelope glycoprotein (E); and precursor membrane (prM)] and non-structural biomolecules (NS1, 2A, 2B, 3, 4A, 4B, and 5) (Henchal and Putnak, 1990). During virus assembly, the C protein encapsidates the viral RNA to form nucleocapsid particles whereas the prM assists the folding of surface-exposed E glycoprotein (Whitehead et al., 2017). Transmission of the endemic virus has been observed in over 100 countries. The incidence rate has expanded 500-fold, spreading from Southeast Asia to the Americas and Western Pacific within just a-half century (Pang and Loh, 2016). Global distribution of dengue disease is strongly influenced by urbanization, demographic, and environmental factors including global warming which has enabled Aedes mosquitoes to survive beyond their usual distribution. Concerns are also driven by increasing movement of travelers (Pang and Loh, 2016). The annual incidence has grown dramatically in recent decades, in which 390 million cases are predicted per annum and 96 million amongst these cases manifest an apparent clinical or sub-clinical severity (Bhatt et al., 2013). Out of these, it was reported that 500,000 people were hospitalized with severe dengue and approximately 2.5% of them would succumb to the disease (World Health Organization [WHO], 2017). The reported figure may be under-estimated due to the passive surveillance system adopted by many countries (Runge-Ranzinger et al., 2014).

To date, no specific medication is available for dengue treatment. Current clinical practices mainly rely on administration of paracetamol and intravenous fluid, together with close monitoring of the haematocrit and platelet levels (Anfasa et al., 2015). The absence of specific drugs and lack of confidence in currently marketed vaccines have also driven the public's reliance on folk remedies that are yet to be scientifically proven. Thus, development of a dengue vaccine is still being aggressively pursued to address the unmet medical needs of people in tropical regions (Pang and Loh, 2017). For subunit vaccine production, the dengue E glycoprotein has been the most studied antigenic determinant. Its structure is organized into three ectodomains (EDIII), and serves to assist attachment and entrance into host cells via receptors (Faheem et al., 2011). The immunoglobulin-like domain III (EDIII) is an ideal immunogen as it harbors receptor binding motifs that can elicit neutralizing monoclonal antibodies production (Crill and Roehrig, 2001). In recent years, EDIII has been expressed as a consensus sequence (cEDIII) aligned between four DENV serotypes (Chiang et al., 2011; Kim et al., 2012, 2015, 2016, 2017; Huy and Kim, 2017). A proof-of-concept study showed that cEDIII could inhibit the infectivity of four dengue serotypes simultaneously following mice immunization (Leng et al., 2009). Therefore, this sequence was adopted with the aim of conferring protection against all four co-circulating dengue serotypes.

Virus-like particles (VLPs) have gradually emerged as vaccine delivery vehicles that are spontaneously assembled from viral structural proteins. These are multimeric structures that can directly stimulate immune cells by mimicking the threedimensional conformation of native viruses. Moreover, VLPs are devoid of infectious genetic material which makes them inherently safer than attenuated or inactivated virus preparations (Pang, 2018). VLPs are known to elicit higher B- and T-cell immune responses and hence lower dosage is usually sufficient (Gamvrellis et al., 2004; Roy and Noad, 2008). The repetitive array of protein subunits in VLPs has the potential to confer superior properties as a stand-alone vaccine when compared to those of recombinant subunit-based ones which may be weak immunogens despite the use of an adjuvant (Noad and Roy, 2003). All these features make VLPs a premium platform for the production of a safe and effective vaccine (Jain et al., 2015).

In this study, hepatitis B core antigen (HBcAg) was exploited for cEDIII epitope display. Early work on HBcAg was done by Clarke et al. (1987) to produce foot and mouth disease virus fusion particles with proven serological response in guinea pigs. The icosahedral VLPs are shaped by association of two HBcAg monomers into 90 (T = 3) or 120 (T = 4) dimers, with a hairpin structure bridged by c/e1 loops to form protruding spike (Crowther et al., 1994). The flexibility of inserting foreign sequence into the immunodominant c/e1 loop for surface exposure, while retaining its antigenic properties, is ideal for antigen presentation (Pumpens and Grens, 1999). In fact, insertion in the c/e1 region was shown to impart a stronger protective response compared to N- and C-terminal fusions to the core particles (Koletzki et al., 2000). Thus, the cEDIII gene was incorporated into the c/e1 loop for the benefit of maximized exposure on protruding spikes of the assembled VLPs. "Tandem Core" technology was adopted here to produce the chimeric HBcAg VLPs. It has been shown that this technology which covalently links two core proteins into dimer forms (Peyret et al., 2015) can alleviate potential steric hindrance between two inserts at each immunodominant c/e1 loop of the dimer interface and thus promotes chimeric VLP assembly. This technology has recently been applied successfully to make other viral vaccine candidates (Ramirez et al., 2018).

In the context of molecular pharming, plants have certain advantages when compared to bacterial and animal expression systems relating to lower production cost, rapid scalability, biocontainment warranty, and eukaryotic processing machinery (Sack et al., 2015; Tschofen et al., 2016; Loh et al., 2017). The emergence of transient expression systems has ultimately sped up the process, whereby rapid candidate screening and largescale production are achievable within days (Thuenemann et al., 2013). Moreover, there is evidence that plants may be a better choice than bacteria for the production of tandem core-based VLPs (Peyret et al., 2015). This study aimed to demonstrate the production of chimeric HBcAg particles displaying dengue cEDIII epitopes (tHBcAg-cEDIII) using a plant-based system,

with the hope of developing a novel VLP-based vaccine against the deadly dengue disease. Given that DENV clade replacements had been detected in recent years (Teoh et al., 2013), the development of a vaccine that can provide a consistent protection in the long run will be highly valued (Pang, 2018). Moreover, generating a vaccine based on the single consensus cEDIII antigen should reduce the underlying cost as it obviates the need to test the best tetravalent formulation from monovalent components of each DENV serotype.

# MATERIALS AND METHODS

#### Recombinant Vector Construction

The 103 amino acid residues of cEDIII consensus sequence used (see **Supplementary Material**) were based on the alignment of four DENV serotypes as described by Leng et al. (2009). The synthesized gene sequence of cEDIII was codon-optimized (GeneArt, United States) for the expression in Nicotiana benthamiana. Primers were designed to incorporate AvrII and SbfI restriction sites (underlined) at 50 and 30 ends of the cEDIII gene (cED3F: 50 - GAATACCTAGGAAGGGAATGTCATACGCTATGTGTACTG GAAAG-30 ; cED3R: 50 -CATTGCCTGCAGGTGAAGATCCCT TCTTGAAC-30 ) for sub-cloning into pEAQ-HT::tHBcAg-VHH2, a plasmid derived from pEAQ-τGFP (GenBank accession number KM396759, Peyret et al., 2015) which contains long glycine-rich linkers [(GGS)n] with unique restriction sites at the c/e1 loop region of the downstream second core (Core II) for ease of cloning. Following heat-shock transformation of competent Escherichia coli, putative clones harboring the expression vector (pEAQ-HT::tHBcAg-cEDIII) were screened and verified by sequencing (Eurofins, Germany). **Figure 1** illustrates the expression cassette and corresponding recombinant vector used in this study. Additional information on the construct sequence can be found in the **Supplementary Material**.

## Agrobacterium tumefaciens Transformation and Plant Infiltration

Nicotiana benthamiana plants were grown on custom-mixed soil comprising of peat, 2.5 kg/m3 dolomite limestone, 1.3 kg/m3 base fertilizer, 2.7 kg/m3 Osmocote R (applied every 3–4 months), 0.3 kg/m3 Exemptor R , and 0.25 kg/m3 wetter in a controlled environment of 16-h photoperiod generated by 400 W sodium lamps, 24◦C and 70% relative humidity. Plants at 5–6 weeks old (until they reached the pre-flowering stage) were used in the study. The recombinant vector, pEAQ-HT::tHBcAg-cEDIII was introduced into Agrobacterium tumefaciens strain LBA4404 via electroporation. Transformed colonies were then selected from agar plates supplemented with 50 µg/ml kanamycin and 50 µg/ml rifampicin. The protocol from Sainsbury et al. (2012) was adopted here. Agrobacterial suspensions were cultured at 28◦C in a shaking incubator (200 rpm) for 24–48 h. The agrobacterial cells were harvested by 4,000 × g centrifugation for 10 min and resuspended in MMA solution [10 mM MES (2-[N-morpholino]ethanesulfonic acid) at pH 5.6, 10 mM MgCl2 and 100 µM acetosyringone] to a final OD600 of 0.4. The abaxial side of the developed N. benthamiana leaves was pricked and infiltrated with the agrobacterial suspensions using a needleless syringe. Time-course evaluation of the selective leaves of infiltrated plants was performed until 9 days post-infiltration (dpi). Control agrobacterial suspensions containing the empty pEAQ-HT vector (Sainsbury et al., 2009) without a gene insert was included to compare with pEAQ-HT::tHBcAg-cEDIII for physical observation of plants post-infiltration.

#### Protein Extraction

Small-scale extraction was conducted to test for protein expression and accumulation. Approximately 100 mg of the infiltrated leaf was harvested and homogenized with a 1 ⁄4-inch ceramic bead (MP Biomedicals, United States) in 3× volume of extraction buffer [100 mM sodium phosphate at pH 6.8, 150 mM NaCl, 0.1% Triton-X and protease inhibitors (Roche, Switzerland)]. The Omni Bead Ruptor 24 homogeniser (Camlab, United States) was adjusted at speed setting 4 for 30 s for tissue homogenization. Samples were then centrifuged at 16,000 × g for 10 min and the supernatant was kept as soluble protein (SP) fraction. Nevertheless, insoluble protein (IP) fraction could be extracted from the pellet through boiling with protein denaturing buffer [NuPAGE LDS buffer (Life Technologies) mixed 3:1 with 2-mercaptoethanol] and centrifugation at 16,000 × g for 10 min. To check for protein integrity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted at 200 V for 50 min and stained with InstantBlue (Expedeon, United Kingdom).

For large-scale extraction, infiltrated leaves were excised, weighed and homogenized with 3× volume of the same extraction buffer in a Waring blender. Crude extracts were filtered through a layer of Miracloth before subjecting to centrifugation (20,000 × g) at 4◦C for 20 min using an SS34 rotor (Thermo Fisher Scientific, United States). The clarified supernatant was filtered over 0.45 µm syringe filters prior to subsequent purification procedures.

# Purification of Virus-Like Particles (VLPs)

Virus-like particles samples were subjected to a two-stage purification process as described by Peyret (2015). Firstly, clarified extracts were overlain above different concentrations of sucrose solution; specifically 6 ml of 25% (w/v) and 2 ml of 70% (w/v). Double-layered sucrose cushions were prepared in UltraClear ultracentrifuge tubes (Beckman Coulter, United States) and centrifuged in a Surespin 630/36 swing-out rotor (Thermo Fisher Scientific, United States) at 167,000 × g, 4 ◦C for 2.5 h. The gradient was fractionated by piercing the bottom of the tube with a needle and recovering the bottom and interface fractions. These fractions were then dialyzed thoroughly against 200 mM ammonium bicarbonate buffer (pH 8.0) overnight. Next, samples were concentrated using SpeedVac (Thermo Fisher Scientific, United States), and loaded onto a Nycodenz step gradient extending from 60 to 20% (w/v), with 2 ml of each concentration in UltraClear ultracentrifuge tubes (Beckman Coulter, United States). High-speed centrifugation was operated at 274,000 × g using the TH-641 swing-out rotor (Thermo Fisher Scientific, United States) for 20 h at 4◦C. Bottom

of the tubes was punctured with a needle and divided into successive fractions. These fractions were assayed with Western blotting to determine the distribution of the desired particles.

#### Purification of cEDIII Protein

Recombinant cEDIII protein was used in this study as an antigen positive control for immunization work. In brief, the plant-expressed protein was harvested at 6 dpi following agroinfiltration with pEAQ-HT::PR1a-cEDIII-sGFPH-KDEL (Pang, 2018). As the recombinant protein was produced as a cleavable fusion to green fluorescent protein (sGFP) with a histidine tag, first-step isolation was achieved via native immobilized metal affinity chromatography (IMAC) (agarose resin derivatized with nickel ion-nitrilotriacetic acid; Qiagen, Germany). Following AcTEVTM protease digestion (Thermo Fischer Scientific, United States), the final product (cEDIII alone) was harvested from the flow-through fraction of a second IMAC procedure. The cEDIII protein was dialysed against phosphate-buffered saline (PBS) prior to the downstream testing.

# Western Blotting Analysis

For immunoblotting analysis, the electrophoresed proteins were electroblotted onto a nitrocellulose membrane (GE Healthcare, United States) via wet transfer at 100 V for 1 h. Blotted membrane was blocked with 5% (w/v) milk powder in PBS containing 0.05% (v/v) Tween-20 (PBST) for at least 1 h. The membrane was subsequently incubated with either mouse anti-HBcAg monoclonal antibody (10E11; Abcam, United Kingdom) (1:4,000 dilution) or mouse anti-DENV 1–4 monoclonal antibody [D1- 11(3); Thermo Fisher Scientific, United States] (1:2,000 dilution) for 1 h. Then, the membrane was washed three times with PBST at 5-min intervals before incubation with mouse immunoglobulin

G (IgG) horseradish peroxidase (HRP)-conjugated secondary antibody (M30107; Invitrogen, United States) (1:10,000 dilution). Washing steps were repeated three times before the blot was soaked in chemiluminescence solution and detected using ImageQuant LAS 500 (GE Healthcare, United States).

# Transmission Electron Microscopy (TEM) Examination

All VLP samples (tHBcAg-cEDIII) were dialyzed against PBS using Float-A-Lyzer (Sigma, United States). Approximately 10 µl of the VLPs sample was adsorbed onto copper-palladium grids, washed with sterile distilled water and negatively stained with 2% (w/v) uranyl acetate. As an experimental control, the tandem core particles without gene insert (tEL, Peyret et al., 2015) was also examined. Both particles (tHBcAg-cEDIII and tEL) were viewed using the FEI Tecnai 20 transmission electron microscope (FEI, United States).

## Protein Quantification

Pierce modified Lowry protein assay kit was used according to the manufacturer's instruction (Thermo Fisher Scientific, United States) for protein quantification. The absorbance of sample replicates and diluted albumin standards was then measured at 750 nm using CLARIOstar microplate reader (BMG LABTECH, Germany). The purified tHBcAg-cEDIII VLPs were also analyzed by SDS-PAGE (refer to **Supplementary Figure S1** for this SDS-PAGE profile).

#### Mouse Immunization

Two independent animal immunization experiments were conducted using female BALB/c mice (Envigo, United Kingdom) at 6–8 weeks old. For each experiment, at least four mice per group were immunized with 5 µg of purified VLP particles containing the cEDIII antigen insert (tHBcAg-cEDIII) mixed with 50 µl of Imject Alum (Thermo Fisher Scientific, United States) in a final volume of 100 µl made up by sterile saline solution. As a positive control group for cEDIII antigen, mice were immunized with 5 µg of recombinant cEDIII protein purified by using nickel ion-nitrilotriacetic acid agarose resin (Qiagen, Germany). Another group of mice was immunized with 5 µg of empty VLPs without cEDIII insert (tEL), which served as a negative control for cEDIII antigen. In addition, there was another control group of mice were injected with 100 µl of sterile normal saline solution. Three immunizations were delivered intra-peritoneally, administered 1 week apart. Tail bleeds were performed to check for seroconversion 4 weeks after primary immunization, and terminal bleeds at the completion of experiment at 9 weeks post-immunization. All animal works were done in compliance with United Kingdom Home Office approved animal protocols under license PPL 70/7376.

# Enzyme-Linked Immunosorbent Assay (ELISA)

Detection of antigen-specific IgG antibodies was performed by ELISA. Briefly, 96-well Nunc Maxisorp plates (Sigma, United States) were coated with 1 µg/ml pure cEDIII protein in carbonate bi-carbonate buffer overnight at 4◦C. Coated plates were washed three times with PBS-Tween 20 (0.05% v/v) and blocked with 10% skimmed milk solution (Sigma, United States) for 1 h at 37◦C. Primary sera were serially diluted (twofold) in 2.5% (w/v) milk solution from 1:100 to 3,200. All samples were added to duplicate test wells, incubated at 37◦C for 1 h then washed three times as mentioned before. Goat-anti-mouse IgG-peroxidase secondary antibody was added at 1:2,500 dilution (Sigma, United States) and incubated at 37◦C for 1 h then washed three times again. 3, 30 , 5, 50 -Tetramethylbenzidine substrate (Sigma, United States) was added to each well for 20 min and reaction was stopped with 1M H2SO4. Absorbance at 450 nm with a 630 nm correction was read using the SpectraMAX 190 plate reader (Molecular Devices, United States). Unpaired Student's t-test was conducted to compare the immunization groups of mice receiving either recombinant cEDIII protein or tHBcAg-cEDIII VLPs against the empty tandem core VLPs control (tEL) in order to determine the significance level of cEDIII-specific IgG produced. The significant difference of cEDIII-specific IgG levels between cEDIII and tHBcAg-cEDIII groups was also compared. The significance levels were denoted at p ≤ 0.001 with ∗∗ and p ≤ 0.0001 with ∗∗∗ in the graph.

# RESULTS

## Expression of Recombinant tHBcAg-cEDIII Construct in N. benthamiana

Following agroinfiltration, N. benthamiana leaves were collected on 6 dpi and analyzed by Western blotting technique to check for expression. Western profiles indicated that the tHBcAgcEDIII proteins had been expressed well in N. benthamiana infiltrated with pEAQ-HT::tHBcAg-cEDIII (**Figure 2**), based

FIGURE 2 | Expression profiles of tHBcAg-cEDIII protein in N. benthamiana analyzed at 6 dpi, which was predominantly obtained in insoluble form. Immunoblot detection of tHBcAg-cEDIII proteins at ∼54 kDa in size (black arrow) was performed using anti-HBcAg monoclonal antibody. Lane M: SeeBlue R Plus2 Pre-Stained Standard; Lane 1: SP extracted from pEAQ-HT::tHBcAg-cEDIII-infiltrated leaf disks; Lane 2: IP extracted from pEAQ-HT::tHBcAg-cEDIII-infiltrated leaf disks; Lane 3: SP extracted from pEAQ-HT-infiltrated leaf disks; Lane 4: IP extracted from pEAQ-HT-infiltrated leaf disks.

on the band at around 54 kDa which was not present in the pEAQ-HT-infiltrated plant leaf sample. However, the yield of soluble tHBcAg-cEDIII (SP) was very low (barely detectable) compared to the insoluble tHBcAg-cEDIII (IP) fraction. It was estimated that over 90% of the tHBcAg-cEDIII produced by the plant was insoluble.

## Kinetic Expression and Physical Observations on the Infiltrated N. benthamiana

A time-course evaluation was performed to determine the optimal harvest time of tHBcAg-cEDIII protein for subsequent extraction and purification procedures. In general, symptoms of leaf chlorosis were observed from 7 dpi onward in all pEAQ-HT::tHBcAg-cEDIII-infiltrated plants, which led to visible necrosis by 9 dpi. This leaf chlorosis symptom was not observed with the empty vector, pEAQ-HT-infiltrated plants. A representative series of a pEAQ-HT::tHBcAg-cEDIIIinfiltrated leaf is shown in **Figure 3A**. Concurrently, infiltrated leaves were harvested on a daily basis to monitor the accumulation of soluble proteins. As illustrated in **Figure 3B**, increasing amounts of tHBcAg-cEDIII SP could be seen from 6 dpi onward. As advanced necrosis was observed on 9 dpi in these infiltrated plants, it was deemed sensible to set 8 dpi as the optimal harvest time for soluble tHBcAgcEDIII protein.

# Purification of Chimeric Tandem Core Particles Displaying cEDIII Epitopes

Leaves infiltrated with the recombinant vector, pEAQ-HT::tHBcAg-cEDIII were harvested on 8 dpi for large-scale purification. The first isolation step via sucrose cushion yielded 70% sucrose and interface fractions, which were collected for maximal recovery of VLPs present in the sample (**Figure 4A**). Further purification by Nycodenz gradient gave rise to a single grayish band (**Figure 4B**). From here, the band was separated from the sedimentation of green impurities toward the top of the gradient. The single band was confirmed to be tHBcAg-cEDIII VLPs distribution following Western blotting analysis (**Figure 4C**), with sedimentation point estimated at around 40% Nycodenz concentration. The yield of purified tHBcAg-cEDIII VLPs was in the range of ∼12–16 mg/kg, which is comparable to that of purified recombinant cEDIII (∼13–14 mg/kg).

Following the purification process, TEM imaging analysis revealed that plant-produced tHBcAg-cEDIII assembled into VLPs (**Figure 5A**), which were visualized as a mixture of corelike particles of slightly different sizes. These particles exhibited an irregular spherical morphology that characterizes tandem core particles displaying a heterologous sequence in the c/e1 loop. The diameters of these particles ranged from 32 to 35 nm, with an average particle size of ∼34 nm. As illustrated in **Figure 5B**, purified tEL sample (empty tandem core VLPs without insert) formed smaller, more evenly sized particles, with an average size of ∼27 nm in diameter. Comparatively, tHBcAg-cEDIII particles appeared to be somewhat larger than the empty tEL particles.

# Immunogenicity of Chimeric Tandem Core Particles Displaying cEDIII Epitopes

The seroconversion results in BALB/c mice immunized with the VLP-based dengue vaccine candidate are presented in **Figure 6**. cEDIII-specific IgG antibody was successfully detected in mice immunized with tHBcAg-cEDIII VLPs and recombinant cEDIII protein alone. However, the IgG antibody level elicited by the recombinant cEDIII protein alone was higher than that of tHBcAg-cEDIII VLPs at all 1:100 to 1:3,200 dilutions tested (**Figure 6A**). The control groups (normal saline and tEL) did not trigger any cEDIII-specific IgG antibody responses and this observation has indicated the absence of pre-exposure of DENV (cEDIII). **Figure 6B** shows that cEDIII-specific IgG antibody level elicited by tHBcAg-cEDIII VLPs was significantly higher than that of the negative control, tEL-immunized mice (p ≤ 0.0001 at

week 4 and week 9). However, when comparing between cEDIII and tHBcAg-cEDIII groups, the specific IgG level of cEDIIIimmunized group mice was in fact significantly higher (p ≤ 0.001 at week 4 and p ≤ 0.0001 at week 9). In general, our data showed that IgG antibody levels appeared to peak after 4-week post-immunization, then began to wane but were still detectable more than 2 months after immunization. These results suggest that the cEDIII antigen inserted into the c/e1 loop of tandem core particles was still immunogenic and able to elicit a specific humoral response.

#### DISCUSSION

Development of VLPs as a highly structured form of subunit vaccine has been increasingly explored in recent years, whereby they present themselves as multimeric structures that mimic native virions. Production of dengue VLPs had been attempted previously (Sugrue et al., 1997; Liu et al., 2010; Zhang et al., 2011; Mani et al., 2013); however, several shortfalls were identified such as the neutralizing antibody responses acquired by mice were generally weak and co-expressing the DENV prM and EDI/EDII can trigger antibody-dependent enhancement (ADE) phenomenon. This is where pre-existing, non-neutralizing antibodies from an initial DENV infection can bind with the new infecting serotype and infect Fc gamma receptor (FcγR) bearing cells to gain entry into host cells (Bäck and Lundkvist, 2013). Hence, ADE is often affiliated with disease aggravation and known to be the strongest risk factor of DHF/DSS development (Kliks et al., 1989). Therefore, generating a durable immunity against all four serotypes of DENV by using serotype-specific VLPs would require the expression of four monovalent VLPs together in the optimal tetravalent formulation, which would represent a significant technical challenge.

Because of this, the rationale for the work presented here was to produce chimeric VLPs which are able to self-assemble while presenting a consensus DENV antigen on their surfaces. In this study, the VLPs derived from HBcAg were utilized for cEDIII epitope display. As the stand-alone stability of DENV domain III has made it intrinsically different from other parts of the E protein (Soares and Caliri, 2013), it is believed that cEDIII can behave as an independent entity and would not interrupt the assembly of viral particles (Pang, 2018). The resulting chimeric HBcAg VLPs are therefore expected to display a high density of cEDIII epitopes on 90 or 120 copies of core protein dimers per particle.

In this study, "Tandem Core" technology was adopted due to the concern that two copies of cEDIII inserts at HBcAg dimer interface might suffer from steric clashes which could abrogate particle assembly. Thus, it is anticipated that this strategy could resolve the steric constraints as VLPs are now assembled from the dimers of HBcAg protein, expressed from a single open reading frame coding for two copies of HBcAg that have been covalently linked (Peyret et al., 2015). The cEDIII gene was inserted into the immunodominant c/e1 loop of Core II (the C-terminal copy of HBcAg), so that this would minimize the disruption to VLPs assembly as the translation moved in 50 → 3 0 direction from the unmodified Core I. With this, the tandem core would have greater flexibility as only one of the two c/e1 loops on each dimer was decorated with cEDIII antigens (Pang, 2018).

The cEDIII gene expressed in this study was codon-optimized based on N. benthamiana preference to boost translation by recoding the rare codon in foreign gene with synonymous codon preferred by the expression host (Angov, 2011). Apart from that, a glycine and serine-rich linker was designed to avoid spurious interaction between the cEDIII inserts and core protein subdomains: this linker consisted of 15 amino acids on either side of the cEDIII insert (the exact sequence is shown in the **Supplementary Material**). As this approach was shown to be useful by Kratz et al. (1999), it was hoped that the (GGS)n linkers can help to minimize the steric constraints and stabilize the chimeric HBcAg VLPs expressing the cEDIII epitopes (Pang, 2018). Indeed, these linkers allowed the display of a nanobody protein inserted in Core II of plant-produced tandem cores

successfully assembled into viral particles.

(Peyret et al., 2015). Nevertheless, the length of the linkers was not optimized in the context of this work, and it is possible that higher yield and better solubility could be achieved upon optimization.

Based on the kinetic expression studies (**Figure 3**), it is postulated that the chimeric VLP construct does not confer significant toxicity to plants as early necrosis or apoptosis was not evident. The profiles were useful to gauge the ideal harvest time which is assessed based on the peak accumulation of soluble target protein, and the post-infiltration morphological distortions of the leaves. Necrotic tissues are usually avoided as they are flaccid and may contain higher amount of phenolics that could be introduced into downstream processing (Tanguy and Martin, 1972). In fact, the antimicrobial exudate produced by necrotic tissues can inhibit efficient colonization and gene delivery by Agrobacterium (Pitzschke, 2013). In this case, the optimal harvest time for the chimeric tHBcAg-cEDIII VLPs was determined to be around 8 dpi. A constant OD600 of 0.4 for the agrobacterial infiltration suspension was used throughout this study in line with the recommended range of OD600 at 0.3–0.4 as reported previously (Li, 2011; Pua et al., 2012; Shamloul et al., 2014). This is because high bacterial density tends to trigger hypersensitive response that can lead to tissue necrosis, whereas low amount of Agrobacterium may result in insufficient gene delivery (Leuzinger et al., 2013). As presented in this study, an acceptable level of transient gene expression was achieved at the chosen OD600 of 0.4, without severely triggering the hypersensitivity responses (Pang, 2018).

To purify the VLPs of interest, the procedures begin with a discontinuous two-step sucrose cushion to enrich the isolation of core particles from clarified lysate in a fast and reproducible manner (Peyret, 2015). After that, an additional isopycnic gradient was applied to complement the earlier technique in preparation of high purity particles (Brakke, 1961). Generally, Nycodenz is an inert chemical that can generate a self-forming gradient (Gugerli, 1984) and it worked well for the purification of tHBcAg-cEDIII VLPs as evidenced in **Figure 4**. TEM observation revealed that the assembly of particles was successful (**Figure 5A**). Instead of being uniformly shaped, tHBcAg-cEDIII particles appeared to be rather "knobbly" due to the epitopes protruding from HBcAg spikes. These VLPs, which ranged from 32 to 35 nm in diameter are somewhat larger than their empty counterpart labeled as tEL (**Figure 5B**). This type of surface morphology is in fact expected for tandem core particles displaying a heterologous sequence on their surface (Peyret et al., 2015). Such finding indicates that cEDIII epitopes were presented on the protruding spikes of HBcAg that retained the inherent propensity to fold into discrete VLPs. In fact, the size range of 35–40 nm for chimeric HBcAg VLPs with dengue EDIII epitopes produced in microbial cells, was previously reported by Arora et al. (2012, 2013).

To our knowledge, this is the first study that reports the successful production of chimeric HBcAg VLPs with dengue protein epitopes in a plant system. It was shown that the cEDIII epitopes remain immunogenic when presented on the VLP scaffold (HBcAg-cEDIII): the specificity of the IgG responses detected is demonstrated by the lack of cEDIIIreactive antibodies in the saline control and empty tHBcAg (tEL) groups (**Figure 6**). The specific IgG antibody levels induced by both HBcAg-cEDIII VLPs and recombinant cEDIII (positive control) were higher at week 4 post-immunization and declined thereafter as detected at week 9, which is consistent with the kinetics of a normal immune response (Leo et al., 2011). As the ELISA plates were coated with purified cEDIII protein, it is possible that the higher IgG antibody level detected in the recombinant cEDIII group could be due to an optimum antigenantibody match as compared to that of tHBcAg-cEDIII group. Additionally, the low antibody response (tHBcAg-cEDIII) could also be explained by the lower molar antigen dose received by the HBcAg-cEDIII-immunized mice as the antigen dose for immunization was normalized to a total protein concentration of 5 µg. Therefore, the actual dosage of cEDIII on the VLPs is much lower (in molar terms) than the subunit recombinant proteins. The tHBcAg-cEDIII is composed mainly of tHBcAg carrier by protein mass, and cEDIII only contributes to 103 out of the total 508 amino acids. Taken the suggestion of Whitacre et al. (2016), the amount of cEDIII presented in

relation to the entire chimeric particle size should be considered in future in order to gauge the optimal dose needed for in vivo study.

In any case, the results presented here highlight potential limitations to chimeric VLP vaccine development strategies. The recovered yield of tHBcAg-cEDIII VLPs is considered relatively low, as it was estimated that less than 10% of the tHBcAg-cEDIII protein formed soluble particles. Extraction under denaturing conditions was not attempted, since denaturing and refolding steps for such a complex structure (an assembly of 90 or 120 copies of a triple protein fusion) are unlikely to result in the proper formation of core-like particles. In this study, the tHBcAg-cEDIII protein was targeted to the cytosol, since this is the strategy that had previously been used successfully with tandem cores (Peyret et al., 2015), whereas, by contrast, the recombinant cEDIII protein alone was targeted to the endoplasmic reticulum (ER). Subcellular localization for tHBcAg-cEDIII protein was not attempted initially due to the concerns that distinct pH conditions in different plant organelles could abolish VLPs assembly as shown by van Zyl et al. (2016). Nevertheless, it has recently been shown that HBcAg VLPs are still capable of assembly despite localization to ER (Yang et al., 2017). In fact, accumulation of tHBcAg-cEDIII VLPs in the plant cytosol may have affected the antigen stability via improper disulphide bond formation in cEDIII. This issue is highlighted because

the correct folding of antibody recognition epitope on DENV EDIII relies upon the disulphide linkage (Suzarte et al., 2014). Given that the redox environment is highly regulated in the ER (Ellgaard, 2004), optimal oxidoreductase activity can be achieved to form proper disulphide bridges. The relatively low antibody response generated in mice immunized with tHBcAg-cEDIII VLPs as compared to recombinant cEDIII immunized group might therefore also be explained by the presence of VLPs with improper folding produced in the plant cytosol.

Hence, as the way forward, future optimizations to modulate protein yield and stability at the post-translational level may include attempting different extraction buffers to improve the solubility of crude extract as well as targeting the heterologous protein to a subcellular compartment. Researchers carrying out the future works would be well-advised to test the pre-immune sera from each individual mouse in order to increase confidence in any post-immunization results. The full benefits of VLP presentation should be explored further, as its three-dimensional structure has a higher potency to activate cell-mediated immunity including the cytotoxic T cell response, which is crucial for better viral clearance as reported by Yang et al. (2017). Besides, since cEDIII represents a consensus antigen, it would also be of great interest to test the cross-reactivity of polyclonal responses as well as the protection levels of tHBcAg-cEDIII VLPs against the different DENV serotypes.

### CONCLUSION

While there has been a previous initiative that used a yeast system to produce a chimeric VLP-based dengue vaccine candidate (Arora et al., 2013); development of a plantderived vaccine can offer scalability and safety advantages that revolutionize the accessibility of dengue vaccines (Pang and Loh, 2017). This is particularly important to address the alarming burden of dengue disease that has yet to meet a promising resolution. The adoption of "Tandem Core" technology was proven to be feasible although there is still room for improvements. Overall, a successful assembly of VLPs displaying a consensus dengue antigen has been achieved. The immunization data have shown that seroconversion to cEDIII is possible when mice are immunized with tHBcAg-cEDIII VLPs. The current findings have validated the viability of using a VLP system as an antigen-presentation platform; this warrants further investigation into its potential as a next-generation dengue vaccine.

# REFERENCES

# ETHICS STATEMENT

All animal works in this study were done in compliance with United Kingdom Home Office approved animal protocols under license PPL 70/7376.

#### AUTHOR CONTRIBUTIONS

H-SL, WMR, and GPL conceived and designed the study. ELP, HP, and AR performed the experiments. C-MF performed the statistical analysis. ELP and H-SL wrote the first draft of the manuscript. HP, AR, K-SL, and C-MF wrote sections of the manuscript. All authors contributed to manuscript revision and read and approved the current version.

# FUNDING

This research work was supported by John Innes Centre, United Kingdom; University of Nottingham Malaysia, Malaysia; and iQur Limited, United Kingdom. At John Innes Centre, this work was supported by the United Kingdom Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/L020955/1, the Institute Strategic Programme Grants, "Understanding and Exploiting Plant and Microbial Secondary Metabolism" (BB/J004596/1) and "Molecules from Nature – Enhanced Research Capacity" (BBS/E/J/000PR9794), and the John Innes Foundation.

# ACKNOWLEDGMENTS

The authors would like to thank the Ministry of Higher Education, Malaysia for supporting ELP in her Ph.D. study. Some of the contents presented in this paper are based on the ELP's Ph.D. thesis (University of Nottingham Malaysia).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.00455/ full#supplementary-material

and protective potential of core-derived particles. Virology 276, 364–375. doi: 10.1006/viro.2000.0540

**Conflict of Interest Statement:** GPL declares that he is a named inventor on granted patent WO 29087391 A1 that describes the system used for transient expression in this manuscript. The Tandem Core vaccine technology described in this paper is covered by the patent application PCT/GB01/01607 licensed by iQur Limited. AR and WMR are the current employees of iQur Limited.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Pang, Peyret, Ramirez, Loh, Lai, Fang, Rosenberg and Lomonossoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide

*Mark A. Jackson1 , Kuok Yap1 , Aaron G. Poth1 , Edward K. Gilding1 , Joakim E. Swedberg1 , Simon Poon2 , Haiou Qu1 , Thomas Durek1 , Karen Harris2 , Marilyn A. Anderson2 and David J. Craik1 \**

*1 Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia, 2Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia*

#### *Edited by:*

*Suvi Tuulikki Häkkinen, VTT Technical Research Centre of Finland Ltd, Finland*

#### *Reviewed by:*

*Joshua Lee Fuqua, University of Louisville, United States Somen Nandi, University of California, United States*

*\*Correspondence: David J. Craik<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science*

*Received: 10 December 2018 Accepted: 24 April 2019 Published: 15 May 2019*

#### *Citation:*

*Jackson MA, Yap K, Poth AG, Gilding EK, Swedberg JE, Poon S, Qu H, Durek T, Harris K, Anderson MA and Craik DJ (2019) Rapid and Scalable Plant-Based Production of a Potent Plasmin Inhibitor Peptide. Front. Plant Sci. 10:602. doi: 10.3389/fpls.2019.00602*