Harnessing Plant Chloroplasts for Oral Delivery of a Multi-epitope HPV Vaccine: Toward Cost-Effective Systemic and Mucosal Immunization

Harnessing Plant Chloroplasts for Oral Delivery of a Multi-epitope HPV Vaccine: Toward Cost-Effective Systemic and Mucosal Immunization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Harnessing Plant Chloroplasts for Oral Delivery of a Multi-epitope HPV Vaccine: Toward Cost-Effective Systemic and Mucosal Immunization Maryam Ehsasatvatan, Bahram Baghban Kohnehrouz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7801011/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Dec, 2025 Read the published version in Journal of Biological Engineering → Version 1 posted 15 You are reading this latest preprint version Abstract Human papillomavirus (HPV) is a major causative agent of cervical and other mucosal cancers, yet the distribution and accessibility of current prophylactic vaccines remain limited, especially in low- and middle-income countries (LMICs), due to high production costs, cold-chain dependency, and limited induction of mucosal immunity. To overcome these challenges, we designed a multi-epitope HPV vaccine (HPV_MEV) incorporating conserved cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes from diverse high- and low-risk HPV genotypes. The construct includes the Toll-like receptor 4 (TLR4) agonist RS09 to enhance innate immune activation and cholera toxin B subunit (CTB) as a mucosal adjuvant to facilitate uptake and presentation at mucosal surfaces. The codon-optimized gene was stably integrated into the chloroplast genome of Nicotiana tabacum using biolistic transformation. Molecular analyses confirmed site-specific integration, homoplasmy, and high-level expression of the recombinant antigen (~ 3.6 mg/g fresh weight; ~20.8% of total soluble protein). Immunogenicity was evaluated in BALB/c mice via intraperitoneal injection of purified antigen or oral gavage of lyophilized transplastomic leaf tissue. Oral administration elicited strong systemic IgG and mucosal IgA responses, with mucosal immunity surpassing that of the injected formulations. The chloroplast-produced HPV_MEV was comparable in immunogenicity to its E. coli-expressed counterpart, validating its structural and functional integrity. This study highlights the potential of plastid biotechnology for producing an effective, thermostable, needle-free oral HPV vaccine. By integrating rational antigen design with a scalable plant-based production and delivery platform, this approach offers a promising solution for accessible immunization against HPV and other mucosal pathogens in resource-limited settings. Human papillomavirus (HPV) Multi-epitope vaccine Chloroplast transformation Plant-based vaccine Oral immunization Mucosal immunity Figures Figure 1 Figure 2 Figure 3 Introduction Human papillomavirus (HPV) is a highly prevalent DNA virus with over 200 identified genotypes, primarily targeting epithelial tissues and predominantly transmitted through sexual contact (Doorbar, 2018 ). Persistent infection with high-risk HPV types, particularly HPV16 and HPV18, is the leading cause of cervical cancer and significantly contributes to other malignancies, such as oropharyngeal, anal, penile, and vulvar cancers (Arbyn et al., 2014 ; de Sanjose et al., 2018 ). Despite widespread screening programs and the availability of prophylactic vaccines, HPV-related diseases continue to pose a major global health burden, especially in low- and middle-income countries (LMICs), where access to vaccine remains limited (Bruni et al., 2021 ). According to the World Health Organization (WHO), more than 300,000 cervical cancer-related deaths occur annually, with over 85% of these deaths occurring in LMICs (SHEET, 2018 ). highlighting the urgent need for affordable and broadly accessible vaccine alternatives. Currently licensed HPV vaccines —Gardasil, Gardasil-9, and Cervarix— are based on virus-like particles (VLPs) composed of the L1 capsid protein produced in yeast or insect cell systems (Schiller & Lowy, 2012 ). These vaccines provide strong protection against the most common high-risk HPV types and have substantially reduced infection rates and precancerous lesions (Kjaer et al., 2009 ). However, their widespread implementation is limited by high production and distribution costs, cold chain requirements, intramuscular delivery, and limited type-specific coverage (Denny, 2012 ; Wang et al., 2022 ). To overcome these limitations, next-generation vaccine strategies aim to provide broader protection, reduce production costs, and enable noninvasive delivery platforms. One promising strategy involves multi-epitope vaccine (MEV) design, which uses immunoinformatics tools to identify conserved B-cell- and T-cell epitopes across multiple HPV genotypes (Chauhan et al., 2011 ). This approach allows for rational antigen design with enhanced immunogenicity, safety, and broad-spectrum coverage (Doytchinova & Flower, 2008 ). In our previous study, we designed a chimeric HPV multi-epitope vaccine (HPV_MEV) incorporating conserved L1-derived CTL, HTL, and B-cell epitopes fused with adjuvants (CTB and RS09 peptide) and a His-tag for purification. In silico analyses, including molecular docking, dynamics simulations, and immune simulations, confirmed the stability and immunogenic potential of the construct (Ehsasatvatan & Kohnehrouz, 2024 ). An effective and scalable expression system is essential to translate this design into a practical vaccine platform. Traditional microbial and mammalian cell expression systems are often limited by high production costs and biosafety concerns (Walsh & Walsh, 2022 ). In contrast, plant-based systems offer a safe, cost-effective, and scalable alternative for producing recombinant vaccines (Rybicki, 2017 ). Among these, chloroplast genetic engineering is particularly advantageous because of the high copy number of plastid genomes, site-specific integration, and absence of transgene transmission through pollen in most species (Daniell, 2007 ; Maliga, 2004 ). Protein yields can reach up to 70% of the total soluble protein (Oey et al., 2009 ), making chloroplasts highly efficient biofactories. Tobacco ( Nicotiana tabacum ) is widely used as a model for chloroplast transformation owing to its well-characterized plastomes and high biomass yield (Bock, 2015 ). Several pharmaceutical proteins, including human somatotropin, interferons, antibodies, and vaccine antigens, have been successfully produced in tobacco chloroplasts at industrially relevant scales (Davoodi-Semiromi et al., 2010 ; Ruhlman et al., 2010 ). Furthermore, chloroplast-expressed vaccine antigens can be delivered orally via the consumption of lyophilized plant tissue, which protects antigens from degradation and promotes uptake by gut-associated lymphoid tissue (GALT), eliciting both mucosal and systemic immune responses (Chan & Daniell, 2015 ; Kwon & Daniell, 2016 ). Oral delivery of chloroplast-derived vaccines has demonstrated efficacy in preclinical models of cholera, tuberculosis, and influenza (Kwon et al., 2013 ; Pantazica et al., 2021 ), and these vaccines also exhibit remarkable thermostability, eliminating the need for refrigeration (Daniell et al., 2019 ; Ehsasatvatan & Kohnehrouz, 2023 ). In this study we aimed to express the immunoinformatics-derived HPV_MEV antigen in tobacco chloroplasts and evaluate its ability to elicit both systemic and mucosal immune responses following oral administration in a murine model. By combining rational immunogen design with high-yield plant expression, this platform critical limitations in current HPV vaccine accessibility and distribution, offering a promising path toward cost-effective, orally administrable vaccines for HPV and other mucosal pathogens. Materials and Methods Design and Construction of Plastidial Expression Vector The HPV_MEV construct (GenBank Accession number: PV057300) was previously designed using an immunoinformatics-based approach, incorporating conserved immunodominant epitopes derived from the L1 protein of both high-risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) and low-risk (HPV6, 11, 42, 43, 44) HPV genotypes. The final vaccine sequence includes cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell linear epitopes, interlinked with appropriate flexible linkers (AAY, GPGPG, and KK), and fused with two molecular adjuvants: the TLR4 agonist RS09 at the N-terminus and the cholera toxin B subunit (CTB) at the C-terminus. A 6×His-tag was appended to facilitate protein detection and purification (Ehsasatvatan & Kohnehrouz, 2024 ). Immunomolecular analyses indicated that the vaccine candidate exhibited promising antigenicity and immunogenicity, lacked allergenic and toxic properties, and demonstrated potential to elicit robust immune responses without adverse effects. In order to high-yield expression of the designed multi-epitope vaccine (HPV_MEV) in the tobacco plastid genome, the codon-optimized nucleotide sequence of the antigen was synthesized based on its amino acid composition using the EMBOSS Backtranseq tool ( https://www.ebi.ac.uk/jdispatcher/st/emboss_backtranseq ) (Madeira et al., 2024 ). The synthetic HPV_MEV gene was flanked with Nco I and Xho I restriction sites and cloned into the pPRV-DARPin vector (Ehsasatvatan et al., 2022b ), replacing the DARPin G3 coding region. This chloroplast transformation vector, derived from pPRV111A (Zoubenko et al., 1994 ), facilitates homologous recombination into the rps12/7–rrn16 intergenic spacer region of the tobacco plastome. For robust transgene expression, the HPV_MEV gene was placed under the control of the plastid ribosomal RNA operon promoter (Prrn), fused to the T7g10 5′ untranslated region (UTR) to enhance translation, and terminated by the E. Coli rrnB 3′ UTR. Selection of transplastomic events was enabled by the spectinomycin resistance gene ( aadA ) driven by appropriate regulatory elements. This chloroplast-compatible expression cassette was subsequently used for biolistic transformation of Nicotiana tabacum chloroplasts. Cloning Expression and Purification of HPV_MEV in Bacterial Vector The HPV_MEV coding sequence was inserted into the pET-28a (+) vector (Novagen) using Nco I and Xho I restriction sites. To obtain purified HPV_MEV, the plasmid was introduced into the BL21 (DE3) cells. Upon the cell culture reaching an optical density (O.D.600) of approximately 0.8, protein expression was induced with 1mM isopropyl β-D-thiogalactoside (IPTG), and the cells were incubated at 37°C with agitation. HPV_MEV protein was subsequently purified using Ni 2+ -NTA affinity chromatography. Fractions containing the purified protein were pooled and dialyzed against 10mM HEPES buffer (pH 8.0) with 10% glycerol. The purified samples were analyzed using 15% SDS-PAGE and electroblotting. Plant material, Growth Conditions and Chloroplast Transformation Nicotiana tabacum cv. Perega plants were grown under sterile conditions on medium devoid of growth regulators. The medium consisted of MS salts, B5 vitamins, 3% (w/v) sucrose, and 0.7% (w/v) agar with a pH of 5.8. Plants were maintained at 24 ± 2°C with a 16-hour light cycle at 40 µmol photons m − 2 s−1 . Fully developed, dark green leaves were subjected to bombardment using tungsten particles (0.7 µm) coated with 1 µg pPRV-HPV_MEV plasmid, using a PDS-1000/He Biolistic gun (Bio-Rad, California, USA) as previously outlined (Ehsasatvatan et al., 2022a ). Shoots resistant to spectinomycin were isolated on the RMOP medium containing 500 mg/l spectinomycin dihydrochloride. These resistant shoots were tested on a medium containing both spectinomycin and streptomycin (500 mg/l each). Shoots that showed positive results underwent three cycles of regeneration on selective medium containing only spectinomycin. The plants were then rooted in a medium composed of MS salt, B5 vitamins, 3% (w/v) sucrose, 0.7% (w/v) agar, and 500 mg/l spectinomycin under aseptic conditions. Molecular Analysis of Putative Transplastomic Plants Leaf samples (100–150 mg) from wild-type and in vitro transplastomic plants expressing HPV_MEV were subjected to DNA extraction using the CTAB method (Murray & Thompson, 1980 ). To detect the inserted HPV_MEV gene in the tobacco plastid genome, PCR amplification was conducted using the primer pair HPV-F (5ˈ-GAAGCTGCTGCTAAAGTATCTGG-3ˈ) and HPV-R (5ˈ-GCAGCAATAGCATGAGGAGTT-3ˈ). The correct insertion of the expression cassette into the targeted plastid genome region was verified through PCR using 16S-F (5ˈ-AACTAAACACGAGGGTTGC-3ˈ) and aadA-R (5ˈ-AAGAATTTGTCCACTACGTGA-3ˈ) prime set. Figure 1 shows the primer locations used in this study. To further confirm the homoplasmy status of the transplastomic plants, Southern hybridization was performed. This technique involved digesting leaf genomic DNA (2 µg) with Bgl II (NEB, USA) and probing it with 232 bp DNA fragments derived from the rrn16 flanking sequence. The DIG-high prime DNA labeling and detection starter kit II was used for this process, following the manufacturer's instructions (Roche, Germany). Plants that were determined to be homoplasmic were selected for protein analysis. Immunoblot Detection of Vaccine Candidate Total soluble protein (TSP) was extracted from transplastomic tobacco ( N. tabacum ) leaves expressing the HPV_MEV antigen. Leaf tissues were harvested and ground to a fine powder in liquid nitrogen. The powdered material was resuspended in extraction buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 0.05% Tween-20, 2 mM PMSF, and a plant-compatible protease inhibitor cocktail (pH 8.0). Homogenates were incubated on ice for 30 minutes and subsequently clarified by centrifugation at 15,000 × g for 30 minutes at 4°C. The total protein concentration was determined using the Bradford protein assay. Protein samples were resolved on 15% SDS and transferred to a PVDF membrane. Western blot analysis was conducted employing rabbit anti-His-tag primary antibody at a dilution of 1:1,000 and goat anti-rabbit conjugated with horseradish peroxidase (HRP) secondary antibody at a dilution of 1:10,000. Protein bands were subsequently visualized using DAB peroxidase substrate solution. The total soluble protein extracted from wild-type plants served as a negative control. Estimation of Vaccine Candidate Concentration in TSP The concentration of HPV_MEV in the transplastomic plants was determined using enzyme-linked immunosorbent assay (ELISA). The assay was conducted by coating ELISA 96-well microplates with 50 ng/well of total soluble proteins extracted from transplastomic and wild-type leaves and incubating them overnight at 4°C. Subsequently, the plates were washed and blocked with a solution containing 1% BSA in 1X PBS buffer and 0.1% Tween 20 for 1 h at 37°C. After three washes, the wells were treated with rabbit anti-His-tag antibody (1:1000 dilution in blocking buffer) for 2 h at 37°C. After additional washing, the plates were incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) at a 1:10,000 dilution in blocking buffer for 1 h at 37°C. The plates were washed three more times before the addition of tetramethylbenzidine (TMB) substrate (Bio-Rad), allowing color development for 10 min. The reaction was terminated using 2 M H 2 SO 4 and the absorbance was measured at 450 nm using an ELISA reader. To estimate the expression levels of the HPV_MEV antigen in transplastomic plant samples, a standard calibration curve was established using serial dilutions of purified E. coli -expressed HPV_MEV protein. All measurements were performed in triplicates. Purification of Vaccine Candidate Total soluble protein from transplastomic tobacco leaves expressing the HPV_MEV antigen was filtered through a 0.45 µm membrane and loaded onto a Ni²⁺-NTA agarose column (Qiagen), pre-equilibrated with 4X binding buffer containing 2 M NaCl, 2X PBS and 10 mM imidazole. Non-specifically bound proteins were removed by washing the column with 10 column volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 30 mM imidazole, pH 8.0). The His-tagged HPV_MEV protein was eluted with elution buffer containing 250 mM imidazole in the same phosphate buffer base. Eluted fractions were pooled and subjected to buffer exchange and imidazole removal using dialysis against phosphate-buffered saline (PBS). To selectively remove the highly abundant Rubisco protein from plant tissue extracts, purified protein fractions were incubated with 10 mM sodium phytate (phytic acid sodium salt hydrate; Sigma) and 10 mM CaCl₂ at 37°C for 10 minutes. Following incubation, the mixtures were centrifuged at 14,000 rpm for 10 minutes at room temperature. The resulting supernatants were collected, dialyzed against PBS, and stored at − 20°C for subsequent analyses. Protein concentration was quantified by the Bradford assay using BSA as the standard. The purity and identity of the purified protein were confirmed via SDS-PAGE. Immunogenicity Analysis Twenty five 9-week-old female BALB/c mice were obtained from Laboratory Animal Research and Development center at Urmia University of Medical Sciences, Urmia, Iran. All experimental procedures were carried out in accordance with the regulations of the Animal Care and Use Committee and the Animal Experimentation Guidelines of the University of Tabriz, Iran. The study protocol was reviewed and approved by the University Ethics Committee (Approval ID: IR.TABRIZU.REC.1404.090). The mice were randomly assigned into five groups (n = 5 per group) and immunized using two different approaches: oral administration of plant-expressed HPV_MEV antigen or intraperitoneal injection of recombinant HPV_MEV purified from E. coli and transplastomic tobacco chloroplasts. The negative control group received 50 mg of freeze-dried wild-type (WT) tobacco leaves via oral gavage. Experimental groups were administered either intraperitoneal injections of 5 µg chloroplast- or E. coli -derived HPV_MEV, or oral gavage of 50 mg or 150 mg of freeze-dried transplastomic tobacco leaves expressing HPV_MEV. Immunizations were performed three times at two-week intervals. The detailed immunization schedule is summarized in Table 1 . Table 1 Immunization schedule with HPV_MEV Group Description Mode of Immunization Schedule (Days) WT-50 50 mg lyophilized wild-type tobacco leaves Oral gavage 0, 14, 28 ORV-50 50 mg lyophilized transplastomic tobacco leaves containing HPV_MEV Oral gavage 0, 14, 28 ORV-150 150 mg lyophilized transplastomic tobacco leaves containing HPV_MEV Oral gavage 0, 14, 28 IPV-Eco 5 µg purified HPV_MEV protein expressed in E. coli Intraperitoneal injection 0, 14, 28 IPV-Chl 5 µg purified HPV_MEV protein expressed in chloroplasts Intraperitoneal injection 0, 14, 28 ORV: Orally vaccinated mice IPV: Intraperitoneal vaccinated mice Serum and Vaginal Fluid Samples Preparation Blood samples were obtained from the tails of the mice, and the serum was immediately isolated and stored at − 20°C for subsequent analysis. Vaginal secretions were collected via repeated washing of the vagina with 100 µL PBS using a micropipette. To mitigate the influence of the estrous cycle on antibody production, vaginal fluids were collected twice, with a five-day interval, and the samples were pooled for analysis. Specimen collection was performed several days prior to the initial oral immunization and two weeks after last immunization. To prevent repeated thawing, all samples were aliquoted and stored at − 20°C until analysis. Estimation of Antibody Responses To evaluate the immunogenic characteristics of the mice immunized with HPV_MEV, a 96-well microtiter plate was coated with 400 ng of purified E. coli -derived HPV_MEV by overnight incubation at 4°C. Subsequently, 200 µL of blocking solution (5% skim milk in PBS) was added to each well and incubated for 1 h at room temperature. The wells were then washed three times with PBST buffer (PBS with 0.1% Tween-20). Pre-immune sera or vaginal wash, sera, or vaginal wash from HPV_MEV-immunized mice (1:4000 dilution) or PBS alone were then introduced into the plate wells. The plate was then incubated in a microtiter plate shaker for 2 h at room temperature. After washing the plate three times with PBST, HRP-conjugated anti-mouse IgG secondary antibodies (1:5000 dilution) were added and incubated for 2 h. Subsequently, 50 µL of TMB substrate was added and the mixture was briefly agitated. The reaction was terminated by adding 50 µl of 0.18 M sulfuric acid. Finally, signal intensity was measured at 450 nm using an ELISA reader. Results Generation of Transplastomic Plants Expressing HPV_MEV To express the codon-optimized HPV_MEV vaccine construct in the chloroplasts of Nicotiana tabacum , the recombinant plastid transformation vector pPRV-HPV_MEV was successfully assembled by replacing the DARPin G3 coding region in the pPRV-DARPin vector with the HPV_MEV sequence flanked by Nco I and Xho I sites (Fig. 1 A). The recombinant vector was confirmed by restriction enzyme digestion and Sanger sequencing (data not shown). Chloroplast transformation was performed via biolistic delivery of the pPRV-HPV_MEV construct into sterile tobacco leaves. Following selection on RMOP medium supplemented with 500 mg/L spectinomycin, putative transplastomic shoots were regenerated and subjected to three successive rounds of selection on RMOP medium containing 500 mg/L of both spectinomycin and streptomycin to ensure homoplasmy. Plants exhibiting stable resistance were rooted and propagated under sterile conditions. Molecular Confirmation of Transgene Integration and Homoplasmy PCR amplification using gene-specific primers (HPV-F/HPV-R) resulted in a distinct amplicon of 743 bp in transplastomic lines, corresponding to a partial fragment of the HPV_MEV gene, while no amplification was detected in wild-type plants, thereby confirming successful transgene integration (Fig. 1 B). To further validate the site-specific integration of the transgene into the rps12/7–rrn16 locus, PCR was conducted using junction-specific primers (16S-F/rps7-R). This analysis produced an amplicon of the expected size (4.4 kb) exclusively in transplastomic lines, whereas a 2 kb fragment was amplified from the wild-type control, consistent with the native plastid genome organization (Fig. 1 C). Southern blot analysis using a probe specific to the rrn16 flanking region confirmed successful site-specific integration of the transgene and homoplasmy in the selected transplastomic lines. A single hybridization signal of the expected size (6.9 kb) was detected in transplastomic plants, whereas a 4.5 kb band corresponding to the native plastid genome was observed in wild-type controls (Fig. 1 D). The absence of WT-specific signals in the transplastomic samples strongly indicates the complete replacement of the native plastid genome with the transformed version, thereby confirming the attainment of homoplasmy in the selected lines. These results collectively demonstrate the stable, site-specific integration and uniform inheritance of the HPV_MEV expression cassette within the plastid genome. Cloning, Expression and Purification of HPV_MEV in E. Coli The HPV_MEV gene, codon-optimized for bacterial expression, was successfully cloned into the pET-28a(+) vector using Nco I and Xho I restriction sites. Transformation into E. Coli strain BL21(DE3) and induction with 1 mM IPTG at an OD₆₀₀ of 0.8 resulted in robust expression of a recombinant protein of ~ 34.4 kDa, as expected based on the predicted molecular weight of the HPV_MEV construct. SDS-PAGE analysis of the induced cultures revealed a prominent band corresponding to the HPV_MEV protein in the soluble fraction (Fig. 2 A), which was absent in uninduced control samples. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni² + -NTA) affinity chromatography via its N-terminal His-tag. Eluted fractions showed high purity and yield, as verified by 12.5% SDS-PAGE (Fig. 2 A). The purified protein was further confirmed by western blot using an anti-His-tag antibody, which specifically recognized a band of ~ 34 kDa in the eluted samples, confirming successful expression and purification of the HPV_MEV construct (Fig. 2 B). The purified recombinant protein was used as a reference antigen in downstream ELISA assays and as an immunogen for the positive control group in the mouse immunization studies. Immunoblot Analysis of Transplastomic Plants To evaluate the expression of the HPV_MEV protein in transplastomic lines, total soluble proteins (TSP) extracted from mature leaves were analyzed by SDS-PAGE followed by immunoblotting. A prominent immunoreactive band of approximately 34.4 kDa, corresponding to the expected size of HPV_MEV, was detected exclusively in transplastomic samples using anti-His-tag antibodies (Fig. 2 B). No such band was observed in wild-type controls, confirming the specific expression of the recombinant antigen in tobacco plastids. Quantification of HPV_MEV by ELISA The amount of HPV_MEV protein expressed in chloroplasts was further quantified using a His-tag-based sandwich ELISA. TSPs from transplastomic and wild-type leaves were coated in 96-well plates, and binding was detected using anti-His-tag antibodies. A strong signal was obtained from transplastomic extracts, while no detectable signal was observed in wild-type controls. Based on the standard curve using purified E. coli -derived HPV_MEV, the chloroplast-produced antigen accumulated to approximately 3.6 mg/g fresh weight, equivalent to 20.8% of total soluble protein. These results confirm robust expression of the vaccine antigen in the plastome. Purification and Characterization of Chloroplast-Expressed HPV_MEV The HPV_MEV antigen produced in transplastomic tobacco leaves was successfully purified using Ni²⁺-NTA affinity chromatography. Following extraction and clarification of total soluble proteins, the antigen was selectively captured by the nickel resin via its N-terminal His-tag. To improve purity, the highly abundant Rubisco protein was effectively removed by treatment with sodium phytate and CaCl₂ prior to further analysis. SDS-PAGE of the eluted fractions revealed a prominent band at approximately 34.4 kDa, consistent with the expected molecular weight of the HPV_MEV construct (Fig. 2 C). Assuming a purification efficiency of approximately 60–70% using Ni-NTA agarose resin (Qiagen), the yield of purified HPV_MEV protein was estimated at ~ 2.2 mg per gram of fresh leaf tissue. This reflects efficient chloroplast-targeted expression and effective affinity-based purification of the recombinant antigen, supporting its applicability in downstream immunological analyses. Evaluation of Antibody Responses To evaluate the immunogenicity of the HPV_MEV antigen in vivo , BALB/c mice were immunized via three different approaches: intraperitoneal injection of 5 µg purified HPV_MEV produced in E. coli , intraperitoneal injection of 5 µg purified HPV_MEV expressed in chloroplasts, or oral gavage with freeze-dried transplastomic tobacco leaves containing HPV_MEV (50 mg or 150 mg per dose). Control mice received 50 mg of freeze-dried wild-type leaves. Immunizations were performed at two-week intervals. Two weeks after the final dose, serum and vaginal wash samples were collected to assess antigen-specific IgG and IgA responses by ELISA using E. coli -derived HPV_MEV as the coating antigen. Mice immunized with HPV_MEV—either via intraperitoneal injection (of E. coli – or chloroplast-derived antigen) or oral gavage with freeze-dried transplastomic tobacco—exhibited significantly elevated levels of HPV L1-specific IgG antibodies in serum (Fig. 3 A, B). These results confirm that the HPV_MEV antigen, regardless of its delivery route, is capable of inducing robust systemic humoral responses, effectively targeting both conformational and linear epitopes. Given the critical role of mucosal IgA antibodies in protective immunity against pathogens, mucosal responses were specifically analyzed following immunization. Oral administration of chloroplast-expressed HPV_MEV via transplastomic tobacco leaves effectively elicited robust mucosal IgA responses, particularly in vaginal secretions (Fig. 3 C), with no statistically significant difference observed between the low (50 mg) and high (150 mg) oral dose groups. In contrast, intraperitoneal immunization with either E. coli – or chloroplast-derived HPV_MEV protein induced robust systemic IgG responses, along with detectable—but comparatively lower—vaginal IgG levels, likely reflecting transudation from circulation. Notably, chloroplast-derived HPV_MEV protein triggered mucosal and systemic antibody responses comparable to its bacterial counterpart, confirming its immunogenicity. However, neither injectable formulation elicited a robust mucosal IgA response, underscoring the advantage of oral delivery for inducing local mucosal immunity. Discussion In recent years, plastid-based molecular farming has emerged as a compelling alternative for the production of recombinant vaccines, offering advantages in biosafety, scalability, cost-effectiveness, and oral delivery potential. The present study demonstrates the successful expression, purification, and immunogenic evaluation of a rationally designed multi-epitope HPV vaccine (HPV_MEV) within tobacco chloroplasts. This work underscores the utility of chloroplast transformation not only as a high-yield production platform but also as a vehicle for oral immunization against mucosal pathogens. The chloroplast expression system utilized in this study offers distinct advantages over traditional nuclear transformation and microbial fermentation platforms. High plastid genome copy numbers and the lack of gene silencing enable robust expression of foreign proteins, with yields exceeding 3.5 mg/g fresh weight—over 20% of total soluble protein. This high-level expression allows for direct oral delivery of lyophilized plant tissue, eliminating the need for large-scale purification. Moreover, antigens expressed in chloroplasts demonstrate remarkable thermostability, maintaining stability and immunogenicity at ambient temperatures for extended periods, which is particularly beneficial for vaccine distribution in resource-limited settings lacking cold-chain infrastructure (Ehsasatvatan & Kohnehrouz, 2023 ). The bioencapsulation of the HPV_MEV antigen within plant cell walls further protects it from gastrointestinal degradation and facilitates antigen uptake and presentation via gut-associated lymphoid tissue (GALT). Consequently, lyophilized transplastomic leaves not only enable needle-free administration but also overcome major logistical barriers, enhancing vaccine accessibility and efficacy in low-resource environments. HPV is a mucosally transmitted pathogen, making the induction of local mucosal immunity a critical target for vaccination strategies. Unlike current licensed HPV vaccines, which are administered intramuscularly and primarily elicit systemic IgG responses with minimal induction of mucosal IgA (Kiamba et al., 2025 ; Schiller & Lowy, 2012 ), orally administered vaccines have the potential to stimulate both serum and mucosal immune responses, particularly secretory IgA at the infection site. In our study, the use of CTB (cholera toxin B subunit) as a mucosal adjuvant was incorporated to enhance antigen uptake via mucosal surfaces (Stratmann, 2015 ). Notably, oral delivery of the plant-derived HPV_MEV resulted in obuomucosal immunity, evidenced by the induction of high titers of vaginal IgA alongside systemic IgG, highlighting the efficacy of bioencapsulated antigens in stimulating both arms of the immune system. In contrast, the same antigen delivered via intramuscular injection failed to elicit a strong mucosal IgA response, underscoring the route-dependent nature of mucosal immunization. These findings are consistent with earlier reports demonstrating that orally delivered plant-derived HPV antigens, such as HPV16-L1 co-expressed with LT-B in transgenic tobacco, can induce both systemic IgG and mucosal IgA responses, highlighting the potential of oral immunization strategies for mucosal pathogens (Hongli et al., 2013 ). HPV is a mucosally transmitted pathogen, and thus the induction of local mucosal immunity—particularly secretory IgA at the site of infection—is a critical goal in vaccine development. Current licensed HPV vaccines are delivered intramuscularly and mainly elicit systemic IgG responses, with limited induction of mucosal IgA (Kiamba et al., 2025 ; Schiller & Lowy, 2012 ). In this study, oral administration of chloroplast-derived HPV_MEV via bioencapsulated plant tissue effectively induced both systemic and mucosal immune responses, as evidenced by high serum IgG and vaginal IgA titers. This outcome reflects the contribution of CTB, a known mucosal adjuvant, which likely enhanced antigen uptake through mucosal surfaces (Stratmann, 2015 ). In contrast, intraperitoneal injection of the same antigen—whether produced in E. coli or chloroplasts—generated robust systemic IgG but failed to elicit strong mucosal IgA responses, highlighting the route-dependent nature of mucosal immunization. Notably, chloroplast-produced HPV_MEV retained its immunogenicity regardless of the delivery route, but only oral administration achieved a balanced humoral response at both systemic and mucosal levels. These findings are consistent with earlier studies using plant-expressed HPV antigens (e.g., HPV16 L1 with LT-B in tobacco) and support the potential of needle-free oral vaccines for mucosal pathogens (Hongli et al., 2013 ). Our findings align with those of Shapiro et al. (Shapiro et al., 2023 ), who demonstrated that oral administration of a plant-derived norovirus VLP vaccine induced both robust systemic IgG and mucosal IgA responses in mice, comparable to the responses achieved via intramuscular injection of the same antigen. Beyond immunogenic performance, the platform offers critical advantages in cost-efficiency, production scalability, and biosafety. Tobacco plants, used here as the production host, offer high biomass and are not part of the food chain, minimizing the risk of unintentional human exposure. The integration of the HPV_MEV gene into the plastid genome via homologous recombination ensures transgene containment due to maternal inheritance, which aligns with biosafety requirements for genetically modified organisms (Adem et al., 2017 ). Furthermore, the direct use of lyophilized leaves eliminates downstream processing costs typically associated with protein purification and formulation. This study introduces a novel chloroplast-based platform that addresses several key limitations of current HPV vaccines, including cold chain dependency, high production costs, and limited genotype coverage due to type specificity (Denny, 2012 ; Kjaer et al., 2009 ; Schiller & Lowy, 2012 ; Wang et al., 2022 ). The multi-epitope design employed enables broad-spectrum immune protection against both high-risk and low-risk HPV genotypes. Simultaneously, chloroplast expression offers a safe, scalable, and cost-effective method of antigen production, positioning this system as an attractive alternative to conventional vaccine platforms. These advantages make plastid-derived oral vaccines strong candidates for integration into global HPV immunization programs, particularly in low- and middle-income countries (LMICs) where vaccine access remains suboptimal (Bruni et al., 2021 ; SHEET, 2018 ). Beyond HPV, this chloroplast-based strategy has shown promising results in preclinical studies targeting other infectious diseases such as malaria, tuberculosis, and SARS-CoV-2 (Chan & Daniell, 2015 ; Shahid & Daniell, 2016 ; Singh et al., 2023 ). Given that HPV is one of the most preventable causes of cancer, especially in underserved populations, developing an effective oral vaccine is not only scientifically valuable but could also significantly reduce global cancer-related morbidity and mortality. In summary, the combination of rational immunogen design, chloroplast-based expression, and oral delivery results in a highly immunogenic, thermostable, and scalable vaccine platform. Given its ability to induce both systemic and mucosal immunity, this HPV_MEV vaccine candidate holds significant promise as a next-generation prophylactic solution. Its applicability could further extend to other mucosally transmitted pathogens, providing a universal strategy for affordable, needle-free immunization in global health contexts. Further studies are warranted to comprehensively evaluate the protective efficacy and durability of the HPV_MEV vaccine. Future work should assess long-term immune memory, neutralizing antibody titers, and T-cell-mediated responses to better understand the breadth and longevity of protection. Additionally, dose optimization and large-scale production strategies should be explored to ensure consistent antigen yield and scalability. Investigating alternative edible plant platforms may also facilitate direct oral consumption without the need for processing, further simplifying vaccine delivery and expanding accessibility in low-resource settings. Conclusion This study presents a significant advancement in the development of a next-generation, broad-spectrum vaccine against human papillomavirus (HPV), combining rational multi-epitope design with high-yield expression in the tobacco chloroplast system. The resulting HPV_MEV vaccine candidate not only targets a wide array of high-risk and low-risk HPV genotypes but also overcomes key limitations of current prophylactic vaccines, such as cold-chain dependency, high production costs, and limited mucosal immunity. The chloroplast-derived HPV_MEV antigen demonstrated robust expression levels and, notably, retained its immunogenicity when delivered orally in the form of lyophilized transplastomic plant tissue. In murine models, oral administration elicited both strong systemic IgG and mucosal IgA responses—an immunological profile highly desirable for combating mucosally transmitted pathogens like HPV. This contrasts with intraperitoneal delivery, which induced systemic but limited mucosal immunity, underscoring the added value of oral, bioencapsulated vaccines for inducing localized immune protection. By integrating rational antigen design with a cost-effective, scalable, and thermostable production and delivery platform, this work lays the foundation for an accessible, needle-free HPV vaccination strategy ideally suited for low- and middle-income countries (LMICs). Furthermore, the successful demonstration of oral immunogenicity opens avenues for broader application of plastid-based vaccines against other mucosal pathogens. In conclusion, the HPV_MEV vaccine platform offers a compelling alternative to conventional HPV vaccines and represents a critical step toward democratizing access to effective immunization globally. Future research should focus on evaluating long-term protection, neutralizing antibody titers, and large-scale production optimization, as well as exploring edible plant hosts for direct oral consumption. Declarations Conflict of interest The authors declare no competing interests. Ethics approval and consent to participate All animal experiments were conducted in compliance with the institutional guidelines for the care and use of laboratory animals at the University of Tabriz, Iran. The experimental protocol was reviewed and approved by the Ethics Committee of the University of Tabriz (Approval ID: IR.TABRIZU.REC.1404.090). All procedures adhered to the Animal Experimentation Guidelines of the University of Tabriz, and every effort was made to minimize animal suffering and the number of animals used. As this study did not involve human participants, consent to participate was not applicable. Consent for publication Not applicable. Funding Not applicable. Author Contribution M. E., designed the construct, performed analyses, and wrote the manuscript. B. B. K., supervised the study, provided specialized scientific and revised the paper. All authors have agreed with the manuscript and provided their consent for publication. Acknowledgment This work is based upon research funded by Iran National Science Foundation (INSF) under project No.4023935. Data Availability The nucleotide and protein sequence data of the expression cassette of HPV_MEV are available in GenBank at NCBI under the accession number PV057300. 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Efficient targeting of foreign genes into the tobacco plastid genome. Nucleic Acids Res. 1994;22(19):3819–24. Additional Declarations No competing interests reported. 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01:18:21","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104795,"visible":true,"origin":"","legend":"","description":"","filename":"ef3c1b7b1ef749588443dd778e3e98ab1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/b763aa4e77a9b348e53b7bed.xml"},{"id":96503075,"identity":"5fe77032-61f4-481a-85b4-91f4577211fb","added_by":"auto","created_at":"2025-11-22 01:18:22","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112792,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/7d89a2eda789cb509ab0aa51.html"},{"id":96503061,"identity":"382133c4-7629-4061-bc9d-0e5f56a1693b","added_by":"auto","created_at":"2025-11-22 01:18:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70240,"visible":true,"origin":"","legend":"\u003cp\u003eA) Physical map of homologous recombination regions of the transplastomic tobacco plastid genome. Nt-Prrn: ribosomal RNA operon promoter from tobacco; T7g10 5′ UTR: 5′ untranslated region of bacteriophage T7 gene 10; HPV_MEV: coding sequence of HPV multiepitope vaccine, TrrnB: rrnB 3′ untranslated region from \u003cem\u003eE. coli; \u003c/em\u003ePpsbA: promoter and 5′ UTR of \u003cem\u003epsbA \u003c/em\u003egene; \u003cem\u003eaadA\u003c/em\u003e: aminoglycoside 3′- adenylytransferase gene; TpsbA: terminator of \u003cem\u003epsbA \u003c/em\u003egene. The transgenes are targeted to the intergenic region between the rrn16 and rps7/12 plastid genes. The expected sizes of the DNA fragments in Southern blot analyses with the restriction enzyme \u003cem\u003eBgl\u003c/em\u003eII are indicated. The location of the Southern blotting probe is shown as a black bar. B) PCR analysis of putative transplastomic plants with primers P1/P2 which land on the HPV-MEV coding sequence, generating a 743 bp fragment. C) PCR analysis of transplastomic plants with primers P3/P4 which land on the rrn16 and rps7/12 flanking sequences, generating 4.4 kb fragment in transplastomic and 2 kb fragment in wild-type plant. D) Southern blot analysis of transplastomic plants. A single band of 6.9 kb after digestion of DNA with \u003cem\u003eBgl\u003c/em\u003eII confirms HPV-MEV gene integration and homoplasmy in transplastomic plants.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/7c180d6e5f61eb0b20b96421.png"},{"id":96503063,"identity":"87c74340-6cbb-42b9-8e4c-0da7144600cb","added_by":"auto","created_at":"2025-11-22 01:18:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":445946,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and purification of HPV_MEV in \u003cem\u003eEscherichia coli\u003c/em\u003eand transplastomic \u003cem\u003eNicotiana tabacum\u003c/em\u003e chloroplasts. (A) SDS–PAGE analysis of total soluble proteins (TSPs) from \u003cem\u003eE. coli\u003c/em\u003e. Lane M: unstained protein molecular weight marker (Thermo Scientific); Lane 1: TSP from non-transformed \u003cem\u003eE. coli\u003c/em\u003e; Lane 2: TSP from \u003cem\u003eE. coli\u003c/em\u003e expressing HPV_MEV; Lane 3: purified HPV_MEV protein from \u003cem\u003eE. coli \u003c/em\u003eexpressing HPV_MEV.\u003cbr\u003e\n(B) Immunoblot analysis of HPV_MEV accumulation in \u003cem\u003eE. coli\u003c/em\u003e and chloroplasts of transplastomic \u003cem\u003eN. tabacum\u003c/em\u003e. Lane M: pre-stained molecular weight markers (kDa); Lane 1: TSP from \u003cem\u003eE. coli \u003c/em\u003eexpressing HPV_MEV; Lane 2: TSP from transplastomic tobacco leaves expressing HPV_MEV; Lane 3: TSP from non-transformed \u003cem\u003eE. coli\u003c/em\u003e; Lane 4: TSP from wild-type tobacco plants. (C) SDS–PAGE analysis of total soluble proteins from transplastomic plants. Lane M: unstained protein molecular weight marker (Thermo Scientific); Lane 1: TSP from wild-type tobacco plants; Lane 2: TSP from transplastomic tobacco leaves expressing HPV_MEV; Lane 3: purified HPV_MEV protein from transplastomic plants. Molecular masses (kDa) are indicated on the left.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/d80368f70cf66aed128875a5.png"},{"id":96503062,"identity":"417565ab-e744-4faa-bade-ef9ef22d7db0","added_by":"auto","created_at":"2025-11-22 01:18:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48714,"visible":true,"origin":"","legend":"\u003cp\u003eHumoral and mucosal immune responses elicited by HPV-MEV immunization in mice. Mice were immunized with different formulations, including lyophilized wild-type tobacco leaves (WT-50, 50 mg, oral gavage), lyophilized transplastomic tobacco leaves expressing HPV-MEV (ORV-50, 50 mg; ORV-150, 150 mg; oral gavage), purified HPV-MEV protein produced in \u003cem\u003eE. coli\u003c/em\u003e(IPV-Eco, 5 µg, intraperitoneal injection), and purified HPV-MEV protein expressed in chloroplasts (IPV-Chl, 5 µg, intraperitoneal injection). (A) HPV-MEV–specific IgG titers in sera. (B) Mucosal IgG responses in vaginal fluid. (C) Mucosal IgA responses in vaginal fluid. The optical density (OD) values in ELISA for all mice are plotted in the graphs. Data represent mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/eeae49fc4ea2b89a18186de0.png"},{"id":99172282,"identity":"cd800ea4-7dd3-4e8f-ba48-8b8e54978e36","added_by":"auto","created_at":"2025-12-29 16:07:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1408013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7801011/v1/26d74464-d8ec-42c4-99a4-d67cad1eb32f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Harnessing Plant Chloroplasts for Oral Delivery of a Multi-epitope HPV Vaccine: Toward Cost-Effective Systemic and Mucosal Immunization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman papillomavirus (HPV) is a highly prevalent DNA virus with over 200 identified genotypes, primarily targeting epithelial tissues and predominantly transmitted through sexual contact (Doorbar, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Persistent infection with high-risk HPV types, particularly HPV16 and HPV18, is the leading cause of cervical cancer and significantly contributes to other malignancies, such as oropharyngeal, anal, penile, and vulvar cancers (Arbyn et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; de Sanjose et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Despite widespread screening programs and the availability of prophylactic vaccines, HPV-related diseases continue to pose a major global health burden, especially in low- and middle-income countries (LMICs), where access to vaccine remains limited (Bruni et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to the World Health Organization (WHO), more than 300,000 cervical cancer-related deaths occur annually, with over 85% of these deaths occurring in LMICs (SHEET, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). highlighting the urgent need for affordable and broadly accessible vaccine alternatives.\u003c/p\u003e\u003cp\u003eCurrently licensed HPV vaccines \u0026mdash;Gardasil, Gardasil-9, and Cervarix\u0026mdash; are based on virus-like particles (VLPs) composed of the L1 capsid protein produced in yeast or insect cell systems (Schiller \u0026amp; Lowy, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These vaccines provide strong protection against the most common high-risk HPV types and have substantially reduced infection rates and precancerous lesions (Kjaer et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, their widespread implementation is limited by high production and distribution costs, cold chain requirements, intramuscular delivery, and limited type-specific coverage (Denny, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To overcome these limitations, next-generation vaccine strategies aim to provide broader protection, reduce production costs, and enable noninvasive delivery platforms.\u003c/p\u003e\u003cp\u003eOne promising strategy involves multi-epitope vaccine (MEV) design, which uses immunoinformatics tools to identify conserved B-cell- and T-cell epitopes across multiple HPV genotypes (Chauhan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This approach allows for rational antigen design with enhanced immunogenicity, safety, and broad-spectrum coverage (Doytchinova \u0026amp; Flower, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In our previous study, we designed a chimeric HPV multi-epitope vaccine (HPV_MEV) incorporating conserved L1-derived CTL, HTL, and B-cell epitopes fused with adjuvants (CTB and RS09 peptide) and a His-tag for purification. In silico analyses, including molecular docking, dynamics simulations, and immune simulations, confirmed the stability and immunogenic potential of the construct (Ehsasatvatan \u0026amp; Kohnehrouz, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAn effective and scalable expression system is essential to translate this design into a practical vaccine platform. Traditional microbial and mammalian cell expression systems are often limited by high production costs and biosafety concerns (Walsh \u0026amp; Walsh, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, plant-based systems offer a safe, cost-effective, and scalable alternative for producing recombinant vaccines (Rybicki, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among these, chloroplast genetic engineering is particularly advantageous because of the high copy number of plastid genomes, site-specific integration, and absence of transgene transmission through pollen in most species (Daniell, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Maliga, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Protein yields can reach up to 70% of the total soluble protein (Oey et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), making chloroplasts highly efficient biofactories.\u003c/p\u003e\u003cp\u003eTobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e) is widely used as a model for chloroplast transformation owing to its well-characterized plastomes and high biomass yield (Bock, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Several pharmaceutical proteins, including human somatotropin, interferons, antibodies, and vaccine antigens, have been successfully produced in tobacco chloroplasts at industrially relevant scales (Davoodi-Semiromi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ruhlman et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Furthermore, chloroplast-expressed vaccine antigens can be delivered orally via the consumption of lyophilized plant tissue, which protects antigens from degradation and promotes uptake by gut-associated lymphoid tissue (GALT), eliciting both mucosal and systemic immune responses (Chan \u0026amp; Daniell, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kwon \u0026amp; Daniell, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Oral delivery of chloroplast-derived vaccines has demonstrated efficacy in preclinical models of cholera, tuberculosis, and influenza (Kwon et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pantazica et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and these vaccines also exhibit remarkable thermostability, eliminating the need for refrigeration (Daniell et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ehsasatvatan \u0026amp; Kohnehrouz, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study we aimed to express the immunoinformatics-derived HPV_MEV antigen in tobacco chloroplasts and evaluate its ability to elicit both systemic and mucosal immune responses following oral administration in a murine model. By combining rational immunogen design with high-yield plant expression, this platform critical limitations in current HPV vaccine accessibility and distribution, offering a promising path toward cost-effective, orally administrable vaccines for HPV and other mucosal pathogens.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDesign and Construction of Plastidial Expression Vector\u003c/h2\u003e\u003cp\u003eThe HPV_MEV construct (GenBank Accession number: PV057300) was previously designed using an immunoinformatics-based approach, incorporating conserved immunodominant epitopes derived from the L1 protein of both high-risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) and low-risk (HPV6, 11, 42, 43, 44) HPV genotypes. The final vaccine sequence includes cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell linear epitopes, interlinked with appropriate flexible linkers (AAY, GPGPG, and KK), and fused with two molecular adjuvants: the TLR4 agonist RS09 at the N-terminus and the cholera toxin B subunit (CTB) at the C-terminus. A 6\u0026times;His-tag was appended to facilitate protein detection and purification (Ehsasatvatan \u0026amp; Kohnehrouz, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Immunomolecular analyses indicated that the vaccine candidate exhibited promising antigenicity and immunogenicity, lacked allergenic and toxic properties, and demonstrated potential to elicit robust immune responses without adverse effects.\u003c/p\u003e\u003cp\u003eIn order to high-yield expression of the designed multi-epitope vaccine (HPV_MEV) in the tobacco plastid genome, the codon-optimized nucleotide sequence of the antigen was synthesized based on its amino acid composition using the \u003cem\u003eEMBOSS Backtranseq\u003c/em\u003e tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/st/emboss_backtranseq\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/st/emboss_backtranseq\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Madeira et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The synthetic HPV_MEV gene was flanked with \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI restriction sites and cloned into the pPRV-DARPin vector (Ehsasatvatan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e), replacing the DARPin G3 coding region. This chloroplast transformation vector, derived from pPRV111A (Zoubenko et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), facilitates homologous recombination into the rps12/7\u0026ndash;rrn16 intergenic spacer region of the tobacco plastome. For robust transgene expression, the HPV_MEV gene was placed under the control of the plastid ribosomal RNA operon promoter (Prrn), fused to the T7g10 5\u0026prime; untranslated region (UTR) to enhance translation, and terminated by the \u003cem\u003eE. Coli\u003c/em\u003e rrnB 3\u0026prime; UTR. Selection of transplastomic events was enabled by the spectinomycin resistance gene (\u003cem\u003eaadA\u003c/em\u003e) driven by appropriate regulatory elements. This chloroplast-compatible expression cassette was subsequently used for biolistic transformation of \u003cem\u003eNicotiana tabacum\u003c/em\u003e chloroplasts.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCloning Expression and Purification of HPV_MEV in Bacterial Vector\u003c/h3\u003e\n\u003cp\u003eThe HPV_MEV coding sequence was inserted into the pET-28a (+) vector (Novagen) using \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI restriction sites. To obtain purified HPV_MEV, the plasmid was introduced into the BL21 (DE3) cells. Upon the cell culture reaching an optical density (O.D.600) of approximately 0.8, protein expression was induced with 1mM isopropyl β-D-thiogalactoside (IPTG), and the cells were incubated at 37\u0026deg;C with agitation. HPV_MEV protein was subsequently purified using Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity chromatography. Fractions containing the purified protein were pooled and dialyzed against 10mM HEPES buffer (pH 8.0) with 10% glycerol. The purified samples were analyzed using 15% SDS-PAGE and electroblotting.\u003c/p\u003e\n\u003ch3\u003ePlant material, Growth Conditions and Chloroplast Transformation\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eNicotiana tabacum\u003c/em\u003e cv. Perega plants were grown under sterile conditions on medium devoid of growth regulators. The medium consisted of MS salts, B5 vitamins, 3% (w/v) sucrose, and 0.7% (w/v) agar with a pH of 5.8. Plants were maintained at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with a 16-hour light cycle at 40 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2 s\u0026minus;1\u003c/sup\u003e. Fully developed, dark green leaves were subjected to bombardment using tungsten particles (0.7 \u0026micro;m) coated with 1 \u0026micro;g pPRV-HPV_MEV plasmid, using a PDS-1000/He Biolistic gun (Bio-Rad, California, USA) as previously outlined (Ehsasatvatan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Shoots resistant to spectinomycin were isolated on the RMOP medium containing 500 mg/l spectinomycin dihydrochloride. These resistant shoots were tested on a medium containing both spectinomycin and streptomycin (500 mg/l each). Shoots that showed positive results underwent three cycles of regeneration on selective medium containing only spectinomycin. The plants were then rooted in a medium composed of MS salt, B5 vitamins, 3% (w/v) sucrose, 0.7% (w/v) agar, and 500 mg/l spectinomycin under aseptic conditions.\u003c/p\u003e\n\u003ch3\u003eMolecular Analysis of Putative Transplastomic Plants\u003c/h3\u003e\n\u003cp\u003eLeaf samples (100\u0026ndash;150 mg) from wild-type and in vitro transplastomic plants expressing HPV_MEV were subjected to DNA extraction using the CTAB method (Murray \u0026amp; Thompson, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). To detect the inserted HPV_MEV gene in the tobacco plastid genome, PCR amplification was conducted using the primer pair HPV-F (5ˈ-GAAGCTGCTGCTAAAGTATCTGG-3ˈ) and HPV-R (5ˈ-GCAGCAATAGCATGAGGAGTT-3ˈ). The correct insertion of the expression cassette into the targeted plastid genome region was verified through PCR using 16S-F (5ˈ-AACTAAACACGAGGGTTGC-3ˈ) and aadA-R (5ˈ-AAGAATTTGTCCACTACGTGA-3ˈ) prime set. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the primer locations used in this study. To further confirm the homoplasmy status of the transplastomic plants, Southern hybridization was performed. This technique involved digesting leaf genomic DNA (2 \u0026micro;g) with \u003cem\u003eBgl\u003c/em\u003eII (NEB, USA) and probing it with 232 bp DNA fragments derived from the rrn16 flanking sequence. The DIG-high prime DNA labeling and detection starter kit II was used for this process, following the manufacturer's instructions (Roche, Germany). Plants that were determined to be homoplasmic were selected for protein analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eImmunoblot Detection of Vaccine Candidate\u003c/h3\u003e\n\u003cp\u003eTotal soluble protein (TSP) was extracted from transplastomic tobacco (\u003cem\u003eN. tabacum\u003c/em\u003e) leaves expressing the HPV_MEV antigen. Leaf tissues were harvested and ground to a fine powder in liquid nitrogen. The powdered material was resuspended in extraction buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 0.05% Tween-20, 2 mM PMSF, and a plant-compatible protease inhibitor cocktail (pH 8.0). Homogenates were incubated on ice for 30 minutes and subsequently clarified by centrifugation at 15,000 \u0026times; g for 30 minutes at 4\u0026deg;C. The total protein concentration was determined using the Bradford protein assay. Protein samples were resolved on 15% SDS and transferred to a PVDF membrane. Western blot analysis was conducted employing rabbit anti-His-tag primary antibody at a dilution of 1:1,000 and goat anti-rabbit conjugated with horseradish peroxidase (HRP) secondary antibody at a dilution of 1:10,000. Protein bands were subsequently visualized using DAB peroxidase substrate solution. The total soluble protein extracted from wild-type plants served as a negative control.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEstimation of Vaccine Candidate Concentration in TSP\u003c/h2\u003e\u003cp\u003eThe concentration of HPV_MEV in the transplastomic plants was determined using enzyme-linked immunosorbent assay (ELISA). The assay was conducted by coating ELISA 96-well microplates with 50 ng/well of total soluble proteins extracted from transplastomic and wild-type leaves and incubating them overnight at 4\u0026deg;C. Subsequently, the plates were washed and blocked with a solution containing 1% BSA in 1X PBS buffer and 0.1% Tween 20 for 1 h at 37\u0026deg;C. After three washes, the wells were treated with rabbit anti-His-tag antibody (1:1000 dilution in blocking buffer) for 2 h at 37\u0026deg;C. After additional washing, the plates were incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) at a 1:10,000 dilution in blocking buffer for 1 h at 37\u0026deg;C. The plates were washed three more times before the addition of tetramethylbenzidine (TMB) substrate (Bio-Rad), allowing color development for 10 min. The reaction was terminated using 2 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and the absorbance was measured at 450 nm using an ELISA reader. To estimate the expression levels of the HPV_MEV antigen in transplastomic plant samples, a standard calibration curve was established using serial dilutions of purified \u003cem\u003eE. coli\u003c/em\u003e-expressed HPV_MEV protein. All measurements were performed in triplicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePurification of Vaccine Candidate\u003c/h3\u003e\n\u003cp\u003eTotal soluble protein from transplastomic tobacco leaves expressing the HPV_MEV antigen was filtered through a 0.45 \u0026micro;m membrane and loaded onto a Ni\u0026sup2;⁺-NTA agarose column (Qiagen), pre-equilibrated with 4X binding buffer containing 2 M NaCl, 2X PBS and 10 mM imidazole. Non-specifically bound proteins were removed by washing the column with 10 column volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 30 mM imidazole, pH 8.0). The His-tagged HPV_MEV protein was eluted with elution buffer containing 250 mM imidazole in the same phosphate buffer base. Eluted fractions were pooled and subjected to buffer exchange and imidazole removal using dialysis against phosphate-buffered saline (PBS).\u003c/p\u003e\u003cp\u003eTo selectively remove the highly abundant Rubisco protein from plant tissue extracts, purified protein fractions were incubated with 10 mM sodium phytate (phytic acid sodium salt hydrate; Sigma) and 10 mM CaCl₂ at 37\u0026deg;C for 10 minutes. Following incubation, the mixtures were centrifuged at 14,000 rpm for 10 minutes at room temperature. The resulting supernatants were collected, dialyzed against PBS, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent analyses. Protein concentration was quantified by the Bradford assay using BSA as the standard. The purity and identity of the purified protein were confirmed via SDS-PAGE.\u003c/p\u003e\n\u003ch3\u003eImmunogenicity Analysis\u003c/h3\u003e\n\u003cp\u003eTwenty five 9-week-old female BALB/c mice were obtained from Laboratory Animal Research and Development center at Urmia University of Medical Sciences, Urmia, Iran. All experimental procedures were carried out in accordance with the regulations of the Animal Care and Use Committee and the Animal Experimentation Guidelines of the University of Tabriz, Iran. The study protocol was reviewed and approved by the University Ethics Committee (Approval ID: IR.TABRIZU.REC.1404.090). The mice were randomly assigned into five groups (n\u0026thinsp;=\u0026thinsp;5 per group) and immunized using two different approaches: oral administration of plant-expressed HPV_MEV antigen or intraperitoneal injection of recombinant HPV_MEV purified from \u003cem\u003eE. coli\u003c/em\u003e and transplastomic tobacco chloroplasts. The negative control group received 50 mg of freeze-dried wild-type (WT) tobacco leaves via oral gavage. Experimental groups were administered either intraperitoneal injections of 5 \u0026micro;g chloroplast- or \u003cem\u003eE. coli\u003c/em\u003e-derived HPV_MEV, or oral gavage of 50 mg or 150 mg of freeze-dried transplastomic tobacco leaves expressing HPV_MEV. Immunizations were performed three times at two-week intervals. The detailed immunization schedule is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eImmunization schedule with HPV_MEV\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMode of Immunization\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSchedule (Days)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWT-50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mg lyophilized wild-type tobacco leaves\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOral gavage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0, 14, 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eORV-50\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 mg lyophilized transplastomic tobacco leaves containing HPV_MEV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOral gavage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0, 14, 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eORV-150\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e150 mg lyophilized transplastomic tobacco leaves containing HPV_MEV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOral gavage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0, 14, 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIPV-Eco\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 \u0026micro;g purified HPV_MEV protein expressed in \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIntraperitoneal injection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0, 14, 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eIPV-Chl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5 \u0026micro;g purified HPV_MEV protein expressed in chloroplasts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIntraperitoneal injection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0, 14, 28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eORV: Orally vaccinated mice\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eIPV: Intraperitoneal vaccinated mice\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eSerum and Vaginal Fluid Samples Preparation\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eBlood samples were obtained from the tails of the mice, and the serum was immediately isolated and stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent analysis. Vaginal secretions were collected via repeated washing of the vagina with 100 \u0026micro;L PBS using a micropipette. To mitigate the influence of the estrous cycle on antibody production, vaginal fluids were collected twice, with a five-day interval, and the samples were pooled for analysis. Specimen collection was performed several days prior to the initial oral immunization and two weeks after last immunization. To prevent repeated thawing, all samples were aliquoted and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eEstimation of Antibody Responses\u003c/h2\u003e\u003cp\u003eTo evaluate the immunogenic characteristics of the mice immunized with HPV_MEV, a 96-well microtiter plate was coated with 400 ng of purified \u003cem\u003eE. coli\u003c/em\u003e-derived HPV_MEV by overnight incubation at 4\u0026deg;C. Subsequently, 200 \u0026micro;L of blocking solution (5% skim milk in PBS) was added to each well and incubated for 1 h at room temperature. The wells were then washed three times with PBST buffer (PBS with 0.1% Tween-20). Pre-immune sera or vaginal wash, sera, or vaginal wash from HPV_MEV-immunized mice (1:4000 dilution) or PBS alone were then introduced into the plate wells. The plate was then incubated in a microtiter plate shaker for 2 h at room temperature. After washing the plate three times with PBST, HRP-conjugated anti-mouse IgG secondary antibodies (1:5000 dilution) were added and incubated for 2 h. Subsequently, 50 \u0026micro;L of TMB substrate was added and the mixture was briefly agitated. The reaction was terminated by adding 50 \u0026micro;l of 0.18 M sulfuric acid. Finally, signal intensity was measured at 450 nm using an ELISA reader.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of Transplastomic Plants Expressing HPV_MEV\u003c/h2\u003e\u003cp\u003eTo express the codon-optimized HPV_MEV vaccine construct in the chloroplasts of \u003cem\u003eNicotiana tabacum\u003c/em\u003e, the recombinant plastid transformation vector pPRV-HPV_MEV was successfully assembled by replacing the DARPin G3 coding region in the pPRV-DARPin vector with the HPV_MEV sequence flanked by \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The recombinant vector was confirmed by restriction enzyme digestion and Sanger sequencing (data not shown).\u003c/p\u003e\u003cp\u003eChloroplast transformation was performed via biolistic delivery of the pPRV-HPV_MEV construct into sterile tobacco leaves. Following selection on RMOP medium supplemented with 500 mg/L spectinomycin, putative transplastomic shoots were regenerated and subjected to three successive rounds of selection on RMOP medium containing 500 mg/L of both spectinomycin and streptomycin to ensure homoplasmy. Plants exhibiting stable resistance were rooted and propagated under sterile conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Confirmation of Transgene Integration and Homoplasmy\u003c/h2\u003e\u003cp\u003ePCR amplification using gene-specific primers (HPV-F/HPV-R) resulted in a distinct amplicon of 743 bp in transplastomic lines, corresponding to a partial fragment of the HPV_MEV gene, while no amplification was detected in wild-type plants, thereby confirming successful transgene integration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo further validate the site-specific integration of the transgene into the rps12/7\u0026ndash;rrn16 locus, PCR was conducted using junction-specific primers (16S-F/rps7-R). This analysis produced an amplicon of the expected size (4.4 kb) exclusively in transplastomic lines, whereas a 2 kb fragment was amplified from the wild-type control, consistent with the native plastid genome organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eSouthern blot analysis using a probe specific to the rrn16 flanking region confirmed successful site-specific integration of the transgene and homoplasmy in the selected transplastomic lines. A single hybridization signal of the expected size (6.9 kb) was detected in transplastomic plants, whereas a 4.5 kb band corresponding to the native plastid genome was observed in wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe absence of WT-specific signals in the transplastomic samples strongly indicates the complete replacement of the native plastid genome with the transformed version, thereby confirming the attainment of homoplasmy in the selected lines. These results collectively demonstrate the stable, site-specific integration and uniform inheritance of the \u003cem\u003eHPV_MEV\u003c/em\u003e expression cassette within the plastid genome.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCloning, Expression and Purification of HPV_MEV in\u003c/b\u003e \u003cb\u003eE. Coli\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe HPV_MEV gene, codon-optimized for bacterial expression, was successfully cloned into the pET-28a(+) vector using \u003cem\u003eNco\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI restriction sites. Transformation into \u003cem\u003eE. Coli\u003c/em\u003e strain BL21(DE3) and induction with 1 mM IPTG at an OD₆₀₀ of 0.8 resulted in robust expression of a recombinant protein of ~\u0026thinsp;34.4 kDa, as expected based on the predicted molecular weight of the HPV_MEV construct.\u003c/p\u003e\u003cp\u003eSDS-PAGE analysis of the induced cultures revealed a prominent band corresponding to the HPV_MEV protein in the soluble fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which was absent in uninduced control samples. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni\u0026sup2;\u003csup\u003e+\u003c/sup\u003e-NTA) affinity chromatography via its N-terminal His-tag. Eluted fractions showed high purity and yield, as verified by 12.5% SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe purified protein was further confirmed by western blot using an anti-His-tag antibody, which specifically recognized a band of ~\u0026thinsp;34 kDa in the eluted samples, confirming successful expression and purification of the HPV_MEV construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The purified recombinant protein was used as a reference antigen in downstream ELISA assays and as an immunogen for the positive control group in the mouse immunization studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblot Analysis of Transplastomic Plants\u003c/h2\u003e\u003cp\u003eTo evaluate the expression of the HPV_MEV protein in transplastomic lines, total soluble proteins (TSP) extracted from mature leaves were analyzed by SDS-PAGE followed by immunoblotting. A prominent immunoreactive band of approximately 34.4 kDa, corresponding to the expected size of HPV_MEV, was detected exclusively in transplastomic samples using anti-His-tag antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). No such band was observed in wild-type controls, confirming the specific expression of the recombinant antigen in tobacco plastids.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eQuantification of HPV_MEV by ELISA\u003c/h2\u003e\u003cp\u003eThe amount of HPV_MEV protein expressed in chloroplasts was further quantified using a His-tag-based sandwich ELISA. TSPs from transplastomic and wild-type leaves were coated in 96-well plates, and binding was detected using anti-His-tag antibodies. A strong signal was obtained from transplastomic extracts, while no detectable signal was observed in wild-type controls. Based on the standard curve using purified \u003cem\u003eE. coli\u003c/em\u003e-derived HPV_MEV, the chloroplast-produced antigen accumulated to approximately 3.6 mg/g fresh weight, equivalent to 20.8% of total soluble protein. These results confirm robust expression of the vaccine antigen in the plastome.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003ePurification and Characterization of Chloroplast-Expressed HPV_MEV\u003c/h2\u003e\u003cp\u003eThe HPV_MEV antigen produced in transplastomic tobacco leaves was successfully purified using Ni\u0026sup2;⁺-NTA affinity chromatography. Following extraction and clarification of total soluble proteins, the antigen was selectively captured by the nickel resin via its N-terminal His-tag. To improve purity, the highly abundant Rubisco protein was effectively removed by treatment with sodium phytate and CaCl₂ prior to further analysis. SDS-PAGE of the eluted fractions revealed a prominent band at approximately 34.4 kDa, consistent with the expected molecular weight of the HPV_MEV construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Assuming a purification efficiency of approximately 60\u0026ndash;70% using Ni-NTA agarose resin (Qiagen), the yield of purified HPV_MEV protein was estimated at ~\u0026thinsp;2.2 mg per gram of fresh leaf tissue. This reflects efficient chloroplast-targeted expression and effective affinity-based purification of the recombinant antigen, supporting its applicability in downstream immunological analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of Antibody Responses\u003c/h2\u003e\u003cp\u003eTo evaluate the immunogenicity of the HPV_MEV antigen \u003cem\u003ein vivo\u003c/em\u003e, BALB/c mice were immunized via three different approaches: intraperitoneal injection of 5 \u0026micro;g purified HPV_MEV produced in \u003cem\u003eE. coli\u003c/em\u003e, intraperitoneal injection of 5 \u0026micro;g purified HPV_MEV expressed in chloroplasts, or oral gavage with freeze-dried transplastomic tobacco leaves containing HPV_MEV (50 mg or 150 mg per dose). Control mice received 50 mg of freeze-dried wild-type leaves. Immunizations were performed at two-week intervals. Two weeks after the final dose, serum and vaginal wash samples were collected to assess antigen-specific IgG and IgA responses by ELISA using \u003cem\u003eE. coli\u003c/em\u003e-derived HPV_MEV as the coating antigen.\u003c/p\u003e\u003cp\u003eMice immunized with HPV_MEV\u0026mdash;either via intraperitoneal injection (of \u003cem\u003eE. coli\u003c/em\u003e\u0026ndash; or chloroplast-derived antigen) or oral gavage with freeze-dried transplastomic tobacco\u0026mdash;exhibited significantly elevated levels of HPV L1-specific IgG antibodies in serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These results confirm that the HPV_MEV antigen, regardless of its delivery route, is capable of inducing robust systemic humoral responses, effectively targeting both conformational and linear epitopes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the critical role of mucosal IgA antibodies in protective immunity against pathogens, mucosal responses were specifically analyzed following immunization. Oral administration of chloroplast-expressed HPV_MEV via transplastomic tobacco leaves effectively elicited robust mucosal IgA responses, particularly in vaginal secretions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with no statistically significant difference observed between the low (50 mg) and high (150 mg) oral dose groups. In contrast, intraperitoneal immunization with either \u003cem\u003eE. coli\u003c/em\u003e\u0026ndash; or chloroplast-derived HPV_MEV protein induced robust systemic IgG responses, along with detectable\u0026mdash;but comparatively lower\u0026mdash;vaginal IgG levels, likely reflecting transudation from circulation. Notably, chloroplast-derived HPV_MEV protein triggered mucosal and systemic antibody responses comparable to its bacterial counterpart, confirming its immunogenicity. However, neither injectable formulation elicited a robust mucosal IgA response, underscoring the advantage of oral delivery for inducing local mucosal immunity.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, plastid-based molecular farming has emerged as a compelling alternative for the production of recombinant vaccines, offering advantages in biosafety, scalability, cost-effectiveness, and oral delivery potential. The present study demonstrates the successful expression, purification, and immunogenic evaluation of a rationally designed multi-epitope HPV vaccine (HPV_MEV) within tobacco chloroplasts. This work underscores the utility of chloroplast transformation not only as a high-yield production platform but also as a vehicle for oral immunization against mucosal pathogens.\u003c/p\u003e\u003cp\u003eThe chloroplast expression system utilized in this study offers distinct advantages over traditional nuclear transformation and microbial fermentation platforms. High plastid genome copy numbers and the lack of gene silencing enable robust expression of foreign proteins, with yields exceeding 3.5 mg/g fresh weight\u0026mdash;over 20% of total soluble protein. This high-level expression allows for direct oral delivery of lyophilized plant tissue, eliminating the need for large-scale purification. Moreover, antigens expressed in chloroplasts demonstrate remarkable thermostability, maintaining stability and immunogenicity at ambient temperatures for extended periods, which is particularly beneficial for vaccine distribution in resource-limited settings lacking cold-chain infrastructure (Ehsasatvatan \u0026amp; Kohnehrouz, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The bioencapsulation of the HPV_MEV antigen within plant cell walls further protects it from gastrointestinal degradation and facilitates antigen uptake and presentation via gut-associated lymphoid tissue (GALT). Consequently, lyophilized transplastomic leaves not only enable needle-free administration but also overcome major logistical barriers, enhancing vaccine accessibility and efficacy in low-resource environments.\u003c/p\u003e\u003cp\u003eHPV is a mucosally transmitted pathogen, making the induction of local mucosal immunity a critical target for vaccination strategies. Unlike current licensed HPV vaccines, which are administered intramuscularly and primarily elicit systemic IgG responses with minimal induction of mucosal IgA (Kiamba et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Schiller \u0026amp; Lowy, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), orally administered vaccines have the potential to stimulate both serum and mucosal immune responses, particularly secretory IgA at the infection site. In our study, the use of CTB (cholera toxin B subunit) as a mucosal adjuvant was incorporated to enhance antigen uptake via mucosal surfaces (Stratmann, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Notably, oral delivery of the plant-derived HPV_MEV resulted in obuomucosal immunity, evidenced by the induction of high titers of vaginal IgA alongside systemic IgG, highlighting the efficacy of bioencapsulated antigens in stimulating both arms of the immune system. In contrast, the same antigen delivered via intramuscular injection failed to elicit a strong mucosal IgA response, underscoring the route-dependent nature of mucosal immunization. These findings are consistent with earlier reports demonstrating that orally delivered plant-derived HPV antigens, such as HPV16-L1 co-expressed with LT-B in transgenic tobacco, can induce both systemic IgG and mucosal IgA responses, highlighting the potential of oral immunization strategies for mucosal pathogens (Hongli et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHPV is a mucosally transmitted pathogen, and thus the induction of local mucosal immunity\u0026mdash;particularly secretory IgA at the site of infection\u0026mdash;is a critical goal in vaccine development. Current licensed HPV vaccines are delivered intramuscularly and mainly elicit systemic IgG responses, with limited induction of mucosal IgA (Kiamba et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Schiller \u0026amp; Lowy, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, oral administration of chloroplast-derived HPV_MEV via bioencapsulated plant tissue effectively induced both systemic and mucosal immune responses, as evidenced by high serum IgG and vaginal IgA titers. This outcome reflects the contribution of CTB, a known mucosal adjuvant, which likely enhanced antigen uptake through mucosal surfaces (Stratmann, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, intraperitoneal injection of the same antigen\u0026mdash;whether produced in \u003cem\u003eE. coli\u003c/em\u003e or chloroplasts\u0026mdash;generated robust systemic IgG but failed to elicit strong mucosal IgA responses, highlighting the route-dependent nature of mucosal immunization. Notably, chloroplast-produced HPV_MEV retained its immunogenicity regardless of the delivery route, but only oral administration achieved a balanced humoral response at both systemic and mucosal levels. These findings are consistent with earlier studies using plant-expressed HPV antigens (e.g., HPV16 L1 with LT-B in tobacco) and support the potential of needle-free oral vaccines for mucosal pathogens (Hongli et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our findings align with those of Shapiro et al. (Shapiro et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who demonstrated that oral administration of a plant-derived norovirus VLP vaccine induced both robust systemic IgG and mucosal IgA responses in mice, comparable to the responses achieved via intramuscular injection of the same antigen.\u003c/p\u003e\u003cp\u003eBeyond immunogenic performance, the platform offers critical advantages in cost-efficiency, production scalability, and biosafety. Tobacco plants, used here as the production host, offer high biomass and are not part of the food chain, minimizing the risk of unintentional human exposure. The integration of the HPV_MEV gene into the plastid genome via homologous recombination ensures transgene containment due to maternal inheritance, which aligns with biosafety requirements for genetically modified organisms (Adem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, the direct use of lyophilized leaves eliminates downstream processing costs typically associated with protein purification and formulation.\u003c/p\u003e\u003cp\u003eThis study introduces a novel chloroplast-based platform that addresses several key limitations of current HPV vaccines, including cold chain dependency, high production costs, and limited genotype coverage due to type specificity (Denny, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kjaer et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Schiller \u0026amp; Lowy, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The multi-epitope design employed enables broad-spectrum immune protection against both high-risk and low-risk HPV genotypes. Simultaneously, chloroplast expression offers a safe, scalable, and cost-effective method of antigen production, positioning this system as an attractive alternative to conventional vaccine platforms.\u003c/p\u003e\u003cp\u003eThese advantages make plastid-derived oral vaccines strong candidates for integration into global HPV immunization programs, particularly in low- and middle-income countries (LMICs) where vaccine access remains suboptimal (Bruni et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; SHEET, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Beyond HPV, this chloroplast-based strategy has shown promising results in preclinical studies targeting other infectious diseases such as malaria, tuberculosis, and SARS-CoV-2 (Chan \u0026amp; Daniell, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shahid \u0026amp; Daniell, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Given that HPV is one of the most preventable causes of cancer, especially in underserved populations, developing an effective oral vaccine is not only scientifically valuable but could also significantly reduce global cancer-related morbidity and mortality.\u003c/p\u003e\u003cp\u003eIn summary, the combination of rational immunogen design, chloroplast-based expression, and oral delivery results in a highly immunogenic, thermostable, and scalable vaccine platform. Given its ability to induce both systemic and mucosal immunity, this HPV_MEV vaccine candidate holds significant promise as a next-generation prophylactic solution. Its applicability could further extend to other mucosally transmitted pathogens, providing a universal strategy for affordable, needle-free immunization in global health contexts.\u003c/p\u003e\u003cp\u003eFurther studies are warranted to comprehensively evaluate the protective efficacy and durability of the HPV_MEV vaccine. Future work should assess long-term immune memory, neutralizing antibody titers, and T-cell-mediated responses to better understand the breadth and longevity of protection. Additionally, dose optimization and large-scale production strategies should be explored to ensure consistent antigen yield and scalability. Investigating alternative edible plant platforms may also facilitate direct oral consumption without the need for processing, further simplifying vaccine delivery and expanding accessibility in low-resource settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a significant advancement in the development of a next-generation, broad-spectrum vaccine against human papillomavirus (HPV), combining rational multi-epitope design with high-yield expression in the tobacco chloroplast system. The resulting HPV_MEV vaccine candidate not only targets a wide array of high-risk and low-risk HPV genotypes but also overcomes key limitations of current prophylactic vaccines, such as cold-chain dependency, high production costs, and limited mucosal immunity.\u003c/p\u003e\u003cp\u003eThe chloroplast-derived HPV_MEV antigen demonstrated robust expression levels and, notably, retained its immunogenicity when delivered orally in the form of lyophilized transplastomic plant tissue. In murine models, oral administration elicited both strong systemic IgG and mucosal IgA responses\u0026mdash;an immunological profile highly desirable for combating mucosally transmitted pathogens like HPV. This contrasts with intraperitoneal delivery, which induced systemic but limited mucosal immunity, underscoring the added value of oral, bioencapsulated vaccines for inducing localized immune protection.\u003c/p\u003e\u003cp\u003eBy integrating rational antigen design with a cost-effective, scalable, and thermostable production and delivery platform, this work lays the foundation for an accessible, needle-free HPV vaccination strategy ideally suited for low- and middle-income countries (LMICs). Furthermore, the successful demonstration of oral immunogenicity opens avenues for broader application of plastid-based vaccines against other mucosal pathogens.\u003c/p\u003e\u003cp\u003eIn conclusion, the HPV_MEV vaccine platform offers a compelling alternative to conventional HPV vaccines and represents a critical step toward democratizing access to effective immunization globally. Future research should focus on evaluating long-term protection, neutralizing antibody titers, and large-scale production optimization, as well as exploring edible plant hosts for direct oral consumption.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e All animal experiments were conducted in compliance with the institutional guidelines for the care and use of laboratory animals at the University of Tabriz, Iran. The experimental protocol was reviewed and approved by the Ethics Committee of the University of Tabriz (Approval ID: IR.TABRIZU.REC.1404.090). All procedures adhered to the Animal Experimentation Guidelines of the University of Tabriz, and every effort was made to minimize animal suffering and the number of animals used. As this study did not involve human participants, consent to participate was not applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. E., designed the construct, performed analyses, and wrote the manuscript. B. B. K., supervised the study, provided specialized scientific and revised the paper. All authors have agreed with the manuscript and provided their consent for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e\u003cp\u003eThis work is based upon research funded by Iran National Science Foundation (INSF) under project No.4023935.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe nucleotide and protein sequence data of the expression cassette of HPV_MEV are available in GenBank at NCBI under the accession number PV057300. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdem M, Beyene D, Feyissa T. Recent achievements obtained by chloroplast transformation. 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Nucleic Acids Res. 1994;22(19):3819\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Human papillomavirus (HPV), Multi-epitope vaccine, Chloroplast transformation, Plant-based vaccine, Oral immunization, Mucosal immunity","lastPublishedDoi":"10.21203/rs.3.rs-7801011/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7801011/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman papillomavirus (HPV) is a major causative agent of cervical and other mucosal cancers, yet the distribution and accessibility of current prophylactic vaccines remain limited, especially in low- and middle-income countries (LMICs), due to high production costs, cold-chain dependency, and limited induction of mucosal immunity. To overcome these challenges, we designed a multi-epitope HPV vaccine (HPV_MEV) incorporating conserved cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes from diverse high- and low-risk HPV genotypes. The construct includes the Toll-like receptor 4 (TLR4) agonist RS09 to enhance innate immune activation and cholera toxin B subunit (CTB) as a mucosal adjuvant to facilitate uptake and presentation at mucosal surfaces. The codon-optimized gene was stably integrated into the chloroplast genome of \u003cem\u003eNicotiana tabacum\u003c/em\u003e using biolistic transformation. Molecular analyses confirmed site-specific integration, homoplasmy, and high-level expression of the recombinant antigen (~\u0026thinsp;3.6 mg/g fresh weight; ~20.8% of total soluble protein). Immunogenicity was evaluated in BALB/c mice via intraperitoneal injection of purified antigen or oral gavage of lyophilized transplastomic leaf tissue. Oral administration elicited strong systemic IgG and mucosal IgA responses, with mucosal immunity surpassing that of the injected formulations. The chloroplast-produced HPV_MEV was comparable in immunogenicity to its E. coli-expressed counterpart, validating its structural and functional integrity. This study highlights the potential of plastid biotechnology for producing an effective, thermostable, needle-free oral HPV vaccine. By integrating rational antigen design with a scalable plant-based production and delivery platform, this approach offers a promising solution for accessible immunization against HPV and other mucosal pathogens in resource-limited settings.\u003c/p\u003e","manuscriptTitle":"Harnessing Plant Chloroplasts for Oral Delivery of a Multi-epitope HPV Vaccine: Toward Cost-Effective Systemic and Mucosal Immunization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-22 01:18:16","doi":"10.21203/rs.3.rs-7801011/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-08T16:27:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T16:01:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T15:06:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-21T17:36:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T09:10:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305859677761357730103083147876772419209","date":"2025-11-17T17:40:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166199042673510415786636411264388715506","date":"2025-11-17T13:32:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309049397734337890289399386349081980569","date":"2025-11-14T15:17:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294820182708657450535580321853512469681","date":"2025-11-13T17:37:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298576827552215480419467862913226975378","date":"2025-11-12T11:54:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73877323790417274030404238643509955199","date":"2025-11-11T19:28:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-11T17:34:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-08T11:13:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T11:13:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Biological Engineering","date":"2025-10-07T15:23:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8cf0bcb-54a2-46ab-b12a-dcf59a46944f","owner":[],"postedDate":"November 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:00:45+00:00","versionOfRecord":{"articleIdentity":"rs-7801011","link":"https://doi.org/10.1186/s13036-025-00618-5","journal":{"identity":"journal-of-biological-engineering","isVorOnly":false,"title":"Journal of Biological Engineering"},"publishedOn":"2025-12-27 15:57:38","publishedOnDateReadable":"December 27th, 2025"},"versionCreatedAt":"2025-11-22 01:18:16","video":"","vorDoi":"10.1186/s13036-025-00618-5","vorDoiUrl":"https://doi.org/10.1186/s13036-025-00618-5","workflowStages":[]},"version":"v1","identity":"rs-7801011","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7801011","identity":"rs-7801011","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}