Optimal size of DNA encapsidated by plant produced human papillomavirus pseudovirions.

Human papillomaviruses (HPVs) are known to be the cause of anogenital and oropharyngeal cancers as well as genital and common warts. HPV pseudovirions (PsVs) are synthetic viral particles that are made up of the L1 major and L2 minor HPV capsid proteins and up to 8 Kb of encapsidated pseudogenome dsDNA. HPV PsVs are used to test novel neutralising antibodies elicited by vaccines, for studying the virus life cycle, and potentially for the delivery of therapeutic DNA vaccines. HPV PsVs are typically produced in mammalian cells, however, it has recently been shown that Papillomavirus PsVs can be produced in plants, a potentially safer, cheaper and more easily scalable means of production. We analysed the encapsidation frequencies of pseudogenomes expressing EGFP, ranging in size from 4.8 Kb to 7.8 Kb, by plant-made HPV-35 L1/L2 particles. The smaller pseudogenomes were found to be packaged more efficiently into PsVs as higher concentrations of encapsidated DNA and higher levels of EGFP expression were obtained with the 4.8 Kb pseudogenome, compared to the larger 5.8-7.8 Kb pseudogenomes. Thus, smaller pseudogenomes, of 4.8 Kb, should be used for efficient plant production of HPV-35 PsVs.

Recovery from Beak and Feather Disease Virus Infection in a Cape Parrot (Poicephalus robustus) Population in South Africa.

Psittacine beak and feather disease (PBFD) is one of the most important viral diseases affecting parrot species worldwide. Outbreaks of PBFD have been reported in wild endemic and endangered South African Cape Parrots (Poicephalus robustus), most recently in 2008. A previous study of wild Cape Parrots in the Eastern Cape region of South Africa in 2010-11 found 34/49 birds positive for beak and feather disease virus (BFDV), the causative agent of PBFD, showing that the outbreak was still ongoing. The present study (2015-16) screened 30 blood samples from the same Cape Parrot population for BFDV infection by PCR: all parrots were found to be BFDV DNA-negative, which showed both that BFDV infection in the region has declined and that the parrot population has recovered. Our data contribute to the important negative data set which permits monitoring the progress of BFDV infections in wild Psittaciformes. We recommend a PCR method with universal BFDV primers as a quick, easy, and consistent diagnostic test for BFDV detection.

Plant expression systems as an economical alternative for the production of iELISA coating antigen AHSV VP7.

African horse sickness (AHS) is a debilitating and highly infectious arthropod-borne disease affecting all species of Equidae. The causative agent of AHS is the non-enveloped dsRNA African horse sickness virus (AHSV), belonging in the genus Orbivirus, family Reoviridae. The identification and surveillance of AHSV by simple and reliable diagnostic tools is essential for managing AHS outbreaks. Indirect ELISAs utilising soluble AHSV antigen or recombinant VP7, an immunodominant and serogroup-specific major core structural protein, are commonly used for serological diagnostic assays. Plant production systems are a significant alternative for recombinant protein production, as they are safe, easily scalable, production rates are rapid and upstream processes are more cost-effective than more traditional expression systems. This pilot study reports the successful production of AHSV-5 VP7 quasi-crystals in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated transient expression using the self-replicating pRIC3.0 plant expression vector. After purification by means of density gradient ultracentrifugation, yields of pure VP7 of 2.66 µg/g fresh leaf mass (FLM) were achieved. Purified plant-produced AHSV-5 VP7 detected AHSV-specific antibodies in horse sera in an indirect ELISA and was able to distinguish between AHSV-positive and negative sera. Additionally, plant-produced AHSV-5 VP7 detected AHSV-specific antibodies to the same degree as E. coli-produced VP7. These results justify further investigation into the diagnostic capability of plant-produced AHSV VP7 quasi-crystals. To the best of our knowledge, this is the first report of AHSV VP7 quasi-crystal production in N. benthamiana and the first time that plant-produced VP7's potential as a diagnostic has been assessed.

Humoral and cell-mediated immune responses to plant-produced African horse sickness virus VP7 quasi-crystals.

African horse sickness (AHS) is a devastating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS, and commercially used live-attenuated vaccines have various undesirable side effects. Previous studies have shown that mice inoculated with insoluble African horse sickness virus (AHSV) VP7 crystals are protected from live challenge with a lethal dose of AHSV. This study investigates the humoral and cell-mediated immune responses in guinea-pigs to a safer monovalent vaccine alternative based on AHSV-5 VP7 quasi-crystals produced in plants. Guinea-pigs received prime- and boost-inoculations of between 10 and 50 µg of purified plant-produced AHSV VP7. Western immunoblot analysis of the humoral response showed stimulation of high titres of anti-VP7 antibodies 28 days after the boost-inoculation in sera from three of the five experimental animals. In addition, RNA-seq transcriptome profiling of guinea-pig spleen-derived RNA highlighted thirty significantly (q ≤ 0.05) differentially expressed genes involved in innate and adaptive immunity. Differential expression of genes involved in Th1, Th2 and Th17 cell differentiation suggest a cell-mediated immune response to AHSV-5 VP7. Upregulation of several important cytokines and cytokine receptors were noted, including TNFSF14, CX3CR1, IFNLR1 and IL17RA. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. While further investigation is needed to validate these findings, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic and induce both humoral and cell-mediated responses.

Immunogenicity of Plant-Produced Human Papillomavirus (HPV) Virus-Like Particles (VLPs).

Cervical cancer is ranked fourth among the top cancers in women and is the second most common cancer in low- and middle-income regions, with ~570,000 new cases reported in 2018, which attributed to 84% of worldwide cervical cancer cases. Three commercially available prophylactic Human papillomavirus (HPV) vaccines are effective at preventing HPV infections. However, these vaccines are expensive due to their complex production systems, therefore limiting their use in developing countries. Recently, the use of plants to produce vaccines has emerged as a cost-effective alternative to conventionally used expression systems. Here, L1 proteins of eight high-risk (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and two low risk (HPV 6 and 34) HPV types were successfully expressed in Nicotiana benthamiana, and transmission electron microscopy (TEM) analysis showed the presence of VLPs and/or capsomeres. Immunogenicity studies were conducted in mice utilizing HPV 35, 52, and 58 and showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous HPV pseudovirions in pseudovirion-based neutralization assays (PBNAs). This work demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries.

Extended Set of GoldenBraid Compatible Vectors for Fast Assembly of Multigenic Constructs and Their Use to Create Geminiviral Expression Vectors.

Methods for simple and fast assembly of exchangeable standard DNA parts using Type II S restriction enzymes are becoming more and more popular in plant synthetic and molecular biology. These methods enable routine construction of large and complex multigene DNA structures. Two available frameworks emphasize either high cloning capacity (Modular Cloning, MoClo) or simplicity (GoldenBraid, GB). Here we present a set of novel α-level plasmids compatible with the GB convention that extend the ability of GB to rapidly assemble more complex genetic constructs, while maintaining compatibility with all existing GB parts as well as most MoClo parts and GB modules. With the use of our new plasmids, standard GB parts can be assembled into complex assemblies containing 1, 5, 10 and up to theoretically 50 units in each successive level of infinite loop assembly. Assembled DNA constructs can be also combined with conventional binary GB-assemblies (1, 2, 4, 8… units). We demonstrate the usefulness of our framework on single tube assembly of replicating plant expression constructs based on the geminivirus Bean yellow dwarf virus (BeYDV).

Transient protein expression in tobacco BY-2 plant cell packs using single and multi-cassette replicating vectors.

KEY MESSAGE: This is the first evidence that replicating vectors can be successfully used for transient protein expression in BY-2 plant cell packs. Transient recombinant protein expression in plants and recently also plant cell cultures are of increasing interest due to the speed, safety and scalability of the process. Currently, studies are focussing on the design of plant virus-derived vectors to achieve higher amounts of transiently expressed proteins in these systems. Here we designed and tested replicating single and multi-cassette vectors that combine elements for enhanced replication and hypertranslation, and assessed their ability to express and particularly co-express proteins by Agrobacterium-mediated transient expression in tobacco BY-2 plant cell packs. Substantial yields of green and red fluorescent proteins of up to ~ 700 ng/g fresh mass were detected in the plant cells along with position-dependent expression. This is the first evidence of the ability of replicating vectors to transiently express proteins in BY-2 plant cell packs.

African Horse Sickness: A Review of Current Understanding and Vaccine Development.

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines that utilize viral vectors or are based on reverse genetics or virus-like particle technologies. This review summarizes the current understanding of AHSV structure and the viral replication cycle and also evaluates existing and potential vaccine strategies that may be applied to prevent or control the disease.

Substitution of Human Papillomavirus Type 16 L2 Neutralizing Epitopes Into L1 Surface Loops: The Effect on Virus-Like Particle Assembly and Immunogenicity.

Cervical cancer caused by infection with human papillomaviruses (HPVs) is the fourth most common cancer in women globally, with the burden mainly in developing countries due to limited healthcare resources. Current vaccines based on virus-like particles (VLPs) assembled from recombinant expression of the immunodominant L1 protein are highly effective in the prevention of cervical infection; however, these vaccines are expensive and type-specific. Therefore, there is a need for more broadly protective and affordable vaccines. The HPV-16 L2 peptide sequences 108-120, 65-81, 56-81, and 17-36 are highly conserved across several HPV types and have been shown to elicit cross-neutralizing antibodies. To increase L2 immunogenicity, L1:L2 chimeric VLPs (cVLP) vaccine candidates were developed. The four L2 peptides mentioned above were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or in the C-terminal region at position 431 (SAE) to generate HPV-16-derived L1:L2 chimeras. All eight chimeras were transiently expressed in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated DNA transfer. SAC chimeras predominantly assembled into higher order structures (T = 1 and T = 7 VLPs), whereas SAE chimeras assembled into capsomeres or formed aggregates. Four SAC and one SAE chimeras were used in vaccination studies in mice, and their ability to generate cross-neutralizing antibodies was analyzed in HPV pseudovirion-based neutralization assays. Of the seven heterologous HPVs tested, cross-neutralization with antisera specific to chimeras was observed for HPV-11 (SAE 65-18), HPV-18 (SAC 108-120, SAC 65-81, SAC 56-81, SAE 65-81), and HPV-58 (SAC 108-120). Interestingly, only anti-SAE 65-81 antiserum showed neutralization of homologous HPV-16, suggesting that the position of the L2 epitope display is critical for maintaining L1-specific neutralizing epitopes.

Immunogenicity of plant-produced porcine circovirus-like particles in mice.

Porcine circovirus type 2 (PCV-2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus-associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV-2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV-2 capsid protein (CP) from plants is an essential first step towards the goal of a plant-produced PCV-2 vaccine candidate. In this study, the PCV-2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV-2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self-assembled into virus-like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant-produced PCV-2 VLPs elicited specific antibody responses to PCV-2 CP. This is the first report describing the expression of PCV-2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.

Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses.

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.

Optimizing a Human Papillomavirus Type 16 L1-Based Chimaeric Gene for Expression in Plants.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5' region of the L1 gene with the wild type (wt) viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5' end of the L1 gene back to its wt sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5' end of L1 are inadvertently destroyed by changing the codon usage, which enhances protein expression.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Therapeutic vaccines for high-risk HPV-associated diseases.

Cancer is the second leading cause of death worldwide, and it is estimated that Human papillomavirus (HPV) related cancers account for 5% of all human cancers. Current HPV vaccines are extremely effective at preventing infection and neoplastic disease; however, they are prophylactic and do not clear established infections. Therapeutic vaccines which trigger cell-mediated immune responses for the treatment of established infections and malignancies are therefore required. The E6 and E7 early genes are ideal targets for vaccine therapy due to their role in disruption of the cell cycle and their constitutive expression in premalignant and malignant tissues. Several strategies have been investigated for the development of therapeutic vaccines, including live-vector, nucleic acid, peptide, protein-based and cell-based vaccines as well as combinatorial approaches, with several vaccine candidates progressing to clinical trials. With the current understanding of the HPV life cycle, molecular mechanisms of infection, carcinogenesis, tumour biology, the tumour microenvironment and immune response mechanisms, an approved HPV therapeutic vaccine seems to be a goal not far from being achieved. In this article, the status of therapeutic HPV vaccines in clinical trials are reviewed, and the potential for plant-based vaccine production platforms described.

LALF32-51 -E7, a HPV-16 therapeutic vaccine candidate, forms protein body-like structures when expressed in Nicotiana benthamiana leaves.

High-risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV-16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF32-51 ), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF32-51 -E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF32-51 -E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF32-51 -E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast-targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB-like structures were also seen for the cytoplasmic LG. PB-like structure formation was confirmed for both LG and LALF32-51 -E7 by TEM. LALF32-51 -E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB-like structures. This study could open a new avenue for the use of LALF32-51 as a PB-inducing peptide.

Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine.

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live-attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost-effective recombinant vaccine. Here, we report the plant-based production of a virus-like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens-mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell-based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.

Complete Genome Sequence of Bos taurus Papillomavirus Type 1, Isolated in Morocco.

Bos taurus papillomaviruses infect cattle, which has both animal health and economic consequences. This is the first report and sequence of Bos taurus papillomavirus type 1 isolated from warts in cattle in Morocco. The double-stranded DNA (dsDNA) genome was 7,945 bp in size, and eight open reading frames were identified.

Xenogenic rolling-circle replication of a synthetic beak and feather disease virus genomic clone in 293TT mammalian cells and Nicotiana benthamiana.

The preparation of infectious beak and feather disease circovirus virions (BFDV) has until now relied on the extraction of virus from whole tissue of deceased or euthanized parrots known to be infected with the virus. Extraction from diseased tissue is necessary, as the virus has yet to be grown in vitro using tissue-cultured cells from any source. While infectious DNA clones have been synthesized for porcine and duck circoviruses, and both replicate in host cells and result in active viral infection in animals, this has not been shown for BFDV. The aim of this study was to prepare an infectious BFDV genomic clone that could be used as challenge material in birds for vaccine testing. A putatively infectious BFDV genomic clone was designed and tested in mammalian cell culture, and in the plant Nicotiana benthamiana in the presence of plant-specific ssDNA geminivirus replication components. Replication was assessed using rolling-circle amplification, qPCR, replication-deficient clones and rescue plasmids. We showed that a synthetic partially dimeric BFDV genomic clone self-replicated when transfected into 293TT mammalian cells, and was also replicated in N. benthamiana in the presence of geminivirus replication elements. This is the first report of a BFDV genome replicating in any cell system, and the first report of a circovirus replicating with the aid of a geminivirus in a plant. Both of these developments could open up possibilities for making reagents and vaccines for BFDV, testing vaccine efficacy and investigating viral replication using rationally designed artificial genomes.

Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana.

BACKGROUND: Beak and feather disease virus (BFDV) is an important disease causing agent affecting psittacines. BFDV is highly infectious and can present as acute, chronic or subclinical disease. The virus causes immunodeficiency and is often associated with secondary infections. No commercial vaccine is available and yields of recombinant BFDV capsid protein (CP) expressed in insect cells and bacteria are yet to be seen as commercially viable, although both systems produced BFDV CP that could successfully assemble into virus-like particles (VLPs). Plants as expression systems are increasingly becoming favourable for the production of region-specific and niche market products. The aim of this study was to investigate the formation and potential for purification of BFDV VLPs in Nicotiana benthamiana. METHODS: The BFDV CP was transiently expressed in N. benthamiana using an Agrobacterium-mediated system and plant expression vectors that included a bean yellow dwarf virus (BeYDV)-based replicating DNA vector. Plant-produced BFDV CP was detected using immunoblotting. VLPs were purified using sucrose cushion and CsCl density gradient centrifugation and visualised using transmission electron microscopy. RESULTS: In this study we demonstrate that the BFDV CP can be successfully expressed in N. benthamiana, albeit at relatively low yield. Using a purification strategy based on centrifugation we demonstrated that the expressed CP can self-assemble into VLPs that can be detected using electron microscopy. These plant-produced BFDV VLPs resemble those produced in established recombinant expression systems and infectious virions. It is possible that the VLPs are spontaneously incorporating amplicon DNA produced from the replicating BeYDV plant vector. CONCLUSIONS: This is the first report of plant-made full-length BFDV CP assembling into VLPs. The putative pseudovirions could be used to further the efficacy of vaccines against BFDV.

Expression optimization of a cell membrane-penetrating human papillomavirus type 16 therapeutic vaccine candidate in Nicotiana benthamiana.

High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors. The highest expression levels of LALF32-51-E7 were obtained by using a self-replicating plant expression vector and chloroplast targeting, which increased its accumulation by 27-fold compared to cytoplasmic localization. The production and extraction of LALF32-51-E7 was scaled-up and purification optimized by affinity chromatography. If further developed, this platform could potentially allow for the production of a more affordable therapeutic vaccine for HPV-16. This would be extremely relevant in the context of developing countries, where cervical cancer and other HPV-related malignancies are most prevalent, and where the population have limited or no access to preventative vaccines due to their typical high costs.