Fig. 11. Antibody (A) and cytotoxic T-lymphocytes (B) induction by L1-VPLs. Response elicited by control plant extracts (mice 1 - 5), control plant extract from *Nicotiana benthamiana*  containing purified VLPs from insect cells (mice 6 - 10) and crude plant extracts from leaves expressing L1 (mice 11 - 25) in C57BL/6 mice. Collected sera from individual mice were tested by ELISA for induction of specific antibody (y-axis in OD 450 units). CTLs induction was measured by ELISPOT analysis of splenocytes recovered from scarified animals. The number of CTLs spots was recorded by an ELISPOT reader and expressed as a mean per 106 splenocytes

Plant Production of Vaccine Against HPV: A New Perspectives 163

40% of mice immunized with E7 containing crude leaf extract were protected from growth of cancer induced by E7-expressing C3 cells

80% of mice immunized with E7 containing crude leaf extract were protected from growth of cancer induced by E7-expressing C3 cells

Mice vaccinated orally with freezedried E7-containing leaf extract mixed with feed produced high titer of anti-E7 antibodies

Dendritic cells pulsed with E7 containing leaf extract were able to prime naive lymphocytes to induce E7 specific CTLs

Purified protein injected into mice induced IgG and CTL response and protected them against challenge with E7-expressing tumor cells in both prophylactic and therapeutic vaccination regimen

Mice vaccinated with the protein were protected against tumor cells expressing E7

DNA vaccine was able to protect vaccinated mice from the growth of tumors induced by E7-expressing TC-1 cells

a) DNA vaccine tested in therapeutic setting was able to block tumor growth in the 40% of challenged mice b) Not performed

ND

Mice injected with chimeric VLPs were able to develop neutralizing antibodies and specific CTLs

**Antigen Production system and yield Efficacy data**

*N. benthamiana* tobacco leaves infected with PVX–E7 3–4 μg/g fresh leaves

*N. benthamiana* tobacco leaves infected with PVX–E7; protein targeted to secretory pathway 15–20 μg/g fresh leaves

*N. benthamiana* tobacco leaves infected with PVX–E7; protein targeted to secretory pathway

infected with PVX–E7

*N. benthamiana* tobacco leaves infected with LicKM–E7GGG, using a launch vector expression system 400 μg purified protein per gram of fresh leaves

PVX CP–HPV-16 E7 Tobacco chloroplast ND

b) Expressed in N. benthamiana

Expressed by PVX in Nicotiana tabacum, N. benthamiana using Agrobacterium tumefaciensmediated inoculation

Tomato seedling cotyledons HPV-16 VLPs carrying L1 fused to string of epitopes from E6 and E7 using A. tumefaciens-mediated inoculation

proteins from *Saponaria officinalis;* VLP: Virus-like particle. (Giorgi et al., 2010)

Table 3. Plant-derived HPV antigens for the development of therapeutic vaccines. CP: Coat protein; CTL: Cytotoxic T lymphocyte; HPV: Human papillomavirus; ND: No data; PVA: Potato virus A; PVX: Potato virus X; SAP-KQ: Mutagenized type I ribosome inhibiting

The important proof of the L2 plant-produced nonhuman papillomavirus vaccine efficiency was published in connection with the purified rTMV particles displaying cottontail rabbit papillomavirus (CRPV) or rabbit oral papillomavirus (ROPV) L2 protein peptides on their

HPV-16 E7 *N. benthamiana* tobacco leaves

peptide–E7 mut *N. benthamiana* via agroinfiltration

PVX CP–HPV-16 E7GGG DNA vaccine

SAP-KQ–E7GGG a) DNA vaccine

HPV-16 E7

HPV-16 E7

HPV-16 E7

LicKM–E7GGG

11-kDa Zera zein-derived

HPV-16 L2–PVA CP–E7 epitope fused protein

HPV-16 VLPs carrying L1 fused to a string of epitopes from E6 and E7

Fig. 12. (A) The specificity of mAb E2 toward various L1 assembly structures. VLPs, T1 particles (12 L1-pentamers) and capsomeres produced in insect cells were absorbed on the microtiter plates and analyzed by ELISA. Interactions of hybridoma E2 supernatant is compared to antibodies obtained after immunization with insect cell-derived VLPs (<IP_ADDRESS>, E10 and 25C). (B) Neutralization assay. Sera of mice and of E2 hybridoma supernatant were tested for neutralization of infection of 293T cells by HPV pseudovirions (Psv). Percent of neutralization obtained by incubation with mice sera diluted 1:50 and that of E2 hybridoma supernatant diluted 1:5, 1:20, 1:100, 1:500. Neutralization activity of mice sera was compared to a high titer rabbit polyclonal anti-L1 antiserum 4543 (100 %) and to the mAb's 1.3.1 and 1.3.2 as additional positive controls.

### **2.4.5 Plant based therapeutic and second-generation vaccines**

Capsomeres can be used as a potential cost-saving substitute of VLPs, as L1-pentameric capsomeres are considered thermo-stable, the advantageous feature for the use in developing countries where cold chain administration and delivery of vaccine is difficult to maintain (Stanley et al., 2008). A promising step towards a capsomeres-based vaccine was described by Yuan et al. (2001) when dogs were completely protected against canine oral papillomavirus (COPV) infection by capsomeres vaccination. Capsomeres have been also reported to induce neutralizing antibodies and L1-specific cytotoxic T-lymphocytes (CTLs) upon oral, intranasal and subcutaneous immunization (Dell et al., 2006; Thönes & Müller, 2007; Schadlich et al., 2009). To increase immunogenicity, L1\_2xCys mutant version of HPV16 L1 protein was fused with LTB as an adjuvant and expressed in chloroplasts (Waheed et al, 2011a, 2011b).

Plant expressed HPV antigens to be used as therapeutic vaccine to date are summarized in Table 3:

Human Papillomavirus and Related Diseases 162 – From Bench to Bedside – A Clinical Perspective

Fig. 12. (A) The specificity of mAb E2 toward various L1 assembly structures. VLPs, T1 particles (12 L1-pentamers) and capsomeres produced in insect cells were absorbed on the microtiter plates and analyzed by ELISA. Interactions of hybridoma E2 supernatant is compared to antibodies obtained after immunization with insect cell-derived VLPs (<IP_ADDRESS>, E10 and 25C). (B) Neutralization assay. Sera of mice and of E2 hybridoma supernatant were tested for neutralization of infection of 293T cells by HPV pseudovirions (Psv). Percent of neutralization obtained by incubation with mice sera diluted 1:50 and that of E2 hybridoma supernatant diluted 1:5, 1:20, 1:100, 1:500. Neutralization activity of mice sera was compared to a high titer rabbit polyclonal anti-L1 antiserum 4543 (100 %) and to the mAb's 1.3.1 and

Capsomeres can be used as a potential cost-saving substitute of VLPs, as L1-pentameric capsomeres are considered thermo-stable, the advantageous feature for the use in developing countries where cold chain administration and delivery of vaccine is difficult to maintain (Stanley et al., 2008). A promising step towards a capsomeres-based vaccine was described by Yuan et al. (2001) when dogs were completely protected against canine oral papillomavirus (COPV) infection by capsomeres vaccination. Capsomeres have been also reported to induce neutralizing antibodies and L1-specific cytotoxic T-lymphocytes (CTLs) upon oral, intranasal and subcutaneous immunization (Dell et al., 2006; Thönes & Müller, 2007; Schadlich et al., 2009). To increase immunogenicity, L1\_2xCys mutant version of HPV16 L1 protein was fused with LTB as an adjuvant and expressed in chloroplasts

Plant expressed HPV antigens to be used as therapeutic vaccine to date are summarized in

**2.4.5 Plant based therapeutic and second-generation vaccines**

1.3.2 as additional positive controls.

(Waheed et al, 2011a, 2011b).

Table 3:

Table 3. Plant-derived HPV antigens for the development of therapeutic vaccines. CP: Coat protein; CTL: Cytotoxic T lymphocyte; HPV: Human papillomavirus; ND: No data; PVA: Potato virus A; PVX: Potato virus X; SAP-KQ: Mutagenized type I ribosome inhibiting proteins from *Saponaria officinalis;* VLP: Virus-like particle. (Giorgi et al., 2010)

The important proof of the L2 plant-produced nonhuman papillomavirus vaccine efficiency was published in connection with the purified rTMV particles displaying cottontail rabbit papillomavirus (CRPV) or rabbit oral papillomavirus (ROPV) L2 protein peptides on their

Plant Production of Vaccine Against HPV: A New Perspectives 165

significant number of compounds tested for adjuvant effect are clearly more effective than alum, albeit usually accompanied with a higher toxicity as e.g. Freund's complete adjuvant. This is the main reason preventing their use as adjuvants in human vaccine formulations. As adjuvants were successfully tested low toxic mutants of the cholera toxin (CT) (Yamamoto et al., 1997) and *E.coli* heat-labile enterotoxin (LT) (Chong et al., 1998). The inactive B-subunits of these toxins proved to be a strong mucosal (oral, nasal, vaginal, etc.) adjuvants (e.g. Salmonella toxin B subunit is used in commercial Cervarix vaccine) for a wide variety of antigens in mice and other animal species so far tested, however their use in humans is limited (Chong et al., 1998). This is why there is a growing interest to develop new adjuvants eliciting high mucosal, humoral and cellular immune response accompanied by

**Antigen delivery systems Immunopotentiators**  Insoluble aluminum compounds MPL and synthetic derivates Calcium phosphate MDP and derivatives

Microparticles (e.g., PLG) Saponins (Quils, QS-21)

Virus-like particles & viral vectors Cytokines & chemokines

resistant products mimicking the effect of LPS in foliar extract (Di Bonito et al, 2009).

Probably the most studied plant compound with adjuvant effect is the saponin fractions isolated from *Quillaja saponaria* (Newman et al., 1992). The mechanism of saponin effect is complex and, apart from direct cellular stimulation, there is also evidence that saponins may

Table 4. Examples of adjuvant classes (O'Hagan & Rappuoli, 2004)

Emulsions (e.g., MF59, Montanides) Small-molecule immune potentiators ( SMIPs)

Plant extracts are known to contain various compounds, which supposedly have immunostimulatory and immunosuppressive effects (Wagner & Proksh, 1985). Plant crude extracts and their components were tested for their adjuvant capacity. The extract from leaves of *Nicotiana bethamiana* co-administrated subcutaneously with HPV16E7 (Franconi et al., 2002) or HPV16L1 VLP´s (Maclean et. al, 2007; Šmídková et al., 2010) enhanced specific humoral and cellular immune response in tested mice. Freund's adjuvant added to the plant extract did not increased noticeably humoral response elicited by HPV16L1 VLP´s in subcutaneously immunized mice and results indicate that the addition of Freund's adjuvant to plant extract might be even deleterious (Maclean et. al, 2007). The study of Isfar et al. (2004) compares adjuvant effect of CT and of aqueous extract of *Solanum torvum* (STE). STE was shown to evoke an increase in IgA titer comparable to that of CT when coadministrated with ovalbumin intraperitoneally. No acute toxic effects were evident with the used dose range. Plant extract has been shown to induce DC maturation of dendritic cells. This effect was not caused by lipopolysaccharide (LPS) but rather by presence of heat-

Liposomes Oligonucleotides (CpG, etc.) Virosomes™ Double-stranded RNA (dsRNA)

> Alternative pathogen-associated Molecular patterns (PAMPs) (*E. coli* heat Labile enterotoxin (LTB); flagellin)

> > (e.g., resiquimod [R848])

negligible or low toxicity.

ISCOMS®

**3.1.2 Adjuvant effect of plant extracts**

surface (Palmer et al., 2006). This pseudovirion system was also used to express entire native *L1* gene of CRPV. Intramuscular injection with of CRPV L1-containing concentrated plant extract derived from transgenic tobacco protected rabbits against CRPV challenge (Kohl et al., 2006).

Since VLPs based vaccines are not effective in the therapy of diseases, an important goal is development of anti-HPV vaccines with either therapeutic or both prophylactic and therapeutic properties. Few studies were performed with viral oncoproteins expressed in plants. HPV-16 E7 was transiently expressed using a viral vector based on potato virus X (PVX) in the *Nicotiana benthamiana, Nicotiana rustica, Nicotiana tabacum, Chenopodium quinoa*  and the tomato *Solanum lycopersicum L.* cv. Micro-Tom. The highest expression of HPV-16 E7 3–4 μg/g of fresh leaves was achieved in *Nicotiana benthamiana* and the expressed E7 induced specific humoral and cell-mediated immune responses in mice (Franconi et al., 2002, 2006). The efficiency of chimeric constructs when E7 is fused to other proteins and expressed in plants was also reported. The expression of HPV-16 E7 fused with the PVX CP in tobacco chloroplasts has been recently reported. The expression of the fusion protein in this system was higher than E7 alone (Morgenfeld et al., 2009). Mutated E7, E7GGG, which lacks the retinoblastoma binding site, and thus the native transformation potential, was fused to the *Clostridium thermocellum* b-1,3–1,4-glucanase (LicKM) as a carrier molecule for expression in plants. The expression of fusion protein in *Nicotiana benthamiana* yielded 400 μg of purified protein per gram of leaf (Musiychuk et al., 2007). Injection of the purified LicKM–E7GGG fusion protein into mice induced both E7-specific IgG and cytotoxic T-cell responses, and protected mice against challenge with E7-expressing tumor cells (Massa et al., 2007). The successful expression of chimeric HPV-16 L1 protein fused to a string of three E7 and one E6 epitopes in transgenic tomatoes demonstrates that a combination prophylactic therapeutic HPV vaccine could be produced in plants (Paz de la Rosa et al., 2009; Monroy-García et. al, 2011). Unfortunately, while the produced VLPs stimulated both antibody and T-cell responses, yields were low in the range 0.05 – 0.1% of total soluble protein. Similarly, combined vaccine based on minor capsid protein L2 and an epitope of E7 oncoprotein was successfully expressed in plants, when an epitope of the L2 protein and an epitope of E7 oncoprotein were fused to the N'- and C'-end of PVA CP, respectively. The construct was cloned into a PVX-based vector and transiently expressed in plants using *Agrobacterium*-mediated inoculation (Čeřovská et al., 2008).

### **3. Vaccine formulation**

### **3.1 Parenteral administration**

### **3.1.1 Adjuvants**

The goal of vaccination is to generate a strong immune response to the administered antigen. Papillomavirus VLPs themselves are good "inducers" of immune response and antigen determinants present on their surface are able to activate dendritic cells (DCs) for triggering T-cell activation (Bontkes et al., 2005; Yang et al., 2005). Nevertheless, for efficient clinical use additional adjuvants are needed not only to enhance the immune response, but also assuring achievement of appropriate type of protective immunity in each situation.

The aluminum (alum) salts or gel-based adjuvant formulations used e.g. in HPV Gardasil vaccine are currently approved in vaccines licensed for human use in the US. Nevertheless a Human Papillomavirus and Related Diseases 164 – From Bench to Bedside – A Clinical Perspective

surface (Palmer et al., 2006). This pseudovirion system was also used to express entire native *L1* gene of CRPV. Intramuscular injection with of CRPV L1-containing concentrated plant extract derived from transgenic tobacco protected rabbits against CRPV challenge (Kohl et

Since VLPs based vaccines are not effective in the therapy of diseases, an important goal is development of anti-HPV vaccines with either therapeutic or both prophylactic and therapeutic properties. Few studies were performed with viral oncoproteins expressed in plants. HPV-16 E7 was transiently expressed using a viral vector based on potato virus X (PVX) in the *Nicotiana benthamiana, Nicotiana rustica, Nicotiana tabacum, Chenopodium quinoa*  and the tomato *Solanum lycopersicum L.* cv. Micro-Tom. The highest expression of HPV-16 E7 3–4 μg/g of fresh leaves was achieved in *Nicotiana benthamiana* and the expressed E7 induced specific humoral and cell-mediated immune responses in mice (Franconi et al., 2002, 2006). The efficiency of chimeric constructs when E7 is fused to other proteins and expressed in plants was also reported. The expression of HPV-16 E7 fused with the PVX CP in tobacco chloroplasts has been recently reported. The expression of the fusion protein in this system was higher than E7 alone (Morgenfeld et al., 2009). Mutated E7, E7GGG, which lacks the retinoblastoma binding site, and thus the native transformation potential, was fused to the *Clostridium thermocellum* b-1,3–1,4-glucanase (LicKM) as a carrier molecule for expression in plants. The expression of fusion protein in *Nicotiana benthamiana* yielded 400 μg of purified protein per gram of leaf (Musiychuk et al., 2007). Injection of the purified LicKM–E7GGG fusion protein into mice induced both E7-specific IgG and cytotoxic T-cell responses, and protected mice against challenge with E7-expressing tumor cells (Massa et al., 2007). The successful expression of chimeric HPV-16 L1 protein fused to a string of three E7 and one E6 epitopes in transgenic tomatoes demonstrates that a combination prophylactic therapeutic HPV vaccine could be produced in plants (Paz de la Rosa et al., 2009; Monroy-García et. al, 2011). Unfortunately, while the produced VLPs stimulated both antibody and T-cell responses, yields were low in the range 0.05 – 0.1% of total soluble protein. Similarly, combined vaccine based on minor capsid protein L2 and an epitope of E7 oncoprotein was successfully expressed in plants, when an epitope of the L2 protein and an epitope of E7 oncoprotein were fused to the N'- and C'-end of PVA CP, respectively. The construct was cloned into a PVX-based vector and transiently expressed in plants using

The goal of vaccination is to generate a strong immune response to the administered antigen. Papillomavirus VLPs themselves are good "inducers" of immune response and antigen determinants present on their surface are able to activate dendritic cells (DCs) for triggering T-cell activation (Bontkes et al., 2005; Yang et al., 2005). Nevertheless, for efficient clinical use additional adjuvants are needed not only to enhance the immune response, but also assuring achievement of appropriate type of protective immunity in each situation.

The aluminum (alum) salts or gel-based adjuvant formulations used e.g. in HPV Gardasil vaccine are currently approved in vaccines licensed for human use in the US. Nevertheless a

*Agrobacterium*-mediated inoculation (Čeřovská et al., 2008).

**3. Vaccine formulation**

**3.1.1 Adjuvants**

**3.1 Parenteral administration**

al., 2006).

significant number of compounds tested for adjuvant effect are clearly more effective than alum, albeit usually accompanied with a higher toxicity as e.g. Freund's complete adjuvant. This is the main reason preventing their use as adjuvants in human vaccine formulations. As adjuvants were successfully tested low toxic mutants of the cholera toxin (CT) (Yamamoto et al., 1997) and *E.coli* heat-labile enterotoxin (LT) (Chong et al., 1998). The inactive B-subunits of these toxins proved to be a strong mucosal (oral, nasal, vaginal, etc.) adjuvants (e.g. Salmonella toxin B subunit is used in commercial Cervarix vaccine) for a wide variety of antigens in mice and other animal species so far tested, however their use in humans is limited (Chong et al., 1998). This is why there is a growing interest to develop new adjuvants eliciting high mucosal, humoral and cellular immune response accompanied by negligible or low toxicity.

Table 4. Examples of adjuvant classes (O'Hagan & Rappuoli, 2004)

### **3.1.2 Adjuvant effect of plant extracts**

Plant extracts are known to contain various compounds, which supposedly have immunostimulatory and immunosuppressive effects (Wagner & Proksh, 1985). Plant crude extracts and their components were tested for their adjuvant capacity. The extract from leaves of *Nicotiana bethamiana* co-administrated subcutaneously with HPV16E7 (Franconi et al., 2002) or HPV16L1 VLP´s (Maclean et. al, 2007; Šmídková et al., 2010) enhanced specific humoral and cellular immune response in tested mice. Freund's adjuvant added to the plant extract did not increased noticeably humoral response elicited by HPV16L1 VLP´s in subcutaneously immunized mice and results indicate that the addition of Freund's adjuvant to plant extract might be even deleterious (Maclean et. al, 2007). The study of Isfar et al. (2004) compares adjuvant effect of CT and of aqueous extract of *Solanum torvum* (STE). STE was shown to evoke an increase in IgA titer comparable to that of CT when coadministrated with ovalbumin intraperitoneally. No acute toxic effects were evident with the used dose range. Plant extract has been shown to induce DC maturation of dendritic cells. This effect was not caused by lipopolysaccharide (LPS) but rather by presence of heatresistant products mimicking the effect of LPS in foliar extract (Di Bonito et al, 2009).

Probably the most studied plant compound with adjuvant effect is the saponin fractions isolated from *Quillaja saponaria* (Newman et al., 1992). The mechanism of saponin effect is complex and, apart from direct cellular stimulation, there is also evidence that saponins may

Plant Production of Vaccine Against HPV: A New Perspectives 167

A good nonparenteral alternative for vaccine delivery could be nasal immunization, which already proved to be effective in tests with animals. The obstacles imposed by the normal process of mucociliary clearance limiting residence time of applied antigens could be circumvent by presence of an inert *in situ* gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen (Hefferon, 2010). The nasal cavity is a promising site for vaccine delivery because it is easy to access, is highly vascularized, has a relatively large surface area, has low proteolytic activity, and is able to induce systemic immunity as well as both local and distal mucosal immunity via the Common Mucosal

The major reason for the vaccine production in plants is that the vaccine antigen production is safe and could be potentially cheap and both transient and transgenic productions are scalable. Biologically active proteins can be produced more easily in plants than in other eukaryotic systems; and that the use of food plants could eventually allow edible and/or oral vaccines to be produced cheaper. The recent reports indicate very high yields of human vaccine candidates to be obtained via plastid transformation or large scale transient expression what could enable to meet the expected requirement of antigen for oral route as is required parenterally for the same immune response. A recent review on human trials of plant-based oral vaccines summarizing human studies of oral transgenic plant derived vaccines against enterotoxigenic *E. coli* infection, norovirus and HBV adds weight to the growing body of evidence that plant-made oral vaccines to these viruses are not only feasible, but could effective (Rybicki, 2010). Nevertheless there is still long way to go from improvement of antigen yields, to formulation of the vaccine including auxiliary factors improving efficacy and stability, to translation of the proposed vaccines into clinical trials

This work was supported by the Grant Agency of the Czech Republic contract 521/01/1418 and 521/04/0971, Grant Agency of the Czech Academy of Sciences A6038201 and S5038304 and by Ministry of Education, Youth and Sports of the Czech Republic projects 1M0505.

Alvarez-Salas, L.M. (2008). Amolimogene bepiplasmid, a DNA-based therapeutic encoding

the E6 and E7 epitopes from HPV, for cervical and anal dysplasia. *Current opinion in molecular therapeutics*, Vol.10, No.6, (December 2008), pp. 622-6288, ISSN 1464-8431 Armstrong, E. P. (2010). Prophylaxis of cervical cancer and related cervical disease: a review

of the cost-effectiveness of vaccination against oncogenic HPV types. *Journal of managed care pharmacy*, Vol.16, No.3, (April 2010), pp. 217-230, ISSN 1083-4087 Bardor, M.; Faveeuw, C.; Fitchette, A.C.; Gilbert, D.; Galas, L.; Trottein, F.; Faye, L. &

Lerouge, P. (2003). Immunoreactivity in mammals of two typical plant glycoepitopes, core alpha(1,3)-fucose and core xylose. *Glycobiology,* Vol.13, No.6, (June

and, not least, governmental and/or regulatory body approvals.

2003), pp.427-434, ISSN 0959-6658

Immune System (CMIS)

**5. Acknowledgements**

**6. References**

**4. Conclusions**

enhance oral immunization by protection of antigen from degradation by digestive enzymes and by increasing permeability of the intestine to macromolecules (Campbell, 1995).

All these findings are promising for development of needle-free administration route of immunization as an alternative to intramuscular vaccine application. For this purposes intranasal, intravaginal, transdermal, sublingual and intramuscular administration routes were tested for systemic immune responses against HPV16L1 using (Cho et al., 2010). The sublingual route provided the most effective mucosal secretory IgA (sIgA) and serum IgG responses, cholera toxin subunit B (CTB) showed the most promising adjuvant activity.

### **3.2 HPV L1 antigens as an edible vaccine?**

The majority of currently licensed vaccines are administered parenterally, even though they have the disadvantages of patient reluctance to tolerate needle sticks and lack of mucosal immune induction (Velasquez et al 2010). Edible vaccine represents further approach to selfadministrated nonparenteral vaccine that could solve the problem of high cost and need for appropriate storage of currently available preventive HPV vaccines.

Thönes & Müller (2007) investigated the oral immunogenicity of different assembly forms of HPV 16 L1: T7-VLPs, T1 particles and capsomeres produced from Baculovirus expression vector in insect cells and showed that all three assembly forms induce humoral and cellular immune responses after oral vaccination of mice. The anti-L1 antibodies were conformationspecific and showed neutralizing activity in a pseudovirion-based assay. They also investigated whether adjuvants have an effect on oral immunogenicity when coadministrated with different L1 forms. Besides saponins, which were significantly toxic if applied orally, co-administration of either CpG DNA or *Escherichia coli* heat-labile enterotoxin LT(R192G) had no apparent enhancing effect on the production of anti-L1 antibodies. Compared to capsomeres, VLPs induced stronger humoral immune responses while the CTL responses were induced at comparable levels.

To establish an edible HPV16 vaccine Sasagawa et al., (2005) constructed a recombinant HPV16 L1-expressing *Schizosaccharomyces pombe* yeast strain to be administrated as freezedried yeast powder orally as an edible vaccine, with or without the mucosal adjuvant heatlabile toxin LT (R192G), to mice. After the third immunization, none of the mice that received the edible HPV16 vaccine showed specific antibody responses, whereas all of the positive controls that were administered intranasally with 5 μg of HPV16-virus-like particles (VLP) had serum IgG, and genital IgA and IgG that reacted with HPV16-VLP in enzymelinked immunosorbent assays (ELISAs).

HPV L1 antigens that proof to be highly immunogenic when administrated parenterally induce only mild or none response when administrated orally. In light of these experiments it seems unlikely that current design of L1 based HPV vaccines will reach the market as an edible replacement of existing vaccines. More research is needed to establish vaccine concentration and formulation to boost its effect. It is also obvious that the highly phrased concept of edible vaccine administrated as plants or fruits for direct consumption in the less developed countries is rather romantic dream than reality and have to be corrected. It is now clear that if there will be an edible vaccine, it will have complex formulation that will be strictly controlled.

A good nonparenteral alternative for vaccine delivery could be nasal immunization, which already proved to be effective in tests with animals. The obstacles imposed by the normal process of mucociliary clearance limiting residence time of applied antigens could be circumvent by presence of an inert *in situ* gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen (Hefferon, 2010). The nasal cavity is a promising site for vaccine delivery because it is easy to access, is highly vascularized, has a relatively large surface area, has low proteolytic activity, and is able to induce systemic immunity as well as both local and distal mucosal immunity via the Common Mucosal Immune System (CMIS)

### **4. Conclusions**

Human Papillomavirus and Related Diseases 166 – From Bench to Bedside – A Clinical Perspective

enhance oral immunization by protection of antigen from degradation by digestive enzymes

All these findings are promising for development of needle-free administration route of immunization as an alternative to intramuscular vaccine application. For this purposes intranasal, intravaginal, transdermal, sublingual and intramuscular administration routes were tested for systemic immune responses against HPV16L1 using (Cho et al., 2010). The sublingual route provided the most effective mucosal secretory IgA (sIgA) and serum IgG responses, cholera toxin subunit B (CTB) showed the most promising adjuvant activity.

The majority of currently licensed vaccines are administered parenterally, even though they have the disadvantages of patient reluctance to tolerate needle sticks and lack of mucosal immune induction (Velasquez et al 2010). Edible vaccine represents further approach to selfadministrated nonparenteral vaccine that could solve the problem of high cost and need for

Thönes & Müller (2007) investigated the oral immunogenicity of different assembly forms of HPV 16 L1: T7-VLPs, T1 particles and capsomeres produced from Baculovirus expression vector in insect cells and showed that all three assembly forms induce humoral and cellular immune responses after oral vaccination of mice. The anti-L1 antibodies were conformationspecific and showed neutralizing activity in a pseudovirion-based assay. They also investigated whether adjuvants have an effect on oral immunogenicity when coadministrated with different L1 forms. Besides saponins, which were significantly toxic if applied orally, co-administration of either CpG DNA or *Escherichia coli* heat-labile enterotoxin LT(R192G) had no apparent enhancing effect on the production of anti-L1 antibodies. Compared to capsomeres, VLPs induced stronger humoral immune responses

To establish an edible HPV16 vaccine Sasagawa et al., (2005) constructed a recombinant HPV16 L1-expressing *Schizosaccharomyces pombe* yeast strain to be administrated as freezedried yeast powder orally as an edible vaccine, with or without the mucosal adjuvant heatlabile toxin LT (R192G), to mice. After the third immunization, none of the mice that received the edible HPV16 vaccine showed specific antibody responses, whereas all of the positive controls that were administered intranasally with 5 μg of HPV16-virus-like particles (VLP) had serum IgG, and genital IgA and IgG that reacted with HPV16-VLP in enzyme-

HPV L1 antigens that proof to be highly immunogenic when administrated parenterally induce only mild or none response when administrated orally. In light of these experiments it seems unlikely that current design of L1 based HPV vaccines will reach the market as an edible replacement of existing vaccines. More research is needed to establish vaccine concentration and formulation to boost its effect. It is also obvious that the highly phrased concept of edible vaccine administrated as plants or fruits for direct consumption in the less developed countries is rather romantic dream than reality and have to be corrected. It is now clear that if there will be an edible vaccine, it will have complex formulation that will

and by increasing permeability of the intestine to macromolecules (Campbell, 1995).

**3.2 HPV L1 antigens as an edible vaccine?**

appropriate storage of currently available preventive HPV vaccines.

while the CTL responses were induced at comparable levels.

linked immunosorbent assays (ELISAs).

be strictly controlled.

The major reason for the vaccine production in plants is that the vaccine antigen production is safe and could be potentially cheap and both transient and transgenic productions are scalable. Biologically active proteins can be produced more easily in plants than in other eukaryotic systems; and that the use of food plants could eventually allow edible and/or oral vaccines to be produced cheaper. The recent reports indicate very high yields of human vaccine candidates to be obtained via plastid transformation or large scale transient expression what could enable to meet the expected requirement of antigen for oral route as is required parenterally for the same immune response. A recent review on human trials of plant-based oral vaccines summarizing human studies of oral transgenic plant derived vaccines against enterotoxigenic *E. coli* infection, norovirus and HBV adds weight to the growing body of evidence that plant-made oral vaccines to these viruses are not only feasible, but could effective (Rybicki, 2010). Nevertheless there is still long way to go from improvement of antigen yields, to formulation of the vaccine including auxiliary factors improving efficacy and stability, to translation of the proposed vaccines into clinical trials and, not least, governmental and/or regulatory body approvals.

### **5. Acknowledgements**

This work was supported by the Grant Agency of the Czech Republic contract 521/01/1418 and 521/04/0971, Grant Agency of the Czech Academy of Sciences A6038201 and S5038304 and by Ministry of Education, Youth and Sports of the Czech Republic projects 1M0505.

### **6. References**

Plant Production of Vaccine Against HPV: A New Perspectives 169

Davidson, E.J.; Boswell, C.M.; Sehr, P.; Pawlita, M.; Tomlinson, A.E.; McVey, R.J.; Dobson, J.;

Dell, K.; Koesters, R.; Linnebacher, M.; Klein, C. & Gissmann, L. (2006). Intranasal

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**8**

*Venezuela*

**Development of Vaccines and Gene Therapy**

*1Instituto de Oncología y Hematología, Laboratorio de Genética Molecular, Ciudad*

*2Universidad Central de Venezuela, Instituto de Investigaciones Económicas y Sociales,*

From the establishment of etiologic relationship between HPV and cervical cancer, researchers have emphasized the importance of prevention by education of people, especially teenagers. This virus is associated with diseases of the cutaneous and mucosal human epithelia, including benign warts and invasive cancer that affect different anatomical regions as skin, cervix, vagina, vulva, anus, penis, head and neck. Controversial evidence suggests a relationship between HPV infection and breast cancer (de Villiers et al., 2005;

Verrucae vulgares, verrucae palmares et plantares 1, 2, 4

Epidermodysplasia verruciformis (EV) 3, 5, 8 EV- squamous cell carcinoma 5, 8 Condylomata acuminata 6, 11

carcinomas of the anogenital tract <sup>16</sup>

Buschke-Lowenstein tumor 6, 11 Respiratory Papillomatosis 6, 11 Heck´s disease 13, 32

Breast cancer? 16, 18

Bowenoid papulosis, erythroplasia of Queyrat 16

Head and neck cancers (larynx, tonsils, tongue, sinuse, lung) 16, 18, 6, 11

Table 1. Clinical manifestations and associated HPV types (taken and modified of

Squamous cell carcinoma of the finger, Bowen´s disease 16

High grade-squamous intraepithelial neoplasias and invasive

**Clinical manifestation HPV types often detectable**

Verrucae planare 3, 10 Butcher´s warts 7

**1. Introduction**

Handisurya et al., 2009).

Khan et al., 2005; Heng et al., 2009) (Table 1).

**Against HPV Infection and Cervical Cancer**

Zoraya De Guglielmo Cróquer1 and Armando Rodríguez Bermúdez2

*Universitaria, Calle Minerva, Los Chaguaramos, Caracas*

*Ciudad Universitaria, Los Chaguaramos, Caracas*

### **Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer**

Zoraya De Guglielmo Cróquer1 and Armando Rodríguez Bermúdez2 *1Instituto de Oncología y Hematología, Laboratorio de Genética Molecular, Ciudad Universitaria, Calle Minerva, Los Chaguaramos, Caracas 2Universidad Central de Venezuela, Instituto de Investigaciones Económicas y Sociales, Ciudad Universitaria, Los Chaguaramos, Caracas Venezuela*

### **1. Introduction**

Human Papillomavirus and Related Diseases 176 – From Bench to Bedside – A Clinical Perspective

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From the establishment of etiologic relationship between HPV and cervical cancer, researchers have emphasized the importance of prevention by education of people, especially teenagers. This virus is associated with diseases of the cutaneous and mucosal human epithelia, including benign warts and invasive cancer that affect different anatomical regions as skin, cervix, vagina, vulva, anus, penis, head and neck.

Table 1. Clinical manifestations and associated HPV types (taken and modified of Handisurya et al., 2009).

Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer 179

formation of warts and benign condylomata, and types 16 and 18 of high cancer risk, associated with cancerous and precancerous lesions, which are responsible for approximately 70% of all cervical cancers worldwide (Muñoz et al., 2004).Initially it was suggested to produce a vaccine based on attenuated virus, but its implementation and evaluation in humans was a very high risk due to the presence of oncogenic viral DNA; in addition the growth of virus in *in vitro* culture had been limited until recently when a researchers group managed to establish a reproducible and highly efficient production of HPV type 18 in human keratinocytes, which has a potential value for establishing research

Moreover, although the studies on the immunology of HPV have shown antibodies against many different viral products, the best characterized and most type-specific antibodies are those directed against conformational epitopes of the L1 capsid protein. In the 90's it was possible to produce *in vitro*, genetically engineered virus-like particles or VLPs, which consist of L1 or L1 + L2 recombinant protein, obtained by introducing one or both genes, respectively, in cultures of eukaryotic cells (yeast, insect or bacteria). These recombinant proteins have the ability to self-assemble to form three-dimensional structures that are morphological and antigenically identical to the original HPV virions, but not containing the viral genome, so these structures can not replicate or cause infection or cancer(Muñoz et al.,

There have been studies in experimental animal models and humans, where there was a good tolerance to systemic vaccination with L1- VLPs, and induction of serum antibody titers of at least 40 times higher than the titles produced in an natural infection (Lowy & Frazer, 2003). The first large multicenter, double-blind study, with phase III results, was published in 2002, on a monovalent vaccine developed by Merck Laboratories HPV type 16 (Brull & Carrera, 2005). This company created another quadrivalent vaccine called Gardasil, synthesized in the yeast *Saccharomyces cerevisiae*, based on L1- VLPs of oncogenic types most commonly found in cervical dysplasia (HPV types 16 and 18) and non-oncogenic types responsible for approximately 90% of warts genitals and recurrent respiratory papillomatosis (types 6 and 11), which is considered to act on two different hyperproliferative diseases (Schiller & Lowy, 2006). It was approved by the U.S. Food and Drug Administration (FDA) in 2006 and is administered in 3 doses, spread over 6 months (0, 2 and 6 months). Follow-up studies for 3 ½ years after vaccination showed an effectiveness of 94% in persistent infection with HPV types 16, as well as 100% in preventing high-grade intraepithelial lesions associated with types 16 and 18, and prevention of genital lesions related to HPV types 6 and 11 (Mao et al., 2006; Villa et al., 2005). The efficacy against vulvar and vaginal neoplasia grade II and III was 72-100% (Joura et al., 2007). In well designed clinical trials in young women aged 15-25 years who were HPV 16/18 seronegative and DNA negative to 14 HPV high-risk types, high levels of immunogenicity and protection were sustained for follow-up periods of up to 8.4 years (McKeage & Romanowski, 2011).

Each 0.5-mL dose contains 20 *µ*g HPV 6 L1 protein, 40 *µ*g HPV 11 L1 protein, 40 *µ*g HPV 16 L1 protein, and 20 *µ*g HPV 18 L1 protein. VLPs are adsorbed on an aluminum-containing adjuvant. Each 0.5-mL dose contains 225 *µ*g amorphous aluminum hydroxyphosphate sulfate. The formulation also includes sodium chloride, L-histidine, polysorbate 80, sodium borate, and water for injection. The quadrivalent HPV vaccine contains no thimerosal or

antibiotics. The vaccine should be stored at 2°C--8°C (36°F--46°F) and not frozen.

models of *ex vivo* viral expression (Castellsagué et al., 2006; Wang et al., 2009).

2008).

Noted that a variety of diseases caused by HPV is largely due to viral tropism, i.e., the preference of a certain type of HPV by a tissue or cell group in particular, depending on factors of virus and host, mainly receptors, transcriptional activators, enhancers, and tissuespecific promoters (Graham, 2010). Based on the tropism, HPV has been classified into two main phylogenetic genera, the α-HPV and the β-HPV, which correspond to the mucosal and cutaneous infective HPV, respectively (de Villiers et al., 2004). The temporal organization of the virus replication cycle is also different between different HPV types perhaps reflecting differences in sites of infection and transmission modes.

Traditionally, cancer and associated lesions have been treated with surgery, radiotherapy and chemotherapy, these treatments produced widely known adverse effects (American Cancer Society, 2007; Instituto Nacional del Cáncer, 2008). However, many efforts have been done in order to find effective preventive and curative options less invasive and with minimal or no side effects. These options are mainly based on molecular biology techniques to develop vaccines and the use of molecules that stimulate the immune and cytotoxic response against HPV infection and cervical cancer. The development of these vaccines and therapeutic procedures is based on *in vitro* culture and knowledge of the life cycle, genome and regulation of viral transcription, which has allowed the identification of potential targets to control genes expression in infectious and / or neoplastic processes (Table 2).

### **2. Prophylactic vaccines to prevent HPV infection**

Prophylactic vaccines currently exist to prevent the spread of HPV infections; these vaccines have the objective to create antigens capable to induce neutralizing antibodies that prevent the entry of virus into host cells. They are based on preventing infection of HPV types most prevalent around the world: types 6 and 11, of low oncogenic risk, associated with the Human Papillomavirus and Related Diseases 178 – From Bench to Bedside – A Clinical Perspective

Noted that a variety of diseases caused by HPV is largely due to viral tropism, i.e., the preference of a certain type of HPV by a tissue or cell group in particular, depending on factors of virus and host, mainly receptors, transcriptional activators, enhancers, and tissuespecific promoters (Graham, 2010). Based on the tropism, HPV has been classified into two main phylogenetic genera, the α-HPV and the β-HPV, which correspond to the mucosal and cutaneous infective HPV, respectively (de Villiers et al., 2004). The temporal organization of the virus replication cycle is also different between different HPV types

Traditionally, cancer and associated lesions have been treated with surgery, radiotherapy and chemotherapy, these treatments produced widely known adverse effects (American Cancer Society, 2007; Instituto Nacional del Cáncer, 2008). However, many efforts have been done in order to find effective preventive and curative options less invasive and with minimal or no side effects. These options are mainly based on molecular biology techniques to develop vaccines and the use of molecules that stimulate the immune and cytotoxic response against HPV infection and cervical cancer. The development of these vaccines and therapeutic procedures is based on *in vitro* culture and knowledge of the life cycle, genome and regulation of viral transcription, which has allowed the identification of potential targets to control genes expression in infectious and / or neoplastic processes (Table 2).

site Function

E2 Helps E1 to locate the origin of replication in

E4 Cell Cycle arrest, virion assembly, remodels

E5 Control of cell growth and differentiation,

E6 Inhibits apoptosis and differentiation E7 Cell cycle control, controls centrosome

L2 Minor capsid protein, recruits L1, virus

region (LCR) Binds many cellular transcription activators,

Prophylactic vaccines currently exist to prevent the spread of HPV infections; these vaccines have the objective to create antigens capable to induce neutralizing antibodies that prevent the entry of virus into host cells. They are based on preventing infection of HPV types most prevalent around the world: types 6 and 11, of low oncogenic risk, associated with the

ATP- dependent DNA helicase; unique enzyme expressed by the virus, which is essential for viral replication

LCR, cell cycle and apoptosis regulation

cytokeratin network

immune modulation

duplication

Major capsid protein

assembly

confers keratinocyte specificity to transcription

perhaps reflecting differences in sites of infection and transmission modes.

Expression

Basal, parabasal and intermedia te cells of the host

L1 Superficial

**2. Prophylactic vaccines to prevent HPV infection**

cells

Genome region

Early

Late

Long control

Table 2. Role of HPV genome regions

Gen/ protein

E1

formation of warts and benign condylomata, and types 16 and 18 of high cancer risk, associated with cancerous and precancerous lesions, which are responsible for approximately 70% of all cervical cancers worldwide (Muñoz et al., 2004).Initially it was suggested to produce a vaccine based on attenuated virus, but its implementation and evaluation in humans was a very high risk due to the presence of oncogenic viral DNA; in addition the growth of virus in *in vitro* culture had been limited until recently when a researchers group managed to establish a reproducible and highly efficient production of HPV type 18 in human keratinocytes, which has a potential value for establishing research models of *ex vivo* viral expression (Castellsagué et al., 2006; Wang et al., 2009).

Moreover, although the studies on the immunology of HPV have shown antibodies against many different viral products, the best characterized and most type-specific antibodies are those directed against conformational epitopes of the L1 capsid protein. In the 90's it was possible to produce *in vitro*, genetically engineered virus-like particles or VLPs, which consist of L1 or L1 + L2 recombinant protein, obtained by introducing one or both genes, respectively, in cultures of eukaryotic cells (yeast, insect or bacteria). These recombinant proteins have the ability to self-assemble to form three-dimensional structures that are morphological and antigenically identical to the original HPV virions, but not containing the viral genome, so these structures can not replicate or cause infection or cancer(Muñoz et al., 2008).

There have been studies in experimental animal models and humans, where there was a good tolerance to systemic vaccination with L1- VLPs, and induction of serum antibody titers of at least 40 times higher than the titles produced in an natural infection (Lowy & Frazer, 2003). The first large multicenter, double-blind study, with phase III results, was published in 2002, on a monovalent vaccine developed by Merck Laboratories HPV type 16 (Brull & Carrera, 2005). This company created another quadrivalent vaccine called Gardasil, synthesized in the yeast *Saccharomyces cerevisiae*, based on L1- VLPs of oncogenic types most commonly found in cervical dysplasia (HPV types 16 and 18) and non-oncogenic types responsible for approximately 90% of warts genitals and recurrent respiratory papillomatosis (types 6 and 11), which is considered to act on two different hyperproliferative diseases (Schiller & Lowy, 2006). It was approved by the U.S. Food and Drug Administration (FDA) in 2006 and is administered in 3 doses, spread over 6 months (0, 2 and 6 months). Follow-up studies for 3 ½ years after vaccination showed an effectiveness of 94% in persistent infection with HPV types 16, as well as 100% in preventing high-grade intraepithelial lesions associated with types 16 and 18, and prevention of genital lesions related to HPV types 6 and 11 (Mao et al., 2006; Villa et al., 2005). The efficacy against vulvar and vaginal neoplasia grade II and III was 72-100% (Joura et al., 2007). In well designed clinical trials in young women aged 15-25 years who were HPV 16/18 seronegative and DNA negative to 14 HPV high-risk types, high levels of immunogenicity and protection were sustained for follow-up periods of up to 8.4 years (McKeage & Romanowski, 2011).

Each 0.5-mL dose contains 20 *µ*g HPV 6 L1 protein, 40 *µ*g HPV 11 L1 protein, 40 *µ*g HPV 16 L1 protein, and 20 *µ*g HPV 18 L1 protein. VLPs are adsorbed on an aluminum-containing adjuvant. Each 0.5-mL dose contains 225 *µ*g amorphous aluminum hydroxyphosphate sulfate. The formulation also includes sodium chloride, L-histidine, polysorbate 80, sodium borate, and water for injection. The quadrivalent HPV vaccine contains no thimerosal or antibiotics. The vaccine should be stored at 2°C--8°C (36°F--46°F) and not frozen.

Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer 181

2005). Some researchers have pointed out as risk of vaccination the possible selection of not included HPV types in vaccines or the increase in the prevalence of rare oncogenic HPV types, which can only be known over time, after the mass vaccination had been effective.

Other weaknesses of the prophylactic vaccine against HPV is that it only protects against specific HPV types, leaving out a wide range of viral types, although rare, are also present, and have also been reported conflicting results regarding the existence of cross protection. In addition, the vaccine is preventive (not cure existing infections or injuries) and there is a low percentage of cervical cancer and precursor lesions in which no association has been established with HPV, existing other factors associated with the development of this malignancy, as exposure to mutagens, genetic susceptibility, hormonal status and immune status (Tirado-Gómez et al., 2005; del Amo et al., 2005); unknown the duration of protection provided by the vaccine, it may be necessary to administer a booster dose (Brull & Carrera, 2005; Diestro Tejeda et al., 2007). In this regard, an evaluation showed that the quadrivalent vaccine provides strong and sustained protection against condiloma and vulvovaginal and cervical low grade neoplasia, related to types 6, 11, 16 and 18, for more than four years. The same study suggests that the effectiveness of the vaccine might be less in cases where

It is important to take into account the high cost of the vaccines listed, however, this is not comparable to the loss of life or money spending for the treatments of lesions associated with HPV infection.  In this sense, the ACIP has recommended the introduction of HPV vaccine in national immunization programs of governments worldwide. It is therefore essential to determine the HPV types circulating in each region and assess the potential impact of preventive vaccination in the respective populations.

Various researchers have emphasized that even with the globalization of preventive vaccination, screening schemes should continue due to multifactorial nature of cervical cancer, only 2 of 15 oncogenic HPV types are included in the vaccine and vaccination programs initially cover women into a limited range of ages, anticipating that at least during 2-3 decades unvaccinated sexually active women remain at risk for the disease (Giuliano,

The efficacy of L2 vaccination has been proved in pre-clinical and clinical studies. Natural infection does not induce anti-L2 antibodies and many L2 epitopes are not on the virus surface, but during the infection cellular protease furin removes an L2 N-terminal sequence rendering L2 accessible on the capsid surface and displaying the L2-neutralizing epitopes. The L2-neutralizing epitope was inserted on the surfaces of VLPs increasing the titers of neutralizing antibodies approximately 10-fold. A synthetic L2 lipopeptide with concatenated multitype L2 fusion proteins from different papillomavirus types have already been utilized in inducing cross-neutralizing antibodies against several clinically relevant HPV types. This polymeric L2 approach gives rise to antisera, that neutralize at higher titers, not only the types included in the multimeric immunogen but also other types. So, immunization against L2 could be a candidate prophylactic pan-human papillomaviruses

coinfection with HPV types absent in the vaccine is present (Dillner, 2010).

2007; Stanley, 2008; Hutchinson & Klein, 2008).

vaccine (Alphs et al., 2008; Jagu et al., 2009).

GlaxoSmithKline laboratories developed a bivalent vaccine called Cervarix for HPV types 16 and 18, produced in insect cells with baculovirus as expression system, which is also administered in three doses (0, 1 and 6 months). Studies reported 100% efficacy in preventing infections with involved HPV types, with an almost absolute immunogenicity for 4 ½ years after vaccination and the detection of antibody titers from 16 to 26 times higher than detected after natural infection (McKeage & Romanowski, 2011; Bhatla et al., 2010; Muñoz et al., 2008; Harper et al., 2004; Kahn, 2005).

Both vaccines (Gardasil and Cervarix) use aluminum-based adjuvants, which reduces the dose required to induce antibodies peak titer and helps to stabilize the vaccine during storage (Schiller & Lowy, 2006). Overall adverse effects reported in vaccination protocols are limited and include reactions at the injection site (erythema, pain and swelling) and systemic adverse effects (headache, fever and nausea) which were of middle nature, transitory and common for individuals receiving the vaccine or placebo (Paavonen et al., 2007).

Whereas the onset of sexual activity during adolescence, the FDA approved the vaccine Gardasil for girls and women aged between 9 to 26 years, while the Advisory Committee on Immunization Practices of the USA Center for Disease Control and Prevention (ACIP) recommended vaccination in females aged between 11 to 26 years and added males to the population who can benefit from Gardasil vaccination (FDA, 2010).

There has been controversy regarding the vaccination of women alone or the inclusion of men. In this regard, it has been noted that vaccinating girls aged 12 years old, can reduce cases of cervical cancer associated with types 16 and HPV 18 in about 95% and the introduction of vaccination in children would increase this figure three points (Taira et al., 2004). We must also take into account the potential role of vector that man can exert on the transmission of HPV infection, so the inclusion of the male population in vaccination programs could contribute to optimal control of transmission (Tirado-Gómez et al., 2005).

Currently the vaccine is not recommended for pregnant women. The long-term effects of the vaccine on fertility are not known, but no effects are anticipated. Although studies on the toxicity to the fetus are inconclusive, FDA has classified the HPV vaccine as a pregnancy Category B medication, meaning that the medication does not appear to cause harm to the fetus in animal studies. Initially, the trials for Gardasil and Cervarix excluded pregnant women. Pregnancy was determined by a sensitive human chorionic gonadotropin (HCG) test on the day of expected vaccination. However, some women became pregnant during the few weeks or months following the receipt of a vaccine or placebo injection. Overall, the proportions of pregnancies with an adverse outcome were comparable in subjects who received Gardasil and subjects who received placebo. However, the clinical trials had a relatively small sample size. Though receiving the HPV vaccine while pregnant is generally considered safe, it is still recommended to wait until after pregnancy to get the vaccine (American Cancer Society, 2008).

Vaccination at early age has led to concerns of parents, researchers and health specialists because of the need to talk with children about sexuality and sexually transmitted diseases, the vaccination charges received by children, the possibility that immunization may lead to the adoption of risky sexual behaviors and concerns about the safety of vaccines (Kahn, Human Papillomavirus and Related Diseases 180 – From Bench to Bedside – A Clinical Perspective

GlaxoSmithKline laboratories developed a bivalent vaccine called Cervarix for HPV types 16 and 18, produced in insect cells with baculovirus as expression system, which is also administered in three doses (0, 1 and 6 months). Studies reported 100% efficacy in preventing infections with involved HPV types, with an almost absolute immunogenicity for 4 ½ years after vaccination and the detection of antibody titers from 16 to 26 times higher than detected after natural infection (McKeage & Romanowski, 2011; Bhatla et al., 2010;

Both vaccines (Gardasil and Cervarix) use aluminum-based adjuvants, which reduces the dose required to induce antibodies peak titer and helps to stabilize the vaccine during storage (Schiller & Lowy, 2006). Overall adverse effects reported in vaccination protocols are limited and include reactions at the injection site (erythema, pain and swelling) and systemic adverse effects (headache, fever and nausea) which were of middle nature, transitory and common for individuals receiving the vaccine or placebo (Paavonen et al.,

Whereas the onset of sexual activity during adolescence, the FDA approved the vaccine Gardasil for girls and women aged between 9 to 26 years, while the Advisory Committee on Immunization Practices of the USA Center for Disease Control and Prevention (ACIP) recommended vaccination in females aged between 11 to 26 years and added males to the

There has been controversy regarding the vaccination of women alone or the inclusion of men. In this regard, it has been noted that vaccinating girls aged 12 years old, can reduce cases of cervical cancer associated with types 16 and HPV 18 in about 95% and the introduction of vaccination in children would increase this figure three points (Taira et al., 2004). We must also take into account the potential role of vector that man can exert on the transmission of HPV infection, so the inclusion of the male population in vaccination programs could contribute to optimal control of transmission (Tirado-Gómez et al., 2005).

Currently the vaccine is not recommended for pregnant women. The long-term effects of the vaccine on fertility are not known, but no effects are anticipated. Although studies on the toxicity to the fetus are inconclusive, FDA has classified the HPV vaccine as a pregnancy Category B medication, meaning that the medication does not appear to cause harm to the fetus in animal studies. Initially, the trials for Gardasil and Cervarix excluded pregnant women. Pregnancy was determined by a sensitive human chorionic gonadotropin (HCG) test on the day of expected vaccination. However, some women became pregnant during the few weeks or months following the receipt of a vaccine or placebo injection. Overall, the proportions of pregnancies with an adverse outcome were comparable in subjects who received Gardasil and subjects who received placebo. However, the clinical trials had a relatively small sample size. Though receiving the HPV vaccine while pregnant is generally considered safe, it is still recommended to wait until after pregnancy to get the vaccine

Vaccination at early age has led to concerns of parents, researchers and health specialists because of the need to talk with children about sexuality and sexually transmitted diseases, the vaccination charges received by children, the possibility that immunization may lead to the adoption of risky sexual behaviors and concerns about the safety of vaccines (Kahn,

population who can benefit from Gardasil vaccination (FDA, 2010).

Muñoz et al., 2008; Harper et al., 2004; Kahn, 2005).

(American Cancer Society, 2008).

2007).

2005). Some researchers have pointed out as risk of vaccination the possible selection of not included HPV types in vaccines or the increase in the prevalence of rare oncogenic HPV types, which can only be known over time, after the mass vaccination had been effective.

Other weaknesses of the prophylactic vaccine against HPV is that it only protects against specific HPV types, leaving out a wide range of viral types, although rare, are also present, and have also been reported conflicting results regarding the existence of cross protection. In addition, the vaccine is preventive (not cure existing infections or injuries) and there is a low percentage of cervical cancer and precursor lesions in which no association has been established with HPV, existing other factors associated with the development of this malignancy, as exposure to mutagens, genetic susceptibility, hormonal status and immune status (Tirado-Gómez et al., 2005; del Amo et al., 2005); unknown the duration of protection provided by the vaccine, it may be necessary to administer a booster dose (Brull & Carrera, 2005; Diestro Tejeda et al., 2007). In this regard, an evaluation showed that the quadrivalent vaccine provides strong and sustained protection against condiloma and vulvovaginal and cervical low grade neoplasia, related to types 6, 11, 16 and 18, for more than four years. The same study suggests that the effectiveness of the vaccine might be less in cases where coinfection with HPV types absent in the vaccine is present (Dillner, 2010).

It is important to take into account the high cost of the vaccines listed, however, this is not comparable to the loss of life or money spending for the treatments of lesions associated with HPV infection.  In this sense, the ACIP has recommended the introduction of HPV vaccine in national immunization programs of governments worldwide. It is therefore essential to determine the HPV types circulating in each region and assess the potential impact of preventive vaccination in the respective populations.

Various researchers have emphasized that even with the globalization of preventive vaccination, screening schemes should continue due to multifactorial nature of cervical cancer, only 2 of 15 oncogenic HPV types are included in the vaccine and vaccination programs initially cover women into a limited range of ages, anticipating that at least during 2-3 decades unvaccinated sexually active women remain at risk for the disease (Giuliano, 2007; Stanley, 2008; Hutchinson & Klein, 2008).

The efficacy of L2 vaccination has been proved in pre-clinical and clinical studies. Natural infection does not induce anti-L2 antibodies and many L2 epitopes are not on the virus surface, but during the infection cellular protease furin removes an L2 N-terminal sequence rendering L2 accessible on the capsid surface and displaying the L2-neutralizing epitopes. The L2-neutralizing epitope was inserted on the surfaces of VLPs increasing the titers of neutralizing antibodies approximately 10-fold. A synthetic L2 lipopeptide with concatenated multitype L2 fusion proteins from different papillomavirus types have already been utilized in inducing cross-neutralizing antibodies against several clinically relevant HPV types. This polymeric L2 approach gives rise to antisera, that neutralize at higher titers, not only the types included in the multimeric immunogen but also other types. So, immunization against L2 could be a candidate prophylactic pan-human papillomaviruses vaccine (Alphs et al., 2008; Jagu et al., 2009).

Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer 183

After treatment it was possible the elimination of pre-cancerous lesions and even cancer (cancer *in situ*), with an efficiency of 95% in the first and 40% in the latter. All patients developed antibodies to the vaccine and a specific cytotoxic response against HPVtransformed cells. These results were compared with those obtained in a similar group of patients treated with cryosurgery, a technique that is able to eliminate low-grade lesions in

As for of the virus detection, there was no evidence in 50% of the treated sample after treatment and in the remaining 50% was only detected 10% of the original viral load. Assessments of MVAE2 vaccine in women with cervical cancer are in Phase III, consisting of a multicenter study in 250 patients with cancer *in situ* in the Juarez Hospital of the Ministry

This vaccine was also tested in men for the treatment of urethral condilomas, showing the stimulation of the immune response against HPV and regression of lesions in 93% of cases, 4 weeks after therapy. These assessments are in phase II. The results are promising and show that local therapeutic vaccination with MVAE2 is an effective tool for stimulating the immune response to HPV infection and the presence of virus-transformed cells, as well as

On the other hand, researchers have highlighted the potential utility of gene gun used in the administration of DNA-based vaccines as part of the antigenic systems strategy for the control of cancer and infectious diseases, projecting itself as an important tool in antigen-

In the particular case of uterine cancer, Kim et al. (2008b), in a mouse model, used a Helio gun to dispense gold microparticles coated with E6 DNA of HPV type 16 attached to an expression regulator of major histocompatibility complex class I molecules (human calreticulin). They observed an increased cellular and humoral immune response and antitumor effect, from the increased processing and presentation of antigens to T cells, together with the regression of tumors, enhanced antigen-specific memory and prolonged survival of vaccinated mice. The authors highlight the potential clinical benefits of this therapeutic strategy in humans, which may include co-administration of molecules with other properties, e.g. DNA encoding anti-

This vaccine was combined with E7 and L2 proteins of HPV, also observed significant therapeutic effects against E6/E7 expressing tumor cells, and generate a potent L2-antigen specific response, thereby protecting against pseudovirion infection. These results highlight

Ahn et al. (2004) performed in mice, direct intratumoral injection of an adenoviral vaccine carrier *E7* sequence of HPV type 16 and interleukin 12 (IL-12) as adjuvant, which induces cellular immune responses for protection against tumor formation. They observed partial or complete regression of the tumors and long-term immunity against recurrence of the malignancy, and this effect was much greater with the vaccine formed with the all components, compared with the injection of any of the separate components. The IL-12 is one of the most widely used cytokine on gene therapy against cervical cancer, due to its effect in inhibiting tumor growth and experimental metastasis, dependent on the activation of NK cells. However, Sin (2009) reported that IL-12 and *E7* HPV type 16 cDNA-based vaccine lost its antitumor and immunoprotective effect when it was combined with nitric oxide (used and

all patients; but there was not observed cytotoxic activity against cancer cells.

regression of high and low grade cancer lesions (Albarran y Carvajal et al., 2007).

apoptotic or angiogenic proteins (Kim et al., 2004; Kim et al., 2008b).

the potential clinical benefits of this vaccine (Kim et al., 2008b).

of Health of Mexico.

specific immunotherapy (Kim et al., 2008a).

### **3. Therapeutic vaccines and gene therapy for treatment of cervical cancer**

Another line in the study of cervical cancer treatment has pursued in the implementation of therapeutic vaccines and / or gene therapy to cure existing cases, through the transfer of DNA, inserting the normal gene or gene expression regulation.

Therapeutic vaccines are composed of peptides homologous to the viral proteins, as indicated in the treatment of dysplasia and invasive cervical cancer or as adjuvant therapy for locally recurrent or metastatic (Diestro Tejada et al., 2007). DNA vaccines have also been developed, which are considered as stable, safe, can be prepared in large quantities and repeatedly administered without significant adverse effects. In addition, the DNA tends to be preserved in the receptor cells, ensuring long-term expression of the encoded antigen and reinforcing the maintenance of immunological memory.

Generally, the immune response generated from DNA alone is weak, so it has been tested the adjuvant effect of several molecules, and the combination of specific genes (Kim et al., 2004). However, vaccines have been developed based on naked DNA, viral or bacterial vector, tumor cells and dendritic modified cells.

Other molecules in use for gene therapy of cervical cancer are cytokines which has immunoregulatory effect that promotes maturation, activation and migration of effector cells of the immune response to the tumor site. Of particular interest are interferons, interleukin 2 or IL-2 (which activates T cells, NK cells, macrophages and the release of other cytokines) and the aforementioned IL-12 (whose anti-tumor effect individually or in combination with *E6* and *E7* is dependent on the activation of CD8 + cytotoxic T lymphocytes and NK cells at the site of immunization). Intratumoral administration of these molecules significantly reduced the progression of HPV-associated tumors and inhibited recurrent tumor formation after being removed by surgery (Frechtel, 2005; Bubenik et al., 2003).

In 1999, a group of researchers developed and tested the vaccine TA-GW based on the L2 and E7 fusion proteins of HPV type 6, with Alhydrogel as adjuvant, for the treatment of condilomas, reporting immunogenicity and cure in approximately 62% of lesions 8 weeks after vaccination, without considerables adverse effects (Lacey et al., 1999).

In 2002, Kaufmann et al. performed one of the first multicenter and multinational studies with a recombinant vaccine, TA-HPV, consisting of attenuated vaccinia virus genetically engineered to express the E6 and E7 proteins of HPV types 16 and 18. The protocol included two doses of the vaccine in patients with early-stage cervical cancer, and the induction of humoral and cell-mediated response with low side and/or toxic effects was observed.

In 2004, Gutierrez et al. evaluated the effect of the recombinant vaccine MVAE2, consisting of attenuated vaccinia virus and the E2 gene of HPV, in squamous intraepithelial lesions of high and low grade. Treatment consisted in the administration of six doses, one every week, injected directly into the cervix. During the observation of treatment results, the reduction of injuries was monitored by colposcopy and histological analysis. The immune response was determined by measuring of antibodies againts MVAE2 and analysis of cytotoxic lymphocyte activity against cancer cells with oncogenic human papillomavirus. The presence of viral DNA and viral load were determined using the Hybrid Capture method (Digene).

Human Papillomavirus and Related Diseases 182 – From Bench to Bedside – A Clinical Perspective

Another line in the study of cervical cancer treatment has pursued in the implementation of therapeutic vaccines and / or gene therapy to cure existing cases, through the transfer of

Therapeutic vaccines are composed of peptides homologous to the viral proteins, as indicated in the treatment of dysplasia and invasive cervical cancer or as adjuvant therapy for locally recurrent or metastatic (Diestro Tejada et al., 2007). DNA vaccines have also been developed, which are considered as stable, safe, can be prepared in large quantities and repeatedly administered without significant adverse effects. In addition, the DNA tends to be preserved in the receptor cells, ensuring long-term expression of the encoded antigen and

Generally, the immune response generated from DNA alone is weak, so it has been tested the adjuvant effect of several molecules, and the combination of specific genes (Kim et al., 2004). However, vaccines have been developed based on naked DNA, viral or bacterial

Other molecules in use for gene therapy of cervical cancer are cytokines which has immunoregulatory effect that promotes maturation, activation and migration of effector cells of the immune response to the tumor site. Of particular interest are interferons, interleukin 2 or IL-2 (which activates T cells, NK cells, macrophages and the release of other cytokines) and the aforementioned IL-12 (whose anti-tumor effect individually or in combination with *E6* and *E7* is dependent on the activation of CD8 + cytotoxic T lymphocytes and NK cells at the site of immunization). Intratumoral administration of these molecules significantly reduced the progression of HPV-associated tumors and inhibited recurrent tumor formation after being removed by surgery (Frechtel, 2005; Bubenik et al.,

In 1999, a group of researchers developed and tested the vaccine TA-GW based on the L2 and E7 fusion proteins of HPV type 6, with Alhydrogel as adjuvant, for the treatment of condilomas, reporting immunogenicity and cure in approximately 62% of lesions 8 weeks

In 2002, Kaufmann et al. performed one of the first multicenter and multinational studies with a recombinant vaccine, TA-HPV, consisting of attenuated vaccinia virus genetically engineered to express the E6 and E7 proteins of HPV types 16 and 18. The protocol included two doses of the vaccine in patients with early-stage cervical cancer, and the induction of humoral and cell-mediated response with low side and/or toxic effects was observed.

In 2004, Gutierrez et al. evaluated the effect of the recombinant vaccine MVAE2, consisting of attenuated vaccinia virus and the E2 gene of HPV, in squamous intraepithelial lesions of high and low grade. Treatment consisted in the administration of six doses, one every week, injected directly into the cervix. During the observation of treatment results, the reduction of injuries was monitored by colposcopy and histological analysis. The immune response was determined by measuring of antibodies againts MVAE2 and analysis of cytotoxic lymphocyte activity against cancer cells with oncogenic human papillomavirus. The presence of viral DNA and viral load were determined using the Hybrid Capture method

after vaccination, without considerables adverse effects (Lacey et al., 1999).

**3. Therapeutic vaccines and gene therapy for treatment of cervical cancer**

DNA, inserting the normal gene or gene expression regulation.

reinforcing the maintenance of immunological memory.

vector, tumor cells and dendritic modified cells.

2003).

(Digene).

After treatment it was possible the elimination of pre-cancerous lesions and even cancer (cancer *in situ*), with an efficiency of 95% in the first and 40% in the latter. All patients developed antibodies to the vaccine and a specific cytotoxic response against HPVtransformed cells. These results were compared with those obtained in a similar group of patients treated with cryosurgery, a technique that is able to eliminate low-grade lesions in all patients; but there was not observed cytotoxic activity against cancer cells.

As for of the virus detection, there was no evidence in 50% of the treated sample after treatment and in the remaining 50% was only detected 10% of the original viral load. Assessments of MVAE2 vaccine in women with cervical cancer are in Phase III, consisting of a multicenter study in 250 patients with cancer *in situ* in the Juarez Hospital of the Ministry of Health of Mexico.

This vaccine was also tested in men for the treatment of urethral condilomas, showing the stimulation of the immune response against HPV and regression of lesions in 93% of cases, 4 weeks after therapy. These assessments are in phase II. The results are promising and show that local therapeutic vaccination with MVAE2 is an effective tool for stimulating the immune response to HPV infection and the presence of virus-transformed cells, as well as regression of high and low grade cancer lesions (Albarran y Carvajal et al., 2007).

On the other hand, researchers have highlighted the potential utility of gene gun used in the administration of DNA-based vaccines as part of the antigenic systems strategy for the control of cancer and infectious diseases, projecting itself as an important tool in antigenspecific immunotherapy (Kim et al., 2008a).

In the particular case of uterine cancer, Kim et al. (2008b), in a mouse model, used a Helio gun to dispense gold microparticles coated with E6 DNA of HPV type 16 attached to an expression regulator of major histocompatibility complex class I molecules (human calreticulin). They observed an increased cellular and humoral immune response and antitumor effect, from the increased processing and presentation of antigens to T cells, together with the regression of tumors, enhanced antigen-specific memory and prolonged survival of vaccinated mice. The authors highlight the potential clinical benefits of this therapeutic strategy in humans, which may include co-administration of molecules with other properties, e.g. DNA encoding antiapoptotic or angiogenic proteins (Kim et al., 2004; Kim et al., 2008b).

This vaccine was combined with E7 and L2 proteins of HPV, also observed significant therapeutic effects against E6/E7 expressing tumor cells, and generate a potent L2-antigen specific response, thereby protecting against pseudovirion infection. These results highlight the potential clinical benefits of this vaccine (Kim et al., 2008b).

Ahn et al. (2004) performed in mice, direct intratumoral injection of an adenoviral vaccine carrier *E7* sequence of HPV type 16 and interleukin 12 (IL-12) as adjuvant, which induces cellular immune responses for protection against tumor formation. They observed partial or complete regression of the tumors and long-term immunity against recurrence of the malignancy, and this effect was much greater with the vaccine formed with the all components, compared with the injection of any of the separate components. The IL-12 is one of the most widely used cytokine on gene therapy against cervical cancer, due to its effect in inhibiting tumor growth and experimental metastasis, dependent on the activation of NK cells.

However, Sin (2009) reported that IL-12 and *E7* HPV type 16 cDNA-based vaccine lost its antitumor and immunoprotective effect when it was combined with nitric oxide (used and

Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer 185

The second, based on double-stranded RNA has proven to be more powerful than the first,

The antisense oligonucleotides have shown effectiveness in inhibiting the expression of *E6* and *E7* oncogenes, and also produced the release and / or activation of molecules involved in defense mechanisms (such as cytochrome c and procaspases 3 and 9), the induction of apoptosis and inhibition of telomerase activity. However, it has been reported that these molecules are unstable and design and management are very

RNA interference (RNAi) is a process of RNA-based gene silencing, which relies on nucleotide sequence complementarity and is involved in the mobilization of transposable genetic elements, in defense mechanisms and in different cellular events (such as differentiation, metabolism , stress response, propagation and apoptosis). This natural RNAdependent gene silencing process is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-RNA molecules in a cell´s cytoplasm, where they interact with the catalytic RISC component, protein argonauta, and the enzyme Dicer

This process occurs through effector molecules identified in many eukaryotes, called microRNAs (miRNAs), highly conserved in orthologous species, indicating their importance in basic cellular processes. miRNAs are endogenous short RNA molecules with space-time independents expression patterns that determine inhibition of translation or degradation of target mRNAs when complementarity is incomplete or perfect, respectively (Raia & Calin, 2011). Hence, this methodology has a potential therapeutic for various diseases, additionally it can be used in the evaluation of molecular and metabolic pathways. MiRNAs originate from populations of non-coding small RNAs (they are one of several small non-coding RNAs, including ribosomal RNA, transfer RNA and small nuclear RNA) resulting from transcription of DNA sequences by RNA polymerase II and form secondary structures hairpin loop type. Several investigators have found alterations of these molecules (particularly single nucleotide polymorphism or SNP) in all cancers studied to date and have indicated that miRNAs are expressed abnormally in these patologies and are involved in predisposition, development and progression of cancer, so they can be used for diagnostic and prognostic purposes. In this regard, miRNAs have been detected in body fluids, which favors its use as biomarkers, because their assessment would be less invasive compared with other conventional markers, such as Pap smears and biopsies. Also, they can become as tumor suppressor, inhibiting cancer development, and as oncogenes, stimulate their development, depending on its expression pattern (Patel & Sauter 2011, Vitale et al.

In the biogenesis of mature miRNAs, act two type III Rnases, Drosha and Dicer, which cut precursor RNAs in double-stranded RNA (dsRNA) molecules with a length of 21 to 25 nucleotides, which will separated to generate single strand molecules (Ketting et al., 2001). In addition to miRNAs, in the RNAi mechanism have also identified other endogenous small RNAs called short interfering RNAs (siRNA) which, like miRNAs, originate from endogenous complementary dsRNA transcripts, but have an exact length of 21 nucleotides and most interesting is that they can derive from mRNA-coding sequences, transposons and

based on single-stranded RNA (Mao et al., 2007).

expensive (Choo et al., 2000).

(Humayun et al., 2008) (figure 1).

heterochromatin (Ghildiyal et al., 2008).

2011).

known for its adjuvant effect in routine protocols for vaccination), which demonstrates an immunosuppressive effect of the compound (nitric oxide) in the system used.

Peng et al. (2010) in a preclinical model about recurrent respiratory papillomatosis (RRP), generated a DNA vaccine that encodes the HPV-11 *E6* and *E7* genes in a pcDNA3 backbone plasmid. Vaccinated mice generated strong CD8+ T cell response against the E6aa44-51 peptide, which is presented by the major histocompatibility complex class I molecule. Results revealed that the E6aa44-51 peptide contains the most immunogenic region for HPV-11 viral type, making it a candidate for the development and evaluation of novel vaccine strategies targeting the RRP patient population.

On the other hand, it has been established that an L1 molecule of various HPV types contains several cysteine residues at markedly similar relative positions, strongly suggesting that these cysteine residues play important roles in the structure and function of the HPV capsids, especially in the viral capsid assembly. Ishii et al. (2007) , in an *in vitro* model (HeLa cells), observed that HPV type 16-pseudovirions lost their infectivity after incubation with thiol-reactive reagents that bound to the free thiol of pseudovirions major capsid protein L1, due to conformational changes that result in the inhibition of the entry and trafficking of this molecules. Therefore, the authors suggest that these reagents might function as practical inhibitors of HPV infection. These reagents could be used in drug design or in combination with preventive and/or therapeutic strategies. It would be necessary further evaluation on this topic.

### **3.1 Antisense molecules and RNA interference in cervical cancer treatment**

Another line of investigation for the treatment of cervical cancer by gene therapy has been successful in testing antisense molecules as the ribozyme R434 (which, through its catalytic activity, specifically destroys the HPV types 16 *E6* and *E7* mRNA and prevents the growth of immortalized cells in the presence of virus), antisense oligonucleotides (AS-ODN) that hybridize with viral messenger blocking viral translation, and interference RNA (RNAi) (Álvarez Salas, 2006; Hamada et al., 1996; Hall & Alexander, 2003) (table 3).

Both technologies, antisense and RNAi, consist of gene silencing (interruption or suppression of the expression of a gene at transcriptional or translational levels).

Table 3. Comparison of different gene silencing strategies.

Human Papillomavirus and Related Diseases 184 – From Bench to Bedside – A Clinical Perspective

known for its adjuvant effect in routine protocols for vaccination), which demonstrates an

Peng et al. (2010) in a preclinical model about recurrent respiratory papillomatosis (RRP), generated a DNA vaccine that encodes the HPV-11 *E6* and *E7* genes in a pcDNA3 backbone plasmid. Vaccinated mice generated strong CD8+ T cell response against the E6aa44-51 peptide, which is presented by the major histocompatibility complex class I molecule. Results revealed that the E6aa44-51 peptide contains the most immunogenic region for HPV-11 viral type, making it a candidate for the development and evaluation of novel vaccine

On the other hand, it has been established that an L1 molecule of various HPV types contains several cysteine residues at markedly similar relative positions, strongly suggesting that these cysteine residues play important roles in the structure and function of the HPV capsids, especially in the viral capsid assembly. Ishii et al. (2007) , in an *in vitro* model (HeLa cells), observed that HPV type 16-pseudovirions lost their infectivity after incubation with thiol-reactive reagents that bound to the free thiol of pseudovirions major capsid protein L1, due to conformational changes that result in the inhibition of the entry and trafficking of this molecules. Therefore, the authors suggest that these reagents might function as practical inhibitors of HPV infection. These reagents could be used in drug design or in combination with preventive and/or therapeutic strategies. It would be necessary further evaluation on

**3.1 Antisense molecules and RNA interference in cervical cancer treatment**

(Álvarez Salas, 2006; Hamada et al., 1996; Hall & Alexander, 2003) (table 3).

suppression of the expression of a gene at transcriptional or translational levels).

Another line of investigation for the treatment of cervical cancer by gene therapy has been successful in testing antisense molecules as the ribozyme R434 (which, through its catalytic activity, specifically destroys the HPV types 16 *E6* and *E7* mRNA and prevents the growth of immortalized cells in the presence of virus), antisense oligonucleotides (AS-ODN) that hybridize with viral messenger blocking viral translation, and interference RNA (RNAi)

Both technologies, antisense and RNAi, consist of gene silencing (interruption or

**dependent ODNs** Hybridize to target mRNA Degradation of the mRNA

**DNA enzymes** Catalyze cleavage of target mRNA Degradation of the mRNA

Hybridize to target mRNA by its antisense strand and guide it into endoribonuclease enzyme complex RISC

Table 3. Comparison of different gene silencing strategies.

Hybridize to target mRNA Inhibition of translation of

the target protein

by RNase H

Degradation of the mRNA

**Agent Mechanism Result Most drugs** Bind to target protein Protein inhibition

immunosuppressive effect of the compound (nitric oxide) in the system used.

strategies targeting the RRP patient population.

this topic.

**RNase Hindependent ODNs**

**RNase H-**

**Ribozymes and**

**siRNA**

The second, based on double-stranded RNA has proven to be more powerful than the first, based on single-stranded RNA (Mao et al., 2007).

The antisense oligonucleotides have shown effectiveness in inhibiting the expression of *E6* and *E7* oncogenes, and also produced the release and / or activation of molecules involved in defense mechanisms (such as cytochrome c and procaspases 3 and 9), the induction of apoptosis and inhibition of telomerase activity. However, it has been reported that these molecules are unstable and design and management are very expensive (Choo et al., 2000).

RNA interference (RNAi) is a process of RNA-based gene silencing, which relies on nucleotide sequence complementarity and is involved in the mobilization of transposable genetic elements, in defense mechanisms and in different cellular events (such as differentiation, metabolism , stress response, propagation and apoptosis). This natural RNAdependent gene silencing process is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-RNA molecules in a cell´s cytoplasm, where they interact with the catalytic RISC component, protein argonauta, and the enzyme Dicer (Humayun et al., 2008) (figure 1).

This process occurs through effector molecules identified in many eukaryotes, called microRNAs (miRNAs), highly conserved in orthologous species, indicating their importance in basic cellular processes. miRNAs are endogenous short RNA molecules with space-time independents expression patterns that determine inhibition of translation or degradation of target mRNAs when complementarity is incomplete or perfect, respectively (Raia & Calin, 2011). Hence, this methodology has a potential therapeutic for various diseases, additionally it can be used in the evaluation of molecular and metabolic pathways. MiRNAs originate from populations of non-coding small RNAs (they are one of several small non-coding RNAs, including ribosomal RNA, transfer RNA and small nuclear RNA) resulting from transcription of DNA sequences by RNA polymerase II and form secondary structures hairpin loop type. Several investigators have found alterations of these molecules (particularly single nucleotide polymorphism or SNP) in all cancers studied to date and have indicated that miRNAs are expressed abnormally in these patologies and are involved in predisposition, development and progression of cancer, so they can be used for diagnostic and prognostic purposes. In this regard, miRNAs have been detected in body fluids, which favors its use as biomarkers, because their assessment would be less invasive compared with other conventional markers, such as Pap smears and biopsies. Also, they can become as tumor suppressor, inhibiting cancer development, and as oncogenes, stimulate their development, depending on its expression pattern (Patel & Sauter 2011, Vitale et al. 2011).

In the biogenesis of mature miRNAs, act two type III Rnases, Drosha and Dicer, which cut precursor RNAs in double-stranded RNA (dsRNA) molecules with a length of 21 to 25 nucleotides, which will separated to generate single strand molecules (Ketting et al., 2001). In addition to miRNAs, in the RNAi mechanism have also identified other endogenous small RNAs called short interfering RNAs (siRNA) which, like miRNAs, originate from endogenous complementary dsRNA transcripts, but have an exact length of 21 nucleotides and most interesting is that they can derive from mRNA-coding sequences, transposons and heterochromatin (Ghildiyal et al., 2008).

Development of Vaccines and Gene Therapy Against HPV Infection and Cervical Cancer 187

sequence (Schubert et al., 2005); studies of cervical cancer reported the silencing of the gene of interest and the production of inflammatory cytokines and interferon, simultaneously. This was particularly evident when using high concentrations of siRNA. That is the reason because the researchers recommend dose-response studies on a given system in order to select the lowest concentration of siRNA to observe the desired result, eliminating or reducing non-specific responses (Yamato et al., 2008). These nonspecific responses may also be caused by mismatches present in a siRNA in a tolerable level with its mRNA target (Haley et al., 2004). All these elements must be taken into account to design siRNA sequences and protocols in order to obtain the greatest effectiveness and specificity.

The DNA sequence to silence can also be introduced into a vector that allows the transcription of siRNA. These vectors include siRNA expression plasmids, in which the DNA insert is placed under the control of the promoter of RNA Pol III (H1 gene or U6 gene) and form hairpin secondary structures that are processed by RISC and hybridize to target

Another type of widely used vectors are viruses, because they have a wide capacity of cell infection, especially retroviruses and adenoviruses. The former have the ability to integrate into the genome of replicating cells and therefore are useful for stable transfection of cancer cells. However, it has been reported that the transfection rate is low (de Felipe & Izquierdo, 2000). Adenoviruses have the ability to infect quiescent cells and dividing cells and produce a higher transfection rate than the retroviruses, but are more difficult to handle because of

Noted that the use of this technology in the treatment of cervical cancer is possible because while *E6* and *E7* sequester the activities of p53 and pRb proteins in HPV-induced malignancies, copies of *p53* and *pRb* wild genes are usually conserved, which confers a reversible character of the malignancy, whereas reducing the expression of *E6* and *E7* in HPV-transformed cells could restore the activity of endogenous tumor suppressor and thus

It has also been shown that RNAi technology against *E6* and *E7* genes induces senescence, apoptosis or inhibition of cancer cell growth in cell models (Webster et al., 2000; Butz et al., 2003; Niu et al., 2006) and destroys or suppresses the growth of tumors in mice injected directly with short molecules of RNAi (siRNA) against E6/E7 (Niu et al., 2006; Fujii et al., 2006). The first work on gene silencing by RNAi in uterine cancer was conducted by Jiang and Millner (2002). The researchers, in an *in vitro* model, administered synthetic siRNAs against *E6* and *E7* oncogenes of HPV 16. They observed the degradation of the *E6* and *E7* mRNA, with consequent expression of *p53*, decreased cell proliferation and induction of cell death by apoptosis. In particular, the induction of apoptosis or senescence in tumor cells has been effective since the introduction of the transcriptional regulator *E2* and the reduction or

At the molecular level, it was observed that *E6* silencing induced accumulation of p53 cellular protein and transactivation of *p21* cell cycle control gene (Jiang & Milner, 2005). On the other hand, has been demonstrated the specificity of the technique since it was found that *E6* RNAi of HPV type 16 was less efficient in silencing *E6* gene in cells infected with other HPV types (Niu et al., 2006). These findings support the usefulness of this technique as

Currently there is software that facilitates the design of these molecules.

mRNA for its degradation (Brummelkamp et al., 2002).

prevent the proliferation of these cells (Webster et al., 2000).

inhibition of *E6* and *E7* genes expression (Butz et al., 2003).

the size of its genome (Carette et al., 2004).

Fig. 1. A simplified model for the RNAi pathway.