REVIEW. *Corresponding author: Arrigo Fruscalzo, St. Franziskus Hospital, Münster, Germany. ABSTRACT

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1 V O L U M E No. 1. F E B R U A R Y

2 2016 EDIZIONI MINERVA MEDICA The online version of this article is located at Minerva Medica 2016 February;107(1):26-38 REVIEW Second-generation prophylactic HPV vaccines: current options and future strategies for vaccines development Arrigo FRUSCALZO 1, 2 *, Ambrogio P. LONDERO 3, Serena BERTOZZI 4, Ralf J. LELLÉ 2 1Obstetrics and Gynecology, St. Franziskus Hospital Münster, Germany; 2Obstetrics and Gynecology University of Münster Münster, Germany; 3 Unit of Obstetrics and Gynecology, S. Polo Hospital, Monfalcone, Italy; 4Surgical Oncology Department, IRCSS CRO, Aviano, Italy *Corresponding author: Arrigo Fruscalzo, St. Franziskus Hospital, Münster, Germany. fruscal@libero.it. ABSTRACT Two vaccines focused on the prevention of HPV-related diseases have been introduced in the last decade, the quadrivalent vaccine Gardasil and the bivalent vaccine Cervarix. They are targeted to prevent precancerous and cancerous lesions not only of the cervix, but also of the vulva, vagina, anal and head-neck region. Furthermore, the protection of the quadrivalent vaccine Gardasil includes also genital warts and recurrent respiratory Papillomatosis, two benign conditions with high socio-economic impact. Although their efficacy in reducing the burden of HPV-related pathologies has been already documented, second-generation HPV vaccines are being developed in order to overcome major limitations, above all the cost of production, distribution and acceptance, thus promoting an easier access to vaccination, especially in developing countries. Recently a new multivalent VLP vaccine active against nine HPV subtypes, called Gardasil 9 (Merck & Co., Inc., Whitehouse Station, NJ, USA), has been approved, showing promising preliminary results. In this article, we outline the strategies adopted for second-generation HPV vaccine engineering, the latest HPV vaccines available at this time, as well as those currently in development. (Cite this article as: Fruscalzo A, Londero AP, Bertozzi S, Lellé RJ. Second-generation prophylactic HPV vaccines: current options and future strategies for vaccines development. Minerva Medica 2016;107:26-38) Key words: Papillomaviridae - Vaccination - Infection - Neoplasms - Condylomata acuminata - Uterine cervical dysplasia. Human Papilloma virus (HPV) is a DNA virus belonging to the papillomavirus family. Several HPV subtypes have been reported, but only a part of them demonstrates a specific affinity for the genital tract. These genital HPV types have been categorized according to their ability to lead to malign transformation in high-risk or low-risk HPVs. According to a study conducted by Munoz et al., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered high-risk varieties, HPV 26, 53, and 66 are likely high-risk types, and HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP are classified as lowrisk. 1 High-risk HPV subtypes are thought to be responsible for virtually 100% of cervical cancers, 40% of cases of cancer of the vulva, vagina, and penis, and no less than 12% of oropharyngeal malignancies and 3% of other oral cancers. On the Other hand, genital warts and recurrent respiratory papillomatosis are two benign conditions caused by low-risk HPV types infections, which are thought to lead to cancer very rarely. 2, 3 26 MINERVA MEDICA February 2016

3 SECOND-GENERATION PROPHYLACTIC HPV VACCINES FRUSCALZO Table I. Function of the most important HPV viral proteins. L1 Major capsid protein, which builds the capsid. It has the capability of self-assembling into a three dimensional structure named the capsomer which is similar to viral capsids (VLPs). L2 Minor capsid protein, contribute to the building of the capsid and bonds the viral DNA. E6 Inhibits the action of the tumor-suppressor protein p53. E7 Inactivates the retinoblastoma tumor-suppressor protein Rb. Adapted from Lelle RJ, Küppers V. Kolposkopie in der Praxis. Berlin: Springer Verlag; HPV biology HPV In order to comprehend completely the mechanism of the action of HPV vaccines, further explanation of some of the basic biologic processes of HPV is required. Papillomaviruses are small non-enveloped, twofold strand DNA viruses with a specific tropism for mucosal and cutaneous epithelia. A genome is made up of around 7900 basesets, including a variety of genes, encoding for the synthesis of ten viral proteins, and a control region, named LCR (long control region). The viral proteins are categorized into early and late proteins (E= early : E1 to E8 and L= late : L1 and L2) which are contingent upon the order of the expression pattern during the replication cycle. L1 and L2 are structural proteins that can self-unite to form the viral capsid. E proteins assist in viral transcription and interact with the host genome. 4 The main layer containing effectively dividing cells is called the basal layer, which is the area where infection is started. It is most likely that very little trauma to the outer kerantinized layer is needed for an HPV infection to occur. From that point, the infection uses the epithelial cell infrastructure for viral replication amid keratinocyte differentiation. E-proteins and L1/L2 capsid proteins are then created for viral replication. The E-proteins of most significance in influencing the potential cancer-causing properties of high-risk HPV are E6 and E7. E6 has the ability to restrict the pro-apoptotic protein p53. Meanwhile, E7 allows for inactivation of the cell-cycle control protein Rb. In this manner, epithelial cells that generally would be non-dividing enter in an uncontrolled multiplying state. HPV genome can stay in epithelial cells in an episomal structure; however, can likewise integrate in the cell host chromosome at an arbitrary site. At the point when the viral genome is integrated, the E1 and E2 genes, that manage E6 and E7 expression, are inhibited, leading to an uncontrolled E6/E7 oncoprotein expression. 4-6 It seems that the integration of the HPV DNA into the host DNA is the determining factor in the cancer transformation process (Table I). 7, 8 HPV immunology HPV infects and reproduces in basal epithelial cells during the entirety of their differentiation. Unfortunately, because of its specific pattern of infection and proliferation, HPV disease remains virtually undetected by the host immune system. Indeed, viral release occurs in the final stages of epithelial cell life, when the cells become keratinized, die and then exfoliate. As infection during this cell phase occurs with minimal or even no inflammation, commonly there is no natural immune response. Additionally, exposure of viral proteins to the T-lymphocytes by the antigen-presenting cells (APCs) of squamous epithelia in these conditions induces only a low or retarded activation of the adaptive immune system. 9 In case of an HPV infection, antibody production is low and is contingent upon viral load and persistence. The neutralizing antibodies against capsid proteins L1 and L2 is mostly type specific, which offers an amount of protection against reinfection and appears to promote some cross reactivity to heterologous HPV infection. On the other hand, a specific cell-mediated immune system is the determining factor in the clearance of infected cells by way of APCs and stimulation of cytotoxic T-lymphocytes. Vol No. 1 MINERVA MEDICA 27

4 FRUSCALZO SECOND-GENERATION PROPHYLACTIC HPV VACCINES Despite the fact that pervasiveness of HPV infection is quite high in younger and sexually active women (approximately 80%), persistent HPV disease and progression to cervical cancer is not that common, which leads to the conclusion that the immune system protects against and clears most HPV infections Strategies for vaccine development Prophylactic versus therapeutic HPV vaccines Vaccines are compounds that have the capacity to elicit a specific antigen reaction of the host immune system through its own administration or in conjunction with other compounds. Prophylactic vaccines are included, which offer protection against new infections, as well as therapeutic vaccines, which offer the capability to resolve already present infections. Prophylactic vaccines arrest viruses before they are able to infect and enter cells by activating the humoral immunity for the production of antigen neutralizing antibodies. Development of effective vaccines able to prevent HPV infection will have a dramatic impact on HPV related disease, above all cervical cancer, but also other types of cancers, genital warts and recurrent respiratory papillomatosis. The gains will be even more consistent in non-industrialized nations, given the disadvantages of these countries to prevent cancer development through cost-intensive screening programs. 12 However, amount and temporal onset of expected positive effects will largely depend on the diffusion pattern of vaccination programs. On the other hand, scientists have been also trying for many years to create vaccines that will therapeutically act against already infected cells. Even though promising results have been gained, these are unfortunately still not conclusive. These vaccines should have the ability to target specific antigen and destroy viruses within the host cells through cellular immunological response. 13, 14 Ideally, they should prevent lesions from progressing to high grade dysplasia or cancer or, optimally, even activate cancer regression. 13, 15 Currently, various HPV vaccines are under evaluation by providing E6 and/or E7 proteins to host immune system through live vectors, or in form of peptides or protein, or as nucleic acid, or chimeric VLPs vaccines. A third fascinating strategy is the development of a chimeric prophylactic and therapeutic vaccine. Such a vaccine could combine the advantages of a therapeutic vaccine with those obtained with a prophylactic one, thus inducing a faster impact on HPV related diseases. 12 Antigen selection There are two important considerations to make when developing a cervical cancer vaccine: The first regard the HPV epitopes selected as target antigen for the host immune system being stimulated and the second regard the HPV type or types targeting the vaccine. Possible HPV epitopes are relevant proteins produced by the virus that the humoral or cellular immune system can recognize. Focusing on the late proteins constituting the viral capsid, will prevent new infections, before the virus enter the cells. Focusing on HPV early proteins (E1-E7) will activate the cellular immune system to act against HPV infected cells. 14 On the other side, by choosing some HPV types instead of other ones, it should be taken into consideration which subtypes are more dangerous for the community because of the high-risk transformation potential or because of their widespread epidemiologic pattern. The inclusion of more HPV subtypes in the vaccines will in fact offer a broader protection spectrum. However, it must also be taken into account a rising in production costs, thereby resulting in a reduction of accessibility, particularly in developing nations. 14 For example, it is estimated that a vaccine targeting the most common high-risk subtypes, i.e. HPV 16 and HPV 18, could prevent worldwide up to 71% of cervical cancer cases. Otherwise, including the two or even the seven further most common high-risk HPV types, respectively the HPV 45 and HPV 31, and the HPV 16, 18, 45, 28 MINERVA MEDICA February 2016

5 SECOND-GENERATION PROPHYLACTIC HPV VACCINES FRUSCALZO 31, 33, 52, and 58, could potentially prevent respectively about 80% and between 85% and 91% cervical cancers. 16 Another viable alternative would be to target cross protective antibodies against different HPV types. This would in turn reduce the difficulty of producing a multivalent HPV vaccine. 17, 18 Route of administration It is also important to consider the route of administration when focusing on HPV vaccines, as this can have a profound impact on the evoked immunization. Parenteral vaccination. The intravenous (i.v.) and intramuscular (i.m.) injection is the most commonly administration route used for vaccination. The parenteral administration has the advantage to elicit a systemic immunologic response. Counter to that, the injection is thought to be a poor stimulator of secretory IgA, which is an important form of immunologic response involved in local mucosal immunity The route of application of all three currently available HPV vaccines is the intramuscular one. Mucosal vaccination (oral, aerosol, nasal, vaginal). Vaccines delivered via mucosa are needle-free, and thus easier and safer to administer than parenteral ones. Even more, mucosal absorption of this type of formulation causes an induction of the local production of IgA. This could potentially improve the effectiveness of these types of vaccines. In addition, mucosal vaccines seem to be especially compatible with the use of viruses or bacteria, as a large amount of clinical experience has been already done with live recombinant vectors vaccines. 14 Mucosal vaccines can be administered in form of purified VLPs, or as yeast or plant extracts. Edible vaccines are administered orally, while intranasal vaccination can be obtained by delivering it in a water solution that has been nebulized into the upper respiratory tract. Oral vaccination and intranasal vaccination require the preparation of fairly large amounts of HPV-VLPs. 22 Nonetheless, these vaccines might be a commonly acceptable and cheaper route of vaccination. 23 There is also the alternative of vaginal vaccination; however, it is doubtful this would be accepted for use, particularly in adolescent patients. 24 Skin vaccination. There are several different means of delivering vaccines into the skin. These include the use of injections, particle bombardments or jet injectors, a mean in which pressurized liquid is forced into the skin. The advantage of this route of administration is that some components of the immune system are particularly active in tissues, such as skin, that serve as barriers to infection. 14 By introducing these particles into the skin, the result is the direct transfection of the epidermal Langerhans cells and the epidermal cells. With DNA vaccines, the DNA should be sufficiently coated into particles that are enough dense in order to penetrate the epithelium, for example by means of gold beads. Compared to intramuscular injections only a minimal amount of DNA is required in order to induce an adequate immune response. 25 Immunologic enhance of HPV vaccines: adjuvants One of the major problems in vaccine engineering is the difficulty to produce a valid immune reaction by providing only one or few epitopes. This reaction should be ideally at least similar to that obtained in the host recipient following a real infection. Enhancing the efficacy of vaccine response can be obtained with adjuvants, which can be given either alone or in conjunction with the vaccine. They also offer the possible benefit of reducing the required amount of antigen dosages and of enhancing the strength and duration of immunologic activity. Presently, over 100 substances are used or are being developed as vaccine adjuvants. 10 Most commonly, these are aluminum-based formulations. They have been shown to act as a means of antigen transport to the lymphoid tissue in the injection site after vaccination. 26 Vol No. 1 MINERVA MEDICA 29

6 FRUSCALZO SECOND-GENERATION PROPHYLACTIC HPV VACCINES Table II. Characteristics of prophylactic first generation HPV vaccines actually on the market. Commercial name Gardasil Cervarix Manufacturer Merck & Co. GlaxoSmithKline (Sanofi-Pasteur MSD) Vaccine L1 VLP L1 VLP Adjuvant Aluminum salt ASO4 HPV types Quadrivalent HPV 6, 11, 16, 18 bivalent HPV 16 und 18 Route of administration, Interval Intramuscular, Month 0, 2 and 6 Intramuscular, Month 0, 1 and 6 Adapted from Fruscalzo A, Londero AP, Bertozzi S, Lelle RJ. First generation prophylactic HPV vaccines: The state of the art. Minerva Med [Epub ahead of print on July 17, 2015]. 29 A new substance has been used as adjuvant by GlaxoSmithKline (GSK) Biologicals in its first generation VLPs vaccine Cervarix. It is called AS04 and is made with aluminum hydroxide and monophoshoryl lipid A (MLP ). It has the capability to activate directly the immune system thus enhancing the immune response to antigens within the vaccine. The experiment conducted by GSK showed that a vaccine that contains AS04 had better strength and a more durable immune response in comparison to that containing aluminum salt alone. 27 Antigen incorporation into HPV-vaccines The way the HPV antigens are incorporated into vaccines is the other cornerstone of HPV vaccine engineering. There are many ways to offer a vehicle of transport into the host recipient. These are generally classified in virus-like particles (VLP), proteins and peptides or naked DNA, as well as using recombinant livevectors (bacteria, DNA and RNA viruses), or plant and yeast extracts. 4, 6, 14, 28 Their most important molecular features and clinical applications will be extensively described in the next chapter reserved to the prophylactic first and second-generation vaccines. Fist and second-generation vaccines The two HPV vaccines currently commercialized are both virus-like particles (VLP) vaccines (Table II). 29 For this reason, they are commonly considered as first-generation vaccines. The first one is a bivalent vaccine, targeting the two most relevant types of high- risk HPV, the HPV 16 and 18 (commercial name: Cervarix ). The second one is a quadrivalent vaccine that, along with HPV 16 and 18, includes two other low-risk HPV types, the HPV 6 and 11 (commercial name: Gardasil ). Beside this first example of HPV vaccines researchers are developing further vaccines in order to ameliorate and overcome some limitations typical of first-generation vaccines. This class of HPV vaccines belongs to the so-called second-generation HPV vaccines. First-generation prophylactic HPV vaccines First-generation virus-like particles (VLP) vaccines VLPS (virus-like particles) are empty viral capsids that lack genetic material. In the early 1990s Kirnbauer reported the ability of viral L1 proteins to self-assemble and form a viral capsid morphologically and antigenic comparable to HPV virus. 30 VLPs can be attained in vivo by mammals and insects, but they can also be formed spontaneously from purified recombinant L1 proteins. VLPs are ideal options for HPV vaccines because they have the capability to activate high-titer, type specific antibodies. 31 To date there are two extremely efficacious HPV vaccines available internationally. Gardasil (Merck & Co., Inc., Whitehouse Station, NJ, USA) and Cervarix (GlaxoSmith- Kline [GSK] Biologicals, Rixensart, Belgium) (Table II). 29 Both of them are indicated for the protection against cervical dysplasia and 30 MINERVA MEDICA February 2016

7 SECOND-GENERATION PROPHYLACTIC HPV VACCINES FRUSCALZO cervical cancer, but also against other forms of cancers, mainly connected to the high-risk HPV subtypes 16 and 18. In addition, Gardasil is effective in the protection against genital warts and recurrent respiratory papillomatosis, mainly connected to the low-risk HPV subtypes 6 and 11. These two vaccines were shown to be extremely efficient in the prevention of cervical, vulvar, and vaginal lesions in HPV negative populations Unfortunately, no therapeutic effect on HPV infections or on dysplastic or invasive cervical lesion could be demonstrated. 38 Limitations of first-generation VLP vaccines Although efficacy of first generation VLPs vaccine has been documented, some limitations reduce its widespread, above all in low resource setting countries. These include above all the injection schedule, as well as the relatively advanced techniques required for production, storage and administration. In addition, relatively a few HPV subtypes are targeted by these vaccines (HPV 16 and 18, and, for the quadrivalent HPV vaccine, the types 6 and 11). 39 Second-generation prophylactic HPV vaccines Development of second-generation prophylactic HPV vaccines is aimed at improving first-generation HPV vaccines and to overcome their most relevant limitations. Under investigation are different techniques. These include the refining of VLPs vaccines (second generation VLPs vaccines) or developing different classes of vaccines using others methods of antigen incorporation into vaccines and delivery systems (protein and peptide vaccines, DNA vaccines, live-vectors vaccines and plant-based vaccines). 4, 17, 28 It is important that this research targets the development of a vaccine, which can be widely used in countries with low resources, where the burden of cervical cancer is high and population screening for early diagnosis is not widespread due to affordability. There are several ideal conditions to deal with when assessing a second-generation vaccine. First, they should be as safe and effective as the first-generation vaccines. Second, their administration must be orally or mucosally as opposed to via injection and lastly, it must be easily manufactured, and able to be stored in a variety of temperatures for easier distribution. Moreover, there could be a definite advantage attained if the targeted HPV types were amplified, or even through the creation of a vaccine having at the same time prophylactic and therapeutic properties. Although several potential HPV vaccines have been created, their application to date still appears mostly experimental. Some examples are listed below. Second generation VLPs vaccines The process of refining first generation VLPs vaccines appears to be a reasonable way to create a vaccine that allows for the combination of well-established techniques with new strategies to improve effectiveness and overcome existing constraints. This includes the targeting of other HPV antigens and HPV subtypes or the use of immunomodulators and other routes of administration. Multivalent VLPs vaccines Improving the range of protection offered by the vaccine could be done by broadening the number of targeted HPV types. However, the advantages of the increased range of vaccinal protection should compensate an improvement in the cost of vaccine production. Furthermore, there could be an effect on type-specific antibody production referred to as immune interference. An in-vitro study demonstrated that a trivalent vaccine consisting of HPV 16, 18, and 58 L1 VLPs was able to induce lower specific neutralizing antibody titers when compared to the monovalent form of the vaccine. This was also observed when the HPV 6 and HPV 11 L1 VLPs were introduced into a pentavalent vaccine. This disadvantage, however, was counterbalanced by the combination of a proper adjuvant into the vaccine. 40 Vol No. 1 MINERVA MEDICA 31

8 FRUSCALZO SECOND-GENERATION PROPHYLACTIC HPV VACCINES Very recently, a ninevalent vaccine, which includes the HPV types 31, 33, 45, 52, and 58 over the usual HPV 6, 11, 16, and 18, was introduced on the American market. 21 This vaccine is manufactured by Merck Sharp & Dohme Corp (Merck & Co., Inc., Whitehouse Station, NJ, USA) with the commercial name of Gardasil 9 and has been approved in the USA by the Food and Drug Administration (FAD) for females aged 9-26 years and males aged 9-15 years. This vaccine has the ability to protect against about 90% of HPV-related conditions, such as vulvar, vaginal, cervical, and anal cancers. An initial report showed a very high efficacy (96.7%) of this vaccine against pathologies related to HPV 31, 33, 45, 52, and 58 in a susceptible cohort of patients. 41 Another interesting strategy is the production of VLP made up of L2 HPV viral proteins. L2 is a minor capsid protein having a broad cross-neutralizing effect for many phylogenetic divergent HPV serotypes. In animal model studies, it was shown that the chimeric VLPs showing the L2 peptides on their surface maintained the ability to activate the neutralization of HPV16 and gain the ability to invoke antibodies that cross neutralized other HPV serotypes. 42 Cross neutralizing antibody titer obtained was lower compared to the typespecific titer. Nonetheless, this vaccination strategy seems to hold promise for covering in a relative simple way a wide variety of highrisk HPVs Chimeric VLPs Chimeric VLPs are VLP formed through self-assembling of a L1 or L2 protein fused with all or part of another viral protein. This vaccine strategy elicits the induction of a high level of L1 antibodies, like a traditional VLP, combining the advantage of targeting the immune system to others viral epitopes. 48 Another possible strategy is the augmentation of VLPs vaccine efficacy by combining adjuvant proteins to L1 or L2 proteins. Xu et al. reported the improvement of the immunogenicity of virus-like proteins of HPV 16 by combining an adjuvant, the mltk63, to the HPV 16 capsid protein L2 (see also 3.1.3: New adjuvants for VLPs vaccines). 49 In addition, it was shown that when VLPs are engineered with immunogenic epitopes from E6 or E7 protein, a simultaneous prophylactic and therapeutic effect against established infections or even cancer can be achieved, as this vaccine formulation can produce both a serologic and a cellular immune response. 50 A Chimeric VLP combining a new fusion protein comprising an amino acid sequence of L1 protein and E7 was constructed. The vaccine showed a very good safety profile and a good humoral and cellular immunogenicity against HPV16 virus. 51 Similar results were obtained generating a chimeric VLP vaccine including HPV 16 L1 protein, as well as E6 and E7 epitopes. 52 New adjuvants for VLPs vaccines Generally, vaccines contain an adjuvant aimed to enhance the host immune response and heighten the endurance of protection. Currently, several adjuvants are being investigated. One such example was obtained encapsidating the interleukin-2 gene as adjuvant, which induced a better mucosal and systemic immunogenicity to VLPs. 53 Another research conducted in mice showed an enhanced immunogenicity of nasal or aerosol-like HPV16 VLPs vaccine when coadministrating with a mucosal adjuvant, the Escherichia coli heat-labile enterotoxin (HLT). 54 Xu et al. reported an enhanced vaccine immunization by fusing an adjuvant (a modified LTK63) into HPV-16 chimeric virus-like particles. 49 In addition, a different form of aluminium-based adjuvants was tested, the amorphous aluminum hydroxyphosphate sulfate (AAHS), evidencing a greater immune responses in mice immunized with HPV16 vaccine. 55 Finally, the coadministration of a lipopolysaccharide derivate (CIA05) to the quadrivalent vaccine Gardasil was recently tested in Balb/c mice. This substance was shown to have an antitumor effect and to enhance the immunologic reaction to the vaccine. This adjuvant promoted an enhanced immune response, 32 MINERVA MEDICA February 2016

9 SECOND-GENERATION PROPHYLACTIC HPV VACCINES FRUSCALZO suggesting its potential use in the creation of more effectual second-generation prophylactic HPV vaccines. 56 Mucosal administration of VLPs vaccines Another route of administration of vaccine is the mucosal one (intranasal, aerosol, oral, vaginal) which might be especially viable in low-resource countries by avoiding parenteral injection. The following research findings reinforce the possibility of this route of administration of HPV VLPs as a possible alternative to parenteral injection. In a study on HPV-16 L1 VLPs, a comparison between different routes of administration has been performed: Via nasal spray, aerosol, and a combination of injection and aerosol (a priming dose followed by a boosting dose, respectively). The results showed that the mucosal routes of vaccination were safe and well tolerated. Moreover, aerosol administration induced, besides the humoral immunity, also a mucosal immune response with the production of anti-hpv16 VLP IgA. 23 The same research group found that adding a mucosal adjuvant, the non-toxic Escherichia coli heat-labile enterotoxin (HLT) to the HPV16 VLPs could enhance the immunological competence in mice. 54 Oral immunization with purified VLPs was also studied. This route of administration was shown to induce a good immunologic reaction in mice even though a 100 fold higher VLPs concentration was required in comparison to parenteral injection. 57 This implicates for example the requirement of adding a mucosal adjuvant in order to potentiate the immunologic vaccine efficacy. In other studies it was shown that oral administration of specific assembly form of HPV 16 L1 (T7-VLPs, T1 particles and capsomeres) activated both humoral and cellular responses in mice. Additionally, the coadministration with an adjuvant, a synthetic short single stranded DNA molecule, caused a substantially stronger recall response after the initial vaccination, even though there was no enhancing effect on anti-l1 antibodies production. 58 Stabile formulations Stabilization of VLPs formulas will allow for the simplification of the purification process and a reduction in production costs, which is important particularly for economically disadvantaged countries. One example includes the addiction of non-ionic surfactants into HPV VLP solutions, which provided for better stabilization. 59 Protein and peptide vaccines These particular kinds of vaccines are founded on protein or peptides that act as viral antigens able to activate an immunogenic response. Genetic engineering techniques provide for the possibility of designing molecules, which are composed of one or more HPV epitopes of immunologic significance. Afterwards, targeted HPV genetic regions are introduced into bacterial or yeast systems for large amount production. Because these epitopes induce only a weak immune response, they must be combined with adjuvants. 17 This sort of vaccine production offers a flexible way to generate vaccines with various target functions or even vaccines displaying a combined prophylactic and therapeutic function. L1-based protein and peptide vaccines The production of fusion proteins seems to offer a more cost efficient way than the production of VLPs vaccines, especially in regions with limited resources. Furthermore, a serological and cellular immune response can be evoked, theoretically allowing for concurrent prophylactic and therapeutic action. It has been shown that L1 capsid protein fused to glutathione S-transferase allows the antigen to selfassemble in pentamers and to keep its immunogenicity in mice models. While systemic and vaginal antibody response was less potent than VLPs response, this vaccine was shown to be a potent induction of cellular responses with a regression of established tumor in mice. 60 L2-based protein and peptide vaccines Supported by some initial evidence indicating a very broad cross reactive action related to Vol No. 1 MINERVA MEDICA 33

10 FRUSCALZO SECOND-GENERATION PROPHYLACTIC HPV VACCINES some L2 epitopes when taken out of the VLP context, some researchers have proved the efficacy and safety of administering synthetic L2 peptide as cross-protective vaccines. 61 Kondo et al. identified cross neutralizing epitopes in the L2 protein sequence of HPV16 serotype. It was shown that the antisera against three different peptides in these regions exhibited the capability to evoke in rabbits a cross-neutralization activity against the HPV16, 18, 31 and In a study conducted by other researchers, the synthetic P25-P2C-HPV lipopeptide elicited a strong L2-specific response in mice either when delivered subcutaneously or intranasally. 63 Lastly, Jagu et al. were able to develop a concatenated multitype L2 fusion protein to be tested as prophylactic pan-hpv vaccine. This can overcome the problem of a weaker neutralizing antibody response of L2 vaccines against heterologous types. When mice were given a vaccination with the multiype L2 fusion protein, a high serum heterologous titer was produced against a wide band of non-tested HPV types An experimental vaccine composed of HPV16 L2 protein, as well as of the early proteins E6 and E7 was tested coadministered with the adjuvant GPI-0100 in mice. 67 The possible benefits this vaccine is that it is able to offer both a wide cross protection against HPV infection (through the L2 cross neutralizing anti-hpv antibodies), and of cellular immunity induced (through the T-cell activation induced by the HPV16 E6 and E7 proteins). The authors showed an improvement in both antibody and cell-mediated immune responses after subcutaneous administration together with the adjuvant GPI Recently, a clinical study evaluating the effectiveness of a combination of imiquimod and TA-CIN in reported a complete resolution of precancerous lesions of the vulva in 63% of patients. 68 DNA vaccines DNA based vaccines are new candidates for prophylactic vaccines. Of interest is their ability to remain stable at room temperature and to have durable storage capabilities. They can be also quite simply produced by combining circular engineered DNA structure (plasmids) into bacteria. After that, a concentrated solution of plasmids can be conveyed into host cells by intramuscular injection or encapsulated in microparticles or introduced into skin cells by gene gun technology. Some concerns have been raised regarding their safety given that delivered DNA has theoretically the ability to stably integrate in the host genome and interfere with its functionality. Mutating the E7 proteins but leaving the critical epitopes unchanged or injecting DNA in cells that only have a short lifespan, such as skin cells, could reduce concerns about a possible host cell oncogenic transformation. 14 It has been shown that the administration of DNA-based vaccines can induce both a cellmediated and an antibody-mediated response to HPV antigens. Nevertheless, it seems that DNA vaccines could be more effective in the development of therapeutic vaccines than in in the development of prophylactic ones. Indeed, a research on HPV-16 L1 vaccines administered in monkeys showed a strong cellular immunity albeit only an insubstantial antibody response of DNA-based vaccines. 69 The fusion to a modified HPV16 L1 DNA of a cdna of a chemokine (immune cell-recruiting RANTES) was carried out in order to ameliorate the humoral immune response to the vaccine. The researchers were able to show the production in mice of high levels of HPV16 L1-specific IgG antibodies coupled to a T-cell response. 70 Similarly, Hu et al. investigated the mucosal immunity induced by administering intranasally HPV16L1 DNA vaccine in conjunction with an adjuvant, the heat-labile enterotoxin B subunit (LTB). Results showed a favorable specific IgG and IgA antibody response in serum and a specific IgA production in the vaginal tract. 71 Finally, the HPV-16L1 DNA gene was used in a recombinant baculovirus as a vector in mice by Lee et al. When compared to Gardasil, this DNA vaccine demonstrated, in addition to a reduced cost of production, similar safety and humoral IgG, as well as IgA immunity, and higher cellular immunogenicity MINERVA MEDICA February 2016

11 SECOND-GENERATION PROPHYLACTIC HPV VACCINES FRUSCALZO Recombinant live-vector vaccines Viral proteins responsible for immune system activation can be directly produced by host cells when transfected with live-vector vaccines. These vectors are bacteria and viruses manufactured with late viral proteins, normally L1. After infection in the host has occurred, the viral proteins are formed and self-assemble in the immunogenic VLPs. In comparison to naked DNA vaccines, they provide the advantages to be safe and induce a strong and protracted cell-mediated and antibody-mediated immunity. When compared to peptide vaccines, they have the convenience of being easier to be delivered and processed within the host. Furthermore, they can be administered mucosally and are moderately inexpensive to produce and distribute. 17 Conversely, there is the possibility that a pre-existing immunity to vectors or even an acquired immunity induced after the first vaccinal dose administration could diminish the immunologic response to vaccine. 73 Bacteria Researchers in Lausanne first created an enhanced recombinant Salmonella organism able to assemble HPV16 VLPs in mice. Results showed that a single oral or nasal administration could elicit a good humoral immune response. 74 The same group of research also designed a new plasmid expressing L1 protein (named kanl1s), and showed that intravaginal immunization with HPV16 VLPs vaccine administration via live attenuated Salmonella caused both humoral and cellular immunity in mice. This kind of immunization was also able to protect against subcutaneously implanted tumor cells. 24 Also an attenuated L1-expressing Shigella vaccine was demonstrated to be suitable in guinea pigs for eliciting an IgG immune response to HPV16 L1 VLP after conjunctival vaccination. 75 Viruses These vaccines are based on a viral genome encoding for HPV immunologic epitopes, gen- erally L1, E1, E2, and E7 proteins or modified forms of them. This allows producing viral vectors that are able to advocate both a humoral and a cellular immunological response into the recipient organism. Berg et al. created a recombinant adenovirus expressing the L1 protein of canine oral papillomavirus (COPV) and showed that L1 capsid protein could assemble into VLPs, able to match to specific antibodies. 76 Another research group reported the development of a modified adenovirus codifying the HPV 16 L1 capsid protein. The yielded neutralizing L1 antibodies were both stronger and more persistent in comparison to the DNA vaccine, but the titers of these antibodies were lower when compared to the L1 VLP vaccine. However, when a covaccination with an adenovirus that was encoded the adjuvant GM-CSF occurred, the neutralizing L1 antibodies were comparable with those of VLP vaccine. 77 A recombinant baculovirus that was not able to be replicated in mammals but produced an envelope protein charring for HPV-16L1 DNA vaccine (AcHERV-HP16L1) was also recently introduced. Higher cellular immunogenicity and reduced production costs in comparison to a vaccine with only Gardasil and to a combined immunization with Gardasil as a booster was shown in vivo tests in mice. 72 Plant-and yeast-based vaccines Transgenic plants or yeast has the ability to produce HPV antigens when viral vectors infect them or when viral antigens are directly inserted into them. Creating an adequate plant- or bacteria-based vaccine would be beneficial because it would provide for an adequately cost-effective instrument of the mass producing, oral administration and heat stabilization of vaccines and would thus remove the high costs of vaccine manufacture and distribution. 22, 78 Nonetheless, a multidisciplinary effort would be required in order to design and create transgenic plants or bacteria with the capability to produce large quantities of L1 proteins assembling in VLPs. In addition, initial findings in mice and rabbits produced only a weak immunogenic response. 17 Vol No. 1 MINERVA MEDICA 35

12 FRUSCALZO SECOND-GENERATION PROPHYLACTIC HPV VACCINES Sasagawa discovered that freeze-dried yeast cells could safely be introduced into and digested by the intestines of mice, but fell short of showing a definite antibody response. 79 Intranasal boosting with a suboptimal dose of HPV-VLP was able to regain the induced tolerance in the vaccinated mice. An interesting HPV-16 L1-based chimera was engineered including a cross-protective epitopes from the L2 minor capsid protein. Acceptable amounts of self-assembled VLPs were produced which evoked anti-l1 and anti-l2 immune responses in mice. 80 Conclusions Further clinical development of new vaccines and heightened research efforts are warranted, given the encouraging results demonstrated with the use of first-generation VLPs HPV vaccines. 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