Human Papilloma Virus vaccines a review of advances in the development of HPV vaccines
Leighton Davies MD, MSc for HIV i-Base
It is now clearly established that individuals infected with HIV are at increased risk of human papillomavirus (HPV)-related anogenital neoplasia.
HIV-positive women have a higher prevalence of HPV infection of the cervix and anus as well as high- and low-grade squamous intraepithelial lesions (HSIL and LSIL) at these sites compared to HIV-negative women, matched for age & risk factors. Similarly HIV-positive men who have sex with other men (MSM) have a higher prevalence of anal HPV infection and anal SIL and carcinoma than HIV negative MSMs. During the last decade it has been shown conclusively that HPV infection is implicated in more than 99% of cases of invasive cervical carcinoma. 
The prevalence of anogenital SIL as well as the proportion infected with multiple HPV types increases with decreasing CD4 count. This may reflect the failure of the impaired immune response to deal with HPV antigens or may possibly be due to local interactions between HIV and HPV at the tissue or cellular level.
Now in the third decade of the HIV pandemic, we are seeing increasing numbers of SIL progressing to invasive carcinoma. This is despite HAART, which by prolonging survival may increase the risk of progression of SIL to invasive carcinoma if these lesions do not resolve spontaneously or remain untreated.
There are now over a 100 different HPV subtypes with new types being identified annually. Broadly speaking most types can be divided into two groups: low risk (non-oncogenic) types which are rarely, if ever implicated in neoplastic lesions; and high risk (oncogenic) types.
Of the low risk types (6, 11, 42, 43 and 44), types 6b and 11 are responsible for the majority of cases of condyloma acuminata (genital warts) and are infrequently associated with cases of cervical intraepithelial neoplasia (CIN) type 1. Of the high-risk types, types 16 and 18 (and to a lesser extent types 31 and 45) are found in 50-80% of CIN 2 and 3 and up to 90% of invasive cervical cancer. [2, 3]
Many other types are also associated with cervical neoplasia, though with much less frequency, including types 33, 35, 39, 52, 56, 58, 59 and 68.
Papilloma viruses are members of the papovavirus family: double stranded DNA viruses that replicate in their hosts nuclei. The virions icosahedral capsid comprises an outer protein coat, that consists of two different proteins; a major (L1) and a minor (L2) capsid protein, which encloses a circular 7900 base-pair genome divided into three groups of (1) early, (2) late and (3) control genes. Additionally, there is a non-coding region called the Long Control Region (LCR), which regulates the expression of the open reading frames (ORFs). Papilloma viruses with less than 90% sequence homology in E6, E7 and L1 ORFs to any of the known HPV types are classified as a new type. The function of these three genes is crucial to the virus oncogenicity. E6 is responsible for cell transformation through p53 degradation, as is E7 through its binding to the retinoblastoma protein (pRB). The L1 gene is responsible for the coding of the major capsid protein.  The E6 protein of high-risk HPV interferes with p53 function and deregulates the cell cycle. E6 binds to p53, forming a stable complex that undergoes proteolysis by an ubiquitin-dependant ligase known as E6AP.  The degradation of p53 targets the transcriptional co-activator CBP/p300, which has a role in cell cycle and differentiation. 
Meanwhile, the E7 protein forms inactivating complexes with the pRB anti-oncoprotein through competitive binding with the retinoblastoma pocket. This binding releases a transcription factor E2F, which accelerates DNA synthesis and cell cycle progression.  Thus E6 has an anti-apoptotic effect, whilst E7 promotes cell proliferation. In high-risk HPV, viral gene integration occurs in the E1/E2 region, disrupting the E2 gene, which represses the promoter from which E6 and E7 genes are transcribed. This leads to accelerated expression of E6 and E7, ultimately leading to the accumulation of damaged DNA and the development of the cancer phenotype over an extended period of time.
It should be noted, however, that infection with a high-risk HPV by itself is insufficient to trigger the chain of events that ultimately leads to the development of invasive carcinoma. Other co-factors have been proposed, including becoming sexually active at an earlier age, number of sexual partners (increasing likelihood of exposure to an oncogenic HPV type), smoking, the number of children that a women has given birth to, duration of use of oral steroid contraceptives, and co-infection with other sexually transmitted organisms (particularly chlamydia trachomatis).
Whilst attempts to arrest SIL from progressing to invasive lesions, through the use of immunomodulating drugs such as Imiquimod are progressing, exciting advances in the development of vaccines directed towards oncogenic HPV types are also in development.
Prophylactic HPV vaccines
Initial attempts using recombinant vaccines based on the major capsid protein L1, were largely unsuccessful in animal models, as these proved to be of insufficient immunogenicity to augment the bodys natural immune response. Whilst compelling evidence from animal studies showed that neutralising antibodies against L1 were able to block new infection, it was deemed important to obtain a source of conformationally correct L1 protein. A breakthrough was made by Kirnbauer and colleagues, and later by Zhou and colleagues, who discovered that L1 self-assembles into virus-like particles (VLPs), when expressed at high levels in cultured insect cells.  Moreover these VLPs induced the production of neutralising antibodies to conformational epitopes. HPV VLPs can now be produced using several expression systems including vaccinia viruses, baculoviruses and yeast systems.
Several groups are using VLPs against HPV types 6, 11, 16 and 18 in clinical trials.
Phase I and II studies have shown VLP vaccines to be well tolerated, safe and capable of inducing high titres of both binding and neutralising antibodies. These are often 50 times higher than titres induced by naturally occurring infection. In some studies, T-cell responses were also reported, suggesting possible therapeutic as well as prophylactic uses.
In 2002, Koutsky and colleagues published the results of a large randomised double-blind study to establish the efficacy of a HPV-16 VLP vaccine, developed by Merck, in preventing infection in women aged 16-23. 
In this study, 2392 women were eligible, but nearly 36% were excluded because they were HPV-16 seropositive or had HPV-16 DNA detected in the genital tract either at enrolment or at the last vaccination. This shows not only the need for a vaccine, but raises the issues of providing early protection against a sexually transmitted infection, long before young people actually become sexually active. This clearly raises issues of moral and political ethics that will vary by country.
Vaccinations of HPV-16 VLP (40 micrograms per dose) were given at day 0, month 2 and month 6. Follow-up at 6-monthly intervals, looked for evidence of persistent HPV-16 infection, defined as detection of HPV-16 by PCR at two visits, at least 24 months apart. An interim analysis was performed after a fixed number of persistent HPV-16 cases were detected, although all women will be followed up to 48 months following completion of the vaccination regimen. The results were impressive. In the placebo group (n=765), 41 cases of persistent HPV-16 including nine cases of HPV-16 associated SIL. With no cases in the vaccinated group (n=768) this established 100% efficacy (95% CI 90-100; p<0.001). Furthermore, an additional 33 women (6 in the vaccine group and 27 in the placebo group) were positive for HPV-16 DNA at a single visit. However, none of whom went on to develop SIL suggesting the possibility that this vaccine may provide sterilising immunity. With the median time of follow-up was 17.4 months, in is impossible to access the durability of protection. To be completely efficacious the vaccine would need to provide protection from adolescence for several decades.
More recently, Harper and colleagues published results of a trial of the efficacy of a bivalent VLP vaccine developed by GlaxoSmithKline, to prevent infection with HPV-16 and HPV-18. 
This was a randomised, double blind, placebo-controlled trial in women aged 15-25 years old, performed at numerous centres in North America and Brazil. In addition to the primary trial objective, efficacy against cytological abnormalities and CIN as well as vaccine immunogenicity, safety and tolerability were assessed.
Two study phases were evaluated: an initial phase for vaccination and follow-up to 18 months, and a blinded follow-up extension phase that ended at month 27. Inclusion criteria for the initial phase of the study included pevious history of no more than six sexual partners, no history of an abnormal cervical smear or ablative or excisional treatment of the cervix, no ongoing treatment for external condylomata, and who were cytologically negative, seronegative for HPV-16 and HPV-18 antibodies by ELISA and HPV-DNA negative by PCR for 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) within 90 days of study entry. Women who did not have any surgical treatment of the cervix or uterus were eligible to participate in the extension phase.
The bivalent vaccine contained 20mcg of HPV-16 VLP and 20mcg of HPV-18 VLP combined with an AS04 adjuvant (500mcg aluminium hydroxide and 50mcg 3-deacylated monophosphoryl lipid A) to stimulate dendritic cells. The placebo, identical in appearance to the vaccine contained only 500mcg of aluminium hydroxide. All study participants received a 0.5ml dose of either vaccine or placebo at 0, 1 and 6 months.
Cervical smears for cytology and HPV-DNA testing were obtained at screening and months 6, 12 and 18. The study participants self-obtained cervicovaginal samples at months 0 and 6 and every 3 months thereafter for HPV DNA testing.
Colposcopy was recommended after two reports of atypical squamous cells of undetermined significance (ASCUS), or after one report of atypical glandular cells of undetermined significance, LSIL, HSIL, squamous cell carcinoma, adenocarcinoma in situ or adenocarcinoma.
Incident cervical infection with HPV-16 or HPV-18 was defined as at least one positive PCR result. Persistent infection was defined as at least two positive PCR assays, for the same viral genotype, separated by at least 6 months. Serological testing was carried out by ELISAs to HPV-16/18 VLPs, with seropositivity defined as a titre greater than or equal to the assay cut-off titre (8 ELISA units/ml for HPV-16 and 7 ELISA units/ml for HPV-18). These were compared to the typical natural titres obtained from women in a previous epidemiology study, found to be seropositive for HPV-16/18.
Of the 1113 women enrolled and randomised, 560 received the vaccine and 553 the placebo. Demographic characteristics were similar between both groups, including similar patterns of risk factors for HPV acquisition (smoking, number of sexual partners and age at sexual debut). Reasons for elimination from the according-to-protocol (ATP) efficacy analysis were abnormal cytology, high-risk HPV DNA positivity or seropositivity for HPV-16 or 18 at enrolment.
A total of 958 women (85%) completed the initial phase, with similar proportions of women from both groups dropping out of the study.
In the ATP analyses, vaccine efficacy was determined to be 91.6% (95% CI 5-98.0) against incident infection (not statistically significant) but was found to be 100% (47.0-100) against persistent infection (statistically significant). In the intention-to-treat analyses vaccine efficacy was 95.1% (63.5-99.3) against persistent cervical infection with HPV16/18 and 92.9% (70.0-98.3) against cytological abnormalities associated with HPV-16/18 infection.
The vaccine was generally determined to be safe with only a few reports of injection site symptoms, which were transient and mild. Among the vaccinated women in the ATP cohort from month 0 to month 7, 100% seroconverted to HPV-16 positive and 99.7% seroconverted to HPV-18 positive after 3 doses of vaccine. By 18 months 100% of women had seroconverted to HPV-16 and HPV-18 positive. Geometric mean titres for vaccine-induced antibodies to HPV were over 80- and 100-fold greater than those seen in natural infections with HPV-18 and HPV-16 respectively. These titres remained substantially raised at 18 months, being 10-16-fold higher than those seen in natural infection with HPV-16 and HPV-18 respectively. This suggests that the immune responses induced in vaccinated women may provide a longer duration of protection than natural HPV infection. A protective antibody level has however not yet been established.
SanofiAventisPasteur are currently conducting trials of a tetravalent vaccine directed against HPV subtypes 6,11,16 and 18. The results of which are eagerly anticipated, as this vaccine would not only confer protection against the neoplastic subtypes but also against the subtypes most commonly responsible for condylomata acuminata.
Therapeutic HPV vaccines
The purpose of a therapeutic HPV vaccine is to eradicate or reduce the number of infected cells by inducing specific cell-mediated immunity (CMI) that prevents the development of lesions and eliminates existing lesions or even malignant neoplasms. There are many strategies to generate cytotoxic T lymphocytes (CTLs), which all involve causing antigen presenting cells (APCs) to process the tumour or viral antigen and present it in the context of an MHC receptor, along with adhesion and co-stimulatory molecules to stimulate anti-tumour lymphocytes. The induced specific CMI can directly target HPV viral products, HPV-induced cellular products or a combination of both.
Although HPV VLPs can induce L1-specific CMI responses, in addition to inducing high titres of neutralising antibodies, L1 capsid proteins are not expressed at a detectable level in the proliferating basal keratinocytes of virus producing lesions, or in the dedifferentiated calls of HPV-induced dysplasias and carcinomas. It is therefore unlikely that CMI responses to L1 proteins will induce regression of established lesions; HPV VLPs have been generated in which polypeptides of non-structure viral proteins are incorporated into the VLPs as fusion proteins of L1 or L2. Since E6 and E7 are consistently expressed in most cervical cancers and their precursor lesions, but are absent from normal tissues, most efforts have focused on eliciting CTLs directed against these viral oncoproteins. While most tumour-specific antigens are derived from normal or mutated proteins, E6 and E7 are completely foreign viral proteins and may therefore harbour more antigenic peptides/epitopes than a mutant cellular protein. Furthermore, since E6 and E7 are required for the induction and maintenance of the malignant phenotype of cancer cells, cervical cancer cells are unlikely to evade an immune response through antigen loss.
Finally animal studies suggest that targeting oncoproteins such as E7 can generate therapeutic as well as preventive effects.
Potential therapeutic vaccine strategies
It is useful to now provide a short overview of the following eight vaccine approaches:
- Vector-based vaccines
- Peptide- and protein-based vaccines
- DNA vaccines
- Chimeric VLP vaccines
- Dendritic cell-based vaccines
- Tumour cell-based vaccines
- Self-replicating RNA vector vaccines
- HPV pseudovirion vaccines
Using viral vectors to introduce genes for vaccination is an effective way to stimulate many arms of the immune system. Vaccinia virus vectors have the advantage of being able to accommodate large recombinant gene insertions, and do not persist in the host. They also offer high efficiency of infection and high levels of recombinant gene expression. Finally vaccinia virus is a lytic virus and thus the chance of integration into the host genome is extremely small. One downside is that older people may have pre-existing antibodies to vaccinia virus that limits the elicited immune response. A recombinant vaccinia virus comprising mutated HPV-16/18 E6 and E7 genes (to remove their oncogenic potential) was created and in an initial study the vaccine was reported to be safe when administered to nine patients with late-stage cervical cancer. Most of these patients were immuno-suppressed and only one developed CTLs in addition to a clinical remission. 
In a more recent trial, 29 patients with stage Ib or IIa cervical cancer were vaccinated. After a single vaccination 4 patients developed CTLs and 8 developed serological responses to the HPV proteins.  This approach may offer some promise for future developments, however it emphasises the difficulties in eliciting therapeutic responses in immuno-compromised individuals.
More recently a vaccinia virus has also been utilised to explore tumour vaccine strategies employing intracellular sorting signals. Endosomal and lysosomal compartments, associated with MHC-II processing and presentation are characterised by the presence of a number of compartment-specific membrane proteins, including the lysosomal associated membrane protein (LAMP-1). In one study the sorting signals of LAMP-1 were linked to the HPV-16 E7 antigen to create the Sig/E7/LAMP-1 chimera. It was found that expression of this chimera with a recombinant vaccinia virus targeted E7 to endosomal and lysosomal compartments and enhanced MHC class II presentation to CD4+ T cells compared to vaccinia virus expressing wild-type E7. Furthermore, the Sig/E7/LAMP-1 vaccinia virus vaccine cured established tumours containing the E7 antigen whilst the wild-type E7 vector showed no effect on the established tumour. These experiments demonstrate that modifications rerouting cytosolic antigen to the endosomal/lysosomal compartment can profoundly improve the in vivo therapeutic potency of recombinant vaccinia vaccines. Phase I/II clinical trials using intramuscular administration of attenuated Sig/E7/LAMP-1 are underway at the Johns Hopkins Hospital in the USA (13).
Other viral delivery systems include recombinant adenoviruses and RNA-based alphavirus vaccines that have been constructed to express E7 or polyepitope proteins and are in early clinical trials.
Bacteria can also be used to deliver recombinant gene products. Listeria monocytogenes has recently emerged for use as a recombinant vaccine for human cancers due to its ability to elicit both CD8+ and CD4+ responses and induce regression of established tumours expressing a model antigen. This gram-positive intracellular bacterium usually infects macrophages, whereupon it is phagocytosed and taken up into a phagosome. Unlike other intracellular bacteria it escapes into the macrophage cytoplasm by secreting a factor listeriolysin O, that disrupts the phagosomal membrane. Because of its presence in both endosomal compartments and cytoplasm it can deliver its antigens or carry foreign antigens into both the MHC-I and MHC-II pathways, thus inducing strong CMI responses. A recombinant L. monocytogenes secreting HPV-16 E7 has recently been shown to lead to regression of pre-existing E7-expressing tumours and is currently undergoing phase I/II trials (14). Other bacterial carrier systems have been investigated for delivering HPV vaccines, including Salmonella, Shigella and Escherichia coli.
Peptide- and protein-based vaccines
The characterisation of many CTL-defined antigenic determinants has opened the possibility of developing antigen-targeted vaccines. Several HPV-16 E7-specific CTL epitopes have been characterised for the HLA A-0201 haplotype and clinical trials have been conducted on patients possessing this genotype and whose HPV tumour type matched the viral peptide epitopes. Mixed results however, were obtained from these trials. Preclinical data has suggested that longer peptides that contain a helper T-cell epitope linked to the CTL epitope are more efficient at eliciting CTLs than the minimal epitope. The potency of HPV-16 E7 peptide based vaccines can be further enhanced by the use of a dendritic cell activating adjuvants such as immune stimulatory complexes (ISCOMs) and immunostimulatory carriers (ISCARs). Other strategies include modifying the CTL epitopes using lipid conjugation to form an immunogenic lipopeptide vaccine. An additional problem with using peptides is that one must know the HLA haplotype of the patient and the HPV genotype of the tumour. This has prompted many investigators to consider full-length E6 and/or E7 proteins, or fusions with other proteins e.g an E6/E7 fusion protein in a saponin-based adjuvant. To increase the immunogenicity of the E7 protein, one group has fused E7 to the BCG heat shock protein 65 (HSP65), which stimulates immunity through engaging the Toll-like receptor 4. This fusion has been used to immunise men with anal HSIL, some of whom, also had condylomata acuminata, in an open-label trial. 
Of 14 patients with genital warts, three had a complete resolution of symptoms and ten had a reduction in size of 70-95%. At least 95% of the men with HSIL showed a reduction in histopathological findings to at least LSIL with 44% having complete remission. These results obviously need to be confirmed in double-blind randomised controlled trials but are nevertheless encouraging and suggest that HSP65-E7 fusion protein elicits cross-reactive immunity.
Not all peptide based vaccines generate CTL responses and tumour protection; although interestingly the same epitopes loaded onto dendritic cells (DCs) can generate protective immunity, indicating that it might not be the peptides per se, but rather the method of presenting the epitope to T-cells that determines the outcome of vaccination with peptide based vaccines. It is therefore important to choose the appropriate adjuvants and route of administration for peptide based vaccines in order to determine their immunising or tolerising properties in vivo before clinical use. It is evident that the application of peptide-based vaccines is limited by MHC restriction and the necessity to define specific CTL epitopes. In fact, most epitopes of HPV-16 E6 and/or E7 in patients with HLA other than HLA-A0201, remain undefined, making it difficult to use peptide-based vaccines in such situations. As intimated above, the use of protein-based vaccines can present all possible epitopes of a protein to the immune system, thereby bypassing the MHC restriction. Additionally, with a protein vaccine, serious side effects such as insertional gene activation and transformation (a possible concern with the use of recombinant viruses and DNA vaccines), are not an issue.
Several strategies devised to increase the potency of protein-based vaccines include:
- Association of the E7 protein with various adjuvants to enhance E7-specific CTL activities.
- The fusion of antigen with heat shock protein (vide supra) represents another strategy for enhancing CTL priming.
- Linking GM-CSF to an antigen, can target the antigen to DC and other GM-CSF responsive cells, after the chimeric molecule binds to the GM-CSF receptor, generating enhanced immune responses in these cells. In the context of HPV immunotherapy it has been possible to take peripheral blood monocytes (PBMCs) of cervical cancer patients, differentiate the cells in culture using IL-4 and GM-CSF into dendritic cells, mix the DCs with an HLA-A0201 E7 epitope and sensitise the autologous PBMCs from the cancer patients. 
A case report, of a woman with an HPV-18-containing adenocarcinoma, treated over 10 months with DCs that had been pulsed with an HPV-18 E7 protein, suggested an inhibition of metastatic disease for at least three years. 
DNA vaccines can be prepared inexpensively and rapidly on a large scale, and allow for expression of antigen for a sustained period of time. Consequently, the availability of antigen to be processed and presented as MHC-peptide complexes is likely to be more prolonged than peptide-based vaccines. Furthermore, DNA transduced into antigen inside APCs, enables the synthesised peptides to be presented by the patients own HLA molecules. DNA vaccines targeting many different types of HPV can be mixed and effectively administered together, thus providing an efficient method of treating a variety of HPV-associated infections and tumours. Although the efficacy of DNA vaccination is important, safety is also a critical issue. DNA present in the vaccine may integrate into the host genome, potentially inactivating tumour suppressor genes or activating oncogenes, ultimately inducing malignant transformation of the host cells. Fortunately, it is estimated that the frequency of integration is much lower than that of spontaneous mutation, and integration should not pose any real risk. 
Other potential risks are associated with the presence of HPV-16 E7 oncoprotein in host cells. It is feared that the presence of E7 in the host nuclei may lead to accumulation of genetic aberrations and eventual malignant transformation of the host cells. Strategies such as employing the endosomal/lysosomal-targeting Sig/E7/LAMP-1 DNA vaccine, may be sufficient to divert E7 away from the nucleus to regions such as the endosomal and lysosomal compartments. This would physically separate E7 from pRB, thus abrogating the transformation activity of E7.
Ultimately DNA vectors employed in human clinical trials could utilise a minimally mutated E7 gene in which critical epitopes are preserved whilst eliminating potential oncogenic transformation.
Chimeric VLP vaccines
In order to create a preventive and therapeutic VLP-based vaccine, several E7 chimeric VLPs consisting of the L2 minor capsid protein plus the E7 protein or the n-terminus of E7 fused to L1 have been created. These E7 chimeric VLPs have been shown to generate significant E7-specific CTL activities and E7-specific anti-tumour effects. Furthermore E7 chimeric VLPs are indistinguishable from parental VLPs in their ability to elicit high titres of neutralising antibodies in murine models.
The anti-tumour immune response to the chimeric VLPs appears to be primarily mediated by CD8+ cytotoxic lymphocytes. In vitro E7-specific CTL activity was detected in lymphocytes from chimeric VLP-vaccinated mice. Furthermore, good protection was observed in MHC class II knockout or natural killer cell-depleted mice, but no protection was seen in beta-2_microglobulin or perforin knockout mice. It is unclear how the VLPs are routed for class I presentation it might involve an endocytic pathway that the virus normally uses to enter the cell during the infectious process.
L1 and L2 chimeras for E7 produced similar results in mice, so it is unclear whether L1 or L2 chimeric VLPs would be preferable for testing in humans. L1 chimeras have the theoretical advantage of delivering more copies of the target antigen per VLP than L2 chimeras (360 for L1 versus 12 for L2). L2 chimeras on the other hand have the theoretical advantage of being able to incorporate larger polypeptides and thereby increasing the number of epitopes for immune recognition. It would seem reasonable to continue testing both types of chimeras. Currently, clinical grade HPV-16 L1/L2-E2-E7 fusion chimeras, which contain four HPV-encoded proteins (L1, L2, E2 and E7) as target antigens are undergoing phase I/II clinical trials. 
Chimeric HPV VLPs containing polypeptides of non-HPV targets are also being investigated in pre-clinical studies. One approach is to incorporate polypeptides of other sexually transmitted infections (STIs). With the provision that induction of neutralising antibodies is sufficient for protection against genital HPV infection, this strategy could produce a vaccine that provides protection against both HPV and another STI at little or no increase in the cost of production or administration. A second approach involves incorporating cellular tumour antigens into the VLPs. This strategy was recently shown to induce therapeutic anti-tumour immune responses in a murine model. 
Immunisation of mice with an immuno-dominant peptide derived from the P815 tumour-associated antigen P1A induces specific T-cell tolerance, resulting in progressive outgrowth of a normally regressing P815 tumour line. In contrast immunisation with an L1 chimera that contained the same P1A peptide did not induce tolerance rather it protected mice from lethal challenge with a progressor P815 line. Vaccination with this chimeric VLP also functioned therapeutically to suppress the growth of established tumours and to increase survival of the tumour-bearing mice.
Cell-based vaccines for cancer immunotherapy can be conceptually divided into two broad categories: DC-based vaccines, and cytokine-transduced tumour cell-based vaccines.
DCs are the most potent professional APCs, specialised to prime helper and killer T-cells in vivo. Ex vivo preparation and modification of DCs, therefore represent an attractive vaccine strategy that is capable of enhancing T-cell mediated immunity against tumours. The understanding that DCs can be generated from haematopoietic progenitors in the setting of various cytokines, mainly GM-CSF and Flt3-ligand, has created the opportunity to use a tumour cell-based vaccine transduced with GM-CSF or Flt3-ligand cytokines to expand and prime DCs in vivo (see under Peptide- and Protein based vaccines, point 3 above).
Dendritic cell-based vaccines
A lack of information about DC maturation and their lineage-specific markers, which define their cellular differentiation state, previously hindered the generation of a large number of DCs. Recent advances have revealed the origins of DCs, their antigen uptake mechanisms, and the signals that stimulate their migration and maturation into immuno-stimulatory APCs (21, 22). DCs derived from cultured haematopoietic progenitors appear to have an APC function similar to purified mature DCs. Broadly speaking; two strategies are employed to generate DCs ex vivo.
Dendritic cells pulsed with peptides/proteins.
Syngeneic spleen DCs pulsed with E7-specific T cell epitopes can generate protective E7-specific anti-tumour T CTLs. Treatment of tumours with peptide-pulsed DCs had resulted in sustained tumour regression in several different tumour models.  Similarly DCs pulsed with whole E7 proteins are able to elicit potent E7-specific CTL responses in vivo, which are associated with protection against a challenge with syngeneic HPV-16 induced tumour cells.  Another study demonstrated that DCs derived from patients could be pulsed with fusion proteins such as E6/E7 and used to generate E6/E7-specific CTLs in vitro. 
Dendritic cells transduced with HPV E6 and/or E7 genes.
Gene-transduced DC-based vaccines represent an attractive alternative to peptide pulsed DC-based vaccines since MHC restriction may be bypassed by directly transducing genes coding for E6 and/or E7 inside DCs, allowing synthesized peptides to be presented by any given patients HLA molecules. Gene transfer into DCs can be accomplished by a variety of methods involving either naked DNA or the use of viral vectors. The major limitation of naked DNA transfer into DCs is poor transfection efficiency using various physical methods. Various studies indicate that the potency of DC-based vaccines may ultimately depend on their route of administration be it subcutaneous, intramuscular or intravenous. 
Tumour cell-based vaccines
The use of tumour cell-based vaccines may not be suitable for the treatment of early-stage, pre-cancerous HPV-associated lesions, because of the risks and controversy associated with administering modified tumour cells to these patients.
Tumour cell-based vaccination is therefore better reserved for patients with advanced HPV-associated carcinomatous lesions. Transduction of tumour cells with genes encoding co-stimulatory molecules or cytokines may enhance immunogenicity leading to T-cell activation and anti-tumour effects after vaccination. 
In murine studies strong anti-tumour effects have been demonstrated with tumour cells transduced with IL-2 and IL-12 genes that indicate that tumour cell-based vaccines may be useful for the control of minimal residual disease in patients with advanced HPV-associated cervical carcinomas.
Self-replicating RNA vector vaccines
Nucleic acid vaccines using RNA replicons have recently been shown to significantly enhance vaccine potency.  RNA replicon vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA, which is then transcribed into RNA replicons in transfected cells or in vivo.  The self-replication allows the expression of the antigen of interest at high levels for an extended period, optimising vaccine potency. Since such replicons ultimately cause lysis of transfected cells, the concern associated with naked DNA vaccines of integration into the host genome is lessened, particularly important when the oncogenic E6 and E7 proteins are targeted.
The potency of a self-replicating RNA vaccine can be further enhanced by applying the LAMP-1 targeting strategy, creating a Sig/E7/LAMP-1 RNA replicon. 
DNA based RNA replicons also known as suicidal DNA – share the advantages of both the RNA replicons and naked DNA vaccines, being both stable and easily prepared. Furthermore they are significantly more potent than conventional DNA vaccines. Since cells transfected with DNA-launched RNA replicons are eventually lysed (hence the term suicidal) there is little concern for malignant transformation commonly associated with naked DNA vaccines.
Recently a DNA-launched RNA replicon vaccine demonstrated significant E7-specific CTL activity and anti-tumour effects. 
HPV pseudo-virion vaccines
The encapsulation of naked DNA by HPV capsids forming HPV pseudovirions has been achieved using various expression systems including recombinant vaccinia viruses, Semiliki Forest virus, baculoviruses and even in yeast systems. The target DNA can be packaged into HPV-16 VLPs expressed for example in yeast cells and transduced into different primary and established cells in culture and in vivo via receptor-mediated endocytosis, establishing a quantitative system to assess HPV-16 VLP infection, thus providing a safe, nonreplicative and improved delivery of therapeutic DNA vaccines to target cells. The enhanced delivery of DNA vaccine to professional APCs may be due to several reasons.
The capsid can protect the DNA from nuclease activity and may also act as an adjuvant. Additionally alpha-6-integrin has been proposed as the cell surface receptor for HPV and is highly expressed by DCs of the skin (Langerhans cells) and lymph nodes. Hence HPV pseudovirions may represent an ideal method to deliver therapeutic DNA vaccines to DCs to prime MHC-I-restrictedCD8+ cytotoxic T cells and MHC-II-restricted CD4+ helper T cells, and these are the most potent effector cells in anti-tumour immune responses. Pseudovirions may therefore act as ideal prophylactic and therapeutic vaccines.
In the rapidly expanding fields of immunology, molecular biology and vaccinology there is clearly an abundance of on-going research into delivering the most potent, effective and inexpensive prophylactic and therapeutic Human Papilloma Virus vaccines. Later this year, two or more licensed prophylactic HPV vaccines are likely to be approved. It is hopefully only a matter of time before the ideal therapeutic vaccine is produced capable of eradicating early HPV-associated malignant disease, as well as curing established advanced carcinomatous disease.
From the evidence on prophylactic vaccines, and the exclusion rate during screening for these trials, if these vaccines are approved, then deciding when and how to offer the vaccination and in which populations will introduce new challenges for public health programmes.
It is as yet unclear how these vaccines will benefit adults already infected with HPV, and specifically those coinfected with HIV. It they have a therapeutic role, then optimal time for successful vaccination is likely to be linked to CD4 count and possibly CD4 nadir.
- Fontaine et al. High levels of HPV-16 DNA are associated with high-grade cervical lesions in women at risk or infected with HIV. AIDS, 2005, 19 (8) 785-794.
- Munoz N et al. Current views on the epidemiology of HPV and cervical cancer. In Lacey C et al. Papilloma virus reviews: current research on papillomavirus. Leeds. Leeds University Press, 1996: 227-37
- Schiffman MH et al. Epidemiological evidence showing that human HPV infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 85, 958-64, 1993.
- Furomoto H et al. Human Papilloma Virus and cervical cancer. J Med. Invest. 49, 124-133, 2002
- Scheffner M et al. The HPV-16 E6 and E6-AP complex functions as an ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495-505, 1993
- Zimmerman H et al. The HPV-16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional co-activator CBP/p300. J Virol 73, 6209-6219, 1999
- Dyson N et al. The HPV-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science, 243, 934-937, 1989.
- Zhou J et al. Expression of vaccinia recombinant HPV16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV VLPs. Virology 185, 251-7, 1991
- Koutsky L A et al. A controlled trial of a human papillomavirus type 16 vaccine, NEJM, 347,21,1645-1651
- Harper D et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet, 2004, 364, 1757-1765.
- Borysiewicz L K et al. A recombinant vaccinia virus encoding HPV types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer. Lancet, 1996, 347,1523- 1527.
- Kaufmann AM et al. Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified HPV 16 and 18 E6 and E7 genes in women wit progressive cervical cancer. Clin Cancer Res. 2002,8, 3676-3685.
- Wu T C et al. Engineering an intracellular pathway for MHC class II presentation of antigens. Proc Natl Acad Sci USA, 1995, 92, 11671-11675.
- Pan ZX et al. Regression of established tumours in mice mediated by the oral administration of a recombinant Listeria Monocytogenes vaccine. Cancer Res, 1995, 55,4776-4779.
- Goldstone SE et al. Activity of HspE7, a novel immunotherapy in patients with anogenital warts. Dis Colon Rectum. 2002, 45, 502-507.
- Santin AD et al. Induction of HPV-specific CD4 (+) and CD8 (+) lymphocytes by E7-pulsed autologous dendritic cells in patients wit HPV-16 and 18 positive cervical cancer. J Virol. 1999, 73, 5402-5410.
- Santin AD et al. Vaccination with HPV-18 E7-pulsed dendritic cells in a patient with metastatic cervical cancer. NEJM, 2002, 346, 1752-1753.
- Nichols WW et al. Potential DNA vaccine integration into host cell genome. Ann NY Acad Sci, 1995, 772, 30-39.
- Schafer K et al. Immune response to HPV-16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumour protection. Int J Cancer, 1999, 81, 881-888.
- Nieland JD et al. Chimeric HPV VLPs induce a murine self-antigen-specific protective and therapeutic anti-tumour immune response. J Cell Biochem, 1999, 73, 145-152.
- Cella M et al. Origin, maturation and antigen presenting function of dendritic cells. Curr Opin Immunol, 1997, 9 10-16.
- Hart DN. Dendritic cells: Unique leucocyte populations, which control the primary immune response. Blood, 1997, 90, 3245-3287.
- Mayordomo Ji et al Bone marrow derived dendritic cells serve as potent adjuvants for peptide-based anti-tumour vaccines. Stem Cells, 1997, 15, 94-103.
- De Bruijn ML et al. Immunisation with HPV-16 oncoprotein-loaded dendritic cells as well as protein in adjuvant induces MHC class I-restricted protection to HPV-16 induced tumour cells. Cancer Res. 1998, 58, 724-731.
- Murakami M et al. Induction of specific CD8+ T-lymphocyte responses using a HPV-16 E6/E7 fusion protein and autologous dendritic cells. Cancer Res. 1999, 59 1184-1187.
- Arthur JF et al. A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther, 1997,4, 17-25.
- Chen CH et al. Experimental vaccine strategies for cancer immunotherapy. J Biomed Sci, 1998, 5, 231-252.
- Ying H et al. Cancer therapy using a self-replicating RNA vaccine. Nat Med, 1999, 5 823-827
- Berglund P et al. Enhancing immune responses using suicidal DNA vaccines. Nat Biotechnol, 1998, 16, 562-565.
- Wang TL et al. A LAMP-1 targeting strategy enhances the anti-tumour immunity of Semiliki Forest virus self-replicating RNA vaccines against E7-expressing murine tumours (manuscript in preparation)
- Hsu KF et al. Enhancement of suicidal DNA vaccine potency by linkage of antigen gene to an HSP70 gene (manuscript in preparation