Cervical Cancer: Beyond Today’s HPV Vaccine

Overview of Cervical Cancer and HPV

Cervical cancer was once the leading cause of death for women, but progress has shown how successful screening for early disease can be in reducing deaths from cancer. Thanks to the studies of George Papanicolaou aided by his assistant Andromache Mavroyenous (who was also his wife), vaginal cytology preparations now known as the Pap test have become part of many women’s routine gynecological examination.^1,2 Although the number of new cases diagnosed has fallen steadily since 1992, the death rate related to cervical cancer has remained fairly constant.³ Today, nearly a quarter million American women live with cervical cancer.³ In 2014, an estimated 12,360 women will be diagnosed with cervical cancer, and more than 4000 will die from it.³ The 5-year survival rate overall is nearly 68%; however, this varies widely by stage, from nearly 91% for localized cancer to 16.1% for metastasized disease.³ Cervical cancer is likely to affect women in their active reproductive years and immediately following; those aged 20 to 44 years account for 38.7% of cases. Women in perimenopausal and menopausal years (ages 45 - 64 years) account for 41.2% of cases.³ The median age at diagnosis is 49 years, and the median age at death is 57 years.³ Risk factors include early sexual activity, multiple sexual partners, history of sexually transmitted disease, and having a weakened immune system.³ Having a circumcised male partner decreases the risk of human papillomavirus (HPV) infection in the penis and thus reduces its spread to female partners.⁴

By the 1980s, it was noted that the DNA of certain types of HPV were strongly associated with cervical neoplasia.⁵ Today we know that the majority of cervical cancer cases are due to 2 types, HPV 16 and HPV 18.⁶ In one study across a large population of women, the type of HPV samples used for hybridization (a state-of-the-art technology used before the polymerase chain reaction was invented) depended to some extent on the geographic locale in which the patient lived. For example, HPV type 16 was more prevalent in samples from German women but not common in samples in Kenyan or Brazilian women.⁵ Furthermore, isolates not readily hybridizing with then known HPV types were noted; these may represent early detection of HPV 18, which had not been described at the time the paper was published.⁵ Studies of American women confirmed that previous HPV infection causes most cases of cervical cancer, and that HPV types 16 and 18 are the most prevalent.⁷ Indeed, previous infection with oncogenic types of HPV is so tightly linked to cervical cancer at all stages that statistical adjustment for HPV infections explains why sexual risk factors are associated with cervical cancer.⁸ It should be noted that whereas HPV is the causative agent for the vast majority of cervical cancer cases, there is evidence that previous infection with Chlamydia trachomatis may play a role in the development of the disease as well.⁹ One possible explanation could be that while HPV is the most common cause for cervical cancer, not every woman carrying HPV, even one of the more oncogenic types, will develop cervical cancer. It is possible that a secondary infection, such as with C trachomatis or some other source of chronic inflammation, induces malignant transformation.¹⁰

Currently, more than 90% of cervical cancer cases are diagnosed through Pap tests, HPV screening for oncogenic types, or a combination of both.¹¹ Treatment depends on staging and surgery, and ranges from cone bi­opsy to radical hysterectomy. Depending on the case, chemotherapy with cisplatin may be given; pelvic radiation therapy either as external beam radiation therapy or as vaginal brachytherapy is another option.¹²

The association of viruses with cancer dates back to 1908, when Ellerman and Bang reported the transmission of chicken leukemia by a filterable agent.¹³ In 1910, Rous described the first tumor that could spread by direct transplant of tumor cells or by a cell-free filtrate.¹⁴ At the time of Rous’s work, it was already known that human warts were contagious and could be transmitted by a filterable agent. By the 1970s, scientists hunted for a virus that might cause cervical cancer, possibly spreading from males to females. At first, herpes virus of some type was suspected,¹⁵ but this was dismissed when zur Hausen and colleagues failed to find herpes viral DNA in cervical cancer specimens. Instead, zur Hausen and colleagues consistently found papillomavirus in their specimens.⁵ By 1984, zur Hausen’s team had found papillomavirus types 16 and 18 in a preponderance of their specimens.¹⁶ Large epidemiologic studies followed zur Hausen’s reports confirming the role of HPV 16 and HPV 18 in the development of precancer lesions and cervical cancer.^15,17

Preventing cervical cancer is now the major focus for disease management through the use of approved vaccines for HPV. This review will discuss current HPV vaccines, and also will present the latest research for future cervical cancer immunotherapy that was presented at the 2014 American Society of Clinical Oncology annual meeting.

HPV Vaccines Today

Currently, 2 vaccines are available that are approved by the FDA and recommended by the Centers for Disease Control and Prevention (CDC): Cervarix, approved in October 2009, and Gardasil, approved in June 2006.^18,19 Both are given in 3 doses over a 6-month span of time.^20,21 Although cervical cancer is, by nature, a disease specific to women, male-transmitted HPV plays a role in spreading the disease. Circumcision, as mentioned previously, helps prevent the male-to-­female transfer of the virus,⁴ but circumcision is not universally practiced. Therefore, in 2011, the CDC changed the HPV vaccine recommendation from solely girls and women to include males aged 13 to 26 years.²² Multivalent vaccines for HPV have the potential to prevent the vast majority of cervical cancer cases worldwide; it is estimated that vaccinating for HPV types 16 and 18, the most frequent types found in cervical cancer specimens, would prevent 71% of all cervical cancer cases worldwide.⁶ If a vaccine could be created to prevent HPV infection caused by the 7 most common oncogenic types, then possibly 87% of all cases worldwide could be prevented.⁶ Although not a focus of this review, it should be noted that HPV is also the cause of oropharyngeal cancer and that HPV vaccination prevents this disease as well.¹⁵

Gardasil is also known as Human Papillomavirus Quadrivalent (Types 6, 11, 16, and 18) Vaccine, Recombinant.²⁰ As the name discloses, it is a quadrivalent vaccine for HPV types 6, 11, 16, and 18, with virus-like particles (VLPs) serving as immunogens.²³ Multiprotein structures, VLPs mimic viruses sufficiently to be quite immunogenic. Formation of VLPs by the immunogenic L1 and L2 surface (capsid) proteins is a feature of HPV, and important for vaccine production against an oncogenic virus.¹⁵ VLPs self-assemble and elicit strong T-cell and B-cell responses while being noninfectious.¹⁵

Because the vaccines lack viral genomes, there is no potential to cause disease or transformation.²⁴ Immunization with the L1 VLPs used for this vaccine is longlasting; a study of 552 women showed that serum antibodies stabilized 5 years postimmunization.²⁵ Moreover, the vaccine was effective in preventing not only squamous cell carcinoma (the most common form of cervical cancer), but also cervical intraepithelial neoplasia (CIN) and adenocarcinoma in situ.²⁶ CIN grade 2+ is the accepted surrogate end point for licensing HPV vaccines.²⁷ As for types 6 and 11, they are classified as low risk, meaning they are nononcogenic or have very low risk of inducing malignant transformation. Instead, they cause low-grade abnormalities (dysplasias) of cervical cells, as well as condylomata acuminata and anogenital warts.^28,29

Long-term follow-up studies showed that Gardasil provides safe and effective prophylaxis from infection with HPV types 6, 11, 16, and 18.³⁰ Six years postimmunization, among more than 1300 women aged 24 to 45 years included in the study, there were no cases of CIN or external genital lesions noted.³⁰ A large Canadian study of more than 10,000 women revealed that vaccination is most effective prior to HPV exposure and in women with normal cytology.³¹

Cervarix is the bivalent vaccine, also called Human Papillomavirus Bivalent (Types 16 and 18) Vaccine, Recombinant.²¹ Like Gardasil, it is composed of L1 VLPs.²¹ A study of more than 4600 young women conducted across England showed that immunization with the bivalent vaccine successfully prevented infection with HPV types 16 and 18, with some herd immunity benefits noted.³² The phase 3 PATRICIA study confirmed that Cervarix prevented CIN grades 1+ and 2+ (efficacies were 94.6% and 97.7%, respectively); the vaccine efficacy was more than 90% against 6-month persistent infections. The vaccine had no effect on HPV infections present at the time of immunization, as expected.³³

Head-to-head studies comparing the 2 vaccines in young adult women showed that seroconversion (titers of >1:320) were reached by day 95 postimmunization.³⁴ In adolescent girls, serum neutralizing antibody responses were somewhat more robust following Cervarix than following Gardasil.³⁵ In this randomized trial, 100% of immunized patients had strong neutralizing antibody responses regardless of which vaccine they received. Titers peaked 7 months postimmunization and were stable a year later.³⁵ Because infection with human immunodeficiency virus (HIV) is a risk factor for cervical cancer, a study was done to examine immunogenicity of both vaccines. In this population of adults, both vaccines were immunogenic, although Cervarix induced somewhat more vigorous responses among women. Among men, the responses were equivalent.³⁶ A meta-analysis of clinical trial data available from several databases demonstrated that the slightly superior immunogenicity of the bivalent vaccine is not statistically different from that of the quadrivalent vaccine.³⁷ End-of-study analyses of the phase 3 trials showed that both vaccines have comparable and excellent efficacy against HPV infection.³⁸ Although both the quadrivalent and bivalent vaccines have superb safety profiles, the bivalent vaccine caused adverse events somewhat more frequently, possibly due to the larger number of clinical trials conducted for it.³⁹ The most common adverse events noted for both vaccines were local pain, redness and swelling, and also fever, none of which was serious.^20,21,30,39 Both vaccines were well-tolerated by HIV-infected adults.³⁶

Further studies revealed the extent to which both vaccines induce cross-neutralizing antibodies. HPV types genetically related to type 16 comprise the Alpha-9 group: types 31, 33, 35, 52, and 58. The Alpha-7 group comprises those that are HPV type 18–like: types 39, 45, 59, and 68. Cervarix induced a range of cross-neutralizing antibodies against the Alpha-9 group. This study did not look for cross-neutralizing antibodies against the Alpha-7 group.⁴⁰ Both Gardasil and Cervarix induced antibodies against HPV types 31, 33, and 45 in HIV-positive adults⁴¹; in young females, Cervarix induced higher titers against types 31 and 45 than did Gardasil, possibly due to different age ranges in the 2 cohorts.³⁵ A meta-­analysis of trials from Medline and Embase databases noted substantial heterogeneity with regard to vaccine efficacy for the bivalent vaccine against persistent infections with HPV type 31 or 45.³⁷ The efficacy of Cervarix against HPV types 31 and 45, moreover, seemed to decrease on follow-up, although initial responses appeared more vigorous than those resulting from Gardasil. The authors speculate that some of the differences between the vaccines may be due to trial design.³⁷

Future Immunotherapy for Cervical Cancer

Although the efficacies of the 2 approved vaccines is quite impressive, together they prevent only about 70% of cervical cancer cases worldwide.⁶ Thus, there is a need for improved vaccines and other new approaches to increase prevention tactics. One approach to improving outcomes is to create a vaccine that would clear HPV types 16 and 18 before cancer develops but after infection occurs. Neither Gardasil nor Cervarix is effective in clearing HPV once infection takes hold, and neither is effective once cervical dysplasia is noted.^20,21 A vaccine that could clear HPV and, in essence, reverse cervical dysplasia before it develops into cancer could bridge the current gap. To this end, methods to induce immunity by introducing viral antigens into patients’ cells are being developed to create a novel HPV vaccine. In this approach, 2 major HPV-expressed antigenic proteins, E6 and E7, were identified.⁴² In an early prototype vaccine, a protein created by fusing the genes for E6 and E7 was 5 times more potent in driving specific anti-HPV immune responses, and experimental results showed that the prototype vaccine gave 100% protection against HPV.⁴³ Further experiments showed that immunization with this prototype vaccine helped overcome immune tolerance, resulting in antitumor responses.⁴³ The prototype vaccine was refined further to increase immunogenicity against HPV types 6 and 11.⁴⁴ Experiments confirmed that immunization with these vectors elicited strong T-cell responses, as measured by interferon gamma levels.⁴⁴ It should be noted that this HPV vaccine is not composed of VLPs, but is a synthetic vaccine that is meant to be administered by electroporation into patients’ cells, so that the patients’ cells express the viral antigens efficiently and therefore increase the vaccine’s immunogenicity up to 100-fold over bare DNA alone.⁴⁵ This synthetic vaccine, now in clinical trials, is called VGX-3100.⁴⁶ In a phase 1 trial in which 18 women enrolled who were previously treated for cervical dysplasia or cervical intracellular neoplasia, no grade 3 adverse events were ascribed to VGX-3100, and all patients completed the study protocol.⁴⁶ VGX-3100 induced strong and lasting antibody responses, as well as robust HPV-specific helper T-cell type 1 responses.⁴⁶ Moreover, VGX-3100 induced antigen-specific cytotoxic CD8+ T cells.⁴⁶ Thirteen of the original 18 women enrolled in a follow-up phase 1 trial that entailed a booster administration of VGX-3100; these results were presented at the recent ASCO meeting.⁴⁷ Following the booster dose, the women exhibited increased immunologic reactivity by several determinants, including cytokine quantifications and T-cell phenotyping by flow cytometry. Furthermore, increased Fas ligand and activation marker CD137 expression were noted on CD8+ T cells after the booster dose.⁴⁷ These results show promise for VGX-3100, prompting the advancement to a phase 2 trial. The trial information can be found at www.clinicaltrials.gov as NCT01188850 for phase 1 and NCT01304524 for phase 2 trials.

Beyond targeting E6 and E7, an additional approach to improving HPV vaccines is to target cellular p16^Ink4A, a protein strongly overexpressed in HPV-associated cancers and necessary for the survival of cervical carcinoma cells.⁴⁸ The p16^Ink4A tumor suppressor normally activates the RB tumor suppressor, but in cancer cells, it is dysregulated and no longer inhibits D-type cyclin-dependent kinases.⁴⁹ In specimens taken from cone biopsies, the extent of p16^Ink4A expression roughly correlated with the severity of the dysplasia going from low-grade CIN to carcinoma.⁵⁰ In phase 1/2 trials, 26 patients with HPV-associated cancers (not limited to cervical cancer) were enrolled. The vaccine consisted of a p16^Ink4A peptide plus a proprietary adjuvant called Montanide ISA-51 VG.^51,52 In the phase 1 trial, no toxicity was observed during or after immunization among the 10 patients enrolled.⁵³ Anti-p16^Ink4A immune responses were noted in the patients following immunization; these consisted of both antibodies and T-cell responses.⁵³ One patient with lung metastases due to head and neck carcinoma completed the study protocol, maintaining stable disease for 18 months.⁵³ Importantly, no autoimmune symptoms were noted.⁵³ Therefore, the trial has advanced to phase 2a; its clinical trial information can be found at www.clinicaltrials.gov as NCT01462838.⁵³

Another future approach to immunotherapy for cervical cancer is to design vaccines that incorporate L2 capsid proteins into VLPs. Although less immunogenic than L1 capsid proteins, L2 capsid proteins have immunogenic epitopes that are highly conserved across HPV types and thus may comprise vaccines of broader-type specificity.⁵⁴ In addition, L2 VLP-based vaccines may be lower in cost than prevailing L1 VLP-based vaccines, and may prevent cancers caused by types rarer in the developed world but more prevalent in developing nations.⁵⁵ Finally, these vaccines will be delivered intranasally, obviating much of the logistical problems possible through intramuscular delivery.⁵⁵ To this end, efforts are under way to maximize the immunogenicity of multi­meric HPV L2-based vaccines⁵⁶ and to create L1/L2 chi vaccines.⁵⁷

At the 2014 ASCO annual meeting, a description of adoptive cell transfer (ACT) of tumor-infiltrating T cells using T cells recognizing 2 HPV epitopes was presented. The report presented the patient cohort with cervical cancer. Three of 6 patients having HPV reactivity reported objective tumor regression characteristics specified by RECIST (response evaluation criteria in solid tumors); 1 was a partial response and 2 were complete responses.⁵⁸ Two complete responses were in patients with widespread metastases; these responses were durable, lasting 18 months.⁵⁸ This ACT protocol is still under investigation; the phase 2 trial information can be found as NCT01585428 at www.clinicaltrials.gov/show/NCT01585428.

Using a virus to fight cancer is a novel approach, and one such approach was described at ASCO for other types of cancer. Here, investigators designed an oncolytic herpesvirus vector carrying the immune-stimulating cytokine interleukin-12 (IL-12), compared with the same vector without IL-12.⁵⁹ Adding IL-12 to the oncolytic herpesvirus vector (identified as an armed vector by the authors) enhanced antitumor activity in glioblastoma models, compared with the unarmed vector; the IL-12–modified vector prolonged survival, inhibited angiogenesis, and effected tumor regression in vivo, especially when another vector armed with angiostatin was added.⁶⁰ Activity of the vectors was assessed in vitro and in vivo. In a cervical cancer model, both vectors were highly cytotoxic and both had antitumor effects in vivo.⁵⁹ Mice given the herpesvirus vector had an increased infiltrate of CD8+ cells in their spleen, compared with control mice receiving no vector.⁵⁹ These results are very preliminary; however, this approach is one that might be applied to other types of cancer and for which new results are expected in the near future.

Conclusion

In light of the above discussion, current HPV vaccines have room for improvement in order to prevent cervical cancer more effectively. New vaccines that can be given after HPV infection would protect even more women, and perhaps even help treat the disease. The research presented at the recent ASCO annual meeting demonstrates some exciting possibilities for immunotherapy looking forward in the treatment and prevention of cervical cancer.

References

1. Papanicolaou GN. A new procedure for staining vaginal smears. Science. 1942; 95:438-9.
   
2. Vilos GA. The history of the papanicolaou smear and the odyssey of George and Andromache Papanicolaou. Obstet Gynecol. 1998;91:479-483.
   
3. Howlader N, Noone AM, Krapcho M, et al, eds. Cervix Uteri Cancer, SEER Cancer Statistics Review, 1975-2011. Bethesda, MD: National Cancer Institute; 2014. [27 June 2014]. Accessed http://seer.cancer.gov/csr/1975_2011/results_merged/sect_ 05_cervix_uteri.pdf.
   
4. Castellsagué X, Bosch FX, Muñoz N, et al. Male circumcision, penile human papillomavirus infection, and cervical cancer in female partners. N Engl J Med. 2002; 346:1105-1112.
   
5. Dürst M, Gissmann L, Ikenberg H, et al. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad Sci USA. 1983;80:3812-3815.
   
6. Muñoz N, Bosch FX, Castellsagué X, et al. Against which human papillomavirus types shall we vaccinate and screen? the international perspective. Int J Cancer. 2004; 111:278-285.
   
7. Schiffman MH, Bauer HM, Hoover RN, et al. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst. 1993;85:958-964.
   
8. Schiffman MH, Castle P. Epidemiologic studies of a necessary causal risk factor: human papillomavirus infection and cervical neoplasia. J Natl Cancer Inst. 2003;95:E2.
   
9. Anttila T, Saikku P, Koskela P, et al. Serotypes of Chlamydia trachomatis and risk for development of cervical squamous cell carcinoma. JAMA. 2001;285:47-51.
   
10. Samoff E, Koumans EH, Markowitz LE, et al. Association of Chlamydia trachomatis with persistence of high-risk types of human papillomavirus in a cohort of female adolescents. Am J Epidemiol. 2005;162:668-675.
    
11. National Cancer Institute website. Cervical Cancer Treatment: PDQ. Last modified August 15, 2014. http://www.cancer.gov/cancertopics/pdq/treatment/cervical/HealthProfessional/page4. Accessed June 27, 2014.
    
12. Koh WJ, Greer BE, Abu-Rustum NR, et al. Cervical cancer. J Natl Compr Canc Netw. 2013;11:320-343. hnern.
    
13. Ellerman V, Bang O. Experimentelle Leukämie bei Hühnern. Centralblt J Bakt Abt I. 1908;46:595-609.
    
14. Rous P. A transmissible avian neoplasm. (sarcoma of the common fowl.). J Exp Med. 1910;12:696-705.
    
15. Palmer AK, Harris AL, Jacobson RM. Human papillomavirus vaccination: a case study in translational science. Clin Transl Sci. Epub May 19, 2014.
    
16. Boshart M, Gissmann L, Ikenberg H, et al. A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 1984;3:1151-1157.
    
17. Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) study group. J Natl Cancer Inst. 1995;87:796-802.
    
18. Food and Drug Administration. Cervarix. 2013. www.fda.gov/biologicsblood vaccines/vaccines/approvedproducts/ucm186957.htm. Accessed June 27, 2014.
    
19. Food and Drug Administration. Gardasil. 2014.www.fda.gov/biologicsblood vaccines/vaccines/approvedproducts/ucm094042.htm. Accessed June 27, 2014.
    
20. Gardasil (Human Papillomavirus Quadrivalent (Types 6, 11, 16, 18) Vaccine, Recombinant) [package insert]. Whitehouse Station, NJ: Merck & Co, Inc; June 2014.
    
21. Cervarix (Human Papillomavirus Types 6, 11, 16, 18 Vaccine) [package insert]. Philadelphia, PA: GlaxoSmithKline; 2011.
    
22. Centers for Disease Control and Prevention. Recommendations on the use of quadrivalent human papillomavirus vaccine in males—Advisory Committee on Immunization Practices (ACIP), 2011. MMWR Morb Mortal Wkly Rep. 2011;60:1705-1708.
    
23. Villa LL, Ault KA, Giuliano AR, et al. Immunologic responses following administration of a vaccine targeting human papillomavirus Types 6, 11, 16, and 18. Vaccine. 2006;24:5571-5583.
    
24. Roldão A, Mellado MCM, Castilho LR, et al. Virus-like particles in vaccine development. Expert Rev Vaccines. 2010;9:1149-1176.
    
25. Olsson SE, Villa LL, Costa RLR, et al. Induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (HPV) types 6/11/16/18 L1 virus-like particle (VLP) vaccine. Vaccine. 2007;25:4931-4939.
    
26. Ault KA. Effect of prophylactic human papillomavirus L1 virus-like-particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: a combined analysis of four randomised clinical trials. Lancet. 2007; 369:1861-1868.
    
27. Lehtinen M, Paavonen J, Wheeler CM, et al. Overall efficacy of HPV-16/18 AS04-adjuvanted vaccine against grade 3 or greater cervical intraepithelial neoplasia: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lancet Oncol. 2012;13:89-99.
    
28. Gissmann L, Boshart M, Durst M, et al. Presence of human papillomavirus in genital tumors. J Invest Dermatol. 1984;83:26s-28s.
    
29. Hariri L, Dunne E, Saraiya M, et al. Chapter 5: Human papillomavirus (HPV). Manual for the Surveillance of Vaccine-Preventable Diseases. Centers for Disease Control and Prevention. Atlanta, GA: 2011.
    
30. Luna J, Plata M, Gonzalez M, et al. Long-term follow-up observation of the safety, immunogenicity, and effectiveness of Gardasil in adult women. PloS One. 2013; 8:e83431.
    
31. Mahmud SM, Kliewer EV, Lambert P, et al. Effectiveness of the quadrivalent human papillomavirus vaccine against cervical dysplasia in Manitoba, Canada. J Clin Oncol. 2014;32:438-443.
    
32. Mesher D, Soldan K, Howell-Jones R, et al. Reduction in HPV 16/18 prevalence in sexually active young women following the introduction of HPV immunisation in England. Vaccine. 2013;32:26-32.
    
33. Szarewski A, Poppe WAJ, Skinner SR, et al. Efficacy of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine in women aged 15–25 years with and without serological evidence of previous exposure to HPV-16/18. Int J Cancer. 2012; 131:106-116.
    
34. Nelson EAS, Lam HS, Choi KC, et al. A pilot randomized study to assess immunogenicity, reactogenicity, safety and tolerability of two human papillomavirus vaccines administered intramuscularly and intradermally to females aged 18–26 years. Vaccine. 2013;31:3452-3460.
    
35. Draper E, Bissett SL, Howell-Jones R, et al. A randomized, observer-blinded immunogenicity trial of Cervarix and Gardasil human papillomavirus vaccines in 12-15 year old girls. PloS One. 2013;8:e61825.
    
36. Toft L, Storgaard M, Müller M, et al. Comparison of the immunogenicity and reactogenicity of cervarix and gardasil human papillomavirus vaccines in HIV-infected adults: a randomized, double-blind clinical trial. J Infect Dis. 2014;209:1165-1173.
    
37. Malagon T, Drolet M, Boily M-C, et al. Cross-protective efficacy of two human papillomavirus vaccines: a systematic review and meta-analysis. Lancet Infect Dis. 2012; 12:781-789.
    
38. Schiller JT, Castellsagué X, Garland SM. A review of clinical trials of human papillomavirus prophylactic vaccines. Vaccine. 2012;30:F123-F138.
    
39. Gonçalves AK, Cobucci RN, Rodrigues HM, et al. Safety, tolerability and side effects of human papillomavirus vaccines: a systematic quantitative review. Braz J Infect Dis. 2014. doi: 10.1016/j.bjid.2014.02.005.
    
40. Bissett SL, Draper E, Myers RE, et al. Cross-neutralizing antibodies elicited by the cervarix human papillomavirus vaccine display a range of alpha-9 inter-type specificities. Vaccine. 2014;32:1139-1146.
    
41. Toft L, Tolstrup M, Müller M, et al. Comparison of the immunogenicity of cervarix and gardasil human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults. Hum Vaccin Immunother. 2014;10:1147-1154.
    
42. Yan J, Harris K, Khan AS, et al. Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein in mice and rhesus macaques. Vaccine. 2008;26:5210-5215.
    
43. Yan J, Reichenbach DK, Corbitt N, et al. Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen. Vaccine. 2009;27:431-440.
    
44. Shin T, Pankhong P, Yan J, et al. Induction of robust cellular immunity against HPV6 and HPV11 in mice by DNA vaccine encoding for E6/E7 antigen. Hum Vaccin Immunother. 2012;8:470-478.
    
45. Sardesai NY, Weiner DB. Electroporation delivery of DNA vaccines: prospects for success. Curr Opin Immunol. 2011;23:421-429. Epub May 3, 2011.
    
46. Bagarazzi ML, Yan J, Morrow MP, et al. Immunotherapy against HPV16/18 generates potent TH1 and cytotoxic cellular immune responses. Sci Transl Med. 2012;4:155ra38.
    
47. Morrow MP, Kraynyak K, Shen X, et al. Boosting of cellular and humoral immune responses to HPV16/18 antigens by VGX-3100: a follow-on phase I trial. J Clin Oncol. 2014;32:Abstract 3101.
    
48. McLaughlin-Drubin ME, Park D, Munger K. Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines. Proc Natl Acad Sci U S A. 2013;110:16175-16180.
    
49. Ohtani N, Yamakoshi K, Takahashi A, Hara E. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression. J Med Invest. 2004; 51:146-153.
    
50. Reuschenbach M, Seiz M, von Knebel Doeberitz C, et al. Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells. Int J Cancer. 2012;130:388-394.
    
51. Bonhoure F, Gaucheron J. Montanide ISA 51 VG as adjuvant for human vaccines. J Immunother. 2006;29:647-648.
    
52. Rosenberg SA, Yang JC, Kammula US, et al. Different adjuvanticity of incomplete freund’s adjuvant derived from beef or vegetable components in melanoma patients immunized with a peptide vaccine. J Immunother. 2010;33:626-629.
    
53. Reuschenbach M, Rafiyan M-R, Karbach J, et al. Phase I/IIa study of therapeutic p16INK4a vaccination in patients with HPV-associated cancers. J Clin Oncol. 2014;32: Abstract 3097.
    
54. Tyler M, Tumban E, Chackerian B. Second-generation prophylactic HPV vaccines: successes and challenges. Expert Rev Vaccines. 2014;13:247-255.
    
55. Karanam B, Jagu S, Huh WK, et al. Developing vaccines against minor capsid antigen L2 to prevent papillomavirus infection. Immunol Cell Biol. 2009;87:287-299.
    
56. Jagu S, Kwak K, Karanam B, et al. Optimization of multimeric human papillomavirus L2 vaccines. PloS One. 2013;8:e55538.
    
57. McGrath M, de Villiers GK, Shephard E, et al. Development of human papillomavirus chimaeric L1/L2 candidate vaccines. Arch Virol. 2013;158:2079-2088.
    
58. Hinrichs CS, Stevanovic S, Draper L, et al. HPV-targeted tumor-infiltrating lymphocytes for cervical cancer. J Clin Oncol. 2014;32:Abstract LBA3008.
    
59. Kagabu M, Miura Y, Saito T, et al. Impact of new oncolytic herpes simplex virus vector armed with interleukine-12 for cervical cancer therapy. J Clin Oncol. 2014;32: Abstract 3102.
    
60. Zhang W, Fulci G, Wakimoto H, et al. Combination of oncolytic herpes simplex viruses armed with angiostatin and IL-12 enhances antitumor efficacy in human glioblastoma models. Neoplasia. 2013;15:591-599.