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Received: 10/03^th 2015

Accepted: May 15^th 2015

Immune response in cervical cancer. Strategies for the development of therapeutic vaccines

María de Lourdes Mora-García,^a Alberto Monroy-García^b

^aLaboratorio de Inmunobiología, Unidad de Investigación en Diferenciación Celular y Cáncer, Unidad Multidisciplinaria de Investigación Experimental Zaragoza (UMIEZ), Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México

^bLaboratorio de Inmunología y Cáncer, Unidad de Investigación Médica en Enfermedades Oncológicas, Hospital de Oncología, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social

Distrito Federal, México

Communication with: María de Lourdes Mora-García

Email: mogl@servidor.unam.mx

High-risk human papillomaviruses (HR-HPV), as HPV-16, evade immune recognition through the inactivation of cells of the innate immune response. HPV-16 E6 and E7 genes down-regulate type I interferon response. They do not produce viremia or cell death; therefore, they do not cause inflammation or damage signal that alerts the immune system. Virus-like particles (VLPs), consisting of structural proteins (L1 and L2) of the main HR-HPV types that infect the genitourinary tract, are the most effective prophylactic vaccines against HR-HPV infection. While for the high grade neoplastic lesions, therapeutic vaccines based on viral vectors, peptides, DNA or complete HR-HPV E6 and E7 proteins as antigens, have had limited effectiveness. Chimeric virus-like particles (cVLPs) that carry immunogenic peptides derived from E6 and E7 viral proteins, capable to induce activation of specific cytotoxic T lymphocytes, emerge as an important alternative to provide prophylactic and therapeutic activity against HR-HPV infection and cervical cancer.

Keywords: Cervical cancer, Human papillomavirus, Immune response antigens, Vaccines.

Infection with h

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Human papillomavirus (HPV) in the genitals is one of the sexually transmitted diseases that occurs most commonly in the population. In 2008, it was estimated that 610,000 (4.8%) of the 12.7 million cancer cases reported worldwide were attributable to HPV infection, of which 530,000 cases were cervical cancer (CC), and the remaining were cancer of the penis, vulva, vagina, and oropharynx.1 CC is third in incidence and fourth in cancer mortality in women worldwide (more than 270,000 deaths per year). It is the most common cancer in developing countries, where 85% of cases occur (in Mexico CC ranks second in incidence and mortality from cancer), which represents a public health problem in these countries due the huge economic burden related to health care costs.¹ The presence of HPV in virtually all (99.7%) carcinomas of the uterine cervix suggests that this virus is a requirement for the development of this malignancy.² In the last two decades, the study of the immune response to the structural and nonstructural proteins of these viruses has created a model for the development of prophylactic and therapeutic vaccines to prevent and avoid the development of cancer associated with HPV infection.   

HPV and cervical cancer

HPVs are a family of over 100 different viral genotypes made of double-stranded DNA. About 40 types of HPV can infect the genitourinary tract and, based on the spectrum of lesions that they produce, they are divided into low-risk HPV (LR-HPV) and high-risk HPV (HR-HPV).³ HPV- 6 and HPV-11, belonging to the LR-HPV, produce more than 90% of genital warts and respiratory papillomas; they are also found in a large number of low-grade lesions: cervical intraepithelial lesions of the anus (AIN), penis (PIN), vagina (VAIN), and vulva (VIN); while the HR-HPV group, including HPV types -16, -18, -31, -33, -45, -52, and -58, among others, is associated with the development of anogenital cancers including cervical, vaginal, head and neck, penile, and anal cancers. In the case of cervical cancer, more than 99% of tumors present HR-HPV infection, and HPV-16 is found in about 50% of cases.² 

The HPV genome contains open reading frames that may result in eight different proteins: six early transcription proteins (E1, E2, E4, E5, E6, and E7) and two late transcription proteins (L1 and L2). E1 and E2 early transcription proteins regulate DNA and viral RNA replication; whereas E4, E5, E6, and E7 proteins are involved in cell cycle control and in cellular transformation.⁴ The L1 and L2 proteins are associated in a ratio of 30:1 to form the structure of the viral envelope (capsid) surrounding the double-stranded circular DNA, thus forming infectious viral particles (virions).⁵ The entire structure of the virion is icosahedral, with a diameter of 50-60 nm, and contains a total of 72 L1 protein subunits (60 hexameric and 12 pentameric); while the L2 protein is an internal protein that helps the stability of the particle. HPV has as infection target the mucous and epithelial cells, where it carries out its replication. Infectious particles enter baseline or germ cells of stratified epithelium through a lesion or microtrauma. Alpha-6/beta-4 integrin has been proposed as the membrane receptor to bind to the virus and to the heparan sulfate proteoglycans present in the plasma membrane, to permit entry of the virus into germinal cells.6 Within the cells, the viral genome is maintained in episomal state (complete DNA) in low copy number (10 to 200 copies); initially the E1 and E2 proteins are expressed to facilitate proper separation of genomes during cell division. The initial infection is followed by a proliferative phase leading to increased number of basal cells containing the viral genome (Figure 1), which may require the expression of E6 and E7 proteins, which stimulate the progress of the cell cycle from phase G1 to S. For infectious virions to be produced, the papillomavirus must amplify its genome and package it in the protein particle.⁶ This occurs in the upper layers of the epithelium, in the stratum spinosum, where the transcriptional activity of proteins involved in viral DNA replication increases, such as E1, E2, E4, and E5, as well as the constituents of the capsid, L1 and L2. The assembly of viral particles occurs in the granular layer of the epithelium and eventually infected cells are shed from its uppermost layer. The infectious process and amplification of viral DNA can occur between six and twelve weeks, while malignant lesions develop in two years or more (Figure 1).⁶

Immune response to HPV

Host defense against viral infection is a collaboration between innate immunity (phagocytes, soluble proteins, and epithelial barriers) and adaptive immunity (antibodies, helper and cytotoxic cells). The innate immune system through macrophage, Langerhans cells, and dendritic cells detects the virus particles and acts as a first line of defense, eliminating most of the intrusion; it has no specific memory, but is responsible for activating the adaptive immunity, which through exquisitely specific cellular and humoral effector mechanisms towards foreign antigens can generate long-lived memory cells. In the case of HPV infection, this pathogen has developed mechanisms to evade the host’s immune response (Figure 1). The virus’ infection cycle occurs on its own (it is a means of evading the immune system), because HPV is a purely intraepithelial pathogen; it does not have a bloodborne or viremia phase in its life cycle, and only trace amounts of virus are exposed to immune defenses.⁷ In addition, there is no cytopathic death or cytolysis from virus replication; therefore, there is no inflammation, and during most of the HPV infection cycle, there appears to be little or no release of proinflammatory cytokines important for activation and migration of antigen presenting cells (APC) in the local microenvironment.⁷ For example, it is known that type I interferons, especially IFN-alpha and IFN-beta, have antiviral, antiproliferative, and antiangiogenic activity, besides participating in the activation of adaptive response by innate immunity.⁸ HR-HPV has mechanisms that inhibit the synthesis of interferon and intracellular signaling pathways; for example, E6 and E7 HPV-16 proteins may interact directly with components of the interferon signaling pathways, which favors the evasion of the immune response (Figure 1).⁹

Despite the best efforts of the virus to evade host defenses, at least 80 to 90% of genital HPV infections resolve with the passage of time.¹⁰  

Anogenital warts and low-grade intraepithelial neoplasia (CIN-1) revert due to the immune response mediated by cells, which is directed against early transcription viral proteins, especially against E2 and E6 proteins.^11,12 This process is accompanied by a massive infiltration of mononuclear cells (CD4+, CD8+, CD56, and macrophages) in the lesion and the expression of Th1 cytokines.¹³ In a prospective histological study over 12 months on CIN-1 lesions, regression observed during the study period was strongly correlated with the presence of cytotoxic cells (granzyme B+ CD8+ and granzyme B+ CD56+) and CD8+ T lymphocytes expressing alpha-4/beta-7 integrin in koilocytic cervical lesions and CIN-1 lesions, but they are absent or present in reduced numbers in high-grade CIN lesions (CIN-2/3).¹³ However, despite this intense local response, systemic responses of antigen-specific T cells are weak and often fleeting. The cellular immune response against HPV is closely followed by seroconversion and antibody production, specifically towards the L1 protein of the viral capsid.¹⁴ Antibody titers following natural HPV infection are low, and it has been found for the most-studied papillomaviruses (HPV-6, -11, -16, and -18) average seroconversion times exceed six months; it appears that between 30 and 50% of patients with evidence of persistent genital HPV infection never acquire antibodies, which may be associated with a state of progression of the disease.¹⁵ In CC, seropositivity for HR-HPV is generally reported between 30 and 50% of patients.¹⁶ Moreover, humoral immune responses against E6 and E7 proteins are considered a tumor progression marker.¹⁷ This is because integration of E6 and E7 oncogenes of the HR-HPV into the host cell causes genomic instability, which can lead to malignant progression.¹⁷ In the infected epithelium and underlying microvascular endothelium, the expression of the major cytokines, adhesion molecules, chemokines, and chemokine receptors is deregulated. Therefore, even if specific cytotoxic T lymphocytes against HPV have been generated, their penetration into the epithelium is poor, and regulatory T cells increasingly dominate the lesions and suppress the killing effector response.¹⁸

Vaccines against HPV infection

In 1991 the Zhou and Frazer research group¹⁹ for the first time succeeded in expressing the protein L1 (the main protein of HPV capsid) in eukaryotic cells and with this the researchers showed that these proteins could self-assemble into virus-like particles (VLP), which marked the basis for developing effective first-generation vaccines to prevent HPV infection. In fact, commercial prophylactic vaccines that include VLP of HPV types -16 and -18 (Glaxo SK) and HPV-6, -11, -16 and -18 (Gardasil, Merck Co.) generate high titers of neutralizing antibodies with specificity to the L1 protein (between 100 and 1000 times higher than those found in natural infections) and a robust immune memory.²⁰  

Current clinical trials have shown that these vaccines provide sustained protection against grade I intraepithelial neoplasia in the cervix (CIN-1), vulva (VIN) and vagina (VAIN), as well as against condyloma lesions attributable to various types of HPV (-6, -11, -16, and -18) in over 90% of cases, and a significant reduction in the incidence of these diseases. Furthermore, although the immunogenic capacity of first-generation VLPs is usually specific, currently prophylactic second-generation vaccines are being generated that include in their composition sequences of the structural protein L2, which are conserved between the different HPV genotypes, which will result in the generation of more effective polyvalent prophylactic vaccines.²¹ However, the efficacy of prophylactic vaccines against lesions with actual presence of HPV-16 or HPV-18 DNA is poor.²² Therefore, the development of therapeutic vaccines designed to induce a cellular immune response becomes a priority, particularly against E6 and E7 oncoproteins, as these are expressed predominantly in the malignant cells and also are important for maintaining the transformed state. Consequently, in recent years therapeutic vaccines have been generated that include the E6 and E7 proteins as total antigens or in peptides, either associated with recombinant viral vectors and activated dendritic cells, or containing nucleic acids encoding for these proteins (Table I).²³    

Evaluations of these vaccines in healthy people or patients with cervical cancer, CIN, VIN, VAIN, or perianal lesions, as well as people affected by genital warts and laryngeal papillomas, have shown safety, feasibility, tolerability, and immunogenicity.²³ However, despite optimistic preclinical data, evidence of therapeutic benefit of responses induced by T lymphocytes in humans has been limited, probably because the first clinical trials were performed with patients in the end stages of the disease who were immunocompromised both by the disease and their previous treatment. Moreover, there are promising results in cases with less advanced intraepithelial lesions (CIN, VIN, VAIN); however, neither study was designed to show a statistically significant difference between the vaccinated group and the placebo, in addition to a weak correlation between clinical response and immune response in patients when T lymphocytes from peripheral blood or cytokines were analyzed.²⁴

Recently a strategy has been reported based on the construction of chimeric VLPs (cVLPs) merging the L1 and L2 structural proteins with antigenic sequences of the E6 and E7 proteins, with which a more complete particle is achieved in terms of antigenic equivalence with virions.²⁵ In a first trial, cVLP were applied for the treatment of patients infected with HPV-16 who had high grade CIN2/3.²⁶ The treatment consisted of 2 parenteral doses of 75 or 250 mg, in volunteer patients who were evaluated for shrinkage of their lesions, degree of CIN, and loss of viral DNA. Vaccination resulted in an increase in the concentration of antibodies against L1 up to 100 times greater than normal titers in the placebo group. Stimulation of cellular immune response against the two proteins was also observed.26 This treatment was safe and therefore set the pattern for the generation of other strategies in the design and development of cVLP. 

Following this strategy, our research group has generated cVLP using plants as biofactories producing antigenic proteins.²⁷ The cVLPs generated consist of the structural protein L1 and immunogenic peptides of E6 and E7 HPV-16 proteins capable of stimulating cytotoxic T lymphocytes.²⁸ Mice immunized with these particles produced IgG antibodies, which persisted for more than one year and were able to specifically recognize particles constituted of HPV-16 protein L1 and with strong neutralizing activity.²⁹ Immunization with these cVLPs avoided tumor growth in previously immunized mice and inhibited between 50 and 70% of tumor growth when mice were immunized simultaneously or after tumor challenge.²⁹ These results suggest that cVLP generated in plants may be a viable option for producing prophylactic and therapeutic vaccines for the treatment of infections and lesions caused by HR-HPV. Therefore, the prophylactic and therapeutic properties of cVLP open an important perspective for their widespread use in the clinic, so we believe that this type of strategy can be very useful to counteract the development of malignancies caused by HR-HPV and, above all, to gain access in low-income countries, where infection by papilloma virus has strong repercussions as a public health problem.

Acknowledgements

The authors acknowledge the research funding granted by CONACYT (projects 83732, 82827, and 84071) and the FIS/IMSS (protocols No. 60, 617, and 876).

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