Vaccines and immunization against human papillomavirus.

Treatment and Prevention Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 252–264 (DOI: 10.1159/000356183)

Vaccines and Immunization against Human Papillomavirus Neil D. Christensen · Lynn R. Budgeon Departments of Pathology and Microbiology and Immunology, Penn State Hershey Medical Center, College of Medicine, Hershey, Pa., USA

Abstract Prophylactic and therapeutic immunization strategies are an effective method to control human papillomavirus (HPV)-associated diseases and cancers. Current protective virus-like particle and capsid-based vaccines are highly protective against vaccine-matched HPV types, and continued improvements in second-generation vaccines will lead to broader protection and cross-protection against the cancer-associated types. Increasing the effectiveness of broadly cross-protective L2based immunogens will require adjuvants that activate innate immunity to thus enhance adaptive immunity. Therapeutic immunization strategies are needed to control and cure clinical disease and HPV-associated cancers. Significant advances in strategies to improve induction of cell-mediated immunity to HPV early (and capsid) proteins have been pretested in preclinical animal papillomavirus models. Several of these effective protocols have translated into successful therapeutic immunemediated clearance of clinical lesions. Nevertheless, there are significant challenges in activating immunity to cancer-associated lesions due to various immune downregulatory events that are triggered by persistent HPV infections. A better understanding of immune responses to HPV lesions in situ is needed to optimize immune effector T cells that efficiently locate to sites of infection and which should lead to an effective immunotherapeutic management of this important human viral pathogen. The most effective immunization strategy may well require combination antiviral and immunotherapeutic treatments to achieve complete clearance of HPV infections and associated cancers.

Human papillomaviruses (HPVs) infect epithelial cells of the skin and mucosa and produce active infections of the skin, anogenital region, oral cavity and larynx [for reviews, see 1, 2]. The pathogenesis of HPV infections manifests itself as increased epithelial proliferation that can progress to local, then invasive cancers in some cases. HPV-associated cancers of the cervix, anogenital and oral sites still account for more than 250,000 deaths annually worldwide [3]. HPV DNA has been occasionally located at other anatomical sites such as lung tissues, the ovary, prostate, breast, tropho-

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blasts and brain but there is no clear evidence of malignant conversion in these tissues caused by HPV, although there are recent intriguing possible correlates with epilepsy [4]. Nevertheless, HPVs represent a major human pathogen and thus warrant immunological interventions for management. Current immunization strategies to combat HPV infections include activation of protective anti-capsid-neutralizing antibodies [5–7] and activation of postinfection therapeutic cell-mediated immunity (CMI) [8, 9]. Two Food and Drug Administration (FDA)-approved capsid vaccines comprising virus-like particles (VLPs) of the major capsid protein L1 induce strong, long-lasting, type-specific protection against the two most frequent cancer-associated HPV types [7, 10]. HPVs are noncytolytic and counteract natural immunity by a variety of strategies including induction of minimal inflammatory responses at the primary site of infection, reduced immunological signaling and reduction of some natural host antiviral mechanisms [11–14]. The failure to trigger inflammatory responses is a consequence of the natural life cycle of the virus which occurs in differentiating epithelial cells which in turn are programmed for natural death or anoikis. A major challenge to study immune responses to HPV directly is the strict species restriction of these viruses. Thus, animal models have been used to great effect for assessing various immunization protocols [15, 16]. Given the pathological nature of some HPV types and the failure of naturally developing host immunity to clear these infections in some patients, immunization strategies to activate protective and clearing immunity are a major goal of current basic and applied research.

HPVs express up to 7 early (E) proteins and 2 late (L) or capsid proteins during their life cycle [for a review, see 17]. These proteins are all potential targets of host adaptive immunity, and several HPV viral products can also activate innate immunity. To maximize immunization strategies to control HPV infections, a complete understanding of both the viral life cycle and the anatomy of a papilloma is required (fig. 1). HPVs are strictly epitheliotropic with viral early proteins E1, E2, E5, E6 and E7 expressed at low levels in the infected basal epithelial cells [17]. As differentiation occurs, there are increased levels of the viral oncogenes E6 and E7 in the middle layers of the papilloma, and in the upper layers, E4, L1 and L2 are now highly expressed. In cancerous lesions, the viral DNA is often integrated leading to upregulated viral oncogene expression and loss of viral regulatory and capsid proteins. The anatomical segregation of these viral proteins increases the complexity of targeting by the adaptive immune response and requires T cells in particular to migrate from the vasculature and dermis into the lower and middle layers of the papilloma epithelium for effective clearance. Interestingly, the viral protein levels expressed in the lower layers are low due to the relative abundance of rare codons that

HPV Immunization Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 252–264 (DOI: 10.1159/000356183)

253


Life Cycle

Capsid proteins L1, L2 and viral DNA

Koilocytes

Early proteins E4, viral DNA

Early proteins E6, E7 (higher levels)

Early proteins E1, E2, E5, E6, E7 (increasing expression)

Basement membrane

Type IV collagen, laminin, nidogen/entactin and perlecan

Fig. 1. Anal papilloma showing features of benign HPV infections. Viral protein and DNA expression is organized in different departments of the infected epithelium with the highest levels of capsid proteins and viral DNA in the fully differentiated upper layers.

make up most HPV genes [18]. Standard immunohistological assays therefore often fail to detect these proteins. Despite low protein expression levels, there is the likelihood that T cells recognizing processed viral peptide-major histocompatibility (MHC) complexes can still respond immunologically to HPV-infected keratinocytes.

HPVs cause hyperproliferative lesions in epithelial tissues of the skin and mucosa. In cervical cancers, graded stages of pathology have been defined including cervical intraepithelial neoplasia grades I, II, III and IV or invasive carcinoma; and the Bethesda system of high- and low-grade squamous intraepithelial lesions. An additional category includes atypical cells of undetermined significance commonly associated with cervical cytology in PAP smears. These categories define both stages and treatment options for patients with HPV infections of the cervicovaginal region. Interestingly, not all HPV infections produce clinical disease and/or cancerous progression, and immunological strategies to boost immunity to HPV infections in

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Christensen · Budgeon Ramírez-Fort MK, Khan F, Rady PL, Tyring SK (eds): Human Papillomavirus: Bench to Bedside. Curr Probl Dermatol. Basel, Karger, 2014, vol 45, pp 252–264 (DOI: 10.1159/000356183)


Disease Symptoms

general depend upon disease severity. HPVs are genetically characterized into major groups with common pathophysiological features [19]: (a) Alphapapillomaviruses: these HPV types infect predominantly mucosal surfaces and are responsible for all cases of HPV-associated mucosal malignancies. HPV16 is the major oncogenic type accounting for up to 50% of cervical cancers and a higher proportion of HPV-associated head-and-neck and anal cancers [20]. These viruses synthesize the E proteins (E1, E2, E4, E5, E6 and E7) and in precancerous lesions can produce the capsid proteins L1 and L2. The capsid proteins are targets for neutralizing antibody, whereas all the E proteins together with the capsid proteins are potential targets of CMI. Upon infection, innate immune responses are triggered minimally due to the absence of virus-induced inflammation at primary sites. (b) Betapapillomaviruses: these HPV types infect predominantly cutaneous tissues and result in mostly benign lesions that often resolve immunologically. Several types are associated with skin cancers in immunocompromised patients and those with a rare genetic disorder known as epidermodysplasia verruciformis [21]. Recent studies have shown the oral cavity also as a site for these HPV types [22]. (c) Gamma-, Mu- and Nupapillomaviruses: these HPV types include several skintropic types as well as recently discovered mucosotropic viruses that produce minimal disease in the oral cavity [22]. No known pathological disease is associated with these latter types, and the natural host immune responses are subsequently understudied. In summary, the pathogenesis (or lack thereof) of infections of the various HPV types will dictate the need for immunization strategies to control this diverse set of human pathogens. A large number of recently discovered cutaneous and oral papillomaviruses produce no demonstrable lesions and are considered by some investigators to be equivalent to commensal microflora [23]. It is unclear yet as to whether these infections are immunologically ‘silent’ or whether they negatively regulate potential cross-protective immunity to the more pathological HPV types.

Immunization to Activate Innate Immunity to HPV Infections and Viral Products Activation of innate immune responses to HPV infections should also amplify adaptive immunity. Questions of interest include whether HPV infections either activate and/or downregulate innate immunity and whether HPVs have the capacity to thwart host natural antiviral mechanisms. HPVs are DNA viruses and thus have the potential to trigger various innate antiviral immune responses that recognize double-stranded DNA and/or viral RNA. Successful immunization strategies (summarized in table 1) may therefore include innate immune activators for the dual purpose of increasing natural antiviral host mechanisms as well as improved adaptive immunity. HPV can combat both innate immunity and natural antiviral host cell responses. Several recent studies have documented the impact of HPV infections on innate im-


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Immunization Strategies to Control Human Papillomavirus Infections

Viral target

Potential immune response

Immunization strategy

References

Viral DNA/RNA

Innate immunity via intracellular sensors

Generic adjuvants

No clinical studies

Capsid proteins L1 (in VLP)

Neutralizing antibodies

VLP with alum and ASO4 adjuvants

6, 55, 56; 7, 10, 56

Capsid proteins L2 (fragments and fusion proteins)

Neutralizing antibodies

Peptide and/or fusion proteins with various adjuvants (preclinical studies; clinical trials under way)

32, 47

Early proteins (E6 and E7)

CTL and T helper cells

Long peptides (clinical studies); various recombinant strategies (preclinical models)

48, 49, 57, 58; 39

Early proteins (E1, E2)


Recombinant vaccines (clinical and preclinical studies)

40, 43; 36, 49

Modified early proteins


DNA vaccines, recombinant strategies aimed at improving antigen presentation and overcoming MHC downregulation (clinical and preclinical studies)

40, 44, 58; 52, 53

mune activation or suppression. Thus, HPV capsids and pseudoviruses can enter skin and cervical Langerhans cells which are the primary antigen-presenting cells on site for HPV infection. The uptake of HPV VLPs by Langerhans cells can trigger immunesuppressing or activating responses. The minor capsid protein L2 in VLPs and pseudoviruses is responsible for preventing functional maturation of skin Langerhans cells leading to a potential local immune escape for HPV infection [24]. In the absence of L2, HPV VLP antigens potently activate both antigen-presenting cells and subsequent adaptive immunity including humoral and CD8+ cytotoxic T lymphocytes (CTLs). Other HPV viral mechanisms of innate immune interference include downregulation of the antiviral interferon response by the viral oncogene E7 [25], reduced MHC-I expression via E5 proteins [14] and inactivation of the EVER1/2 host cell proteins that modulate zinc concentrations in the cells necessary for viral DNA replication [26]. Recent keratinocyte-profiling studies demonstrated that the presence of HPV leads to broad deregulation of keratinocyte inflammatory networks focused on IL-1β pathways [27]. Interestingly, various intracellular antiviral sensors such as TLR-3, RIG-I, MDA5 that sense viral DNA and RNA were not downregulated in cells harboring replicating HPV. Collectively, these findings demonstrate that HPVs have developed a variety of strategies to escape immune detection. Thus, successful immunization to control HPV infections will need to overcome these negative regulatory events that circumvent both innate and adaptive immunity.

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Table 1. Summary of immunization targets and strategies for HPV infections

Adjuvants that activate innate immunity are included in current prophylactic and therapeutic vaccines. Thus, VLP vaccines now include TLR-4 activators such as monophosphoryl lipid A (Cervarix), and second-generation preclinical capsid vaccines are testing TLR-5 activators [28] for improved antigenicity. Peptide-based vaccines that activate adaptive T cell immunity have used montanide ISA-51 adjuvant that is used to make antigen emulsions [29]. Strategies to overcome other HPV-induced negative regulatory events such as downregulation of MHC-I expression, EVER1/2 and reduced levels of inflammation are under investigation in preclinical animal papillomavirus models.

Cell-Mediated Immunity All HPV early and late proteins are potential targets of CMI. The production of these proteins in the infected keratinocyte provides clear targets for CD8+ CTLs that have the capacity to eliminate HPV-containing cells.


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Immunization to Activate Adaptive Immunity to HPV Humoral Immunity Pathogenic HPV infections are mostly located at mucosal sites. Viral entry is predicted to occur through microtrauma of the epithelium, and virions thus gain access to basal cells assisted by a wound-healing environment and migrating keratinocytes. Strong protective immunity develops upon systemic immunization with HPV VLPs containing the L1 protein [6, 10]. Neutralizing antibodies are hypothesized as the mechanism of protection; however, a role for anti-capsid-specific CMI in this protection is unresolved. The neutralizing antibodies are predominantly type specific with limited cross-protection to closely related vaccine types. The mechanism of this limited cross-protection is poorly understood. It should be noted that these VLP vaccines are potent immunogens, and strong long-lasting protection can be induced even in the absence of adjuvants [30]. Given that mucosal-targeting HPV types are the most pathogenic, there is considerable interest in the activation of secretory IgA HPV-neutralizing antibodies at the site of infection [31]. Current capsid vaccines delivered systemically activate high levels of serum IgG neutralizing antibodies that are highly type specific and are believed to target HPV virions at the site of microtrauma by means of egress from the vasculature at these wounded sites. It is not clear yet whether improved cross-protection against related HPV types would benefit from an immunization strategy that induces strong IgA-specific antibodies at mucosal sites. In contrast, the minor capsid protein L2 develops more broadly cross-protective neutralizing antibodies although the antibody titers and the antigenicity of L2 are several orders of magnitude lower than VLP vaccines [32]. Adjuvants are thus included in these L2-based vaccines which are currently being tested in clinical trials. Another distinct advantage of L2-based neutralizing antibodies is that the epitopes recognized as neutralization inducing are linear in nature and thus easily manufactured.

E6 and E7 Proteins The viral oncogenes E6 and E7 have been studied extensively for transforming function and antigenicity. These proteins represent the favored targets of immunotherapy for HPV-associated cancers given their persistent expression in cancer cells. In several immunotherapeutic trials, these antigens have been tested as immunogens by systemic delivery as peptides, whole proteins and as DNA-based vaccines using either recombinant viruses or in vivo electroporation [8]. The small size of E7 presents some challenges such as a possible lack of effective CD8-stimulating epitopes for some MHC-I molecules. Failure to activate an E7-specific CTL response was noted in HLA transgenic mouse lines [38]. The E6 protein appears to be a better immunological target as measured by in vitro responses of immune cells from patient samples [39] and in preclinical papillomavirus models [15]. Current clinical trials that have shown promise have used overlapping long peptides for the therapeutic treatment of HPV16+ persistent vaginal intraepithelial neoplasia [9]. Other clinical studies are using DNA-based vaccinations together with in vivo electroporation to enhance immune responses [8].

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Capsid Proteins. The capsid proteins L1 and L2 are produced predominantly in fully differentiated keratinocytes that are in essence already dead. A role for CD8+ CTLs targeting capsid protein epitopes in protection and clearance appears obscure. However, there is the possibility that low levels of the capsid proteins are produced in basal and intermediate cells in the infection that can then be targeted by capsid-specific CMI. Indeed, in both preclinical and clinical studies, L1-specific CTL/CMI can be demonstrated and which may well play a role in disease management [33]. There is clear evidence for the induction of L1-specific CD4+ T cells. In some viral systems CD4+ cytolytic T cells have been identified [34]. Capsid-specific CD4+ helper T cells can be measured by various in vitro assays, and a role for supporting the production of capsid antigen-specific B cells in vivo is self-evident. Unknown as yet is the impact of these CD4+ T cells in maintaining a strong CD8+ CTL response to clear HPV infections and activation of both memory B cells and memory CTLs. Early Proteins. The E proteins of HPV are all potential targets of CMI responses. The strict requirement for continued viral oncogene (E6 and E7) expression in HPVassociated cancers makes them excellent candidates for immunological targets. In addition, the E proteins are foreign antigens and not immediately subject to developmental tolerance as are many nonviral tumor antigens. Assessment of HPV-specific immunity in patient populations identified 2 major responsive groups. Those patients who have evidence of past exposure and have cleared HPV infections show robust CMI responses as measured in a variety of in vitro and in vivo assays [35, 36]. In contrast, patients with persistent infections show limited CMI to HPV antigens and evidence of immune suppression via regulatory T cells and immunosuppressive cytokines [37].

E1, E2, E4 and E5 Proteins The regulatory and ‘other’ early viral proteins (E1, E2, E4, E5) can also be considered viable targets for CMI. Currently there are no therapeutic clinical vaccine trials using these proteins as targets, although these proteins have been tested in situ to measure skin reactions [36]. However, extensive testing in preclinical animal models indicates that these proteins represent strong immunological targets for control of precancerous papillomavirus infections [40–42]. Comparative studies in rabbits using persistent cottontail rabbit papillomavirus infections indicated that E1, E2 and E6 strongly activate protective CMI responses [43, 44]. Therapeutic vaccinations with these antigens showed less effective immunity indicating (as found in clinical trials) that clearing persistent HPV infections by immunization strategies will be challenging.

Preclinical Papillomavirus Models for Testing Immunization Protocols

What have we learnt from preclinical papillomavirus infection models to help design successful immunization strategies for HPV infections? In essence, several preclinical papillomavirus models (rabbit, dog and bovine) were essential for the design, testing and subsequent FDA approval of Gardasil and Cervarix [16, 45]. These 3 models together with modified mouse models continue to help shape second-generation protective capsid-based vaccines that induce neutralizing antibodies [46]. In addition, these preclinical models were first to demonstrate the feasibility of L2-based vaccines for cross-protection against different HPV types [47]. All CMI responses to HPV antigens were also pretested in papillomavirus preclinical models. Findings in these models have helped develop T-cell-based therapeutic vaccines for clinical trials, and some success has recently been obtained in patients with vulvar intraepithelial neoplasia [9]. The impact of various delivery systems, vaccine modifications and adjuvants is best tested in these preclinical models and will greatly assist second-generation vaccines that are focused on providing broad protection against the cancer-associated HPV types that currently number 15 antigenically unique types [19].

Prophylactic Vaccines Protective HPV vaccines targeting capsid proteins have met with considerable success. The ‘failures’ have basically arisen from the lack of strong cross-protection against the many HPV types associated with malignancies. Second-phase capsidbased vaccines are in clinical trials and include strategies such as increasing the valency of the current VLP vaccine from 4 to 9 types, and targeting the more cross-


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Strategies for the Future

protective L2 capsid protein. A combination of VLP and L2 vaccines is likely to improve and enhance coverage against the current panel of 15 oncogenic or high-risk types. Combination of Antiviral and Immunotherapy Current clinical trials testing therapeutic immunization against persistent HPV infections and HPV-associated cancers have met with limited success. Thus far, vaccinations with long peptides of HPV-16 E6 and E7 have shown promise against vulvar intraepithelial neoplasia [9] and heat shock protein 70-E7 fusion proteins against precancerous cervical lesions [48]. Other vaccination programs have demonstrated activation of antigen-specific immune responses and are part of ongoing clinical trials [49, 50]. The impact on host immunity in patients with persistent infections often manifests itself as various immunosuppressive events such as the localized presence of regulatory T cells and negatively regulating cytokines [11, 37]. Given these observations, the need for improved strategies is evident. One such improvement in outcome can be achieved by combination treatments that have proven successful in the management of HIV and HCV infections. For example, preclinical papillomavirus models have shown improved efficacy using combination immunotherapy and antiviral treatments [51], low-dose irradiation [52] and monoclonal antibody therapy [53]. Recent clinical trials using low-dose cyclophosphamide improved the clinical outcome of condylomatous disease [54] and paved the way for combination immunotherapy against HPV disease. Future opportunities to improve resolution of persistent HPV infections and associated cancers may therefore well include similar combinations of antiviral treatments and therapeutic vaccination. The challenge will be to determine the kinetics of the treatments that lead to optimized clearance and control of HPV infections in patient populations.

There are currently 2 FDA-approved capsid-based vaccines, Gardasil and Cervarix, for the protection against HPV infections. Gardasil is a quadrivalent VLP-based vaccine containing L1-only VLPs for HPV types 6, 11, 16 and 18, whereas Cervarix is a bivalent vaccine containing L1-based VLPs for HPV types 16 and 18. These vaccines generate a high-titer neutralizing antibody to the vaccine-containing HPV types, and some reduced cross-protection to closely related HPV types such as HPV-31 and -45 [7, 10]. Gardasil is recommended for both females and males aged 9–26 years, and Cervarix currently for females aged 9–29 years. Many clinical trials both completed and active are detailed on the clinical website (http://clinicaltrials.gov/). Future modifications of capsid-based protective vaccines are under development and testing. For example, there are trials under way, testing nonavalent (VLP for the 9 most common cervical-cancer-associated HPV types) vaccines as well as L2-based vaccines.

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Vaccination

Conclusion

Immunization against HPV infections is the most effective method to control these important human pathogens. Strong immunity to infectious virus can be induced using capsid-based vaccines that induce neutralizing antibodies. Numerous preclinical and clinical studies show strong protection against both experimental and natural infections. The next challenge to overall immunological control of HPV-associated disease is the development of postinfection T-cell-based vaccines that target existing HPV infections and HPV-associated cancers. There are a number of E7- and E6-based peptide, dendritic-cell-peptide-pulsed and rDNA-based clinical studies under way to treat HPV-associated cervical, anal and head-and-neck cancers (http://clinicaltrials. gov/). Taken together, there is much promise (and much additional research into mechanisms of tumor regression in situ) in the immunological management of HPV infections. Preclinical models will continue to be both valuable and valid models to assess both new treatments and combination therapies.

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Neil D. Christensen, PhD Jake Gittlen Cancer Research Foundation Department of Pathology, Rm C7800, H059 Penn State College of Medicine, 500 University Drive, Hershey, PA 17033 (USA) E-Mail [email protected]

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