Diane M. Da Silva

Human Dendritic Cells Are Activated by Chimeric Human Papillomavirus Type-16 Virus-Like Particles and Induce Epitope-Specific Human T Cell Responses In Vitro

Human papillomavirus (HPV)-derived chimeric virus-like particles (VLPs) are the leading candidate vaccine for the treatment or prevention of cervical cancer in humans. Dendritic cells (DCs) are the most potent inducers of immune responses and here we show for the first time evidence for binding of chimeric HPV-16 VLPs to human peripheral blood-derived DCs. Incubation of immature human DCs with VLPs for 48 h induced a significant up-regulation of the CD80 and CD83 molecules as well as secretion of IL-12. Confocal microscopy analysis revealed that cell surface-bound chimeric VLPs were taken up by DCs. Moreover, DCs loaded with chimeric HPV-16 L1L2-E7 VLPs induced an HLA-*0201-restricted human T cell response in vitro specific for E7-derived peptides. These results clearly demonstrate that immature human DCs are fully activated by chimeric HPV-16 VLPs and subsequently are capable of inducing endogenously processed epitope-specific human T cell responses in vitro. Overall, these findings could explain the high immunogenicity and efficiency of VLPs as vaccines.

Human Papillomavirus Virus-Like Particles Do Not Activate Langerhans Cells: A Possible Immune Escape Mechanism Used by Human Papillomaviruses

High-risk human papillomaviruses are linked to several malignancies including cervical cancer. Because human papillomavirus-infected women do not always mount protective antiviral immunity, we explored the interaction of human papillomavirus with Langerhans cells, which would be the first APCs the virus comes into contact with during infection. We determined that dendritic cells, normally targeted by vaccination procedures and Langerhans cells, normally targeted by the natural virus equally internalize human papillomavirus virus-like particles. However, in contrast to dendritic cells, Langerhans cells are not activated by human papillomavirus virus-like particles, illustrated by the lack of: up-regulating activation markers, secreting IL-12, stimulating T cells in an MLR, inducing human papillomavirus-specific immunity, and migrating from epidermal tissue. Langerhans cells, like dendritic cells, can display all of these characteristics when stimulated by proinflammatory agents. These data may define an intriguing immune escape mechanism used by human papillomavirus and form the basis for designing optimal vaccination strategies.

Cervical cancer vaccines: emerging concepts and developments.

Certain human cancers are linked to infection by oncogenic viruses that are able to cause transformation of the normal host cell into a cancerous cell. Human papillomavirus (HPV) DNA and expression of viral transforming proteins are found in virtually all cervical cancer cells, indicating an important role of this virus in the pathogenesis of the disease. Evidence exists that the immune response to cancer cells can play a major role in determining the outcome of disease. The fact that HPV is a necessary cause for cervical cancer provides a clear opportunity to develop a therapeutic vaccine against the virus to treat patients with cervical cancer at its early and late stages. Development of a prophylactic vaccine for HPV would also reduce the incidence of cervical neoplasias by preventing virus infection. Various candidate HPV vaccines are being developed and tested in animal models and/or in human clinical trials. These HPV vaccines, both preventive and therapeutic, are the subjects of this review. J. Cell. Physiol. 186:169–182, 2001.

The S100A10 Subunit of the Annexin A2 Heterotetramer Facilitates L2-Mediated Human Papillomavirus Infection

Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108–120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.

Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response.

The use of chimeric virus‐like particles represents a new strategy for delivering tumor antigens to the immune system for the initiation of antitumor immune responses. Immunization of DBA/2 mice with the P1A peptide derived from the P815 tumor‐associated antigen P1A induced specific T‐cell tolerance, resulting in progression of a regressor P815 cell line in all animals. However, immunization with a human papillomavirus type 16 L1 virus‐like particle containing the P1A peptide in the absence of adjuvant induced a protective immune response in mice against a lethal tumor challenge with a progressor P815 tumor cell line. Additionally, we demonstrated that these chimeric virus‐like particles could be used therapeutically to suppress the growth of established tumors, resulting in a significant survival advantage for chimeric virus‐like particle‐treated mice compared with untreated control mice. Chimeric virus‐like particles can thus be used as a universal delivery vehicle for both tolerizing and antigenic peptides to induce a strong protective and therapeutic antigen‐specific antitumor immune response. J. Cell. Biochem. 73:145–152, 1999.

Human Papillomavirus Can Escape Immune Recognition through Langerhans Cell Phosphoinositide 3-Kinase Activation

Human papillomavirus (HPV) infection of cervical epithelium is linked to the generation of cervical cancer. Although most women infected with HPV clear their lesions, the long latency period from infection to resolution indicates that HPV evolved immune escape mechanisms. Dendritic cells, which are targeted by vaccination procedures, incubated with HPV virus-like particles induce an HPV-specific immune response. Langerhans cells (LC), which are located at the sites of primary infection, do not induce a response implicating the targeting of LC as an immune escape mechanism used by HPV. LC incubated with HPV virus-like particles up-regulate the phosphoinositide 3-kinase (PI3-K) pathway and down-regulate MAPK pathways. With the inhibition of PI3-K and incubation with HPV virus-like particles, LC initiate a potent HPV-specific response. PI3-K activation in LC defines a novel escape mechanism used by HPV, and PI3-K inhibition may serve as an effective clinical target to enhance HPV immunity.

Cervical cancer vaccines: recent advances in HPV research.

Carcinomas of the anogenital tract, particularly cancer of the cervix, account for almost 12% of all cancers in women, and so represent the second most frequent gynecological malignancy in the world (48). It is well established that chronic infection of cervical epithelium by human papillomaviruses (HPV) is necessary for the development of cervical cancer. In fact, HPV DNA has been demonstrated in more than 99.7% of cervical cancer biopsy specimens, with high-risk HPV16 and HPV18 sequences being most prevalent (45,73). Therefore, an effective vaccine that would mount an immune response against HPV-related proteins might contribute to the prevention or elimination of HPV expressing lesions. This review will concentrate on the most recent advances in vaccine-mediated prevention and immunotherapy of HPV-induced cervical cancer, including presentations from the 20(th) International HPV Conference held in October 2002 in Paris.

Heterologous boosting increases immunogenicity of chimeric papillomavirus virus-like particle vaccines.

Chimeric human papillomavirus virus-like particles (HPV cVLPs), containing the HPV16 non-structural protein E7, are potent vaccines for inducing antigen-specific protective immunity against HPV-transformed tumors in animal models. Previous data demonstrated that the effectiveness of cytotoxic T lymphocyte (CTL) induction after repetitive vaccination with the same cVLP, and thus vaccine efficacy, is limited by the presence of neutralizing antibodies induced after the first application. Here, we determined if altering the route of vaccine delivery or incorporation of the target antigen into VLPs of a heterologous papillomavirus type could overcome inhibition of MHC class I antigen presentation by neutralizing antibodies, resulting in a boosting of CD8(+) T-cell responses against the incorporated antigen, HPV16 E7. Mucosal delivery of cVLPs resulted in detection of systemic E7-specific CD8(+) T cells, however, these routes were not able to bypass the inhibitory effect of circulating antibodies against homologous VLP types. In contrast, mice immunized and boosted with heterologous cVLPs containing HPV16 E7 showed a higher frequency of E7-specific T cells in vitro and displayed reduced tumor growth in a therapeutic setting compared to mice treated with homologous cVLPs. The data indicate that the use of different cVLP types for prime/boost regimens is a promising strategy to increase the efficacy and usefulness of cVLP-based vaccines for the treatment of cervical neoplasia.

Recent advances in strategies for immunotherapy of human papillomavirus-induced lesions

Human papillomavirus (HPV)‐induced lesions are distinct in that they have targetable foreign antigens, the expression of which is necessary to maintain the cancerous phenotype. Hence, they pose as a very attractive target for “proof of concept” studies in the development of therapeutic vaccines. This review will focus on the most recent clinical trials for the immunotherapy of mucosal and cutaneous HPV‐induced lesions as well as emerging therapeutic strategies that have been tested in preclinical models for HPV‐induced lesions. Progress in peptide‐based vaccines, DNA‐based vaccines, viral/bacterial vector‐based vaccines, immune response modifiers, photodynamic therapy and T cell receptor based therapy for HPV will be discussed.

Uptake of Human Papillomavirus Virus-Like Particles by Dendritic Cells Is Mediated by Fcγ Receptors and Contributes to Acquisition of T Cell Immunity

Chimeric human papillomavirus virus-like particles (HPV cVLP) are immunogens able to elicit potent CTL responses in mice against HPV16-transformed tumors; however, the mechanism of T cell priming has remained elusive. HPV VLP bind to human MHC class II-positive APCs through interaction with FcγRIII, and immature dendritic cells (DC) become activated after incubation with HPV VLP; however, it is unclear whether FcγR on DC are involved. In mice, FcγRII and FcγRIII are homologous and bind similar ligands. In this study, we show that binding and uptake of VLP by DC from FcγRII, FcγRIII, and FcγRII/III-deficient mice are reduced by up to 50% compared with wild-type mice. Additionally, maturation of murine DC from FcγRII/III-deficient mice by VLP is also reduced, indicating that DC maturation, and thus Ag presentation, is diminished in the absence of expression of FcγR. To investigate the in vivo contribution of FcγR in the induction of cellular immunity, FcγR single- and double-knockout mice were immunized with HPV16 L1/L2-E7 cVLP, and the frequency of E7-specific T cells was analyzed by tetramer binding, IFN-γ ELISPOT, and cytotoxicity assays. All readouts indicated that the frequency of E7-specific CD4+ and CD8+ T cells induced in all FcγR-deficient mice after immunization with cVLP was significantly diminished. Based on these results, we propose that the low-affinity FcγR contribute to the high immunogenicity of HPV VLP during T cell priming by targeting VLP to DC and inducing a maturation state of the DC that facilitates Ag presentation to and activation of naive T cells.