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.

Human papillomaviruses (HPVs) infect epithelia and can lead to the development of lesions, some of which have malignant potential. HPV type 16 (HPV16) is the most oncogenic genotype and causes various types of cancer, including cervical, anal, and head and neck cancers. However, despite significant research, our understanding of the mechanism by which HPV16 binds to and enters host cells remains fragmented. Over several decades, many HPV receptors and entry pathways have been described. This review puts those studies into context and offers a model of HPV16 binding and entry as a framework for future research. Our model suggests that HPV16 binds to heparin sulfate proteoglycans (HSPGs) on either the epithelial cell surface or basement membrane through interactions with the L1 major capsid protein. Growth factor receptors may also become activated through HSPG/growth factor/HPV16 complexes that initiate signaling cascades during early virion-host cell interactions. After binding to HSPGs, the virion undergoes conformational changes, leading to isomerization by cyclophilin B and proprotein convertasemediated L2 minor capsid protein cleavage that increases L2 N terminus exposure. Along with binding to HSPGs, HPV16 binds to ␣6 integrins, which initiate further intracellular signaling events. Following these primary binding events, HPV16 binds to a newly identified L2-specific receptor, the annexin A2 heterotetramer. Subsequently, clathrin-, caveolin-, lipid raft-, flotillin-, cholesterol-, and dynamin-independent endocytosis of HPV16 occurs. Since the discovery of human papillomaviruses (HPVs), researchers in the field have sought to identify the mechanism by which these viruses enter host cells. Although much work has been done to date and many possible receptors have been identified, a clearly defined description of the entry of HPVs has remained controversial. In many cases of viral infection, our understanding of simple binding and uptake through a singular mechanism has given way to a model of a more complex interaction between several specific receptors, coreceptors, and cofactors (1-3). The purpose of this review is to synthesize the known data regarding HPV type 16 (HPV16) binding proteins at the cell surface, and their associated molecules, and attempt to connect them, if possible, into a testable framework of binding and entry. Due to the fact that HPVs are a diverse group of over 150 viruses, this review focuses primarily on the most common of the cancer-causing genotypes, HPV16, while making it clear when non-HPV16 genotypes are reviewed. The development of several HPV particle production systems has allowed researchers to begin to delineate the mechanisms behind viral entry; however, important differences between particles developed in vitro made direct comparisons challenging. Therefore, not only HPV16 structure but also the multiple forms the virus structure takes in the laboratory are discussed below. Finally, due to the tropism of HPV16 for human epithelial cells during a natural infection, this rev...

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.

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 (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 HPVinduced 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. ' 2007 Wiley-Liss, Inc.Key words: human papillomavirus; immunotherapy; therapeutic vaccines; immunomodulation; animal models; clinical trialsThe human papillomaviruses (HPVs) are a family of sexually transmitted, double-stranded DNA viruses with over 100 different genotypes identified till date. HPV genotypes are divided into the low-risk and high-risk categories based on the spectrum of lesions they induce. Fifteen HPV types are classified as high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82); 3 are classified as probable high-risk types (26, 53 and 66) and 12 are classified as low-risk types (6,11,40,42,43,44,54,61,70,72, 81 and CP6108). 1 The low-risk types primarily induce benign genital condylomas and low-grade squamous intraepithelial lesions whereas the high-risk types are most frequently associated with the development of anogenital cancers and can be detected in 99% of cervical cancers, 2 with HPV16 found in about 50% of cases. 3 Infection by the low-risk types is not confined to the anogenital area and can cause other diseases such as recurrent respiratory papillomatosis. Similarly, infection by the high-risk types is also not confined to the anogenital area, since 18.3% of cancers of the oropharynx contain DNA from these types. 4 In the United States, an estimated 75% of the sexually active general population ages 15-49 years acquires at least one genital HPV type during their lifetime. 5 Though most individuals remain asymptomatic and spontaneously clear their infections, a small percentage of patients develop clinically or histologically recognizable lesions that develop into invasive cancer. The widespread use of cervical cytological screening using Papanicolaou (Pap) smear tests has reduced the mortality rate from cervical cancer in developed countries. However, in developing countries where screening programs are minimal, cervical cancer remains the second leading cause of cancer-related deaths among women. 6 Furthermore, Pap smear tests will not help identify HPV infection in males, where it can cause penile and anal cancers as well as cancers of the head and neck. Thus, it is essential to develop effective prophylactic and therapeutic strategies directed against HPV. Prophylactic vaccinesWhile therapeutic vaccines aim to develop a strong ...