Graham R. Leggatt

Potential strategies utilised by papillomavirus to evade host immunity.

Summary: The co‐evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non‐specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus‐induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced lo PV antigens, resting keratinocytes (KC) appear resistant to interferon‐γ‐enhanced mechanisms of cytotoxic T‐lymphocyte (CTL)‐mediated lysis, and expression of PV antigens by resting KC can tolerise PV‐specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV‐induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag−/− mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Role of intratumoural heterogeneity in cancer drug resistance: molecular and clinical perspectives

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over‐expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new‐targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy.

Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Prevention and Treatment of Papillomavirus-Related Cancers Through Immunization

Cervical and other anogenital cancers are initiated by infection with one of a small group of human papillomaviruses (HPV). Virus-like particle-based vaccines have recently been developed to prevent infection with two cancer-associated HPV genotypes (HPV16, HPV18) and have been ∼95% effective at preventing HPV-associated disease caused by these genotypes in virus-naive subjects. Although immunization induces virus-neutralizing antibody sufficient to prevent infection, persistence of antibody as measured by current assays does not appear necessary to maintain protection over time. Investigators have not identified a reliable surrogate immunological marker of protection against disease following immunization. The prophylactic vaccines are not therapeutic for existing infection. Trials of HPV-specific immunotherapy have shown some efficacy for existing disease, although animal modeling suggests that a combination of immunization and local enhancement of innate immunity may be necessary for optimal therapeutic outcome. HPV prophylactic vaccines are the first vaccines designed to prevent a human cancer and are the practical outcome of a global collaborative effort between basic and applied scientists, clinicians, and industry.

Serological evaluation of the 12 kDa subunit of antigen B in Echinococcus granulosus cyst fluid by immunoblot analysis

This study evaluated the 12 kDa (smallest) subunit of Echinococcus granulosus antigen B as a diagnostic molecule. Using immunoblotting, 90.9% of cystic hydatid patients, 40% of alveolar hydatid patients and 5.5% of cysticercosis patients showed sero-reactivity to this subunit. Human antibody response to the 12 kDa molecule appeared independent of factors such as parasite strain or host population responsiveness. The majority of infection sera, and some normal human controls, also recognized the 38 kDa subunit of antigen 5.

The number of long-lasting functional memory CD8+ T cells generated depends on the nature of the initial nonspecific stimulation

The mechanism of generation of memory cytotoxic T cells (CTL) following immunization remains controversial. Using tumor protection and IFN‐γ ELISPOT assays in mice to detect functional CTL, we show that the initial effector CTL burst size after immunization is not directly related to the amount of functional memory CTL formed, suggesting that memory CTL are unlikely to arise stochastically from effector CTL. Induction of MHC class II‐restricted T helper cells at the time of immunization by inclusion of a T helper peptide or protein in the immunogen, is necessary to generate memory CTL, although no T helper cell induction is required to generate effector CTL to a strong MHC class I‐binding peptide. Host protective T cell memory correlates with the number of CTL epitope responsive IFN‐γ‐secreting memory T cells as measured in an ELISPOT assay at the time of tumor challenge. We conclude that a different antigen presenting environment is required to induce long‐lasting functional memory CTL, and non‐cognate stimulation of the immune system is essential to allow generation of a long‐lasting host protective memory CTL response.

Regulation of immune responses to HPVinfection and during HPV‐directed immunotherapy

Summary:  The recent development of vaccines prophylactic against human papillomavirus (HPV) infection has the potential to reduce the incidence of cervical cancer globally by up to 70% over the next 40 years, if universal immunization is adopted. As these prophylactic vaccines do not alter the natural history of established HPV infection, immunotherapies to treat persistent HPV infection and associated precancers would be of benefit to assist with cervical cancer control. Efforts to develop immuno‐therapeutic vaccines have been hampered by the relative non‐immunogenicity of HPV infection, by immunoregulatory processes in skin, and by subversion of immune response induction and immune effector functions by papillomavirus proteins. This review describes HPV‐specific immune responses induced by viral proteins, their regulation by host and viral factors, and highlights some conclusions from our own recent research.

Invariant NKT Cells in Hyperplastic Skin Induce a Local Immune Suppressive Environment by IFN-γ Production

NKT cells can promote or inhibit adaptive immune responses. Cutaneous immunity is tightly regulated by cooperation between innate and adaptive immune processes, but the role of NKT cells in regulating cutaneous immunity is largely unknown. In this study, we show, in a mouse model, that skin-infiltrating CD1d-restricted NKT cells in HPV16-E7 transgenic hyperplastic skin produce IFN-γ, which can prevent rejection of HPV16-E7–expressing skin grafts. Suppression of graft rejection is associated with the accumulation of CD1dhi-expressing CD11c+F4/80hi myeloid cells in hyperplastic skin. Blockade of CD1d, removal of NKT cells, or local inhibition of IFN-γ signaling is sufficient to restore immune-mediated graft rejection. Thus, inhibition of NKT cell recruitment or function may enable effective immunity against tumor and viral Ags expressed in epithelial cells.

IL-10 Mediates Suppression of the CD8 T Cell IFN-γ. Response to a Novel Viral Epitope in a Primed Host

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-γ effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as priming to virus without CTL induction is associated with inhibition. However, IL-10−/− mice, in contrast to IL-10+/+ mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-γ-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.