1. Immunology of HPV Infection Craig Woodworth Department of Biology Clarkson University Potsdam, NY

2. The Natural History of HPV Infection Suggests that Immunity is Effective Most HPV infections are cleared without clinical disease. When lesions develop, they regress after several months Individuals who have decreased cell mediated immunity (transplant recipients and patients with HIV) have an increased prevalence and persistence of HPV infections The humoral immune response accompanies papilloma regression, and is effective in preventing reinfection Immunity to HPV is effective but often delayed

3. The Papillomavirus Life Cycle has Evolved to Evade the Immune Response low level episomal replication capsid assembly and virus release expression of late genes persistence of HPV and malignant progression E6/E7 expression DNA replication

4. The Innate Immune System is the First Line of Defense Against HPV type I interferons proinflammatory cytokines epithelial barrier macrophages NK cells toll-like receptors HPV-infected keratinocytes

5. Type I Interferons Exert Direct Antiviral Effects Interferons  and  are produced by HPV-infected keratinocytes Inhibit cell proliferation and immortalization of HPV- expressing keratinocytes Down regulate expression of the HPV-16 E6 and E7 proteins in immortal keratinocytes Stimulate expression of HLA class I proteins However, therapy using type I interferons has had variable success….

6. HPV-16 E6 and E7 Proteins Directly Inhibit Interferon Signaling IFN interferon  R2 STAT2 nucleus cytoplasm STAT1 TYK2 p48 ISRE ISGF3 E7 inhibits IRF-3 and IRF-1 interferon  R1 E6 E7 E6

7. Inflammation is Important for Innate and Adaptive Immunity cell lysis & release of bioactive IL1-  IL1IL18TLRsTNF NF-kB cytokines (IL1, TNF-  ) IL-12, IFN- , chemokines, & ‘danger’ signals

8. Imiquimod Activates the Toll-Like Receptor 7 cell lysis & release of bioactive IL1-  imiquimod cytokines (IL1, TNF-  ) IL-12, IFN- , chemokines, & ‘danger’ signals NF-kB TLR-7

9. HPV-16 E6 and E7 Proteins Interfere with Proinflammatory Signaling cell lysis & release of bioactive IL1-  IL1TLRsTNF NF-kB E6 E6/E7 cytokines (IL1, TNF-  ) IL-12, IFN- , chemokines, & ‘danger’ signals

10. HLA Class I and II Proteins HLA class I proteins are expressed on the surface of keratinocytes and contribute to immune recognition HLA class II proteins are expressed by Langerhans cells and are required for cross presentation of antigens Specific HLA haplotypes are associated with protection from cervical cancer (HLA DRBI * 13 / DBQI * 0603) Cervical cancers show down regulation of HLA class I and up regulation of class II proteins

11. HPV Early Proteins Alter Expression of MHC Class I Genes HPV antigens proteasome TAP-1 class I HLA E5/E7 ER E5 E5/E7 HLA class I transcription

12. NK Cells and Macrophages NK cells target HPV-infected keratinocytes that express low levels of class I protein HPV-infected keratinocytes are relatively resistant to NK mediated lysis, but sensitive to LAK cells NK activity is decreased in patients with precancerous or malignant anogenital disease Macrophages are present in regressing papillomas, they produce 1L-12, and they contribute to elimination of HPV-infected cells Dysregulation of MCP-1 gene expression and lack of macrophage infiltration may contribute to HPV- linked carcinogenesis

13. Adaptive Immune Response to HPV Langerhans cells mature and migrate to lymph nodes where they cross present HPV antigens stimulation of a Th1 type cell mediated immune response activated macrophages Th1 helper T cells secretion of IgG memory T cells HPV-infected keratinocytes

14. Langerhans Cells Cross Present HPV Antigens to the Immune System HPV-infected keratinocyte Langerhans cell lymphocyte HPV antigens + ‘danger signal’ IL-10 TGF-  1

15. A Th1 Helper T Cell Response is Associated with Papilloma Regression IL-12 IFN-  Th1 helper T cell response regressing HPV infections IFN-  IL-2, TNF-  Th2 helper T cell response progressing HPV infections IL-4, IL-5, IL-10 IL-4 TGF-  Th1 Th2

16. Immune Events in Regressing Papillomas Non-regressing papillomas contain few immune cells and no inflammation Regression is associated with a Th1 type cell mediated immune response and increased expression of IFN- , IL-12, and TNF-  There is a large infiltration of mononuclear cells in the epithelium and stroma. This consists of CD4+ cells, macrophages, and CD8+ cells Infected keratinocytes express HLA class II proteins and ICAM-1

17. The Humoral Immune Response Protects Against Reinfection An antibody response to the L1 protein occurs commonly after HPV infection. Seroconversion is delayed for several months Both IgG and IgA are secreted. The IgG response to the L1 capsid protein is type-specific and long- lasting (over 10 years). The IgA response may be as long lived Antibodies are neutralizing and are directed to conformational epitopes on L1. The humoral immune response protects against reinfection, but does not cause regression of existing papillomas

18. Summary Most HPV infections are not apparent or regress, suggesting that the host’s immune response is effective The HPV life cycle has evolved to evade the host’s immune response, and HPV early proteins directly inhibit specific components of immunity Papilloma regression is mediated by a Th1 type cell mediated immune response with infiltration of macrophages and CD4+ cells Humoral immunity is protective and is mediated by neutralizing antibodies to conformation specific epitopes of the L1 protein

19. Questions How do HPV-16 early genes alter the ability of infected keratinocytes to contribute to the innate and adaptive immune responses? What is the most effective method for preventing tolerance or stimulating a strong Th1 helper T cell response? Do the innate and adaptive immune responses differ in the cervical transformation zone where most carcinomas develop?