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Papillomaviruses

Papillomaviruses. Chris Buck Email: buckc@nih.gov National Cancer Institute, Bethesda, Maryland. Papillomaviruses. National Cancer Institute*, Bethesda, Maryland. *The views expressed in this talk are my own and do not necessarily represent the views of the NCI

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Papillomaviruses

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  1. Papillomaviruses Chris Buck Email: buckc@nih.gov National Cancer Institute, Bethesda, Maryland

  2. Papillomaviruses National Cancer Institute*, Bethesda, Maryland *The views expressed in this talk are my own and do not necessarily represent the views of the NCI *I receive royalties from inventions related to HPV vaccines

  3. Medical Significance of Human Papillomaviruses (HPVs) >160 named HPV types •Ubiquitous. Infection can be completely asymptomatic, commensal However •Some types cause benign skin warts (papillomas) •Other types cause cancer of the uterine cervix or other epithelial cancers non-enveloped, DNA genome

  4. Papillomaviruses - Historical Overview •1894-1924: Ciufo, Variot and others show that genital and skin warts can be transmitted between individuals by a filterable infectious agent

  5. Papillomaviruses - Historical Overview •1894-1924: Ciufo, Variot and others show that genital and skin warts can be transmitted between individuals by a filterable infectious agent •1933 (Shope) rabbit papillomas have viral etiology •1935 (Rous) papillomas can progress to carcinoma •Papillomaviruses found to cause skin warts and other tumors in vertebrates ranging from birds to people Infection with Shope cottontail rabbit papillomavirus

  6. Papillomaviruses versus Human Cancer •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns

  7. Papillomaviruses versus Human Cancer •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer

  8. Papillomaviruses versus Human Cancer •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer •1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer

  9. Papillomaviruses versus Human Cancer •1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns •1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer •1928: GeorgiousPapanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer •1983: Harald zur Hausen discovers new HPV types (types 16 and 18) lurking in HeLa cells and other cervical cancer cells. •2008: Harald zur Hausen wins Nobel Prize for his work establishing a causal link between HPVs and cervical cancer Harald zur Hausen

  10. HPV Vaccine: Not Just for Girls Anymore •Summer 2006: FDA and CDC recommend new HPV vaccine “Gardasil” (Merck) for girls and women age 9 - 26 •October 2009: A second vaccine, “Cervarix” (GlaxoSmithKline) approved by FDA October 2009: Gardasil approved for use in boys and young men

  11. HPV Vaccine: Highly Effective

  12. HPV Vaccine: What Is It? •Recombinant virus-like particles based on HPV capsid protein L1 •Produced in yeast (Merck) or baculovirus (GSK) •Elicits HPV-neutralizing antibody responses that protect against initial infection

  13. “Pap” smear Common Cancers in Women Adapted from Parkin et al, Eur J Cancer 37:S4, 2001

  14. Worldwide Incidence of Cancers Attributable to HPV Estimates from IARC

  15. HPV and Non-Cervical Cancers •About half of cancers of the upper throat (for example, tonsils) are caused by HPV infection. A great majority of HPV-associated throat cancers contain HPV type 16 (covered by the vaccine) •Recent work by Maura Gillison and others has shown that oral sex, particularly with multiple partners correlates with risk of throat cancer •Most cases of anal cancer are caused by HPV. As with throat cancer, HPV16 accounts for a great majority of cases. No clear connection between anal sex and anal cancer, though overall history of multiple sex partners is a clear risk factor.

  16. HPV Vaccine: Not Just for Girls Anymore

  17. 70% of cervical cancers HPV16 HPV18 HPV6&11 90% of genital warts HPVs Are a Diverse Family Skin, hair follicles mostly commensal foot warts genital mucosa, cancer- associated hand warts >75% lifetime infection risk! genital warts

  18. Distribution of HPV Types in Cervical Cancer by Geographical Region % of cervical cancers Bosch et al, JNCI, 1995

  19. partial HPV31? no yes (weak?), Cervarix may offer greater cross-protection? HPV45 Some Degree of Cross-Protection? HPV16 “high risk” types HPV18 Harper (2006) Lancet 367:1247

  20. Current HPV Vaccine - What It Will Do •Protect against two HPV types that cause 70% of all cervical cancer •Gardasil (but not Cervarix) will protect against two HPV types that cause 90% of all genital warts •Protect against other HPV-induced cancers, such as throat cancer and anal cancer

  21. Current HPV Vaccine - What It Won’t Do •Won’t initially be affordable for women in developing countries •Won’t protect against all of the dozen or so HPV types that can cause cervical cancer •Won’t protect against a non-carcinogenic HPV types that may still cause abnormal Pap smear •Won’t protect women who are already infected (more on why this is so coming up later in the talk)

  22. Physical Characteristics / Genetic Organization

  23. Papillomavirus Virion •Non-enveloped icosahedral shell formed by 72 pentamers of a single protein, L1 (basis of current HPV vaccine) •60 nanometer diameter •A second capsid protein, L2, is present at up to 72 copies (basis of future HPV vaccine?) •Many features in common with polyomaviridae (e.g., SV40), but the two families probably never shared a common viral ancestor

  24. Genome •8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) E6 E7 7904/1 1000 7000 L1 2000 6000 E1 5000 3000 E4 4000 L2 E2 •One coding strand. Genome is divided into Late and Early regions E5

  25. 7904/1 1000 7000 2000 6000 5000 3000 4000 Capsid Genes •virion formation •cell association E7 E6 L1 E1 L2 E4 E2 •genome encapsidation •membrane penetration •post-entry trafficking E5

  26. 7904/1 1000 7000 2000 6000 5000 3000 4000 Oncogenes E6 E7 •Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle. L1 E1 L2 E4 E2 E5

  27. Adenovirus E1B SV40 T Ag Adenovirus E1A •Other types of DNA virus also disrupt p53 and pRB. This promotes entry into the S phase of the cell cycle, allowing replication of the viral DNA Cell Cycle Disruption •The E6 and E7 genes of cancer-causing HPVs mediate destruction of the tumor-supressor genes p53 and pRB (respectively)

  28. E6 & E7 - Other Functions E6: Activates telomerase. Interferes with about a dozen other known cellular targets, resulting in p53-independent effects. E7: Triggers chromosomal instability Interferes with about a dozen other known cellular targets, resulting in pRB-independent effects. Ongoing E6 and E7 expression is crucial for maintenance of HPV-transformed cells. For example, the famous HeLa cell line dies if E6 and/or E7 expression is knocked down.

  29. 7904/1 1000 7000 2000 6000 5000 3000 4000 DNA Handling Genes LCR/Ori E6 E7 •Helicase, recruits cellular DNA polymerase L1 E1 L2 E4 •Transcriptional regulation •recruit E1 to Ori, •tethers viral DNA to chromosomes during cell division E2 E5

  30. Life Cycle: Tissue Tropism

  31. Life Cycle - Initial Infection (Skin) virions micro-trauma Epidermis (Keratinocytes) Basal Cells Basement Membrane Dermis Slide courtesy M. Kast

  32. Gene Expression (Wart) Progeny virions Viral Proteins Capsid & Early Epidermis Early only Basement Membrane Dermis None

  33. Immune Evasion Epidermis Langerhans cells Basement Membrane Dermis Dendritic cells Draining Lymphatics

  34. Cryo Therapy, Wounding Epidermis Langerhans cells Basement Membrane Dendritic cells Dermis Draining Lymphatics DC & LC Activation = HPV Specific Immunity

  35. Draining Lymphatics Vaccination Epidermis Langerhans cells Basement Membrane Dendritic cells Dermis DC Activation = HPV-specific Response

  36. Viral Lifestyles •“Live fast, die young” - high-level viremia. Virus attempts to outrun the adaptive immune response. Examples: Ebola, influenza •“Tiptoe past the graveyard” - virus can linger and lie low in a safehouse cell type (neurons, stem cells). Latently infected cells express few or no viral proteins. Example: HPVs, polyomaviruses, herpesviruses. Immunosuppression can lead to emergence from latency and high level virion production •Second strategy is somewhat more common among DNA viruses (RNA is hard to preserve in a latent state?)

  37. Progression to Cancer is Accompanied by Deregulation of Viral Gene Expression CIN 1 CIN 2 CIN 3 Doorbar, J Clin Virol 32:7-15, 2005 Common molecular events: •Viral genome integration into cellular DNA •Loss of E2 leads to increased E6/E7 expression •Loss of L1, L2 expression. Therefore, current vaccine can’t clear pre-cancerous lesions.

  38. Life Cycle: Assembly

  39. Genome Packaging in DNA Viruses •DNA is stuffed into the capsid through a ring-shaped portal vertex. Packaging is coupled to ATP hydrolysis. Examples: bacteriophages and herpesviruses •A packaging sequence in viral genome provides handlebars for viral structural proteins. Examples: practically all other DNA viruses •HPV doesn’t have a well-defined packaging signal. Virions easily take up non-viral DNA, possibly by interacting with cellular histone proteins

  40. Phage HK97 Phage Lambda Phage P22 HSV HBV SV40 adenovirus HPV DNA Density for Different Viruses 0.40 0.30 basepairs per cubic nanometer 0.20 0.10 0 Name Mugshot (to scale) Portal Vertex? Packaging Sequence?

  41. Capsid Maturation More mature Less mature many hours •Maturation requires formation of extensive disulfide crosslinks between L1 molecules. •Mature capsid is very stable (bovine papillomaviruses vs fence posts) •Hypothesis: conserved, neutralizable motifs on the virion surface may become buried during maturation to hide them from B cells / antibody recognition.

  42. MCV Reporter Vectors (Pseudoviruses) L2 L1 GFP GFP reporter (8 kb) codon-modified L1+L2 (helper plasmid) SV40 Ori transfect transduce T Ag “293TT”

  43. HPV-Based Gene Transfer Vectors •L1 and L2 can package non-viral DNA and deliver packaged DNA to cells with high efficiency. HPV-derived DNA sequences are not required for efficient packaging (though some types of non-viral DNAs are packaged better than others) •HPV vectors may become useful for genetic delivery of vaccine immunogens (e.g., HIV vaccines)

  44. Life Cycle: Entry

  45. - - - Infectious Entry Pathway Step 1: Attachment + + + + heparan sulfate proteoglycans (=surface proteins decorated with sulfated polysaccharide side-chains) cell membrane

  46. - - - Infectious Entry Pathway Step 1: Attachment Heparin, carrageenan

  47. Topical Microbicides •Several brands of sex lube happen to use carrageenan as the principal gelling agent

  48. Topical Microbicides •Recently-announced clinical trial results suggest that a carrageenan gel protects women from HPV infection when applied before sex

  49. Infectious Entry Pathway conformational shift? Step 2: L2 exposure

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