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Papillomaviruses Chris Buck National Cancer Institute, Bethesda, Maryland.

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Presentation on theme: "Papillomaviruses Chris Buck National Cancer Institute, Bethesda, Maryland."— Presentation transcript:

1 Papillomaviruses Chris Buck 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 Baltimore Classification Scheme

4 No integration into host cell DNA. Viral genome only exists as an “episome”

5 Papillomavirus Virion Non-enveloped icosahedral shell formed by 72 pentamers of a single protein, L1 (basis of current HPV vaccine) 60 nanometer diameter Many features in common with polyomaviridae (e.g., SV40). Most notably: the viral genome is a small circular dsDNA molecule decorated with host cell histones (chromatin) Human papillomavirus type 16

6 Taxonomy “Crown Group” hypothesis: the last common ancestor of placental mammals already had several distinct types of papillomavirus. Each ancestral virus has co-speciated with its host animal. Little or no evidence of papillomaviruses transmission between distantly related animals

7 Taxonomy Genus: Mu Species: 1 Type: HPV1 causes warts on the soles of the feet Genus: Mu Species: 1 Type: HPV1 causes warts on the soles of the feet

8 Taxonomy Genus: Alpha Species: 4 Type: HPV2 causes warts on the palms of the hand Genus: Alpha Species: 4 Type: HPV2 causes warts on the palms of the hand

9 Taxonomy Genus: Gamma Species: 1 Type: HPV4 Caused a wart on the palm of my hand. Grrr! Genus: Gamma Species: 1 Type: HPV4 Caused a wart on the palm of my hand. Grrr!

10 Taxonomy Genus: Alpha Species: 10 Type: HPV6 causes genital warts. Does NOT cause cervical cancer Genus: Alpha Species: 10 Type: HPV6 causes genital warts. Does NOT cause cervical cancer

11 Taxonomy Genus: Alpha Species: 9 Type: HPV16 Generally asymptomatic, but causes cervical cancer in a small minority of infected individuals Genus: Alpha Species: 9 Type: HPV16 Generally asymptomatic, but causes cervical cancer in a small minority of infected individuals

12 /1 E6E6 E7E7 E1E1 E2E2 E4E4 E5E5 L2L2 Genome 8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) L1L1

13 /1 E6E6 E7E7 E1E1 E2E2 E4E4 E5E5 L2L2 Genome 8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) L1L1 One coding strand. Genome is divided into Late and Early regions

14 virion formation cell association /1 E6 E7 E1 E2 E4 E5 Capsid Genes L1 L2

15 genome encapsidation membrane penetration post-entry trafficking /1 E6 E7 E1 E2 E4 E5 Capsid Genes L2 L1

16 genome encapsidation membrane penetration post-entry trafficking /1 E6 E7 E1 E2 E4 E5 Capsid Genes L2 L1

17 /1 E1 E2 E4 Oncogenes L1 L2 E6 E7 E5 Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle.

18 /1 E1 E2 E4 Oncogenes L1 L2 E6 E7 Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle. E5 Small genome! No room for fancy stuff like DNA polymerase, DNA primase, ribonucleotide reductase, etc. The host cell must be forced to provide these factors.

19 Cell Cycle Disruption The E6 and E7 genes of cancer-causing HPVs mediate destruction of the tumor-supressor genes p53 and pRB (respectively) Adenovirus E1B Adenovirus E1A SV40 T Ag 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

20 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. E6 & E7 - Other Functions 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.

21 /1 E1 E2 E4 DNA Handling Genes L1 L2 E6 E7 Helicase, recruits cellular DNA polymerase E5 LCR/ Ori Transcriptional regulation recruit E1 to Ori, tethers viral DNA to chromosomes during cell division

22 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?)

23 Life Cycle - Initial Infection (Skin) micro-trauma Epidermis(Keratinocytes) BasalCells BasementMembrane Dermis virions Slide courtesy M. Kast

24 Gene Expression (Wart) BasementMembrane Epidermis Dermis Viral Proteins Early only Capsid & Early None Progeny virions

25 Immune Evasion BasementMembrane Draining Lymphatics Epidermis Dermis Langerhanscells Dendriticcells

26 Immune Evasion BasementMembrane Draining Lymphatics Epidermis Dermis Langerhanscells Dendriticcells In traditional cell culture monolayers, the virus thinks it’s in a basal keratinocyte, where hiding from the immune system is top priority. The only way to trick the virus into late phase (virion production) in vitro is through the use of keratinocyte raft cultures

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

28 T Ag GFP SV40 Ori GFP reporter (8 kb) L1 L2 transduce codon-modified L1+L2 (helper plasmid) “293TT” HPV Reporter Vectors (Pseudoviruses) transfect Neutralizing antibody

29 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., vaccines capable of eliciting cytotoxic T cells against HIV or other viruses)

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

31 Basement Membrane Epidermis Langerhans cells Dendritic cells Dermis Draining LymphaticsVaccination DC Activation = HPV-specific Response

32 Doorbar, J Clin Virol 32:7-15, 2005 Progression to Cancer is Accompanied by Deregulation of Viral Gene Expression CIN 1CIN 2CIN 3 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.

33 Doorbar, J Clin Virol 32:7-15, 2005 Therapeutic Vaccines CIN 1CIN 2CIN 3 Goal: recruit T cells against HPV E6 and/or E6 Goal: eradicate transformed cells (including pre- cancerous lesions)

34 Gene Expression (Wart) BasementMembrane Epidermis Dermis Viral Proteins Early only Capsid & Early None Progeny virions Bad news: stably infected keratinocyte stem cells don’t produce L1. Therefore, the L1 vaccine can’t possibly clear existing infections. This is why you must vaccinate kids before they’re exposed.

35 Therapeutic Vaccines Expert Opinion on Emerging Drugs, November 2012 Bad news: effective T cell immunity is a lot more difficult to elicit than effective antibody responses Good news: cervical cancer cells depend on ongoing E6/E7 expression

36 : Ciufo, Variot and others show that genital and skin warts can be transmitted between individuals by a filterable infectious agent History and Medical Significance

37 : 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 History and Medical Significance

38 1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns History and Medical Significance

39 1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns 1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer History and Medical Significance

40 1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns 1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer 1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer History and Medical Significance

41 1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervical cancer than nuns 1928: Georgious Papanicolaou develops the “Pap smear” technique for microscopic detection of cervical cancer and pre-cancer 1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancer Harald zur Hausen 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 History and Medical Significance

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43 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

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

45 HPV Vaccine: Highly Effective

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

47 Worldwide Incidence of Cancers Attributable to HPV Estimates from IARC

48 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) Work by Maura Gillison and others has shown that oral sex, particularly with multiple partners correlates with risk of throat cancer (Michael Douglas: not crazy! actually kind of heroic!) 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.

49 HPV Vaccine: Not Just for Girls Anymore

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

51 HPV Vaccine

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

53 HPV Vaccine

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58 HPV Vaccine - Theory

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60 Other virologists should be trying to figure out how to apply this magic to their favorite virus. We’re looking at you, HIV biologists!

61 Protect against two HPV types that cause 70% of all cervical cancer. Given the amazingly strong antibody responses, protection will probably be life-long 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 Current HPV Vaccine - What It Will Do

62 Won’t 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 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 lecture) Current HPV Vaccine - What It Won’t Do

63 Life Cycle: Tissue Tropism

64 Life Cycle: Entry

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

66 Infectious Entry Pathway Heparin, carrageenan Step 1: Attachment

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

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

69 Infectious Entry Pathway conformational shift? Step 2: L2 exposure

70 Infectious Entry Pathway furin protease Step 3: Furin cuts L2

71 Infectious Entry Pathway furin protease Step 3: Furin cuts L2 Note: many viruses use furin during assembly. Some toxins (anthrax toxin) use furin during entry

72 Infectious Entry Pathway Unidentified second receptor? conformational shift?

73 Infectious Entry Pathway Receptor = Annexin-A2? conformational shift?

74 Infectious Entry Pathway Receptor? conformational shift? Many hours nAb

75 Recent Developments Infection of mouse female genital tract reveals that the virus initially attaches to heparan sulfate on the basement membrane furin Day et al (2009) PNAS 106:20458

76 footwarts genitalmucosa,cancer-associated cutaneous, mostly asymptomatic handwarts genital warts L2-Neutralization - Chop the Whole Tree? Richard Roden Diana Pastrana (2005) Virol 337:365 Immunogen: BPV1 L2 aa 1-88 HPV16, 31 HPV18 CRPV HPV6, 11 neutralization BPV1

77 Infectious Entry Pathway Step 5: Endocytosis clathrin PO 4 ? 2 to 8 hours?

78 Infectious Entry Pathway Step 6: Endosome H+H+ H+H+ H+H+ H+H+ H+H+ protease(s)? disulfide reduction?

79 Infectious Entry Pathway Step 7: Membrane disruption L2 carboxy-terminus Syntaxin-18? Sortin Nexin 17? ~8 to 20 hours?

80 Infectious Entry Pathway Step 8: L2 drags viral genome to nucleus nuclear pore DNA Tubulin Actin? L2

81 Infectious Entry Pathway Step 8: L2 drags viral genome to ND-10 ND-10 mRNA 24 to 72 hours

82 Antibody responses to certain portions of L2 can be broadly cross-neutralizing. For example, antibody response to HPV16 L2 can neutralize other genital and skin-tropic HPV types. Maybe also true for conserved portions of L1? Single immunogen might protect against all HPV types. Would eliminate the need for expensive Pap testing Added bonus: protection against skin warts might facilitate implementation of childhood vaccination Future Directions: L2-Based Vaccines

83 T cell responses to E6 and E7 might cure existing cervical cancer and pre-cancerous lesions The fact that E6 and E7 are likely required for maintenance of transformed phenotype in cervical cancers makes them excellent cancer vaccine targets Future Directions: Therapeutic Vaccines

84 Practicalities Unvaccinated college-age adults should consider getting the HPV vaccine. This advice now applies to both men and women In contrast to other STIs, condoms provide limited (possibly no) protection against HPV transmission. Large areas of genital surfaces can be productively infected Papanicolaou (“Pap”) testing is highly effective for detecting cervical pre-cancer Newer HPV DNA testing is also a highly effective screening method Topical microbicides are in trials. Carrageenan, which is already on store shelves, appears to be effective for protecting women against HPV infection in an early clinical trial

85 the end


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