1. Speaker: 孙云帆 Speaker: 孙云帆 Group members: 陈红艳、李剑、 沈丽、王皓晨、洪璇阳、赵彬彬 Group members: 陈红艳、李剑、 沈丽、王皓晨、洪璇阳、赵彬彬 HPV Vaccine

2. Cancer = Death Cancer = Unpreventable

3. Human Papillomavirus (HPV) Vaccine The First Vaccine Against Cancer ------The Anti-Cervical Cancer Vaccine

4. This happens in an HPV infected patient Hey, long time no see. How are you doing recently Couldn’t be worse! What’s up, buddy? Have you watched BBC(Brain Broadcast) last night? It reported that HPV army had already captured the vagina. Really? How could that possible! You never give us warning about HPV invasion. Yeah! That’s why i am gonna be fired. Damn! I patrol in the tissue every day. Yesterday i just passed by vagina and every thing is OK! Well, maybe the HPV is unlike influenza virus. So I don’t think it’s your fault. But my boss said it was my fault, and if I can’t catch a HPV to the Lymph node and present antigens to T cell guys, I am gonna loose my job! Jesus Christ!! Hang on buddy, one of my fellow told me that there was going to be a lecture about HPV Vaccine in Shandong University right now. What about we have a look at it? You better not joke!

5. Contents The Overview of Vaccine The Features of HPV The Mechanism of HPV infection Strategies for Prophylactic ( 预防的 ) Therapeutic HPV Vaccine Discussions

6. What is Vaccine? Suspension of weakened, killed, or fragmented microorganisms or toxins or of antibodies or lymphocytes that is administered primarily to prevent disease. Encyclopaedia Britannica Encyclopaedia Britannica （不列颠百科全书） （不列颠百科全书）

7. Vaccine Approaches Type of vaccineExamples Live attenuated or killed bacteria BCG, cholera Live attenuated virusesPolio, rabies Subunit(antigen) vaccine Tetanus toxoid, diphtheria toxoid Conjugate Vaccine Haemophilus influenzae, pneumococcus ✓ Synthetic Vaccine Hepatitis(recombinant proteins) Viral Vectors Clinical trials of HIV antigens in canarypox vector ✓ DAN Vaccines Clinical trials ongoing for several infections

8. Characteristics of HPV Small (55 nm) Icosahedral ( 二十面体的 ) capsid, double-stranded non-enveloped DNA virus, 72 Capsomeres of L1(Capsid protein) non-enveloped DNA virus, 72 Capsomeres of L1(Capsid protein) HPV-18 and HPV-16 are highly associated with cervical cancer Species specific, site specific & Epithelitropic Species specific, site specific & Epithelitropic Not yet Isolated in Lifeless culture Media Not yet Isolated in Lifeless culture Media The epitope that can be recognized by corresponding The epitope that can be recognized by corresponding immunocytes locates on capsid protein(L1 protein) immunocytes locates on capsid protein(L1 protein)

9. Capsid Protien L1 has high immunogenicity

10. ORFs include six early transcript units(E1, E2, E4, E5, E6, E7), and two late transcript region(L1, L2) two open reading frames(ORFs) one upper regulatory region(URR) one Long control region(LCR)

11. Normal Epithelium More than 70 types of human papillomavirus (HPV) have been described. Some cause benign papillomas of the skin (warts). Other strains, particularly HPV-16 and HPV-18, are linked to genital and anal cancers. These viruses are sexually transmitted. HPV-16 and HPV-18 are found in the majority of squamous-cell carcinomas of the uterine cervix. Genital warts with low malignant potential are associated with HPV-6 and HPV-11. When transforming DNA viruses infect a cell, they integrate their DNA into the genome of the host. At this point the virus does not reproduce but only produces the proteins necessary to commandeer the DNA synthesis machinery of the host cell. Two of these viral genes, E6 and E7, can act as oncogenes. The proteins they encode bind to the protein products of two important tumour suppressor genes, p53 and RB, respectively, knocking these proteins out of action and allowing the cell to grow and divide. The E6 and E7 proteins of HPV-16 and HPV-18 bind to the RB and p53 proteins very tightly; in contrast, the E6 and E7 proteins of HPV-6 and HPV- 11 (the low-risk types) bind RB and p53 with low affinity. The differences in binding ability of these proteins correlate with their ability to activate cell growth, and they are consistent with the differences in malignant potential of these virus strains. More than 70 types of human papillomavirus (HPV) have been described. Some cause benign papillomas of the skin (warts). Other strains, particularly HPV-16 and HPV-18, are linked to genital and anal cancers. These viruses are sexually transmitted. HPV-16 and HPV-18 are found in the majority of squamous-cell carcinomas of the uterine cervix. Genital warts with low malignant potential are associated with HPV-6 and HPV-11. When transforming DNA viruses infect a cell, they integrate their DNA into the genome of the host. At this point the virus does not reproduce but only produces the proteins necessary to commandeer the DNA synthesis machinery of the host cell. Two of these viral genes, E6 and E7, can act as oncogenes. The proteins they encode bind to the protein products of two important tumour suppressor genes, p53 and RB, respectively, knocking these proteins out of action and allowing the cell to grow and divide. The E6 and E7 proteins of HPV-16 and HPV-18 bind to the RB and p53 proteins very tightly; in contrast, the E6 and E7 proteins of HPV-6 and HPV- 11 (the low-risk types) bind RB and p53 with low affinity. The differences in binding ability of these proteins correlate with their ability to activate cell growth, and they are consistent with the differences in malignant potential of these virus strains.

12. Capsid peptides bind with cell surface receptors in basal cell layer Alpha-7 integrin molecule is one of the candidate receptors

13. Early proteins that regulate the virus are expressed in lowest layers of epithelium

14. Late proteins are expressed in upper layers of epithelium Viral nucleic acid can be found in basal stem cells, but late gene expression(capsid proteins) is restricted to the uppermost layer of differentiated keratinocytes.

15. HPV Infection Causes Epithelial Neoplasia ( 肿瘤 形成 ) And it also results in that few APCs can catch these viruses.

16. Why can’t our immune system respond to HPV infection adequately?

17. HPV replication and release do not cause cell death, since the differentiating keratinocyte is already programmed to die, and this “death by natural causes” does not present as a danger signal to the immune system. Thus, for most of the HPV infectious cycle, there is little or no release of the proinflammatory cytokines important for dendritic cell activation and migration into the local milieu High-risk HPV viruses downregulate IFN- -inducible gene expression, and the HPV 16 E6 and E7 oncoproteins directly interact with components of the interferon signaling pathways, abrogating these pathways. Capsid entry is usually an activating signal for dendritic cells, but there is evidence that Langerhans cell, unlike stromal dendritic cells, are not activated by uptake of HPV capsids, a phenomenon that would inhibit both LC migration and maturation, and the priming of the immune response against the capsid proteins.

18. In Summary, HPV efficiently evades the innate immune response and delays the activation of the adaptive immune response.

19. E2 protein and YY1 can inhibit the transcription of E6, E7; E2 Binding siteLCR E2 ProteinTranscription protein YY1 E6E7 E6 and E7 are inactive E6 and E7 are activated HPV infection

20. HPV virology

21. Worldwide Infection of Cervical Cancer Cervical cancer kills around 250,000 women every year worldwide and is the second most common type of cancer in women. Two high-risk HPV types (HPV 16/18) are responsible for an estimated 70% of cases of cervical cancer.

22. Strategies for the Prevention of Cervical Cancers ✴ Therapeutic Vaccine ✴ Prophylactic Vaccine Prophylactic Vaccine

23. Strategies for prophylactic HPV vaccine ๏ HPV L1 VLP(virus-like particles) vaccine Now approved by FDA and available in USA: Cervarix and Gardasil Elicit mainly humoral immunity--- neutralizing antibody and partially adaptive immunity

24. In June 8 2006, the Food and Drug Association (FDA) approved the first vaccine to prevent cancer. The vaccine approved by the FDA prevents infection by human papillomavirus, or HPV, which is the cause of nearly all cases of cervical cancer. Dr Schlegel is Chair of the Georgetown University Medical Center Department of Pathology C. Richard Schlegel, MD, PhD, C. Richard Schlegel, MD, PhD,

25. HPV L1 VLP vaccine Gardasil is a quadrivalent HPV-16/18/6/11 L1 VLP vaccine developed by Merck and Co. Inc. Cervarix is a bivalent HPV-16/18 L1 VLP vaccine developed by GlaxoSmithKline(GlaxoSmithKline Biologicals, Rixensart, Belgium)

26. HPV L1 VLP vaccine Mimic natural virion structure generate potent immune response

27. HPV L1 VLP vaccine IgG APC present MHC-Ag complex and co- stimulation signal Plasma cell Ig switch IgG Immunized by HPV L1 VLP vaccine The protection against challenge with infectious virus is elicited by high titers of neutralizing serum antibody(IgG) induced by these vaccine Future HPV infection Adaptive Immunity

28. HPV Vaccine Trial 99.7% Seroconversion

29. HPV Vaccine Trial Number of Women Over 17 Months

30. Therapeutic Vaccine ✤ DNA based vaccination protocol aimed at inducing an efficient anti-E7 immune response in the vivo. ✤ DNA vaccine serving in treatment of cervical cancer is at the cutting edge of tummor immunotherapy.

31. HPV DNA Recombinant Vaccine Human papillomavirus type 16 is commonly implicated in cervical cancers. The viral genome encodes potential targets like the oncoprotein E7, expressed in transformed cells but thought to represent a poorly immunogenic antigen. Recent researches are focused on a DNA-based vaccination protocol aimed at inducing an efficient anti-E7 immune response in vivo.

32. Brief Mechanism of HPV Recombinant Vaccine E7 protein encoded by vitro recombinant plasmid

33. Brief Mechanism of HPV Recombinant Vaccine ubiquitinated form of E7

34. Some research results and discussions Many in vivo studies indicate that only mice vaccinated using a plasmid-encoded IiE7 fusion protein are protected against in vivo tumor challenge. Researches demonstrated that protection against tumor growth is associated with the development of a CD4-regulated, E7- specific CTL activity mediated by CD8 cells. In contrast to proteasome-mediated degradation, the IiE7 protein appears to be located in the endosomal compartment and displays increased stability upon tansfection. Collectively, many studies indicate that expression of E7 in the endosomal pathway provides prolonged protein expression and immmunogenicity in vivo, paving the way for novel DNA-based immunization strategies against HPV

35. Keep our minds clear! AdvantagesDefects HPV VLP Vaccine No viral DNA No viral DNA High Immunogenicity High Immunogenicity elicit high titer of neutralizing Abs elicit high titer of neutralizing Abs Only one VLP vaccine aimed at protection against its corresponding type of HPV Only one VLP vaccine aimed at protection against its corresponding type of HPV DNA Vaccine induce strong adaptive immunity induce strong adaptive immunity No HPV type restriction No HPV type restriction Long term protection-- T, B memory cell Long term protection-- T, B memory cell Plasmid DNA may infect host DNA and results of DNA mutation Plasmid DNA may infect host DNA and results of DNA mutation

36. Discussions Idea 1: Building a plasmid where can insert different types of antigenic gene. In terms of the pros and cons of VLP and DNA vaccine, we have the following ideas Idea 3: Adding a control gene segment to the plasmid, which make sure the expression of those oncogenes(E6, E7) in the right time. Idea 2: Designing a protein molecule which will carry all types of HPVs’ epitopes. Idea 4: Developing a RNA vaccine, which is safer than DNA vaccine

37. Speaker: 孙云帆 Speaker: 孙云帆 Group members: 陈红艳、李剑、 沈丽、王皓晨、洪璇阳、赵彬彬 Group members: 陈红艳、李剑、 沈丽、王皓晨、洪璇阳、赵彬彬 Thank you!