0
HPV: Cervical CancerHPV: Cervical Cancer
and the Vaccineand the Vaccine
Abhijit Chaudhury.Abhijit Chaudhury.
HistoryHistory
► Warts were known to the ancient GreeksWarts were known to the ancient Greeks
and Romans.and Romans.
► The...
HistoryHistory
► Richard Shope(1933):Richard Shope(1933):
First identified theFirst identified the
animal papillomavirusan...
HistoryHistory
► 1842: Rigoni-Stern reports that prostitutes1842: Rigoni-Stern reports that prostitutes
have much higher i...
HistoryHistory
► Mid 1970s: Meisels and FortinMid 1970s: Meisels and Fortin
recognized on morphologicalrecognized on morph...
HPV----- BiologyHPV----- Biology
►Originally classified together withOriginally classified together with
polyomavirus in t...
HPV----- BiologyHPV----- Biology
►Small, non enveloped, icosahedral particles.Small, non enveloped, icosahedral particles....
HPV----- BiologyHPV----- Biology
► Capsid contains 2 structural proteins:Capsid contains 2 structural proteins:
L1 (major ...
HPV----- Biology: GenomeHPV----- Biology: Genome
► All the Open Reading Frames are located on oneAll the Open Reading Fram...
10001000
20002000
30003000
40004000
50005000
60006000
70007000
7904/17904/1
E6 E7
E1
E2
E4
E5
L2
GenomeGenome
•8 kilobase
...
•genome encapsidation
•membrane penetration
•post-entry trafficking
•virion formation
•cell association
1000
2000
3000
400...
1000
2000
3000
4000
5000
6000
7000
7904/1
E1
E2
E4
OncogenesOncogenes
L1
L2
E6
E7
•Together the
viral oncogenes
immortaliz...
1000
2000
3000
4000
5000
6000
7000
7904/1
E1
E2
E4
DNA Handling GenesDNA Handling Genes
L1
L2
E6
E7
•Helicase,
recruits
ce...
HPV Replication: The Life CycleHPV Replication: The Life Cycle
Attachment, Entry, UncoatingAttachment, Entry, Uncoating
Vi...
Attachment, Entry, UncoatingAttachment, Entry, Uncoating
► Basal kerationocytes are infected first----Basal kerationocytes...
Attachment, Entry, UncoatingAttachment, Entry, Uncoating
► Dis-assembly and exposure of viral genomeDis-assembly and expos...
Infectious Entry PathwayInfectious Entry Pathway
Step 8: L2 drags viralStep 8: L2 drags viral
genome to nucleusgenome to n...
Immortalization andImmortalization and
TransformationTransformation
► A few HPV types can immortalize primaryA few HPV typ...
Immortalization andImmortalization and
TransformationTransformation
►Transformation is "Transformation is "the introductio...
Immortalization andImmortalization and
TransformationTransformation
The transformed cells have abnormal growth parameters ...
The cell cycle: Role ofThe cell cycle: Role of RbRb
Cell growth regulated
by an internal timer:
cell cycle
Divided into 4 ...
Cell cycle is controlled by the cyclin-Cdk machinery
Different cyclins and cyclin dependent kinases expressed at different...
RbRb
► pRb functions in growth control by its bindingpRb functions in growth control by its binding
ability and inhibition...
Cell Regulation:Cell Regulation: p53p53
► p53 is a tumor suppressor genep53 is a tumor suppressor gene
► Determines respon...
HPV and CancerHPV and Cancer
► Genital Cancers: Cervix, Vulva, Vagina,Genital Cancers: Cervix, Vulva, Vagina,
Penis, Anus....
Worldwide Incidence ofWorldwide Incidence of
Cancers Attributable to HPVCancers Attributable to HPV
Cervical cancer: 0.500 million in 2002
1
High-grade precancerous lesions:
10 million
2
Low-grade cervical lesions:
30 mill...
HPV and CancerHPV and Cancer
►Greater than 100 subtypesGreater than 100 subtypes
 About 15-20 cancer related types (type ...
The HPV Oncoproteins:The HPV Oncoproteins: E6E6
► 150 amino acids in size.150 amino acids in size.
► Both high- and low-ri...
The HPV Oncoproteins:The HPV Oncoproteins: E6E6
► The E6-E6 AP complex targets ubiquitin targetedThe E6-E6 AP complex targ...
The HPV Oncoproteins:The HPV Oncoproteins: E7E7
► E7 protein is small nucleoprotein of 100E7 protein is small nucleoprotei...
The HPV Oncoproteins:The HPV Oncoproteins: E7E7
► E7 binds preferentially to the hypo (de-)E7 binds preferentially to the ...
HPV virologyHPV virology
NonproliferatingNonproliferating cellcell
p53p53
pRBpRB
G0: quiescent stateG0: quiescent state
G1...
Normal
Cervix
HPV
Infection
Persistent
HPV
Infection
Cervical
Dysplasia
(pre-cancer)
Cervical Cancer
Cervical Cancer Biolo...
Classification of Histological Findings:Classification of Histological Findings:
Cervical Intraepithelial NeoplasiaCervica...
Life Cycle - Initial Infection (Skin)Life Cycle - Initial Infection (Skin)
micro-traumamicro-trauma
EpidermisEpidermis
(Ke...
Gene Expression (Wart)Gene Expression (Wart)
BasementBasement
MembraneMembrane
EpidermisEpidermis
DermisDermis
Viral Prote...
Immune EvasionImmune Evasion
BasementBasement
MembraneMembrane
Draining LymphaticsDraining Lymphatics
EpidermisEpidermis
D...
Doorbar, J Clin Virol 32:7-15, 2005
Progression to Cancer is Accompanied byProgression to Cancer is Accompanied by
Deregul...
Natural History of HPV Infection:Natural History of HPV Infection:
Surrogate Markers for Cervical CancerSurrogate Markers ...
HPV and Cancer: TheHPV and Cancer: The
ConclusionConclusion
Cervical and other genital cancers are composed of cells that...
HPV and Cancer: TheHPV and Cancer: The
ConclusionConclusion
► E6 and E7 themselves contribute to the accumulation ofE6 and...
THE VACCINETHE VACCINE
►Work started in 1980s, CollaborationWork started in 1980s, Collaboration
between Georgetown Univer...
The VaccineThe Vaccine
► Subunit Vaccines, containing hollow Virus LikeSubunit Vaccines, containing hollow Virus Like
Part...
The VaccineThe Vaccine
► CervarixCervarix has 20 mcg each of HPV 16 and HPV 18has 20 mcg each of HPV 16 and HPV 18
L1 prot...
The VaccineThe Vaccine
► Injection Site:Injection Site:
 Upper arm (Deltoid region)Upper arm (Deltoid region)
 Thigh (hi...
Prophylactic VaccineProphylactic Vaccine
► Both are quite effective at preventing HPV 16/18Both are quite effective at pre...
MECHANISM OF ACTION
HPV L1 VLP vaccineHPV L1 VLP vaccine
Mimic natural virion structure generate potent immune responseMimic natural virion st...
HPV L1 VLP vaccineHPV L1 VLP vaccine
APC
HPV L1 VLP
IgG
B T
APC present
MHC-Ag complex
and co-stimulation
signal
Plasma ce...
Protective EfficacyProtective Efficacy
► The FUTURE I (Females United to Unilaterally
Reduce Endo/Ectocervical Disease)Stu...
Protective EfficacyProtective Efficacy
► FUTURE II trial, showed that the quadrivalent
vaccine is 98 percent effective in ...
Measurement of induced antibodies
to HPV 16 and 18 by Cervarix and
Gardasil one month after the 3rd
dose (month 7) among 1...
Protective Efficacy: Cross-ProtectionProtective Efficacy: Cross-Protection
► Gardasil potentially offers disease
protectio...
Duration of ProtectionDuration of Protection
► At least five years.At least five years.
► Phase II trials provide the long...
Sustained clinical efficacySustained clinical efficacy and antibody titerand antibody titer
forfor at leastat least 5 year...
Duration of ProtectionDuration of Protection
► Induced antibodies against the L1 VLP for HPVInduced antibodies against the...
SafetySafety
► Autoimmune demyelinating neurologic sequelae afterAutoimmune demyelinating neurologic sequelae after
Gardas...
Controversy: IndiaControversy: India
► Gardasil dissemination programs in AndhraGardasil dissemination programs in Andhra
...
Future Direction: TherapeuticFuture Direction: Therapeutic
VaccineVaccine
The oncoprotein E7, is expressed in transformedT...
Future Direction: TherapeuticFuture Direction: Therapeutic
VaccineVaccine
► Researches demonstrated that protectionResearc...
Eradication of Cervical Cancer andEradication of Cervical Cancer and
HPV Related DiseasesHPV Related Diseases
Not a Dream ...
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Human Papilloma Virus : Cervical Cancer and Vaccines

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  • Messages
    100% efficacy lasting for 5 years with antibody titres (neutralising, HPV type specific) plateauing at high level will not drop off tomorrow
    We therefore expect that Gardasil will protect a long time, 20, may be 30, may be 40 years, may be life-long
    Comments/Additional information
    Prevention of Human Papillomavirus infection is essentially through antibodies (neutralising, specific) and not through cell mediated immunity
    The formulation with 225 μg of Merck’s aluminium phosphate adjuvant has proven to stimulate high antibody response for Gardasil
    It is very unusual that vaccines show a higher immune response than natural infection. Gardasil shows ~50 times higher immune response (note that the scale on the left is a log scale!) which is extremely high and exceptional already. Given the 100% efficacy this again shows that there is no sense in trying to induce even higher immune response.
  • Transcript of "Human Papilloma Virus : Cervical Cancer and Vaccines"

    1. 1. HPV: Cervical CancerHPV: Cervical Cancer and the Vaccineand the Vaccine Abhijit Chaudhury.Abhijit Chaudhury.
    2. 2. HistoryHistory ► Warts were known to the ancient GreeksWarts were known to the ancient Greeks and Romans.and Romans. ► Their infectious nature was also known.Their infectious nature was also known. ► 1894-1924: Ciufo, Variot and others1894-1924: Ciufo, Variot and others showed that genital and skin warts can beshowed that genital and skin warts can be transmitted between individuals by atransmitted between individuals by a filterable infectious agentfilterable infectious agent..
    3. 3. HistoryHistory ► Richard Shope(1933):Richard Shope(1933): First identified theFirst identified the animal papillomavirusanimal papillomavirus and showed itsand showed its transmissible naturetransmissible nature using the cottontailusing the cottontail rabbit.rabbit. ► 1950s,60s: Physico-1950s,60s: Physico- chemical analysis andchemical analysis and replication studies.replication studies.
    4. 4. HistoryHistory ► 1842: Rigoni-Stern reports that prostitutes1842: Rigoni-Stern reports that prostitutes have much higher incidence of cervicalhave much higher incidence of cervical cancer than nunscancer than nuns ► 1935 (Rous) papillomas can progress to1935 (Rous) papillomas can progress to carcinoma.carcinoma. ► 1951: George Otto Gey establishes in vitro1951: George Otto Gey establishes in vitro culture of HeLa (Henrietta Lacks) cellsculture of HeLa (Henrietta Lacks) cells derived from a lethal cervical cancerderived from a lethal cervical cancer
    5. 5. HistoryHistory ► Mid 1970s: Meisels and FortinMid 1970s: Meisels and Fortin recognized on morphologicalrecognized on morphological grounds an associationgrounds an association between HPV infection ofbetween HPV infection of cervix and CIN.cervix and CIN. ••1983: Harald zur Hausen1983: Harald zur Hausen discovers new HPV typesdiscovers new HPV types (types 16 and 18) in HeLa(types 16 and 18) in HeLa cells and other cervical cancercells and other cervical cancer cells.cells. ••2008: Harald zur Hausen wins2008: Harald zur Hausen wins Nobel Prize for his workNobel Prize for his work establishing a causal linkestablishing a causal link between HPVs and cervicalbetween HPVs and cervical cancer.cancer.
    6. 6. HPV----- BiologyHPV----- Biology ►Originally classified together withOriginally classified together with polyomavirus in the familypolyomavirus in the family Papovaviridae.Papovaviridae. ► Assigned to the new familyAssigned to the new family PapillomaviridaePapillomaviridae in 2004.in 2004. ► 12 genera, of which 5 infect humans, other12 genera, of which 5 infect humans, other seven infects only animals.seven infects only animals. ► Human papillomavirus belong to generaHuman papillomavirus belong to genera αα,,ββ,,γγ,,μμ,,νν..
    7. 7. HPV----- BiologyHPV----- Biology ►Small, non enveloped, icosahedral particles.Small, non enveloped, icosahedral particles. ► Replicate in squamous epithelial cellsReplicate in squamous epithelial cells ► 52-55 nm size.52-55 nm size. ► Genome consists of a single molecule of dsGenome consists of a single molecule of ds circular DNA appx. 8000 bp size.circular DNA appx. 8000 bp size. ► Capsid contains 72 capsomers.Capsid contains 72 capsomers.
    8. 8. HPV----- BiologyHPV----- Biology ► Capsid contains 2 structural proteins:Capsid contains 2 structural proteins: L1 (major capsid protein): 55kd, 80% ofL1 (major capsid protein): 55kd, 80% of total viral proteinstotal viral proteins L2 (Minor): 70kdL2 (Minor): 70kd Expression of L1 alone or a combination ofExpression of L1 alone or a combination of L1 and L2 will result in production of VirusL1 and L2 will result in production of Virus like particles (VLP) without the DNA.like particles (VLP) without the DNA.
    9. 9. HPV----- Biology: GenomeHPV----- Biology: Genome ► All the Open Reading Frames are located on oneAll the Open Reading Frames are located on one strand of viral DNAstrand of viral DNA ► Ten designated translational ORF, classified intoTen designated translational ORF, classified into E and L ORF.E and L ORF. ► E Region: Encode viral regulatory proteins, andE Region: Encode viral regulatory proteins, and those needed for initiating viral DNA replication. Inthose needed for initiating viral DNA replication. In non-productively infected cells.non-productively infected cells. ► L Region: Viral capsid proteins in productivelyL Region: Viral capsid proteins in productively infected cells.infected cells.
    10. 10. 10001000 20002000 30003000 40004000 50005000 60006000 70007000 7904/17904/1 E6 E7 E1 E2 E4 E5 L2 GenomeGenome •8 kilobase circular dsDNA. Persists as episome (doesn’t integrate into cellular DNA) L1 •One coding strand. Genome is divided into Late and Early regions
    11. 11. •genome encapsidation •membrane penetration •post-entry trafficking •virion formation •cell association 1000 2000 3000 4000 5000 6000 7000 7904/1 E6 E7 E1 E2 E4 E5 Capsid GenesCapsid Genes L1 L2
    12. 12. 1000 2000 3000 4000 5000 6000 7000 7904/1 E1 E2 E4 OncogenesOncogenes L1 L2 E6 E7 •Together the viral oncogenes immortalize the cell and prime it for viral DNA replication by disrupting the cell cycle. E5
    13. 13. 1000 2000 3000 4000 5000 6000 7000 7904/1 E1 E2 E4 DNA Handling GenesDNA Handling Genes L1 L2 E6 E7 •Helicase, recruits cellular DNA polymerase E5 LCR •Transcriptional regulation •recruit E1 to Ori, •tethers viral DNA to chromosomes during cell division Long Control Region/Upstream Regulator region: Origin of DNA replication.
    14. 14. HPV Replication: The Life CycleHPV Replication: The Life Cycle Attachment, Entry, UncoatingAttachment, Entry, Uncoating Viral TranscriptionViral Transcription Virus Assembly and ReleaseVirus Assembly and Release
    15. 15. Attachment, Entry, UncoatingAttachment, Entry, Uncoating ► Basal kerationocytes are infected first----Basal kerationocytes are infected first---- wounds, abrasions.wounds, abrasions. ► Receptor/s to which HPV binds is not veryReceptor/s to which HPV binds is not very clear:clear: αα6 integrin, heparan, cell surface6 integrin, heparan, cell surface glycosaminoglycansglycosaminoglycans ► Enters the cell by clathrin dependentEnters the cell by clathrin dependent receptor mediated endocytosis.receptor mediated endocytosis.
    16. 16. Attachment, Entry, UncoatingAttachment, Entry, Uncoating ► Dis-assembly and exposure of viral genomeDis-assembly and exposure of viral genome occurs within endosomesoccurs within endosomes ► L2 and the genome escape into cytoplasm andL2 and the genome escape into cytoplasm and enter the nucleus ------enter the nucleus ------ L2 Functions:L2 Functions: Enhances nuclear import of viralEnhances nuclear import of viral genomegenome;; localization of the genome to thelocalization of the genome to the transcriptionally active nuclear domain 10;transcriptionally active nuclear domain 10; mediates endosomal escapemediates endosomal escape;; interacts withinteracts with syntaxin 18(End Ret Protein) needed for transportsyntaxin 18(End Ret Protein) needed for transport across cytoplasm.across cytoplasm.
    17. 17. Infectious Entry PathwayInfectious Entry Pathway Step 8: L2 drags viralStep 8: L2 drags viral genome to nucleusgenome to nucleus nuclear pore DNA Tubulin Actin?L2
    18. 18. Immortalization andImmortalization and TransformationTransformation ► A few HPV types can immortalize primaryA few HPV types can immortalize primary human fibroblasts, human foreskinhuman fibroblasts, human foreskin kerationcytes or human cervical epithelialkerationcytes or human cervical epithelial cells.cells. ► The resulting cell lines display alteredThe resulting cell lines display altered growth properties and resistant in thegrowth properties and resistant in the response to signals for terminalresponse to signals for terminal differentiation.differentiation.
    19. 19. Immortalization andImmortalization and TransformationTransformation ►Transformation is "Transformation is "the introduction ofthe introduction of inheritable changes in a cell causing changes ininheritable changes in a cell causing changes in the growth phenotype and immortalisation". "thethe growth phenotype and immortalisation". "the introduction of inheritable changes in a cellintroduction of inheritable changes in a cell causing changes in the growth phenotype andcausing changes in the growth phenotype and immortalisation".immortalisation".
    20. 20. Immortalization andImmortalization and TransformationTransformation The transformed cells have abnormal growth parameters andThe transformed cells have abnormal growth parameters and behaviors:behaviors: ► Immortality: can grow indefinitelyImmortality: can grow indefinitely ► Reduced requirement for serum growth factorsReduced requirement for serum growth factors ► Loss of capacity for growth arrest upon nutrient deprivationLoss of capacity for growth arrest upon nutrient deprivation ► High saturation densitiesHigh saturation densities ► Loss of contact inhibitionLoss of contact inhibition ► Anchorage independent (can grown in soft agar)Anchorage independent (can grown in soft agar) ► Altered morphology (rounded and refractile)Altered morphology (rounded and refractile) ► Tumorogenic: can cause tumors when transplanted intoTumorogenic: can cause tumors when transplanted into animalsanimals
    21. 21. The cell cycle: Role ofThe cell cycle: Role of RbRb Cell growth regulated by an internal timer: cell cycle Divided into 4 phases 1. G1: cell growth, restriction point 2. S: DNA synthesis 3. G2: preparation for cell division 4. M: Mitosis
    22. 22. Cell cycle is controlled by the cyclin-Cdk machinery Different cyclins and cyclin dependent kinases expressed at different stages of the cell cycle. Rb protein: phosphorylation status of Rb used to control cell cycle •Rb phosphorylation: allows passage of G1 restriction point, entry into S-phase •Rb dephosphorylation: signals end of M phase. This stage represents the active stage of Rb because it inhibits cell cycle progression.
    23. 23. RbRb ► pRb functions in growth control by its bindingpRb functions in growth control by its binding ability and inhibition of various transcription factorsability and inhibition of various transcription factors namely E2 F-1and cMyc proteins.namely E2 F-1and cMyc proteins. ► These cMyc and E2 F-1 proteins activate some ofThese cMyc and E2 F-1 proteins activate some of the essential genes which are required for DNAthe essential genes which are required for DNA synthesis.synthesis. ► The HPV infected cell typically shows that pRb isThe HPV infected cell typically shows that pRb is inactive throughout in association with binding ofinactive throughout in association with binding of the E7 proteins.the E7 proteins. ► This decreased or inappropriate release ofThis decreased or inappropriate release of transcription factors favours the deregulatedtranscription factors favours the deregulated expression of genes which are related with normalexpression of genes which are related with normal control and cell divisioncontrol and cell division ..
    24. 24. Cell Regulation:Cell Regulation: p53p53 ► p53 is a tumor suppressor genep53 is a tumor suppressor gene ► Determines response of cells to DNA damage andDetermines response of cells to DNA damage and hypoxiahypoxia ► p53 promotes eitherp53 promotes either  Cell cycle arrest (until problem is fixed)Cell cycle arrest (until problem is fixed)  Apoptosis (unfixable problem)Apoptosis (unfixable problem) ► Virus infection is a stress that turns on p53Virus infection is a stress that turns on p53 ► Proteins from many viruses mislocalize or blockProteins from many viruses mislocalize or block p53 e.g. Adenoviruses, papillomaviruses,p53 e.g. Adenoviruses, papillomaviruses, polyomavirusespolyomaviruses
    25. 25. HPV and CancerHPV and Cancer ► Genital Cancers: Cervix, Vulva, Vagina,Genital Cancers: Cervix, Vulva, Vagina, Penis, Anus.Penis, Anus. ► Skin Cancer in patients withSkin Cancer in patients with epidermodysplastic verruciformis.epidermodysplastic verruciformis. ► Oral and tonsillar CancersOral and tonsillar Cancers ► Malignant progression of RecurrentMalignant progression of Recurrent Respiratory Papillomatosis.Respiratory Papillomatosis.
    26. 26. Worldwide Incidence ofWorldwide Incidence of Cancers Attributable to HPVCancers Attributable to HPV
    27. 27. Cervical cancer: 0.500 million in 2002 1 High-grade precancerous lesions: 10 million 2 Low-grade cervical lesions: 30 million 2 Genital warts: 30 million 3 AttributabletooncogenicHPVtypes HPV infection without detectable abnormalities: 300 million 2 Attributabletonon-oncogenicHPVtypes Very high global burden of HPV-related diseases HPV disease burdenHPV disease burden 1.Parkin et al. 2005 2.WHO 1999 3.WHO 1990 HPV 16, 18 and othersHPV 16, 18 and others HPV 6, 11 and othersHPV 6, 11 and others
    28. 28. HPV and CancerHPV and Cancer ►Greater than 100 subtypesGreater than 100 subtypes  About 15-20 cancer related types (type 16 andAbout 15-20 cancer related types (type 16 and 18 associated with 67% of cervical cancer)18 associated with 67% of cervical cancer)  All of these 15-20 subtypes associated withAll of these 15-20 subtypes associated with 99.7% of cervical cancer99.7% of cervical cancer ►Types 16,18, 31,33,35, 39, 45,Types 16,18, 31,33,35, 39, 45, 51,52,56,58,59,68, 72, (26,53,66): HIGH51,52,56,58,59,68, 72, (26,53,66): HIGH RISKRISK
    29. 29. The HPV Oncoproteins:The HPV Oncoproteins: E6E6 ► 150 amino acids in size.150 amino acids in size. ► Both high- and low-risk HPV E6 have similarBoth high- and low-risk HPV E6 have similar transcription activity, those from low risk typestranscription activity, those from low risk types have little/no transforming activity.have little/no transforming activity. ► Complexes with p53 and blocks theComplexes with p53 and blocks the transcriptional activity of p53. For binding with p53transcriptional activity of p53. For binding with p53 it needs a cellular protein of 100KD ----E6-AP.it needs a cellular protein of 100KD ----E6-AP.
    30. 30. The HPV Oncoproteins:The HPV Oncoproteins: E6E6 ► The E6-E6 AP complex targets ubiquitin targetedThe E6-E6 AP complex targets ubiquitin targeted substratessubstrates ► This results in ubiquitin targeted degradation of p53This results in ubiquitin targeted degradation of p53 protein.protein. ► The host cell is allowed to go from the p53 associatedThe host cell is allowed to go from the p53 associated negative growth control.negative growth control. ► E6 also activate expression of hTERT, the catalyticE6 also activate expression of hTERT, the catalytic subunit of telomerase : Activation of cellular Telomerasesubunit of telomerase : Activation of cellular Telomerase (synthesis of telomere) resulting in maintenance of(synthesis of telomere) resulting in maintenance of telomere length.telomere length.
    31. 31. The HPV Oncoproteins:The HPV Oncoproteins: E7E7 ► E7 protein is small nucleoprotein of 100E7 protein is small nucleoprotein of 100 amino acidsamino acids ► It can bind to zinc and is phosphorylated byIt can bind to zinc and is phosphorylated by casein kinase IIcasein kinase II ► Can bind to cellular regulatory proteinCan bind to cellular regulatory protein product of retinoblastoma tumourproduct of retinoblastoma tumour suppressor gene pRB and related proteinssuppressor gene pRB and related proteins p107 and p130.p107 and p130.
    32. 32. The HPV Oncoproteins:The HPV Oncoproteins: E7E7 ► E7 binds preferentially to the hypo (de-)E7 binds preferentially to the hypo (de-) phosphorylated form of pRb resulting in itsphosphorylated form of pRb resulting in its functional inactivation through the release of E2Ffunctional inactivation through the release of E2F transcription factor. This permits the cell to entertranscription factor. This permits the cell to enter into the S phase of cell cycle resulting in cellinto the S phase of cell cycle resulting in cell proliferation.proliferation. ► It also interacts with cyclin dependent kinaseIt also interacts with cyclin dependent kinase inhibitors like p21, p27 which helps the cell ininhibitors like p21, p27 which helps the cell in overcoming TGF-overcoming TGF-ββ associated growth arrest.associated growth arrest.
    33. 33. HPV virologyHPV virology NonproliferatingNonproliferating cellcell p53p53 pRBpRB G0: quiescent stateG0: quiescent state G1: gapG1: gap phasephase G2: gapG2: gap phasephase Cells exitCells exit cycle herecycle here M phase:M phase: mitosismitosis S phase: DNAS phase: DNA synthesissynthesis HPV stimulates proliferationHPV stimulates proliferation HPVHPV G1: gapG1: gap phasephase G2: gapG2: gap phasephase M phase:M phase: mitosismitosis Non-stopNon-stop proliferationproliferation P53P53++E6E6 pRBpRB++E7E7
    34. 34. Normal Cervix HPV Infection Persistent HPV Infection Cervical Dysplasia (pre-cancer) Cervical Cancer Cervical Cancer BiologyCervical Cancer Biology
    35. 35. Classification of Histological Findings:Classification of Histological Findings: Cervical Intraepithelial NeoplasiaCervical Intraepithelial Neoplasia ► Cervical intraepithelial neoplasia (CIN)Cervical intraepithelial neoplasia (CIN)11  CIN 1: Mild dysplasia; includes condyloma (anogenitalCIN 1: Mild dysplasia; includes condyloma (anogenital warts)warts)  CIN 2: Moderate dysplasiaCIN 2: Moderate dysplasia  CIN 3: Severe dysplasia; includes CISCIN 3: Severe dysplasia; includes CIS 1. Bonnez W. In: Richman DD, Whitley RJ, Hayden FJ, eds. Washington, DC: American Society for Microbiology Press; 2002:569–612. 2. Ostor AG. Int J Gynecol Pathol. 1993;12:186–192. CINCIN11 NormalNormal CIN 1CIN 1 (condylom(condylom a)a) CIN 1CIN 1 (mild(mild dysplasia)dysplasia) CIN 2CIN 2 (moderate(moderate dysplasia)dysplasia) CIN 3CIN 3 (severe dysplasia/CIS)(severe dysplasia/CIS) InvasiveInvasive CancerCancer Histology ofHistology of squamoussquamous cervicalcervical epitheliumepithelium11 CIN caused by HPV can clear without treatment.2
    36. 36. Life Cycle - Initial Infection (Skin)Life Cycle - Initial Infection (Skin) micro-traumamicro-trauma EpidermisEpidermis (Keratinocytes)(Keratinocytes) BasalBasal CellsCellsBasementBasement MembraneMembrane DermisDermis virionsvirions Slide courtesy M. Kast
    37. 37. Gene Expression (Wart)Gene Expression (Wart) BasementBasement MembraneMembrane EpidermisEpidermis DermisDermis Viral ProteinsViral Proteins Early only Capsid & Early NoneNone Progeny virionsProgeny virions
    38. 38. Immune EvasionImmune Evasion BasementBasement MembraneMembrane Draining LymphaticsDraining Lymphatics EpidermisEpidermis DermisDermis LangerhansLangerhans cellscells DendriticDendritic cellscells
    39. 39. Doorbar, J Clin Virol 32:7-15, 2005 Progression to Cancer is Accompanied byProgression to Cancer is Accompanied by Deregulation of Viral Gene ExpressionDeregulation of Viral Gene Expression CIN 1 CIN 2 CIN 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.
    40. 40. Natural History of HPV Infection:Natural History of HPV Infection: Surrogate Markers for Cervical CancerSurrogate Markers for Cervical Cancer 0 to 5 Years Up to 20 Years Initial HPV Infection CIN 1 Cleared HPV Infection Sq. Cell Carcinoma CIN 2 Persistent Infection CIN 3 Adeno- CarcinomaAIS
    41. 41. HPV and Cancer: TheHPV and Cancer: The ConclusionConclusion Cervical and other genital cancers are composed of cells that have lost the ability to differentiate, a process necessary for productive replication of HPVs.  In addition, cervical cancers can often contain integrated HPV genomes rather than episomes and viral DNA needs to exist episomally in order to be productively replicated.  Not surprisingly, cancer cells do not produce HPV virions.  Since a primary goal of infection is to generate new virions, the induction of cancers by HR HPV types is a ‘dead end’ for the virus.  In fact, it would seem to be in the interest of the virus to avoid promoting malignancy.  However, the persistent nature of HPV infections allows cellular genetic changes to accumulate over extended periods of time until a combination of genetic abnormalities has occurred allowing cancer to develop.
    42. 42. HPV and Cancer: TheHPV and Cancer: The ConclusionConclusion ► E6 and E7 themselves contribute to the accumulation ofE6 and E7 themselves contribute to the accumulation of mutations in the cellular genome by promoting genomicmutations in the cellular genome by promoting genomic instability during persistent infectionsinstability during persistent infections ► E6 and E7 induce centrosomal abnormalities resulting inE6 and E7 induce centrosomal abnormalities resulting in abnormal segregation of chromosomes and aneuploidy.abnormal segregation of chromosomes and aneuploidy. E7 causes centrosome reduplication leading to abnormalE7 causes centrosome reduplication leading to abnormal numbers of centrosomesnumbers of centrosomes ► In addition, abrogation of cell cycle checkpoints throughIn addition, abrogation of cell cycle checkpoints through the targeting of p53 and pRb family members allowsthe targeting of p53 and pRb family members allows retention of cells with chromosomal abnormalitiesretention of cells with chromosomal abnormalities ► What advantage the virus gains by promoting cellularWhat advantage the virus gains by promoting cellular genome instability is not clear.genome instability is not clear.
    43. 43. THE VACCINETHE VACCINE ►Work started in 1980s, CollaborationWork started in 1980s, Collaboration between Georgetown University medicalbetween Georgetown University medical centre, Rochester University, University ofcentre, Rochester University, University of Queensland, and National Cancer Institute.Queensland, and National Cancer Institute. ► GARDASIL (Merck) approved by FDAGARDASIL (Merck) approved by FDA (USA) in 2006.(USA) in 2006. ► CERVARIX (GSK) approved in Australia,CERVARIX (GSK) approved in Australia, EU (2007) and in USA in 2009.EU (2007) and in USA in 2009.
    44. 44. The VaccineThe Vaccine ► Subunit Vaccines, containing hollow Virus LikeSubunit Vaccines, containing hollow Virus Like Particles (VLPs) containing the L1 Capsid protein..Particles (VLPs) containing the L1 Capsid protein.. ► Generated by recombinant genetic engineering inGenerated by recombinant genetic engineering in yeasts.yeasts. ► Amorphous Aluminum Hydroxyphosphate SulfateAmorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (225Adjuvant (225 μμg per dose)g per dose) ► No Mercury preservativeNo Mercury preservative ► Storage: 2 to 8Storage: 2 to 8 OO CC ► Gardasil: HPV 6,11,16, 18.Gardasil: HPV 6,11,16, 18. ► Cervarix: HPV 16,18.Cervarix: HPV 16,18.
    45. 45. The VaccineThe Vaccine ► CervarixCervarix has 20 mcg each of HPV 16 and HPV 18has 20 mcg each of HPV 16 and HPV 18 L1 proteins;L1 proteins; GardasilGardasil has 20 mcg of HPV 6, 40has 20 mcg of HPV 6, 40 mcg of HPV 11, 40 mcg of HPV 16, and 20 mcg ofmcg of HPV 11, 40 mcg of HPV 16, and 20 mcg of HPV 18HPV 18 ► Target Population: Routine ImmunizationTarget Population: Routine Immunization Cervarix: Females 10-25 yrs, Gardasil: Females 9-Cervarix: Females 10-25 yrs, Gardasil: Females 9- 26 yrs.26 yrs. ► ACIP: Non routine immunization of males 9-18ACIP: Non routine immunization of males 9-18 yrs.yrs. ► Both Vaccines: THREE Doses; 0, 1-2 mts, 6 mts.Both Vaccines: THREE Doses; 0, 1-2 mts, 6 mts.
    46. 46. The VaccineThe Vaccine ► Injection Site:Injection Site:  Upper arm (Deltoid region)Upper arm (Deltoid region)  Thigh (higher anterolateral area)Thigh (higher anterolateral area) ►0.5 mL volume0.5 mL volume IM injectionIM injection ►Package:Package:  Prefilled syringePrefilled syringe  Shake well before useShake well before use ► Appx Cost: ~ US$ 450 for full vaccinationAppx Cost: ~ US$ 450 for full vaccination
    47. 47. Prophylactic VaccineProphylactic Vaccine ► Both are quite effective at preventing HPV 16/18Both are quite effective at preventing HPV 16/18 persistent infections and the associated CIN 2+persistent infections and the associated CIN 2+ caused in women of 15-26 years oldcaused in women of 15-26 years old with nowith no current HPV infection at the time of initialcurrent HPV infection at the time of initial vaccinationvaccination ► However, if the girl/woman is currently infectedHowever, if the girl/woman is currently infected with and producing the HPV 16 or 18 virus at thewith and producing the HPV 16 or 18 virus at the time of vaccination, the vaccines will not stop thetime of vaccination, the vaccines will not stop the HPV from progressing to CIN 2+.HPV from progressing to CIN 2+. ► They show no therapeutic efficacy against currentThey show no therapeutic efficacy against current infectioninfection
    48. 48. MECHANISM OF ACTION
    49. 49. HPV L1 VLP vaccineHPV L1 VLP vaccine Mimic natural virion structure generate potent immune responseMimic natural virion structure generate potent immune response
    50. 50. HPV L1 VLP vaccineHPV L1 VLP vaccine APC HPV L1 VLP IgG B T APC present MHC-Ag complex and co-stimulation signal Plasma cellPlasma cell Plasma cellPlasma cell Ig switch IgG 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
    51. 51. Protective EfficacyProtective Efficacy ► The FUTURE I (Females United to Unilaterally Reduce Endo/Ectocervical Disease)Study: Three- year phase 3 trial examined the effectiveness of the quadrivalent vaccine in preventing genital diseases associated with HPV types 6, 11, 16, and 18. ► The vaccine was nearly 100 percent effective in preventing precancerous lesions (CIN 2 and CIN 3) and genital warts when given to unexposed women. (NEJM 2007)
    52. 52. Protective EfficacyProtective Efficacy ► FUTURE II trial, showed that the quadrivalent vaccine is 98 percent effective in preventing high- grade precancerous cervical lesions (HPV 16, 18) in unexposed women over a three-year period ( NEJM 2007) ► PATRICIA (Papilloma Trial Against Cancer in Young Adults) trial : The bivalent vaccine is 98 percent effective in preventing precancerous cervical lesions (CIN 2 and CIN 3) caused by HPV types 16 and 18, and may provide some cross- protection against types 31, 33, and 45.38 (Lancet, 2009)(Lancet, 2009)
    53. 53. Measurement of induced antibodies to HPV 16 and 18 by Cervarix and Gardasil one month after the 3rd dose (month 7) among 18-45 year old women. This figure shows that Cervarix induces a significantly higher antibody response after three doses of vaccine than Gardasil
    54. 54. Protective Efficacy: Cross-ProtectionProtective Efficacy: Cross-Protection ► Gardasil potentially offers disease protection against additional oncogenic HPV types 31, 33, 45, 52, 58 (43-62%) ► Cervarix (Bivalent): Cross protection 31- 60%.
    55. 55. Duration of ProtectionDuration of Protection ► At least five years.At least five years. ► Phase II trials provide the longest follow-upPhase II trials provide the longest follow-up data for Gardasil at 5 years and for Cervarixdata for Gardasil at 5 years and for Cervarix at 8.4 years (Villa et al., 2006; The GSKat 8.4 years (Villa et al., 2006; The GSK HPV Vaccine 007 Study Group, 2010).HPV Vaccine 007 Study Group, 2010). ► Modeling has shown that HPV vaccinesModeling has shown that HPV vaccines must maintain their near 100% efficacy for amust maintain their near 100% efficacy for a full 15 years, at minimum, in order forfull 15 years, at minimum, in order for cervical cancers to be preventedcervical cancers to be prevented
    56. 56. Sustained clinical efficacySustained clinical efficacy and antibody titerand antibody titer forfor at leastat least 5 years5 years 10 000 1 000 100 10 GMT (mMU/mL) GARDASIL Natural infection 0 7 12 18 24 30 36 54 60 months GARDASIL 100% 1st 2nd 3rd Dose Clinicalefficacy* Neutralisingantibodies (HPVtype16) 18 5 years * against infection, CIN (Cervical intraepithelial neoplasia) and genital warts due to HPV types 6,11,18; 5 yrs follow up (after dose 1) of a subset (241 women, vaccine & placebo) from a phase II efficacy study Villa L High Sust Eff Proph Quad HPV Vacc 5 Year Followup Br J Can 2006 95 1459
    57. 57. Duration of ProtectionDuration of Protection ► Induced antibodies against the L1 VLP for HPVInduced antibodies against the L1 VLP for HPV 16 are high and are sustained for both Cervarix16 are high and are sustained for both Cervarix and Gardasil for their 8.4 years and 5 years ofand Gardasil for their 8.4 years and 5 years of protection, respectivelyprotection, respectively ► Induced antibodies for HPV 18 from Gardasil fallInduced antibodies for HPV 18 from Gardasil fall quickly after peak with 35% of women having noquickly after peak with 35% of women having no measurable antibody titers to HPV 18 at 5 years.measurable antibody titers to HPV 18 at 5 years. Cervarix titers remain several fold above naturalCervarix titers remain several fold above natural infection titer for HPV 18.infection titer for HPV 18. ► Natural infection titers are not protective againstNatural infection titers are not protective against future type specific infections.future type specific infections.
    58. 58. SafetySafety ► Autoimmune demyelinating neurologic sequelae afterAutoimmune demyelinating neurologic sequelae after Gardasil administration, resulting in blindness, paralysis,Gardasil administration, resulting in blindness, paralysis, and death (GB Syndrome): 13 Casesand death (GB Syndrome): 13 Cases ► Deaths: 11 casesDeaths: 11 cases ► Causal relationship between the events and theCausal relationship between the events and the vaccinationvaccination has not beenhas not been establishedestablished ► From the studies, number of death cases wereFrom the studies, number of death cases were notnot differentdifferent between vaccine groups and placebo groupsbetween vaccine groups and placebo groups ► Total doses: >18 millionTotal doses: >18 million ► CDC and FDA were satisfied with the safety dataCDC and FDA were satisfied with the safety data ► Large scale clinical trials have also shown well toleratedLarge scale clinical trials have also shown well tolerated safety profile.safety profile.
    59. 59. Controversy: IndiaControversy: India ► Gardasil dissemination programs in AndhraGardasil dissemination programs in Andhra Pradesh and Gujarat were suspended in AprilPradesh and Gujarat were suspended in April 2010 by the Indian government despite an annual2010 by the Indian government despite an annual cervical cancer incidence rate of 28 women percervical cancer incidence rate of 28 women per 100,000 which is over three times that of the US100,000 which is over three times that of the US and seven times that of Finland.and seven times that of Finland. ► The most disquieting objection to the program,The most disquieting objection to the program, was the lack of information provided to the publicwas the lack of information provided to the public so that each participant could be afforded theso that each participant could be afforded the opportunity for informed decision making aboutopportunity for informed decision making about their cervical cancer protection.their cervical cancer protection.
    60. 60. Future Direction: TherapeuticFuture Direction: Therapeutic VaccineVaccine The oncoprotein E7, is expressed in transformedThe oncoprotein E7, is expressed in transformed cells but thought to represent a poorlycells but thought to represent a poorly immunogenic antigen.immunogenic antigen. ► Many in vivo studies indicate that miceMany in vivo studies indicate that mice vaccinated using a plasmid-encoded E7 fusionvaccinated using a plasmid-encoded E7 fusion protein are protected against in vivo tumorprotein are protected against in vivo tumor challenge.challenge.
    61. 61. Future Direction: TherapeuticFuture Direction: Therapeutic VaccineVaccine ► Researches demonstrated that protectionResearches demonstrated that protection against tumor growth is associated with theagainst tumor growth is associated with the development of a CD4-regulated, E7-specificdevelopment of a CD4-regulated, E7-specific CTL activity mediated by CD8 cellsCTL activity mediated by CD8 cells ► Recent researches are focused on aRecent researches are focused on a DNA-DNA- based vaccinationbased vaccination protocol aimed at inducingprotocol aimed at inducing an efficient anti-E7 immune response in vivo.an efficient anti-E7 immune response in vivo.
    62. 62. Eradication of Cervical Cancer andEradication of Cervical Cancer and HPV Related DiseasesHPV Related Diseases Not a Dream Anymore…Not a Dream Anymore…
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