Lecture 18- HBV pathogenesis Flashcards
what cancer does HBV cause?
liver cancer
HBV history
1965- australian antigen in Aboriginal, reacted with serum from a haemophilia patient
1968- antigen was particulate consisting predominantly of spherical particles and filamentous forms
1970- additional double shelled particles found
1979- full genome sequence published
HBV is part of what family?
Hepadnaviridae virus family
1st member
route of infection for HBV
blood
what type of virus is HBV?
pararetrovirus- DNA genome with an RNA intermediate
HBV-like viruses that have been discovered
orthohepadnaviruses
avihepadnaviruses
what bird HBV can infect human cells?
bat HBV
where is chronic HBV highly prevalent?
asia pacific and africa
rates of HBsAg positivity in indigenous australians
10x higher
- novel C4 subgenotype (not present anywhere else in the world)
how many people in australia with chronic HBV?
239 000 person
-90 000 unaware they are infected
why wont our preventative vaccination program help?
only helps for those that have not been infected
- negligible impact on the number of people with chronic HBV infection
what is HBsAg
a marker of chronic infection
4 phases of the natural history of CHB
- immunotolerance- no liver damage, no activity of immune system high levels of DNA
- immunoactive phase- DNA levels fluctuate, maybe clearing of infection
- low replicative phase- nothing appears to be happening, inactive carriers (not treated at this stage), viral integration occurs (5x more likely to develop liver disease)
- high replicative phase- immune system wakes up again, stimulates immune response, liver damage
when is HBeAg + and -
immunoactive phase- HBeAg +
high replicative phase- HBeAg -
natural history of chronic HBV infection
acute infection chronic hepatitis cirrhosis liver cancer death 30-50 years
under normal circumstances, HBV is not…
cytopathic
- liver damage is caused by host’s cellular immune response to HBV infected hepatocytes as part of immune clearance phase (cytotoxic T cell clearance)
HBV pathogenesis represents?
the outcome of the interplay between the virus, the hepatocyte, and hosts immune response
how many major genotypes in CHB?
9 (A to I)
which genotype has more severe disease progression?
genotype C
C> B
D>A
what is perinatal/vertical transmission? which genotypes?
mother to baby- shortly after birth will develop unless treated
genotypes B and C
which genotypes for horizontal transmission?
genotypes A, D-H
what is unique about genotype J?
combination of orangutan and human virus (Japan)
- created his own virus
what is unique about A1 genotype ?
Genotype A prevalent in sub-saharan africa
- but also rapid progression to liver cancer in black american boys NOT girls
which genotype is the most widespread?
genotype D
HCC
hepatocellular carcinoma
mechanisms by which we think HBV causes HCC
- high levels of HBV replication
- persistent HBsAg protein (envelope protein)
- truncated HBV proteins (preS deletions)
- integrated HBV DNA (viral DNA integration)
- genotype (C is most oncogenic)
- splicing/novel proteins?
pgRNA (pregenomic RNA) role
RNA intermediate
- plays a critical role in viral replication
- the mRNA for core and polymerase proteins
- transcriptional template for DNA synthesis
cause of high mutation frequency
absence of proof reading during reverse transcription
- results in quasispecies pool of mutant viruses dependent on immune pressure
shape of HBV genome
what does this allow for?
circular
- makes longer RNA than genome
- 3.5kb RNA (longest piece of RNA)
HBsAg
Hep B surface antigen
- envelope protein
HBcAg
Hep C core antigen
- capsid protein
HBeAg
Hep B e antigen
- secreted truncated form of HBcAg
Hep B polymerase
- multi function enzyme
DNA polymerase, reverse transcriptase, RNase H
HBxAg
hepB x antigen
- generalised transactivator
- only occurs in mammalian hepadnaviruses
- critical for expression of cccDNA (viral expression)
what is the Pre-S1 domain important for?
Pre-S1 domain of the HBV large envelope protein critical for entry
cellular receptor that facilitates entry through binding to Pre-S1 protein
sodium taurocholate co-transporting polypeptide (NTCP)
other role of NTCP
uptake of conjugated bile salts, metabolites, and drugs from portal blood into hepatocytes
the HBV replication cycle
- virus binds to NTCP receptor and enters hepatocyte
- de-envelopment- envelope of virus removed, nuclear capsid remains, encodes dsDNA
- dsDNA enters nucleus
- circular genome has gap in positive strand, forms a complete dsDNA
- converted into cccDNA then converted to minichromosome
- cccDNA- transcriptional template for all the viral RNAs or
- pgRNA- encapsulated in nuclear capsid with polymerase protein
- pgRNA reverse transcribed into minus sense DNA
- RNAsH chops up DNA, plus sense DNA forms
- replication cycle occurs within nuclear capsid
- coated in envelope protein, leave cell and infect more cells OR
before coating, goes back to nucleus, make more of this cccDNA due to nucleus localisation signal (replenishment pathway)
why is cccDNA bad?
covalently closed circular DNA
- very important, major barrier to cure
cccDNA in those who have cleared acute infection
people who have cleared their acute infection still have cccDNA in nucleus
- go into immunosuppressive drug therapy in future, hepB emerges
what do current therapies for hepB target and not target?
target- viral replication outside in the cytoplasm
not target- cccDNA hiding away in nucleus
why are drugs for HepC more successful (curable) compared to HepB?
HepC- whole lifecycle out in cytoplasm, no reservoir
HepB- has cccDNA in nucleus
major integration pathway in HBV replication cycle
HepB can make linear DNA, integrate into our DNA, 10% of the time, integration associated with liver cancer
virion production
10*12-13 virions per day
mutational frequency
high mutational rate; lack of proofreading capacity of HBV Reverse transcriptase
biological implications of quasispecies
- escape mutants to immune response
- escape mutants to vaccination
- resistance to antiviral therapy
current treatment for HBV
- nucleoside/nucleotide RT inhibitors (NAs)
- immune modulators - IFN-alpha
what is bad about current treatment?
these are not cures (due to persistence of cccDNA)
- does not target the viral nuclear reservoir of cccDNA
- HBV readily develops resistance to most NAs in setting of monotherapy
- also doesnt impact integrated HBV DNA
recent drug that has shown no resistance
tenofovir
where do the phases of HBV lifecycle occur?
nuclear (cccDNA) and cytoplasmic phases
what does cccDNA produce?
continual source of viral mRNAs and proteins which are associated with disease progression
- HBsAg, HBx antigen
two major barriers to curing CHB
viral - cccDNA - HBsAg -continual source immunological - T cell exhaustion - emerging role of inadequate B cell responses
explain the immunological barrier to CHB cure
T cell exhaustion
- T cells dont work very well to clear infected cells, wake up now and then, causing immune system to flare, produce T cell activity, in some people this clears infections, most people not enough to clear infection
inadequate B cell responses
functional cure
cleared the viral load- undetectable serum DNA
no activity of virus
but still has cccDNA- not transcriptionally active, therefore allow treatment cessation
complete cure
cccDNA clearance
what suggests that functional cure is a realistic goal?
functional HBV cure (HBsAg seroclearance) is observed in a small number of patients, either spontaneously or in setting of antiviral therapy
viral targets to cure HBV infection
- RNA interference
- targeting HBsAg
- polymerase inhibitors
- inhibition of nucleocapsid assembly (prevent recycling to nucleus to make more cccDNA)
- targeting cccDNA (tried with CRISPR-Cas9, but making new molecule, dont know what it will do)
- entry inhibitors (block entry of HepB)
- likely need a combination that is going to hit various parts of the viral replication cycle
combination therapy for HBV cure
combination therapy with direct antiviral and immune restoration
immune restoration as therapy for HBV
positive and problem?
stimulate the immune response
- restore phenotype of exhausted T cell response
PROBLEM
- if you have liver cancer, want to activate every cell for an immune response, you will activate liver cells and kill liver as well
continued expression of HBsAg is associated with?
disease progression–>liver cancer
- failure to clear HBsAg prior to age 50, associated with increased risk of HCC, even in absence of detectable viremia
HBV splice variants are associated with?
liver cancer
how are spliced transcripts replicated?
by WT polymerase in trans (virus) as spliced transcripts do not encode Pol
how much viral RNA and DNA is produced by splicing?
50%- huge part of viral pool
what is the most common transcript and what does it encode?
Sp1 encodes a novel protein- hepatitis B splice protein (HBSP)
what does splicing remove and what is the consequence?
removes cis-acting sequences which promote circular DNA, leading to linear DNA
- linear DNA integrates into host genome
- integration strongly associated with liver cancer
what is HBSP associated with?
increased cell proliferation
increased with liver disease progression
promotes carcinogenic effects in the liver
results from a pilot study to assess association between splice variants and cancer?
patients with liver cancer had higher median secrete Sp/wt HBV levels than those with chronic HBV infection alone
results from taiwanese cohort study to assess association between splice variants and cancer?
subjects with spHBV DNA level were 8.5 times more likely to develop liver cancer
- if splice variants formed more than 20% of viral pool, then 23.3 times more likely to get HCC
potential biomarker of liver cancer?
spliced DNA
