GI & Hepatology JC057: I Am A Hepatitis B Carrier Flashcards
Hepatitis B structure
Structure of virion (Dane particle)
- 42 nm (tiny)
- Circular surface
- Hexagonal core (HBcAg)
- Double-stranded DNA, long Minus strand, short Plus strand
- Excessive HBsAg in Circular (22nm) / Tubular form
Hepatitis B molecular virology
- smallest DNA virus
- approx. 3,200 nucleotides
- overlapping ***open reading frame for “economical” manufacture of proteins
- regulatory sequences within the genes, i.e. nucleotide both encode for proteins + regulate their production e.g. Glucocorticoid responsive element (GRE), Enhancer (enhance replication of virus)
Proteins + Regulatory sequences
- Gene S
- produces HBsAg - Pre-S2
- encodes polyalbumin-binding sites
- encodes peptides ***binding to cell surface receptors —> allow virus to enter host cell
- Pre-S2 + S produces “middle” protein - Pre-S1
- encodes peptides ***recognised by cell surface receptors for HBV
- Pre-S1 + Pre-S2 + S produces “large” protein
3 different types of HBsAg:
1. Pure S
2. Middle protein (Pre-S2 + S)
3. Large protein (Pre-S1 + Pre-S2 + S)
- Role in hepatocarcinogenesis of pre-S1 / S2 uncertain (probable)
- Gene C
- encodes HBcAg
- HBcAg found inside virus / hepatocytes, **NOT in serum (∴ can only detect Ab against HBcAg but not HBcAg itself)
- **HBeAg, its “derivative” —> secreted in serum (can be detected) - Pre-C
- 1st 19 a.a. constitutes signal peptide to change Pre-core to **HBeAg via ER, Golgi —> secreted in serum (can be detected)
- Mutant strain (Pre-C mutation) with stop codon (TAG) causes inability to produce HBeAg —> even when virus is replicating, but cannot detect level of HBeAg
- Mutant strain exist around **e-seroconversion ∵ host immune response act against HBeAg —> select for mutant strains which does not produce HBeAg —> HBeAg-deficient mutants emerge —> so host immune response cannot recognise virus, virus can reproduce without attacked by host immune response
- IMPORTANT: patients with be HBeAg -ve, **Anti-HBe +ve, HBV DNA +ve —> but virus still replicating: “*False e-seroconversion”
- among Anti-HBe patients, >90% in Mediterranean countries, ~55% in HK, China, TW - Gene P
- encodes **polymerase (exactly identical to RT) + **reverse transcriptase (RT)
- overlap with most reading frames - Glucocorticoid responsive element
- regulates the “Enhancer” and is stimulated by **steroid (i.e. Steroid stimulates replication of HBV)
—> **Enhancer 1 stimulates **protein expression (esp. **Core protein) - Enhancer 2
- stimulates ***Surface gene promoters - Region X
- encodes products for ***transactivation (activate adjacent genes when incorporated into host DNA)
- ?related to carcinogenesis (e.g. insert near oncogene) - Direct repeat 1 (DR1)
- 11 nucleotides (exactly same as DR2), initiates **long strand synthesis
- together with DR2 are preferential sites for **integration into host DNA
(NB DR1, 2 coincide with Region X) - Direct repeat 2 (DR2)
- 11 nucleotides (exactly same as DR1), initiates ***short strand synthesis
***Replication of HBV
Virion envelop surrounds nucleocapsid which contains:
- ***Relaxed circular HBV DNA
—> partially double-stranded
—> Minus strand: full length
—> Plus strand: variable length
Process:
- Entrance of virus by Adsorption via cell surface receptors
—> release of relaxed circular HBV DNA
—> **completion of Plus strand DNA (complete double-stranded circular DNA / cccDNA)
—> transport to nucleus
—> conversion of relaxed circular DNA to **supercoiled covalently closed circular DNA (cccDNA) exclusively inside the **nucleus of hepatocytes (make virus very hard to be attacked directly)
—> transcription of **4 mRNA classes including **“pregenomic mRNA” of greater than one unit length of virus
—> mRNA transportation to cytoplasm
—> cytoplasmic mRNA translated into **viral proteins (e.g. HBsAg, HBcAg) (which hide inside ER)
—> pregenomic mRNA translated to form **HBV DNA polymerase (DNA pol) / reverse transcriptase (RT)
—> **encapsidation of DNA pol bound to pregenomic mRNA
—> reverse transcription of pregenomic mRNA to produce full length Minus strand DNA (造翻d viral DNA出黎)
—> Plus strand DNA synthesis by HBV DNA polymerase (RT act as DNA pol as well)
—> partially double-stranded relaxed circular DNA
—>
1. migration to ER to collect viral proteins (e.g. HBsAg, HBcAg)
—> forms whole virion
—> acquisition of envelops
—> exocytosis
- Migration to nucleus and completion of Plus strand DNA
—> cccDNA
—> amplification / replenishment of cccDNA
—> whole reproductive process all over again
—> virion multiply rapidly
cccDNA:
- 10-50 copies per cell
- no. of copies probably regulated by viral envelop protein(s)
- no direct replication (only replicates when form pregenomic mRNA) + hide inside nucleus —> ∴ difficult to attack cccDNA
簡單而言:
Transport to nucleus
—> relaxed circular HBV DNA conversion to cccDNA
—> transcription of mRNA
—> some mRNA form HBsAg, HBcAG
—> pregenomic RNA encapsidated in cytoplasm
—> formation of Minus-strand HBV DNA by RT
—> formation of Plus-strand HBV DNA by DNA pol
—> double-stranded DNA acquires HBsAg —> exocytosis
OR
—> double-stranded DNA replenishes cccDNA
Epidemiology of HBV
- 257 million chronic HBV carrier
- ***75% Chinese
- high prevalence in China, Africa, Australia
- estimated HBsAg carrier in China: 83,864,139
- **very low prevalence in younger subjects currently (∵ **vaccination at birth)
Transmission of HBV
***Parenteral only (i.e. ∵ at least require mucosal breach —> saliva cannot transmit HBV)
- At birth
- Early postnatal period
- Playmates
- Needles: IV addicts, Acupuncture (disposable needles now), Tattoo
- Sexual contact
- Other instruments e.g. toothbrushes, razors
HBsAg +ve mothers
—> HBsAg +ve infants (50%) (father can also transmit to infants)
—> Daughters
—> becomes HBsAg +ve mothers
—> transmit to next generation
—> 14% HBsAg +ve mothers die of liver death (cirrhosis / liver cancer)
—> Sons
—> 50% die of liver death (cirrhosis / liver cancer)
Transmission by Saliva
- Report of high levels of HBV DNA in saliva of HBeAg +ve children / adults (though still lower than in plasma)
- Potential modes of transmission include premastication of food, sharing sweets
- No definite proof of transmission
Level of saliva required to transmit HIV: 1L
Amount of saliva produced per day: 1.5L
***Immune response to HBV
Virion uptake by Hepatocyte (i.e. infected)
—> Replication
—> Viral peptide display on surface of Hepatocyte (in infants covered up by maternal Anti-HBc)
—> T-cell response
—> T-cell cytolysis / Anti-virion Ab —> Phagocytosis
Chances of chronicity in HBV infection
- Neonates and 1st year: 90%
- 1-6 yo: 30%
- > 6 yo: 2% (probably <1%)
Possible reasons for Chronicity in Neonates
Host factors:
1. Failure of host to recognise infected hepatocytes (e.g. covering of viral Ag by maternal Anti-HBc —> fetal T cells cannot detect the hepatocyte as being infected —> maternal Anti-HBc can exist in infants for up to 1 year)
Viral factors:
1. Excessive production of HBsAg
- acts as “decoy” for HBV specific humoral + T cell response
- leads to modulation of immune signalling pathways with suppression of inflammatory cytokines (T cells cannot recognise hepatocytes as being infected)
- ***HBx protein
- inhibits degradation of viral protein thus ↓ Ag presentation (Ag require degradation of viral protein first) on surface of hepatocytes - ***Polymerase protein
- suppress myeloid differentiation protein —> ↓ Toll-like receptor (TLR) function - ***Pre-core / HBeAg
- down-regulate TLR-2 expressions on Kupffer cells, hepatocytes, monocytes (of infant)
- down-regulate CD28 on T cells, CD86 on monocytes, Kupffer cells (of infant)
Typical profile of Hep B serological markers
- Incubation period (4-12 weeks)
—> Acute infection (2-12 weeks) (Symptoms occur)
—> HBsAg ↑ then ↓
—> HBeAg ↑ then ↓
—> Anti-HBc ↑ - Recent acute infection (12-16 weeks)
—> Anti-HBc level off
—> Anti-HBe ↑
—> Anti-HBs ↑ (~16 weeks)
—> Recovery
(NB: Anti-HBs is protective, Anti-HBc is not protective)
Hep B ***Chronic carrier serological markers profile
No seroconversion at all (***No Anti-HBs)
- HBsAg ↑ + remains high
- HBeAg ↑ + remains high before **Anti-HBe ↑ after **several decades (e-seroconversion)
- Anti-HBc ↑ + remains
- Anti-HBc IgM ↑ then ↓
***Serological markers for HBV
HBsAg: +
Anti-HBs: -
Anti-HBc: +
1. Acute infection —> check IgM Anti-HBc
2. Chronic carrier (Anti-HBs not appear at all even after decades, Anti-HBe appear decades later)
HBsAg: +
Anti-HBs: -
Anti-HBc: -
- Uncommon, in very early phase of incubation period of acute infection (before production of Anti-HBc)
HBsAg: -
Anti-HBs: +
Anti-HBc: -
1. Past infection
2. Post vaccination
HBsAg: -
Anti-HBs: -
Anti-HBc: +
1. If IgM Anti-HBc +ve —> Acute infection (acute exacerbation of chronic Hep B —> IgM Anti-HBc can also become +)
2. Past infection
3. ***Occult Hep B infection (more common with use of potent immunosuppressants e.g. Anti-CD20)
HBsAg: -
Anti-HBs: +
Anti-HBc: +
1. Past infection
2. ***Occult Hep B infection (more common with use of potent immunosuppressants e.g. Anti-CD20)
(Occult Hep B: HBsAg -ve but have HBV DNA in serum / liver tissue)
(記住: different types of Hep B Ag are just markers!!! No specific indication and less sensitive than HBV DNA (most sensitive))
Hepatitis B pathogenesis
Damage to liver:
- HBV **NOT directly cytopathic
- Damage to liver through **cytolytic T cells + **cytokines (TNFα, IL1β)
- Proteolytic cleavage of viral proteins in infected hepatocytes (probably HBcAg) —> peptides carried and presented to cell surface by **class 1 HLA —> T cell response
Chronic HBsAg carrier:
- 25% die of liver diseases
—> 50% for male
—> 14% for female
***Natural history of Chronic Hepatitis B
- Viral tolerance phase
- **minimal host reaction
- **high EBV DNA, HBeAg +ve
- **normal AST, ALT (∵ very little reaction from host against virus —> little damage to liver)
- only mild histologic abnormality
- **lasts 2-3 decades - Viral clearance phase
- host tolerance suddenly decrease (?∵ HBV-specific thymocytes from thymus)
- **cytotoxic T cells kill HBV-infected hepatocytes
- **fluctuating HBV DNA
- **fluctuating AST, ALT
- histology shows **intermittent active hepatitis (aka chronic active hepatitis) +/- **cirrhosis onset
- T cell kill hepatocytes —> AST, ALT ↑ —> no. of infected cell ↓ —> immune reaction ↓ —> no. of infected hepatocyte ↑ (reinfection of hepatocytes) —> T cell kill hepatocyte ↑ (aka **exacerbation of chronic Hep B)
- multiple exacerbations before e-seroconversion - Late / Residual phase
- **e-seroconversion: HBeAg -ve, Anti-HBe +ve
- host response variable
- **HBV DNA levels relatively low
- ALT levels normal with ~30% having reactivation (HBeAg-negative hepatitis)
- **>70% of HCC / cirrhosis complications occur in Anti-HBe +ve phase, even with “normal” ALT
- e-seroconversion median age: **35, onset of all types of complications: **57 (already Anti-HBe +ve, in fact most complications occur **after e-seroconversion (∵ liver damaged during e-seroconversion))
Development of Cirrhosis
Occur more frequently with
1. Age
- Hepatic ***decompensation
- ***Repeated severe acute exacerbation
- AFP >100 ng/ml (produced by young liver cells, high level when regeneration of new liver cells)
- Bridging necrosis on biopsy
- Unsuccessful / Prolonged HBeAg seroconversion - HBV reactivation with ***HBeAg reversion after e-seroconversion (uncommon)
***Hepatitis episodes in Chronic Hep B
Can be with / without symptoms (symptoms of acute hepatitis)
Causes:
1. HBV-related
- “Spontaneous” reactivation: IgM Anti-HBc ↑ (but not as high as in Acute Hep B)
—> HBeAg clearance (spontaneous / during therapy)
OR
—> e-seroreversion (HBeAg -ve / Anti-HBe +ve/-ve —> HBeAg +ve) (uncommon)
- ***Emergence of resistant variants / Non-compliance during nucleoside analogue therapy
- ***Corticosteroid / other immune suppressants, esp. anti-CD20 —> withdrawal
- Superinfection by ***Hepatitis D
- Superinfection by other viral agents (e.g. HAV, HEV)
- Drug-induced hepatic injury (e.g. alcohol, TCM, herbal tea (涼茶))
***Factors associated with disease progression
- HBeAg seroconversion (Anti-HBe development)
- ALT levels
- HBV DNA levels
- HBsAg seroclearance +/- seroconversion (Anti-HBs development)
- 記住: HBsAg跌唔代表Anti-HBs一定出現
***1. HBeAg seroconversion (Anti-HBe development)
- Acute exacerbation probably triggered off by viral replication + viral protein
- ***Disease progresses after HBeAg seroconversion in patients who acquire HBV infection at birth / early childhood (i.e. Asians / some Mediterraneans)
—> Development of cirrhosis complications / HCC - Patients acquiring disease at birth / early childhood: disease progresses after e-seroconversion in a proportion of patients
—> Majority of complications occur after e-seroconversion - ***HBeAg-negative disease (active viral replication despite HBeAg -ve, Anti-HBe +ve)
—> Mediterraneans >90% precore mutation
—> Asian: 45-56.5% precore mutation; 41-69.5% core promoter mutations, ~10% precore + core promoter wild type
記住: 就算係唔係hidden Hep B (HBeAg-negative disease) —> disease都會progress —> 都會有complications
Conclusion:
- Disease can continue to progress after e-seroconversion
- Cirrhosis-related complications and HCC:
—> peak age ~55
—> >2/3 Anti-HBe +ve
- Disease progression likely related to ***prolonged continuing inflammatory destruction even at low-viral levels
- ALT levels
Normal (QMH):
- Male: <53
- Female: <45
Normal (American Liver Association 2018):
- Male: **<35
- Female: **<25
Except when ALT levels too high (100-300) / extremely high (>300)
—> Risk of complications is less than that of ALT 50-100 (∵ ?stronger immune response to clear away virus more rapidly)
Conclusions:
- ***Lower ALT levels —> Lower risk of complications
- ALT 25-50 has increased risk
- ALT 50-100 has highest risk
—> Implications: decide when to treat patients
- HBV DNA levels
REVEAL (Risk Evaluation of Viral Load Elevation Associated Liver Disease / Cancer) study
Conclusions:
- Higher HBV DNA levels —> Higher risk of developing Cirrhosis / HCC
- 80% of them even have HBeAg -ve + normal ALT
—> HBV DNA levels recognised as important for disease progression
Problems:
- Patients in immune tolerance phase has very high HBV DNA levels —> but disease not yet progressive —> liver has very little damage
—> so when to start treatment?
—> when patients are in ***immune clearance phase (HBe clearance phase) (but hard to define)
When to stop treatment?
- see “treatment” section
- HBsAg seroclearance +/- seroconversion (Anti-HBs development)
Spontaneous HBsAg seroclearance associated with:
- progressively more patients with undetectable HBV DNA (only 13.4% detectable after 1 year)
- majority have normal ALT levels
- near normal histology
- 100% had intrahepatic HBV DNA, but only 1 had detectable mRNA —> signifying low transcriptional level
Study showed:
- HBsAg clearance <50 yo —> almost 0% develop HCC (∵ less significant fibrosis of liver)
- HBsAg clearance >=50 yo —> higher risk of developing HCC (∵ significant fibrosis of liver)
—> although HBV can still cause HCC without going through cirrhosis stage (∵ Oncogenic virus itself: Region X, Pre-S2, Pre-S1)
(vs HCV / Alcoholic liver disease: must go through cirrhosis before HCC (CL Lai))
Conclusion:
- HBsAg seroclearance is the ideal end-point but still require HCC monitoring
Aims for Treatment of Chronic Hep B
Prevent / Decrease cirrhotic complications + HCC
Objectives:
1. Viral suppression + reduce liver damage
- maximal suppression of HBV DNA (PCR undetectable level)
- ↓ AST, ALT
- improvement in liver histology
- Viral eradication
- difficult ∵ cccDNA and Viral integration (hide inside host cell nucleus)
- loss of HBsAg occurs in <10% —> esp. difficult for carriers acquiring disease early in childhood (loss of HBsAg: commonly known as “functional endpoint”)
- loss of cccDNA
Current guidelines for indications of treatment
NEJM:
Patients with
1. Active viral replication (HBV DNA)
- HBV DNA levels indicated for treatment currently progressively lowered
- Active disease (↑ ALT levels)
- currently recognised that disease may be active with relatively low ALT level
—> recommended ALT levels for treatment: male >35, female >25
European Liver Association 2017 (least restrictive)
1. (**記) ALL patients
- e +ve / -ve
- with **ALT > ULN
- **DNA >2000 IU/ml
- with **moderate necroinflammation / fibrosis
- (**記) Patients with **cirrhosis with any detectable HBV DNA irrespective of ALT levels
- Patients with family history of HCC / Cirrhosis
- HBeAg +ve patients (not yet e-seroconversion) with high HBV DNA but persistently normal ALT, if ***>30 yo (∵ damage start to be done)
Current endpoints for treatment
記: 4個criteria: HBeAg seroconversion, HBsAg clearance, HBV DNA, ALT level
- HBeAg seroconversion for HBeAg +ve patients
- NOT sufficient as sole endpoint (not very useful) - HBsAg loss
- Ideal (so-called “functional endpoint”)
- but only achieved in <10% - HBV DNA undetectable by PCR
- ideally permanently - ALT normalisation
- ideally <50% ULN
