Module 3.2: Viral Hepatitis Flashcards

Question

Describe the Model for Biogenesis of Quasi-enveloped HEV (eHEV)

Answer 1

Yin X et al, Viruses, 2016 1. HEV with ORF3 on capsid 2. Docking with ESCRT (endosomal sorting complexes required for transport) proteins 3. Formation of eHEV in MVB (multivesicular bodies) 4. Transport of eHEV in MVB to plasma membrane 5. Release of eHEV, resistant to neutralisation by anti-ORF2 antibodies Summary: thought to be a more efficient way for the virus to transmit to other hepatocytes in different sites of liver. Note: although most of this virus is released via the basolateral membrane, some is similarly released into the biliary tract like HAV  small intestine  faeces.

Answer 2

* Avoid drinking water (and beverages with ice) of unknown purity uncooked shellfish and uncooked fruit/vegetables not peeled or prepared by traveller * Immunoglobulin prepared from donors in developed countries does not prevent infection * Unknown efficacy of Ig prepared from donors in endemic areas * Vaccine (?) --> trialled in China but not successful (possibly due to IgG responses?)

Answer 3

• Incubation period is 2-3 months • Leading to clinical illness in a proportion of individuals o <5 years of age: 10% o >5 years of age: 30-50% • Acute case-fatality rate: 0.5-1% • Chronic infection o Age<5 years: 30-90% Depending on the age of the individual the virus can become chronic  more likely to occur if you are infected before the age of 5 due to immature immune system (in neonates it can especially be recognised as being a part of the “self” hence reduced response from host immune system) o Age>5 years: 2-10% • Mortality from chronic liver disease: 15-25% o Mortality is dependent on the length of infection o A lot of morbidity associated with chronic Hep B infection

Answer 4

* Around 250 million chronic infections * 10-20% develop cirrhosis * Can cause primary liver cancer * Highest prevalence in Subsaharan Africa and South Asia with fewer but significant number of chronic cases in developed world

Answer 5

• Damage is immunopathic o Host immune response destroys infected liver cells o Cytotoxic T-cells kill infected cells o Loss of liver function o Hepatocytes regenerate from liver stem cells o Chronic infection hepatocyte replaced by scar tissue – cirrhosis ``` • Types of infection (age>5) o Acute = 90%  5-6m duration  Self-limiting (90%)  Fulminant (rare) – very extensive damage o Chronic = 10%  Quiescent  Active --> cirrhosis ```

Answer 6

SEE GRAPH p52 • After 1-3m incubation, the HBsAg rises in concentration, with subsequent rise in HBeAg sustained immunologic response to - Anti-HBs - Anti-HBe - Anti-HBc • The concentrations of both the surface and e antigens fall within about 6m

Answer 7

SEE GRAPH p52 • Again, 1-3m incubation period * Acute viraemia happens in a similar way, with the rise of the surface and e antigens in the serum * However, there is no response to the surface antigen * The e antigen may also reside for many years, and eventually are cleared by a delayed antibody response * Overall failure of immune response to sufficiently clear the infection * Chronic infection leads to cirrhosis, and possibly HCC

Answer 8

Outside surface layer o Three surface proteins – large, medium, small o Small surface protein is the most abundant, accounts for the viral structure o The large surface protein is the least abundant, accounts for receptor binding Inside there is the icosahedral capsid, comprising of the core protein which encapsulates the viral DNA with a polymerase attached to DNA o 180 copies of core protein o 27nm nucleocapsid

Answer 9

• Partial double stranded DNA structure + viral polymerase protein • HBV genome has a partial missing strand o Quite a small DNA – 3200 nucleotides (one of the smallest viruses infecting humans) o 4 open reading frames, which can encode 4 separate protein structures o Partial overlap of the open reading frames – which allows multiple protein synthesis from same information LEARN HOW TO DRAW THIS. p53 • Core sequence o Core + pre-core o Pre-core is the signal sequence for the production of e antigen • Surface sequence o Pre-S1 + Pre-S2 + S o Translation of just S = small antigen o Translation of Pre-S2 + S = medium antigen o Translation of Pre-S1 + Pre-S2 + S = large antigen • Polymerase sequence o Overlaps surface sequence completely, and partially overlaps both core and X sequences o This means that the information is compact to encode for different proteins o This is an evolutionary advantage, however, it constrains potential mutation ability of the surface antigen as it would ALSO affect the polymerase protein, which is essential for HBV function

Answer 10

* Small, medium and large antigens all have similar intermembrane structures (4 units) * Medium antigen has a longer NH2 terminus than small * Large antigen has an even longer terminus with MYR (myristoyl group) bound  lipid group that allows the virus to interact with hepatocytes and is involved in its stabilisation (along with small and medium antigens) • HBV also produces aggregates of surface proteins called empty non-infectious subviral particles (SVP): spheres and tubules o These are the surface envelopes floating around within the infected cell that do not contain HBV itself o 100-100,000X more present than HBV virion o Acts as a decoy mechanism  immune system is “tricked” into attacking these, hence virus survives for a longer period of time

Answer 11

* The core gene features two potential start codes * If translated from first start code o The pre-core/core protein produced o This contains a signal sequence (hydrophobic region) which is secreted into the lumen of the ER o In the ER, they encounter proteases, which remove the signal sequences + binding domains for nucleic acids  this produces e-antigen o The e-antigen is secreted into the circulation o Again, the e-antigens have no part in the viral lifecycle, and only act as a down regulatory mechanism against immune response by acting as another decoy protein to inhibit immune response • If translated from second start codon (ribosomes initiate translation from here) o Only the core protein without pre-core is produced o The core protein spontaneously forms the viral capsid, forming hexon and penton 3D structures SEE FIGURE

Answer 12

* Begins as core protein monomers that dimerise, and eventually form an icosahedral structure * However, unlike other hepatitis viruses, although it has icosahedral geometry, it has projections out of its main icosahedral structure (interact with lipid bilayer in final outer structure of the virus and also with cytoplasmic domains of surface proteins) and pores to allow molecules through (remember it has reverse transcription occurring in replication cycle, and so it has to be able to allow material such as nucleotides for conversion from RNA precursor to DNA)

Answer 13

* HBV is a non-retrovirus that still uses reverse transcription in their replication * The virus gains entry into the cell by binding to NTCP on the surface --> endocytosed o Peptide corresponding to HBs-L protein o Cross-linked to cell receptor o Purified and characterised o NTCP (Sodium taurocholate cotransporting polypeptide) o Functional receptor for HBV  BUT it is not efficient so it is proposed that there are likely other receptors involved in viral entry • The viral genomic DNA is transferred into the nucleus by host proteins called importins (this does not allow capsid to enter the nuclear pore) • The partially double stranded viral DNA is made fully double stranded by the host repair mechanism • This is then transformed into supercoiled covalently closed circular DNA (cccDNA) that serves as a template for transcription of the four viral mRNAs o Structure of cccDNA (right) is very similar to chromosome/cellular DNA so there is no host response to this • This DNA is then transcribed because it looks like host chromosome material • This is then translated by RNA Polymerase II in the cytoplasm to form the viral proteins (capsid and surface proteins) • An mRNA is reverse transcribed to form extra viral DNA copies SEE FIGURE p54

Answer 14

• Core o 3.5kb (greater than genome length) o Translation  core, e-antigen, polymerase proteins o Template for reverse transcription for the replication of viral DNA • S1 o 2.4kb o Translation  Pre-S1 (large S) • S2 o 2.1kb RNA o Translation  Pre-S2 (medium) + S (small) • X o 0.7kb RNA o Translation  X protein

Answer 15

• The polymerase structure is encoded by 4 regions o Terminal domain – interacts with various viral nucleic acid structures (binds to stem-loop) o Spacer – holds domains together (allows re-orientation of TP and RT domains) o Reverse transcriptase – performs reverse transcription and DNA polymerase actvities o RNAseH – ribonuclease H domain degrades RNA in dsDNA-RNA hybrid • Heat shock proteins (HSP70 and HSP40) have been found to bind and remodel domains during priming interactions with stem-loop HBV --> i.e. they have been found to restructure polymerase at different stages of viral replication • The HBV pregenomic RNA is required for the viral DNA replication • HBV reverse transcriptase binds to the stem-loop recognition site near the 5’ end of the pregenome • The stem-loop structure offers a landing platform for the polymerase • It initiates reverse transcription to the 5’ end • It then detaches and is able to rebind onto the second stem-loop, again reverse transcribing • While the DNA is being produced from the polymerase, the RNA is nearly entirely degraded • Process: referred to as primer strand switching • This process forms the first (-ve) strand of the HBV DNA • The second replication is for the +ve strand o From the –ve strand formed, the positive DNA primer attach and are primed o The negative strand loops on itself to create a circular sequence o There is even a small gap the new strand can jump over o But the polymerase is able to jump over this gap and is able to produce a partially double-stranded molecule  Why it goes around and doesn’t produce a second fully double-stranded DNA molecule is unclear but suggested that this occurs due to the constraint in space inside the viral capsid which only allows for a partially double-stranded DNA molecule

Answer 16

* Has a transactivating activity – a variety of promoters for viral replication * Several biochemical activities – but not DNA binding * Binds p53 * Interferes with DNA repair * Blocks anti-viral action of cellular restriction factor Smc5/6

Answer 17

• Dimerisation region • UVDDB1 binding o Affects DNA repair • P53 binding SEE FIGURE p56

Answer 18

Uneven distribution Common in some parts of America, Eastern Europe and Middle East SIMULTANEOUS INFECTION of HBV and HDV ---> strong host response, eventual loss of both, good prognosis SUPERINFECTION --> induction of HDV into HBV +ve agent --> sustained presence of viran DNA --> chronic infection in 60-70% - causes severe acute hepatitis, HCC can occur

Answer 19

Outer envelope consists of HBsAg from helper virus Nucleocaspid contains 70 copies of HDAg Encloses RNA genome single stranded circular, -ve polarity Forms a rod like structure due to internal base pairing Smallest genome of all known human pathogens

Answer 20

HDV receptor on human hepatocyte UNIDENTIFIED but thought to be same as HBV RNA transferred to nucleus following uncoating Replication of the genome and synthesis of mRNA carried out by host RNA polymerase II (this is because the genomic and anti-genomic RNA mimics dsDNA) (one study implicated RNA pol I and III) Genomic RNA used as the template for production of o An mRNA that is translated into HDAg o An antigenomic RNA that is full-length, and is produced from greater-than-full length transcripts • The antigenomic RNA is also covalently circular and single stranded • Three key features of the genome necessary for transcription o Origin for the start of RNA synthesis [1639] o Polyadenylation site downstream of ORF encoding HDAg o Self-cleavage site capable of self-ligation [ribozyme]  cleaves RNA itself  Self-cleaving RNA molecules  Cleave the RNA at specific sites  Present in both genomic and antigenomic strands  Central to the rolling circle mechanism of replication  Linear multimeric RNA is processed by autocatalytic cleavage to yield unit-length RNAs  These are ligated to form circles, by a host RNA ligase or by reversing the ribozyme cleavage reaction  HDV ribozymes are the fastest known self-cleaving RNAs * So HBV requires the HBsAg coat in order to enter the cell and utilise the cellular machinery * The L-HDAg is the one that interacts with the HBsAg protein

Answer 21

nucleocapsid consists of two polypeptides – a small and large surface antigen o The large one has a farnesylation site – allows binding to lipids (+19aa of small) + suppression of RNA replication o Small surface antigen is required for replication

Answer 22

• The large antigen is produced from the antigenome o The mRNA is modified so that the STOP codon is changed (UAG  UGG) o This change is performed by the host enzyme adenosine deaminase 1 (ADAR1) – where deamination occurs o One in three mRNA are changed by the enzyme • L-HDAg is produced later in the infectious cycle o It suppresses RNA replication and promotes encapsidation o An isoprenylation signal from the L-HDAg is required for interaction between HBsAg o HDV capsids contain both HDAgs, and bud off the ER membrane picking up their HBsAg envelope in the process

Answer 23

1. The virion attaches to the hepatocyte via an interaction between large-HBsAg and an uncharacterised membrane receptor in the host cell; 2. the virion enters the cell and is uncoated; 3. the RNP is targeted to the nucleus; 4. genomic RNA is transcribed in the nucleus to form antigenomic RNA, which forms the template for replication of new transcripts of the circular genome, and mRNA, which contains the open reading frame; 5. the mRNA is exported to the cytoplasm where it is translated at the endoplasmic reticulum to form new molecules of hepatitis D antigen; 6. the new antigen molecules return to the nucleus where the small-HDAg isoform supports further genome replication, and where both forms of hepatitis D antigen associate with new transcripts of genomic RNA to form new RNPs 7. RNPs are exported to the cytoplasm where large-HDAg facilitates association with HBV envelope proteins in the ER to form new virus particles; 8. these particles bud through an intermediate compartment; 9. they are then exported from the hepatocyte via the trans-Golgi network to re-infect further cells.

Answer 24

• Transmission o Blood – transfusion, products, IVDU o Sexual o Sporadic • Characteristics o Only infects humans and chimps o Icosahedral capsid, enveloped o RNA genome (single stranded, +ve sense) o 6 genotypes (genotype 1 most common in developed worlds hence where most action has been developed against)

Answer 25

* 150-200 million chronic infections * UK – approx. 214000 (Public Health England – HCV in UK 2015) * 10-20% lead to cirrhosis * Primary liver cancer implicated

Answer 26

• Course of infection: o Onset 5-12w after infection o Acute: often asymptomatic, raised ALT o Chronic in 90% of cases • Pathology o Liver failure, cirrhosis, liver cancer o 40% of Europeans with HCC have antibodies to HCV o Damage is immunological (+ cytopathic)

Answer 27

• Capsid o Icosahedral o Single protein • Envelope o Two envelope proteins E1 and E2 o The virus forms a complex with serum lipoproteins to form VLDL + viral hybrids These interactions cause difficulty in purifying the viral particle for investigation + the VLDL provides cloaking from immune system.

Answer 28

``` o CD81 o SR-B1 o CLDN1 o OCLN o Also the VLDL hybrid binds to the LDL-R on the surface and GAGs ```

Answer 29

 Receptor binding and interaction leads to invagination into the intracellular compartment – receptor mediated endocytosis  The coated vesicles are pre-lysosomal and have low pH  The change in pH confers conformational change in E (envelope) protein  reveals a fusogenic domain  Often this domain is cleaved and released, which interacts with the vesicle to release the viral RNA into cytoplasm

Answer 30

* Single stranded RNA * Approximately 9,600 nucleotides * Positive polarity = large mRNA * 5’ and 3’ UnTranslated Regions (UTRs)  stem loop structure (important for protein-RNA interactions to occur) o 5’-UTR  Contain Internal ribosomal entry site (IRES)  Stem-Loop structure I-IV  IRES can be used to ensure that viral translation is active during periods when host translation is inhibited o 3’-UTR  Has a Poly U tract (30-90 nucleotides)  Also incorporates stem loop structure  This 3’-UTR aspect is thought to bind to other parts of the genome and have a switch effect – causing it to either make proteins OR undergo replication (I.e. there is high affinity between HCV 3’ UTR and host ribosome, as well as interaction of 3’ UTR to 40S ribosomal subunit)

Answer 31

• Single large Open Reading Frame (ORF) o This translates into a big polyprotein, which is later cleaved to form the individual functioning proteins o There are structural and non-structural proteins  Non-structural proteins are not packaged into the main virus, but are used to assemble it

Answer 32

* Most of the polyprotein cleavage products (in particular NS3-5B) form the replicase complex associated with intracellular membranes * Allows production of viral proteins and RNA in a distinct compartment * The viral replication depends on making the complementary (-ve) copy, which is transcribed again to produce the original (+ve) strand * The positive strand is 10x more concentrated than the negative due to its priority as the functioning strand

Answer 33

* The large polyprotein contains all the viral proteins required * The polyprotein is cleaved at junctions to produce the individual proteins * Two classes of proteases are involved o NS3 protease  Coded by the virus  Cleaves the non-structural domain  This is a potential antiviral target o Host cell signalases  Cleave the structural part (mainly the envelope proteins)  These proteins have strong ER membrane association, where they bind to  These proteins can also bind to lipid droplets, which provides platform for the binding process

Answer 34

• Highly basic which makes up the viral nucleocapsid o Binds viral RNA o Associates with lipid droplets * Nucleocapsid monomer binds to E1 * Associates at cytoplasmic side of ER

Answer 35

• E1 (35 kd) and E2 (72 kd) implicated • The envelope proteins are glycosylated – difficult to create immune response against them • Two regions: homology region + divergence regions • The divergence region is a hypervariable region (HVR) found on N-terminus of E2 o Lots of variants for this region o Antibody development for one E2 will effectively wipe out viral population, but there are other mutated copies, which will repopulate  hence why it is difficult to form a vaccine against HCV ``` • Functions o Receptor binding o Membrane fusion  Can fuse the viral and host vesicular membranes together to release the viral capsin into cytoplasm during initial entry o Virion assembly ```

Answer 36

• Properties o Protease (binds to NS4A as a cofactor)  Amino 1/3 of NS3  Serine protease with catalytic triad of amino acids  Cleaves HCV polyprotein both cis and trans  Targets host interferon stimulation pathway proteins  viral persistence (by targeting specific host defences i.e. RIG-I and TLRs) o Helicase in replication complex  Carboxy 2/3 of NS3  Unwinds double stranded RNA which is produced during viral replication o ATPase (associated with the helicase activity) • The viral polyprotein initially requires cleavage to produce NS3 from the non-structural domain o How can it initially cleave itself without NS3 presence? o Due to types of cleavage mechanisms: cis and trans  Cis-cleavage: intramolecular self-cleavage  cleaves itself out of the polyprotein to begin the process (but this is less efficient)  Trans-cleavage: intermolecular cleavage  occurs once mature protease has been cleaved out • The helicase function is required to unwind the double stranded RNA during the viral replication o However this occurs under a membranous web (proliferation of membranes from the Golgi apparatus) induced by NS4B  this is where viral RNA and non-structural proteins complete RNA replication, so that the only particle released is positive sense single stranded RNA  This is an mechanism induced by the virus to hide from the host immune system (TLRs and RIG-I). Single stranded RNA is able to be detected, but to a lesser degree than double stranded RNA present in these webs. It also prevents nucleases from reaching and targeting RNA.

Answer 37

* RNA dependent RNA polymerase (RdRp) – replicates HCV viral RNA by using the positive RNA strand as template * GDD motif (characteristic of RdRps)

Answer 38

• NS5B + NS3 + (NS4A) + (host proteins?) • Membrane associated o ER/perinuclear • DNA stranded RNA localisation

Answer 39

• NS4A o Protease complex with NS3 and its co-factor o Localisation of RNA replication machinery to membranes • S2 o NS2/NS3 protease • NS5A o Interferon inhibition – PKR o P53 inhibition? • NS4B o Induces membrane web, which is a site of replication o Inhibits cellular translation?

Answer 40

Lanford, Science, 2012 • Liver specific – hence why liver is a primary target for HCV infection o Associated with miRISC  this then binds to HCV 5’ UTR • Required for HCV RNA replication  as it stabilises and initiate process of replication • It has been shown that siRNA against miR122 (small interfering RNA  hence degrades miRNA) completely shuts off viral replication o However, difficult to develop into an anti-viral therapy as it is not easy to deliver and has been shown to have numerous cellular functions (particularly regulating cholesterol metabolism)

Answer 41

• HCV produces double stranded RNA, which normally triggers a big IFN response via TLR pathway • Therefore, the non-structural proteins form a membrane vesicle in the ER-lumen with a narrow entry point (membranous web) o The narrow point allows diffusion of single nucleotides, but big immune related proteins cannot enter o The assembly initiates on the cytosolic side of the ER membrane • In the cytosol, the core protein is assembled which Is associated with lipid droplets (Lipid Droplet Model) o The lipid droplet is brought in close proximity to the membranous web where replication is occurring . o Core protein is released and interacts with RNA o The lipid molecules are utilised to build the viral envelope + budding into the ER lumen o This allows the encapsidation of the positive viral genome that is released into the cytosol for release o The formed vesicles then fuse with the plasma membrane and release the virions from the cell (don’t forget: they will then associated with VLDLs to form viral hybrids)

Answer 42

* Site of RNA replication – ER vesicles * NS3 – inhibits cell monitoring of virus molecules * NS5A – inhibits interferon responses * Variation in genome/protein structure – quasispecies

Answer 43

• HCV RNA Polymerase – no editing mistakes (does not have function to stop it making mistakes) o Error rate: 10-3 – 10-5 nucleotides/replication cycle (approx. 106 cellular mutation rate) – all occurring by chance (hence genes are not functional) • BUT this allows the virus to mutate so that it is able to escape host immunity • This means that host will create an antibody response to envelope proteins e.g. E2 and some virus will be neutralised but those with changes in amino acids in hypervariable region of glycoprotein E2 will escape o This leads to new dominant genome to proliferate  SEQUENCE DIVERGENCE • Hence, a major problem for therapies e.g. HCV vaccine

Answer 44

Vaccine • E1/E2, NS proteins, subunit vaccines, DNA vaccines • HCV is a quasispecies hence a major problem • No vaccine so far Antivirals • Alpha interferon (pegylated  allowed for increase in half-life of interferon) • Ribavirin o Daily treatment over 6-12 months New Antiviral Protease Inhibitors for HCV • NS3/4 protein – protease inhibitors o Telaprevir o Boceprevir o Simeprevir • Blockade of the NS3/4 protease would prevent assembly of replication machinery o Secondary effect: blocking protease that downregulates the immune response  hence boosting immune system as a secondary effect • NS5A protein – polymerase inhibitors o Sofosbuvir • NS5 protein inhibitors o Daclatasvir But it is possible to get re-infected with hepatitis C  especially if continuing high risk behaviour. Hence why a vaccine would be useful.

Answer 45

HCC • HBV and HCV chronic infections (>30-50 years) predispose to HCC • Potential Mechanism: o Continuous liver damage o Continuous repair and turnover of cells o Accumulation of DNA damage o Multi-hit transformation of cells o HCC

Answer 46

Hepatitis C – 2013 • 170 million infected worldwide o Outdated: new WHO statistics show this number has been vastly overestimated and actually only ~70 million infection worldwide • Diagnosis frequency estimated at 50% • Estimates of 500,000 new infections per year • Cumulative mortality rate of 5-7% • Prevalence varies from 0.5-2% in Europe to 20% in Egypt • Infection predominantly through iv drug use and non-screened blood supplies Hepatitis B – 2005 • 400 million infected worldwide • Evidence for 2 billion people exposed to HBV • Estimates of 10-30 million new infections per year • HBV is approximately 100 times more infectious than HIV • Infection predominantly during childhood via blood contact o One of the most infectious viruses that infect humans

Answer 47

Both viruses can ultimately lead to liver failure due to cirrhosis and HCC. But the proportion of those who end up with these clinical manifestations is different for both viruses. In HCV infection, 80% develop chronic disease --> 2/3s of these will develop progressive forms of the disease. (30% of which will develop into cirrhosis, 25% of which will result in liver failure, cancer, transplant or death) In HBV, the proportion of those who develop an acute infection is heavily dependent on the age of the individual (90% of children = chronic, 5% of adults = chronic).

Answer 48

* Environmental factors (exposure of hepatotoxic substances e.g. aflatoxin, drinking) * Viral factors (genotype, viral load, mutants associated with higher pathogenesis) * Host genetic factors (ethnicity, age at infection, others)

Answer 49

* Familial segregation of rare phenotypes * Animal models * Twin studies

Answer 50

• MHC Class II found to be the only genetic association for HBV o Well defined function  cell processes HBV peptides and present them on MHC class II and present to CD4+ T-cells o Well characterised polymorphisms  found in beta-1 chain (peptide interaction groove)  HLA-DRB1*0301 and *0401 (Thursz et al 2011)  associated with vaccine non-responsiveness  HLA-DRB1*1302 (Thursz et al 1994/Hohler et al 1995)  protective MHC allele, i.e. those with this polymorphism they are able to clear infection of HBV better (found in both children and adults) • MHC not found to be related to HCV clearance.

Answer 51

Found a IL28b gene on chromosome 19 to be related to why some people responded to IFN and why some didn’t, as well as why some people cleared infection and others didn’t. IL28B (part of Type III IFNs) • SNP rs12979860 on Chr19 found to be associated with sustained viral response (SVR) of HCV (strongest genetic association found so far linked with outcome of HCV infection) • It has been long recognised that African patients are less likely to achieve SVR – this C allele at rs12979860 is found less frequently among these people – found to explain 50% difference between treatment responses in African Americans compared to Caucasians – Ge et al, Nature, 2009 (Tanaka et al, Nature genetics, 2009) (Rauch et al, Gastroenterology, 2010) o C allele = good, T allele = bad  found that T allele overrides the C allele in genome o There are also differences observed between the response levels – i.e. if both Caucasian and African-American has CC genotype, Caucasian is still more likely to clear it compared to African-American thereby implying that there may be other interactions in the genome that result in this ability to clear infection • Urban et al, Hepatology, 2010 – found that IL28b governs expression of ISGs, but found that TT genotype led to increased expression level of IFN and other anti-viral proteins (counter-intuitive)  so original hypothesis turned out to be that those that have the CC genotype would express more IL-28b and so would clear infection better but this turned out to be disproven. • Prokunina-Olsoon et al, Nat Genet, 2013 + Bibert et al, JEM, 2013 – these individuals found a variant upstream of IL28b  found that those with TT genotype would create a new IFN known as IFNlamda4, which resulted in reduced clearance of HCV o Important: IFN lamda 4 = only present in TT (unfavourable) genotype  this TT variant inserts a frameshift that creates a new gene through a premature stop o Ge et al, Nature, 2009: found that this TT genotype originated in Africa (hence why these individuals have a reduced ability to clear HCV and it may have been due to it being selected against by other predominant infections such as TB)

Answer 52

``` • Lin et al JGH 2005: o 25% of those chronically infected with HBV go on to develop complications  Compensated cirrhosis  Decompensated cirrhosis  HCC ``` • Global Cancer Statistics GLOBOCAN 2008: o Globally 30-53% of liver cancer is due to HBV o Highest regions: Eastern Asia, South-eastern Asia, Middle Africa, Western Africa, Southern Africa • Mokad, BMC Medicine, 2014: o Looked at attributable risk factors to cirrhosis  In LMIC the biggest cause of cirrhosis is HBV

Answer 53

o 30% reduction of new infections by 2020 o 10% reduction in deaths by 2020 o 90% reduction in Hepatitis B + C by 2030 o 65% reduction in number of viral-hepatitis related deaths

Answer 54

• Prevention of transmission o Infant vaccination to prevent childhood infection o Prevention of mother-to-child transmission  Birth dose vaccination within 24 hours of birth  Hepatitis B immunoglobulin  Peripartum antiviral therapy for mothers with high viral load • Antiviral treatment of those infected

Answer 55

* Safe and effective vaccine available since 1982 * Universal infant vaccination WHO recommendation sinc e1992 * Integrated with the EPI (at 1, 2, 6 months) * Impact on horizontal (early childhood transmission) * Cheap (

Answer 56

* Difficult to access areas e.g. conflict/displacement * Difficult to reach populations * Competing health demands and health budget constraints * Fear of vaccination * Reductions in vaccination coverage causes rebound increase in HBV prevalence

Answer 57

Nayagam et al, Lancet Infectious Disease 2016 • Reduction of horizontal transmission is important as well as infant vaccination because infant vaccination only makes up a % of cases o Hence scale up of infant vaccination will mean that, in the future, a greater proportion of new cases will be among neonates What Measures can reduce Perinatal Transmission? • Birth dose vaccination o Birth dose vaccination recommended by WHO since 2009 (not every country implements it) o Chen et al, 2012 – effective at reducing HBV MTCT o Delayed administration beyond 24 hours of birth decreases effectiveness o Cheap + cost-effective (Kingler 2012) o However, global coverage is only 39%  UNICEF/WHO 2015: only 9 countries in SSA have adopted birth dose vaccination policy for HBV • Hepatitis B immunoglobulin o Addition of HBIG within 24 hours of birth to babies of HBsAg+ mothers reduces MTCT further o However not a WHO recommendation because of cost, need for cold chain, problems with lack of availability • Peripartum antiviral therapy for mothers with high viral load o Studies have shown antiviral therapy in 3rd trimester of pregnancy in reducing MTCT further (if high viral load) o Pan et al, NEJM, 2016 – RCT comparing TDF to placebo: given to mothers with HBeAg+ mothers with VL>200,000 IU/ml [intention to treat analysis: 5% vs 18%, p = 0.07]  remember this is including two strategies above as well o AASLD and EASL guidelines now recommend treatment o Successful implementation requires: antenatal screening with HBsAG (not routinely performed in LMICs) and HBeAG + VL testing to stratify risk (limited by cost and availability)

Answer 58

 Low rates of delivery in health care facilities  Difficult access to rural areas  Cultural barriers  Logistical difficulties (transport of vaccination)  Lack of funding support (GAVI do not fund birth dose vaccinations, but fund universal vaccinations)

Answer 59

Nayagam et al, Lancet Infectious Disease 2016 • Incidence will only change if scale-up of prevention interventions are implemented • In the absence of a functional cure  eradication will not change BUT screening and treating with current anti-viral therapies will reduce mortality Safe and Effective Treatment • Interferon • Nucleot(s)ide analogue drugs – Tenofovir and Entecavir - FIRST LINE o Once daily tablets, easily tolerated o High barrier to resistance • Efficay of treatment: Liaw et al NEJM 2004 in Taiwan – 50% reduction in liver disease progression with lamivudine, HCC incidence of 3.9% (lam) vs 7.4% (placebo)

Answer 60

* Requirement for complex diagnostics * Drug cost and availability * Screening * Capacity of health care system

Answer 61

• Benefits o Severity of liver disease/likelihood of disease progression to cirrhosis and liver cancer (see right: yellow = stages of treatment, remainder need monitoring) o Probability of treatment response • Risks o Adverse effects o Drug resistance o Cost

Answer 62

* Haematology * Biochemistry * Viral serology * Viral load testing * Liver biopsy and histology

Answer 63

PROLIFICA – Prevention of Liver Fibrosis and Cancer in Africa Summary: it is feasible to screen and treat (acceptable in community, high uptake, simplified models worked) • Community screening and treatment is likely to also be cost-effective: Nayagam et al, Lancet Global Health 2016 o Also: evolution of price quoted on developing countries  i.e. price is reducing from 400 to 48 dollars

Answer 64

Chronic hepatitis is defined by inflammation of the liver that lasts >6 months (i.e. LFTs not improving).

Answer 65

• Predominantly portal inflammation o Acute HBV hepatitis causes lobular inflammation o Some pathologists may refer to this as piecemeal necrosis  But there is no necrosis as there is no secondary death to the cells – the death is via apoptosis (see right) o So refer to it as periportal inflammation/interface hepatitis (see right) • Immunopathic cell damage o Not cytopathic with the exception of transplanted patients o These patients may develop fibrosing cholestatic hepatitis (FCH) due to their immunocompromised state o In HIV and HBV coinfection, there is no immune response against HBV  no immunopathic damage, but continuous accumulation of HBV o Subsequent treatment with IVIG may cause massive inflammatory response against the built up HBV infection • Ground glass hepatocytes --> characteristic of Hep B o Expression of surface antigen in the ER of liver o The presence of these ground glass hepatocytes imply 1) HBV infection 2) chronic disease • Immunohistochemistry – indications: o DD of ground glass hepatocytes o Assessment of viral replication o Identification of immune escape (precore) mutant  i.e. patients with a stop codon in precore area meaning they don’t make e-antigen but they still make core antigen (hence patients can be very active but may appear inactive as patient is not making e-antigen = indication of activity of disease) o Prominent lobular activity o Hepatitis in immune suppressed patients  Done for both HBsAg and HBcAg (can see HBsAg confirmation)  In active viral replication, there is staining of both the nucleus and the cytoplasm in HBcAg

Answer 66

Remember: need HBV to infect either through co-infection or superinfection (important to recognises due to its significance in clinical practice) • Prominent cytopathic, lobular activity o Lots of lobular liver damage with a lot of inflammation (see right)  remember the inflammation in this case is due to clearing up of dead hepatocytes) • Immunohistochemistry  very important in HDV o Proves HDV presence with HBV

Answer 67

``` • Borderline interface hepatitis • Prominent lobular inflammation o HCV is more complex  even in chronicity it presents both features of HDV and HBV together but milder in format • Lymphoid aggregates • Bile duct damage • Fatty change (genotype 3) • Granulomas? • Immunohistochemistry and in situ? ``` • Lymphoid aggregates (see right) o HCV induces the formation of secondary lymphoid tissue in the liver o H. pylori also does this  lymphoma o i.e. not by any means is this unique to HCV but allows differentiation from HBV  feature of immune response to HCV • Bile duct damage (see right) o The biliary epithelium are deranged and have different sizes – but it does not cause bile duct loss o HCV infects bile ducts and HBV doesn’t – although both are associated with cirrhosis and HCC, only HCV is implicated in cholangiocarcinoma • Fatty change (see right) o Patients with genotype 3 have associated fatty change that lead to cytopathic damage o If you cure genotype 3 HCV, the fat goes away o If it doesn’t go, obviously the cause of steatosis lies somewhere else HCV plus alcohol will show ballooning and Mallory Denk bodies, neutrophils etc, and it would be beyond simple fatty liver. • Granuloma o HCV patients found to have an increased number of granulomas however you must ensure to exclude other causes such as HCV o Not as important to remember

Answer 68

• Acute hepatitis • The other causes of chronic hepatitis o Autoimmune hepatitis  one can have HCV with autoimmune features = more difficult to treat o Drugs  can cause immune damage (oxyphenisatin, nitrofurantoin, methyldopa, NSAIDS) or chronic toxicity (phenytoin, isoniazid, paracetamol) • Other causes of interface hepatitis (I.e. PBC, Alpha-1 antitrypsin deficiency, Wilson’s disease) • Co-existing liver disease (just because you have one liver disease does not mean you cannot have 2  about 5% of those with HCV have haemochromatosis; similar risk factors: HIV or other types of viral hepatitis)

Answer 69

Dysplasia * There is no clearly identified intermediate stage between chronic liver damage and carcinoma (as seen in adenoma-carcinoma sequence of the colon) * But there are associated intermediate changes that occur, which indicate increased risk of cancer development * Large cell change: similar nuclear-cytoplasmic ratio despite large nucleus + no proliferation  just abnormal cells that die and never become cancer (see right) * But they ARE indicators of cancer development * Small cell change: this is actually DYSPLASIA as cells are crowded and do eventually become cancer

Answer 70

* Confirmation/support for the aetiology * Exclude co-existing causes of liver disease * Assess grade and stage * Dysplasia * Response to treatment – look at previous biopsies with newer biopsies to see if there has been any improvement

Answer 71

* Grade: severity of inflammation * Stage: severity of fibrosis METAVIR and ISHAK = allow for scoring stage and grade score. Modified HAI (ISHAK) – Necroinflammatory Score (Ishak et al, J Hepatol, 1995) – used in the UK (METAVIR in France) • Periportal Interface hepatitis • Confluent necrosis (seen in AIH not viral hepatitis) • Focal lytic necrosis, apoptosis and focal inflammation • Portal inflammation

Answer 72

o Severe widespread hepatocellular necrosis o Liver failure – defined by INR (high), PT (low), jaundice, encephalopathy ++ o Regeneration without longstanding injury o Death/Transplantation HBV HEV (especially if pregnant)

Answer 73

• Virus o Protein  nucleic acid amplification through PCR • Antibodies o IgM/IgG response against viral components (ELISA) • Liver cell damage o Serum detection of liver enzymes – ALT, AST, ALP (or by direct liver biopsy but rarely ever done)

Answer 74

Liver Tests ALT/AST Liver inflammation/ damage GGT/ALP/Bili Cholestasis PT/INR/Bili/Albumin Liver failure ++ Viral DNA/ RNA Stool/ blood viral PCR Active viraemia Envelope antigens HBsAg, HBeAg Active viraemia and replication Other viral antigens Proteins Active viraemia Antibody IgM type IgM ++ i.e. anti HBc IgM anti HVA IgM Acute infection (or reactivation of HBV infection) Antibody IgG type HBsAb Resolved infection HCVAb, HBcAb IgG, Anti-HVA IgG Exposed and resolved or ongoing infection

Answer 75

Largest outbreak: 310,000 people in Shanghai in 1988 who caught it from blood clams

Answer 76

o Hepatitis A immune globulin can be given up to 2w after exposure o Immunity is temporary (4-5m) o Also given in travellers leaving for endemic area on short notice

Answer 77

o HAVRIX o 1st dose at time 0, 2nd dose 6-12m after o Inactivated live vaccine o Highly immunogenic: 97-100% of children and adults have protective levels within 1m of first dose, 100% have protective levels after second dose o Highly efficacious: in published studies, 94-100% children protected against clinical hep A

Answer 78

o Travellers to areas with increased rates of HAV o Men who have sex with men o Injecting and non-injecting drug users o Persons with clotting-factor disorders (e.g. haemophilia) o Persons with chronic liver disease (+++ cirrhotics) o Persons with HIV o Military personnel o Day care centre personnel o Close family contacts of index case o Exposed in outbreaks or work-related (e.g. plumbers)

Answer 79

* Stages not necessarily linear * IgM is gold standard for diagnosis • Phase 1 o Viral replication, limited immune response o High viral load, HBsAg+, eAg+, ALT normal • Phase 2 o Immune response, viral replication lower o Medium viral load, HBsAg+, eAg+, ALT fluctuates o Liver inflammation and damage • Phase 3 o Immune suppression o Low/undetectable viral load, HBsAg+, eAb+, ALT normal  Low viral load is defined by: <2000IU/ml o In a proportion, chronic active eAg negative but viral replication – precore mutant- • Phase 4 o Viral clearance o Undetectable VL, HBsAb+, eAb+, ALT normal o Still at risk of liver cancer due to incorporation of viral cccDNA in host genome

Answer 80

• Most vaccines are recombinant non-glycosylated small s protein (Engerix-B, Recombivax-Hb) • Others include Pre-S1 and/or pre-S2 proteins o More immunogenic, not widely available • Available in combination with Hep A vaccine – Twinrix • Also as a multivalent with diphtheria, pertussis, haeophilus and tetanus (GAVI-sponsored immunisation) o Strong debate: in developing countries are infected very early in life with HBV due to vertical transmission hence it is important to provide a dose at birth yet this immunisation is only given after three months of birth  if babies born from HBV mothers do not receive the vaccine early on they will become infected and endemicity will remain • Infants: several options that depend on status of mother o If mother negative: birth + 1-2m + 6-18m o If mother positive: vaccine and hep B immune globulin within 12h of birth + 1-2m + 6m o Plus tenofovir in T3-6w post partum • Adults o 0 + 1 + 6m

Answer 81

o All those aged 0-18 o Those at high risk  Everyone!  240 million people chronically infected with HBV  The most at risk: infants born in endemic areas +++ (Africa and Asia) = should be vaccinated within the first 24 hours, as they are likely to be infants born to HBsAg+ mothers  Persons with multiple sex partners or diagnosis of STDs  Men who have sex with men  Sex contacts of infected persons  Injection drug users  Household contacts of chronically infected persons  Infants/children of immigrants from areas with high rates of HBV infection  Health care and public safety workers  Haemodialysis patients

Answer 82

HBVIg * Infants of surface antigen positive mothers * Exposures to infected blood or infected body fluids in individuals who are unvaccinated, unknown vaccination status or known non-responders * Ideally within 24h (probably not effective >7 days post-exposure)

Answer 83

• In acute phase: IgM Abs to HDV + increased transaminases and detectable HDV RNA o Normally missed as it can be asymptomatic • IgG phase: still need to check for total viral load  patients will not usually clear infection due to double hit on the liver o In this case, the IgG serology is simply a signature of the previous acute infection

Answer 84

* HBV naïve: HBV vaccination | * HBV infected: avoid high risk exposures

Answer 85

• Acute infection: only 30% of those infected will recover o If resolved: lose HCV RNA, normalise transaminases and develop anti-HCV antibodies • Chronic infection: 70% of those infected o Maintain viral load (may reduce a little bit) and detectable HCV RNA o Develop anti-HCV and semi-normalise ALT

Answer 86

* Injecting drug users | * HIV infected men who have sex with men because HIV infection favours acute infection with

Answer 87

* Extensive sequence diversity * 6 genotypes * Individuals infected with large numbers of viral quasispecies – resistance develops quickly * Antibody responses don’t neutralise virus – T cell responses important * Vaccine strategies need to target CD4 and CD8 T cell responses * Lack of small animal models for vaccine development * Several vaccines under development PREVENTION IS KEY

Answer 88

• Incubation period: 2-8 weeks (mean of 40 days) • Right: serology as with HAV BUT remember that it takes time to develop antibodies so if you suspect HEV, always quantify viral load in blood or faeces of infected in dividual • Molecular diagnosis o Serologies: not homogenous  variation in sensitivity o Blood and stool HEV PCR = more accurate as can be detected just before onset of clinical symptoms and persist during the first 3 weeks • Mostly silent in childhood o Mortality is higher in infants <2 years • Acute cholestatic hepatitis o Same as before: jaundice, myalgia, vomiting, weakness, elevated transaminases, bilirubin • Fulminant hepatitis o +++ in pregnant women in third trimester (10-25%) o in patients with underlying chronic liver disease • Chronic hepatitis o Immunocompromised individuals o Solid organ transplantation (liver, kidney)  only one case reported: Dalton, HR NEJM, 2009 • Extra-hepatic manifestations o Neurological complications o Associated with Guillan Barre syndrome, encephalitis

Answer 89

``` • Avoid: o Drinking water o Uncooked shellfish o Uncooked fruit/vegetables not peeled or prepared o Undercooked offal ``` • Screening donor organs for transplantation • Ig prepared from donors in endemic or non-endemic areas does not prevent infection • Vaccine o In China but not available anywhere else (Zhu FC et al, Lancet, 2010)

Answer 90

HAV and then HEV

Answer 91

incubation of 2-6 weeks - often subclinical - faecal-oral spread - notifiable Occupational risks: daycare providers, hospital workers, sewage workers, food handlers HAV in young poeple --> due to travel Older population --> due to food contamination

Answer 92

Shanghai blood clams 1998 - >300000 cases New Zealand Blueberries 2001-2002 - 17 cases - showed that HAV isn't killed by freezing - highlighted need for food safety programmes

Answer 93

Clinical illness correlated with peak in ALT 1-2 prior to clinical illness -> shedding in faeces HAV+ve IgM --> diagnostic of acute infection HAV+ve IgG --> not diagnostic - done to ensure no false positives NO TEST for HAV RNA

Answer 94

inactivated vaccine - immune system develops anti-HAV IgG antibodies 2nd dose after 6-12 months Give within 14 days of exposure (also with human normal immunoglobulin if immunosuppressed)

Answer 95

* Double-stranded DNA virus + unusual features that make it similar to a retrovirus (replicates through an RNA intermediate) * Exists in 3 main forms in serum  dane particle is of particular importance (this is the virion), filamentous and spherical form are composed of HBsAg and hos-derived lipids without viral nucleic acids = non-infectious o Dane particle: spherical, double-shelled structure 42 nm in diameter, consisting of a lipid envelope containing HBsAg that surrounds an inner nucleocapsid composed of hepatitis B core antigen (HBcAg) complexed with virally encoded polymerase and the viral DNA genome

Answer 96

• Incubation period is 2-6 months (hence need to go as back as 6 months when identifying risk factors) • Transmission can be done through sexual, vertical (mother to child), blood products • Can cause both an acute and chronic illness o Chronicity is defined by HBsAg+ persistence > 6months

Answer 97

• Swedish cross country runners, Ringertz and Zetterberg, 1957-1963 o 568 cases of HBV infection skin lesions mainly during washing after the competition adequate protective clothing was made a compulsory regulation. * North London Alternative Clinic * Glenochil Prison, Scotland, 1993

Answer 98

* ALT peak correlates with clinical presentation as with HAV, however the DNA rise may occur before the ALT peak * If acute infection resolves: anti-HBs antibodies would have formed and no HBsAg would be detected in serum (top right) • If chronic infection: o More likely to become infected if you are exposed to HBV at a younger age (hence why vertical transmission is such a potent route) o HBsAg+ is present, no anti-HBs o ALT fluctuating, likely HBeAg+ • If vaccinated: will only have anti-HBs but no anti-HBc o Important to recognise  those who have been infected (i.e. have anti-HBc carry a small risk of reactivation of HBV infection) • Once chronically infected, likely to be at high risk of developing HCC and cirrhosis

Answer 99

Cooke et al, BMJ, 2010 1. Immune tolerant – immune system is tolerating the HBV (hence high viral load and no ALT dysfunction) 2. Immune reactive – immune system is trying to act against the pathogen hence high ALT and active inflammation, viral load reduces due to response of immune system 3. Inactive HBV carrier state – immune system becomes dominant and suppresses the HBV level to low/undetectable levels: hence normal levels ALT and no inflammation 4. HBeAg negative chronic HBV – spontaneous resolution of the hepatitic phase may occur with loss of hepatitis B e antigen (HBeAg) and appearance of anti-HBe. This results in an “asymptomatic” phase with normal liver functions tests and low viral load (<2×103 IU/ml). Many adult patients with chronic hepatitis B virus infection in European settings will present in this phase of disease. In the past, they may have been falsely reassured and discharged BUT now clear that viral reactivation will occur in 15-20% of this group of patients, causing HBeAg negative hepatitis. 5. HBsAg negative phase – rarely occurs SEE TABLE 110

Answer 100

• Safer sex – condoms • Vaccines (ideally universal and given at birth) – UK started 2018 o These can be monovalent or combine A+B o Ultra-rapid vaccination schedule – 0, 1, 3 weeks with booster at 12 months  antiHBs > 10IU in 80% by 4 -12 weeks and 95% by 12 months; aiming for >100 IU o Non-responders  further doses, higher doses, treat HIV, alternative vaccine • Non-responders = new vaccines (FenJrix) and boosters if appropriate

Answer 101

RNA dependent RNA polymerase is ERROR PRONE --> great nucleotide diversity among isolates o Closely related but diverse population of viral variants known as quasispecies is produced within HCV-infected patients (important for outbreaks)

Answer 102

• Spanish Anaesthetist, BMC Biology 2013 • Irish anti-D, 1977-1978 and between 1991-1994 o Led to testing of blood from donors now

Answer 103

• Acute infection usually asymptomatic o 20-40% HCV RNA– (PCR) o 60-80% HCV RNA+ (PCR) * Many will develop antibodies but that does not mean the individual has cleared infection  need to always check viral load through PCR * ALT will fluctuate * If chronic it can progress to cirrhosis and HCC

Answer 104

• Co-infection: when both viruses are acquired together o Acutely unwell with symptoms and elevated ALT o However if you clear HBV then you also clear HDV o Difference between only HBV and co-infection with HDV is increased severity of symptoms with both infections • Superinfection: individual already infected with HBV acquires HDV subsequently o more aggressive disease leading to increased likelihood of cirrhosis and HCC (“double hit on liver”) o Also at risk of developing ALF due to immunodeficiency

Answer 105

three discontinuous and partially overlapping open reading frames (ORFs) o ORF1 encodes a methyltransferase, protease, helicase and replicase o ORF2 encodes the capsid protein o ORF3 encodes a protein of undefined function

Answer 106

• Similarly to HAV it shed is faeces 1-2 weeks prior to symptoms • Similar to HAV in serological course • Incubation period 3-8 weeks (slightly longer than HAV) • High mortality rate in pregnancy (genotype 1) • RARE: o CNS disease – Bell’s palsy, Guillain Barre, other neuropathy • Chronic infection has been observed in immunosuppressed • TREATMENT – supportive, except in chronic infection = ribavirin • VACCINE - EFFECTIVE but not licensed

Answer 107

• Mother-to-child transmission – remains the most important mode of transmission worldwide o How do these viruses get from mother to infant?  Through breast feeding?  In utero?  Intra partum? o If the virus is transmitted from mother before immune deletion takes place, the baby will not develop antibodies against HBV because it will determine it as ‘self’ o >6months – chances of transmission reduce by half but still possible for horizontal transmission due to scratches and bites tends to result in CHRONIC COURSE

Answer 108

Acute Occurs in adults Usually through blood, IVDU, unprotected sex Symptoms: acute severe illness with jaundice, prolonged malaise Majority of patients will recover Potential fatal – especially if co-infected with HIV (immunosuppression) Chronic Almost always acquired extremely early in life Mother-to-child usually Asymptomatic (usually) Majority will remain with lifelong infection Can result in cirrhosis, liver failure, HCC (currently no functional cure) N.B: In acute viral infection, either the virus kills the host or the host kills the virus. In chronic infection, the immune system is unable to clear the virus completely = constant battle.

Answer 109

* 400 million have become chronically infected * Estimated 1 million die each year from HBV and its complications ENTIRELY PREVENTABLE DISEASE

Answer 110

• Cirrhosis o Ascites o Oesophageal varices o Previously would require for liver transplantation  now effectively suppressed with anti-virals • HCC o HBV is an oncogenic virus hence infected individuals can develop HCC without cirrhosis (2nd most common cause of cancer death in Africa) o All other causes of primary liver cancer e.g. HCV, Wilson’s, Haemochromatosis  cirrhosis occurs before HCC, as cirrhosis = cause

Answer 111

• CHB is an imported disease o 97% of those infected were born outside the UK • UK is considered a low-prevalence country, however London is reaching areas of intermediate

Answer 112

see p121 - Immune Tolerance - Immune clearance - Immune control - Reactivation phase

Answer 113

o Because there is no immune response in newborn babies, the virus is able to replicate at will o But because there’s no immune response ALT is normal o At some point the immune system wakes up  Normally in adolescence • Diagnostics and Rationale o Immune system fails to recognise virus as foreign, hence levels of virus are very high (↑eAg+,↑ sAg+, HBV DNA = >>>20000 IU/mL i.e. producing >100000 copies of DNA  usually HBV DNA in billions) but no rise in liver enzymes (normal ALT/AST)  damage only occurs due to immune reaction (immunopathic virus) o None or minimal pathology in liver biopsies o No antibodies formed o Usually asymptomatic • Seen almost exclusively in children/young adults infected at birth by Hepatitis B infected mothers • Management – EASL Guidelines 2009 o No therapy required o Follow-up at least every 3-6 months  this phase is finite hence recognition is important o Argument: immune tolerance phase can last for several years when infected individuals are sexually active, thus from a public health perspective there is an argument that these individuals should be treated as they are highly infectious (↑↑↑ viral load)  prevention of transmission

Answer 114

o As a consequence the immune battle starts  FLARES of ALT whilst the immune system is trying to control the virus • Diagnostics and Rationale o Immune system recognises HBV as foreign and tries to eradicate the virus o During this process, liver inflammation and hepatocyte damage will occur [fluctuating/elevated transaminases (↑ALT/AST), inflammation/fibrosis in liver biopsy] o Viral load will ↓ o Still HBeAg+ and no anti-HBe  “constant battle = virus is still active (replicating) but immune system is mounting response” o Duration of phase is highly variable • Management o Trying to boost the immune system through management – pegylated IFN (naturally produced by immune system to combat virus)  not always successful hence monitoring o Quantitating surface antigen – better than looking just at HBV DNA (Martinot-Peignouz et al, Liver Int. 2014)  Week 12 predictors of sustained response in HBeAg neg patients on IFN therapy = ↓ viral load and ↓ HBsAg  if not observed then treatment discontinued o Side Effects: “flu-like symptoms”: fever, lethargy, headache, nausea, poor appetite

Answer 115

o If you can control the e-antigen, you can control the virus o Viral load drops greatly o ALT normalises • Diagnostics and Rationale o Majority of patients under long-term follow-up are in this phase o Majority of patients would have also independently undergone immune clearance to become HBeAg- and anti-HBe o Immune system is controlling the virus hence: HBsAG+, HBeAg-, anti-HBe, ↓viral load (<2000IU/mL), normal ALT/AST, reduction in liver inflammation • Management o Monitoring is mandatory: LFTs, viral load, HBsAg quantification due to risk of immune escape phase (symptomatically undetectable prior to progression of complications)  HBV has no proof-reading mechanism hence is prone to mutations

Answer 116

o HepB does not have proofreading ability  High levels of mutations • Vast majority is futile and won’t work, but a minority makes a virus that works • The protein configuration is different  e-antibodies no longer work  Leads to immune escape • Diagnostics and Rationale o Often referred to as HBeAg- chronic hepatitis – occurs when a fit HBV mutant escapes immune system o Caused by virions that do not express HbeAg leading to active viral replication (↑ viral load >105), fibrosis and liver inflammation (histology and ↑ ALT/AST) • Management o Patients considered for treatment if ↑HBV DNA, ↑ ALT, proof of fibrosis [previously biopsy now fibroscan] o Nucleotide/nucleoside analogues – inhibit HBV DNA polymerase activity by being incorporated into growing DNA strands and acting as chain terminators thereby reducing viral replication  Tenofovir (nucleotide analogue)  Entecavir (nucleoside analogue) o N.B: this only ↓viral load to ensure that inflammation does not progress in the liver and so, need to be continued indefinitely as they are not a functional cure so simply suppress virus and not completely eliminate it o Marcellin et al, Lancet 2013: fibrosis can regress after 5-years of Tenofovir therapy  Even patients with cirrhosis, 74% of patients no longer had cirrhosis  Initially only ~30% of patients were considered to have mild fibrosis, but at the end of year 5 this number increased to 60%

Answer 117

o Transmission risk is related to maternal viral load  if no intervention is given, 70% of children born to these mothers will acquire HBV o Management:  Active immunisation –HBV vaccine given at birth and subsequent doses (1, 2 and 12 months). However babies can be lost at follow-up in GP.  Passive immunisation – immunoglobulins targeted at HBV however this is difficult to acquire as only very few people are immunised against HBV in the UK. Further risk of transmitting other illnesses to a newborn baby.  Tenofovir in 3rd Trimester given to mothers with high viral loads only

Answer 118

• WHO mandated vaccination against Hepatitis B (now all compliant as of last year) • All babies born on or after 1 August 2017 will receive a hexavalent vaccine which includes HepB • Also protects against Diphtheria, tetanus, pertussis, poliomyelitis and disease caused by HipB • However this is only the universal vaccine o But newborns of mothers infected with HBV still need monovalent vaccine with subsequent doses in the first three months to prevent transmission, which often causes confusion

Answer 119

• Patients in the immune control phase must have their liver assessed (fibroscan or biopsy) to check for cirrhosis • Patients with compensated cirrhosis and detectable HBV DNA should be considered for treatment (EASL Guidelines 2009) as there is evidence that suggests reduction in rates of decompensation and HCC progression (Wong GL et al, Hepatology, 2013) o This reduces cost to NHS and improves survival rate

Answer 120

Acute - Usually asymptomatic - 70% will progress to chronic HCV  quasispecies Rarely diagnosed unless regular monitoring in ‘at risk’ individuals Chronic - Symptoms may be absent or easily dismissed by HCP (fatigue, poor concentration, depression) - Rarely diagnosed based on symptoms – only found accidentally when infected individual tested for abnormal LFTs OR even present with end-stage liver disease (e.g. HCC or varices)

Answer 121

• HCV has 6 distinct genotypes  have different geographical regions (provides evidence that there was a common origin) o 1: US, Northern Europe o 2: Europe and Japan (some America and Italy) o 3: Asia (India, Nepal, Bangladesh, Pakistan) o 4: Egypt (Africa and Middle East) o 5: South Africa o 6: Vietnam (Hong Kong, South East Asia) • Different genotypes respond differently to certain treatments

Answer 122

* Patients progress to cirrhosis especially if undiagnosed, and if other factors e.g. alcoholism, obesity etc * Used to be commonest cause of HCC and liver failure  now curable

Answer 123

• Unlike HIV and HBV, HCV is not a disease for life o RNA virus  so does not integrate into host genome, easier to eradicate o HCV antibodies exist but are ineffective, due to different genotypes, hence patients that partake in ‘at-risk’ behaviours e.g. IVDU are at risk of re-infection • Sustained virological response (SVR) is referred to for most viruses that are controlled however, in the case of HCV it is a CURE • SVR  reduced morbidity and mortality

Answer 124

• Cirrhosis o Ascites o Oesophageal varices o Previously would require for liver transplantation  now effectively cured • HCC o Cirrhosis causes and can lead to HCC

Answer 125

* Skin popping  if IVDU cannot find anymore veins, they will inject with an insulin syringe subcutaneously * Contaminated works  those who purify heroin (for example) will use shared crockery, and because virus can live outside the body for a long period of time, it can be found in spoons etc, used by the same IVDU * Intranasal cocaine  cocaine erodes the mucous membrane (they will bleed) and when IVDUs share straws blood can be mixed and HCV transmitted * Crack pipes  called crack pipes because they crack your lips  they can bleed and glass pipe passed along between users where blood can be mixed and HCV transmitted * IM anabolic steroids  injecting Little Evidence for Sexual Transmission of HCV

Answer 126

o Egypt: 10% of population infected with HCV (~10 million) o Pakistan: 5% of population infected with HCV (~10 million) o Needles being reused  iatrogenic o Tattoos  tribal: not clean o Circumcision  done by elders in a community not by trained doctors, but using the same equipment o Midwifery  small risk of vertical transmission of HCV (5%) but in a lot of rural areas using the same scissors and equipment o Clippers  heads shaved by the same clippers

Answer 127

* People born in countries of high HCV infection form major immigrant routes of HCV * Afghanistan and Northern Pakistan is the major sources of heroin in Europe  hence HCV genotype 3 can be found in IVDUs in Europe * Injecting Drug Use is a growing problem for the immigrant population (cheaper to do drugs than buy alcohol in these countries hence a lot of people who are depressed/unemployed etc will do drugs in order to feel better).

Answer 128

* PWIDs (people who inject drugs) * Prisoners * MSM * Blood transfusion * Iatrogenic from immigrant populations • Most affected age group: 25-34 years old Dore et al INHSU 2013 • PWIDs are 10x more likely to contract HCV than HIV • Prison Reform Trust, The Bromley Briefings 2012 o The majority of addicts in prison will be there for crimes committede related to their addiction  theft, violent crime, supplying or possessing drugs

Answer 129

* Thought to be related to chem-sex and slamming (ketamine, cocaine, crystal meth used to enhance sexual experience) * Initially believed to be associated with traumatic anal sex * Identified as a result of regular HCV monitoring in HIV positive men

Answer 130

o Prevent cirrhosis and decompensation (increased survival rates, and all-cause mortality reduces from other illnesses o To improve quality of life and remove stigma o To lower prevalence and reduce new infection rates o To attempt global eradication of the virus

Answer 131

• Reduce viral load and increasing immune system o Initially treatment was to modulate/strengthen the immune response by giving pegylated-interferon and twice daily ribavirin (mechanism = unsure)  huge side-effect profile and (teratogenic, flu-like symptoms, anaemia)  Plus constant need for monitoring every two weeks  and only ~30% SVR+ rate hence not feasible to implement in the populations who are transmitting the infection o Now: nucleotide analogues inhibit viral replication allowing the immune system to overcome the virus • NS3/4 protease inhibitors (different protease inhibitors to HIV but same principle) o Outcome improved to 80% but really bad side effect profile (worse than immune modulators)  right * Polymerase inhibitors (NS5B inhibitors) * NS5A inhibitors (preventing packaging and release of virus) o Previr  NS3/4 protease o Uvir  Polymerase inhibitors o Asvirs  NS5A inhibitors

Answer 132

* Zepatier (Grazoprevir+Elbasvir)  combination of NS3/4A protease inhibitor and NS5A inhibitor * Epclusa (Sofosbuvir+Velpatasvir)  combination of NS5B and NS5A inhibitor * Maviret (Glecaprevir+Pibrentasvir) * Vosevi (Sofosbuvir+Velpatasvir+Voxelaprevir)  combination of all 3 inhibitors * New drugs are extremely expensive  now cost has reduced to ~9000 in the UK * Still deemed cost-effective by NICE * Solutions needed for resource poor countries  companies now making it as cheap as 900 dollars for those countries * Treatment should be considered a priority in those with a high risk of transmission (e.g. dialysis patients, prisoners, MSM)

Answer 133

• Double stranded RNA retrovirus (replicates via a DNA intermediate) • Genome contains 9 genes which encode 15 proteins • HIV replicates much better in activated CD4+ T cells than in naïve cells o Establishes latent infection in long lived memory CD4 T cell – viral reservoir, inaccessible to current antiviral therapy

Answer 134

- HIV binds to CD4 receptor using gp120, but this is insufficient for it to enter  also needs to bind CCR5 - Upon entry, HIV RNA is reverse transcripted into DNA and integrated to host DNA using integrase - The HIV proteins are transcripted and translated as part of the human genome, and cleaved via protease - RNA genomes are also synthesised from the HIV DNA incorporated into the human genome - HIV particles are formed and budded from the cell to infect other cells • Viral reverse transcriptase (RT) has no proofreading mechanism and makes lots of errors in each viral cycle  driving force for viral diversity • Viral mutations has implications for the evasion of CTL immune responses and the emergence of drug resistant virus in patients with inadequate HIV-1 treatment o This forms the rationale for multidrug therapy • Up to 1010 HIV virions are produced every day in a chronic infected individual which infect 0.001 to 1% of all blood CD4 T cells • Half life of virus is <30min in blood and these originate from recently infected CD4+ cells • Can integrate into memory CD4 T cells  these long-term cells provide a reservoir for latency and makes eradication of HIV difficult

Answer 135

``` • Dendritic cells, monocytes, T cells • Cells expressing CD4 and co-receptor are both used in HIV1 entry: o CD4 receptor o CCR5 in initial transmission  MIP-1a, MIP1b and RANTES inhibit entry of HIV1 o CCX4 occurs in advanced disease  Allows virus to infect naïve cells  50% of cases  SDF1 inhibit entry ```

Answer 136

1. Acute infection 2. Decline in viral load 3. Chronic infection 4. AIDS LEARN GRAPH pg163

Answer 137

* Massive increase in viral replication (predominantly in gastrointestinal tract) * High risk of transmission  although one may possibly have different strains of HIV by the this time (due to error prone RT), one only transmits one strain of virus * In the UK, 10-20% of new HIV cases are recent seroconversion and individuals infected in last 6m * Tracing of this group important

Answer 138

* After some time, there is a decline in viral load, settling 3-6m after infection * This is due to partially effective immune response * CD4 cells are destroyed in the GIT and viruses have less cell reservoirs

Answer 139

• The virus settles into a long asymptomatic phase where viral levels gradually rise

Answer 140

• Viral load increases again

Answer 141

* In acute phase, there is a sudden drop in CD4 cells followed by rebound and progressive loss in AIDS * Over 90% CD4 population in the GIT is lost early in the disease and this does not recover (purple) * The red line shows the concept of immune activation * Associated with an excessive proinflammatory response characterised by cytokines such as TNFa and increased expression of inflammatory markers on CD4 cells pg 163 graph

Answer 142

1. CD4 T cell depletion (more marked in GIT than peripheral blood) 2. Chronic immune activation (increase in inflammatory immune responses) 3. Ineffective T cell regeneration – HIV infects BM stem cells  destruction of thymus + lymphatic architecture  further limits regeneration of T-cells over time 4. Functional T cell exhaustion – (CD8 CTL are present in large numbers but anergic and useless)

Answer 143

• Virus replicates better in memory cells  loss of memory CD4 and CD8 T cells followed by loss of naïve CD4 and CD8 T cells o Why CD8 T-cells? Still not entirely understood – thought to be due to more than just direct infection (see immune activation) • Increase in T cell turnover and accelerated CD4 cell death and impairment of CD4 T cell regeneration • Impairment of CD4 T cell proliferation to new and recall antigens (recall antigens i.e. antigens previously encountered by immune system before, most commonly used experimentally: tetanus) • Loss of CD4 T help to CD8 T cells, B cells (leads to recurrent bacterial pneumonia) and monocytes and macrophages (partly why you get opportunistic infections e.g. pneumocystis jirovecii, cryptosporidium, salmonella) • Progressive loss of killing and cytokine production by CD8 T-cells

Answer 144

* Immune activation is not observed in non-pathogenic HIV primate infection (SIV – Simian immunodeficiency virus) immune activation predict disease progression independently of CD4 and viral load in HIV1 infection * Increases in CD4 T cells post HAART are inversely related to levels of immune activation * Increases in immune activation may result in activation induced cell death of CD4 T cells and immune exhaustion/replicative senescence in CD8 T cells * Laboratory data shows that the % of activated TC (expressing DR+) is much more in viraemic patients and comes down after ART (see below) * One of the better markers for disease progression is the degree of activation of CD8 T cells * Patients not on treatment have increased levels of immune activation markers expressed by CD8 T cells, and these are reduced by ART * Rate of CD4 T cell apoptosis in HIV1 infection is much greater than in healthy controls (Caspase 3 = the marker of apoptosis measured) * 20% of CD4 T cells in HIV patients go through accelerated death

Answer 145

• Immune response to the HIV1 virus • Activation of immune system by HIV1 virus or HIV1 protein products (decoy proteins as seen in HBV infection) • Co-infection with conventional or opportunistic infections (CMV) • Microbial translocation from the GIT o Idea is that damage of GIT leads to access of bacterial constituents to bloodstream  sensed and causes pro-inflammatory response • Loss/dysfunction of counter regulatory immune suppressive responses

Answer 146

* HIV1 infection is associated with reduction in naïve CD4 and CD8 T cell counts * TREC counts (marker of thymus function) reduced in HIV1 infection • Several factors involved: o Accelerated turnover of memory CD4 exceeds capacity of CD4 T cell progenitors to replenish naïve T cell counts o HIV1 infection results in damage to bone marrow thymus and lymph node environment o X4 virus infects and kills naïve T cells

Answer 147

• CD8 T cells play a crucial role in limiting viral replication in EBV and CMV and are probably important in HIV1 infection • Rapid mutation of HIV may evade HIV CTL responses • Cytotoxic CD8 T cell are present in HIV1 however o Capacity to proliferate against HIV1 is impaired o Ability to secrete cytokines which inhibit viral replication is reduced o Cytotoxic activity is lost in progressive disease o CD8 T cell express markers of immune exhaustion such as Programme Death-1 (PD1)

Answer 148

• Consists of discrete inductive and effector compartments o Inductive: encounter with antigen  activation of immune cells o Effector: secretion of antibody  release of cytokines * Lymphocytes leave the inductive compartments and travel to the mesenteric lymph node – then directly go to the blood stream and back to effector compartment * Gastrointestinal mucosa is the largest lymphoid organ in the body * Contains up to 40% CD4 T cells compared to 2-5% found in peripheral blood • Composition of mucosal immune cells differs significantly from blood and lymph nodes o Memory cells far exceed naïve cells o CD4 T cells express high levels of the CCR5 chemokine receptor o Distinct set of CD8 T cells and dendritic cells are found in MALT • The dominant immunoglobulin at mucosal sites: IgA

Answer 149

• Peyer’s patches (PP) + Isolated Lymphoid Follicles (IEL) • Follicular associated epithelium o No afferent lymph node drainage o Antigen uptake occurs via M cells o DC can also take up antigen from lumen of intestine • Within PP and IEL o Antigen presentation takes place to B cells o IgA class switching occurs o IgA plasma cells exit and head for LP

Answer 150

``` • Diffuse collection of immune cells located beneath epithelial lining • Contains activated T and B cells that have been exposed to foreign proteins • Lamina propria cellular network o CD4 T cells  CD8 T cells o Plasma cells which secrete IgA • Intra-epithelial lymphocytes o Mainly CD8aa T cells o Function not fully understood ```

Answer 151

* Damage to the lining of the anorectal mucosa can promote HIV1 transmission * HIV may also be transmitted via M cells * Initial targets of HIV1 infection are dendritic cells and memory CD4 T cells * DC can transfer intact HIV virus to the draining lymph nodes GIT is the major site of HIV replication in acute infection • HIV gp120 molecule can bind to an intestinal homing protein expressed by CD4 T cells (a4b7 integrin) • Within the GIT the interaction of gp120 with a4b7 integrin promotes transfer and spread of HIV • Binding of gp120 to homing protein activates CD4 T cells which makes it easier for the virus to replicate and leads to massive increase in HIV1 viral load seen in acute illness • CCR5+CD4+ cells are much more common in the intestine compared with blood  explains the greater CD4 T cell loss in the gut o There is selective loss of CD4+ in the lamina propria in HIV1 infection

Answer 152

• Damage to the lining of the gut results in microbial translocation and immune activation • Concentration of LPS, a marker of microbial translocation is increased in patients with progressive HIV1 infection and in SIV macaques • PCR to amplify DNA sequences encoding well-conserved rRNA subunit common to most bacteria is used as marker of microbial translocation • Bacteria in plasma correlates with CD4 mucosal depletion in HIV • Microbial translocation is associated with T cell and monocyte immune activation o T cell activation: expressing of DR and CD38 on CD8 T cells o Activated monocytes secrete less TNF and IL1 when stimulated in vitro

Answer 153

* Decreased mucosal IgA production * Loss of epithelial tight junctions * Increased enterocyte apoptosis * Loss of CD4 Th17 cells (which stop bacterial translocation) * Loss of group 3 innate lymphoid cells * Alteration in the composition of intestinal bacteria

Answer 154

• Gap junction protein seal para-cellular junctions • Restrict access of solutes, particulates, bacteria across GIT • Main protein families consist of: o Claudins o Occludins o Zonulins • Claudin 4 o Reduced expression in HIV1 infected (ART naïve) o Partially restored in HIV patients on ART • HIV infection associated with loss of reduced expression of epithelial gap junction proteins and increased breaches in the mucosal barrier – this correlates with systemic immune activation CD4 Th17 cells involved in host defence against extracellular bacterial infections • CD4 Th17 involved in activation and recruitment of neutrophils to mucosal surface via IL17 • Maintenance of integrity of epithelial cells via IL22 • Secretion of mucosal antimicrobial proteins via IL22 • Mucosal CD4 Th17 cytokine secretion (both IL17 and IL22) is reduced in HIV ART naïve patients (i.e those are restored in those who receive ART) • There is a negative correlation between microbial translocation and the % of CD4 Th17 cells in the GIT Summary: for microbial translocation to occur you need “two hits”  both damage to lining of gut and loss of protective CD4+ T-cells

Answer 155

ART • Low level viral replication takes place in the GIT even in patients with undetectable viral load in blood • HIV can exist in a latent state in resting long lived memory CD4 and macrophages in the colon and rectum  forms an important reservoir of infection and formidable barrier in our attempts to cure this infection • Immune reconstitution involving CD4 T cell in the GIT much slower and not as complete as that seen in blood • Concentration of HIV-1 is higher in CD4 T-cells in the gut (GALT) compared to blood even in patients on successful ART o Over-estimates the total burden of latent HIV infection  probably only 5-10% of these cells will be able to produce active virus o There are no good assays to estimate the effectiveness of cure of HIV strategies  can stop ART and then check for viral load however, this can lead to two side effects: rebound of disease or an acute serocoversion illness (headaches, aseptic meningitis etc) hence not widely used. Also time taken for disease to rebound varies between patients and can occur as late as one year after stopping ART.

Answer 156

• Vedolizumab is an 47 monoclonal antibody used in the treatment of Inflammatory Bowel Disease • In non human primates vedolizumab – Reduces risk of transmission IN ART naïve animals – Delays onset of symptomatic disease in ART naïve animals – In combination with ART in acute infection may lead to a functional cure – In HIV-1 individual with IBD humans vedolizumab – Safe and well tolerated – Reduces size and number of lymphoid follicles in terminal ileum – Diminished frequency of activated CD4 T cell in terminal ileum – No change in number of CD4 T cells in the lamina propria

Module 3.2: Viral Hepatitis Flashcards

(181 cards)