Hepatitis Viruses Flashcards
Hepatotropic viruses which induce inflammation of the liver
Hepatitis: inflammation of liver parenchyma, death of hepatocytes
- Symptoms: Ikterus (jaundice), high levels of transaminases in blood serum
Hepatitis A Virus (HAV) Picornaviridae, ca. 7.5 kb ss (+) RNA
Hepatitis B Virus (HBV) Hepadnaviridae, ca. 3 kb RC-DNA
Hepatitis C Virus (HCV) Flaviviridae, ca. 9.5 kb ss (+) RNA
Hepatitis D (Delta) Virus (HDV) “Virusoid”, 1.7 kb circular ss (-) RNA
Hepatitis E Virus (HEV) genus Orthohepevirus, family Hepeviridae ca. 7.5 kb ss (+) RNA (alphavirus-like superfamily)
Pathogenicity
HAV
- route of infection: fecal-oral (enteral) (“traveler hepatitis”-infectious”)
- acute hepatitis: yes
- severe acute-sympt. (fulminant): rare
- chronic hepatitis: no
- cirrhosis HCC: no
HBV
- route of infection: parenteral (“serum”) (blood, -products, body fluids)
- acute hepatitis: yes
- severe acute-sympt (Fulminant): rare
- chronic hepatitis: yes (5-10 %)
- cirrhpsos HCC: yes
HDV
- route of infection: similar to HBV/depends on HBV as helper
- acute hepatitis: yes
- severe acute-sympt. (fulminant): increased
- chronic hepatitis: yes
- cirrhosis HCC: increased
HCV
- route of infection: parenteral (blood, -products, body fluids)
- acute hepatitis: yes
- severe acute-sympt (fulminant): rare
- chronic hepatitis: yes (80 %)
cirrhosis HCC: yes
HEV
- route of infection: fecal-oral
- acute hepatitis: yes
- severe acute-sympt. (fulminant): rare
- chornic hepatits: no
- cirrhosis HCC: no
Chronic hepatitis
ca. 600 million cases worldwide
Chronic Hepatitis B: ca. 400 million Chronic Hepatitis C: ca. 170 million
Cirrhosis: connective tissue replaces hepatocytes
Immune pathology / Viruses are not cytolytic -> (20 - 40 years) -> 20 % of chronic carriers
Health problem: not the virus kills the hepatocytes but the immune system: Immune pathology
Immune reaction against HBV
- severely delayed (after months)
– liver is immuno privileged organ
– non cytopathogenic virus
– vast amount of antigen (1011virions/ml) - Immune pathology
– Cytotoxic T cell response
– Natural killer cells
– Elimination of HBV-producing hepatocytes
– Cytokines (IFN-g, TNF-a)
– Extrahepatic disease manifestation by immune complexes - Arteritis, Vasculitis
Problems of HBsAg Tests
- HBV Genotypes react differently
– WHO provides only heat-inactivated standard serum for genotype A
– worldwide genotypes B, C and D dominate
– until a few years ago genotype F was not recognized by some of the tests used - Suboptimal sensitivity
– best ELISA detetcs 10 pg HBsAg/ml
– or 106 HBV particles/ml
– not reached in early phase of infection - HBsAg is highly variable when under immune pressure /selection
- possible consequence: wrong negative results
Methods for Hepatitis B Virus detection
With Detection limit Particles/mL
1973 Chimps: infectious dose required 10 - 100
1973 detection of endogenous HBV 10^8
-> DNA-polymerase activity
1979 Cloning of HBV genome in E. coli
1980 DNA-Hybridisation, bDNA, dot blot 10^6 - 10^7
Since 1988 PCR 1- 10^3
Hepatitis B Virus: Prophylaxis and Therapy
Diagnostic Test: HBsAg ELISA I
ELISA = Enzyme-linked immuno-sorbant assay
Vaccine: first vaccine 1981(Hepatavax-B) HBsAg purified from chronic carriers since 1986 recombinant vaccine from yeast; highly effective > 97 %
Prophylactic passive immunisation:
human anti-HBsAg immunoglobulin (from vaccinated blood donors) post-exposition-, post-transplantation prophylaxis
block of perinatal transmission: combination of active + passive
Therapy:
Pegylated Interferon alpha (IFN-a)
Three chemical therapeutics: all RT-Inhibitors
-> Lamivudine (3 TC), Adefovir (ADV) and Entecavir
Sustained efficacy (virus elimination and control) only in less than 40%
Problem: Resistance!
Therapeutic Agents Available for Treating HBV Infections
- Interferon alpha
- Lamivudine
- Adefovir Dipivoxil
- Telbivudine (L-dt)
Genotype-Independent Numbering Scheme for HBV Polymerase/RT Domain
Why is this important? To standardise resistance screening!
- I(G): LMV Resistance, ADV Resistance, ETV Resistance, L-dT Resistance
- II(F): LMV Resistance, ADV Resistance, ETV Resistance, L-dT Resistance
- A: rtL80V/I
- B: rtV173L, rtL180M, rtA181T/V, rtS184G
- C: rtM204V/I/S, rtS202I, rtM204I
- D: rtN236T
- E: rtM250V
Viral replication and Mutational Frequency
- High virion production: 10^12-13 virions per day
- High mutational rate-5 substitutions/bases/cycle
- 1010-11 point mutations produced per day
- All possible single base changes can be produced per day
History
1965 “Australia antigen”
History
Blumberg discovered 1965 in the blood of Australian aborigines accidentially a protein of HBV during anthropological studies on polymorphisms in the blood of different human „races“. He called it „Australia antigen“.
The blood of his asistant (Barbara Werner) served as „negative control“; however, after some time her blood became positive for the „Australia antigen“ and she developed an acute hepatitis (but luckily cleared infection). Connection to infection in humans!
Serological blood test!
Baruch S. Blumberg: Nobel Prize 1976 1970 HB virions detected by EM
Antigen infectious in Chimps -> Virus
Dane particles = sub viral filaments and spheres (HBsAg)
1978/79 First DNA clones and sequences of HBV genomes
1979 Recombinant expression of HBV-antigens in E. coli 1984 Recombinant HBsAg Vaccine in yeast (Recombivax HB)
(first recombinant vaccine!)
1980 Duck HBV; DHBV (most relevant model system)
1982 Hepadnaviral replication by reverse transcription
Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate.
Until 1987: no cell lines for HBV genome replication (now HepG2, Huh7)
Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for human hepatitis B and D virus
Expression of NTCP in hepatoma cell lines (e.g. Huh7) paved the way for complete viral replication cycle in cell culture
Otherwise only in primary human hepatocytes!
Structural Components of HBV and HBsAg Particles and their ratio (in serum)
Virus -> 1 PCR
Filaments -> 10 ELISA
Spheres -> 1000 ELISA
HBV - a genome with a gap
- 3.2 kb, small, information enormously condensed
- several unusual features
partially ds DNA:
complete (-)-strand (longer than 1 copy) incomplete (+)-strand (gap)
circular, but not covalently closed: Relaxed Circular (RC)-DNA (vs. cccDNA)
5 ́end of (+)-strand DNA covalently linked to viral RNA with Cap
5 ́end of (-)-strand DNA with covalently linked P-protein
incomplete (+)-strand, structure maintained by hybridization to complementary (+)-strand
HBV genome: enormously condensed genetic information
- each nucleotide has coding function
- overlapping ORFs: > 50 % of bases encode in 2 different ORFs
- all regulatory elements (promoters, enhancers, poly-A-signal, replicational-cis- elements) overlap with ORFs
- ORFs are modular:
PreC/C, PreS1/PreS2/S > C and S modules are used in different proteins
HBV: Pol and env linkage
Consequence of Overlapping ORFs:
- each nucleotide change in envelope gene may at the same time lead to amino acid change in the Pol protein (and the other way round)!
Hepatitis B Virus: Replication cycle
ccDNA Episome (10 - 100 copies): Serves as template for mRNA transcription!
Receptors: sodium taurocholate cotransporting Polypeptide (NTCP)
Hepatitis B Virus: Transcripts and gene products
- 6 mRNAs all transcribed in nucleus by cellular Pol II
- each with Cap and polyA tail
- identical 3 ́ends (only 1 polyA signal)
- usually only first cistron translated (exception is p,
HBV reverse transcriptase: gene product P
Translation of p:
- no separate mRNA for P!
- P protein is encoded in the second cistron of pg (pregenomic) mRNA
- mechanism unclear (Leaky scanning? Reinitiation? Shunting?)
P Domains: includes motifs in Pol/RT and RH which are shared with retroviral RT Additional domain: TP (terminal protein)-domain without know homologs
Hepatitis B Virus: reverse Transcription
-> RT step
- in immature capsid
- requires pg RNA, core and P protein
Specific packaging of pGRNA
- pgRNA is encapsidated with high specificity
- no pgRNA packaging without P-protein
- no pgRNA packaging without intact 5 ́end
- fusion of pgRNA 5 ́-end to heterologous RNA allows it ́s packaging (packaging signal)
RNA stem-loop () at 5 ́end of pgRNA serves as packaging signal recognized by the P-protein (epsilon serves as encapsidation signal)
Reverse transcription takes place in immature capsid
-> Requirements
Specific template RNA (pgRNA) + HBV revers transcriptase (= P-protein) have to be packaged specifically into the capsid (assembled Core proteins)
Specific recognition between pgRNA + P-protein + Core protein
Reverse transcription of pgRNA
Early observations
All DNA-polymerases need a “primer” (3 ́-OH end) for initiation of DNA synthesis
- 5 ́ base of (-)-DNA covalently bound to tyrosine in TP-domain of P > “protein-priming”
- P-protein accepts no other template RNA than pgRNA (<> cDNA-synthesis!)
- 5 ́ end of (-)-DNA localizes in 3 ́proximal DR1 (direct repeat 1)
P-protein required at 5 ́ for pgRNA packaging and at 3 ́ DR1 for DNA synthesis!??
UUC sequence in DR1 exists also in the bulge region (sequence of duck HBV)
Its complementary sequence AAG in the (-)-DNA represents a copy thereof
Transfer/jump of pol (compare?) (TP vs Vpg of Picornaviridae…)
Proof: Mutation of UUC in to UUG; sequence at 5 ́end of (-)-DNA changes to AAC (instead of AAG!)
epsilon contains origin of replication for (-)-DNA
(-)-DNA synthesis is discontinuous (compare Retroviruses)
HBV (-)-DNA Synthesis
Initiation
Priming of (-)-DNA in epsilon at 5 ́-end of pgRNA First template switch to 3 ́DR1
- annealing of primer (linked to P) to 3`DR1
(-)-DNA Synthesis
Elongation of (-)-DNA to 5 ́end Degradation of pgRNA (template) by RNase H (domain of P), with exception of 5 ́-end including DR1
(+)-DNA Synthesis (only side-, not main product)
Remaining 5 ́- end fragment of pgRNA serves as primer for (+)-DNA synthesis
ds linear DNA only side product
in situ priming (5-10% of cases) ds linear DNA (with short RNA at 5 ́end of (+)-DNA)
Regular (productive) HBV (+)-strand DNA-synthesis
Regular (+)-DNA-synthesis > RC-DNA (main product = 90%)
- template switch:
RNA primer transfer from DR1 to DR2
Elongation of (+)-DNA to 5 ́- end of (-)-DNA template (analogous “strong-stop” DNA in retrovirus replication)
“R” identical at 5 ́ and 3 ́-end of (-)-DNA - template switch:
(+)-DNA 3 ́-end of (-)-DNA 5 ́R to 3 ́R
What is the driving force for template switching?
Thermodynamics of strand interactions = genome gymnastics
Genome gymnastics in HBV replication
- Initiation at 5 ́ by P covalently linked to priming nucleotides
- Switch of P-linked primer to DR1, annealing and ( -) strand DNA synthesis
- (+)-RNA genome degradation by RNaseH (part of P)
- 5 ́part of (+) RNA which is not degraded, serves as primer for (+)-DNA synthesis
- Elongation till completion; in 5-10% of cases („in situ priming“; none productive way)
- 90-95% of (+) DNA synthesis: Second template switch leads to elongation to 5 ́R sequence followed by
- Third template switch by hybridization between copy of 5 ́R in (+)-DNA strand and 3 ́R of (-)-DNA strand
(+)-strand synthesis continues for variable distance (-> incomplete (+) strand!)
The resulting partially ds-DNA serves as genome found in mature virions
Formation of stemloop in DR1 in (-) DNA „truncates“ sequence complementary to primer -> longer complementary sequence now in DR2 -> more stable base pairing with DR2
Thermodynamics determine the consecutive annealing steps!
NTCP: sodium taurocholate cotransporting polypeptide - the receptor for HBV
Receptor candidates (binding to aa 21–47 of preS1) published so far:
- human immunoglobulin A receptor
- glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
- interleukin-6
- a 31-kDa protein HBV binding factor
- asialoglycoprotein receptor
- nascent polypeptide-associated complex α polypeptide
- lipoprotein lipase
- hepatocyte-associated heparan sulfate proteoglycans
- glucose-regulated protein 75.
The only bona fide receptor is NTCP (transporter for uptake of bile salts into hepatocytes)
Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein
PreS1 domain binds to NTCP receptor on hepatocyte
Subcutaneous application of myristylated preS1 peptide in humanized uPA-mouse:
- specific accumulation of peptide in liver
- prevention of HBV infection of human liver cells
- prevention of Hepatitis Delta infection
Effective when a HBV-positive person gets a liver transplantation
Peptide drug: Myrcludex B
HDV -> Satellite-virus
- 1.7 kB RNA (neg. polarity)
- needs Hepatitis B Virus capsid proteins for packaging
- codes for hepatitis delta antigen (HD Ag; L and S form)
- S-HD Ag important for genome replication, L for virion assembly
- cell. RNA Pol II (DdRp) is involved in genome replication
- genome slightly larger than in viroids
- Coding one ORF but no capsid proteins
- The virusoid-nucleic-acid is packaged by the capsid proteins of HBV
- HD-Ag forms RNP with RNA genome; import into nucleus via NLS in HD-Ag
- Rolling circle amplification leads to concatameric + strand (anti-genome) which is cut to genome size + RNAs by a ribozyme at the 3 ́end of the anti-genome; circularisation (ligation)
- Rolling circle amplification of - RNA (genomic) concatamer; ribozyme cleavage….
Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: First results of a phase Ib/IIa study
The therapeutic option for patients with chronic hepatitis delta virus infection (CHD) is limited to interferon alpha with rare curative outcome. Myrcludex B is a first-in-class entry inhibitor inactivating the hepatitis B virus (HBV) and hepatitis D virus (HDV) receptor sodium taurocholate co-transporting polypeptide. We report the interim results of a pilot trial on chronically infected HDV patients treated with myrcludex B, or pegylated interferon alpha (PegIFNα-2a) or their combination.
METHODS:
Twenty-four patients with CHD infection were equally randomized (1:1:1) to receive myrcludex B, or PegIFNα-2a or their combination. Patients were evaluated for virological and biochemical response and tolerability of the study drugs at weeks 12 and 24.
CONCLUSIONS:
Myrcludex B showed a strong effect on HDV RNA serum levels and induced ALT normalization under monotherapy. Synergistic antiviral effects on HDV RNA and HBV DNA in the Myr-IFN cohort indicated a benefit of the combination of entry inhibition with PegIFNα-2a to treat CHD patients.
LAY SUMMARY:
Myrcludex B is a new drug to treat hepatitis B and D infection. After 24weeks of treatment with myrcludex B and/or pegylated interferon α-2a, HDV RNA, a relevant marker for hepatitis D infection, decreased in all patients with chronic hepatitis B and D. Two of eight patients which received either myrcludex B or pegylated interferon α-2a, became negative for HDV RNA, and five of seven patients who received both drugs at the same time became negative. The drug was well tolerated.
NEW NAME: Hepcludex
Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo
Hepatitis D virus (HDV) doesn’t encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV.
In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co- infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.
Oral administration of a chimeric Hepatitis B Virus S/preS1 antigen produced in lettuce triggers infection neutralizing antibodies in mice
Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders…
Here we describe the transient expression of the S/preS121-47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121-47 antigen, three times, at 1 μg/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP- expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121-47 antigen as a promising candidate for future development as an edible HBV vaccine.
HVB core as a scaffold to present antigens to the immune system
- core protein self-assembles into particles – easy to purify, highly antigenic
- since long time trials to present foreign antigens on core to immune system
- problem: many large inserts in core protein interfere with particle assembly
- solution: cleavage of core with large insert (by addition of protease) into N-term. and C-term. half followed by assembly into icosahedral hepatitis B virus capsids
- large inserts do no longer interfer with particle formation
- programmable viral nanoparticles
