Chem 195 Lecture 11

1
Chem 195 - Lecture 11

• Infectious viral disease Diagnosis, Therapy and
  Prevention The lessons of Hepatitis - Hepatitis
  C Cloning, Lamivudine, Hepatitis B Vaccine.

2
Housekeeping

• Files on web site this afternoon
• Lecture11.ppt, Hepatitis.pdf
• Project topics - list
• CNS - Alzheimers, Migraine, Schizophrenia
• ID - flu, CCR5/HIV, HBV, malaria
• Allergy, asthma, oral insulin
• Cancer - PDGFR, Histone deacetylase
• Project presentations - 5/10?
• If so, then will discuss new cancer therapeutics
  next week

3
Infectious Diseases

• Hepatitis
• A collection of diseases characterized by liver
  inflammation with a significant immunological
  component, usually viral in origin, with a clear
  link to hepatocellular carcinoma
• Hepatitis viruses
• A - food handling transmission
• B - bodily fluids, vertical transmission
• C - bodily fluids, other?
• D - coinfection with B
• E,F,G, ...

4
Infectious Diseases

• Disease impact
• 1/3 of the population in the world has been
  exposed to HBV! Estimated 1.5 million deaths per
  year. Extremely high incidence in Asia.
• There are 4,000,000 HCV positive people in the US
  alone
• Both diseases result in chronicity in a large
  fraction of cases
• Chronic viral infection leading to liver failure
  is the largest cause of liver transplantation
  today
• HBV is responsible for the large majority of
  liver cancer

5
Infectious Diseases

• Hepatitis B - Discovery
• Pre 1970s major surgery with multiple
  transfusions led to Hepatitis in ca. 50 of
  patients
• Blumberg - a common rare Antigen between a
  hemophiliac in New York and an Australian
  aborigine
• Prince - Correlation of Australia Antigen with
  transfusion Hepatitis
• Dane - first to visualize HBV particles
• Blumberg - 22 nm particles (only Aa/HbSAg)

6
Infectious Diseases

• Hepatitis B - Screening
• Aa recognized to be viral coat protein, renamed
  Hepatitis B surface antigen (HbSAg).
• Radioimmunoassay developed to detect antigen and
  used for blood screening starting 1972
• Inactivated viral preparations shown to have some
  protective efficacy
• Blumberg hypothesizes (and files patent) that 22
  nm particles (no viral nucleic acid) might be
  used to a vaccine

7
Infectious Diseases

• Hepatitis B - First Generation Vaccine
• Maurice Hilleman and colleagues at Merck isolate
  22 nm particles from carriers
• Over 10 years develop a particle based vaccine
  for HBV - shown to be gt 90 effective
• Licensed by FDA in 1981
• Issues with supply (getting serum from carriers)
  and possible contamination with other viruses
• Around the same time recombinant DNA technology
  led to the cloning of the HBV genome

8
Hepatitis
9
Infectious Diseases

• Genome of HBV
• Very small - only 4 proteins
• S (with pre-S1 and S2)
• C (viral core protein, coats NA)
• Pol (viral polymerase)
• X (transcriptional activator - key for
  hepatocarcinogenesis)

10
Infectious Diseases

• Hepatitis B - Second generation vaccine
• Attempts to use recombinant methods for
  expression of viral antigens in E. coli
• Worked for core but not for surface. Why?
• Antigenicity of surface is dependent on particle
  formation. Particles are 1000 fold more
  antigenic
• E.coli doesnt form particles!
• What to do ? Try another expression system!

11
Infectious Diseases

• Hepatitis B - Second generation vaccine
• Expression systems in S. cerevisiae just being
  developed
• Collaboration between Ben Halls group and Bill
  Rutters group led to demonstration that
  particles were formed by yeast expression.
• Chiron started to collaborate with Merck on 2nd
  generation vaccine
• Why and how yeast form particles is still not
  well understood as particles are not secreted
• But yeast particles are as immunogenic as 22 nm
  particles from virus

12
Infectious Diseases

• Hepatitis B - Second Generation Vaccine
• Improvements in expression levels necessary
• First exploration of applied yeast molecular
  biology
• Improved promoters - glycolytic genes
• Hybrid regulated promoters
• Reduced degradation (proteases)
• Transcriptional terminators
• Codon bias/synthetic genes
• Led to approval in 1986 of Recombivax - first
  human vaccine made by recombinant methods

13
Infectious Diseases

• Hepatitis B - Impact of Vaccine and Screening
• Incidence of HBV in infected blood reduced to lt
  11,000,000
• Vaccine now a childhood immunization in many
  countries
• In Asia vaccination reduced carrier rate from ca.
  10 to lt 1 - presumed decrease in hepatocellular
  carcinoma
• Vaccine still expensive for 3rd world
• Combination HAV/HBV vaccine available

14
Infectious Diseases
15
Infectious Diseases

• Hepatitis B - Treatment
• Lamivudine (3TC)
• HBV polymerase/RT inhibitor - also used to treat
  HIV (RT inhibitor)
• Nucleoside analog which causes chain termination
  during virus replication
• After treatment for 1 year resistant virus
  evolves in a significant number of patients
  (25-40)

16

• Molecular Mechanism of Resistance
• Conserved active site motif in all polymerases
• Gly-Met-Asp-Asp at residues 551-554 in HBV
  polymerase reduces Ki for lamivudine 104 fold
• Also reduces viral replication efficiency

17
HBV - Summary

• Major health problem - especially in Asia
• Transmission by bodily fluids or vertically
• Blood screening test has largely eliminated
  transfusion risk
• Vaccine has potential to greatly reduce/eliminate
  disease
• Treatment is difficult because of resistance

18
Infectious Diseases

• HBV blood screening test eliminated most but not
  all transfusion related Hepatitis
• Remaining disease was called NANB Hepatitis
• Early work showed it could be transmitted to
  chimpanzees
• No virus could be isolated or identified but
  serology could recognize NANB

19
NANB Hepatitis

• Questions
• Was it a virus?
• Was it more than one virus?
• What kind of virus?
• Many groups tried to isolate by classical methods
  but were unable to up to the mid 1980s

20
NANB

• New molecular approaches used at Chiron
• Collaboration with CDC obtained NANB acute phase
  serum from a chimpanzee (presumably contained
  virus)
• Prepared cDNA library with random primers after
  RT and cloned in lgt11 expression screening
  vector
• Expression vector clones cDNA in the C-terminus
  of E.coli b-galactosidase - phage lysis on
  nitrocellulose filters enables screening of
  expressed proteins

21
NANB

• cDNA library on filters was then screened with
  serum from a patient who had recovered from NANB
• Out of 106 clones one was reactive with the serum
• Shown not to be encoded in human or chimp DNA
• Subsequently shown that only 1 strand hybridized
  to RNA in original chimp serum - thus
  single-stranded virus
• Hybridization signal was RNAase not DNAase
  sensitive

22
NANB to HCV

• Calculated that of viral RNA in original sample
  was 1107!
• By subsequent cloning experiments showed that
  genome was a single stranded RNA of ca. 10 kB
• Fusion proteins were used to test for antibodies
  in a well characterized panel from the NIH
  containing NANB and other patient sera -
  correctly!

23
HCV

• Subsequent expression of recombinant antigens led
  to a series of improving and highly effective
  tests for HCV antibodies
• Reduced incidence of transfusion mediated HCV to
  very low levels
• Still issue with window between exposure and
  antibody production (seroconversion) - could be
  as long as 16 weeks for HCV

24
HCV

• Newest blood screening methodology
• Detect the viral genome instead of antibodies
• Technology very demanding especially for
  automation required to screen 50 million blood
  donations!
• TMA and PCR as preferred technologies
• Approval of TMA based test this year for genome
  screening of HCV and HIV - reduces window to lt 2
  weeks

25
HCV and Drug Discovery

• Genome sequence showed at least 4 viral enzymes
  (2 proteases, helicase, polymerase)
• Development of infectious clones (1997 - by
  injection of viral RNA into chimp livers) showed
  their necessity for viral infection
• No small animal model until last year (transgenic
  mouse model with human hepatocytes)
• These tools will greatly facilitate HCV antiviral
  drug development

26
HCV Genome and Proteome
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