Hepatitis A Virus

General introduction
Molecular basis of cell culture adaptation
and attenuation
New variants for improved HAV vaccines

Children‘s Hospital of the University of Tübingen, Germany

Bertram Flehmig, Andrea Normann, Judith Graff,
Mechthild Pfisterer-Hunt
 Hepatitis A

Part I General introduction

Part II Development of the HAV vaccine
strain GBM and molecular markers
for cell culture adaptation
Part III Attenuation studies
Part IV Cell culture work
Hepatitis = Jaundice

Symptom:
Destruction of the hepatocytes
leads to elevation of serumbilirubin
and accumulation in sclera and
skin
Hepatitis = Jaundice
 VIRAL HEPATITIS
HISTORICAL PERSPECTIVE

Enterically
“Infectious” A E transmitted

Viral “NANB”
hepatitis C
Parenterally
“Serum” B D transmitted
other
 Hepatitis A Virus
Short scientific history
The illness is well known hundreds of years ago

1973 Detection of hepatitis A virus

1973-1978 Characterisation of the virus
1979/80 Successful growth in cell cultures in vitro
1989 First inactivated hepatitis A vaccine
 Hepatitis A virus

• RNA Picornavirus
 Single serotype worldwide
 Acute disease and asymptomatic infection
• No chronic infection
 Protective antibodies developed in
response to infection - confer lifelong
immunity
 Picornaviruses
• “pico” - small
• “rna” - RNA
• single stranded, positive RNA
• unenveloped, relatively stable
Hepatitis A Virus
•1 Serotype
•7 Genotypes
•>90% of human strains genotype I IA

I
IB

Dendrogram depicting nucleotide

VII
sequence identity among
representative HAV strains. II
III B
Identity was determined by III
III A
pairwise comparison of
sequences within a 168 bp V
segment of the HAV genome.
VI

75% 80% 85% 90% 95% 100%

Nucleotide identity
Hepatitis A Virus Dendrogram
•1 Serotype
•7 Genotypes
•>90% human strains Genotype I
I

VII

II
III
V
VI
75% 80% 85% 90% 95% 100%
Nucleotide identity
HAV Polyprotein Processing
 Hepatitis A – Clinical Features
•Jaundice by age group: <6 yrs <10%
6-14 yrs 40%-50%
>14 yrs 70%-80%
•Rare complications: Fulminant hepatitis
Cholestatic hepatitis
Relapsing hepatitis
•Incubation period: Average 30 days
Range 15-50 days
•Chronic sequelae: None
•Mortality: 0.1% up to 2%
in patients older than 40 years
 Liver failure in hepatitis A infected
children from Pakistan
Study from Shah,Habib, Kleinman in Pediatrics 2000
Aga Khan University, Karachi

2735 Hepatitis A patients children under 15 years

232 Patients admitted to the hospital
30 Patients with liver failure
25 (83.3%) Patients having encephalopathy
10 Patients died
Mortality rate 0.36% this is comparable to the mortality rates
in adults under 40 years in industrialized
countries of Europe and USA
EVENTS IN HEPATITIS A VIRUS INFECTION
Clinical illness

Infection ALT

IgM IgG
Response

Viremia

HAV in stool

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Week
CDC 2003 modified after Normann et al. 2004
Geographic Distribution of
Hepatitis A Virus Infection
 PREVENTING HEPATITIS A

• Hygiene (e.g., hand washing)

• Sanitation (e.g., clean water sources)
• Hepatitis A vaccine (pre-exposure)
• Immune globulin (pre- and post-
exposure)
 PREPARATION OF INACTIVATED
HEPATITIS A VACCINES

• Cell culture adapted virus grown in

human fibroblasts
• Purified product inactivated with
formalin
• Adsorbed to aluminum hydroxide
adjuvant
Development of the Hepatitis A
vaccine strain GBM
Selection scheme of the established cell culture
adapted variants of the wild type HAV strain GBM
GBM/WT isolated from the feces of a patient in the
preicteric phase of infection

8 passages on HEK cells

GBM/HEK
p1-p8
24 passages on HFS cells 64 passages on FRhK-4
cells
GBM/HFS
p8/1-p8/24
GBM/FRhK
p1-p64
What happens in the genome of
HAV strain GBM during the growth
in cell culture ?
Comparison of amino acid exchanges of wild type
HAV to cell culture grown HAV in the 2B region at
nucleotide position 3889

Strain propagated in
GBM HEK HFS
wild type (P0) A A
P1 A A/V
P2 A/V A/V
P3 A/V V
P4 A/V V
P5 V V
Conclusion I

The mutational event at the nucleotide

position 3889 in the 2B region of the HAV
genome, leading to the amino acid
exchange from alanin to valin is a
prerequisit for the growth of HAV in various
cell cultures in vitro and happens in the
first 1 to 3 passages.
What happens in the HAV genome during
further passages in human fibroblast cell
culture ?
Growth characteristics of GBM/WT during
successive propagation on HEK and HFS cells
Secondary structure of the 5‘NCR
Isolated, native Replicationcomplex

100 nm
 Conclusion II

Further passaging (3 to over 20 passages ) of

HAV in cell culture results in faster growing
which is correlated with further mutations in the
5‘ non coding region of the HAV genome
Intravenous inoculation of wild type and
attenuated HAV GBM in chimpanzees
Oral application of attenuated HAV
strain GBM in chimpanzees
Conclusion III

Human fibroblast cell culture adapted HAV strain

GBM is attenuated for chimpanzees
 Genetic variability of Hepatitis A Virus
Evidence for quasispecies distributions in the human hepatitis A virus
genome.
Sánchez G, Bosch A, Gómez-Mariano G, Domingo E, Pintó RM.
Virology. 2003 Oct 10;315(1):34-42.

Analysis of sequential hepatitis A virus strains reveals coexistence of

distinct viral subpopulations.
Costa-Mattioli M, Domingo E, Cristina J.
J Gen Virol. 2006 Jan;87(Pt 1):115-8.
 
Quasispecies distribution in the hepatitis A virus strain GBM genome
Krause B E, Normann A, Flehmig B 2005

Hepatitis A virus mutant spectra under the selective pressure of

monoclonal antibodies: codon usage constraints limit capsid variability.
Aragonès L, Bosch A, Pintó RM.
J Virol. 2008 Feb;82(4):1688-700. Epub 2007 Dec 5.
Conclusion IV

A genetic variability and a quasispecies phenomenon

is existing in hepatitis A virus strains
Development of new and improved
vaccine strains
 Actual HAV yield

required cm2 cell culture for 1 Vaccine-Dosis mean: 16 cm2

Vaccine-Doses/ CF40 (24.000 cm2): 1.500

Vaccine-Doses/ CF10 (6.000 cm2): 375

New GBM/HFS HAV variant: IP3
 Growth comparison of SP and IP3
cell bound HAV-antigen

2500

2000
Titer HAV-antigen

1500 SP
IP3
1000

500

0
0 5 10 15 20 25 30
Days after infection
 Growth Comparison of SP and IP3 in cell culture supernatant

800
700
Titer HAV-antigen

600
500
400
300
200
100
0
0 5 10 15 20 25 30
Days after infection

SP IP3
Newest GBM/HFS HAV variant: SPs
 Results from Flehmig Laboratories Tübingen

HAV-Strain Titer in T25 Vaccine- cm2/ Vaccine-Doses/

GBM/HFS Doses Vaccine-Dose CF10 (6.000cm2)
SP 1:1500 3 8,3 723
IP3 1:2250 4,5 5,5 1091
SPs 1:8666 17,32 1,44 4166

SP = Seedpool GBM/HFS: HKC8/HFS17/MRC-5

3 x end dilution purified

IP 3 = GBM/HFS variant

SPs = GBM/HFS variant

Non-immune humans worldwide 2007
1 Billion
Non-immune humans by region (2007)
Europe: ~ 150 millions
USA: ~ 150 millions
South-America: ~ ?
Asia: ~?
Non-immune humans predicted for 2020
without vaccination

Europe: ~ 600-900 millions

USA: ~ 300-500 millions
South-America: ~ ?
Asia: ~?
February 2013
Hepatitis A epidemic in Pakistan, District of Swat in
Northern Pakistan
several hundred cases

2010
Hepatitis A epidemic in Azad Jammu & Kashmir
>150 cases

? Which genotypes are circulating

in Pakistan?