Transcript Prevention of HBV infections: vaccination and its limitations

Hepatitis B vaccination:
an alternative (re)view
Geert Leroux-Roels
Center for Vaccinology
Ghent University and Hospital
Overview of the presentation
•
•
•
•
•
•
the virus
the infection
the immune response
the HBV vaccine: HBsAg
non-response to HBsAg
strategies to overcome nonresponse
• immune memory
HBV genome organization
The HBV infectious cycle
Reverse transcription
P protein
Capsid
protein
ER/IC
e
Golgi
cap
RNA pregenome
RNA
ccc-DNA
Precore, L, M, S + X
proteins
Viral clearance without destruction of
infected cells during acute HBV infection
Luca Guidotti et al. Science 284:825- 829, 1999
Infectious serum containing ~ 5x107
genome equivalents of HBV (ayw)
from transgenic mice
2 healthy chimps developed typical
acute,self-limited HBV infections
documented with
- serological
- virological
- histopathological
- molecular
analyses on serum specimens and liver
biopsies that were obtained weekly
HBsAg
a-HBs
HBeAg
a-HBe
a-HBc
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
%HBcAg+ hepatocytes
Serum
HBV-DNA
sALT
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
CD3
CD8
CD4
Based on Guidotti et al. Science 284:825, 1999
The adaptive immune response to HBV
a-HBe
a-HBc
a-HBs
B cell
Th2>Th1
cytokines
Lysis
Hepatocyte
CD8+
CTL
CD4+
Th cell
TNF-a
IFN-g
IFN-g
APC
The Hepatitis B vaccine :
choice of the immunogen
• A 1 minute boil of MS2 serum (HBV)
destroyed infectivity, but left
immunogenicity
• HBsAg is an envelope protein
• recovery of acute HBV infection is
characterized by HBsAg/anti-HBs
seroconversion
• passively acquired anti-HBs protects
individuals from infection with HBV
Envelope proteins
of HBV and
HBV vaccines
HBsAg vaccine is effective in
preventing HBV infection
despite the fact that :
• Anti-HBs is not strictly a ‘neutralizing’
antibody, since HBsAg is probably not
the receptor-binding element of HBV
• HBsAg is a poor immunogen
• HBsAg has anti-inflammatory qualities
HBV - Hepatocyte interactions
(1)
HBV
Hepatocyte
• preS1
• Glyceraldehyde-3-P
dehydrogenase
• IgA receptor
• Interleukin 6
• Asialoglycoprotein receptor
• preS2
• Transferrin receptor
• Polymerized-albumin-receptor
• Fibronectin
HBV - Hepatocyte interactions
HBV
Hepatocyte
• HBsAg
• Annexin V
• Apolipoprotein H
• CD14
(2)
Lipids play an important role in these
interactions
Neurath et al. Virology 1994;204:475
Vanlandschoot et al. unpublished
Molecular structure of the major
hydrophilic region of HBsAg
d/y
K
a
122
Mechanisms of action of anti-HBs ?
HBV
Y
Prevent
cell entry
Rapid clearance
of infection
Liver
Anti-HBs
Uptake via FcR
Y
mF
T
Improve antigen presentation
Improve T cell
response
HBsAg is a poor immunogen
Anti-HBs
VAX 1 VAX2 VAX 3
Subject Day 0 W2 W4 W6 W8
16
<1
<1
<1
<1
<1
17
<1
<1
<1
<1
<1
18
<1
<1
<1
<1
<1
19
<1
<1
<1
<1
<1
20
<1
<1
3
6
5
21
2
<1
2
4
8
22
<1
<1
<1
<1
<1
23
<1
<1
<1
<1
<1
24
<1
<1
<1
<1
<1
25
<1
<1
<1
<1
<1
26
<1
<1
<1
<1
<1
27
<1
<1
<1
<1
<1
28
<1
<1
<1
<1
<1
29
<1
<1
<1
<1
<1
30
<1
<1
<1
<1
<1
VAX = 20 µg SL* in PBS
VAX4
W16
<1
10
<1
<1
16
36
<1
<1
<1
2
<1
<1
<1
11
2
VAX5
W18
<1
33
<1
<1
850
132
<1
<1
117
7
8
4
<1
28
7
W20
2
300
<1
1
1420
1250
<1
<1
1410
28
14
102
7
58
66
HBsAg is ‘special’
• Produced by HBV-infected hepatocytes
• Circulates in serum of chronic HBV
patients at 50-300 µg/ml
• HBsAg contains 30% lipids
• HBsAg binds to CD14 expressing cells
– monocytes, macrophages
– suppresses inflammatory responses
Interaction between HBsAg
and CD14+ cells
ISO
CD14
CD3
CD14
Cell
CD19
B
B
B
B
B
SA-PE
CD14
FSC
SA-PE
b-rHBsAg
+
SA-PE
CD14
HBsAg binds to monocytes and
suppresses their activation by LPS
LBP
HBsAg
LPS
Monocyte/mF
TNFa, IL1, ..
Host factors determining
response to HB vaccines
•
•
•
•
Gender
Age
Concomitant illness
Genetic factor - MHC
Influence of H-2 genotype on
the humoral immune response to
HBsAg particles of different compositions
Immunogen
Strain
H-2
Specific antibody titer (1/dilution)
S
PreS2
PreS1
HBsAg (S)
B10.D2
B10.S
B10.M
d
s
f
81,920
0
0
0
0
0
0
0
0
PreS2 + S
B10.D2
B10.S
B10.M
d
s
f
40,960
1,280
0
10,240
10,240
0
0
0
0
PreS1 +PreS2
+S
B10.D2
B10.S
B10.M
d
s
f
81,920
5,120
10,240
5,120
10,240
1,280
640
1,280
10,240
Milich et al. J. Immunol. 1986;137:315
Response to HB vaccine:
multiple HLA genes are involved
GOOD RESPONSE
is associated with
DRB1*010DR5
DPB1*040DQB1*0301
DQB1*0501
NON/POOR RESPONSE
is associated with
DRB1*07
DPB1*1101
DQB1*020-
Desombere et al. Tissue Antigens 1998;51:593-604
Antibody production requires cooperation
between macrophages, T cells and B cells
Peptide fragment
of antigen
HLA
DP, DQ, DR
TCR
Non-response resides at the
level of APC-TCR interaction
Strategies to overcome
nonresponsiveness
• Add preS-epitopes to HBsAg vaccine
• Change vaccine carrier
– DNA vaccines
– HBcAg as carrier
• More immunogenic adjuvants
• Give additional vaccine doses
Influence of H-2 genotype on
the humoral immune response to
HBsAg particles of different compositions
Immunogen
Strain
H-2
Specific antibody titer (1/dilution)
S
PreS2
PreS1
HBsAg (S)
B10.D2
B10.S
B10.M
d
s
f
81,920
0
0
0
0
0
0
0
0
PreS2 + S
B10.D2
B10.S
B10.M
d
s
f
40,960
1,280
0
10,240
10,240
0
0
0
0
PreS1 +PreS2
+S
B10.D2
B10.S
B10.M
d
s
f
81,920
5,120
10,240
5,120
10,240
1,280
640
1,280
10,240
Milich et al. J. Immunol. 1986;137:315
HBV envelope proteins and S-L*
1
126
preS1
175
preS2
12
52
400
HBsAg
175
S-L*
133-145
400
Antibody response to HBsAg following
administration of three additional doses of
Engerix-B or S-L* in poor responders
Month
Seroprotection rate (%)
Engerix-B (n=18)
S-L* (n=14)
Geometric mean titer (mIU/ml)
Engerix-B (n=18)
S-L* (n=14)
0
1
2
3
0
0
83
57
89
71
89
93
3.4
3.7
241
26
385
65
540
198
Leroux-Roels et al. Vaccine 1997;15:1732-6
Persistence of immunity
• Level of anti-HBs declines after vaccination
• How long does protection last ?
• Is booster immunization needed ?
• Very few breakthrough infections occur
• Vaccination induces immune memory
Persistence of anti-HBs
Combined hepatitis A/B vaccine versus Engerix-B
(schedule : 0-1-6 months)
GMTs in mIU/ml (log scale)
10000
1000
100
Twinrix (B)
Engerix-B
10
1
-6
0
6
12
18
24
30
36
TIMING (in months)
42
48
54
60
66
Persistence of immunity
• Levels of anti-HAV and anti-HBs decline after
vaccination
• How long does protection last ?
• Is booster immunization needed ?
• Very few breakthrough infections occur
• Vaccination induces immune memory
Demonstration of CMI towards HAV
Methods - Subjects (1)
• Subjects enrolled in this project were recruited
from 2 follow-up studies of long-term antibody
persistence after the administration of 2 doses
of 1440 EU HAV vaccine
– study HAV-112 : 0-12 month scheme
– study HAV-123 : 0-6 month scheme
• anti-HAV titers were measured on months 24,
36, 48, 60 and 72
Demonstration of CMI towards HAV
Methods - Subjects (2)
• Based on the anti-HAV titers measured on
month 60, two groups were defined
Group H
Group L
anti-HAV > 200 U/L
anti-HAV < 200 U/L
n=20
n=16
• At month 72 blood was drawn to measure
antibodies and HAV-specific cell mediated
immune responses
RESULTS
High Titered Group
HAV CMI response
Study #
Subject # anti-HAV Proliferation
(U/L)
(SI)
10
300
3,1
58
1325
1,5
65
3316
3,1
101
1291
7,9
HAV-112
107
243
1,4
128
339
5,7
135
3731
24,2
149
2020
97,5
176
4738
9,2
184
290
2,2
8
804
15,6
16
287
10,2
18
407
29,5
19
666
8
HAV-123
21
2387
2,5
24
442
2,5
33
2541
47,2
50
377
5,7
55
1459
14,8
57
902
8,6
15/20
IFN-g
(pg/ml)
63
10
1010
1050
10
1400
964
1400
94
70
534
197
492
1400
51
45
1400
65
1388
284
18/20
IL-5
(pg/ml)
5
5
5
20,5
5
214,8
5
49,8
5
5
17,2
5
5
55
5
5
5
5
15,8
5
TT
Proliferation
(SI)
14,6
27,4
13,2
20,9
1,9
8,6
120,3
117,1
60,8
81,4
22,5
119,8
112,3
61,1
12,2
26,1
29,7
28,9
22,6
12,2
RESULTS
Low Titered Group
HAV CMI response
Study #
Subject # anti-HAV Proliferation
(U/L)
(SI)
13
102
1,1
44
197
12
47
10
1,4
51
55
3,6
HAV-112
56
193
2,6
62
185
1,3
77
108
3
86
85
1,1
92
156
1,4
103
186
1,4
9
52
1,1
15
90
3,5
HAV-123
32
106
2
36
127
1,7
59
96
1,5
64
138
1,8
4/16
IFN-g
(pg/ml)
10
349
10
377
399
10
606
10
10
10
10
62
10
10
109
10
6/16
IL-5
(pg/ml)
5
5
5
14,6
5
5
5
5
5
5
5
5
5
5
5
5
TT
Proliferation
(SI)
30,4
12,2
20,5
11,5
26,4
11,3
39,7
66,7
63,1
1,6
8,6
39,8
14,1
12,8
10,1
7,6
Center for Vaccinology
Agnes Vandeputte
Ali Farhoudi
Andrea Verwulgen
Annick Willems
Arsène-Hélène Batens
Cao Tinghua
Frédéric Clement
Freya Van Houtte
Isabelle Desombere
Lieve Van Crombrugge
Lieven Verhoye
Peter Vanlandschoot
Philip Meuleman
Sophia Steyaert
Sybil Couvent
Yvonne Gijbels
Recommendations of the European
Consensus Group in Hepatitis B Immunity
• No boosters for immunocompetent individuals
who have responded to a primary course
• in certain risk groups boosters may be used to
provide reassurance of protective immunity
• for immunocompromised patients regular testing
for anti-HBs and booster injections when titer
falls below 10mIU/ml are recommended
• non-responders to a primary course should
continue to be studied
• long-term monitoring should continue
Lancet 2000;355:561-5
Results
Binding Reactivity of Human anti-HBsAg
mAb with Wild-type and Mutant HBsAg
1600
1400
A450 nm
1200
1000
800
600
Cut-off
400
320
200
0
Wild-type and mutant HBsAg
HBsAg vaccine escape variants
• Point mutations in the second ‘a’ loop,
notably at amino acids 144 and 145, alter
antigenicity dramatically
• these mutations confer escape
characteristics to HBV under pressure
mediated by rHBsAg-induced antibodies
Will escape variants
ever become important ?
Model simulation representing the worse case scenario
with a highly infectious variant and a non-cross-reactive
vaccine
Wilson et al. J.Viral Hepat. 1998;5(suppl2):25-30
T and B cell responses during
acute and chronic HBV infections
HBV antigens
Al/TH/CTL
Acute
infection
Chronic
infection
HBsAg/pre-S
Ab
TH
CTL
+
+
+++ (PBL)
+/+/+ (liver)
HBc/HBeAg
Ab
TH
CTL
++
+++
++ (PBL)
+++
+
+ (liver)
Polymerase
Ab
CTL
+
+++ (PBL)
++
+ (liver)
Vaccination induces memory
In vivo antibody production
In vitro lymphoproliferation
• in vivo humoral and in vitro anti-HBs responses are closely correlated
• booster responses reveal the immune memory
Leroux-Roels et al. Vaccine 1994;12:812-8
The principal actors
B cell
CD8+
CTL
CD4+
Th cell
Hepatocyte
NK
APC
NKT
cells
Strategies to overcome
nonresponsiveness
• Add preS-epitopes to HBsAg vaccine
• Change vaccine carrier
– DNA vaccines
– HBcAg as carrier
• More immunogenic adjuvants
• Give additional vaccine doses
Evolution of anti-HBs in response to three
additional vaccine doses given to 18 subjects
with a poor response to 4 doses of HB vaccine
1
2
4
4
7
Engerix-B
2
S-L*
Leroux-Roels et al. Vaccine 1997;15:1732-6
Severe combined
immunodeficient mouse
Prkdcscid/Prkdcscid (SCID)
– autosomal recessive mutation
in mice
– severe deficiency in mature
lymphocytes
– virtual absence of lypmhoid
cells in the thymus, spleen,
lymph nodes and gut
– no Ab production, no DTH
response, no graft rejection
– innate immune system is intact
In vivo exposure to a recall antigen
activates Ag-specificB cell clones
Anti-HBs
Titer in
IU/L
Donor
HuPBLSCID
HuPBLSCID +
HBsAg
Plasma
6981
180
2502
Culture
supe
0
26
320
Depraetere et al. J Immunol 2001;166:2929