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 non-

response

• immune memory

HBV genome organization

The HBV infectious cycle

Reverse transcription P protein Capsid protein ER/IC Golgi cap e 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 ~ 5x10 7 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 Hepatocyte Lysis TNF -

a

IFN-

g

CD8 + CTL CD4+ Th cell 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

•

preS1

•

preS2 Hepatocyte

• Glyceraldehyde-3-P dehydrogenase • IgA receptor • Interleukin 6 • Asialoglycoprotein receptor • Transferrin receptor • Polymerized-albumin-receptor • Fibronectin

HBV - Hepatocyte interactions

(2) HBV

•

HBsAg Hepatocyte

• Annexin V • Apolipoprotein H • CD14 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 unpublished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 122 a

Mechanisms of action of anti-HBs ?

Y

Prevent cell entry

Anti-HBs

Uptake via FcR

Y HBV

m F Liver Improve antigen presentation

T

Rapid clearance of infection Improve T cell response

HBsAg is a poor immunogen VAX 1 VAX2 VAX 3 Subject Day 0 W2 W4 16

<1 <1 <1

17 18 19 20 21 22 23 24 25 26 27 28 29

<1 <1 <1 <1

2

<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

3 2

<1 <1 <1 <1 <1 <1 <1 <1

30

<1 <1 <1

VAX = 20 µg SL* in PBS Anti-HBs W6

<1 <1 <1 <1

6 4

<1 <1 <1 <1 <1 <1 <1 <1 <1

W8

<1 <1 <1 <1

5 8

<1 <1 <1 <1 <1 <1 <1 <1 <1

VAX4 VAX5 W16 W18 W20

<1 <1

2 10

<1 <1

16 33

<1 <1

850 300

<1 1

1420 36

<1 <1 <1

2

<1 <1 <1

11 2 132

<1 <1

117 7 8 4

<1

28 7 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

ISO CD3 CD19 CD14 FSC SA-PE Interaction between HBsAg and CD14 + cells CD14 CD14 CD14 Cell B B B B B SA-PE b-rHBsAg + SA-PE

HBsAg binds to monocytes and suppresses their activation by LPS LBP HBsAg LPS Monocyte/m

F

TNF

a

, 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

HBsAg (S) B10.D2

B10.S

B10.M

PreS2

+ S B10.D2

B10.S

B10.M

PreS1

+ S +PreS2 B10.D2

B10.S

B10.M

f

d

s f d

s f

d s Specific antibody titer (1/dilution) S PreS2 PreS1

81,920

0 0

0

0 0

0

0 0 40,960

1,280

0 81,920 5,120

10,240

10,240

10,240

0 5,120 10,240

1,280

0

0

0 640 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 NON/POOR RESPONSE is associated with DRB1*010 DR5 DPB1*040 DQB1*0301 DQB1*0501 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

HBsAg (S) B10.D2

B10.S

B10.M

PreS2

+ S B10.D2

B10.S

B10.M

PreS1

+ S +PreS2 B10.D2

B10.S

B10.M

f

d

s f d

s f

d s Specific antibody titer (1/dilution) S PreS2 PreS1

81,920

0 0

0

0 0

0

0 0 40,960

1,280

0 81,920 5,120

10,240

10,240

10,240

0 5,120 10,240

1,280

0

0

0 640 1,280

10,240

Milich et al. J. Immunol. 1986;137:315

HBV envelope proteins and S-L*

1 126 175 preS1 preS2 HBsAg S-L* 12 52 175 133-145 400 400

Antibody response to HBsAg following administration of three additional doses of Engerix-B or S-L* in poor responders

Month

0 1 2 3

Seroprotection rate (%)

Engerix-B (n=18) S-L* (n=14)

Geometric mean titer (mIU/ml)

Engerix-B (n=18) S-L* (n=14) 0 0 3.4

3.7

83 57 241 26 89 71 385 65 89 93 540 198

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 ) 10000 1000 100 10 Twinrix (B) Engerix-B 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 Study # HAV-112 HAV-123 HAV CMI response Subject # anti-HAV Proliferation IFN-

g

IL-5

(U/L) (SI) (pg/ml) (pg/ml) 10 58 65 101 107 128 135 149 176 184 8 16 18 19 21 24 33 50 55 57 300 1325 3316 1291 243 339 3731 2020 4738 290 804 287 407 666 2387 442 2541 377 1459 902

3,1

1,5

3,1 7,9

1,4

5,7 24,2 97,5 9,2

2,2

15,6 10,2 29,5 8

2,5 2,5

47,2 5,7 14,8 8,6 63

10

1010 1050

10

1400 964 1400 94 70 534 197 492 1400 51 45 1400 65 1388 284

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

15/20 18/20 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 Study # HAV CMI response Subject # anti-HAV Proliferation IFN-

g

IL-5

13 (U/L) 102 (SI) 1,1 (pg/ml) 10 (pg/ml) 5

HAV-112 HAV-123

44 47 51 56 62 77 86 92 103 9 15 32 36 59 64 197 10 55 193 185 108 85 156 186 52 90 106 127 96 138

12

1,4

3,6

2,6 1,3

3

1,1 1,4 1,4 1,1

3,5

2 1,7 1,5 1,8

349

10

377 399

10

606

10 10 10 10

62

10 10

109

10 5 5

14,6

5 5 5 5 5 5 5 5 5 5 5 5

4/16 6/16 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 1200 1000 800 600 Cut-off 320 400 200 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 HBsAg/pre-S HBc/HBeAg Polymerase Al/T H /CTL

Ab T H CTL Ab T H CTL Ab CTL

Acute infection

+ + +++ (PBL) ++ +++ ++ (PBL) + +++ (PBL)

Chronic infection

+/ +/ + (liver) +++ + + (liver) ++ + (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

CD8 + CTL The principal actors B cell Hepatocyte CD4+ Th cell NK NKT cells APC

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

Engerix-B S-L*

2 1 7 2 4 4

Leroux-Roels et al. Vaccine 1997;15:1732-6

Severe combined immunodeficient mouse Prkdc scid /Prkdc scid (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 HuPBL SCID HuPBL SCID + HBsAg Plasma

6981 180 2502

Culture supe

0 26 320 Depraetere et al. J Immunol 2001;166:2929