Hepatitis B vaccination:an alternative (re)view

Geert Leroux-RoelsCenter 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

(

(

(

ccc-DNA

RNA

Precore, L, M, S + Xproteins

Capsidprotein

P proteinReverse transcription

ER/IC

Golgi

The HBV infectious cycle

RNA pregenome

cap

Viral clearance without destruction of infected cells during acute HBV infectionLuca Guidotti et al. Science 284:825- 829, 1999

Infectious serum containing ~ 5x107 genome equivalents of HBV (ayw) from transgenic mice2 healthy chimps developed typicalacute,self-limited HBV infectionsdocumented with

- serological- virological- histopathological- molecular

analyses on serum specimens and liver biopsies that were obtained

weekly

HBeAg

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

HBsAg

-HBs

-HBe

-HBc

SerumHBV-DNA

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

sALT

%HBcAg+ hepatocytes

CD3CD8

CD4

Based on Guidotti et al. Science 284:825, 1999

The adaptive immune response to HBV

CD4+Th cell

CD8+

CTLHepatocyte

TNF-IFN-

Lysis

IFN-

APC

YY

B cellY

Th2>Th1cytokines

-HBe

-HBc -HBs

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 interactionsNeurath et al. Virology 1994;204:475Vanlandschoot et al. unpublished

Molecular structure of the major hydrophilic region of HBsAg

K 122d/y

a

Mechanisms of action of anti-HBs ?

Liver

Y

Y

Y

YRapid clearance

of infection

Improve antigen presentation

Improve T cell response

Y

m T

Prevent cell entry

HBV

Anti-HBs

Uptake via FcR

HBsAg is a poor immunogenAnti-HBs

VAX 1 VAX2 VAX 3 VAX4 VAX5Subject Day 0 W2 W4 W6 W8 W16 W18 W20

16 <1 <1 <1 <1 <1 <1 <1 217 <1 <1 <1 <1 <1 10 33 30018 <1 <1 <1 <1 <1 <1 <1 <119 <1 <1 <1 <1 <1 <1 <1 120 <1 <1 3 6 5 16 850 142021 2 <1 2 4 8 36 132 125022 <1 <1 <1 <1 <1 <1 <1 <123 <1 <1 <1 <1 <1 <1 <1 <124 <1 <1 <1 <1 <1 <1 117 141025 <1 <1 <1 <1 <1 2 7 2826 <1 <1 <1 <1 <1 <1 8 1427 <1 <1 <1 <1 <1 <1 4 10228 <1 <1 <1 <1 <1 <1 <1 729 <1 <1 <1 <1 <1 11 28 5830 <1 <1 <1 <1 <1 2 7 66

VAX = 20 µg SL* in PBS

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

CD

14C

D19

CD

3IS

O

FSC SA-PE b-rHBsAg+

SA-PE

Cell

CD14CD14

CD14

B

B

BB

B

SA-PE

Interaction between HBsAgand CD14+ cells

HBsAg binds to monocytes andsuppresses their activation by

LPS

LPS

HBsAg

TNF, IL1, ..-

LBP

Monocyte/m

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.D2B10.SB10.M

dsf

81,92000

000

000

+ S B10.D2

B10.M

d

f

40,960

0

10,240

0

0PreS2B10.S s 1,280 10,240 0

0

PreS1 +PreS2+ S

B10.D2B10.SB10.M

dsf

81,9205,12010,240

5,12010,2401,280

6401,280

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*010-

DR5

DPB1*040-

DQB1*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 cooperationbetween macrophages, T cells and B cells

HLADP, DQ, DR

TCR

Peptide fragmentof antigen

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.D2B10.SB10.M

dsf

81,92000

000

000

+ S B10.D2

B10.M

d

f

40,960

0

10,240

0

0PreS2B10.S s 1,280 10,240 0

0

PreS1 +PreS2+ S

B10.D2B10.SB10.M

dsf

81,9205,12010,240

5,12010,2401,280

6401,280

10,240

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

HBV envelope proteins and S-L*

1 126 175 400

preS1 preS2 HBsAg

12 52

133-145

175 400

S-L*

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)

00

8357

8971

8993

Geometric mean titer (mIU/ml)Engerix-B (n=18)S-L* (n=14)

3.43.7

24126

38565

540198

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)

1

10

100

1000

10000

-6 0 6 12 18 24 30 36 42 48 54 60 66

TIMING (in months)

GM

Ts

in

mIU

/ml

(lo

g s

ca

le)

Twinrix (B)Engerix-B

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 HAVMethods - 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 HAVMethods - Subjects (2)

• Based on the anti-HAV titers measured on month 60, two groups were defined

Group H anti-HAV > 200 U/L n=20

Group L anti-HAV < 200 U/L n=16

• At month 72 blood was drawn to measure antibodies and HAV-specific cell mediated immune responses

High Titered Group HAV CMI response TT

Study # Subject # anti-HAV Proliferation IFN- IL-5 Proliferation(U/L) (SI) (pg/ml) (pg/ml) (SI)

10 300 3,1 63 5 14,658 1325 1,5 10 5 27,465 3316 3,1 1010 5 13,2

101 1291 7,9 1050 20,5 20,9HAV-112 107 243 1,4 10 5 1,9

128 339 5,7 1400 214,8 8,6135 3731 24,2 964 5 120,3149 2020 97,5 1400 49,8 117,1176 4738 9,2 94 5 60,8184 290 2,2 70 5 81,48 804 15,6 534 17,2 22,516 287 10,2 197 5 119,818 407 29,5 492 5 112,319 666 8 1400 55 61,1

HAV-123 21 2387 2,5 51 5 12,224 442 2,5 45 5 26,133 2541 47,2 1400 5 29,750 377 5,7 65 5 28,955 1459 14,8 1388 15,8 22,657 902 8,6 284 5 12,2

RESULTS

15/20 18/20

Low Titered Group HAV CMI response TT

Study # Subject # anti-HAV Proliferation IFN- IL-5 Proliferation(U/L) (SI) (pg/ml) (pg/ml) (SI)

13 102 1,1 10 5 30,444 197 12 349 5 12,247 10 1,4 10 5 20,551 55 3,6 377 14,6 11,5

HAV-112 56 193 2,6 399 5 26,462 185 1,3 10 5 11,377 108 3 606 5 39,786 85 1,1 10 5 66,792 156 1,4 10 5 63,1

103 186 1,4 10 5 1,69 52 1,1 10 5 8,615 90 3,5 62 5 39,8

HAV-123 32 106 2 10 5 14,136 127 1,7 10 5 12,859 96 1,5 109 5 10,164 138 1,8 10 5 7,6

RESULTS

4/16 6/16

Center for Vaccinology

Agnes VandeputteAli FarhoudiAndrea VerwulgenAnnick WillemsArsène-Hélène BatensCao TinghuaFrédéric ClementFreya Van HoutteIsabelle DesombereLieve Van CrombruggeLieven VerhoyePeter VanlandschootPhilip MeulemanSophia SteyaertSybil CouventYvonne 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

ResultsBinding Reactivity of Human anti-HBsAg mAb with Wild-type and Mutant HBsAg

0

200

400

600

800

1000

1200

1400

1600

wt ayw

3

R122K

A128V

Q129R

G130N

M13

3T

Y134F

S136T

K141W

D144H

G145A

G145R

T148A

S154E

K160R

Wild-type and mutant HBsAg

A45

0 n

m

Cut-off320

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 Acuteinfection

Chronicinfection

HBsAg/pre-S AbTH

CTL

+++++ (PBL)

+/-+/-+ (liver)

HBc/HBeAg AbTH

CTL

+++++++ (PBL)

+++++ (liver)

Polymerase AbCTL

++++ (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

B cell

Hepatocyte

CD8+

CTL CD4+Th cell

YY

Y

YY

APCNK

NKTcells

The principal actors

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

Engerix-B

S-L*

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

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

2

2

7

4

4

1

Severe combined immunodeficient

mousePrkdcscid/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-HBsTiter inIU/ L

Donor HuPBL-SCID

HuPBL-SCID +HBsAg

Plasma 6981 180 2502

Culturesupe

0 26 320

Depraetere et al. J Immunol 2001;166:2929