Alpha-interferon for chronic hepatitis B infection: increased efficacy of prolonged treatment

56

Working purties

HEPATITIS B VIRUS E ANTIGEN BUT NOT HBV CORE IS PRESENTED TO SPECIFIC CD4+ T CELLS VIA THE ENDOGENOUS ANTIGEN PROCESSING PATHWAY H. M. Diecolder. G. Ries. M.-C. June. H.-J. Schlicht. J.-T. Gerlach. N. Grilner. H. Hof,xh!&ider. W. H. Caselmann. G.R. Paw Med. Dept. II, Klinkum GmChadern

and lnstilule

for Immunology

University

of Munich,

Germany.

A strong CD4+ T cell response to the HBcJI-IBe sequence has been associated with viral elimination in HBV infection. The majority of CD4+ T cell epitopes are located within the sequence shared by both HBc and HBe, therefore the relative contribution of both antigens for T cell priming and activation is unknow. Via the exogenous antigen processing pathway both antigens are similarly processed for presentation to specific CD4+ T cell clones; however, it is presently unknown whether intracellularly synthesized HBc or HBe is able to enter the endogenous antigen processing pathway. To test the ability of antigen presenting cells (APC) IO present endogenously synthesized HBcRIBe antigen to CD4+ T cells, we infected lymphoblastoid cell lines with recombinant vaccinia viruses containing various HBc (VVconstructs. CD4+ T cell clones specific for aa 93-103 HBc) or HBe (VV-HBe) and aa 145-155 of HBc/HBe were used to detect processing and presentation of HBc/HBe peptide epitopes on HLA class II molecules. T cell activation was detected by double immunofluorescence staining with antibodies against CD4 and CD25 and subsequent FACS analysis. APC infected with the W-HBe were able to strongly stimulate specific

GENERATION OF HIGH AFFINITY, FULLY HUMAN MONOCLONAL ANTIBODIES SPECIFIC TO HEPATITIS B VIRUS IN A HUMAN/MOUSE RADIATION CHIMERA: THE TRIMERA SYSTEM. R Eren*, E. Ban*, I. Lubin*, D. Terkieltaub *, 0. Ben-Moshe’, .I. Arazi* A. Zauberman*, 0. Nussbaum*. S. Barr*, E. Galun+, D. Show&, N. Daudi+, Y. Reisner# , L. 0. MaeniusS and S. Dagan*. *XTL Biopharmaceuticals Ltd., Kiryat Weizmann, Rehovot, +Liver Unit, Hadassah University Hospital, Jerusalem, #Dept. of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel and SDept. of Virology, The Swedish Institute for Infectious Disease Control, Stockholm, Sweden.

contributed to the presentation of HBe epitopes on HLA class II we used specific inhibitors of the endogenous and exogenous antigen processing pathway, brefeldin A and chloroquin, respectively. T cell activation by WHBe was completely inhibited by brefeldin A but not affected by chloroquin. In contrast, T cell activation by exogenous, recombinant HBc or HBe was inhibited by chloroquin but not by brefeldin A. Our findings indicate that processing of endogenously synthesized HBe. possibly also in virus-infected non-professional APC, may be involved in the regulation of the HB&IBe-specific CD4+ T cell response. This finding may be relevant for the pathogenesis of chronic HBV infection and the evolution of HBe-minus variants.

We have generated the Trimera mouse system in which normal strains of mice, lethally irradiated by total body irradiation and radioprotected with SCID mouse bone marrow cells, are rendered permissive for engraflment of human cells and tissues. Employing this system we were able to produce fully human monoclonal antibodies (mAbs) to Hepatitis B virus (HBV). To generate human mAbs to HBV, Trimera mice were transplanted with lymphocytes from donors positive for antihepatitis B surface antigen (HBsAg) antibodies and subsequently immunized with hepatitis B vaccine. Several stable clones secreting IgG specific for HBsAg were isolated and their antibodies were characterized. These mAbs are IgGl and have high affhnty constants for HBsAg in the range Qf IO-10 M. Two of these mAbs, 17.1.41 and 19.79.5 were developed and characterized. Specificity to HBsAg was tested by a competitive inhibition assay and by immunohistostaining of human liver infected with HBV. The hvo antibodies bind to different epitopes on the LIdeterminant of the HBsAg and to all viral subtypes with distinct patterns, indicating diversity and broad reactivity. The genes encoding the variable regions of these mAbs were isolated and sequenced. The biological function and neutralizing activity of these antibodies were tested in our HBV-Trimera mouse model. This model was shown effective for testing anti-HBV drugs such as polyclonal antibodies (Hepatec@) and a reverse transcriptase inhibitor (g-L-5FddC). Treatment of HBV infected Trimera mice with mAbs 17.1.41 and 19.79.5 decreased the rate of infection from SO-85% to 13-35%, whereas treating these mice with Hepatect@’ decreased the rate of infection to 50 60% only. Results obtained with our mAbs were similar to the effect of 8-L-5FddC tested in this model, Treatment cessation resulted in rebound of viral load, indicating HBV replication upon drug withdrawal. However, combinahon therapy of a mAb (17.1.41) with g-L-5FddC could prevent viral load rebound, suggesting potential immunotherapeutic approach.

OVEREXPRESSION

ALPHA-INTERFERON

CD4+

T

cell

clones.

No

diffemnt

W-HBc

or

inhibited

by antibodies

T cell

the

activation,

VV-HCV.

however,

T

to HLA-DR.

ccl1

To exclude

was

observed

activation that reuptake

with

two

W-HBe

by

of secreted

was HBe

antigen

CELLS

IS

OF

THE

HEPATITIS

ASSOCIATED

WITH

H. Lecoeur.

0. Terr adillos.

T. Polbctno.

,

B VIRUS

X GENE

IN

LIVER

APOPTOSIS.

ASED

M. Trioodi*.M.-L.

Gouneon’.

EFFICACY

H.L.A.

P. Tiol_I&.

Janssen’,

S.W. Unite

de Recombinaison

et

d’oncologie

Virale.

Biopatologia

Umana. Universita

BACKGROUND. (HBx)

lnstitut

Expression Pasteur,

cell

function

proliferation

defined.

Recent data fmm our group

activity

of HBx

in the liver

(INSERM

Prance,

and

U163).

‘Unite

di

‘Dipartimento

of the hepatitis

have provided

of transgenic

mice.

evidence The

remain

to

be

for a pro-apoptotic

aim of our smdy was to

characterize the apoptotic

activity of HBx in immortalized hepatocytes derived from transgenic mice and in transiently transfected cells. METHODS AND RESULTS. We generated stable hepatocyte cell lines from p53-null mice and from HBX transgenic/ p53-null mice. The response of these immortalized liver cells to different apoptotic stimuli, such as activation of the Fas pathway, doxorubicin or serum deprivation, was analysed by morphological examination, flow cytomeuy and MIT assays. HBx-expressing hepatocytes showed a markedly increased sensitivity to undergo apoptosis. Identical results were obtained hepatocytes

by comparing

established

the

response

from transgenic

of

highly

mice carrying

differentiated a truncated,

MMHD3

constitutively

gene with that of hepatocytes from HBXlc-Met biuansgenic mice. To further analyze the pro-apoptotic properties of HBX in vitro. we performed transient transfection assays in human hepatoma cells (HepCZ) and in murine MMHD3 hepatocytes using a pCMV/HBx expression vector. Transfeeted cells were identified by co-uansfection with IacZ or H2Ed vectors. The phenotype of uansfected cells was visualized by X-gal and Hoechst staining, and cells undergoing apoptotic death were revealed by TUNBL. Transfection with a wild-type pCMV-HBx vector induced apoptosis within 24-48 hrs in X-gal-stained cells. A activated c-Met

dose-dependent

apoptotic

‘Erasmus

B virus X protein

and carcinogenesis

effect was noted upon transfection

of different

amounts

of pCMV-HBx and of pCMV-WHx. that encodes the related X protein of the woodchuck hepatitis virus. These data were confumed by biparametric FACS analysis and by agarose gel electrophoresis of DNA. showing nucleosomal DNA laddering in HBx-and FADD-transfected cells. CONCLUSION. Our results demonstrate that: 1) constitutive expression of the BBX gene sensitizes immortalized hepatocytes to different apoptotic stimuli independently of p53 expression, 2) in transient transfeetion assays, HBx affects cell viability in a dose dependent manner.

CHRONIC

P. Honkooo’.

and the

HEPATITIS

B INFECTION:

INCRE-

TREATMENT G. Gerken”,

EurODean

V.

Concerted

Carrefid”,

Action

R.A.

on Viral

Heiitink’

Heoatitis

~

(EU-

ROHEP). University

Universitat,

La Sapienza. Rome, Italy.

The exact biological

and its role in liver

Genbtique Paris,

Schalm’

FOR

OF PROLONGED

Hospital

Mains,

Alpha-interferon Standard ved

clinical

was

prolonged

investigate

ducted

continued. whether

a prospective were

during

16

weeks,

willing

to

continue

identical after

16 weeks

Among

the

during

the

standard versus

in 17

(P=O.O4).

In

was

who

7 of

of

could

enhance trial

not

having to

level

the

exhibit

57

(12%)

of viral

patients

in the 8, to

in which 10

of

conall pa-

MU

responded

tiw and

of

the

values

majority

prolongation

a standard

level

of

under

predictor

therapy.

large

after

viral

The

to to

16

pg/ml

tQ was

standard

therapy therapy

of treatment, (Abbott

of response prolonged

randomized response

prolonged

weeks

10

had a response

a

assigned assigned

replication

independent

hepatitis

rate we

or discontinuation

randomization

patients

DNA

prolonged

a IQW

After

(28%)

H8V

only

the

prolongation

therapy)

of

8.

serum,

of a-interferon

randomized tprOlQngad

hepatitis

from

multicenter

those

as long

and acceptability

therapy).

61

is superior

regimen.

were weeks

a retrospective

chronic

HBeAg

regimen

only

therapy.

the

the

tolerated

chronic

weeks

upon

32

prolonged

during

well

European

therapy

by serum

P
where

(standard

Low

indicated

of

B.

to an impro-

of interferon

the efficacy with

Spain.

hepatitis

lead

Since

effect

prolongation

a standard

Chronic

wks

patients.

biological

disappearance

with

up to

of

16

J Gutenberg Madrid,

162 patients who entered the study 27 (17%) first 16 weeks of treatment and 118 were

or

observed

asay),

treatment

Diaz, for

for

in patients

controlled

treated

regimen

30%

tiw

We evaluated

by

Netherlands;”

Jiminez

treatment MU

of the

treatment

as defined

tients

10

in about

a-interferon

response,

of

persistence

the

Fundacion

established

courses

outcome

showed

as therapy To

is the

treatment

analysis

Rotterdam, *..

Germany;

(52%

as

HBV

DNA

versus

0%;

a-interferon

schedule

of patients. of

course

a-interferon of

replication

therapy

16

weeks

at

the

end

up

in those of

the

to

32

patients standard