Interferon antibodies in patients with chronic HBV infection treated with recombinant interferon

351

Interferon

antibodies in patients with chronic HBV infection treated with recombinant interferon

In different trials, a total of 46 adults and 12 children suffering from chronic hepatitis B virus (HBV) and.+bo were HBV-DNA- and HBeAg-positive were treated with recombinant interferon-a (rIMa)-ZA. The interferon was administered intramuscularly in different doses ranging from 1.5 MU to 20 MU/m’ of body suface, two or three times weekly during 4-6 months. Specific detection of anti-IFN antihcdies by enzymoimmuneas~y (EIA), radioimmunoassay (RIA) and biological assays during treatment and follow-up periods were performed. None of the children developed anti-IFN antibodies. During therapy, 12 adult patients (26%) were found to have antiIFN antibodies. A total of five patients became HBV-DNA-negative during therapy, but in three cases a reactivation of viral replication occurred sttbsequen:ly. It! these three patients, the appearance of anti-IFN antibodies occurred prior to or at *he same ti& as HBV-DNA loss. The other seven patients did not respond to therapy. In conclusinn, the development of anti-IFN antibodies during rlFNa treatment of chronic hepatitis B may modify the response to therapy, especially if they apprar before HBV-DNA negativization.

lntrodurtiett In the iast few years, it has been proven that ,tcombinant IFN-a (rIFNa) has an antiviral effect on hepatitis B virus (HBV) chronic infection in adults

and children [l-3]. The development of antibodies against imerferon (anti-IFN), first described by Vakacht et al. [4] is a phenomenon normally associated with treatment with this drug. The percentage of patients developing

Oli8.8278/89/SO3.50~ 1989ElrevierSciencePubsheFsB.V. (BiomedicalDivision)

IS. PORRES er al.

352

study and HBeAg and HBV-DNA-positive at least 6 months prior to starting the treaxnt. The patients had 801 received steroid or mtiviral therapy previously. The basal clinical, epidemiological and histological features from the patter& included are shown in Table 1. None was addicted to drugs. Only we patient was anti-HIV and anti-HD-positive. The studies were approved by the Ethics Committee of our Hospital. All patients agreed to participate in the study and written consent was obtained and signed by all of them. No basal diffrreoces in liver function tests, bistolagical diagnosis and HBV-DNA concentration bctween controls and treated patients were observed in these studies.

anti-IFN antibodies vaties from 0 to 44% [SM. These antibodies way neutralize and block the IFN action with a loss if efficiency of its adtninistrxtioa [?I. However, :his ftct has not been proven in the treatment of chronic hepetitis B. The Rim of this study ws tu determine the freguency of :he appearance of anti-IFN antiiidks during rIFNu-2A ?herapy of chronic hepatitis B in adults and children. Tbu clinic?1 impact of anti-IFN development was observrd by comparing with patients who did not show these aotibodies.

Materials and Methods A total of 84 patients with chronic hepatitis (60 adults and 24 children) were included in four different trials of rIFNa treatment. Twenty one were female and G3male. Forty six adults (mean age 35.8 f 13.4 yean, mnge 18-60 years) and 12 chiidren (mean age 7.3 f 2.9 years, range 3-12 years) were treated with rIFNa-ZA. From tt:: S8 patients treated, 15 were female nnd 43 male. All of them were HBsAgpositive at least 1 year before the beginning of the

rIMa-2A was kindly provided by Hoffmann La Rocbe (98% pure). A total of 58patientswere treated and 26 served as controls distributed as follows. in a first pilot study, twelve adult patients were randomly assigned to one of two groups Doses of either 20 or 10 megaunits (MU)& Lady surface were administered intramuscularly twice a week,

BASAL CLINICAL,EPlDEMlOLOGlCALAND HISTOLOGICALFFATURESOF WE PATtENTS:NCL”DED IN THE STL!D\’

Sex (M/F)

ALTbasal(tl 111) Epidemiology Householdco,,tacts “omorex”ality Parentera!iniection Otherroutes-andunknorm

___

Controlgroup

Treatment group

adults(a = 46) 35.8f 13.4

children(n = 12) 7.3 * 2.9

adults(n = 14) 38.8* 11.2

children(n = 12) ___ 8.3 + 1.8

3M10

7/s

1ON

,312

2.56* 191

155&63

324f 201

149.L:n

11

12

3

i2

: 21

: 6

Histolo~p 23 MildCAH 13 ModerateCAH !D CAH plusCi -______CAH = chronicactivehepatitis:Ci = cirrhosis.

8 4

6

6

3

6

5

ANTI-IFN ANTIBODIES

353

during a period of 6 months. Itt the second study, a total of 24 adult patients were included. Si patients in each of three groups received 2.5,s and 10 M%m* body surface intramuscularly (i.m.) respectively, three times a week during six months; the other group of six patients were considered as controls. In the third study, eight patPnts received 1.5 Ml_1 and eight patients 18 MU im., three times a week during 4 months and eight were included as controls. ‘Twentyfour children were included; 12 of them received 10 MU/m2 body surface i.m., tt.ree times a week during 3 months, and 12 were considered as controls.

anti-HE& anti-HBc, HBeAg and antiHBe w%e tested by radioimmunoassay (RIA) (Abhott Laboratories, North Chicago IL). Anti-HIV antibodies were determined by enzynloimmunoassay (EIA) nnd anti-HD antibodies by RIA (Abbott). Serum HBV-DNA was tested by dot-blot hybridization [S]. using 32P-HBV-DNA isolated from the recombinant plasmid pBH20-HBV and labelled by nick translation. Antibodies to interferon were kindly determined

by Hoffmann La Roche, Basle (Switzerland), using three different methods: RIA, EIA and an antiviral neutralization bioassay; the specificity, sensitivity and the correlation between these three tests !tave been previously reported [?I! Anti-Herpes Zoster-IgM was tested by EIA (Caibiarhem-Behring, La Jolla, CA). Hematoloejcal, urine and liver function tests were performed by standard methods (Coulter, SMAC Technicon, NY). Fo//ow-up A clinical examination and aii the atmve-mentixxd tests were performed monthly during treatment. The post-treatment follow-up period lasted for 9 months.

The whole study lasted IS months from the

beginning

of therapy.

H&A&

Anti-IFN during

the

samples addition,

xntihodies

were

dt?Ua-2A

obtained

determined

rreatment

period,

monthly, in serum

72 h after the last dose of IFN. I.G

the anti-II%

antibodies

were detemtined

at

I, 2,3 asd 6 months oi follow-up. st4Iisliccl1 anarysis

Fisher’s and Wilcoxon’s tests were used for statistical comparisons.

TABLE 2 CLlNiCAL

ANDLABORATORYDATAOF THE PATIENTS WHODEVELOPEDAN%IRI ANTIBODIES

Patient

Sex Age Epidcmio:ogy

.~

ALT

No. 1 JMY

2 CBD 3RTl-l 4 AMC 5 FMS 6 MM” 7 MCI. 8 ILF 9 EMR lo AHG 11 LND 12 IGB

IFNdoses M

M M

26 28

homoscrual neg.wiw* negative

297 37.2 146

M

18

negative

590

hi M M F M M M F

32 36 23 49 18 19 51 19

homosexual ncgntive hamooerual hmPitl?~.wnnel negative” ne@“s negative negatb@*

242 90 193 7fi 267

30

2; 143

CAH = chronicactivehepatitis;Ci = cinhosis. * rlFNa wasadministeredtwicea week. ** Acutehepatitis.

CAHmild

IFN tota, d_

2.5MU/mz 1S3hw

CAH moderaa

IS MU

SfdMu

CAHmoderate CAAH moderate CAWwithCi CAHmild CAHwithCi CAHwithCi CAHmc&ra;i CAH mild CAHmoderate CAHmild

1SMU 2OMUIm” 1SMU 10MU/m’* IOhiUlnl2 10.UUlnG 5 MU/m’ 2.5MU/& 1.5MU 1SMlJ

18MU 272MU S4MU 544MU @aMU 612MU 406MU Z-5MU 3.5MU 72MU

Anti-1FN

RG%tiva &a

3 month 4 month 1 month 1 month 3 month

4 month 2 month 3 month 4 month I ,mm,h 7 month 4 month

2 month 4 month lmontll 2 month 4moiltb n0 110 no no zl* no no

3.54

J.C. PORRES

et

a,.

TABLE 3 ANTIGN LEVELS DURING THE STUDY Resultsareexpressed asthe absorbaxe valueat 492nmobtainedusingEIA. Patient N”.

Time (mo”tbr,

” 1 IMV ZCBD 3RTH 4 AMC 5 FMS 6MMU 7 MCL

2

3

0.251 0.391 -

0.498 0669

0.210 1.563 1.144 0.766

-

0.133 -

0.178 0.167

0.138

i.289

1

-

8 ILF 9 EMR 10AHG 11 LND :* IGB NT = not tested.

4 0.116 1.830 1.250 I.647 0441 0.186 0.328 0.161 0.346 0.169

ReSUltS Development

of an&IFNantrbodies

of the children or control patients developed anti-IF’N antibodies during the study. In addition, none of the adult patients who were treated had antiIFN in the basal sample. During the study, 12 treated patients (26%) developed anti-IFN antibodies (Z/10 females: 20% and lo/36 males: 27.7%), as tested by EIA and RI!.. In all cases, the antibodies were neutralizing as checked by biological assay. The baseline epidemiological, analytical and histological characteristics of the patients who developed anti-IFN are summarized in Table 2. Therapy was not discontinued in any patient in spite of the development of anti-IFN antibodies and the symptoms caused by therapy with interferon (‘flu-like syndrome’, weight loss, etc.) disappeared in relation to the prcsenci: of anti-IFN antibodies. None of the patients developed immune compleres-assoriated illness. The sequence of anti-IFN antibodies’ appearance was as follows (Tables 2 and 3). Two patients (patients 1 and 2) responded to rlFNa ‘xatment with loss of HBV-DNA and IiBcAg. One patient, treated with 2.5 MU/m*, who showed a loss None

5

6

1

0.223

0.286 0.111 l.W8 1.654 1.449 1.549

0.457 0.194 0.202 0.223 0.248

NT 0.225

NT 1.704 NT 1.542 1.857 NT NT NT NT

0.233 1.338 1.353 1.869 1.230

0.110 0.101

0.103 -

8

9

NT NT NT NT 0.992 NT NT NT NT NT NT NT

NT i ,267 0.679 NT 0.287 0.215 NT NT

12 NT NT 1.653 NT NT NT NT

of I-WV-DNA during the second month of therapy, developed anti-IFN antibodies 1 month later, but remained HBV-DNA-negative. The other patient (treated with 18 MU), developed anti-IFN antibodies during the fourth month and simultaneously lost HBV-DNA, and remained so during the follow-up. Three patients (patients 3, 4 and 5) became HBVDNA-negative during therapy, but after the appearance of anti-IFN antibodies, again became HBVDNA-positive. One of the patients treated with only 1.5 MU of dFNa had anti-IFN antibodies dnnng the first month of therapy coinciding with the loss of HBV-DNA. However, 3 months later this patient suffered a reactivation of the viral replication‘ with the reappearance of HBV-DNA. Another patient who received 20 MU rlFNa per m* of body surface developed anti-IFN antibodies 1 month after beginning treatment; he continued receiving rIFNar and during the second month HBV-DNA became ondetectabk dnd later the patient seroconverted to antiHBe. However, the anti-!FN antibody titer continued to increase. At the end of the trea!ment period (6 months), HBV-DNA became positiw again concomitantly with the highest anti-IFN 1~;’ (see Table 2, patient number 4). Finally, HBV-DYA became undetectable at the 15th month of follow-up. One

ANTI-IFNANTlBODlES month after the end of therapy, a new dose of rIFNu was administered to the patient. Sequential serum samples were obtained and an increase in the Levelof anti-IFN antibodies was observed. The third patient (treated with 1.5 MU of dFNa) developed anti-IFN antibodies by the third month, losing HBV-DNA at the fourth month. However, during the seventh month, HBV-DNA again hecame positive and remained so for the durationof the follow-up. Seven patients did not respond to treatment. One patient (patient 6). was treated with 10 MU/n?, and developed anti-IPN antibodiesdoting the fourth mosth of therapy. This patient had a Herpes Zoster infection (anti-Herpes aster-IgM-positive) I month after the appearance of anti-IFN antibodies, but the anti-IM antibody titer increased late,. The other six patients (patients 7-12), treated +th 10, 10, 5, 2.5 and 1.5 (n = 2) MU/& respectively, developed serum antibodies at the second, third, fourth, fdtb and second and fourth month of therapy, respectively, when they were HBV-DNA-positive and they never lost this marker. The mean absorbance value obtained by EIA in tht- first sample which ri.&xi positive to anti-IFN antibodies was higher among patients who suffered a reactivation (mean O.D. + SD.: 0.471 + 0.265) in comparison with the non-responder cases (0.201 f 0.107) or those without reactivation (0.163 + 0.066). In contrast, the maximum absorbance value was significantly higher among patients who reactivated (1.784 k 0.114) with respect to the non-responders (0.294 + 0.094) (PC 0.05) or in relation to those who did not suffer a reactivation(0.259 f 0.037). Changes in HBV-DNA

and HBeAg

during the study

At the end of the study. 17 of the 46 adult patients treated were HBV-DNA- and HBeAg-negative (rIMa responders). As previously mentioned, only two ottt of the twelve (16.6%) cases who developed anti-1FN antibodies responded to therapy, while 15 of the remaining34 patients (44.1%) did. Reactivation episodes only took place among patients with anti-1FN antibodies (3/12: 25%) while nooe of the remainder of the patients suffered this reactivation (P < 0.02).

There were no statistically signifiwtt differences behveen patients who did or did no: d.?velop anti-IPN antibodies with respect to age (ant.-!FN-positive vs. anti-IFN-negative: 29.1 f 11.4 vs. 37.8 f 13.4), sex (10 M/2 F vs. 26 M’S P), homosexuality (3 vs. 7), basal ALT level (229.6 f 149.0 vs. 266.0 f Z’O5.6KX). histological diagnosis (4 mild chrwic active hepatitis, 5 moderate and 3 with cirrhosis v’. 19, 8 and 7, respectively). or duration of rIFN tbxapy (4 months, 5 and 6 months: 7 patients vs. 11 and 23 patients).

Twenty six percent of OUTtreated patients and none of the contrds with chronic hepatitis B deveioped anti-IPN antibodies during the treatment. This result is similar to tbat previously published ir. the treatment of other diseases [lO,lll. It has been reported that the frequency of appearance of anti-IM antibodies depeods on the type of rIPNo used, although this is controversial and it has been suggested that it depends on the tests used to detect the aaribodies [7,12]. We avoided such a pmblem by osiog three different methods all of which gave the same positive results. In addition, the relationship between the development of anti-USN antibodies, the rWN dos: and the duration of therapy ht patiants with cancer during rIFN treatment has been published [7,13]. In our experience, with chronic hepatitis patients, !w correlation between these parameters was observed, although there is no achml explanation for these diiferences. Perhaps the type of the disease cou:d influence the appearance of the antibodies. Wuw i,tr p?&ouls developed anti-IPN antibo~:is, the secondary effects caused by IPN adminiitration disappeared. Thii suggests that anti-IF’N antibodies neutralize the IPN that is given exogenously to the patient [14]. Although the anti-IFN and FN have been involved in the pathogenesis of several autoknmune diseases (systemic lupus erythentatosus, rheumatoid arthritis, etc.) it should be noted that none of our anti-IFN-positive patients developed any im-

I.C. PORRES et at.

356 mune complexes disease or deterioration of the liver disease. This is important and demonstrates the safety of the IFN :herapy in the presence of anti-EN antibodies. Tine majority of patients who developed anti-IFN when they were still HBV-DNA-positive did not respond to therapy. Significant differences between patients who did and those who did not develop antiIFN anqibodies, with respect to the reactivation of viral replication were observed. Furthermore, the patients who suffered a reactivation had anti-IFN antibodies prior to or at the same time as the loss of HBV-DNA. In contrast, patients who developed anti-II74 antibodies after HBV-DNA negativization did not suffer B -eactivatioo Caii~g the foilcw-tip. In addition, the reactivation of viral replication cciw tided with the maximum anti-IFN antibodies level. One possible explanation is that among patients who developed acti-IFN antibodies prior to the negativization of serum HBV-DNA, only a transient and not a total loss of * 1replication takes place and, subsequently, when the level of anti-IM increases. the reappea;aoce of circulating HBV-DNA occurs. However, at least in chronic hepatitis B patients, rIFN treatment should probably be stopped as soon as anti-IFN is detected and the effect of changing the type of interferon should be determined in the future.

1 BsssendmeMF, Ch-dwick RG. Sabneren J. Shipton V. Thomas HC. Sherlock S. Adeninc arrbinostdetberaovin HBsAg-positive chronic liver dircr+se:a controlled s;idy. Gaslroenterology1981:80: 1016-1022. 2 Mora I, Porres JC. BanolomC J, Quiroaa JA. Gurus J. Hernkv+.zGuio C. Bas C, Carreao V. Changesor hcpatitis B virus (HBV) markers during prolongedrecombinant interferon alpha-2A ueaimem of chronicHBV infection. J Hepatol L987:4: 29-36. 3 La Bsnda F, Ruin M, Cane60 V, Bartolom6 J, Gutier J, Ramdn Y Caial S, Moreno A, Moral PorresJC. Recomoinant a2:inte;feron treatment in children with chronichepatitis B. Lancet1988:i: 2% 4 Vallbracht A, Treuner T, Mechmig IB, JoesterK-E. Niethammer D. tnrerkron oenirvlizinga&b&k in a paticm treated with humanfibrobl~stinterferon. Nature 1981;287: 496-498

concluded :hat if anti-IFN antibodies appear when HBV-DNA is already negative, the antibodies will not influence the response to iherapy. Another interesting aspect is that one of our patien:s developed a Herpes Zoster infection when he had a high anti-EN antibody titer and was still receiving 20 ,MU:m* of IFN. Since IFN is active agaix: Herpes Zoster virus, the explanation for this phenomenon is not clear, hut suggests that the exogenous IFN had been blocked in this patients by antiIFN antibodies. Finally, it should be noted that none of the children with chronic hepatitis B treated with IFN developed anti-IFN antibodies. The reason for the lack of antibodies in children is not clear. However, in a similar It may also be

irlal performed in our uni!, none of the 15 children treated developed anti-IFN antibodies (personal observation). It could be that differences in the immune system between children and adults could explain t%is finding, ;;lthougb this hypothesis should be proven in the fixture. In summary, if anti-TN antibodies develop when patients are HBV-DNA-positive, a lack of response to therapy will probably be obtained. The anti-IFN antibodies wili not influence the response if Fltiettts are already HBV-DNA-negative

S JonesGJ, Itri L. Safetyand toleranceof recombinantinterferon affa-2A (Roleron-A) in cancerpnrients.Cancer 1988G: 57(S”pplt): ,7lx-,715. 6 Spiegel BJ, SpicehandlerJR. JacahsSL, Ode” EM. Low incidenceof serumneutralizingfactorsin parientrreceiving recombinant alfa-2b interferon flntron A). Am J Med

1986;80:223-228. 7 ,tri l_M,CampionM. DenninMA. eaueroni A’!. ciuwrman,“. GroapmsnJE, TrownPW. Incidcncr and clinical rignificenceof neutralizingentibndier in paricnrsreceiving recombinantinterferon alfw2a by intramuxular injection. Cancer1987;59: 668-674. 8 Wel!erlVD.FowlerMJF. Monjardino J, ThamasHC. The detectionof HBV-DNA in serumby molecular hyhridiza. lion: a more sensitivemethod for detection of complete HBV particles.J Med Viral 1982;9: 273-280. 9 Hcancs “, Juckeer W, FischerEA, et at. The delcctio” of antibodiesto recombinantinterferon alfa-la in human serum. J FJialStand 1987:15:231-244.

ANTI-IFN

ANTlBODfES

IO Gutterma” JU. Fine S. Quesada JR, Xorning SI, i4”r IF. Akxania” R, Bernhardt L, Kramer M, Spiegel H. Calburn W. Trow” P. Meriga” 7. Dziewanowki Z. Recombs“ant leukucy!e A interferon: pbarmaco-kinetics en&_dose toierance. and biologic eifrcte in ca”ccr yrrientr. A”” Inter” Mcd ,982: 96: 549-556. 11 Gauci I.. Management of cancer ptientr receiving ip e.. feron alfa-2A. Im J Cancer 1987: (Suppl 1): 21-30. 12 Van Wvssaw P. Freund M. Block 8, Diedrich H. Pohwoda H. Deicher H. Clinical significance of anti-In\‘-n antibody

357

titres during interferon therapy. Laneet 1987; ii: 635-636. 13 Kawade Y, Watanabe Y. Neutralizadon of interferon by antibody: apprairalr of methods of del’smini) and enprcsring Ihe “eutralizado” riler. I I”rcrfefem” RFS 1984; 4: 571-584. ‘4 Schmidt H. H&&ma”” E. Vallbraeht A. Wallcr HD. Antibodies m human leukocyte interfere” a-A aftn half a year li interferon treatment. Tuumor Diagnostik & Tberapic ,165: 6: X7-41.