(~) INSTITUTPASTEUR/ELsEVIER Paris 1990
Res. ViroL
1990, 141, 563-570
ANTIBODk RESPONSE TO preSl IN HEPATITIS-B-VIRUS-INDUCED LIVER DISEASE AND AFTER IMMUNIZATION
P. Coursaget (1) (,), y. Buisson (2), C. Bourdil (1), B. Yvonnet (1), C. Molini~ (3), M.T. Diop (4), j.p. Chiton (l), O. Bao (4) and I. Diop-Mar (4) (1) lnstitut de Virologic de Tours et Laboratoire de Microbiologie, Facultd de Pharmacie, 2 bis boulevard Tonnelld, 37042 Tours Cedex (France), (2) H6pital d'Instruction des armdes du Val de Grace, Paris, (3) H6pital d'Instruction des armies Begin, St Mandd (France) and (4) Facultd de Mddecine et de Pharmacie, Dakar (Sdndgal)
SUMMARY Antibodies to the preSl-encoded sequence of hepatitis B virus (HBV) envelope were detected by ELISA using a synthetic peptide analogue of preS1 proteins, in different groups of HBV-infected subjects and also in hepatitis B vaccine recipients. Such antibodies were specifically found in only 1 °70 of HBsAg chronic carriers including patients with cirrhosis and primary liver cancer. Anti-preS1 were detected in patients with acute hepatitis; in 13 °70 of the HBsAg + sera obtained before recovery and in 37 07o of the sera obtained after recovery. Anti-preS1 antibodies were detected in recipients of a plasma-derived vaccine, but not in those receiving a recombinant vaccine. The results indicate that anti-preS1 is an earlier serum marker of HBV clearance than anti-preS2 and anti-S antibodies. KEY-WORDS: Hepatitis B, Protein preS1; Vaccine, Immunity. INTRODUCTION The preS region of hepatitis B virus (HBV) genome comprises two subregions, preSl and preS2, the translation products of which appear in the composition of the surface antigen of HBV (Neurath et al., 1985; 1986c). Submitted July 13, 1989, accepted July 4, 1990. (*) Correspondingauthor.
564
P. C O U R S A G E T
ET AL.
Studies to date have concentrated on the preS2 region. Antigenic determinants located in the preS2 sequences are more immunogenic than S-protein determinants (MiUich et al., 1985), enhance the immune response to S protein (Millich et al., 1985; Coursaget et al., 1985), elicit virus-neutralizing antibodies and protective immunity in the chimpanzee (Neurath et al., 1986a; Itoh et al., 1986), and have the potential to provide T-cell heip for an anti-S antibody response (Millich et al., 1986). Moreover anti-preS2 antibody is a reliable marker of HBV clearance (Coursaget et al., 1988a; Alberti et al., 1988; Zanetti et al., 1988). Such observations form the rationale for the hypothesis that inclusion of preS2 epitopes in a vaccine would improve its efficacy, particularly in subjects with poor anti-S response. The immunogenicity of peptides from the preS 1 sequence and the recognition of the HBV envelope by the corresponding anti-peptide sera, indicate the presence of additional T- and B-cell epitopes in the preS1 region (Millich et al., 1987). The fact that the HBV-binding site for hepatocyte receptor is localized in preS1 (Neurath et al., 1988; 1986b) strongly suggests that it also carries protective epitopes. As anti-preS1 antibodies have been detected early on during infection (Klinkert et al., 1986; Alberti et al., 1990), the preS1 antibody could also represent an early serological marker for HBV clearance and may play a role in the neutralization of the virus. Thus, like the presence of preS2 epitopes, the presence of preS1 in addition to S protein could improve the potency and efficacy of hepatitis B vaccine (Gerlich et al., 1988). in this study, anti-preS1 antibodies were detected in different population groups of HBV-infected individuals in order to investigate the immune response to this protein during the course of natural infection and also in individuals immunized with different hepatitis B vaccines. SUBJECTS AND METHODS Patients with acute hepatitis.
Blood sampleswere taken from 61 acute hepatitis patients during HBs antigenaemia and from 51 patients after recovery from acute hepatitis. Individuals with chronic HBV infection.
The anti-preS1 antibody was studied in (1) sera from 41 heaithy chronic carriers from Senegal, (2) 11 French patients with chronic hepatitis, (3) 36 Senegalesepatients suffering from cirrhosis and 70 from primary liver cancer.
ELISA HBV
= enzyme-linked immunosorbent assay. = hepatitis B virus.
[ [
HBsAg = hepatitis B surface antigen. RIA = radioimmunoassay.
HEPA TITIS B ANTIBODY
RESPONSE
565
TO preS1
Hepatitis B vaccine recipients. Anti-prcS1 antibody was studied in 178 infants immunized with plasma-derived hepatitis B vaccine (Hevac B, Pasteur Vaccins). These infants received 3 or 4 doses of 5 or 2 ttg of HBsAg (Yvonnet et al., 1987). The anti-HBs geometric mean titre after booster dose was 460 mIU/ml in these infants. Moreover, the presence of anti-preS1 was tested in 23 French adults (Coursaget et al., 1988b) receiving 20 btg doses of a recombinant vaccine (Adamowicz et al., 1988) (Gen-Hevac B, Pasteur Vaccins). The anti-HBs geometric mean titre after booster dose was 15,500 mIU/ml in these individuals. The presence of anti-preS1 was tested in vaccine recipients after they received the booster dose.
Controls. Sera obtained before hepatitis B immunization from 72 healthy subjects, negative for HBsAg, anti-HBs and an~.i-HBc were used as controls.
Detection of anti-preS1 antibodies. Microplate wells (Nunc-immuno I) were coated with 6 ~tg of a preS1 synthetic peptide. Microplates were then post-coated with bovine serum albumin. The synthetic peptide corresponded to the sequence 12-49 in the lareS region of HBsAg of ayw subtype. Wells were incubated with sera diluted 1/2 in 5 x phosphate~bufferred saline (PBS). Anti-preS1 antibodies in the sample bound to the synthetic peptide and were recognized by a monoclonal anti-human IgG antibody covalently linked to horseradish peroxidase. To confirm the specificity of the anti-preS1 antibodies, det~tion sera were tested after a 1-h incubation at 37°C with PBS or with 12 ~tg of preS1 peptide.
OD
OD.
2.4 "
24
2.2
2.2
2.0
2.0
1.8
! .8
1,0 "
1.0
08
0.8"
=
0.6 -
0.6 -
-
0.4 -
0.4
0.2 -
0.2 -
,
~,
,
,
2
4
0
t6
~
O|lut|on
FIG. 1.
- -
, 64
,
,
12,; 256
~t
. 2
.
. ,;
. 8
~6
,
,
32
64
,
,
..
~24 , ~
O~lut|on
Detection of anti-preS1 antibodies in two sera obtained during the convalescent phase from two patients (A and B) with a ~ t e hepatitis B.
m = sera incubated in buffer; - - = sera incubated with preSl peptide.
566
P. C O U R S A G E T E T A L .
The enzyme linked to the solid phase was detected by incubation with orthophenylene-diaminedihydrochloride. The results were obtained by photometric comparison at 486 mm (Kontron SLT-210) of the colour intensities of the samples compared to the colour intensities of negative controls. Samples were considered to be positive when the absorbance value exceeded the mean OD of the control sera by two standard deviations and when a 50 °70 reduction in OD was observed after incubation with preS1 synthetic peptide (fig. 1). Serological markers.
HBsAg, anti-HBs and anti-HBc antibodies were tested in serum by commercial radioimmunoassays (Ausria, Ausab and Corab, Abbott Laboratories, North Chicago, USA). Some sera were also tested for anti-HBs by ELISA (Abbott Laboratories). Anti-preS2 antibodies were tested by an ELISA similar to the one used for antipreS1 detection (Coursaget et al., 1988a). RESULTS
Anti-preSl antibodies were detected in none o f the 72 H B V - controls. In HBsAg + individuals (table I), anti-preSl antibodies were noted in 13. 1 % o f patients with acute hepatitis but not in patients with chronic hepatitis. In HBsAg + samples from Senegal, anti-preS1 was detected in none o f the healthy chronic carriers and patients suffering from chronic hepatitis or cirrhosis. Anti-preSl were detected in two patients (2.9 °70) suffering from primary liver cancer.
TABLE I. - - Anti-preS1 antibodies in HBV-infected patients and in vaccine recipients.
Subjects
No.
No. positive for anti-preS1 No. (%)
Control group
72
0
(0.0 %)
Acute hepatitis: before recovery (HBsAg+) - - after recovery (HBsAg-)
61 51
8
19
(13.1%) (37.3 °70)
Chronic carriers: healthy chronic carriers chronic hepatitis cirrhosis - - primary live cancer
41 11 36 70
0 0 0 2
HB vaccine recipients: plasma-derived vaccine recombinant vaccine
178 23
16 0
(0.0 (0.0 (0.0 (2.9
%) %) %) %)
(9.0 o7o) (0.0 %)
H E P A T I T I S B A N T I B O D Y RESPONSE TO preS1
567
TABLE II. - - H B V m a r k e r s in 4 p a t i e n t s w i t h a c u t e h e p a t i t i s B. Weeks after first HBsAg detection 6 8 10-12 18-24
Patients
HBV markers Method
4
1
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
+ +
2
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
+ +
3
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
Anti-preS 1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
4
+ + +
+ + + -
+ + + + -
+ + + + -
-
+ +
+
-
+ + + + -
+ + + -
+ + +
+ +
+ + + + m
Anti-preSl were detected in 37 % o f the sera obtained after recovery from acute hepatitis B a n d in 9 % o f the infants immunized with Pasteur plasma vaccine, but not in the 23 adults who received the recombinant vaccine. Detection o f anti-preSl was compared to the detection o f anti-preS2 a n d anti-HBs by E L I S A a n d r a d i o i m m u n o a s s a y (RIA) in 4 patients with acute hepatitis (table II). Anti-preSl was detected before anti-HBs (anti-S) antibodies were detected by E L I S A . It must be noted that in patient no. 1, anti-preS1 antibodies were not detected 3 m o n t h s after the last HBsAg detection.
DISCUSSION Antibodies directed against preS1 proteins were generally not detected in sera f r o m chronic carriers o f HBsAg. However, such sera frequently gave false-positive reactions, as observed with anti-preS2 detection (Coursaget et al., 1988a). After recovery from acute hepatitis, 37 % o f the patient sera tested were f o u n d to be anti-preSl positive a n d anti-preS! antibodies generally appeared earlier t h a n anti-preS2 and anti-S antibc ~es. The characteristics o f the preSl protein as well as the very early detection o f these a.ntiviral envelope antibodies in patients convalescing after acute hepa-
568
P. C O U R S A G E T E T AL.
titis, and the lack of specific anti-preS1 response in persistently infected individuals, suggest that the immune response against preSl protein may be as essential as preS2 and S proteins for recovery from type B hepatitis and may play an important role in the host mechanism of virus neutralization. Anti.preS1 is the earliest HBV marker of HBV clearance, and may be shorter lasting than anti-preS2 and anti-S since its detection could become negative a few months after recovery from acute hepatitis. After immunization with "Hevac B" Pasteur vaccine (plasma-derived), 9 070 of the recipients developed anti-preS1 antibodies, thus indicating that this vaccine contained preS1. This result confirms that HBsAg 20 nm particles carry preS1 epitopes, though in very small quantities (Heermann et aL, 1984). The recipients of the "Gen-Hevac B" Pasteur vaccine (recombinant), did not develop anti-preS1 antibodies in contrast to the high anti-HBs and anti-preS2 responses (Adamowicz et a~.., 1988; Coursaget et al., 1988a), thus confirming the absence of preSl protein in this vaccine as well as in all other recombinant vaccines currently developed. In view of the observation that in natural infection anti-preS1 antibodies are linked to virus clearance and protective immunity, as has been recently demonstrated in chimpanzee immunized with a preS 1 pe.ptide (Neurath et al., 1989), inclusion of these epitopes in the composition of hepatitis B vaccine would be of some interest.
RI~SUMI~ DI~TECTION DES ANTICORPSANTI-pr6SI CHEZ LES SUJETSATTEINTS D'HI~PATITEU ET APRI~SVACCINATION
Les anticorps dirig6s contre la prot6ine cod~e par la s6quence pr6S1 du g~ne d'enveloppe du xirus de l'h~patite B ont ~t~ recherch~s dans diff6rents groupes de sujets infect6s par le virus de l'h6patite B et chez des sujets vaccin6s contre l'h6patite B. Ces anticorps ont 6t6 d~tect~s par un test immunoenzymatique utilisant un peptide de synth6se analogue A la p~ot~ine pr6S1. Les anticorps anti-pr6Sl ont ~t¢ d~tect6s chez seulement 1 070des sujets porteurs chroniques de l'antig~ne HBs y compris les sujets presentant une cirrhose ou un cancer primitif du foie. Par contre, les anticorps anti-prOS1 sont d6tect6s chez 13 °70des sujets HBsAg+ pr6sentant une h6patite aigu~. Apr6s disparition de I'HBsAg, les anticorps anti-prOS1 sont d6tect~s chez 37 070de ces sujets. Les anticorps anti-pr6S1 ont 6t6 d6tect~s chez 9 070des sujets recevant un vaccin plasmatique mms chez aucun des sujets recevant un vaccin recombinant. Les r6sultats indiquent que les anticorps anti..pr~S1 sont un marqueur s6rologique plus pr6coce de la gu6rison que ne le sont les anticorps anti-pr6S2 et anti-HBs. MOTS-CLI~S: H6patite B, Prot~ine pr6S1 ; Vaccination, Immunit6. ACKNO'~,I LEDGEMENTS
Tb~s work was supported by a grant N ° 8722303E from the Caisse Nationale de l'Assurance Maladie des Travaiileurs Salari6s.
H E P A T I T I S B A N T I B O D Y R E S P O N S E TO preS1
569
The authors are grateful to E. Tortey, T. Diop, M.T. Sow and J.E. Bocande (Dakar, Seneoal) for their help in obtaining serum samples from Senegal, and to P. Adamow~¢z (Pasteur Vac¢ins) for providing monoclonal anti-human IgG antibody covalently linked to horseradish peroxidase. They are also grateful to N. Sornais for secretarial assistance. REFERENCES ADAMOWICZ,P., TRON,F., VINAS,R., MEVELEC,M.N., DIAZ, I., COURROUC~,A.M., MAZERT,M.C., LAGARDE,D. & GIRARD,M. (1988), Hepatitis B vaccine containing the S and pre-S2 antigens produced in chinese hamster ovary cells, , in "Viral hepatitis and liver disease" (A.J. Zuckerman) (pp, 1087-1090). Alan R. Liss, New York. ALBERTI,A., CAVALLETTO,D., PONTISSO,P , CHEMELLO,L., TAGARIELLO,G. & BELLUSSl,F. (1988), Antibody response te pre-S2 and hepatitis-B-virus-induced liver damage. Lancet, I, 1421-1422. ALBERTI,A., CAVALLETTO,D. & PONTiSSO,P. (1990), Anti-preSl in HBV infection and in HB vaccine recipients, in ' Progress in hepatitis B immunization" (P. Coursaget & N.J. Tong) (p. 105). Coltoque, INSERM/John Libbey Eurotext N ° 194, Paris. COURSAt~ET,P., BARR~S,J.L., CHmON, J.P. & ADAMOWICZ,P. (1985), Hepatitis B vaccines with and without polymerized albumin receptors. Lancet, I, 1152. COURSAGET, P., ADAMOWlCZ, P., BOURDIL, C., YVONNET, B., BUISSON, Y., BARRI~S,J.L., SALIOU,P., CHIRON,J.P. & DloP-MAR, I. (1988a), Anti-preS2 antibodies in natural hepatitis B virus infection and after immunization. Vacc~;ne, 6, 357-361. COURSAGET~ P., YVONNET, B., ANTHONIOZ, P., CHOTARD, J., BOURDIL, C., ADAMOWICZ,P., TRON, F. & GIRARD, M. (1988b), Immunogenicity of an hepatitis B vaccine obtained by genetic recombination and containing S and pre-S2 gcne produc~:. Presse Med., 17, 1150-1152. GERUCH, W.H., HEERMAN,K.H., KRUSE,F., MARQUARDT,O. & SEWER,M. (1988), Immunogenicity of gene S and pre-S domains in hepatitis virions and in recombinant hepatitis B surface antigen filaments, in "Viral hepatitis and liver disease" (A.J. Zuckerman) (pp. 1091-1093). Alan R. Liss, New York. HEERMANN,K.H., GOLDMANN,U., SCHWARTZ,W., SEYFFARTH,T., BAUMGARFEN,H. & GERLICH,W.H. (1984), Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol., 52, 396-402. ITOH, Y., TAKAI, E., OHNUMA, I-L, KITAJIMA, K., TOUDA, F., MACHIDA, A,, MISHIRO, S., NAKAMURA,T., MIYAKAWA,Y. & MAYUNI,M. (1986), A synthetic peptide vaccine involving the products of the pre-S2 region of hepatitis B virus DNA: protective efficacy in chimpanzees. Proc. nat. Acad. Sci. (Wash.), 83, 9174-9178. KLINKERT,M.Q., THEILMANN,L., PFAFF,E. & SCHALLER,H. (1986), Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infections. J. ViroL, 58, 522-525. MILUCrt, D.R., THORNTON, G.B., NEURATH, A.R., KENT, S.B., MICHEL, M.L., TIOLLAIS, P. & CHISARI, F.W. (1985), Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen. Science, 228, 1195-1199. MILLICH, D.R., MCLACHLAN,A., CHISARI,F.W. & THORNTON,Ci.]~. (1986), Nonoverlapping T- and B-cell determinants on an hepatitis B surface antigen pre-S2 region synthetic peptide, d. exp. Ned., 164, 532-547. MILL'CH, D.R., MCLACHLAN,A., MORIARTY,A. & THORNTON,G.B. (1987), A single 10 residue pre-Si peptide can prime T-cell help for antibody production to multiple opitopes within the pre-Sl, pre-S2 and S regions of HBsAg. J. ImmunoL, 138, 445%4465.
570
P. C O U R S A G E T E T A L .
NEURATH, A.R., KENT, S.B.H., STRICK,N., TAYLOR,P. & STEVENS,C.E. (1985), Hepatitis B virus contains pre-S-gene-encoded domains. Nature (Lond,), 315, 154-156. NEURATH,A.R., KENT,S.B.H,, PARKER,K,, PRINCE,A.M., STRICK,N., BROTMAN,B. & SPROUL, P. (1986a), Antibodies to a synthetic peptide from the pre-S i20-145 region of the hepatitis B virus envelope are virus-neutralizing. Vaccine, 4, 35-37. NEURATn,A.R., KENT, S.B.H., STRICK,N. & PARKER,K. (1986b), Identification and chemical synthesis of a host cell receptor-binding site on hepatitis B virus. Cell, 46, 429-436. NEURATH,A.R., KENT, S.B.H. & STRICK,N. (1986c), Detection of antiviral antibodies with predetermined specificity using synthetic peptide-[3-1actamase conjugates: application to antibodies specific for the pre-S region of the hepatitis B virus envelope proteins. J. gen. ViroL, 67, 453-461. NEURATH,A.R., KENT, S.B.H., STruCK,N. & PARKER,K. (1988), Delineation of contiguous determinants essential for biological functions of the pre-S sequence of the hepatitis B virus envelope protein. Its antigenicity, immunogenicity and cell-receptor recognition. Ann. Inst. Pasteur/Virol., 139, 13-38. NEURATH,A.R., SETO,B. & STruCK,N. (1989), Antibodies to synthetic peptides from the pre-Sl region of the hepatitis B virus (HBV) envelope (env) protein are virus-neutralizing and protective. Vaccine, 7, 234-236.
YVONNET,B., COURSAGET,P., LEBOULLEUX,D., BARR~S,J.L., FR1TZELL,B., SARR,G.,
CHIRON, J.P. & DIoP-MAR, I. (1987), Low-dose hepatitis B vaccine immunization in children. Lancet, I, 169. ZANETTI,A.R., TANZI,E., COLOMBO,M. & MANNUCCI,P.M. (1988), Anti-preS2 antibodies in clearance of hepatitis B virus. Lancet, II, 447-448.
Res. ViroL
1990, 141, 563-570
ANTIBODk RESPONSE TO preSl IN HEPATITIS-B-VIRUS-INDUCED LIVER DISEASE AND AFTER IMMUNIZATION
P. Coursaget (1) (,), y. Buisson (2), C. Bourdil (1), B. Yvonnet (1), C. Molini~ (3), M.T. Diop (4), j.p. Chiton (l), O. Bao (4) and I. Diop-Mar (4) (1) lnstitut de Virologic de Tours et Laboratoire de Microbiologie, Facultd de Pharmacie, 2 bis boulevard Tonnelld, 37042 Tours Cedex (France), (2) H6pital d'Instruction des armdes du Val de Grace, Paris, (3) H6pital d'Instruction des armies Begin, St Mandd (France) and (4) Facultd de Mddecine et de Pharmacie, Dakar (Sdndgal)
SUMMARY Antibodies to the preSl-encoded sequence of hepatitis B virus (HBV) envelope were detected by ELISA using a synthetic peptide analogue of preS1 proteins, in different groups of HBV-infected subjects and also in hepatitis B vaccine recipients. Such antibodies were specifically found in only 1 °70 of HBsAg chronic carriers including patients with cirrhosis and primary liver cancer. Anti-preS1 were detected in patients with acute hepatitis; in 13 °70 of the HBsAg + sera obtained before recovery and in 37 07o of the sera obtained after recovery. Anti-preS1 antibodies were detected in recipients of a plasma-derived vaccine, but not in those receiving a recombinant vaccine. The results indicate that anti-preS1 is an earlier serum marker of HBV clearance than anti-preS2 and anti-S antibodies. KEY-WORDS: Hepatitis B, Protein preS1; Vaccine, Immunity. INTRODUCTION The preS region of hepatitis B virus (HBV) genome comprises two subregions, preSl and preS2, the translation products of which appear in the composition of the surface antigen of HBV (Neurath et al., 1985; 1986c). Submitted July 13, 1989, accepted July 4, 1990. (*) Correspondingauthor.
564
P. C O U R S A G E T
ET AL.
Studies to date have concentrated on the preS2 region. Antigenic determinants located in the preS2 sequences are more immunogenic than S-protein determinants (MiUich et al., 1985), enhance the immune response to S protein (Millich et al., 1985; Coursaget et al., 1985), elicit virus-neutralizing antibodies and protective immunity in the chimpanzee (Neurath et al., 1986a; Itoh et al., 1986), and have the potential to provide T-cell heip for an anti-S antibody response (Millich et al., 1986). Moreover anti-preS2 antibody is a reliable marker of HBV clearance (Coursaget et al., 1988a; Alberti et al., 1988; Zanetti et al., 1988). Such observations form the rationale for the hypothesis that inclusion of preS2 epitopes in a vaccine would improve its efficacy, particularly in subjects with poor anti-S response. The immunogenicity of peptides from the preS 1 sequence and the recognition of the HBV envelope by the corresponding anti-peptide sera, indicate the presence of additional T- and B-cell epitopes in the preS1 region (Millich et al., 1987). The fact that the HBV-binding site for hepatocyte receptor is localized in preS1 (Neurath et al., 1988; 1986b) strongly suggests that it also carries protective epitopes. As anti-preS1 antibodies have been detected early on during infection (Klinkert et al., 1986; Alberti et al., 1990), the preS1 antibody could also represent an early serological marker for HBV clearance and may play a role in the neutralization of the virus. Thus, like the presence of preS2 epitopes, the presence of preS1 in addition to S protein could improve the potency and efficacy of hepatitis B vaccine (Gerlich et al., 1988). in this study, anti-preS1 antibodies were detected in different population groups of HBV-infected individuals in order to investigate the immune response to this protein during the course of natural infection and also in individuals immunized with different hepatitis B vaccines. SUBJECTS AND METHODS Patients with acute hepatitis.
Blood sampleswere taken from 61 acute hepatitis patients during HBs antigenaemia and from 51 patients after recovery from acute hepatitis. Individuals with chronic HBV infection.
The anti-preS1 antibody was studied in (1) sera from 41 heaithy chronic carriers from Senegal, (2) 11 French patients with chronic hepatitis, (3) 36 Senegalesepatients suffering from cirrhosis and 70 from primary liver cancer.
ELISA HBV
= enzyme-linked immunosorbent assay. = hepatitis B virus.
[ [
HBsAg = hepatitis B surface antigen. RIA = radioimmunoassay.
HEPA TITIS B ANTIBODY
RESPONSE
565
TO preS1
Hepatitis B vaccine recipients. Anti-prcS1 antibody was studied in 178 infants immunized with plasma-derived hepatitis B vaccine (Hevac B, Pasteur Vaccins). These infants received 3 or 4 doses of 5 or 2 ttg of HBsAg (Yvonnet et al., 1987). The anti-HBs geometric mean titre after booster dose was 460 mIU/ml in these infants. Moreover, the presence of anti-preS1 was tested in 23 French adults (Coursaget et al., 1988b) receiving 20 btg doses of a recombinant vaccine (Adamowicz et al., 1988) (Gen-Hevac B, Pasteur Vaccins). The anti-HBs geometric mean titre after booster dose was 15,500 mIU/ml in these individuals. The presence of anti-preS1 was tested in vaccine recipients after they received the booster dose.
Controls. Sera obtained before hepatitis B immunization from 72 healthy subjects, negative for HBsAg, anti-HBs and an~.i-HBc were used as controls.
Detection of anti-preS1 antibodies. Microplate wells (Nunc-immuno I) were coated with 6 ~tg of a preS1 synthetic peptide. Microplates were then post-coated with bovine serum albumin. The synthetic peptide corresponded to the sequence 12-49 in the lareS region of HBsAg of ayw subtype. Wells were incubated with sera diluted 1/2 in 5 x phosphate~bufferred saline (PBS). Anti-preS1 antibodies in the sample bound to the synthetic peptide and were recognized by a monoclonal anti-human IgG antibody covalently linked to horseradish peroxidase. To confirm the specificity of the anti-preS1 antibodies, det~tion sera were tested after a 1-h incubation at 37°C with PBS or with 12 ~tg of preS1 peptide.
OD
OD.
2.4 "
24
2.2
2.2
2.0
2.0
1.8
! .8
1,0 "
1.0
08
0.8"
=
0.6 -
0.6 -
-
0.4 -
0.4
0.2 -
0.2 -
,
~,
,
,
2
4
0
t6
~
O|lut|on
FIG. 1.
- -
, 64
,
,
12,; 256
~t
. 2
.
. ,;
. 8
~6
,
,
32
64
,
,
..
~24 , ~
O~lut|on
Detection of anti-preS1 antibodies in two sera obtained during the convalescent phase from two patients (A and B) with a ~ t e hepatitis B.
m = sera incubated in buffer; - - = sera incubated with preSl peptide.
566
P. C O U R S A G E T E T A L .
The enzyme linked to the solid phase was detected by incubation with orthophenylene-diaminedihydrochloride. The results were obtained by photometric comparison at 486 mm (Kontron SLT-210) of the colour intensities of the samples compared to the colour intensities of negative controls. Samples were considered to be positive when the absorbance value exceeded the mean OD of the control sera by two standard deviations and when a 50 °70 reduction in OD was observed after incubation with preS1 synthetic peptide (fig. 1). Serological markers.
HBsAg, anti-HBs and anti-HBc antibodies were tested in serum by commercial radioimmunoassays (Ausria, Ausab and Corab, Abbott Laboratories, North Chicago, USA). Some sera were also tested for anti-HBs by ELISA (Abbott Laboratories). Anti-preS2 antibodies were tested by an ELISA similar to the one used for antipreS1 detection (Coursaget et al., 1988a). RESULTS
Anti-preSl antibodies were detected in none o f the 72 H B V - controls. In HBsAg + individuals (table I), anti-preSl antibodies were noted in 13. 1 % o f patients with acute hepatitis but not in patients with chronic hepatitis. In HBsAg + samples from Senegal, anti-preS1 was detected in none o f the healthy chronic carriers and patients suffering from chronic hepatitis or cirrhosis. Anti-preSl were detected in two patients (2.9 °70) suffering from primary liver cancer.
TABLE I. - - Anti-preS1 antibodies in HBV-infected patients and in vaccine recipients.
Subjects
No.
No. positive for anti-preS1 No. (%)
Control group
72
0
(0.0 %)
Acute hepatitis: before recovery (HBsAg+) - - after recovery (HBsAg-)
61 51
8
19
(13.1%) (37.3 °70)
Chronic carriers: healthy chronic carriers chronic hepatitis cirrhosis - - primary live cancer
41 11 36 70
0 0 0 2
HB vaccine recipients: plasma-derived vaccine recombinant vaccine
178 23
16 0
(0.0 (0.0 (0.0 (2.9
%) %) %) %)
(9.0 o7o) (0.0 %)
H E P A T I T I S B A N T I B O D Y RESPONSE TO preS1
567
TABLE II. - - H B V m a r k e r s in 4 p a t i e n t s w i t h a c u t e h e p a t i t i s B. Weeks after first HBsAg detection 6 8 10-12 18-24
Patients
HBV markers Method
4
1
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
+ +
2
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
+ +
3
Anti-preS1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
Anti-preS 1 Anti-preS2 Anti-S Anti-S HBsAg
ELISA ELISA ELISA RIA RIA
4
+ + +
+ + + -
+ + + + -
+ + + + -
-
+ +
+
-
+ + + + -
+ + + -
+ + +
+ +
+ + + + m
Anti-preSl were detected in 37 % o f the sera obtained after recovery from acute hepatitis B a n d in 9 % o f the infants immunized with Pasteur plasma vaccine, but not in the 23 adults who received the recombinant vaccine. Detection o f anti-preSl was compared to the detection o f anti-preS2 a n d anti-HBs by E L I S A a n d r a d i o i m m u n o a s s a y (RIA) in 4 patients with acute hepatitis (table II). Anti-preSl was detected before anti-HBs (anti-S) antibodies were detected by E L I S A . It must be noted that in patient no. 1, anti-preS1 antibodies were not detected 3 m o n t h s after the last HBsAg detection.
DISCUSSION Antibodies directed against preS1 proteins were generally not detected in sera f r o m chronic carriers o f HBsAg. However, such sera frequently gave false-positive reactions, as observed with anti-preS2 detection (Coursaget et al., 1988a). After recovery from acute hepatitis, 37 % o f the patient sera tested were f o u n d to be anti-preSl positive a n d anti-preS! antibodies generally appeared earlier t h a n anti-preS2 and anti-S antibc ~es. The characteristics o f the preSl protein as well as the very early detection o f these a.ntiviral envelope antibodies in patients convalescing after acute hepa-
568
P. C O U R S A G E T E T AL.
titis, and the lack of specific anti-preS1 response in persistently infected individuals, suggest that the immune response against preSl protein may be as essential as preS2 and S proteins for recovery from type B hepatitis and may play an important role in the host mechanism of virus neutralization. Anti.preS1 is the earliest HBV marker of HBV clearance, and may be shorter lasting than anti-preS2 and anti-S since its detection could become negative a few months after recovery from acute hepatitis. After immunization with "Hevac B" Pasteur vaccine (plasma-derived), 9 070 of the recipients developed anti-preS1 antibodies, thus indicating that this vaccine contained preS1. This result confirms that HBsAg 20 nm particles carry preS1 epitopes, though in very small quantities (Heermann et aL, 1984). The recipients of the "Gen-Hevac B" Pasteur vaccine (recombinant), did not develop anti-preS1 antibodies in contrast to the high anti-HBs and anti-preS2 responses (Adamowicz et a~.., 1988; Coursaget et al., 1988a), thus confirming the absence of preSl protein in this vaccine as well as in all other recombinant vaccines currently developed. In view of the observation that in natural infection anti-preS1 antibodies are linked to virus clearance and protective immunity, as has been recently demonstrated in chimpanzee immunized with a preS 1 pe.ptide (Neurath et al., 1989), inclusion of these epitopes in the composition of hepatitis B vaccine would be of some interest.
RI~SUMI~ DI~TECTION DES ANTICORPSANTI-pr6SI CHEZ LES SUJETSATTEINTS D'HI~PATITEU ET APRI~SVACCINATION
Les anticorps dirig6s contre la prot6ine cod~e par la s6quence pr6S1 du g~ne d'enveloppe du xirus de l'h~patite B ont ~t~ recherch~s dans diff6rents groupes de sujets infect6s par le virus de l'h6patite B et chez des sujets vaccin6s contre l'h6patite B. Ces anticorps ont 6t6 d~tect~s par un test immunoenzymatique utilisant un peptide de synth6se analogue A la p~ot~ine pr6S1. Les anticorps anti-pr6Sl ont ~t¢ d~tect6s chez seulement 1 070des sujets porteurs chroniques de l'antig~ne HBs y compris les sujets presentant une cirrhose ou un cancer primitif du foie. Par contre, les anticorps anti-prOS1 sont d6tect6s chez 13 °70des sujets HBsAg+ pr6sentant une h6patite aigu~. Apr6s disparition de I'HBsAg, les anticorps anti-prOS1 sont d6tect~s chez 37 070de ces sujets. Les anticorps anti-pr6S1 ont 6t6 d6tect~s chez 9 070des sujets recevant un vaccin plasmatique mms chez aucun des sujets recevant un vaccin recombinant. Les r6sultats indiquent que les anticorps anti..pr~S1 sont un marqueur s6rologique plus pr6coce de la gu6rison que ne le sont les anticorps anti-pr6S2 et anti-HBs. MOTS-CLI~S: H6patite B, Prot~ine pr6S1 ; Vaccination, Immunit6. ACKNO'~,I LEDGEMENTS
Tb~s work was supported by a grant N ° 8722303E from the Caisse Nationale de l'Assurance Maladie des Travaiileurs Salari6s.
H E P A T I T I S B A N T I B O D Y R E S P O N S E TO preS1
569
The authors are grateful to E. Tortey, T. Diop, M.T. Sow and J.E. Bocande (Dakar, Seneoal) for their help in obtaining serum samples from Senegal, and to P. Adamow~¢z (Pasteur Vac¢ins) for providing monoclonal anti-human IgG antibody covalently linked to horseradish peroxidase. They are also grateful to N. Sornais for secretarial assistance. REFERENCES ADAMOWICZ,P., TRON,F., VINAS,R., MEVELEC,M.N., DIAZ, I., COURROUC~,A.M., MAZERT,M.C., LAGARDE,D. & GIRARD,M. (1988), Hepatitis B vaccine containing the S and pre-S2 antigens produced in chinese hamster ovary cells, , in "Viral hepatitis and liver disease" (A.J. Zuckerman) (pp, 1087-1090). Alan R. Liss, New York. ALBERTI,A., CAVALLETTO,D., PONTISSO,P , CHEMELLO,L., TAGARIELLO,G. & BELLUSSl,F. (1988), Antibody response te pre-S2 and hepatitis-B-virus-induced liver damage. Lancet, I, 1421-1422. ALBERTI,A., CAVALLETTO,D. & PONTiSSO,P. (1990), Anti-preSl in HBV infection and in HB vaccine recipients, in ' Progress in hepatitis B immunization" (P. Coursaget & N.J. Tong) (p. 105). Coltoque, INSERM/John Libbey Eurotext N ° 194, Paris. COURSAt~ET,P., BARR~S,J.L., CHmON, J.P. & ADAMOWICZ,P. (1985), Hepatitis B vaccines with and without polymerized albumin receptors. Lancet, I, 1152. COURSAGET, P., ADAMOWlCZ, P., BOURDIL, C., YVONNET, B., BUISSON, Y., BARRI~S,J.L., SALIOU,P., CHIRON,J.P. & DloP-MAR, I. (1988a), Anti-preS2 antibodies in natural hepatitis B virus infection and after immunization. Vacc~;ne, 6, 357-361. COURSAGET~ P., YVONNET, B., ANTHONIOZ, P., CHOTARD, J., BOURDIL, C., ADAMOWICZ,P., TRON, F. & GIRARD, M. (1988b), Immunogenicity of an hepatitis B vaccine obtained by genetic recombination and containing S and pre-S2 gcne produc~:. Presse Med., 17, 1150-1152. GERUCH, W.H., HEERMAN,K.H., KRUSE,F., MARQUARDT,O. & SEWER,M. (1988), Immunogenicity of gene S and pre-S domains in hepatitis virions and in recombinant hepatitis B surface antigen filaments, in "Viral hepatitis and liver disease" (A.J. Zuckerman) (pp. 1091-1093). Alan R. Liss, New York. HEERMANN,K.H., GOLDMANN,U., SCHWARTZ,W., SEYFFARTH,T., BAUMGARFEN,H. & GERLICH,W.H. (1984), Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol., 52, 396-402. ITOH, Y., TAKAI, E., OHNUMA, I-L, KITAJIMA, K., TOUDA, F., MACHIDA, A,, MISHIRO, S., NAKAMURA,T., MIYAKAWA,Y. & MAYUNI,M. (1986), A synthetic peptide vaccine involving the products of the pre-S2 region of hepatitis B virus DNA: protective efficacy in chimpanzees. Proc. nat. Acad. Sci. (Wash.), 83, 9174-9178. KLINKERT,M.Q., THEILMANN,L., PFAFF,E. & SCHALLER,H. (1986), Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infections. J. ViroL, 58, 522-525. MILUCrt, D.R., THORNTON, G.B., NEURATH, A.R., KENT, S.B., MICHEL, M.L., TIOLLAIS, P. & CHISARI, F.W. (1985), Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen. Science, 228, 1195-1199. MILLICH, D.R., MCLACHLAN,A., CHISARI,F.W. & THORNTON,Ci.]~. (1986), Nonoverlapping T- and B-cell determinants on an hepatitis B surface antigen pre-S2 region synthetic peptide, d. exp. Ned., 164, 532-547. MILL'CH, D.R., MCLACHLAN,A., MORIARTY,A. & THORNTON,G.B. (1987), A single 10 residue pre-Si peptide can prime T-cell help for antibody production to multiple opitopes within the pre-Sl, pre-S2 and S regions of HBsAg. J. ImmunoL, 138, 445%4465.
570
P. C O U R S A G E T E T A L .
NEURATH, A.R., KENT, S.B.H., STRICK,N., TAYLOR,P. & STEVENS,C.E. (1985), Hepatitis B virus contains pre-S-gene-encoded domains. Nature (Lond,), 315, 154-156. NEURATH,A.R., KENT,S.B.H,, PARKER,K,, PRINCE,A.M., STRICK,N., BROTMAN,B. & SPROUL, P. (1986a), Antibodies to a synthetic peptide from the pre-S i20-145 region of the hepatitis B virus envelope are virus-neutralizing. Vaccine, 4, 35-37. NEURATn,A.R., KENT, S.B.H., STRICK,N. & PARKER,K. (1986b), Identification and chemical synthesis of a host cell receptor-binding site on hepatitis B virus. Cell, 46, 429-436. NEURATH,A.R., KENT, S.B.H. & STRICK,N. (1986c), Detection of antiviral antibodies with predetermined specificity using synthetic peptide-[3-1actamase conjugates: application to antibodies specific for the pre-S region of the hepatitis B virus envelope proteins. J. gen. ViroL, 67, 453-461. NEURATH,A.R., KENT, S.B.H., STruCK,N. & PARKER,K. (1988), Delineation of contiguous determinants essential for biological functions of the pre-S sequence of the hepatitis B virus envelope protein. Its antigenicity, immunogenicity and cell-receptor recognition. Ann. Inst. Pasteur/Virol., 139, 13-38. NEURATH,A.R., SETO,B. & STruCK,N. (1989), Antibodies to synthetic peptides from the pre-Sl region of the hepatitis B virus (HBV) envelope (env) protein are virus-neutralizing and protective. Vaccine, 7, 234-236.
YVONNET,B., COURSAGET,P., LEBOULLEUX,D., BARR~S,J.L., FR1TZELL,B., SARR,G.,
CHIRON, J.P. & DIoP-MAR, I. (1987), Low-dose hepatitis B vaccine immunization in children. Lancet, I, 169. ZANETTI,A.R., TANZI,E., COLOMBO,M. & MANNUCCI,P.M. (1988), Anti-preS2 antibodies in clearance of hepatitis B virus. Lancet, II, 447-448.
