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Anti-pre-S2 antibody response in subjects vaccinated against hepatitis B and in naturally immunized subjects
(~) INSTITUT PASTEUR/ELsEVIER
Res. Viro/.
Paris 1991
l.~. l., . . . .L 'HL"f,~,
363-371
Anti-pro-S2 antibody response in subjects vaccinated against hepatitis B and in naturally immunized subjects D. Lavanchy it) (*), B. Fritzell (2), B. Geudelin (3), R. Peitrequin O) and P.C. Frei in) (/) Division d'Immunologie et d'Allergie, Centre Hospitalier Universitaire Vaudois, CH-IOl l Lausanne (Switzerland), c2j Pasteur Vaccins, 3, Avenue Pasteur, F-92430 Marnes-la-Coquette (France), and (~) Sanofi Pharma, 2, Jurastrasse, CH-4142 M~nchenstein (Switzerland)
SUMMARY Serum samples from individuals immunized with a pepsinized or non-pepsinized vaccine and from patients who had recovered from acute hepatitis B or who developed a chronic form of the disease, were analysed for the presence of antibody against the pro-S2 epitope of the hepatitis B virus. Anti-pre-S2 antibody was absent in all but one individual immunized with the pepsinized vaccine. Thirty-eight percent of the subjects who responded by anti-HBs production to the non-pepsinized preparation showed anti-pre-S2 antibody o n e year after complete vaccination. Among subjects who did not produce anti-HBs after immunization with this vaccine, I single individual produced anti-pro-S2 antibody. Anti-proS2 antibody was detectable after one year in 38 % of the patients who recovered from acute hepatitis B, but in none of those with chronic hepatitis B. The kinetics of anti-pre-S2 antibody response to a booster injaction was also analysed 1 month and 1 year after the 3rd injection and 1 month after the 4th injection of the non-pepsinized vaccine.
Key-words: HBV, Hepatitis B, Pre-S2 epitope; Pepsinized and non-pepsinized vacqir~es, Anti-pre-S2 antibodies.
INTRODUCTION The hepatitis B virus (HBV) envelope gone codes for the three major proteins, whicl~:constitute the hepatitis B envelope. One of them is the " m i d d l e " protein (p33, gp36), encoded by the pro-S2 and S regions of the envelope gene (Dejean et al., 1983; Heermann et al., 1984; Neurath et al., 1985a; Stibbe and Gerlich, 1983; Wong et al., 1985), corresponding to the "small" protein with 55 additional amino acids (120-174).
Submitted April 18, 1991, accepted August 1, 1991. (*) Correspondingauthor.
The "middle", or pro-S2 protein, is present at a concentration of 5-10 °70 on the surface of the complete virion, on the 22-nm and on the filamentous HBsAg particles in infectious scca, or on particles synthesized by recombinant vectors (Michel et al., 1984; Hadziyannis et al., 1987; Hess et al., 1987a; Hess et al., 1987b; Molnar-Kimber et al., 1988). The "middle" protein contains the domain binding specifically to polymerized human albumin (Ishihara et al., 1987; Machida et al., 1984; Michel et al., 1984;
364
D. LA V A N C H Y E T AL.
Okamato et aL, 1985; Persing et al., 1985; Pontisso et al., 1986; Takahashi et al., 1986) and was shown to be immunogenic in chimpanzee, mouse and man (Heermann et al., 1987; Milich et aL, 1985a; Neurath et al., 1984, 1986; Vento et al., 1987). It may therefore play a crucial role in determining the hepatotropism o f the virus, by mediating its attachment to the hepatccyte via the receptor for polymerized h u m a n serum albumin (Imai et al., 1979; Ishihara et al., 1987; Machida et aL, 1984). It has been demonstrated by kinetic studies (Heermann et al., 1987; Imai et al., 1979; Milich et aL, 1985a; Vento et al., 1987) that the development of antibody directed against the pre-S2 epitope of the virion may play an important role in virus neutralization and clearance (Alberti et al., 1986; Budkowska et al., 1985; Milich et al., 1986) and it was shown to be protective in chimpanzees (Itoh et al., 1986). It may also be of importance in the design of future vaccines (Budkowska et al., 1985; Coursaget et aL, 1985), to be produced in order to better immunize individuals unresponsive to the hepatitis B vaccines containing the HBV surface antigen ( " m a j o r " protein) only. Commercial vaccines of plasma origin either contained some pre-S2 (no treatment by pepsine in the course o f the production procedure) or were devoid of pre-S2 foP.owing a pepsinization step introduced as an additional inactivation measure for viral particles. In this study, we investigated serum samples from individuals immunized by a vaccine either devoid of (pepsinized) or containing (nonpepsinized) pre-S2, as well as samples from subjects after recovery from acute hepatitis B or with chrome hepatitis B, for the presence of antibody to the pre-S2 epitope of the HBV surface antigen (Machida et aL, 1984; Tron et aL, 1989).
BSA CAH CPH EIA HBV
= = = = =
bovine serum albumin. c h r o n i c a c t i v e hepatitis. c h r o n i c p e r s i s t e n t hepatitis. enzyme immunoassay. h e p a t i t i s B virus.
MATERIALS
AND
METHODS
Subjects All subjects were screened for conventional HBV markers (HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe) prior to entering the study.
Vaccine recipients A total of 213 subjects were divided into 4 groups: 1) 27 individuals immunized with a pepsinized vaccine preparation (HB-Vax MSD, 3 injections at 0, 1 and 6 months with 1.0 ml i.m. in the deltoid region) all being responders to HBsAg, with a geometric mean anti-HBs antibody titre of 229 IU/1 (+ SD = 42-1240 IU/I); 2) 17 subjects similarly vaccinated, but nonresponders (anti-HBs-negative at the end of the vaccination schedule and at the time of the study); 3) 130 subjects immunized with a non-pepsinized vaccine preparation (Hevac B Pasteur), according to the schedule established by the manufacturer, i.e. 3 i.m. injections (deltoid) at 0, 1 and 2 months, followed by a booster at 1 year, all responders to HBsAg with a mean titre of anti-HBs of 457 IU/I (+ SD 28-7569 IU/I), measured one year after th~ end of the complete vaccination schedule; 4) 39 subjects similarly vaccinated but non.~j, . . . . . . . . . . . ,, ,,,, not produce ,mL~-~o~n--' up_ : am~t,odies at the end of the vaccination schedule and remained negative one year later; in all the vaccinees, the anti-pre-S2 antibody measurement was performed one year after complete vaccination, if not indicated otherwise.
Naturally acquired immunity Twenty-one individuals who recovered from acute hepatitis B were included in the study. All were clearly anti-HBs-positive (mean 430 IU/I___SD 852181 IU/I) and anti-HBc-positive, appeared in good health and had normal values of serum liver enzymes.
i.m. OD OPD PBS S/N
= = = = ---
intramusc;dar. o p t i c a l density. orth~phenylene diamine. p h o s p h a t e - b u f f e r e d saline_ s a m p l e / d e g a f i v e c o n t r o l s ratio.
ANTI-PRE-S2 ANTIBOD Y RESPONSE
Chronic hepatitis B This group consisted of 52 patients: 26 were positive for HBsAg, HBeAg and anti-HBc, and diagnosed as chronic active hepatitis (CAH), 22 were positive for HBsAg, anti-HBe and anti-HBc and diagnosed as chronic persistent hepatitis (CPH); 3 other cases of CPH were positive for HBsAg, antiHBc and both HBe/anti-HBe. The last case had CPH and was positive for HBsAg and anti-HBc, but negative for both HBe and anti-HBe markers.
~
zyme immunoassay (EIA) from Hoffmann-La Roche, Switzerland).
Anti-pre-S2 EIA The principle of the assay used for the determination of anti-pre-S2 antibody was a sandwich enzyme immunoassay (EIA), using a synthetic pre-S2 peptide as coating material.
Coating Antigen A synthetic pre-S2 peptide (amino acid: 120-150), MW 3,450 daltons, subtype ayw was used. Amino acid 150 was a lysine instead of the naturally occurring tyrosine.
Buffer solutions Solution 1 was made by adding KH2PO 4 2.72 g, K2HPO4 18.26 g, NaC1 90.0 g and Tween-20 1.0 ml to H20 ad 1,000 ml. Solution 2 was made by adding 10 ml of buffer I to 0.04 ml of Tween-20, 1 g of bovine serum albumin (BSA) fraction V and H20 at 100 ml. Solution1 3 was phosphate-buffered saline (PBS) with 0.1 o70of Tween-20. Solution 4 was BSA 1 g diluted in 100 ml of bidistilled H20 and filtered through a 0.2-~tm membrane (Nalgene).
Conjugate Peroxidase conjugated monoclonal anti-human lgG (Janssen, MH 16-01-ME4) was diluted 1/60,000 in buffer 2.
Substrate Seventy mg of O-phenylene diamine (OPD) from Sigma (P3888) was diluted immediately before use in a buffer containing Na 3 citrate.2 H20 11.67 g, citric acid.1 H20 2.168 g, H20 2 30 070 1.0 ml and H20 ad 1,000 ml.
Methods The hepatitis B markers were determined by using commercially available EIA techniques (antiHBs, anti-HBc IgM and anti-HAV IgM respectively by " A u s a b " , "Corzyme-M" and " H A V A B - M " from Abbott Laboratories Chicago; HBsAg, antiHBc, HBeAg and anti-HBe by the corresponding en-
Ninety-six-well microtitre plates (Nunc) were coated with 200 ~tl of a solution containing 250 ng/ml of pre-S2 peptide diluted in bidistilled water (pH 9.6), incubated for 75 rain at 37°C, emptied and subsequently washed (3 x ) in solution 3. At this stage, the plates can be kept for one week. Immediately prior to use, a postcoating step was performed by incubation of the plates in 250 i~1 of buffer 4, for 90 rain at room temperature, followed by 3 washings in buffer solution 3. EIA Solution 1 (100 ~1) was dispensed into each well, followed by the addition of 100 ~tl of undiluted serum test sample or negative serum control samples, and incubated for 90 min at 37°C. After extensive washing (3 x ) in solution 3,200 ~d/well of conjugate were added for 90 rain at 37°C and replaced, after another extensive washing step (3 x ), by 200 IA of the chromogenic substrate. After 30-min incubation, the reaction was stopped by the addition of 50 I~! of a 4 N sulphuric acid solution. Reading was performed at 492 nm. A sample to negative control ratio of > 2.1 (mean of 5 negative serum control samples) was considered pos.~t.ive; if the mean negative control OD was found below 0.030, the cutoff was determined as the negative control plus 0.030. The results were expressed in sample/negative controls ratio (S/N).
Inhibition assay The specificity of the assay was confirmed by competitive inhibition with the synthetic pre-S2 peptide, as follows: serum samples found positive for anti-pre-S2 antibody by the previous assay were preincubated for 30 min at room temperature with 2500 ng of the synthetic pre-S2 peptide dissolved in H20 pH 9.6 and were subsequently tested in the EIA as mentioned above; an inhibition greater than 50 070 of the reaction was considered positive, and therefore specific for pre-S2.
D. L A V A N C H Y E T A L .
366 RESULTS
Table I shows that none of the 27 subjects who produced anti-HBs antibody u p o n immunization with the pepsinized vaccine preparation had antibody against pre-S2 one year after the first injection. The 17 subjects immunized with this vaccine, but who did not produce anti-HBs antibody, did not produce antibody against pre-S2 either, with one notable exception. This case was also negative for all other HBV markers including anti-HBc; the subject belonged to a risk group with high potential exposure to HBV. Inhibition by the pre-S2 peptide was 74 070. In the group of the 130 anti-HBs responders to immunization with the non-pepsinized vaccine, 50 ( = 38 070)were found positive for antipre-S2 antibody. A m o n g the 39 subjects immunized with this vaccine, but who did not produce anti-HBs antibo,~5, one was found positive for anti-pre-S2 antibody in spite of undetectable anti-HBs and anti-HBc antibodies. This person also belonged to an exposed risk group. Inhibition by the pre-S2 peptide was 95 °70. All others were negative for hepatitis B markers, including detectable anti-HBs antibody, at the end of the vaccination and one year after, i.e. at the time of this study.
The presence of anti-pre-S2 antibody was found in 8 out of the 21 subjects (38 °70) who recovered from clinically and serologically documented acute hepatitis B. The measure was performed 12 months after serum liver enzymes had returned to normal values. Anti-pre-S2 antibody was found in none of the 52 patients with chronic hepatitis B tested. Table II shows the anti-HBs and anti-pre-S2 levels measured one m o n t h and one year after the 3rd and one m o n t h after the 4th injection in recipients of the non-pepsinized vaccine. A significant decline in both anti-HBs and anti-pre$2 antibody levels was seen one year after the 3rd injection as compared to one m o n t h after. The 4th booster injection induced a significant rise in both anti-HBs and anti-pre-S2 titres. However, the booster effect was by far more pronounced on the anti-HBs than on the antipre-S2 titres. Figures 1 and 2 show the respective kinetics of anti-HBs and anti-pre-S2 antibody titres measured in 12 subjects individually followed one m o n t h and one year after the 3rd and one m o n t h after the 4th injection.
Table I. Comparison of anti-HBs and anti-pre-S2 antibody response in subjects
vaccinated against hepatitis B and in naturally immunized subjects.
Subjects Negative control Pepsinized vaccine: anti-HBs responders anti-HBs non-responders Non-pepsinized vaccine: anti-HBs responders anti-HBs non-responders Acute hepatitis B
No. subjects Anti-HBs : Anti-pre-S2 : tested mean IU/1_+ SD S/N +_SD
T-test
Anti-pre-S2 positive subjects : total number 070 of pos S/N_+ SD positive
18
0
0.8 ± 0.9
0
27 17
229 (42-1240) 0
0.5 _+0.7 0.7 _+1.0
ns
0 1 (*)
4.5
6 070
130 39 21
457 (28-7569) 0 430 (85-2181)
3.3 +_5.3 0.3 _+0.5 3.5_+4.2
p < 0.001
50 1 (*) 8
8.9_+6.2 2.9 7.5 _+4.2
38 07o 3 070 38 °70
52
0
0.8 +_1.0
P < 0.001 Chronic hepatitis B
0
(*) Both subjects belonged to risk groups, were negative for all HBV markers and showed clear inhibition in the inhibition assay with the pre-S2 peptide (74 and 95 % inhibition, respectively). OD = optical density at 492 nm; SD = standard deviation; ns = not significant. S / N = sample/negative control ratio.
ANTI-PRE-S2 A N T I B O D Y RESPONSE Table
367
If. Anti-HBs and anti-pre-S2 antibody response measured after vaccination w i t h a n o n - p e p s i n i z e d h e p a t i t i s B vaccine. A 1 month after 3rd i n j e c t i o n
No. subjects tested Anti-HBs mean (IU/1) +SD IU/I _ Anti-pre-S2 S/N+SD No. subjects anti-pre-S2 + 070 o f t o t a l p o s i t i v e S / N o f a n t i - p r e - S 2 p o s _+ S D
32 244 I U / I 23-2578 2.1___3.5 10 31 070 6.2 _+4 . 9
T-test A/B -P < 0.04 ns --~
B 1 year after 3rd i n j e c t i o n 30 75 I U / I 11-529 0.9+2.2 5 17 % 5.1 + 4.0
T-test B/C -P < 9001 p < 0.001 -~ --
C 1 month after 4th injection 59 788 I U / I 43-33941 5.1+6.7 34 58 °70 8.5 + 7.0
T-test A/C
p < 0.001 p < 0.0127
OD = optical density at 492 nm ; SD = standard deviation; ns = not significant. S/N = sample/mean negative control ratio.
IU/I 1000000
100000
10000 | mn
1000
100
10 1 month after 1 year after vaccination
1 month alter booster
Fig. 1. Follow-up o f anti-HBs antibody titres in 12 individuals immunized with the non-pepsinized vaccine. The titres were meast, red 1 month and 1 year after the complete vaccination, as well as 1 month after the booster injection given 5 years after the first injection.
368
D. L A V A N C H Y E T A L .
OD 1
|
.e Q, e-
,< .01
.001 1 month after
1 year after
vaccination
1 month after booster
. . . . .• Follow-up of anti-proS2 ~u...,.:o"*m-'~-" pig ;- the same ,.'~ .lul.iuUa.~ ~Luu.~uin figure I. using the u. same serum samples. Results are expressed in OD.
DISCUSSION The results of the present study confirm the hypothesis that the development of anti-pre-S2 antibody is associated with recovery from acute hepatitis B infection, since this antibody ~,as found in 38 07o of the cases who recovered and in none of those with CAH (chronic active hepatitis). Its absence in 62 °7o of the subjects studied after recovery from acute hepatitis B is probably due to the timing of the study (one year later) and to the rapid clearance of the anti-pre-S2 antibody, as shown in table II and figures 1 and 2, and as suggested by others (Petit et al., 1986).
This rapid decline may also explain why some contradictory results have been reported (Hellstr6m and Sylvan, 1986). These data also confirmed that the pepsinization of HBsAg particles destroyed the pre-S2 epitope (Neurath et al., 1985a) and that nonpepsinized particles possess a pre-S2 epitope sufficiently intact (Petit et ai., 1986) to elicit a specific immune response in the same proportion of the subjects (38 %) as does natural immunization. This indicates that better protection can be expected from preparations including the pre-S2 epitope. The absence of anti-pre-S2 in the remaining 62 °7o of subjects immunized with the
ANTI-PRE-S2 ANTIBODY RESPONSE
non-pepsinized vaccine may be explained in the same way as for subjects recovering from acute hepatitis B. The seroconversion rate of 3 1 % obtained by us one month after the 3rd injection (table II) is lower than that observed by Tron et al. (1989). A possible explanation for this finding is that Tron et al. used a recombinant vaccine containing a higher proportion of pre-S2 protein than the plasma-derived vaccine used here. We showed that most low- and non-producers of anti-HBs were unable to mount an antibody response against pre-S2 (table II). The almost complete absence (but two exceptions) of antipre-S2 formation in anti-HBs non-responders to both the pepsinized and the non-pepsinized vaccine preparations may indicate that a genetic linkage of the antibody responses to HBsAg and pre-S2 may exist in man, unlike what has been observed in the mouse. Thus, non-responsiveness to HBsAg in man may not be circumvented by an adequate immunization with pre-S2 in order to obtain a protective immune response against HBV. Though it has been shown that the immune response to both antigens is not genetically linked in most strains of mice so far investigated (Milich et a!., 1985a, 1985b; Neurath e t a ! , 1985b), an H-2f-haplotype mouse strain was described as non-responsive to both S and pre$2 antigens (Milich et al., 1985a). Since, in our study, two subjects with high risk of exposure nonetheless had anti-pre-S2 antibody (confirmed by inhibition assays) in the absence of anti-HBs and anti-HBc antibodies, any definite conclusion on the genetic linkage of the immune response to S and pre-S epitopes in man cannot yet be drawn. Further studies must be undertaken in order to establish whether such a genetic linkage exists in m-n. It should also be considered that the test used here for anti-pre-S2 antibody detection may have some limits. With an non-glycosylated synthetic peptide as antigen, the possibility exists that some antibody specificities are not detected, and therefore that the natural and induced immune responses might be underestimated. This test is nevertheless useful for estimating the immune response against hepatitis B vaccines containing
369
the pre-S2 epitope. Its use as diagnostic procedure to differentiate acute self-limiting from chronic hepatitis B at an early stage of the acute infection remains to be established. Further studies are needed to better delineate the significance of the anti-pre-S2 antibody response and its predicting diagnostic value as compared to that of anti-HBs. The wider use of vaccines containing the pre-S2 epitope (Itoh et al., 1986; Kniskern et al., 1988) will probably be helpful for future studies.
La r~ponse anticorps anti-pr~-S2 chez des sujets vaccines contre I'h~pafite B et des sujets ~naturellement>> immunis~s
L'anticorps sp~cifique de l'~pitope pr6-S2 du virus de l'h~patite Best mesur~ dans des ~chantillons de s~rum pr~lev~s d'une part chez des sujets immunis~s, soit avec un vaccin d'origine plasmatique pepsinis~, soit avec un vaccin d'origine plasmatique non pepsinis~, et d'autre part chez des sujets apr~s gu~rison d'une h6patite virale aigu~ B ou porteurs d'une h6patite chronique B. L'anticorps anti-pr6-S2 n'6tait d6tectable chez aucun des sujets immunis~s avec le vaccin pepsinis~, une exception pr6s. Les 38 °70 des sujets ayant r~ponau ~ rinjection du vaccin non pepsinis~ par la production d'anticorps anti-HBs poss6daient l'anticorps anti-pr~-S2 une ann6e apr~s la fin du protocole de vaccination. Chez les sujets qui n'ont pas produit d'anticorps anti-HBs apr~s immunisation avec ce vaccin, un seul a produit une concentration d6tectab!e d'anticorps anti-pr6-S2. Cet anticorps a 6t~ trouv6, apr~s une ann6e, chez 38 % des sujets ayant gu6ri d'une h~patite aigu~ B, mais chez aucun de ceux souffrant d'h~patite chronique B. On a ~galement analys6 la cin~tique de la r6ponse anti-pr~$2 apr/:s injection de rappel des vaccins, un mois et une annie apr~s la troisi~me injection et un mois apr~s la quatri~me injection avec le vaccin non pepsinis~. Mots-cl6s: HBV, H6patite B, Epitope pr6-S2; Vaccins pepsinis6s ou non, Anticorps anti-pr6-S2.
370
D. L A V A N C H Y E T A L .
References Alberti, A., Pontisso, P. & Fraiese, A. (1986), Pre-S2 antibodies in hepatitis B. Lancet, II, 1457. Budkowska, A., Briantais, M.J., Dubreuil, P., Capel, F., Grangeot-Keros, L. & Pillot, J. (1985), Detection of antibodies directed against the pre-S gene product of hepatitis B virus: relationship between anti-pre-S response and recovery. Ann. Inst. Pasteur/Immunol., 136D, 57-65. Coursaget, P., Barres, J.L., Chiron, J.P. & Adamowicz, P. (1985), Hepatitis B vaccine with and without polymerized albumin receptors. Lancet, I, 1153-1154. Dejean, A., Br6chot, C., Tiollais, P. & Wain-Hohson, S. (1983), Characterization of integrated hepatitis B viral DNA cloned from a human hepatoma and the hepatoma-derived cell line PLC/PRF/5. Proc. nat. Acad. Sci. (Wash.), 80, 2505-2509. Hadziyannis, R.G., Papioannou, C., Anastassakos, C., Wong, D., Sninsky, J. & Shafritz, D. (1987), Expression of pre-S gene-encoded proteins in liver and serum during hepatitis B virus infection in comparison to other markers of active virus replication. J. Hepatol., 5, 253-259. Heermann, K.H., Goldmarm, U., Schwartz, W., Seyffarth, T., Baumgarten, H. & Geflich, W.H. (1984), Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol., 52, 396-402. Heermann, K.H., Kruse, F., Seifer, M. & Gerlich, W.H. (1987), lmmunogenicity of the gene S and pre-S domain.~ in hepatitis B virions and HBsAg f'flaments. Intervirology, 28, 14-25. Hellstrtm, IJ. & Sylvan, S. (1986), Should pre-S coded peptides be used in hepatitis B vaccines? Lancet, I, 389-390. Hess, G., Gerken, G., Weber, C., Knolle, J., Dormeyer, H.H., Arnold, W., Manns, M. & Meyer zum Buschenfelde, K.H. (1987a), Detection of hepatitis B virus markers in sera of asymptomatic hepatitis B surface antigen carriers with special emphasis to pre-Sencoded proteins. Digestion, 38, 90-95. Hess, G., Gerlich, W.H., Gerken, G., Manns, M. & Meyer zum Buschenfelde, K.H. (1987b), Relationship of pre-S encoded antigens in liver and clinical manifestations of chronic hepatitis B infection. Liver, 7, 245-250. Imai, M., Yanase, Y., Nojiri,T., Miyakawa, Y. & Mayumi, M. (1979),A receptor for polymerized human and chimpanzee albumins on hepatitis B virus particles cooccurring with HBeAg. Gastroenterology, 76, 242-247. Ishihara, K., Waters, J.A., Pignatelli, M. & Thomas, H.C. (1987), Characterization of the polymerized and monomeric human serum albumin binding sites on hepatitis B surface antigen. J. meal. Virol., 21, 89-95. Itoh, Y., Takai, E., Ohnuma, H., Kitajima, K., Tsuda, E, Machida, A., Mishiro, S., Nakamura, T., Miyakawa, Y. & Mayumi, M. (1986),A syntheticpeptidevaccine involving the product of the pre-S(2) region of hepatitis B virus D N A : protectiveefficacy in chimpanzees. Proc. nat. Acad. Sci. (Wash.), 83, 9174-~ 78. Kniskeru, P.J., Hagopian, A., Burke, P., Dunn, N., Lmini, E.A., Miller, W.J., Yamazaki, S. & Ellis, R.W. (1988), A candidate vaccine for hepatitis B contain-
ing the complete viral surface protein. Hepatology, 8, 82-87. Machida, A., Kishimoto, S., Ohnuma, H., Baba, K., Itoh, Y., Miyamoto, H., Funabu, G., Oda, K., Usuda, S., Togami, S., Nakamura, T., Miyakawa, Y. & Mayumi, M. (1984), A polypeptide containing 55 amino acid residues coded by the pre-S region of hepatitis B virus deoxyribonucleic acid bears the receptor for polymerized human as well as chimpanzee albumins. Gastroenterology, 86, 910-918. Michel, M.L., Pontisso, P., Sobczak, E., Malpiece, Y., Streeck, R.E. & Tiollais, P. (1984), Synthesis in animal cells of hepatitis B surface particles carrying a receptor for polymerized human serum albumin. Proc. nat. Acad. Sci. (Wash.), 81, 7708-7712. Milich, D.R., McLachlan, A., Chisari, F.V., Kent, S.B.H. & Thornton, G.B. (1986), immane re~poh~e to the pre-S(l) region of the hepatitis B surface antigen (HBs-Ag): a pre-S(l)-specific T-cell response can bypass nonresponsiveness to the pre-S(2) and S regions of HBsAg. J. ImmunoL, 137, 315-322. Milich, D.R., Thornton, G.B., Neurath, A.R., Kent, S.BL, Michel, M.L., Tioliais, P. & Chisari, F.V. (1985a), Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen. Science, 228, 1195-1199. Milich, D.R., McNamara, K.K., McLachlan, A., Thornton, G.B. & Chisari, F.V. (1985b), Distinct H-2-1inked regulation of T-cell responses to the pre-S and S regions of the same hepatitis B surface antigen polypeptide allows circumvention of nonresponsiveness to the S region. Proc. nat. Acad. Sci. (Wash.), 82, 8168-8172. Molnar-Kimber, K.L., Jarocki-Witeck, V., Dheer, S.K., Vernon, S.K., Conley, A.J., Davis, A.R. & Hung, P.P. (1988), Distinctive properties of the hepatitis B virus envelope proteins. J. Virol., 62, 407-416. Neurath, A.R., Kent, S.B.H. & Strick, N. (1984), Location and chemical synthesis of a pre-S-gene-coded immunodominant epitope of hepatitis B virus. Science.. 224, 392-395. Neurath, A.R., Kent, S.B.H. & Strick, N. (1986), Hepatitis B vaccine trial study groups. 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., Strick, N., Taylor, P. & Stevens, C.E. (1985a), Hepatitis B virus contains pre-S gene-encoded domains. Nature (Lond.), 315, 154-156. Neurath, A.R., Kent, S.B.H., Strick, N., Stark, D. & Sproul, P. (1985b), Genetic restriction of immune responsiveness to synthetic peptides corresponding to sequences in the pre-S region of the hepatitis B virus (HBV) envelope gene. J. reed. Virol., 17, i19-125. Okamoto, H., Imai, M., Usuda, S., Tanaka, E., Tachibana, K., Mishiro, S., Machida, A., Nakamura, T., Miyakawa, Y. & Mayumi, M. (1985), Hemagglutination assay of polypeptide coded by the pre-S region of hepatitis B virus DNA with monoclonal antibody: correlation of pre-S polypeptide with the receptor for polymerized human serum albumin in serums containing hepatitis B antigens. J. Immunol., 134, 1212-1216. Persing, D.H., Varmus, H.E. & Ganem, D. (1985), A frameshift mutation in the pre-S region of the hepa-
ANTI-PRE-S2 ANTIBOD ~ RESPONSE
titis B virus genome allows production of surface antigen particles but eliminates binding to polymerized albnm~rt. Proc. nat. Acad. Sci. (Wash.), 82, 3440-3444. Petit, M.A., Maillard, P., Capel, F. & Pillot, J. (1986), lmmunochemical structure of the hepatitis B surface antigen vaccine. - - II. Analysis of antibody responses in human sera against the envelope proteins. MoL Immuno[, 23, 511-523. Pontisso, P., Schiavon, E., Fraiese, A., Pornaro, E., Realdi, G. & Alberti, A. (1986), Antibody to the hepatitis B virus receptor for polymerized albumin in acute infection and in hepatitis B vaccine cecepients. J. Hepatol., 3, 393-398. Stibbe, W. & Gerlich, W.H. (1983), Structural relationship between minor and major proteins of hepatitis B surface antigen. J. ViroL, 46, 626-628. Takahashi, K., Kishimoto, S., Ohnuma, H., Machida, A., Takai, E., Tsuda, F., Miyamoto, H., Tanaka, T., Matsushita, K., Oda, K., Miyakawa, Y. & Mayumi, M. (1986), Polypeptides coded for by the region pre-S
371
and gene S of hepatitis B virus DNA with the receptor for polymerized human serum albumin: expression on hepatitis B particles produced in the HBeAg or anti-HBe phase of hepatitis B virus infection. J. Immunol., 136, 3467-3472. Tron, F., Degos, F., Br~chot, C., Courouce, A.M., Goudeau, A., Made, F.N., Adamowicz, P., Saliou, P., Laplanche, A., Benhamou, J.P. & Girard, M. (1989), Randomized dose range study of a recombinant hepatitis B vaccine produced in mammalian cells and containing the S and preS2 sequences. J. infect. D/s., 160, 199-204. Vento, S., Rondanelli, E.G., Ranieri, S., O'Brien, C.J., Williams, R. & Eddleston, A.L.W.F. (1987), Prospective study of cellular immunity to hepatitis-B-virus antigens from the early incubation phase of acute hepatitis B. Lancet, II, 119-122. Wong, D.T., Nath, N. & Sinsky, J.J. (1985), Identification of hepatitis B virus polypeptides encoded by the entire pre-S open reading frame. J. ViroL, 55, 223-231.
Res. Viro/.
Paris 1991
l.~. l., . . . .L 'HL"f,~,
363-371
Anti-pro-S2 antibody response in subjects vaccinated against hepatitis B and in naturally immunized subjects D. Lavanchy it) (*), B. Fritzell (2), B. Geudelin (3), R. Peitrequin O) and P.C. Frei in) (/) Division d'Immunologie et d'Allergie, Centre Hospitalier Universitaire Vaudois, CH-IOl l Lausanne (Switzerland), c2j Pasteur Vaccins, 3, Avenue Pasteur, F-92430 Marnes-la-Coquette (France), and (~) Sanofi Pharma, 2, Jurastrasse, CH-4142 M~nchenstein (Switzerland)
SUMMARY Serum samples from individuals immunized with a pepsinized or non-pepsinized vaccine and from patients who had recovered from acute hepatitis B or who developed a chronic form of the disease, were analysed for the presence of antibody against the pro-S2 epitope of the hepatitis B virus. Anti-pre-S2 antibody was absent in all but one individual immunized with the pepsinized vaccine. Thirty-eight percent of the subjects who responded by anti-HBs production to the non-pepsinized preparation showed anti-pre-S2 antibody o n e year after complete vaccination. Among subjects who did not produce anti-HBs after immunization with this vaccine, I single individual produced anti-pro-S2 antibody. Anti-proS2 antibody was detectable after one year in 38 % of the patients who recovered from acute hepatitis B, but in none of those with chronic hepatitis B. The kinetics of anti-pre-S2 antibody response to a booster injaction was also analysed 1 month and 1 year after the 3rd injection and 1 month after the 4th injection of the non-pepsinized vaccine.
Key-words: HBV, Hepatitis B, Pre-S2 epitope; Pepsinized and non-pepsinized vacqir~es, Anti-pre-S2 antibodies.
INTRODUCTION The hepatitis B virus (HBV) envelope gone codes for the three major proteins, whicl~:constitute the hepatitis B envelope. One of them is the " m i d d l e " protein (p33, gp36), encoded by the pro-S2 and S regions of the envelope gene (Dejean et al., 1983; Heermann et al., 1984; Neurath et al., 1985a; Stibbe and Gerlich, 1983; Wong et al., 1985), corresponding to the "small" protein with 55 additional amino acids (120-174).
Submitted April 18, 1991, accepted August 1, 1991. (*) Correspondingauthor.
The "middle", or pro-S2 protein, is present at a concentration of 5-10 °70 on the surface of the complete virion, on the 22-nm and on the filamentous HBsAg particles in infectious scca, or on particles synthesized by recombinant vectors (Michel et al., 1984; Hadziyannis et al., 1987; Hess et al., 1987a; Hess et al., 1987b; Molnar-Kimber et al., 1988). The "middle" protein contains the domain binding specifically to polymerized human albumin (Ishihara et al., 1987; Machida et al., 1984; Michel et al., 1984;
364
D. LA V A N C H Y E T AL.
Okamato et aL, 1985; Persing et al., 1985; Pontisso et al., 1986; Takahashi et al., 1986) and was shown to be immunogenic in chimpanzee, mouse and man (Heermann et al., 1987; Milich et aL, 1985a; Neurath et al., 1984, 1986; Vento et al., 1987). It may therefore play a crucial role in determining the hepatotropism o f the virus, by mediating its attachment to the hepatccyte via the receptor for polymerized h u m a n serum albumin (Imai et al., 1979; Ishihara et al., 1987; Machida et aL, 1984). It has been demonstrated by kinetic studies (Heermann et al., 1987; Imai et al., 1979; Milich et aL, 1985a; Vento et al., 1987) that the development of antibody directed against the pre-S2 epitope of the virion may play an important role in virus neutralization and clearance (Alberti et al., 1986; Budkowska et al., 1985; Milich et al., 1986) and it was shown to be protective in chimpanzees (Itoh et al., 1986). It may also be of importance in the design of future vaccines (Budkowska et al., 1985; Coursaget et aL, 1985), to be produced in order to better immunize individuals unresponsive to the hepatitis B vaccines containing the HBV surface antigen ( " m a j o r " protein) only. Commercial vaccines of plasma origin either contained some pre-S2 (no treatment by pepsine in the course o f the production procedure) or were devoid of pre-S2 foP.owing a pepsinization step introduced as an additional inactivation measure for viral particles. In this study, we investigated serum samples from individuals immunized by a vaccine either devoid of (pepsinized) or containing (nonpepsinized) pre-S2, as well as samples from subjects after recovery from acute hepatitis B or with chrome hepatitis B, for the presence of antibody to the pre-S2 epitope of the HBV surface antigen (Machida et aL, 1984; Tron et aL, 1989).
BSA CAH CPH EIA HBV
= = = = =
bovine serum albumin. c h r o n i c a c t i v e hepatitis. c h r o n i c p e r s i s t e n t hepatitis. enzyme immunoassay. h e p a t i t i s B virus.
MATERIALS
AND
METHODS
Subjects All subjects were screened for conventional HBV markers (HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe) prior to entering the study.
Vaccine recipients A total of 213 subjects were divided into 4 groups: 1) 27 individuals immunized with a pepsinized vaccine preparation (HB-Vax MSD, 3 injections at 0, 1 and 6 months with 1.0 ml i.m. in the deltoid region) all being responders to HBsAg, with a geometric mean anti-HBs antibody titre of 229 IU/1 (+ SD = 42-1240 IU/I); 2) 17 subjects similarly vaccinated, but nonresponders (anti-HBs-negative at the end of the vaccination schedule and at the time of the study); 3) 130 subjects immunized with a non-pepsinized vaccine preparation (Hevac B Pasteur), according to the schedule established by the manufacturer, i.e. 3 i.m. injections (deltoid) at 0, 1 and 2 months, followed by a booster at 1 year, all responders to HBsAg with a mean titre of anti-HBs of 457 IU/I (+ SD 28-7569 IU/I), measured one year after th~ end of the complete vaccination schedule; 4) 39 subjects similarly vaccinated but non.~j, . . . . . . . . . . . ,, ,,,, not produce ,mL~-~o~n--' up_ : am~t,odies at the end of the vaccination schedule and remained negative one year later; in all the vaccinees, the anti-pre-S2 antibody measurement was performed one year after complete vaccination, if not indicated otherwise.
Naturally acquired immunity Twenty-one individuals who recovered from acute hepatitis B were included in the study. All were clearly anti-HBs-positive (mean 430 IU/I___SD 852181 IU/I) and anti-HBc-positive, appeared in good health and had normal values of serum liver enzymes.
i.m. OD OPD PBS S/N
= = = = ---
intramusc;dar. o p t i c a l density. orth~phenylene diamine. p h o s p h a t e - b u f f e r e d saline_ s a m p l e / d e g a f i v e c o n t r o l s ratio.
ANTI-PRE-S2 ANTIBOD Y RESPONSE
Chronic hepatitis B This group consisted of 52 patients: 26 were positive for HBsAg, HBeAg and anti-HBc, and diagnosed as chronic active hepatitis (CAH), 22 were positive for HBsAg, anti-HBe and anti-HBc and diagnosed as chronic persistent hepatitis (CPH); 3 other cases of CPH were positive for HBsAg, antiHBc and both HBe/anti-HBe. The last case had CPH and was positive for HBsAg and anti-HBc, but negative for both HBe and anti-HBe markers.
~
zyme immunoassay (EIA) from Hoffmann-La Roche, Switzerland).
Anti-pre-S2 EIA The principle of the assay used for the determination of anti-pre-S2 antibody was a sandwich enzyme immunoassay (EIA), using a synthetic pre-S2 peptide as coating material.
Coating Antigen A synthetic pre-S2 peptide (amino acid: 120-150), MW 3,450 daltons, subtype ayw was used. Amino acid 150 was a lysine instead of the naturally occurring tyrosine.
Buffer solutions Solution 1 was made by adding KH2PO 4 2.72 g, K2HPO4 18.26 g, NaC1 90.0 g and Tween-20 1.0 ml to H20 ad 1,000 ml. Solution 2 was made by adding 10 ml of buffer I to 0.04 ml of Tween-20, 1 g of bovine serum albumin (BSA) fraction V and H20 at 100 ml. Solution1 3 was phosphate-buffered saline (PBS) with 0.1 o70of Tween-20. Solution 4 was BSA 1 g diluted in 100 ml of bidistilled H20 and filtered through a 0.2-~tm membrane (Nalgene).
Conjugate Peroxidase conjugated monoclonal anti-human lgG (Janssen, MH 16-01-ME4) was diluted 1/60,000 in buffer 2.
Substrate Seventy mg of O-phenylene diamine (OPD) from Sigma (P3888) was diluted immediately before use in a buffer containing Na 3 citrate.2 H20 11.67 g, citric acid.1 H20 2.168 g, H20 2 30 070 1.0 ml and H20 ad 1,000 ml.
Methods The hepatitis B markers were determined by using commercially available EIA techniques (antiHBs, anti-HBc IgM and anti-HAV IgM respectively by " A u s a b " , "Corzyme-M" and " H A V A B - M " from Abbott Laboratories Chicago; HBsAg, antiHBc, HBeAg and anti-HBe by the corresponding en-
Ninety-six-well microtitre plates (Nunc) were coated with 200 ~tl of a solution containing 250 ng/ml of pre-S2 peptide diluted in bidistilled water (pH 9.6), incubated for 75 rain at 37°C, emptied and subsequently washed (3 x ) in solution 3. At this stage, the plates can be kept for one week. Immediately prior to use, a postcoating step was performed by incubation of the plates in 250 i~1 of buffer 4, for 90 rain at room temperature, followed by 3 washings in buffer solution 3. EIA Solution 1 (100 ~1) was dispensed into each well, followed by the addition of 100 ~tl of undiluted serum test sample or negative serum control samples, and incubated for 90 min at 37°C. After extensive washing (3 x ) in solution 3,200 ~d/well of conjugate were added for 90 rain at 37°C and replaced, after another extensive washing step (3 x ), by 200 IA of the chromogenic substrate. After 30-min incubation, the reaction was stopped by the addition of 50 I~! of a 4 N sulphuric acid solution. Reading was performed at 492 nm. A sample to negative control ratio of > 2.1 (mean of 5 negative serum control samples) was considered pos.~t.ive; if the mean negative control OD was found below 0.030, the cutoff was determined as the negative control plus 0.030. The results were expressed in sample/negative controls ratio (S/N).
Inhibition assay The specificity of the assay was confirmed by competitive inhibition with the synthetic pre-S2 peptide, as follows: serum samples found positive for anti-pre-S2 antibody by the previous assay were preincubated for 30 min at room temperature with 2500 ng of the synthetic pre-S2 peptide dissolved in H20 pH 9.6 and were subsequently tested in the EIA as mentioned above; an inhibition greater than 50 070 of the reaction was considered positive, and therefore specific for pre-S2.
D. L A V A N C H Y E T A L .
366 RESULTS
Table I shows that none of the 27 subjects who produced anti-HBs antibody u p o n immunization with the pepsinized vaccine preparation had antibody against pre-S2 one year after the first injection. The 17 subjects immunized with this vaccine, but who did not produce anti-HBs antibody, did not produce antibody against pre-S2 either, with one notable exception. This case was also negative for all other HBV markers including anti-HBc; the subject belonged to a risk group with high potential exposure to HBV. Inhibition by the pre-S2 peptide was 74 070. In the group of the 130 anti-HBs responders to immunization with the non-pepsinized vaccine, 50 ( = 38 070)were found positive for antipre-S2 antibody. A m o n g the 39 subjects immunized with this vaccine, but who did not produce anti-HBs antibo,~5, one was found positive for anti-pre-S2 antibody in spite of undetectable anti-HBs and anti-HBc antibodies. This person also belonged to an exposed risk group. Inhibition by the pre-S2 peptide was 95 °70. All others were negative for hepatitis B markers, including detectable anti-HBs antibody, at the end of the vaccination and one year after, i.e. at the time of this study.
The presence of anti-pre-S2 antibody was found in 8 out of the 21 subjects (38 °70) who recovered from clinically and serologically documented acute hepatitis B. The measure was performed 12 months after serum liver enzymes had returned to normal values. Anti-pre-S2 antibody was found in none of the 52 patients with chronic hepatitis B tested. Table II shows the anti-HBs and anti-pre-S2 levels measured one m o n t h and one year after the 3rd and one m o n t h after the 4th injection in recipients of the non-pepsinized vaccine. A significant decline in both anti-HBs and anti-pre$2 antibody levels was seen one year after the 3rd injection as compared to one m o n t h after. The 4th booster injection induced a significant rise in both anti-HBs and anti-pre-S2 titres. However, the booster effect was by far more pronounced on the anti-HBs than on the antipre-S2 titres. Figures 1 and 2 show the respective kinetics of anti-HBs and anti-pre-S2 antibody titres measured in 12 subjects individually followed one m o n t h and one year after the 3rd and one m o n t h after the 4th injection.
Table I. Comparison of anti-HBs and anti-pre-S2 antibody response in subjects
vaccinated against hepatitis B and in naturally immunized subjects.
Subjects Negative control Pepsinized vaccine: anti-HBs responders anti-HBs non-responders Non-pepsinized vaccine: anti-HBs responders anti-HBs non-responders Acute hepatitis B
No. subjects Anti-HBs : Anti-pre-S2 : tested mean IU/1_+ SD S/N +_SD
T-test
Anti-pre-S2 positive subjects : total number 070 of pos S/N_+ SD positive
18
0
0.8 ± 0.9
0
27 17
229 (42-1240) 0
0.5 _+0.7 0.7 _+1.0
ns
0 1 (*)
4.5
6 070
130 39 21
457 (28-7569) 0 430 (85-2181)
3.3 +_5.3 0.3 _+0.5 3.5_+4.2
p < 0.001
50 1 (*) 8
8.9_+6.2 2.9 7.5 _+4.2
38 07o 3 070 38 °70
52
0
0.8 +_1.0
P < 0.001 Chronic hepatitis B
0
(*) Both subjects belonged to risk groups, were negative for all HBV markers and showed clear inhibition in the inhibition assay with the pre-S2 peptide (74 and 95 % inhibition, respectively). OD = optical density at 492 nm; SD = standard deviation; ns = not significant. S / N = sample/negative control ratio.
ANTI-PRE-S2 A N T I B O D Y RESPONSE Table
367
If. Anti-HBs and anti-pre-S2 antibody response measured after vaccination w i t h a n o n - p e p s i n i z e d h e p a t i t i s B vaccine. A 1 month after 3rd i n j e c t i o n
No. subjects tested Anti-HBs mean (IU/1) +SD IU/I _ Anti-pre-S2 S/N+SD No. subjects anti-pre-S2 + 070 o f t o t a l p o s i t i v e S / N o f a n t i - p r e - S 2 p o s _+ S D
32 244 I U / I 23-2578 2.1___3.5 10 31 070 6.2 _+4 . 9
T-test A/B -P < 0.04 ns --~
B 1 year after 3rd i n j e c t i o n 30 75 I U / I 11-529 0.9+2.2 5 17 % 5.1 + 4.0
T-test B/C -P < 9001 p < 0.001 -~ --
C 1 month after 4th injection 59 788 I U / I 43-33941 5.1+6.7 34 58 °70 8.5 + 7.0
T-test A/C
p < 0.001 p < 0.0127
OD = optical density at 492 nm ; SD = standard deviation; ns = not significant. S/N = sample/mean negative control ratio.
IU/I 1000000
100000
10000 | mn
1000
100
10 1 month after 1 year after vaccination
1 month alter booster
Fig. 1. Follow-up o f anti-HBs antibody titres in 12 individuals immunized with the non-pepsinized vaccine. The titres were meast, red 1 month and 1 year after the complete vaccination, as well as 1 month after the booster injection given 5 years after the first injection.
368
D. L A V A N C H Y E T A L .
OD 1
|
.e Q, e-
,< .01
.001 1 month after
1 year after
vaccination
1 month after booster
. . . . .• Follow-up of anti-proS2 ~u...,.:o"*m-'~-" pig ;- the same ,.'~ .lul.iuUa.~ ~Luu.~uin figure I. using the u. same serum samples. Results are expressed in OD.
DISCUSSION The results of the present study confirm the hypothesis that the development of anti-pre-S2 antibody is associated with recovery from acute hepatitis B infection, since this antibody ~,as found in 38 07o of the cases who recovered and in none of those with CAH (chronic active hepatitis). Its absence in 62 °7o of the subjects studied after recovery from acute hepatitis B is probably due to the timing of the study (one year later) and to the rapid clearance of the anti-pre-S2 antibody, as shown in table II and figures 1 and 2, and as suggested by others (Petit et al., 1986).
This rapid decline may also explain why some contradictory results have been reported (Hellstr6m and Sylvan, 1986). These data also confirmed that the pepsinization of HBsAg particles destroyed the pre-S2 epitope (Neurath et al., 1985a) and that nonpepsinized particles possess a pre-S2 epitope sufficiently intact (Petit et ai., 1986) to elicit a specific immune response in the same proportion of the subjects (38 %) as does natural immunization. This indicates that better protection can be expected from preparations including the pre-S2 epitope. The absence of anti-pre-S2 in the remaining 62 °7o of subjects immunized with the
ANTI-PRE-S2 ANTIBODY RESPONSE
non-pepsinized vaccine may be explained in the same way as for subjects recovering from acute hepatitis B. The seroconversion rate of 3 1 % obtained by us one month after the 3rd injection (table II) is lower than that observed by Tron et al. (1989). A possible explanation for this finding is that Tron et al. used a recombinant vaccine containing a higher proportion of pre-S2 protein than the plasma-derived vaccine used here. We showed that most low- and non-producers of anti-HBs were unable to mount an antibody response against pre-S2 (table II). The almost complete absence (but two exceptions) of antipre-S2 formation in anti-HBs non-responders to both the pepsinized and the non-pepsinized vaccine preparations may indicate that a genetic linkage of the antibody responses to HBsAg and pre-S2 may exist in man, unlike what has been observed in the mouse. Thus, non-responsiveness to HBsAg in man may not be circumvented by an adequate immunization with pre-S2 in order to obtain a protective immune response against HBV. Though it has been shown that the immune response to both antigens is not genetically linked in most strains of mice so far investigated (Milich et a!., 1985a, 1985b; Neurath e t a ! , 1985b), an H-2f-haplotype mouse strain was described as non-responsive to both S and pre$2 antigens (Milich et al., 1985a). Since, in our study, two subjects with high risk of exposure nonetheless had anti-pre-S2 antibody (confirmed by inhibition assays) in the absence of anti-HBs and anti-HBc antibodies, any definite conclusion on the genetic linkage of the immune response to S and pre-S epitopes in man cannot yet be drawn. Further studies must be undertaken in order to establish whether such a genetic linkage exists in m-n. It should also be considered that the test used here for anti-pre-S2 antibody detection may have some limits. With an non-glycosylated synthetic peptide as antigen, the possibility exists that some antibody specificities are not detected, and therefore that the natural and induced immune responses might be underestimated. This test is nevertheless useful for estimating the immune response against hepatitis B vaccines containing
369
the pre-S2 epitope. Its use as diagnostic procedure to differentiate acute self-limiting from chronic hepatitis B at an early stage of the acute infection remains to be established. Further studies are needed to better delineate the significance of the anti-pre-S2 antibody response and its predicting diagnostic value as compared to that of anti-HBs. The wider use of vaccines containing the pre-S2 epitope (Itoh et al., 1986; Kniskern et al., 1988) will probably be helpful for future studies.
La r~ponse anticorps anti-pr~-S2 chez des sujets vaccines contre I'h~pafite B et des sujets ~naturellement>> immunis~s
L'anticorps sp~cifique de l'~pitope pr6-S2 du virus de l'h~patite Best mesur~ dans des ~chantillons de s~rum pr~lev~s d'une part chez des sujets immunis~s, soit avec un vaccin d'origine plasmatique pepsinis~, soit avec un vaccin d'origine plasmatique non pepsinis~, et d'autre part chez des sujets apr~s gu~rison d'une h6patite virale aigu~ B ou porteurs d'une h6patite chronique B. L'anticorps anti-pr6-S2 n'6tait d6tectable chez aucun des sujets immunis~s avec le vaccin pepsinis~, une exception pr6s. Les 38 °70 des sujets ayant r~ponau ~ rinjection du vaccin non pepsinis~ par la production d'anticorps anti-HBs poss6daient l'anticorps anti-pr~-S2 une ann6e apr~s la fin du protocole de vaccination. Chez les sujets qui n'ont pas produit d'anticorps anti-HBs apr~s immunisation avec ce vaccin, un seul a produit une concentration d6tectab!e d'anticorps anti-pr6-S2. Cet anticorps a 6t~ trouv6, apr~s une ann6e, chez 38 % des sujets ayant gu6ri d'une h~patite aigu~ B, mais chez aucun de ceux souffrant d'h~patite chronique B. On a ~galement analys6 la cin~tique de la r6ponse anti-pr~$2 apr/:s injection de rappel des vaccins, un mois et une annie apr~s la troisi~me injection et un mois apr~s la quatri~me injection avec le vaccin non pepsinis~. Mots-cl6s: HBV, H6patite B, Epitope pr6-S2; Vaccins pepsinis6s ou non, Anticorps anti-pr6-S2.
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D. L A V A N C H Y E T A L .
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ANTI-PRE-S2 ANTIBOD ~ RESPONSE
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