Synthesis in cho cells of hepatitis B surface antigen containing the PRE-S2 region expression product

9 ELSEVIER Paris 1985

Ann. Inst. Pasleur/Virol. 1985, 136 E, 495-502

SYNTHESIS IN CHO CELLS OF HEPATITIS B SURFACE ANTIGEN CONTAINING THE PRE-S2 REGION EXPRESSION PRODUCT by M. L. Michel (1), p. Pontisso (~), E. Sobzack (s), y . Malpiece (~), R. Streeck (a), D. Milich (4), F. Chisari (~) and P. Tiollais (1)

(1) Unitd de Recombinaison et Expression G~n~tique ( I N S E R M U163, CNRS UA271), Institut Pasteur, 75724 Paris Cedex 16, (3) Istituto di Clinica Medica 2, Universitd di Padova, 35100 Padova (Italy), (8) Groupement de Gdnie G~n~tique, Institut Pasteur, 75724 Paris Cedex 1"5, (4) Scripps Clinic and Research Foundation, Department o/Basic and Clinical Research, 10666 North Torrey Pines Road, La Jolla, CA 92037 ( U S A )

I. - - Introduction. The world-wide importance of hepatitis B, the development of chronic forms and the association between hepatitis B virus (HBV) and hepatocellular carcinoma imply the need for an efficient and safe vaccine. The absence of a cell culture system capable of propagating the virus has greatly impeded the development of a x accine and has led to the production of a vaccine from the serum of HBV chronic carriers. Krugman et al. [1, 2] were the first to show that a crude inactivated extract from an HBV chronic carrier serum was capable of protecting children from hepatitis B. Later, Maupas et al. [3] reported the first positive results of clinical trials using hepatitis B surface antigen (HBsAg) particles purified from human serum. Today, several laboratories have developed plasma-derived vaccines composed of defective 22-nm HBsAg viral particles, and two vaccines have been licensed. The method of purification of HBsAg includes inactivation steps to eliminate residual virus. The efficacy of these vaccines has been demonstrated both in healthy adult populations at high risk of HBV infection [4, 5, 6] and in newborn infants in mass vaccination programs to prevent perinatal HBV transmission [7, 8, 9]. Vaccination of dialysis patients is more difficult to obtain. This is perhaps because these patients are immunosuppressed [10, 121. More than ten years of vaccination programs in different countries is enough to conclude the safety of such vaccines. Despite the proven efficacy and safety of the plasma-derived vaccine, alternative methods of subunit vaccine production are needed. The available supply of human serum, the technical procedures of HBsAg purification, the steps of virus inactivation and the tests of innocuity, repeated for each batch of serum, impose cost restriction upon the use of the vaccine. In this context, DNA recombinant technology is of primary importance for developing new vaccines. Several approaches can be used and the choice of the technique depends on considerations concerning the structure of the HBV envelope, the immunogenicity of the epitopes of the envelope proteins and the localization of their coding sequences on the HBV genome.

496

M. L. M I C H E L AND COLL.

II. - - The H B V envelope and the related coding sequences. The HBV envelope contains proteins, carbohydrates and lipids. The carbohydrate residues are covalently linked to the proteins and the proteins are anchored into a lipid bilayer. As is the rule for virus envelopes, the hydrophobic sequences of the proteins are intramembranous and the hydrophilic sequences containing the carbohydrate are exposed on the outside and carry the antigenic determinants. Gerlich and his colleagues [13, 14] have recently presented a complete analysis of the envelope proteins. According to these authors, the protein composition of the envelope is different for the complete virion and for the 22-nm particles. The envelope of the virion contains probably three proteins, each of them existing in two forms according to glycosylation. The first, called the ,, major protein ,,, is the smallest and is present in two forms, glycosylated (GP27) and non-glycosylated (P24)115]. The second, called the ~ middle protein ,~, exists in two glycosylated forms: the first (GP33) contains only one glycan, whereas the second ((;P36) contains two glycosidic residues. The last, called the c~, is present in a glycosylated form (GP42) and a non-glycosylated form (P39) [14]. These three proteins are encoded by one open reading frame of the HBV genome, called the S region, and which contains three AUG initiation codons (lig. 1). The

b 1

~1~" {eceptor

Fro. 1. - - Region S (region pre-S+gene S ) o[ H B V .

T h e S region c o n t a i n s t h r e e A U G w h i c h define t h e pre-S1 a n d pre-S2 r e g i o n s a n d t h e S gene. T h e t h r e e p r o t e i n s , t h e m a j o r p r o t e i n , t h e m i d d l e p r o t e i n a n d t h e large protein, correspond to t h e s e t h r e e A U G i n i t i a t i o n codons. T h e d a r k p o i n t s indicate t h e p o s i t i o n s of t h e c a r b o h y d r a t e s . T h e two i n i t i a t i o n sites of t r a n s c r i p t i o n , r e s p e c t i v e l y , u p s t r e a m f r o m t h e pre-S1 region a n d u p s t r e a m f r o m t h e pre-S2 r e g i o n , are indicated. T h e m a j o r t r a n s c r i p t is a 2 . 1 - K b R N A .

DHFR EDso HBV HBeAg

= = = =

dihydrofolate reductase. 5 0 % effective dose. hepatitis B virus. h e p a t i t i s Be a n t i g e n .

t

H B s A g = h e p a t i t i s B s u r f a c e antigen. HSA h u m a n serum a l b u m i n .

i MMTV = mouse mammary tumour virus. pHSA = polymerized HSA.

HBsAg SYNTHESIS AND PRE-S2 REGION PRODUCT

497

major protein is initiated at the third AUG and is encoded by the S gene [16]. The middle protein is initiated at the second AUG and is encoded by the pre-S2 sequence and by the S gene [13]. The large protein is probably initiated at the first AUG and is encoded by the complete pre-S sequence and by the S gene [14]. The three proteins therefore have in common a 226-amino-acid sequence corresponding to the S gene translation product. Compared to the major protein, the middle protein has an extra sequence of 55 amino acids at the N terminus. The additional sequence of the large protein would be of 163 to 219 amino acids, depending on the subtype of the virus. One virions contains around 200 to 250 molecules of major protein and approximately the same number of molecules (75 to 100) of middle and large protein. The protein composition of the HBsAg 22-nm particles is different from that of the virion; moreover, this composition varies according to HBV multiplication. HBsAg 22-nm particles purified from HBeAg-positive serum containing complete virion, have no or very few large proteins. HBsAg 22-nm particles from HBeAgnegative serum contain mostly major proteins (around 100 molecules per particle) with few (around 1%) middle proteins [14]. The reason for the variable protein composition of the viral envelope related to virus multiplication is not understood. A possible mechanism of regulation at the transcriptional level of the S region has been proposed [14]. The S gene translation product contains two hydrophilic sequences, and one of them carries a glycan at position 146. We argue that these two hydrophilic sequences contain most or all of the antigenic determinants of HBsAg [16]. Mishiro el al. [17] have shown that full antigenicity and immunogenicity of HBsAg need assembly of two protein molecules linked by disulphide bonds. Moreover, oligopeptides corresponding to either of the two hydrophilic regions induce a low immune response. HBsAg can therefore be considered as a conformational antigen. The N-termini of both the middle and the large antigens are hydrophilic and probably exposed outside of the envelope. One major epitope encoded by the pre-S2 sequence is apparently more immunogenic than the epitopes of HBsAg [18]. The HBV particles have a receptor for polymerized human serum albumin (pHSA) which may be involved in the hepatotropism of the virus [19]. This receptor is carried by the pre-S2 encoded amino acid sequence of the middle protein [20, 21]. Receptor activity is therefore absent or very low in HBsAg 22-nm particles of HBeAg-negative serum. III. - - Recombinant D N A technology /or juture vaccines. Because full immunogenicity of HBsAg is highly dependent on its conformation, the first approach consists of using recombinant DNA technology to produce HBsAg particles with a structure (( as close as possible )) to that of the native virus particles. Contrary to the core antigen, HBsAg is not synthesized as particles in bacteria. This is probably because the envelope assembly needs a lipid bilayer which is not synthesized in bacteria. Production of tlBsAg particles can be obtained either in animal cells [22, 23] or in yeast [24, 25] and these two systems can therefore be used for vaccine production. HBsAg particles produced in animal cells are homogeneous 22-nm particles, whereas particles synthesized in yeast are heterogeneous in size. They are glycosylated principally in animal cells, and it was recently reported that carbohydrate could influence the immune response to glycoprotein antigens [26]. The structure of particles synthesized in animals cells, and especially the presentation of the HBsAg determinants, could therefore be closer to that of virus particles of human origin than particles produced in yeast. This could have consequences on the quality of the immune response. Moreover, in animal cells, HBsAg particles are secreted without lysis of the cells. This facilitates the collection and the purification of HBsAg. The possibility of assembling and secreting HBsAg particles in any animal cells

498

M. L. MICHEL AND COLL.

also offers an opportunity of contructing a ((live vaccine )) using a virus which multiplies in man as vector. Up to now, only the vaccinia virus has been developed [27]. A recombinant vaccinia virus containing the S gene injected in chimpanzees multiplies and induces antibodies to HBsAg. The animals are protected against HBV infection. IV. - - Synlhesis in CHO cells of HBsAg parlicles carrying a receplor /or polymerized human serum albumin. For the reasons presented above, we have chosen the animal cell system to synthesize HBsAg particles. Because the appearance of antibodies to the pHSA receptor is observed in the serum during recovery from hepatitis B, but never during the evolution towards chronicity, thus suggesting that antibodies to ptISA recel)tors are important for viral clearance [28, 29], we have chosen to express both the S gene and the pre-S2 region which codes for the pHSA receptor. To obtain a level of HBsAg production high enough to be compatible with industrial exploitation, we amplified the HBV coding sequences. This was obtained by using the dihydrofolate reductase (DHFt0 gene are by selection of methotrexateresistant clones [30]. The plasmid pSVSdhfr used to transfect CHO D H F R - cells carries two transcription units (fig. 2) [31]. The first consists of the HBV 2.3-Kb BglII DNA fragment (including the pre-S region, the Sgene and the HBsAg mRNA polyadenylation site) placed under the control of the SV40 early promoter. The second unit consists of murine DHFR cDNA placed under the control of the MMTV-LTR promoter, the SV40 t-antigen splice site and the SV40 early mRNA polyadenylation site.

EcoRl Pvul~ ~M/L~.r,, ~ HindIII"~ H~i n d

BgIu lU

pMTVdhfr

P v u ~ I/BamHI)

J PvuII ~ (HindIII/1~1II)

(Bglll/BarnHI)

pSVS FIG. 2. - - Struclure of the pSVSdhfr plasmid. H B V and D H F R sequences are indicated b y open segments, SV40 sequences b y hatched s e g m e n t s and pBR322 sequences b y lines. The SV40 DNA preceding t h e H B V sequences contains the early promoter.

HBsAg S Y N T H E S I S AND P R E - S 2 R E G I O N P R O D U C T

499

CHO D H F R - cells were transfected b y pSVSdhfr and D H F R + cells were selected. Cells producing HBsAg were chosen for gene amplification and methotrexateresistant cells were selected. One close (resistant to 50 nm of methotrexate) producing around 1 ~g per l0 s cells and per day was chosen for further study. The organization of amplified sequences was studied by Southern blot analysis. Complex patterns were observed, suggesting t h a t DNA rearrangement occurred. The HBV-specific transcripts were studied b y RNA blot analysis and S1 nuclease mapping. Two HBV-specific poly(A)+ RNA were characterized, a major one of 2.1 Kb and a minor one of 2.5 Kb. The major RNA initiated in the pre-S1 region 15 nucleotides upstream from the second AUG of the pre-S region, and the minor RNA initiated in the SV40 sequence [31]. V. m Structure and immunogenicity o/ the H B s A g 22-rim parlicles.

HBsAg purified from cell culture supernatant of CHO cells consisted of 22-nm diameter particles with a density of 1.21 g/cm 3 in CsCI. The electrophoretic analysis of the proteins present in the particles showed t h a t the HBsAg particles contain both the major and the middle proteins [31] (fig. 3). The major protein exists in two forms, glycosylated (GP28S) and non-glycosylated (P24S). The middle protein is glycosylated and represents about 30% of the total proteins. The particle contains both group and subtype HBsAg determinants. The presence of pHSA receptor activity was shown using the haemagglutination test and solid-phase RIA. Immunogenicity was studied in mice. The particles elicited anti-HBs antibodies. The EDS0 was 0.04t~g, identical to t h a t obtained w i t h p a r t i c l e s of human orilglin. Moreover, the H B s ~ g particles were able to elicit antibodies to the pHSA receptor in mice [31].

FIo. 3. - - Eleelrophoretic analysis of the HBsAg parliele polypeplides. a) Polypeptides of purified particles visualized by silver staining; b) Autoradiography of 35Slabelled polypeptides from CHO supernatants immunoprecipitated with a rabbit anti-I-IBs anti-serum. Lane 1: HBsAg-produeing CHO clone. Lane 2: HBsAg-producing CHO clone in the presence of tunicamycin. Lane 3: CHO DHFR- cell line. Ann. Inst. Pasteur/Virol., 136 E, n ~ 4 , 1 9 8 5 .

34

500

M. L. M I C H E L AND COLL.

We compared the immune response to pre-S2-region-encoded determinants in terms of immunogenicity, specificity, H-2-1inked regulation and possible overlapping regulatory mechanisms [32]. For this purpose, groups of mice from a panel of H-2 congenic strains were immunized intraperitoneally with 1 ~g of HBsAg 22-nm particles (fig. 4). Ten days after immunization, the HBsAg responder mice (strain B10) contained IgG specific for the pre-S2-region-encoded polypeptide, but none for the S-region-encoded sequence. At 24 days, the response to the pre-S2 region was 25-fold greater than t h a t to the S region. After secondary immunization, the response to the pre-S2 region remained greater. Immunization of HBsAg total nonresponder mice (strain B10S) elicited an IgG response to the pre-S2 region 24 days after injection of viral particles. Furthermore, upon secondary immunization, antibodies to IIBsAg were detected. Cumulatively, these results indicate that the pre-S2 region is an immunogen superior to the S region with respect to the time of tile l)rimary response and the level of specitic IgG. Furthermore, the pre-S2 region can have a positive iniluence on the S region response and circumvent non-responsiveness to the S region.

A

B 100,000 50,000

10.000

5,000

/

.

t II

r-. 1000

100 5O V___,(,~

10

24

20

10 24

2~

DAYS POST-IMMUNIZATION F ~ . 4. - - Immunogenieilg o/ HBsAg 22-nm parlicles containing the pre-S2 region translation product.

A) Eleetron microscopy picture of HBsAg 22-nm particles synthesized in CHO cells, t3) and C) Production of antibodies to HBsAg (S) and the pre-S2 region translation products (pre-S) in an HBsAg responder (B) and non-responder (C) mouse strain. I - - - I = anti-HBs response of mice immunized with HBsAg particles lacking the pre-S2 region determinants.

These data have implications for the development of alternative vaccines. Inclusion of the pre-S2 region product in HBsAg 22-rim particles could augment the effectiveness of future recombinant hepatitis B vaccine.

HBsAg SYNTHESIS AND PRE-S2 REGION PRODUCT

501

VI. - - Conclusion. The HBsAg particles produced in CH0 cells are homogenous and have the same diameter (22 nm) and the same density (1.21) in CsC1 as HBsAg particles from human serum. They contain both the major protein and the middle protein of the viral envelope. The major protein exists in its two forms, glycosylated and nonglycosylated. In addition to the HBsAg determinants, these particles contain a receptor for pHSA carried by the N-terminus of the middle protein and which corresponds to the pre-S2 translation product. The structure and properties of the HBsAg 22-nm particles therefore seem to be identical to those of 22-nm particles present in human serum during virus multiplication. The choice of using HBsAg particles produced in CHO cells as a vaccine depends on three parameters: the immunogenicity of the particles, the safety of the final product and the transfer possibility of the technology of HBsAg production to the industry. Injected into mice, these particles induced a high level of both anti-HBs and anti-pHSA receptor antihodies. Epitopes of the translation products of both gene S and the pre-S2 region are therefore present in an immunoreactive form on the HBsAg particles. This, with the value of the EDL0, strongly suggests that the particles from CHO cells may elicit a strong and efficient immune response in man and can be used as a vaccine. MOTS-CLES : Hepatite B, Vaccin, HBsAg ; R6cepteur pHSA, Region pre-S2 Mise au point. REFERENCES

[1] KRUGMAN, S., GILES, J. P. & HAMOND, J., J. in[ect. Dis., 1970, 122, 432. [2] KRUGMAN,S., GILES, J. P. & HAMOND,J., J. Amer. med. Ass., 1971, 217, 41. [3] MAUPAS, P., GOUDEAU, A., 4OURSAGET, P., DRUCKER, J. & BAGROS, P., Lancet, 1976, I, 1367. [4] CROSNIER, J., JUNGERS, P., COUROUCE, A. M. & al., Lancet, 1981, I, 455. [5] LAPLANCUE,A., 4OUROUCE, A. M., BENHAMOU,E. & al., Lancel, 1982, I, 222. [6] SZMUNESS, W., STEVENS, C. E., HARLEY, E. J. & al., New Engl. d. Med., 1982, 307, 1481. [7] MAUPAS, P., CHmON, J. P., BAroN, F. & al., Lancet, 1981, I, 289. [8] LEE, G. C. Y., HWAN6, L. Y., BEASLEY, R. P. & al., d. in]ect. Dis., 1983, 148, 526. [9] PROZESKY, O. W., STEVENS, C. E., SZMUNESS,W. & al., J. in/ed. Dis., 1983, 7 (suppl. 1), 53, [10] CROSNmR, d., JUN6EnS, P. & COUROUCE, A. M., Lancet, 1981, I, 797. [11] DESMYTER, J., COLAERT, J., DE GROOTE, G. & al., Lancet, 1983, II, 1323. [12] STEVENS, 4. E., ALTER, H. J., TAYLOR, P. E. & al., New Engl. J. Med., 1984, 311, 496. [13] STIBBE, W. & GERLICH, W. H., J. Virol., 1983, 46, 626. [14] HEERMAN, K. H., GOLDMAN, U., SCHWARTZ, W., SEYFFARTH, T., BAUEMGARTEN, H. & GERLICH, W . H., J. Virol., 1984, 52, 396. [15] PETERSON,D. L., ROBERTS, J. M. & VYAS, G. N., Proc. nal. Acad. Sci. (Wash.), 1977, 74, 1530. [16] TIOLLAIS, P., CHARNAY, P. & YYAS, G. N., Science, 1981, 213, 406. [17] MISHIRO, S., IMAI, M., TAKAHASKI, K., MACHIDA, A., GOTANDA, T., MIYAKAWA, Y. & MAYUMI, M., J. Immunol., 1980, 124, 1589. [18] NEURATH,A.~R., KENT, S. B. H. & STRIKE, N., Science, 1984, 224, 392. [19] IMAI,M., YANASE,Y., NOJIRI, T., MIYAKAWA,Y. & MAYUMI,M. A., Gastroenlerologg, 1979, 76, 242.

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M. L. MICHEL AND COLL.

[20] MACHIDA,A., KISHIMOTO, S., OHNUMA,H., MIYAMOTO,H., BABA, K., ODA, K., NAKAMURA, T., MIYAKAWA, Y. r MAYUMI, M., Gastroenterology, 1983, 85, 268. [21] MACHIDA,A., KISHIMOTO,H,, OHNUMA,H., BABA, K., ITO, Y., MIYAMOTO,H., FUNATSU, G., ODA, K., USUDA, S., TOGAMI, S., NAKAMURA,T., MIYAKAWA, Y. r MAYUMI, M., Gaslroenlerology, 1984, 86, 910. [22] ])uBom, M. F., POUR('EL, C., ROUSSET, S., CHANY, C. & TIOLLAIS, P., Proc. nat. Acad. Sci. (Wash.), 1980, 77, 4549. [23] MOIIIARTY,A. M., HOYER,B. H., WAI-KUOSHIH,J., GERIN,J. L. & HAMER, D. H., Proc. hal. Acad. Sci. (Wash.), 1981, 78, 2606. 124] V.XI.ENZUEI.A, l~., MEI)INA, A. & RUTTEIL W..l., Nature (Lond.), 1982, 298, 347. 1251 I~[IYANOHAI/A, A., TOH-E, A., NOZAKI, C., HAMAI)A, F., OHTOMO, N. & MATStmAI~, K., Proc. nat. Acad. Sci. (Wash.), 1983, 80, 1. [26] AI.I';XAN1)EIL S. & I~L1)EI~, .]. |1., Science, 1(.)84, 226, 1328. [27[ SMVrH, G. L., MA(:KETT, M. & Moss, B., Nature (Lond.), 1983, 302, 490. 128} PONTISSO, P., ALm.:lvrL A., BORTOI.OT'rI, F. & I{EALDI, G., Gaslroenteroloyy, 1983, 84, 220. [291 AI.BERTI, A., PONTISSO, P., SCHIAVON, E. & REALm, G., Hepatology, 1984, 4, 220. 130] I{IN(;OLD, G., DIECKMAN, B. & I,VE, F., J. tool. Applied Genel., 1981, 1, 165. [31] ~{ICHEL, M. I,., PONTISSO, P., SOBCZAK, E., MALPIECE, Y., STREECK, R. E. & TIOLLAIS, P., Proc. nat. Acad. Sci. (Wash.), 1984, 81, 7708. ]32] MILICH, D. I~., THORTON, G. B., NEURATH, A. R., KENT, S. B., MICHEL, M. I,., TIOLLAIS, 1~ & CHISAH[, F. W., Science, 1985, 228, 1195.