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A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice
ELSEVIER
Virus Research
Virus Research 43 (1996) 91 96
Short communication
A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice Said A. SeiP,*, Kouichi
M o r i t a ~'b, A k i r a
Igarash?
~Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, 852, Japan bRegional Offiee Jbr the Western Pacific, World Health Organization, United Nations Avenue, P.O. Box 2937. 1099 Manila, The Philippines
Received 9 January 1996; accepted 14 March 1996
Abstract
We have recently shown that neutralizing epitope(s) exist near the C-terminal of JE virus E-protein by expressing the coding gene cDNA fragments as fusion proteins with protein A. Among four cDNA fragments, the fragment (B3) carrying the coding sequence of amino acid number 373-399 of E protein elicited the highest neutralizing (N) antibody titer (1:75). To exclude the possible influence of protein A contained in the expressed gene products on the mouse immune response, we expressed (B3) using pGEX-3X expression vector as fusion with glutathione-S transferase (GST). The mice immunized with recombinant GST-B3 fusion protein induced an immune response (mean average ELISA: 3364; N: 1:75) almost similar to that by recombinant protein A-B3 fusion protein (mean average ELISA: 3476; N: 1:75). While hemagglutination-inhibition (HI) antibodies were not induced by this fusion protein. These results indicate that 27 amino acid sequence on the E protein (373-399) was sufficient to induce neutralizing antibodies without association with protein A moiety. Keywords: JE virus; Molecular cloning; Fusion protein; Gene expression; Neutralizing antibodies
Japanese encephalitis (JE) is a serious inflammatory disease of public concern with significant mortality in m a n y countries in Asia (Umenai et al., 1985). The causative agent, JE virus, is a mosquito-borne flavivirus (Westaway et al., 1985). It has a single stranded positive-sense R N A genome, approximately 11 kb containing a single
* Corresponding author. P.O. Box 3824 Postal code 112, Ruwi-Muscat, Oman. Tel.: + 968 621514.
long open reading frame (ORF), Translation of the genome into a polyprotein precursor, coupled with co- and post-translational proteolytic processing, results in the production of 10 viral gene products. They are structural proteins: the capsid protein C (Mr approximately 14000), membrane protein M (Mr approximately 8000), and envelope protein E (Mr approximately 50000-60000) as well as seven non-structural (NS) proteins. The order of these products in the polyprotein has been documented as 5'-C-preM-E-NS1-NS2A-
0168-1702/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII SO168-1702(96)01323-8
92
S.A. Seif et al. / Virus Research 43 (1996) 91 96
NS2B-NS3-NS4A-NS4B-NS5-3' where preM is a precursor to membrane protein M. The envelope (E) protein of JE virus contains 500 amino acid residues and is glycosylated (Rice et al., 1985; Westaway, 1987; Sumiyoshi et al., 1987). Neutralizing antibodies against flaviviruses recognize the envelope protein E (Russell et al., 1980), which plays important roles in the initial steps of virushost cell interaction. For instance, it has been shown to be involved in the fusion of virus membrane with host cellular membranes after low pH-induced conformational changes (Koblet et al., 1990) and immunization by E protein has also been shown to induce neutralizing antibodies which are crucial in the protective immunity against JE virus infection. Although, immunization appears to be the only reasonable and practical way to control Japanese encephalitis, the cost of the current commercial JE vaccine is still too high for use in large-scale vaccination in developing countries. WHO had recommended the development of the second generation JE vaccine by recombinant gene technology. This kind of vaccine is expected to be inexpensive, easy to produce, efficacious and safe. In the past few years several different recombinant DNA strategies have been employed to generate candidate flavivirus vaccines. These strategies included E. coli fusion proteins (Mason et al., 1989; Seif et al., 1995), recombinant yeast plasmid (Fujita et al., 1987), crude lysate from moth cells infected with recombinant baculovirus (Deubel et al., 1991), live recombinant vaccinia viruses (Yasuda et al., 1990) and Sindbis virus recombinant (Konstantin et al., 1995). We have previously shown that different regions near C-terminal of JE virus E protein expressed as fusion proteins with protein A are immunogenic in mice (Seif et al., 1995). But there are documented reports that peptides containing protein A moiety are likely to interact and/or combine with a wide variety of antibodies (Williams et al., 1995), and its ability to bind with IgG might influence the cell attachment or antigen presentation in vivo. To obtain further insight into the influence of protein A in murine immune system, we have expressed the 27 amino acids (B3) peptide using pGEX-3X vector. This vector
can be used in bacterial systems to express foreign polypeptides as fusions with glutathione-S-transeferase (GST). Japanese encephalitis virus strain JaOArS982 used for cDNA cloning, ELISA and neutralization test was obtained from our department and was passaged several times in C6/36 cells. Aedes albopictus clone C6/36 cell line was grown and maintained by sub-cultures at 28°C using Eagle's medium supplemented with 9% heat-inactivated fetal calf serum and 0.2 mM non-essential amino acids. BHK-21 cells were grown at 37°C using the same growth medium as C6/36 cells. pGEX-3X vector was selected for the viral protein expression in this study as we had determined that enzyme digestion and ligation procedures will not introduce any frame shift in the insert. This vector was used to transform competent E. coli cells (strain JM 109). The purification of the recombinant plasmid was done as described before (Seif et al., 1995). Then the recombinant plasmid was digested by EcoR|, examined by 0.9% agarose gel electrophoresis and stained by ethidium bromide. The correct size band was excised, electro-eluted, de-phosphorylated by calf intestines alkaline phosphotase (CLAP), purified by gene clean kit, extracted by phenol/chloroform and precipitated by ethanol. The digested plasmid was stored at - 2 0 ° until used. B3 gene fragment corresponding to the amino acid residues 373-399 of JE virus E protein was obtained from recombinant pRIT2TB3 (Seif et al., 1995) after EcoRI digestion. The B3 gene fragment was examined and purified as above then cloned into pGEX-3X resulting into pGEX-3X-B3 recombinant plasmid. The nucleotide sequence at the junction between the inserted cDNA and GST gene was examined for its reading frame and orientation by a modification of dedeoxynucleotide chain termination method (Sanger et al., 1977). The inframe colonies were used in the expression work. Fusion protein expression was induced by isopropyl-fl-D thiogalactoside (IPTG), the product was purified by glutathione Sepharose 4B and eluted by reduced glutathione buffer pH 8.0. The negative control specimen was similarly prepared from E. coli transformed with pGEX-3X plasmid only.
93
S.A. Seif et al. / Virus' Research 43 (1996) 91 96
A
M
1
2
3
4
5
6
M
1
2
$
4
|
8
33.5 kDa
B
Mdl I~l
-----
Fig. 1. Detection of GST/fusion proteins by Commassie Brilliant Blue and Western blotting. Two mini-gels were prepared for SDS-PAGE and samples were subjected in duplicate as described in the Materials and methods. (A) CBB staining. Lanes: M, molecular weight marker; 1, GST-B3 (1:100 dilution); 2, GST-B3 (1:200 dilution); 3, GST-B3 induced whole cell (1:2 dilution); 4, GST-only (1:100 dilution); 5, GST-only (1:100 dilution); 6, GST induced whole cell (1:2 dilution). (B) Western blotting. Same arrangement of the specimens as in (A). Only GST-B3 fusion proteins reacted against anti-JE serum but not GST alone. G S T / G S T - B 3 expressed proteins were examined by Commassie brilliant blue (Fig. I(A)) and Western blotting (Fig. I(B)). The immunogenicity o f the recombinant fusion protein was determined by mouse inoculation. T w o inoculations (15 /zg protein per mouse with Titer M a x adjuvant) were administered at two week intervals. Three days
after the last injection, mice were sacrified and the collected blood was used for serum a n t i b o d y assays. Serum a n t i b o d y levels were measured by E L I S A and neutralization test as previously described (Seif et al., 1995). B3 polypeptide was expressed from the t a c prom o t e r as fusion to the C terminus o f glutathione-
94
S.A. Seif et al. / Virus Research 43 (1996) 91 96
A M
1
B 2
M
1
C 2
M
1
•..---30 kDa 30kDa..-.
~
~
m Fig. 2. Antigenicity of B3 fusion protein after digestion with Factor Xa. One mini-gel was prepared for SDS-PAGE and samples were subjected in triplicate as described in the Materials and methods. (A) CBB staining. Lanes: M, low molecular weight protein marker; 1, digested GST-B3protein; 2, undigested GST = B3 fusion protein. (B) Western blotting results using anti-GST serum. (C) Western blotting results using anti-JE serum. S-transferase (GST), a 26 kDa cytoplasmic protein of eukaryotes. Expression of the recombinant B3 clone and pGEX-3X vector in E . coli system led to the synthesis of products with expected molecular weights which were 5 visualized at the positions of 29 and 26 kDa respectively after Commassie brilliant blue staining (Fig. 1(A)). The duplicate protein bands in the other gel were transferred onto nitrocellulose membrane and stained by the Western blotting (Fig. I(B)). As clearly shown in Fig. 2(B), GST-B3 fusion proteins but not GST protein reacted against anti-JE serum, confirming not only the efficient synthesis of recombinant fusion proteins, but also its specific antigenicity. These observations were further clarified by the digestion of the recombinant fusion proteins with Factor Xa and stained by Commassie brilliant blue (Fig. 2(A)) and Western blotting using antiGST (Fig. 2(B)) and anti-JE serum (Fig. 2(C)) accordingly. Fig. 2(C) shows that anti-JE polyclonal serum reacted against GST-B3 fusion protein only but not against GST protein. Their apparent molecular weights were those expected from the size of the inserted JE virus c D N A fragments and pGEX-3X vector, respectively. The expressed fusion proteins were synthesized as sin-
gle stable polypeptide and degradation bands were not observed. In the present study we found that GST-B3 fusion protein induced an immune response almost similar to our previous results using recombinant protein A-B3 fusion protein. This clearly indicates that protein A association with expressed viral protein(s) is not an important requirement for immunogenicity to be attained. The titres of the neutralizing antibody immune response induced by recombinant p G E X 3X-B3 were lower when compared with the titers elicited by recombinants with vaccinia virus expressing PreM and E glycoproteins (Yasuda et al., 1990) or JE vaccine (Biken-Japan), or Sindbis virus recombinant (Konstantin et al., 1995). But, in their studies their recombinants contained the whole of E gene and the viral proteins carriers were either live or inactivated virus. One of the major objectives of our study was to try to prepare a basic platform for the production of a peptide vaccine and to completely avoid the use of live, attenuated or inactivated viruses. Some of the reasons which might have led to lower neutralization titre observed have been discussed in our previous publication (Seif et al., 1995). However, the neutralizing antibody level
S.A. Seif et al. / Virus Research 43 (1996) 91-96
elicited by our recombinant B3 fusion protein could be protective in mice because flavivirus-neutralizing antibodies, even at a low titre (1:10) are believed to reflect a protective immunity status against infection by a homologous virus (Oya, 1988; Putnak et al., 1991). In contrast to other E. coli expression systems which often yield denatured or precipitated gene products, one of the advantages in the p G E X - 3 X expression system is that gene products are more easily obtained without denaturation during extraction and purification procedures. Therefore, expressed foreign peptides m a y retain their functional activities and antigenicity. Additional advantages of this system are its high level of expression ( ~ 30 mg/1), rapidity and easiness of purification compared with the p R I T 2 T system we used in our previous study. Fusion proteins typically remained associated within the bacteria cell m e m b r a n e which was then easily extracted and purified by affinity o f G S T moiety to glutathione immobilized on agarose beads. The shortest recombinant B3 lacking transmembrane and cytoplasmic domain sequences produced higher level of gene product compared with other recombinant clones B1, B2, and B4. Deubel et al. (1991) demonstrated that a dengue type 2 virus E protein with a truncation corresponding to 71 amino acids at the C-terminus of the native protein was synthesized at a higher level and is more soluble than the membrane anchored protein. Their findings are in agreement with our present results. This study determined that the 27 amino acid sequence near the C-terminus of JE virus E protein was sufficient to induce neutralizing antibodies without association with protein A. This portion (B3) could be a suitable candidate for a second generation recombinant JE vaccine. However, more studies should be done in order to determine the shortest sequences which are responsible for the neutralizing epitope(s) in the expressed gene product, by using synthetic peptides carrying different sizes, and again, to determine its antigenicity and immunogenicity.
95
Acknowledgements We sincerely thank Drs. Sachiko Matsuo and Futoshi Hasebe for their valuable assistance during the study. This study was financially supported by a Research G r a n t from the World Health Organization, Regional Office for the Western Pacific, for the Development of the Second Generation Japanese encephalitis vaccine. S.A.S. is a grateful recipient of a Monbusho scholarship from the Ministry of Education, Science and Culture, Japan. Animal experiments for this study were performed at the Animal Research Center, Institute of Tropical Medicine, Nagasaki University.
References Deubel, V., Bordier, M., Mergert, F., Gentry, M.K., Schlesinger, J.J. and Girard, M. (1991) Processing, secretion and immuno-reactivity of carboxyl terminally truncated dengue-2 envelope proteins expressed in insect cells by recombinant baculovirus. Virology 180, 442-447. Fujita, H., Sumiyoshi, H., Mori, C., Manabe, S., Takagi, M., Yashida, I. et al. (1987) Studies in the development of Japanese encephalitis vaccine: expression of virus envelope glycoprotein V3 (E) gene in yeast. Bull. WHO 65, 303 308. Koblet, H., Kohler, U. and Igarashi, A. (1990) Fusion of Aedes albopictus ceils, clone C6/36, by Japanese encephalitis virus is triggered by low pH. Trop. Med. 32(4), 145154. Konstantin, V, Pugachev, Mason, P.W., Shope, R.E. and Frey, T.K. (1995) Double Sub-genomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induced significant protection in mice against lethal JEV infection. Virology 212, 587-594. Mason, PW., Dalrymle, J.M., Gentry, M.K., McCown, J.M., Hoke, C.H., Burke, D.S., Fournier, M. and Mason, T.L. (1989) Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoproteins. J. Gen. Virol. 70, 2037-2049. Oya, A. (1988) Japanese encephalitis vaccine. Acta Paediat. Jpn. 30, 175 184. Putnak, R., Feighny, R., Burrous, J., Cochran, M., Hackett, C., Smith, G. and Hoke, C.H. (1991) Dengue-1 virus envelope glycoprotein gene expressed in baculovirus elicit virus neutralizing antibody in mice and protects them from virus challenge. Am. J. Trop. Med. Hyg. 45, 159-167. Rice, C.M., Strauss, E.G. and Strauss, J.H. (1985) Structure of the flavivirus genome. In: S. Schlesinger and M.J. Schlesinger (Eds.), The Togaviridae and Flaviviridae, Plenum, New York, pp. 297-326.
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Russell, D.L., Darlymple, J.M. and Johnston, R.E. (1989) Sindbis virus mutations which coordinately affect glycoprotein processing, penetration and virulence in mice. J. Virol. 63, 1619-1629. Sanger, F., Nicklen, S. and Coulson, R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. Seif, S.A., Morita, K., Matsuo, S., Hasebe, F. and Igarashi, A. (1995) Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E protein expressed in recombinant Escherichla coli system. Vaccine 13, 1515-1521. Sumiyoshi, H., Mori, C., Fuke, I., Morita, K., Kuhara, S., Kondou, J. et al. (1987) Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161,497-510. Umenai, T., Kryzysko, T., Betkimirov, A. and Assad, F.A. (1985) Japanese encephalitis: current world wide status. Bull. WHO 63, 625-631.
Westaway, E.G., Brinton, M.A., Gaidamovich, S.Ya., Horzinek, M.C., Igarashi, A., Kaariainen, L. et al. (1985) Flaviviridae. Intervirology 24, 183-192. Westaway, E.G. (1987) Flavivirus replication strategy. Adv. Virus Res. 33, 45-90. Williams, J.A., Langelang, .J.A., ThaUey, B.S. and Carroll, S.B. (1995) Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies. In: D.M. Glover and B.D. Hames (Eds.), DNA Cloning 2. pp. 15-58, Oxford University Press, New York. Yasuda, A., Kimura-Kuroda, J., Ogimoto, M., Sato, T., Takamura, C., Kurata, T., Kojima, A. and Yasui, K. (1990) Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that expressed pre M and E glycoproteins of Japanese encephalitis virus. J. Virol. 64, 2788-2795.
Virus Research
Virus Research 43 (1996) 91 96
Short communication
A 27 amino acid coding region of JE virus E protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice Said A. SeiP,*, Kouichi
M o r i t a ~'b, A k i r a
Igarash?
~Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, 852, Japan bRegional Offiee Jbr the Western Pacific, World Health Organization, United Nations Avenue, P.O. Box 2937. 1099 Manila, The Philippines
Received 9 January 1996; accepted 14 March 1996
Abstract
We have recently shown that neutralizing epitope(s) exist near the C-terminal of JE virus E-protein by expressing the coding gene cDNA fragments as fusion proteins with protein A. Among four cDNA fragments, the fragment (B3) carrying the coding sequence of amino acid number 373-399 of E protein elicited the highest neutralizing (N) antibody titer (1:75). To exclude the possible influence of protein A contained in the expressed gene products on the mouse immune response, we expressed (B3) using pGEX-3X expression vector as fusion with glutathione-S transferase (GST). The mice immunized with recombinant GST-B3 fusion protein induced an immune response (mean average ELISA: 3364; N: 1:75) almost similar to that by recombinant protein A-B3 fusion protein (mean average ELISA: 3476; N: 1:75). While hemagglutination-inhibition (HI) antibodies were not induced by this fusion protein. These results indicate that 27 amino acid sequence on the E protein (373-399) was sufficient to induce neutralizing antibodies without association with protein A moiety. Keywords: JE virus; Molecular cloning; Fusion protein; Gene expression; Neutralizing antibodies
Japanese encephalitis (JE) is a serious inflammatory disease of public concern with significant mortality in m a n y countries in Asia (Umenai et al., 1985). The causative agent, JE virus, is a mosquito-borne flavivirus (Westaway et al., 1985). It has a single stranded positive-sense R N A genome, approximately 11 kb containing a single
* Corresponding author. P.O. Box 3824 Postal code 112, Ruwi-Muscat, Oman. Tel.: + 968 621514.
long open reading frame (ORF), Translation of the genome into a polyprotein precursor, coupled with co- and post-translational proteolytic processing, results in the production of 10 viral gene products. They are structural proteins: the capsid protein C (Mr approximately 14000), membrane protein M (Mr approximately 8000), and envelope protein E (Mr approximately 50000-60000) as well as seven non-structural (NS) proteins. The order of these products in the polyprotein has been documented as 5'-C-preM-E-NS1-NS2A-
0168-1702/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII SO168-1702(96)01323-8
92
S.A. Seif et al. / Virus Research 43 (1996) 91 96
NS2B-NS3-NS4A-NS4B-NS5-3' where preM is a precursor to membrane protein M. The envelope (E) protein of JE virus contains 500 amino acid residues and is glycosylated (Rice et al., 1985; Westaway, 1987; Sumiyoshi et al., 1987). Neutralizing antibodies against flaviviruses recognize the envelope protein E (Russell et al., 1980), which plays important roles in the initial steps of virushost cell interaction. For instance, it has been shown to be involved in the fusion of virus membrane with host cellular membranes after low pH-induced conformational changes (Koblet et al., 1990) and immunization by E protein has also been shown to induce neutralizing antibodies which are crucial in the protective immunity against JE virus infection. Although, immunization appears to be the only reasonable and practical way to control Japanese encephalitis, the cost of the current commercial JE vaccine is still too high for use in large-scale vaccination in developing countries. WHO had recommended the development of the second generation JE vaccine by recombinant gene technology. This kind of vaccine is expected to be inexpensive, easy to produce, efficacious and safe. In the past few years several different recombinant DNA strategies have been employed to generate candidate flavivirus vaccines. These strategies included E. coli fusion proteins (Mason et al., 1989; Seif et al., 1995), recombinant yeast plasmid (Fujita et al., 1987), crude lysate from moth cells infected with recombinant baculovirus (Deubel et al., 1991), live recombinant vaccinia viruses (Yasuda et al., 1990) and Sindbis virus recombinant (Konstantin et al., 1995). We have previously shown that different regions near C-terminal of JE virus E protein expressed as fusion proteins with protein A are immunogenic in mice (Seif et al., 1995). But there are documented reports that peptides containing protein A moiety are likely to interact and/or combine with a wide variety of antibodies (Williams et al., 1995), and its ability to bind with IgG might influence the cell attachment or antigen presentation in vivo. To obtain further insight into the influence of protein A in murine immune system, we have expressed the 27 amino acids (B3) peptide using pGEX-3X vector. This vector
can be used in bacterial systems to express foreign polypeptides as fusions with glutathione-S-transeferase (GST). Japanese encephalitis virus strain JaOArS982 used for cDNA cloning, ELISA and neutralization test was obtained from our department and was passaged several times in C6/36 cells. Aedes albopictus clone C6/36 cell line was grown and maintained by sub-cultures at 28°C using Eagle's medium supplemented with 9% heat-inactivated fetal calf serum and 0.2 mM non-essential amino acids. BHK-21 cells were grown at 37°C using the same growth medium as C6/36 cells. pGEX-3X vector was selected for the viral protein expression in this study as we had determined that enzyme digestion and ligation procedures will not introduce any frame shift in the insert. This vector was used to transform competent E. coli cells (strain JM 109). The purification of the recombinant plasmid was done as described before (Seif et al., 1995). Then the recombinant plasmid was digested by EcoR|, examined by 0.9% agarose gel electrophoresis and stained by ethidium bromide. The correct size band was excised, electro-eluted, de-phosphorylated by calf intestines alkaline phosphotase (CLAP), purified by gene clean kit, extracted by phenol/chloroform and precipitated by ethanol. The digested plasmid was stored at - 2 0 ° until used. B3 gene fragment corresponding to the amino acid residues 373-399 of JE virus E protein was obtained from recombinant pRIT2TB3 (Seif et al., 1995) after EcoRI digestion. The B3 gene fragment was examined and purified as above then cloned into pGEX-3X resulting into pGEX-3X-B3 recombinant plasmid. The nucleotide sequence at the junction between the inserted cDNA and GST gene was examined for its reading frame and orientation by a modification of dedeoxynucleotide chain termination method (Sanger et al., 1977). The inframe colonies were used in the expression work. Fusion protein expression was induced by isopropyl-fl-D thiogalactoside (IPTG), the product was purified by glutathione Sepharose 4B and eluted by reduced glutathione buffer pH 8.0. The negative control specimen was similarly prepared from E. coli transformed with pGEX-3X plasmid only.
93
S.A. Seif et al. / Virus' Research 43 (1996) 91 96
A
M
1
2
3
4
5
6
M
1
2
$
4
|
8
33.5 kDa
B
Mdl I~l
-----
Fig. 1. Detection of GST/fusion proteins by Commassie Brilliant Blue and Western blotting. Two mini-gels were prepared for SDS-PAGE and samples were subjected in duplicate as described in the Materials and methods. (A) CBB staining. Lanes: M, molecular weight marker; 1, GST-B3 (1:100 dilution); 2, GST-B3 (1:200 dilution); 3, GST-B3 induced whole cell (1:2 dilution); 4, GST-only (1:100 dilution); 5, GST-only (1:100 dilution); 6, GST induced whole cell (1:2 dilution). (B) Western blotting. Same arrangement of the specimens as in (A). Only GST-B3 fusion proteins reacted against anti-JE serum but not GST alone. G S T / G S T - B 3 expressed proteins were examined by Commassie brilliant blue (Fig. I(A)) and Western blotting (Fig. I(B)). The immunogenicity o f the recombinant fusion protein was determined by mouse inoculation. T w o inoculations (15 /zg protein per mouse with Titer M a x adjuvant) were administered at two week intervals. Three days
after the last injection, mice were sacrified and the collected blood was used for serum a n t i b o d y assays. Serum a n t i b o d y levels were measured by E L I S A and neutralization test as previously described (Seif et al., 1995). B3 polypeptide was expressed from the t a c prom o t e r as fusion to the C terminus o f glutathione-
94
S.A. Seif et al. / Virus Research 43 (1996) 91 96
A M
1
B 2
M
1
C 2
M
1
•..---30 kDa 30kDa..-.
~
~
m Fig. 2. Antigenicity of B3 fusion protein after digestion with Factor Xa. One mini-gel was prepared for SDS-PAGE and samples were subjected in triplicate as described in the Materials and methods. (A) CBB staining. Lanes: M, low molecular weight protein marker; 1, digested GST-B3protein; 2, undigested GST = B3 fusion protein. (B) Western blotting results using anti-GST serum. (C) Western blotting results using anti-JE serum. S-transferase (GST), a 26 kDa cytoplasmic protein of eukaryotes. Expression of the recombinant B3 clone and pGEX-3X vector in E . coli system led to the synthesis of products with expected molecular weights which were 5 visualized at the positions of 29 and 26 kDa respectively after Commassie brilliant blue staining (Fig. 1(A)). The duplicate protein bands in the other gel were transferred onto nitrocellulose membrane and stained by the Western blotting (Fig. I(B)). As clearly shown in Fig. 2(B), GST-B3 fusion proteins but not GST protein reacted against anti-JE serum, confirming not only the efficient synthesis of recombinant fusion proteins, but also its specific antigenicity. These observations were further clarified by the digestion of the recombinant fusion proteins with Factor Xa and stained by Commassie brilliant blue (Fig. 2(A)) and Western blotting using antiGST (Fig. 2(B)) and anti-JE serum (Fig. 2(C)) accordingly. Fig. 2(C) shows that anti-JE polyclonal serum reacted against GST-B3 fusion protein only but not against GST protein. Their apparent molecular weights were those expected from the size of the inserted JE virus c D N A fragments and pGEX-3X vector, respectively. The expressed fusion proteins were synthesized as sin-
gle stable polypeptide and degradation bands were not observed. In the present study we found that GST-B3 fusion protein induced an immune response almost similar to our previous results using recombinant protein A-B3 fusion protein. This clearly indicates that protein A association with expressed viral protein(s) is not an important requirement for immunogenicity to be attained. The titres of the neutralizing antibody immune response induced by recombinant p G E X 3X-B3 were lower when compared with the titers elicited by recombinants with vaccinia virus expressing PreM and E glycoproteins (Yasuda et al., 1990) or JE vaccine (Biken-Japan), or Sindbis virus recombinant (Konstantin et al., 1995). But, in their studies their recombinants contained the whole of E gene and the viral proteins carriers were either live or inactivated virus. One of the major objectives of our study was to try to prepare a basic platform for the production of a peptide vaccine and to completely avoid the use of live, attenuated or inactivated viruses. Some of the reasons which might have led to lower neutralization titre observed have been discussed in our previous publication (Seif et al., 1995). However, the neutralizing antibody level
S.A. Seif et al. / Virus Research 43 (1996) 91-96
elicited by our recombinant B3 fusion protein could be protective in mice because flavivirus-neutralizing antibodies, even at a low titre (1:10) are believed to reflect a protective immunity status against infection by a homologous virus (Oya, 1988; Putnak et al., 1991). In contrast to other E. coli expression systems which often yield denatured or precipitated gene products, one of the advantages in the p G E X - 3 X expression system is that gene products are more easily obtained without denaturation during extraction and purification procedures. Therefore, expressed foreign peptides m a y retain their functional activities and antigenicity. Additional advantages of this system are its high level of expression ( ~ 30 mg/1), rapidity and easiness of purification compared with the p R I T 2 T system we used in our previous study. Fusion proteins typically remained associated within the bacteria cell m e m b r a n e which was then easily extracted and purified by affinity o f G S T moiety to glutathione immobilized on agarose beads. The shortest recombinant B3 lacking transmembrane and cytoplasmic domain sequences produced higher level of gene product compared with other recombinant clones B1, B2, and B4. Deubel et al. (1991) demonstrated that a dengue type 2 virus E protein with a truncation corresponding to 71 amino acids at the C-terminus of the native protein was synthesized at a higher level and is more soluble than the membrane anchored protein. Their findings are in agreement with our present results. This study determined that the 27 amino acid sequence near the C-terminus of JE virus E protein was sufficient to induce neutralizing antibodies without association with protein A. This portion (B3) could be a suitable candidate for a second generation recombinant JE vaccine. However, more studies should be done in order to determine the shortest sequences which are responsible for the neutralizing epitope(s) in the expressed gene product, by using synthetic peptides carrying different sizes, and again, to determine its antigenicity and immunogenicity.
95
Acknowledgements We sincerely thank Drs. Sachiko Matsuo and Futoshi Hasebe for their valuable assistance during the study. This study was financially supported by a Research G r a n t from the World Health Organization, Regional Office for the Western Pacific, for the Development of the Second Generation Japanese encephalitis vaccine. S.A.S. is a grateful recipient of a Monbusho scholarship from the Ministry of Education, Science and Culture, Japan. Animal experiments for this study were performed at the Animal Research Center, Institute of Tropical Medicine, Nagasaki University.
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