Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes

Vaccine, Vol. 14, No. 11, pp. 1069-1076, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Prmted in Great Britain 0264-410X/96 $15+0.00

Elsevier PII: 0264-410X(95)00238-3

ELSEVIER

Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes Tomohiko Sugiyama*§, Kazutomo Suzue*, Munehiro Okamotot, Joseph Inselburgl, Kumiko Tai* and Toshihiro Horii*T We expressed two regions of the serine repeat antigen (SERA) protein of Plasmodium falciparum in Escherichia coli by synthesizing the genes with a changed codon usage. One of the synthetic gene sequences encodes amino acid residues 17-382 (SE47’) and the other encodes amino acid residues 586-802 (SESOA). The products produced by the synthetic gene sequences in E. coli accounted for 15-30% of the total bacterial protein. Antisera against both the pkjied gene products prepared in rats inhibited malaria parasite growth in vitro. The anti-SE47’ serum was significantly more inhibitory than the anti-SESOA serum. The described methods provide a large scale preparation of recombinant antigens for improving and producing malaria vaccine. Copyright 0 1996 Elsevier Science Ltd. Keywords:

SERA

protein

of Plasmodium falciparum; malaria

vaccine

Malaria is widespread, and that caused by Plasmodium is the most virulent form. The increasing incidence of drug resistant parasites makes development of a vaccine an urgent objective. The SERA of P. falciparum’, which was identified using parasiteinhibitory monoclonal antibody 43E5’ has also been known as ~140~ or SERP4, ~126~ and ~113~. The isolation and characterization of the SERA cDNA of P. falciparum FCR3 strain revealed that SERA contains 989 amino acids including a repetition of 3.5 serine residues’.‘. Except for the characteristic serine tract, SERA has few repetitive sequences. The protein accumulates in the parasitophorous vacuole of trophozoites and schizonts, and is processed into three fragments (47 kDa, 50 kDa and 18 kDa) coincident with merozoite release5,8. Though the function of SERA is not known, a part has been shown to contain amino acid sequences homologous with those common to the active site of a serine protease’,” and has been shown to become associated with merozoites’ ‘. The SERA of P. falciparum is presently considered a good candidate antigen for malaria vaccine based on the

falciparum

*Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita Osaka 565, Japan. TThe Institute of Experimental Animal Sciences, Osaka University Medical School, Suita Osaka 565, Japan. iDepartment of Microbiology, Dartmouth Medical School, Hanover, NH 03755-3842, USA. $Present address: Section of Microbiology, Hutchison Hall, University of California, Davis, Davis, CA 95616, USA. To whom correspondence should be addressed. (Received 18 September 1995; revised 7 November 1995; accepted 8 November 1995)

candidates;

synthetic

gene: humoral

immune

response

immunogenicity of a recombinant form. The N-terminal fragment of SERA protein expressed in yeast could induce a protective immunity against malaria infection in Aotus monkeys”m’4. Technical problems relating to the expression of SERA in yeast suggested another source of the protein would be useful. An E. coli system offers advantages for the production and purification of recombinant proteins. The cDNA of P. falciparum is, however, often poorly expressed in Escherichia coli. We assumed a principal cause of the poor gene expression was the difference of codon usages between P. falciparum and E. coli. In a preceding paper, we constructed a synthetic gene encoding the DHFR of P. falciparum with altered codons’5, and showed it was expressed and accumulated in E. coli cells in a large amount. In this paper, we describe: the construction of synthetic gene sequences encoding two parts of SERA and their subsequent expression in E. coli; and the parasite-inhibitory effect of antibodies prepared against these purified recombinant proteins in rats.

MATERIALS

AND

METHODS

Bacterial strains and plasmids E. coli XLl-Blue and plasmid pBluescript II SK+ were from STRATAGENE. Other plasmids used were PET-3alh and M 13-pKM2, a derivative of Ml3 phage, which contains the T7 RNA polymerase gene under the control of a lac promoter”.

Vaccine 1996 Volume 14 Number 11 1069

Recombinant SERA for P. falciparum Table 1

Nucleotide

sequences

malaria vaccine: T: Sugiyama et al.

of the synthesized 5’ CCGGGGTACC CCGGTAATAC 5’CCGGGGATCC AGAGCCCGCC TACCGGTCTG 5’ CCGGGGTACC TGGCCATTCT 5’ CCGGGGATCC CAGATTTCAC CTAACGC 3 5’ CCGGGGTACC CATAll-TATA CACCC 3 5’ CCGGGGATCC GATTCAAATG TATATTGG 3 5’ CCGGGGTACC GTACAGGCAC 5’ CCGGGGTACC GTACAGGCAC 5’ CATATGGACT ATCTAGCTCG CCAGCTCATC 5’ GATCCTACGG GAACTGCTGG TGAACTGGAC 5’ CCGGGGTACC CCGTTAAACC TTCAAACTGG 5’ CCGGGGATCC CGCCGTACAC ACCAGTTTGA 5’ CCGGGGTACC ACTAGTATTC 5’ CCGGGGATCC AGCCCAGCGC TTATATGC 3 5’ CCGGGGTACC TTTCAGTGCG 5’ CCGGGGATCC TCGCTTAGGT S’CGCGCTCGAG TGGAAGATCA CATCTGGAAA 5’ GCGCGGATCC AGAGGGCGGA TCCAGATGAT 5’ CGCGCTCGAG TTCTGCAAAT CCG 3’ 5’ GCGCGGATCC CAACCTTCGG GATTCCGCCG 5’ CGCGCTCGAG TGGATGGTAA ATGGACGCGT 5’ GCGCGGATCC CGATCACAGA GCGTCCATGT 5’ CGCGCTCGAG GCGATGATAC GTAAACTCAG 5’ GCGCGGATCC AATAACCTTC TATGACTTTT

471’+ 47l’-

4721+ 4721-

4722+

4722-

4723+ 47234724+

4724-

4731+

4731-

4732+ 4732-

474+ 474501+

501-

502+

502-

503+

503-

504+

504-

DNA@ CATATGAAAA CGGCGG 3’ GTTGACTGGC TGGTCGCCAA GC 3 GTCAACCCGG GTTAGTACCG GGGCCCGTAA CTGAATGGTA

ACGTGATCAA

ATGTACCGGT

GAAAGCCAGA

GCCTGTGCTA CCGTGTTGCC

CCCTGCGGAG TGCCTGACCA

AGCCGCCGGT CCGCCGGTAT

CTCTAGCGAA TTAGCGTTAG CTTTTAAACC TCTTGTTTTT

CCGTCTAACC CCAGACCAGC CATATAATCT CAGAAGAGGT

CAGTGTCTTC ACC 3 TTCAGCAGCG GCTGGTCTGG

GGGCCCGTGT T-TGATGTGGA

AACGAAAATT TACCGAAGAT

TCATCATGTT ACCAATATAG

CCTGGTTCCG AGCTCCGTAC

AAGCTTCACA AGATCGCGTT

TATTTTTTTT GTTGGTTTCT

TTTCCAGTGA TTCAGGGTGG

ACCACTGTTT TACGGAGCTC

AAGCTTCCGT CG 3 AAGCT-KCGT CG 3 CGAGTAGCTC TCTAGCAGTT GAGCGAAAGT AAGACTTTCG ATGAACTGCT GAAGAGCTAC CATATGGAAA CCCGCGTAAC TGG 3 CGTACGTCCA TTTCCATTTA AGTTTTTGCC CGTACGCAAG TAATCCATGC CAATTGGACG GTAGTTTTTA

CAAACGGCAC

CACCGGTGAA

CAGGGTTCAA

CAAACGGCAC

CACCGGTGAA

CAGGGTTCAA

TTCGTCCAGT CATCCAGCAG CTTCCGTAG 3 CTCGATGAGC AGACGAGCTA TCGAGTCCAT GTCTTCCGGC CTGCAGAACA

TCAAGCTCCT TTCTAGCTCG

CTAGCTCGTC TCCTCTAGT-T

TGGAACTAGA GATGACGAGC ATGGTAC 3 GAATGGCCCG TCTGTGAAAC

GGACGAGCTA TAGAGGAGCT

CTTTGTTATT ATGATTAATG GG 3 TATCTGATTA ATATAAAGAA CCAGGGTATC CTT-KGATCA

TTCGGTGGTA TATTCTCCTT

TCTTTGGTTT AATATACACC

ACGAAAAGGA CATAATGGC 3 ACAllll-TCC GGTTGGTGCC

AACCCCGTTT

CAATTGCTTT CGCTGCTGGT TTACTCCGCT AT-ITGTAACA CATATGAAAG GGGTAACTGC cc 3 TATCGATCTT GATT-TTAGTC AClTGC 3 CATATGGATC TATCGAAGAT

CTGAGTGGTA GGAAAAAGAA TTGATCTCCT CACGTCGT-IT ATGAAAACAA GATACCAGCT

ACTTTAACAT AATAAAAACG TGAAATTAGA TTATTTTCTT CTGCAll-TCG GGATCTTCGC

TGAAAAATGC ACG 3 CACAATATCT TTTCC 3 AACCTGCAGG TAGCAAGTAT

TATGTTCGCC GGTTCGTAGC

TTTATAACAG CT-I-KATACA

TTGGCCACAT ACGAATGGTT

GATGTGATGA TATGGCTTTC

AGGTTCTAGT TGCCGGCGGA

CCCATGGAAT ATCTAACTAT

TAGATCTTGC ACACTGTTCG

CGTTATCCCA CCAACTT-I-TA

AAGGTTCATC CATAGTTATA

CAGTGATCTT CGGATAGl-TA

CATATGAAGA GGGCTATACC TTG 3 TAGAATTCGT ACCTTTGTTC TATCG 3 CATATGGAAT GGCTGATCAT AAGGTGAAAA TAAAAATGGC ATCGCCCCAG TTTCACCTTC

TCTTGCATAA GCGTACGAAA

CAAAAACGAA GCGAGCGTTT

CCGAATAGCC TCACGATAAC

AACCCATCAC ATCACTTCGG

GTTTTCCGCT TTTTAATAAT

TTGATATACG TTTAACAAAC

TCAGCGGCAA GCAGTTAACA AAAGTC 3 AGTGGGTCGG TACGGGCCCC TGAG 3

GAAAGTGCAA TTGTGGGTTA

AACCTGTGCG CGGCAACTAT

GCCGTACATG AAGAGTTACG

TCGACTTTAA CACGATCCAG

Construction of the synthetic genes All of the DNAs were synthesized using an Applied Biosystems model 392 DNA/RNA synthesizer. The products were purified by electrophoresis through a 10% polyacrylamide gel containing 50 mM Tris-borate, pH 8.30 mM EDTA/8 M urea. The DNA nucleotide sequences are shown in Table 1. Synthetic genes SE47’ and SESOA were constructed by ligation of 8 and 4 polynucleotide blocks, respectively.

Vaccine

1996

Volume

14 Number

11

GGAATGTCTG ATTATGTTCT

3

BThe nucleotide sequences of the synthesized DNA are described indicates the upper or lower strands respectively

1070

GATTCCCCGA CGGCAAAAAC

in the direction from 5’ to 3’. The symbols + or - after the block number

The restriction sites used for the ligation were introduced at both ends of each block without changing the original amino acid sequence (Figure 1). The synthetic gene sequences were then inserted into PET-3a by Nde I and Barn HI sites to give PET-SE47’ and PET-SESOA. The basic methods for ligation and cloning the DNA fragments were those of Sambrook et al.‘*. Each block was made from a pair of the synthetic DNA fragments listed in Table 1 and cloned as follows. Twenty

Recombinant

fl j/Z 501

r4e.d

i

CM,

502

i

S@ll

503

i ECORl

504

i Sa-n”,

Figure 1 Construction of the synthetic genes. The regions for SE47’ and SE50A proteins are diagrammed. The open boxes represent the processing products of SERA at merozoite release (47 kDa, 50 kDa, and 18 kDa). Broken lines indicate the borders of each block. The restriction sites introduced at the borders are shown. The arrows indicate the synthesized DNA fragments with the polarity from 5’ to 3’. The regions homologous to the active sites of proteases are indicated as shaded boxes

picomoles of each of the synthetic DNAs + and strands were mixed and incubated for 5 min at 85°C in 20 ~1 of 20 mM Tris-HCl, pH 7.0/50 mM NaCV2 mM MgCl,. The temperature of incubation was then continuously reduced at the rate of 5°C per 5 min to 55°C and then 5°C per 10 min to 25°C using a Zymoreactor II (ATTO, Tokyo) to anneal the complementary regions of both DNA fragments. The incubation was continued for another 10 min at 4”C, after which the reaction mixture was treated with DNA polymerase to convert the singlestranded into double-stranded regions. The annealed component was mixed with an equal volume of a buffer containing 20 mM Tris-HCl, pH 7.8/10 mM MgCl,/S mM dithiothreitol (DTT)/l mM of each of four deoxyNTPsl3 U of T4 DNA polymerase and incubated for 5 min at 4°C 5 min at 25”C, and then 120 min at 37°C. Each of the double-stranded DNA fragments used for constructing the SE47’ sequence were digested with Kpn I and Barn HI to clone them in pBluescript II SK+. Those DNA fragments used for constructing the SESOA sequence were digested with Xho I and Barn HI and cloned in the same vector plasmid. The elongation reaction by T4 DNA polymerase was omitted in constructing block 4723. To clone block 504, the insert DNA was digested with Xho I only and inserted between the Xho I and Eco RV sites of pBluescript II SK+. This was done because block 504 lacked a Barn HI site due to the deletion of 14 nucleotide residues at the 5’ end of the 504-DNA fragment which is one of two synthetic fragments used to form the 504 block. Nucleotide sequences were confirmed by dideoxy DNA sequencing”, and all except that of block 504 were identical to the original designed sequences. Block 504 had a deletion at the C-terminal end resulting in the absence of three amino acid residues. Expression and purification of the recombinant proteins XLl-Blue cells, freshly transformed with either PETSESOA or PET-SE47’ were grown in L-broth to 1.Ox lo8 cells mlat 37”C, at which time isopropyl-P-Dthiogalactopyranoside (IPTG, 50 pg ml- ’ final concentration) and M 13-pKM2 phage (multiplicity of infection 20) were added. After incubation for an additional 3 h, cells were harvested and stored at -80°C until further use.

SERA for P. falciparum malaria vaccine: T: Sugiyama et al.

Subsequent operations were carried out at 4°C or on ice. All centrifugations were performed at 17OOOgfor 15 min at 4°C with a RPR 18-2 rotor (HITACHI, Tokyo) except when indicated. SESOA protein was prepared as follows. Frozen cells (4.1 g) containing induced SESOA protein were thawed and suspended in 44 ml of STE buffer (25% sucrose/50 mM Tris-HCl, pH 8.0/5 mM EDTA/S mM 2-mercaptoethanol) containing 0.1 mg ml-’ lysozyme. The cells were lysed by freezing and thawing followed by repeated treatments with an ultrasonic disrupter (Tomy Seiko Model UR-200P) for 15-s periods until no intact cell were observed microscopically. The sonicated mixture was centrifuged and the pellet was resuspended in 44 ml of STE buffer. This mixture was resonicated, centrifuged and the pellet was saved. SESOA protein in the pellet was dissolved in 22 ml of 5 M guanidine-HCl in PBS (137 mM NaCY2.68 mM KCY8.10 mM Na,HPO,/1.47 mM KH,PO,, pH 7.2), and insoluble debris was removed by centrifugation for 30 min. The resulting supernatant was dialyzed against PBS to re-precipitate SESOA protein and the pellet resulting from centrifugation for 30 min was collected. The precipitated SESOA was resuspended in 22 ml of PBS. The suspension was homogenized by sonication before usage. SE47’ protein was prepared from 20 g of frozen cells as follows. The cells were suspended in 35 ml of STE buffer containing 0.1 mg ml-’ lysozyme and incubated for 20 min. The cells were then disrupted as were those in the SESOA preparation. The sonicates were centrifuged at 27000g for 60 min and the collected supernatants were treated with 5000 U of micrococcal nuclease (Sigma) which was added and stirred for 30 min at 37°C. The SE47’ protein was precipitated by adding solid ammonium sulfate with stirring for 30 min to give 30% saturation. The precipitate was collected by centrifugation. Since the precipitate did not dissolve after the removal of ammonium sulfate, it was washed initially in 30 ml of 50 mM Tris-HCl, pH 8.0/5 mM EDTA/S mM 2-mercaptoethanol and then washed in TEGB buffer (10 mM Tris-HCl, pH 7.60 mM EDTA/ 10% glycerol/l0 mM 2-mercaptoethanol) containing 1 M NaCl for 30 min. The pellet that resulted from centrifugation was dissolved in 15 ml of 8 M urea in TEGB buffer. The insoluble materials in the solution were removed by centrifugation and the supernatant was dialyzed overnight against 2 1 of TEG buffer (10 mM Tris-HCl, pH 7.611 mM EDTA/lO% glycerol). The preparations yielded 40.5 mg of SE47’ protein and 113 mg of SESOA protein. The proteins were either kept at 4°C for short period or at - 20°C for prolonged storage. During the SE47’ protein purification steps, we found that the protein precipitated with ammonium sulfate could hardly be re-solubilized after the removal of ammonium sulfate. When, however, the resulting precipitate was solubilized with 6 M urea, it remained in a soluble form even after the removal of urea. We, therefore, prepared SE47’ protein in a soluble or insoluble form. Amino acid sequence analysis The amino acid sequence of the N-terminal end of the protein was determined by Edman degradation with an Applied Biosystems 473A protein sequencer.

Vaccine 1996 Volume 14 Number 11 1071

Recombinant SERA for P. falciparum malaria vaccine: T Sugiyama et al. Table 2

Immunization

of rats with the recombinant

proteinsa

ELISA Tite? Group

Antigen

Adjuvanta

1

SE47’

41000 100000*= 90000 (77000)

2

SE47’

110000 49000 61000 (73000)

3

SESOA

+

SE47

SESOA

1ooooo* 100000 100000 (100000)

4

SESOA

5

Mixed

6

7

8

150000* 60000 100000 (103000)

+

29000 27000 (28,000)

60000* 50000 (55000)

Mixed

59000 15000 50000 (41000)

38000 34000 69000* (47000)

PBS


390* 6500 1100 (270)


640* 600 1000 (750)

PBS

aAntigen indicated was used for with (+) or without (-) Freund’s immune sera were determined protein as antigen. The average “Sera indicated by * were used growth inhibition experiments

immunization of rats in each group adjuvant. ?he ELISA titers of the by using purified SE47’ or SE50A values are given in parentheses. in Western blotting analyses and

Immunization of rats SD rats (female, 8 weeks old) were obtained from CLEA, Japan, Inc. Each immunization group consisted of two or three animals. The antigens were injected subcutaneously on days 0,2 1, and 42 as a suspension in PBS or as an emulsion with Freund’s complete adjuvant in the initial injection followed by Freund’s incomplete adjuvant at a 1:1 ratio (Table 2). Animals in each group (Nos l-6) received 500 ,ug of the indicated protein in the first injection and another 250 pug in the booster injections. The solubilized antigen was used for the Groups 1 and 2, whereas Group 3-6 received the insoluble form of the antigens. Groups 5 and 6 got a mixture of 250 yg of insoluble forms of SE47’ and SESOA in the first injection and a half the initial antigen dose for the booster injections. The control Groups 7 and 8, received PBS with Freund’s adjuvant or PBS only, respectively. The total volume of each protein used for injection was 0.5 ml for the first injection and 0.25 ml for the booster injections. Blood samples were collected on day 49 to prepare sera. Malaria culture P. falciparum FCR3 strain was maintained in culture according to the method of Trager and Jensen”, and Banyal and Inselburg2. The growth inhibition assay was

1072

Vaccine 1996 Volume 14 Number 11

done as follows. The parasitemia was adjusted to 1% by diluting the maintained culture with fresh erythrocytes when 80% of parasites were trophozoites and schizonts. The erythrocyte concentration was then reduced to 1% by dilution with complete medium. Each malaria cultures (200 yl) containing 1% erythrocytes with 1% parasitemia was incubated for 72 h at 37°C in a 96-well microtiter plate in the presence of diluted rat antiserum. Rat antisera used in inhibition experiments were adsorbed with uninfected human erythrocytes by adding 1 ml of a specific rat serum to 4 ml of the medium containing human serum and 1 ml of packed erythrocytes and incubated for 2 h at 37°C. The mixture was then centrifuged at 15OOgfor 5 min at room temperature in a KN-70C rotor (KUBOTA), collected and mixed with 1 ml of fresh packed erythrocytes. The mix was then incubated and centrifuged as above. The supernatant was saved and used in the growth inhibition assay. The parasitized erythrocytes were examined in Giemsa-stained thin smears and the parasitemia was scored by counting over 5000 erythrocytes in a slide. The observed parasitemia was divided by the parasitemia of the culture containing the control serum at the same dilution to give the parasite inhibition (%). ELISA and western blot analyses Vectastain ABC kit (Vector Laboratories) was used for ELISA2’ and Western blot analyses22. The ELISA performed using 2,2’-azino-bis-(3assay was ethylbenzothiazoline-6-sulfonic acid) as substrate and the SE47’ or SESOA protein as antigen. The ELISA titers were read at 405 nm with a microtiter plate reader (Titertek Multiskan MCC/340 MKII) and determined for an absorbance of 0.3. Western blot analysis was done using diaminobenzidine tetrahydrochloride as substrate. Parasite lysates were prepared as follows. Parasitized erythrocytes were made from a culture containing at least 80% mature trophozoites or schizonts. The erythrocytes were lysed with 0.075% saponin for 10 min at 37°C without agitation and the parasites were pelleted by centrifugation at 5000g for 5 min. The resulting cells were suspended in PBS and mixed with the sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)2’. Delplace et a1.5 reported that SERA was released from the parasitophorous vacuole by the treatment with 0.1% Saponin. However, SERA was found in the pellet but not in the supernatant using our method.

RESULTS Design of the artificial SERA genes We were unsuccessful in obtaining good expression of the SERA gene in E. coli when P. falciparum SERA cDNA was used (unpublished results). We therefore decided to synthesize two parts of the gene encoding SERA, rather than the whole gene, by changing the codons used in P. falciparum to those suitable for the E. coli translation machinery. These two gene sequences were inserted into the expression vector PET-3a. One gene sequence includes the region from amino acid residues 17 through 382 (SE47’). This region includes the amino acid sequence that can induce parasite-inhibitory antibody in Aotus monkeys”m’4. It has been reported

Recombinant SERA for P. falciparum malaria vaccine: 77 Sugiyama et al

A 108

~'-ATGAAAM~GTGAT~A~~A~~JGGKAAA OCCADACCGDPMTA~G~~~~A~CAC~~~~A~~~AC~C~CG _&RNVIRCTGESQTGNTG GGQAGNTVGDQAGSTGGSP N CAooOTA~CAGocccCA~CMC~~~A~~C~~MC~~G~~T~~~~AGT~C~A~A~A~C~~C~~~ QGSTGASQPGS SBPSNPVSSGHSVSTVSVSQTSTSS GAAAAACAAGATACCAlTCAGGTGMATCFGCGCT7XXWdAGATTATATGGGTlT BRQDTIQVK SALLKDYMGLKVTGPCNBNFInPLVPH

216

~~TACOOOCCCO~~~T~~~~~C~AT

ATTl'ATATTGATGTGGATACCGAAGATACCAATATAGAGCTCCGl'ACCACCCFGAMGAAACCAACAACGCGATCTCA IYIDVDTBDTNIELRTTLKETNNAISPESNSGSLBK

324

'IlTGAATCMACAGTGGTl'CACTGGAAAAA

AAAAAATATGTGMGCTF CCGTCAMCGGCACCACCGGTGAACAG4%3FTCAAGTACAGGCACCGFTCGCGGCG&TACCGAACCGkTlTCAGACTCGkGTAGCl'CTTCG KKYVKLPSNGTTGBQGSSTGTVRGDTEPISDSSSSS

433

540

648

DOCCC~~CrrCOACCG?TAMCCCCCOCarMCCMCAOMC GPDSPTVKPPRNLQNICETGKNFKLVVYIKBNTLII

756

~T~GT~ACD~~~GATACCACCOC

864

KWKVYGSTKDTTENNKVDVRKYLINBKETPFTSILI CATGCATATAAAGAACATAGGCACCMCCTGATfXAAA~AAAAACTACGC~ HAYKBHNGTNLIBSKNYALGSDIPBKCDTLASNCFL AGTGGTAACTTTAACAlT GAAAAATGCTTTCAGTGCGCGCTGCTGG~ SGNPNIBKCPQCALLVEKENKNDVCYKYLSBDIVSN TTCAAGGAGATCAAAGCGGAGTAA F K E I K A E***

GGGCTCAOACATTCCG

TGTGATACCCTCKXGl'CCAA~GCFTTCTG

972

1080

OAAMT~CDACaC~TACCTMOCOMOATAT~T~MT

1104

B 5'-ATGAAAGATGAMACMCTGCATTTCGAACCTGCAGGTGGAA J._KDBNNCISNLQVEDQGNCDTSWIPASKYHLBTIRC L ATDAM~ACGMCCGA~~~T~~~ATOT HKGYEPTKISALYVANCYKGEHKDRCDBGSSPMBFL CAAATTATCGAAGATFATGGCTTPCTOCCOOCGGM

QIIEDYGPLPAE

~TCA~M~~TACCA~~~C~TA~~A~A~~C~~~~

108

216 TGAAGATCACTGGATGhAXTT

TCPMCTA~~ATM~A~~~~~~G~T~~~ SNYPYNYVKVGEQCPKVBDHWMNL

TGGGATAACGGCAA GA'lCl'TGCATAACAAAAACGAACCGAATAGCC'lGGATcX3TMG=XTATACCGCGTACGAAAGCGA WXlTTTCACXZATMCATGGACGCGl'TT WDNGKILHNKNBPNSLDGKGYTAYBSERFHDNMDAF

TGCGGCGAlW.TACGATCATGCAGlTMCAlTG~ CGDDTADHAVNIVGYGNYVNSBGBKKSYWIVRNSWG

ACGUXACTAT~AAACTCAGM~

CCt?l'ACFGGGGCGAM!lTATTTl'AMG'RXACATGTACWCCCGACCCACTGCCATATCGM~TAG PYWGDEGYPKVDMYGPTHCH~E*"' FN

GMAMAAGTCATACXGA~GCGTMCX'CTFGGGGC

324

432

648

720

Figure 2 The nucleotide sequences and encoded amino acid sequences of SE47’ (A) and SE50A (6) are shown. Amino acid residues different from the original SERA sequence are underlined with the original residue shown in italic

that SERA is processed between amino acid residues 382 and 383, at the time of merozoite release from infected erythrocyte, to produce 47 kDa and 50 kDa fragmentsz4. The SE47’ sequence includes the 47 kDa fragment with an additional six amino-acid residues at the N-terminal end which is absent in the 47 kDa fragment. The other gene sequence includes the region from amino acid residues 568 to 802 (SESOA). The SESOA sequence encodes two amino acid sequences that are homologous with those common at the active sites of serineproteinases in other organisms’*“. The amino acid sequence of the SESOA region has been shown to be well conserved in the P. falciparum strains whose SERA sequences have so far been reported. We designed the DNA sequences encoding the SE47’ and SESOA proteins to reflect an E. coli rather than on a P. falciparum codon preference. If a codon used in the native SERA gene is employed frequently in P. falciparum. we changed that codon to one frequently used in E. coli. When a codon is rarely used in P.

falciparum,

we likewise changed it to that of rarely used codon in E. coli. An exception to the choice of codon changes was made when particular nucleotides defined restriction sites that were necessary for the construction of full nucleotide sequence. The codon usages in P. falciparum and E. coli were previously reported2s. As shown in Figure I, each gene was divided into several blocks. Each block was composed of two synthetic oligonucleotides to facilitate its construction. Single stranded DNA portions of the blocks were converted to double stranded with DNA polymerase and then cloned independently in a plasmid as described in Materials and Methods. After confirming the nucleotide sequences of the cloned blocks, they were ligated to each other using the unique restriction sites. The nucleotide and amino acid sequences of the constructed genes are shown in Figure 2(A) and (B). The constructed gene sequences were inserted into an expression vector PET-3a, to give PET-SE47’ and PET-SESOA.

Vaccine 1996 Volume 14 Number 11 1073

Recombinant SERA for P. falciparum malaria vaccine: T Sugiyama et al.

1234567 WgDsB) 45-

24-

Figure 3 SDS-PAGE of induced cells and final preparations of SE47’ and SESOA proteins. Each sample was electrophoresed in SDS-polyacrylamide gel (12.5%) and stained with Coomassie Brilliant Blue R-250. Lanes 1 and 7, molecular weight markers (bovine serum albumin, 66 kDa; egg albumin, 45 kDa; ttypsinogen, 24 kDa; /Mactoglobulin, 18.4 kDa; and lysozyme, 14.3 kDa). Lanes 2, 3 and 4, the whole cell lysate after induction of XLl-Blue harboring PET-3a, PET-SE47’ and PET-SESOA, respectively. Lanes 5 and 6, the final preparations of SE47’ protein and SE50A protein, respectively

Expression and purification of SE47’ and SESOA protein in E. coli The expression system used was that described by Sano et al.” The induced products produced from the SE47’ and SESOA sequences were 43 kDa and 26 kDa, respectively (Figure 3, lanes 3 and 4). These values were close to the expected molecular weights of the SE47’ (39 kDa) and the SESOA (28 kDa). The apparent molecular weight of SE47’ was higher than the expected value; however, it was ~47 kDa that is a value originally reported by Delplace et ~1.’ In the induced cells, the products of the SE47’ and SESOA genes were accumulated to the amounts of 15 and 30% of the total bacterial protein, respectively (Figure 3, lanes 3 and 4). After the induced expression of the SESOA sequence, four protein bands with similar mobilities were observed on SDS-PAGE (only two major bands are visible in Figure 3). The four electrophoresed products were separately prepared to determine their N-terminal amino acid sequences. Ten cycles of Edman degradation showed that each of these four products had the identical N-terminal sequence to that expected from the nucleotide sequence of SESOA gene (data not shown). The result did not rule out that some modifications might occur after the translation of SESOA gene. When the SE47’ sequence was induced in the E. coli cell, only one protein band was observed on SDS-PAGE. The N-terminal amino acid sequence of the purified product was identical to that expected of the SE47’ sequence. Immunization of rats with the recombinant sera proteins Rats were immunized with either SE47’ or SESOA, or both proteins with or without Freund’s adjuvant. The results of the immunizations are summarized in Table 2. Each group contains two or three animals and the antibody titer of the antisera was determined by ELISA. All the sera obtained from the animals immunized with the recombinant protein (Groups l-6) had antibody titers that were significantly higher than the controls (Groups 7 and 8).

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Vaccine 1996 Volume 14 Number 11

Figure 4 Western blot analysis of the malaria cell lysate by the anti-SERA sera. Each lane contained a lysate prepared from 7~10~ cells of F! falciparum FCRB. The parasite proteins were separated on 12.5% separating gel of SDS-PAGE and transferred to a PVDF membrane. Lanes l-8 correspond to the reaction with each of the representative sera from Groups 1 to 8

Animals immunized with Freund’s adjuvant gave a similar titer value to those immunized without adjuvant. The results also show that animals immunized with the mixture of SE47’ and SESOA proteins had lower antibody titers to the respective antigens than did animals immunized with individual proteins (see Groups 5 and 6). Further analyses were done using the antisera with the highest ELISA titers in each group (marked sera by * in Table 2). We examined whether the antisera raised against the recombinant proteins reacted with SERA prepared from parasite. Figure 4 shows a representative pattern of Western blot analyses obtained using the whole cell lysate of the P. fulciparum FCR3 strain and serum obtained from animal in each Group. The sera from Groups 1 to 6 strongly reacted with one protein band (lanes l-6) while the control sera did not (lanes 7 and 8). The estimated molecular weight of the protein that reacted with the antibodies was about 125000, which corresponded to that of the SERA (111 kDa). We, therefore, concluded that the antisera raised against both of SE47’ and SESOA recombinant proteins could recognize SERA protein. Effects of the antisera on in vitro malaria growth The parasite-inhibitory effects of the prepared sera were examined in in vitro cultures of the FCR3 strain in which 80% of parasites were in the trophozoite-schizont stages. The parasitemia of each culture was counted after 72 h of incubation with daily changes of media containing the diluted antiserum. The per cent inhibition of an antiserum at a particular serum dilutions was determined by correcting for the inhibitory effect of the same dilutions of the nonimmune control sera added to the parasite culture. Depending on the method of antiserum production, all the tested antisera, when diluted between lo- and 30-folds, inhibited parasites (Figure 5). The inhibitory activity of either the anti-SE47’ or anti-SESOA sera, produced in the presence of Freund’s adjuvant, were similar in the least diluted serum preparations

Recombinant SERA for P. falciparum malaria vaccine: 7: Sugiyama et al.

Figure 5 Effects of rat sera on the in vitro growth of P falciparum FGRB. The assays were performed as in Section 2. Sera used in each panel were (A) Groups 1 (o), 2 (O), (B) Groups 3 (o), 4 (O), (C) Groups 5 (o), 6 (0). Each plot was the averaged value from cultures prepared in triplicate

[Figure 5(A) and (B)]. The inhibitory activity of the antiSE47’ antiserum produced in the absence of Freund’s adjuvant was, unexpectedly, greater than the antiserum produced in the presence of the adjuvant [Figure 5(A)]. Conversely, the inhibitory activity of the anti-SESOA serum produced without the adjuvant was less than that of the antiserum produced with the adjuvant [Figure 5(B)]. The inhibitory effects of the Group 5 antiserum prepared from the animals that were vaccinated with adjuvant [Figure 5(C)] was similar to that of the serum produced by either antigen in the presence of adjuvant [Figure 5(A), (B) and (C)l. The inhibitory effect of the Group 6 antiserum at low dilutions was, however, similar to that exhibited by SE47’ without adjuvant [Figure 5(A) and (C)l. It seems likely that the parasite-inhibitory activity exhibited by the Group 6 antiserum was caused primarily by antibodies directed against SE47’. The simultaneous immunization with both antigens produced a two to fourfolds lower antibody titers than did the singleantigen immunization (Table 2). This is consistent with the finding that the inhibitory effect of the Group 2 serum at higher dilutions was significantly higher than those of Group 6 serum (Figure 5).

DISCUSSION

In our previous report, we found that the synthesis of the DHFR of P. falciparum in E. coli could be enhanced by designing a DNA sequence of the malaria DHFR gene with codons used in the DHFR gene of E. coli. We hypothesized that the local velocity of the translation of the gene product could significantly influence the correct formation of secondary and tertiary structures of the protein. We reasoned that the velocity of synthesis and thus folding of the protein could be affected by the presence of tracts of rare or frequent codons15. In this work we designed DNA sequences using E. coli codons to encode parts of SERA to obtain its efficient expression in E. coli. Since E. coli does not contain a SERA related gene, we could not generally follow our previous method. The averaged codon frequency of all P. falciparum genes and that of E. coli genes were, therefore, applied in selecting codons for the synthetic sequences. According to its codon usage in P. fakiparum, a codon in SERA cDNA was replaced by a similarly preferred codon in E. coli. The resulting DNA

sequences could be expressed in our expression system. The products of the SE47’ and SESOA SERA gene sequences accumulated in large amounts in E. coli and appear to provide a method for preparation of the antigens in a large scale. The purified products were immunogenic in both the rat and the mouse (data not shown). In the present study, the antibody titer against either SE47’ or SESOA was not significantly increased by using Freund’s adjuvant. The reason(s) that Freund’s adjuvant was not as effective as previously reported is not understood but may be attributable to the much higher amount of antigen used (500 pug and 250 ,ug for the first and booster injections), the structure of antigen obtained after purification, differences in amino-acid modification, and/or the use of a rat as an immunization model. We observed that anti-SE47’ antibody was more inhibitory than anti-SESOA antibody irrespective to the methods of immunization or the used animal. The amino acid sequence of SESOA protein was derived from the central part of the SERA where the consensus sequences of the serine protease are located. It suggests that the N-terminal domain of SERA is more accessible than the central domain to the antibodies or the binding of antibodies to the N-terminal domain is more toxic to the parasite than the binding to the central domain. The anti-SE47’ antibody induced in the absence of Freund’s adjuvant was more inhibitory than that induced in its presence. The reverse was true for the inhibitory effect of anti-SESOA antibody induced in the same way. These findings raise the problem of how to obtain a maximum parasite inhibitory effect from a mixture of antigens that respond differently to adjuvants. The enhancement of the effectiveness of the immune presentation of epitopes of one antigen may hinder the presentation of the epitopes of the other. The antigen contributing to the level of parasite-inhibitory activity induced by the antigen mixture in the presence of Freund’s adjuvant may be either anti-SE47’ or antiSESOA, in contrast, the antigen contributing to the level of parasite-inhibitory activity induced by the antigen mixture in the absence of Freund’s adjuvant would seem to be mostly attributable to SE47’. Since both the anti-SE47’ and SESOA titers induced in animals receiving a mix of both antigens were between one half to a quarter of that produced in animals immunized with only one of the antigens, a relative enhancement of immune responses directed toward specific epitopes that influence parasite inhibition may be occurring when the antigen mix is used. In an early stage of this work, we prepared mouse antiserum against SE47’ protein by immunizing mice with the minced SDS-polyacrylamide gel containing the SE47’ protein. That anti-SE47’ serum strongly inhibited the parasite growth (unpublished results), suggesting that the naturally folded structure of the protein may not be important for inducing the protective antibodies. It was previously reported that the antiserum prepared in mice with the SERA-1 recombinant protein, whose region was included in SE47’. had a strong inhibitory effect on the parasite growth in 13itro”. Although, the expression and immunization systems used for SERA-1 protein was different from those used in this work, these results are basically consistent with those previous observations. Vaccine 1996 Volume 14 Number 11 1075

Recombinant SERA for P. falciparum malaria vaccine: T: Sugiyama et al.

In the infected red blood cell, SERA is cleaved into at least three fragments at the time of merozoite release. The N-terminal fragment which includes the region of SE47’ and the C-terminal fragment are connected by disulfide bonds; however, the central fragment which includes the region of SESOA remains separate from the other two fragments. The observation that the antiserum against a part of SERA is growth inhibitory suggests one or more of the parts of SERA has an essential role in parasite multiplication. Further study with different anti-SERA antibody may help understand the function of SERA as well as the mechanism of growth inhibition expressed by the antibodies.

ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for International Scientific Research from the Ministry of Education, Science and Culture of Japan to T.H., the Inamori Foundation of Kyoto to T.H. and NIH Grant No. AI20437 to J.I.

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