Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

197

Gene, 46 (1986) 197-205 Elsevier GENE

1741

Conservation and antigenicity falciparum isolates (Recombinant proteins)

DNA;

of N-terminal

sequences

of CPM5

cDNA library; antigenic determinants;

from different Plasmodium

sequence comparison;

merozoite;

fusion

Randall F. Howard*, Feroza Ardeshir and Robert T. Reese Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, IMMll, La Jolla, CA 92037 (U.S.A.) (Received

May 15th, 1986)

(Revision

received

and accepted

August

Tel. (619)455-8057

lst, 1986)

SUMMARY

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichiu coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the Kl isolate [Hall et al., Nature 311 (1984) 379-3821, codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 3 17 (1985) 270-2731. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of 2 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous Kl isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. fafciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15 y0 of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates. Thus, the conserved regions may be important for maintaining the three-dimensional structure or function of GP185.

falcipantm,

* To whom

correspondence

and

reprint

requests

should

be

addressed.

ampicillin; isolate

Abbreviations: against

bp, base

pair(s);

RNA; GP, glycoprotein;

the

DHl [pRHClO] nonexpressing

aa, amino acid(s); &IO and &a sera, sera raised gel-purified extracts control

0378-I 119/86/$03.50

antigen

encoded

in

E. coli

and to a region of the same M, from the E. co/i DHl [pRHCa],

0 1986 Elsevier

respectively;

Science Publishers

Ap,

of P. falciparum;

oligodeoxynucleotide;

cDNA,

Honduras

carrier

polypeptide;

B.V. (Biomedical Division)

Honduras

PA,

to

l/CDC oligo,

polyacrylamide;

of E. coli DNA polymerase

SDS, sodium dodecyl sulfate;

state.

complementary

kb, 1000 bp; nt, nucleotide(s); P,

PolIk, Klenow (large) fragment resistance;

DNA I isolate,

I; R,

[ 1,designates plasmid-

I98

GP185 elicits an immune response

INTRODUCTION

owl monkeys P. falciparum is responsible type of human malaria.

a glycoprotein

lates produce after generically

homologs

fatal

During its asexual erythrocy-

tic cycle, the Honduras synthesizes

for a frequently

I isolate

of this organism

of M, 185 000; other isoof this glycoprotein,

referred to as GP185.

here-

An N-termi-

during

more, rat antibodies, N-terminal

a malarial

set of repeats.

for a nonglycosylated protein of M, 83000 (P83), which is present on the external surface of the

molecule

merozoite (Hall et al., 1984a,b; Holder and Freeman, 1984;1985; Howard et al., 1985; Lyon et al., 1986). In addition, GP185 shares epitopes with GP46, GP40, and GP 19 and may be processed to produce these molecules (Holder and Freeman, 1984; Howard et al., 1985; Stanley and Reese, 1985; Lyon et al., 1986). At least one of the smaller glycoproteins is also a merozoite surface molecule (Howard et al., 1985). By isolating cDNA clones which code for these proteins from an expression library of the Honduras I isolate, we sought to elucidate the structures and relationships of these molecules. GP 185 differs antigenically among various geographical isolates of P. falciparum as demonstrated with monoclonal antibodies (Hall et al., 1983; Howard et al., 1985). Despite this molecular heterogeneity, the successful vaccination of monkeys with GP185 from one isolate followed by challenge with a heterologous isolate of P. falciparum (Hall et al., 1984b; Perrin et al., 1984) have supported our working hypothesis that certain antigenic determinants on GP 185 are conserved among many isolates. During the course of this work, partial and fulllength DNA sequences of the GP185 gene from several different isolates of the parasite were reported (Hall et al., 1984b; Cheung et al., 1985; Holder et al., 1985; Mackay et al., 1985; Weber et al., 1986). Comparison of some of these data at the levels of restriction fragments (Schwarz et al., 1986) and nt sequence (Mackay et al., 1985) supported the idea that the nt sequence of certain regions of GP185 are conserved. Compilation of all the available GP 185 nt sequence data indicates that a prominent conserved region, in what is believed to be the N-terminal 15 y0 of the GP185 primary structure, is interrupted by a region containing isolate-specific aa repeats. We present evidence that this N-terminal region of

MATERIALS

containing

a different

Since this region of GP185

served and immunogenic, for inclusion

in the

react with GP 185 from

isolate

nal portion of GP 185 appears to serve as a precursor

to the

the conserved

set of aa repeats)

form of a fusion polypeptide, geographical

Further-

which were generated

part of GP185 (containing

region with the intervening another

in both man and infection.

is con-

it might be a candidate

in an antimalarial

vaccine.

AND METHODS

(a) Hybridization

with oligo probes

Two oligos with the sequences TGTCAAATACCTTTCAATCTTAAAATTCGTGCAAATGAATTAGACGTACTT and CTTGTGTTCGGATATAGAAAACCA were chosen based on the partial cDNA sequence of GP185 from the Kl isolate (Hall et al., 1984b). The oligos were synthesized by phosphoramidite chemistry on an Applied Biosystems Model 380A DNA synthesizer and gel-purified as described by the manufacturer. Both of the 32P-endlabeled probes hybridized to single restriction fragments of the same size in digests of Honduras I genomic DNA (not shown). The oligos were used separately to screen the Honduras I cDNA library (Ardeshir et al., 1985), and only those clones which hybridized with both probes were selected. A portion of the cDNA library was screened as described by Hanahan and Meselson (1980). The labeled oligos were hybridized to dot blots of the purified plasmid DNAs from the isolated clones to verify the results from the colony screen. (b) Nucleotide

sequence analysis

Intact cDNA fragments and fragments digested with DraI, HincII, HindIII, or RsaI were sequenced by dideoxy chain termination (Sanger et al., 1977) after subcloning into M 13mp8 and M 13mp9 (Vieira and Messing, 1982) using E. coli JMlOl as a recipient. Both strands of representative 770-bp and 960-bp DNA fragments were sequenced over the entire length of the DNA. A 770-bp fragment was

199

also sequenced from its 3’ end to a point upstream of the A,TA, stretch (located at nt 982-998 in Fig. 2) by chemical degradation (Maxam and Gilbert, 1980) following fill-in of the Sal1 site with [a-32P]dCTP and [ a-32P]dTTP by the large fragment of DNA polymerase. The consensus sequence was compiled with the DBUTIL program of Staden (1980) and further analyzed using the University of Wisconsin Genetics Center library of programs (Devereux et al., 1984).

1976). E. coli and Jensen, (Trager nique DHl[pRHClO] or DHl[pRHCa] was grown to A 550 -- 0.5-1.3 in LB plus Ap at 32°C incubated 2 h at 42°C and washed in saline. Both parasite and E. coli antigens were boiled in SDS-PA gel sample buffer. SDS-PA gel electrophoresis and immunoblotting were performed as previously described (Ardeshir et al., 1985).

(c) Protein expression by cDNA fragments

Rat antisera to the Cl0 or Ca antigen were produced using an emulsion of crushed PA gel pieces (containing the appropriate antigen) and Freund’s complete (primary immunization) or incomplete adjuvant (booster injections). Human sera (obtained through the London School of Tropical Medicine and Hygiene), sera from immune owl monkeys (karyotypes III and VI), and rat sera were adsorbed repeatedly with boiled DH l[ pCQV2] or with uninfected human erythrocytes.

Recombinant proteins were expressed by subcloning into the BamHI and Sal1 restriction sites of the expression vector pCQV2 which contains an ApR gene plus the temperature-sensitive repressor gene ~1857, early promoter pR, and a modified version of the cro gene of bacteriophage A (Queen, 1983). Plasmid ~185.15 was digested with EcoRI and then with BAL 31 nuclease (IBI, slow form) at 0.015 units/pg DNA for 5 min at 30°C. PolIk was used to blunt any ragged ends. After ligation of 32P-end-labeled octameric BamHI linkers to the DNA and digestion with BamHI and SalI, DNA and linkers were separated by gel filtration (Maniatis et al., 1982). pCQV2 DNA was treated with BamHI + Sal1 and digested with calf intestinal alkaline phosphatase before ligation with the cDNA and transformation of E. coli strain DH 1 cells to Ap resistance at 32°C (Ham&an, 1983). After shifting from 32” C to 42°C 14 colonies harboring pRHCl-14 and two nonexpressing colonies containing plasmids pRHCa or pRHCb were selected in a colony immunoscreen with monkey immune serum (Ardeshir et al., 1985). All 16 plasmids had inserts distinct from the BamHI-Sal1 fragment of pCQV2. Recombinant protein from DH 1[pRHC lo] (termed Cl0 antigen) was excised from SDS-PA gels after localization with Coomassie brilliant blue and immunoblot analysis. A corresponding region of a gel containing proteins from the nonexpressing clone DHl[pRHCa] (termed Ca antigen) was also isolated.

(e) Sera

RESULTSANDDISCUSSION

(a) Isolation and sequence analysis of GP185 cDNA clones

Using nonoverlapping, 32P-labeled oligo probes in a colony hybridization screen (MATERIALS AND METHODS, section b), 15 cDNA clones carrying p185.1-~185.15 were selected from a pool of 5900 clones. Since the library was amplified, these do not necessarily represent independent clones. Three of these clones had inserts of about 770 bp, and twelve had inserts of about 960 bp (Fig. 1). Data from restriction mapping studies and DNA sequence analyses of repre5’ ,

0 I

H I

960 bp:

pltw-5,

Honduras for HincII, by enzyme

Antigen was prepared from ghosts of parasitized erythrocytes cultured according to standard tech-

RDRC 11111

c

map

digestion.

cDNA fragments

3' -AAA S I

p185.1,.6,.15

and

the

relative

I GP185 cDNA clones. Restriction C; DraI,

R I

.7-.14

770 tip:

Fig. 1. Restriction

(d) Immunoblot analysis

R I

D; HindIII,

s I

positions

of the

sites are indicated

H; and RxzI, R, as determined

The overlap

of the 960-bp

and 770-bp

(solid lines drawn to scale) and the location

EcoRI (E)and Sal1 (S) linkers on these fragments

of

are shown.

Hond Well CAMP Kl-g Kl-c

Hand Well CAMP Kl-g Kl-c

Well CAMP Kl-g Kl-c

Hond

Hond Well CAMP Kl-g Kl-c

Hond Well CAHP Kl-g Kl-c

Hond Well CAMP Kl-g x1-r: .._ -

CAMP Well Kl-g

Hond

Hond Well CAMP Kl-g

Kl-g SGE2

We11 CAMP MAD20

Hond

G

G

C

C

-->

:

T CG

C

*****

C *

*

A

***

AGT

G G G

G CC G

AAAAAAAAAAAAAAAAA****

TG A

TTATA TTATA TTATA AAAA

I/l

1101

TGATT

A TT

______ GAATTGC

TII/ C TA ______ TAATTCAAGGAA***GTAAGAAAACAATTGATAAAAATAdGAA*****TGCAACTAAAAA ***** G A *k* ***** GA TG *** C

*

*

C

1080

1020

230 /II GlyLysMetGluAspTyrIleLysLysLys/// GGAAAAATGGAAGATTACATTAAAAAAAAATAAAAAAACCATAGAAAATATAAA*TGAAT

* *

960

900

840

760

720

660

>C A 200 190 CysAlaAsnAspTyrCysGlnIleProPheAsnLeuLys - ArgAlaAsnGluLeuAsp TGTGCTAATGATTATTGTCAAATACCTTTCAATCTTAAAAXTCGTGCAAATGAATTAGAC T T T T 220 210 ValLeuLysLysLeuValPheClyTyrArgLysProLeuAspAsnIieLysAspAsnVal CTACTTAAAAAACTTGTGTTCGGATATAGAAAACCATTAGACAATATTAAAGATAATGTA

A

180 170 GluLeuLeuTyrLysLeuAsnPheTyrPheAspLeuLeuArgAlaLysLeuAsnAspVal GAATTATTATATAAATTAAACTTTTATTTTGATTTATTAAGAGCAAAATTAAATGATGTA

G 160 150 ThrLeuCysAspAsnIleHlsGlyPheLysTyrLeuIleAspGlyTyrGluGluIleAsn ACTTTGTGTGATAATATTCATGGTTTCAAATATTTAATTGATGGATATGAAGAAATTAAT

C

-->: 140 130 LeuLeuThrIleLysGluLeuLysTyrProGlnLeuPheAspLsuThrAsnHisMetLeu TTGTTAACTATCAAAGAACTCAAATATCCTCAACTCTTTGATTTAACTAATCATATGTTA

G

G

120 110 AsnSerSerAspSerAspAlaLysSerTyrAlaAspLeuLysHiSArgValArgAsnTyr AATTCAAGTGATTCAGATGCTAAATCTTACGCTGATTTAAAACACAGAGTACGAAATTAC

SGE2

MAD20 Kl-g

Well CAMP

Hond

Kl-g SGE2

UADZO

Hond Well CAMP

Kl-g SGE2

MAD20

Well CAMP

Hond

Hond Well CAMP MAD20 Kl-g SGE2

Kl-g SGE2

MAD20

Hond Well CAMP

CAUj’ MAD20 Ki-g

Hond Well

Well CAMP MAD20 Kl-g

Hand

Well CAUP UAD20

Hand

Hond Well CAMP

Hond Well CAMP

AC AC

T T

GA

***************************T

***************************T

AC

A

AC A

A

A CT

G

G

AG

AC

A CA

CA

TC C T

TC C T

AACA

AACA

T CC C

T CC C

AA

AA

C

C

TCTG

TCTG

GGT

G

G

CC

CC

C

C

C

C

A 80 ySerGlyGlyScrValAla GlyGl GGTGG************************************TTCAGGTGGCTCAGTTGCT TTCAGGTGGCTCAGTTGCTTCAGGTGGCTCAGTTGC TACAAGTGCTAcaAGTGCTACAAGTGGTACAAGTGG A A TA AC C A A ******************TTCACTTCCTTGCTTCAGTTGC TCT CC TACAAGTGGTACAAGTGGTACAAGTGGTCCAAGTGG C A A TA AC C A ******************TTCACCTCCTTCACTTCAGTTGC TCT QG 90 100 ScrGlyGlySerValAlaSerGlyGlySsrGlyAsn8erArgArgThrAsnProSsrAsp TCAGGTGGCTCAGTTGCTTCAGGTGGTTCAGGTAATTCAAGACGTACAAATCCTTCAGAT

A GA

A

60 70 GlyThrScrGlyThrAlaValThrThrSerThrProGlySarLysGlySerValAlaSer GGAACAAGTGGAACAGCTGTTACAACTAGTACACCTGGTTCAAAGGGTTCAGTTGCTTCA

G

40 50 GluAspAlaValLeuThrGlyTyrSerLeuPhcClnLysGluLys~stValLeuAsnGlu GAAGATGCAGTATTGACAGGTTATAGTTTATTTCAAAAGGAAAAAATGGTATTAAATGAA

:-->

20 30 AsnThrGlnCysValThrHlsGluSsrTyrGinGluLcuValLysLysLsuGluAlaLsu AATACACAATGTGTAACACATGAAAGTTATCAAGAACTTGTCAAAAAACTAGAAGCTTTA

10 ktLysIleIlePhePheLeuCysSerPheLeuPhePheIleIle AGCTTTAATTCAATAATGAAGATCATATTCTTTTTATGTTCATTTCTTTTTTTTATTATA

: -->

TATTTTTAAATTATTAACTTATTTTATTATTATTATTTTTATTTATATATATTATTTATT

:-->

TATAAAAATAAGGCTAATGTAAAATGCAAAAATAAATGTATACATATTTTTGCTAAGTCA

TAAGGAAAATTATGTGAATAATATTAAAATTATAGTTATGATGTAATAAATAATTTTTAT A T

GAAAAAAAAAAAAAAAAGGGTTGTATATATACTTATAATATATATATATATACATATGTG C TC C C C

600

540

480

420

360

300

240

180

120

60

g 0

201

sentative clones from the two groups indicated that the DNA sequences overlapped about 670 bp. These two groups of cDNA clones allowed us to sequence 1052 bp of the GP 185 gene (Fig. 2), which is thought to be expressed as an approx. 6-kb species of mRNA (Hall et al., 1984b). Both fragments have 3’ poly(A) tails upstream from the Sal1 linkers. The 960-bp cDNA fragment was probably synthesized by reverse transcriptase following internal priming with the synthetic oligo(dT),, used in cDNA synthesis, since the A,TA, region of the DNA sequence (nt 982-998; Fig. 2) coincides with this poly(A) tail. A single nt difference, a C (770-bp fragments) to T (960-bp fragments) transition, was detected between the two groups of cDNA fragments within the 670-bp overlap (nt 881; X in Fig. 2). This polymorphism is consistent with the existence of either two alleles or multiple, nonallelic gene copies within the Honduras I isolate since the asexual erythrocytic stages of the parasite are believed to be haploid, although the exact copy number of this gene is not known (Hall et al., 1984b).

1983). Fourteen reactive colonies (with plasmids pRHCl-pRHC14) and two unreactive ones (with plasmids pRHCa, pRHCb) were isolated in a colony screen with immune monkey serum after temperature induction of the fusion polypeptides. Significantly, nt sequence analysis of the cDNA fragments in three of the reactive clones showed that all expressed antigen from a single reading frame. The first ATG codon in this reading frame (nt 256-258) is shown in Fig. 2 along with the deduced aa sequence. This is consistent with the methionine initiation codon proposed by Holder et al. (1985) and suggests that the Honduras I GP185 mRNA contains a 5’-untranslated region of at least 255 nt. Of these three plasmids, pRHC10 contained the longest cDNA fragment, coding for a single polypeptide (the Cl0 antigen) of M, 28 500 on an immunoblot (Fig. 3B, lane 5), similar to the M, of 25 300 calculated from the deduced aa sequence.

(b) Recombinant fragments

The cDNA and genomic nt sequences of GP185 from five other isolates of P. falcipantm were recently published (Hall et al., 1984; Cheung et al., 1985; Holder et al., 1985; Mackay et al., 1985; Weber et al., 1986). A comparison of these sequences and the Honduras I cDNA sequence is included in Fig. 2 and provides a great deal of information about the heterogeneity ofthe encoded protein in these isolates (obtained from infected patients in Central America, Africa, and Southeast Asia). There are no nt differences among the sequences of five isolates within 181 nt upstream from the proposed N-terminal methionine, and the sequence is conserved for 129 nt (43 aa) into the coding region. We speculate that this part of the gene is conserved

proteins

expressed

by

(c) Comparison

cDNA

The cDNA library was constructed such that the 5’ end of the mRNA was retained as the 5’ end of the cDNA in the expression vector pUC8. However, none of these 15 clones expressed recombinant protein which was recognized by either immune monkey serum or a pool of anti-GP185 monoclonal antibodies. To determine the correct reading frame as well as prepare recombinant parasite protein for further studies, the 5’ end of the 770-bp fragment was digested with BAL 31 nuclease, and the cDNA was subcloned into the temperature-inducible expression vector pCQV2 (Queen,

Fig. 2. Nucleotide.

sequence

given with the derived

for 1,052 bp of the Honduras

aa sequence

above the nt sequence.

Wellcome

(Well; Holder

et al., 1985), SGE2 (Cheung

(genomic

DNA,

Kl-g; Mackay

termed

Honduras

I isolate

sequence.

Gaps (*) were inserted

nt substitutions

are given. Colons (followed

shown.

to maintain

To conserve

space,

(Weber et al., 1986). Possible polyadenylation (///) are given for the Honduras

or preceded greatest

I GPl85

termed

homology.

signals (overlined; sequences.

strand

computer-aligned (Mackay

of the Honduras DNA

genes

of various

indicate

the beginning

Only in cases of divergence in the CAMP Proudfoot

I (Hond)

sequences

nt sequence

for GP185

is

from the

et al., 1985), CAMP (Weber et al., 1986), and KI

Kl-c, Hall et al., 1984b) isolates

by arrows)

a 108-bp insertion

I and Kl cDNA

gene. The coding

For comparison,

et al., 1985), MAD20

et al., 1985; cDNA,

of the GP185

isolates

isolate (between

and Brownlee,

which

overlap

and end, respectively, from the Honduras

with that of the of a partial

I DNA sequence

the ‘ca’ pair at nt 497-8)

1976) and the first two in-frame

gene are

is not shown stop codons

202

47kDa

-

47kDa -

30kDa

3OkDa -

18kDa

18kDa -

12345 Fig. 3. Binding of antibodies (A) or Cl0 antigen M, markers monkey

antibodies.

(panels A and B) indicate

because

antibodies

Sera were from patients

the 5’-noncoding

sera to the Ca and Cl0 antigens. PA gels and transferred

Recombinant

antigen

or immune owl monkey

Bacterial

to nitrocellulose

was detected

using human

extracts

membranes

containing

either Ca

along with prestained

sera at a 30-fold dilution

(lanes l-4)

serum at a 30-fold dilution (lane 5) and ‘251-labeled rabbit anti-owl

infected in Nigeria (lane 1) and India (lanes 3,4). Control human serum was used in lane 2.

the expected

due to anti-E. coli antibodies

monkey

on SDS-13%

in kDa on left margins).

goat anti-human

Arrows probably

and immune

(B) were electrophoresed

(indicated

and “sI-labeled

in human

12345

location

of the Cl0 recombinant

protein.

The other bands,

present

in both panels,

are

in the sera.

region plays a regulatory

role in GP185 expression, the N terminus of the polypeptide is necessary for the function of the expressed protein, or possibly because this region codes for one or more additional gene products. The region from nt 610-970 is also relatively conserved. Only a few nt substitutions are present in the four isolates that have been sequenced in this region. Separating these two regions of interisolate homology is an area of diversity (nt 385-610) which includes a region coding for tandem repeats of three aa. Such tandem repeats appear to be typical of many antigens of the Plasmodium species (Godson, 1985). Although the number and sequence of

the aa triplets in GP185 are isolate-specific, serine is retained as the first aa of each triplet in all six isolates. Relative to the Honduras I DNA sequence in this region, there is a spectrum of variation which ranges from the nearly identical nt sequence of the Wellcome isolate to the widely different sequences of the Kl and CAMP isolates. Since artifactual recombinational events in cloned DNA are especially likely in regions of repetitive DNA, it is possible that variation has occurred in genomic and cDNA clones of the P. falciparum isolates either during initial cloning and amplification or during propagation in Ml3 vectors for DNA sequence determination. However, we detected no differences

203

in the repeat units in the three different Honduras I cDNA fragments which were sequenced. Since two of these were independent clones, these results suggest that the repeat region is relatively stable and that no recombination occurred during the cloning procedures.Instead,thisvariationmightresultfromspecific mechanisms the parasite uses for changing this amino acid triplet repeat region while conserving the sequence on either side of the repeats. Presumably, changes in this region of GP185 either are not deleterious or are actually beneficial to the survival of the parasite. Between nt 841 and 1075, the Wellcome, Kl genomic (Kl-g), and Honduras I DNA sequences are nearly identical (two differences). In contrast, the Kl-cDNA (Kl-c) sequence is quite different from the Honduras I sequence (one deletion, nine insertions, and 16 nt substitutions) and, most surprisingly, therefore quite different from the Kl-g sequence. These differences plus additional substitutions 5’ of this region (Fig. 2) suggest that the Kl-c and Kl-g sequences might represent different copies of the gene, each of which could be independently transcribed. Although the exact copy number of the gene is unknown (Hall et al., 1984b), parasite clones from another isolate, T9, do contain different copies of the GP185 gene (Holder et al., 1985). Despite the numerous differences between the Honduras I and Kl cDNA sequences, both of their open reading frames terminate with multiple stop codons, predicting that they encode truncated polypeptides. In fact, they share the TGA at nt 1016-9 (Fig. 2). The nt sequence in this region of the Honduras I isolate cDNA is not merely an artifact of the dideoxy sequencing method since it was observed by dideoxy sequencing in all three of the 770-bp cDNA fragments and was verified by the chemical degradation method. The shortened Honduras I and Kl cDNAs might be explained by internal oligo(dT) priming and/or artifactual recombinational events during cloning or propagation. Alternatively, since the evidence suggests that these two isolates contain multiple copies of the GP185 gene, it is conceivable that the parasite actually synthesizes an altered transcript which encodes such a truncated polypeptide. Indeed, small amounts of GP185 gene-specific products of approx. 25 kDa were detectable in an immunoprecipitation assay of

radiolabeled parasite antigen with a GP185 monoclonal antibody (not shown). These observations dissuade us from presently dismissing the altered open reading frames as being mere artifacts. (d) Antigenicity

of

the

N-terminal

region

of

GP185

The deduced C-terminal sequences of GP185 from the Kl and Wellcome isolates (not shown in Fig. 2) are nearly identical, differing by six aa (Mackay et al., 1985). In contrast, the sequences from the Wellcome, T9, and CAMP isolates show little homology in the C-terminal region of GP185 (Schwarz et al., 1986, and analyses not shown). Consequently, at this time, the two regions near the N terminus of GP185 appear to be the major conserved sequences within this gene and, as such, may have value as an antimalarial vaccine component. Therefore, it was of interest to determine whether sera from individuals infected with P.falciparum recognized GP185. Sera from immune owl monkeys clearly recognized the Cl0 but not the control Ca antigen (lane 5, Fig. 3, A and B).

200kOa-

~

97kDa-

*il

(*

69kDa43kDa-

#

1 2

Fig. 4. Immunoblots and KI antigens. rocytes formed

3 4 of &lo

Proteins

(Honduras

were separated

qe

w

and &a

rat sera to Honduras

from schizont-stage

I isolate,

supernatants

from

body 4- 13-4B (lanes 2 and 8) using lZ51-rabbit or with a prebleed

antibodies. different

GP185

anti-mouse

with Ca antigen,

(lanes 5 and 11) and immune

of a rat immunized

with Cl0 antigen,

antiantiserum

and with

serum (lanes 6 and 12) using ‘?-rabbit

and PI06 from these two isolates

in size; the lack of P83 in the Honduras

due to the stage of growth

myeloma

monoclonal

(lanes 3 and 9) and immune

(lanes 4 and 10) from a rat immunized a prebleed

were per-

the parent

secreting

I

eryth-

lanes 7-12)

PA gel. Immunoblots

(lanes 1 and 7) and the hybridoma bodies;

parasitized

lanes 1-6; Kl isolate,

on a SDS-S%

with culture

5 6 7 6 9 101112

at which the parasites

anti-rat are slightly

I is probably were harvested.

204

Similarly, known

sera from six out of eight human

to have had malaria

the Cl0 antigen.

also specifically

subjects bound

ported by the Agency for International Contract DPE-0453-C-00-1017-00.

The results with three of these sera

samples

and computer Research

(Fig. 3, A and B, lanes 1,3, and 4) illustrate the range

Clinical

of the reactions

with the recombinant

Division

contrast,

serum at the same dilution

control

failed to bind ClO. These data indicate parasite

infection

an immune

monkey.

Furthermore,

response

determinants

In

(lane 2)

that during

this region of GP185 is capable

inducing antigenic

protein.

time were from the General

Center, Grant

of Research

Development Donor blood

RR00833

Resources.

No. 4394 from the Research

from the

This is publication Institute

of Scripps

Clinic.

of

in man and in the owl

it can be inferred that certain within

this

N-terminal

portion of GP185 are conserved in other isolates because the sera that were used in these experiments were randomly chosen from individuals in Africa and India who were unlikely to have been exposed to the Honduras I isolate. To test whether antibodies to the Cl0 fusion polypeptide recognized GP185, antiserum to Cl0 (aC10 serum) was raised in rats. This antiserum bound specifically to GP185 and P 106 on immunoblots of antigen from the Honduras I and Kl isolates (Fig. 4, lanes 6 and 12) as judged by the parallel use (lanes 2 and 8) of the GP185specific monoclonal antibody 4- 13-4B (Howard et al., 1985). In contrast, serum to the control Ca antigen (&a serum) did not bind these parasite polypeptides (lanes 4 and 10). Thus, some of the antigenic determinants on the Cl0 antigen resembled those on GP185. Furthermore, the c&l0 antibodies react with GP185 from the Kl isolate whose tandemly repeated aa differ from those in the Honduras I isolate. This, together with the observation that c&l0 serum also reacted in immunoblot assays with GP185 from six other isolates (not shown), suggests that the c&l0 serum was recognizing the conserved regions of the expressed Cl0 protein. We are currently examining whether some of these conserved determinants are on the surface of the intact merozoite and whether antibodies to them have a neutralizing capacity.

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