Int. J . Pepiide Protein Rex 36, 1990, 515-521

Synthetic, immunological and structural studies on repeat unit peptides of Plasmodium fakiparum antigens

PARAMJEET KAUR, PAWAN SHARMA, A. KUMAR, and V.S. CHAUHAN

tniernaf~onal Centre for Genetic Engineering and Biotechnology, NII Campus, Shaheed Jeet Singh Marg. New Delhi, India

Received 2 March, accepted for publication 9 June 1990

Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational charac- teristics with a common feature of - 30% and - 50% helical content in water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.

Key words: CD spectroscopy; malaria antigens; repeat sequences; synthetic vaccine

Primary sequence information is now available for several protein antigens from different stages of a malarial parasite, Plasmodium falciparum. One of the most intriguing features of the antigens characterised to date is that they contain tandemly repeated amino acid sequences inserted amongst non-repeated se- quences (1,2). These repeats vary in length, sequence, and number, and are also the immunodominant portions of the protein (3-6). Some antigens (Fig. 1, type 1) contain one continuous stretch of repeat units, whereas others (Fig. 1, type 2) contain dispersed blocks of repeats.

The abbreviations used are in accordance with the recommenda- tions of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (1984) European J . Biochem. 138, 9-37. In addition: Boc, tert.-butoxycarbonyl; DCC, dicyclohexylcarbodiimide; DMF, dimethylformamide; DCM, dichloromethane; DIEA, diisopropyl- ethylamine; TFA, trifluoroacetic acid; TFMSA, tri- fluoromethanesulphonic acid; TFE, 2,2,2-trifluoroethano1; CD, cir- cular dichroism; HPLC, high performance liquid chromatography; CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s ad- juvant; TT tetanus toxoid; BSA, bovine serum albumin.

FIGURE 1 Type 1 and type 2 classes of repetitive antigens of P. .firlcipurimi showing some cross-reactive epitopes. Vertical lines indicate regions of tdndemly repeating polypeptide sequences. The number of amino acids in each repeat is indicated to the right of each protein.

These repeat unit peptides have formed the basis for synthetic subunit malaria vaccines evaluated so far (7-10). However, none of these have proved entirely satisfactory. It has emerged from these studies that although antibodies directed against some repetitive sequences may be protective for the host, not all repeats are targets of protective response (10-12). Indeed, it has been suggested that these repeats may prevent or delay the development of effective immun- ity by interfering with the normal maturation of high affinity antibody responses and thus play a major role

515

P. Kaur et ml.

c E 4

in the parasite’s evasion strategy ( 13-1 5) . Whether or not such shared sequences relate to a biological func- tion such as mimicry, decoy. or immunosuppression, they may have implications for the host’s ability to respond to the parasite.

Antigenic analysis of cloned P . falcipancrn antigens has indicated a series of cross-reactions among the epitopes represented by such repetitive sequences ( 1 4,16). Antibodies cross-react at several different levels involving different epitopes within one block of repeats or in two or more blocks of repeats in the same molecule, and repetitive sequences in different antigens (1 1). Identification of a highly immunogenic. protective repeat unit peptide capable of inducing protective immunity is of current interest in designing a subunit vaccine. An examination of the primary structure indicates that these repeat units are mostly made up of acidic or hydrophobic residues. but primary sequence homology is not present in these sequences (3-6). It is conceivable that the cross- reactivity of antibodies to these repeat sequences arises due to some common secondary structural fea- tures. To address these questions, we have synthesised peptides consisting of repeat units corresponding to four different malaria antigens, namely. circum- sporozote antigen (CS), ring-infected erythrocyte surface antigen (RESA), falciparum interspersed re- petitive antigen (FIRA), and acidic basic repeat antigen (ABRA) (Fig. 2) and have shown that antibo- dies raised against these peptides cross-react signifi- cantly. We have examined by circular dichroism spec- troscopy the conformational preferences of these pep- tides in solution and found that these tend to adopt similar conformations, and that helical conformation appears to be a significant feature.

I I

EXPERIMENTAL PROCEDURES

Peptide synthesis, purification. und cliurocterization Peptides 1-5 were assembled by stepwise solid phase peptide synthesis on phenylacetamidomethyl (PAM)

2 ( E E N V E H D A ) ~ (RESA)

2 ( V TlQE PVTTQ E PVT I E E P ) ( FIR A 1

6 ( V NDE EDTN DE E DTNDDED 1 ( AB R A )

FIGURE 1

Synthetic peptides corresponding to repeat units of malaria antigens.

resin on a manual or automatic peptide synthesizer (Applied Biosystems, Model 430 A) (17). Boc amino acids were coupled as the preformed symmetric anhy- drides in DCM except for Gln and Asn, which were reacted as preformed HOBt esters in 50% DMF in DCM. Completeness of the coupling reactions was checked by the ninhydrin test (18). The protective group removal and the cleavage of peptides from the resin were accomplished with trifluoromethanesul- phonic acid-thioanisole in trifluoroacetic acid ( 19). The deprotected peptides were extracted in 10% acetic acid, lyophilised, and purified by reverse-phase high performance liquid chromatography (RP-HPLC) on Aquapore RP-300, 10.0mm ID x l00mm long column using linear gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid (Fig. 3). The identity of the peptides was established by amino acid sequencing on protein sequencer (Applied Biosystems, Model 477A) with on line PTH detection using standard protocols.

J

P u r i f i e d C r u d e

F I G U R E 3 HPLC profile of peptide 1 o n Aquapore RP-300 (4.6 x 30mm). Solvent A: 0.05% TFA in water. Solvent B: 70% acetonitrile in water. Buffer B was acetonitrile. The peptide was eluted with a linear gradient of 0- 20% B in 20 min, 2040% B in 5 min, at 40% B for 5 min and 40-0% B in 5 min at a flow rate of I . 5 mL/min. The eluent was mouitorcd at 220nm.

516

Malaria antigens

490 nm using Bio-Tek microplate reader. Results of ELISA were expressed as A,, values representing absorbance (A4w) obtained with the immune sera after subtraction of the corresponding preimmune values. The A,,, values in the (ii) and (iii) sets of control wells were in general < 0.05.

Circulur dichroism studies CD measurements were carried out on a Jasco spectropolarimeter in 5 mm cell at room temperature (25’). Molar ellipticities were calculated using the equation:

H x M x 3300 [a4= c 1

where H = observed dichroic absorbance in degrees, M = mean residue wt. of the peptide, C = con- centration of peptide in mg/mL and 1 = pathlength in cm .

Immunological methods For preparation of immunogens, each of the five syn- thetic peptides was conjugated to a carrier protein, bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent essentially as described by Reich- lin (20). Outbred, female albino rabbits, weighing 600-8OOg each were procured from the National In- stitute of Immunology, New Delhi. Five groups of two animals each were immunized with the respective pep- tide-BSA conjugate. Each animal received 600 pg of peptide-BSA conjugate emulsified in the complete Freund’s adjuvant and injected subcutaneously at multiple sites (three to four). Animals were boosted on day 49 with a similar dose of respective peptide-BSA conjugate emulsified in the incomplete Freund’s ad- juvant (IFA).

Sera obtained a week after the boost (day 56) were tested in an ELISA for the presence of antibodies to the immunizing agent as well as for those cross-react- ing with the other synthetic peptides used in this study (21). Initial experiments showed that peptides con- jugated to tetanus toxoid (TT) coated the wells more uniformly and efficiently than the carrier-free pep- tides, as evident from the marked improvement in reproducibility and sensitivity of the assay (data not presented). Subsequently, only the peptide-TT con- jugates were used to coat the wells of the microtiter plates. Each serum at a dilution of 1: lOOO was in- cubated in duplicate wells coated respectively with each of the five peptide-TT conjugates, followed by incubation with the affinity purified swine anti-rabbit immunoglobulins labeled with horseradish peroxidase (Dakopatts A/S, Denmark; 1:2000 dilution). Appro- priate controls included in each plate of every experi- ment were (i) a positive and a negative reference serum, (ii) three sets of duplicate wells in which in- cubation with coating antigen, test serum or enzyme- labeled second antibody was omitted, and (iii) a set of duplicate wells coated with antigen but not incubated with any antibody. The enzyme reaction was de- veloped using a freshly prepared solution of o-phenyl- enediamine dihydrochloride and hydrogen peroxide in 0.1 M citrate buffer, pH 5.0. The reaction was stopped with 8~ H, SO, and absorbance (A 490) recorded at

RESULTS

Peptides 1 and 2 correspond to different repeat regions of RESA. Peptide 3 belongs to the hexapeptide repeat region of FIRA while 4 represents the corresponding hexapeptide repeat region of ABRA. Peptide 5 repre- sents the tetrapeptide repeat region of the most well studied CS protein. Peptides 1-5 were synthesised by solid phase methodology and purified to homogeneity on RP-HPLC. Each peptide was conjugated to BSA and the conjugate was used as immunogen. All syn- thetic peptide-conjugates turned out to be highly im- munogenic. In most cases antibody levels remained detectable even at a dilution of

Several blood stage antigens have been shown to cross-react immunologically but a clear reason for function of this phenomenon remains unclear except that all malarial proteins identified to date contain repeat sequences (14, 16). Interestingly antibodies raised against the synthetic repeat sequence peptides also showed remarkable cross-reactivity (Fig. 4). A quantitative estimate of this reactivity using ELISA test is detailed in Table 1. It is noteworthy that the peptides corresponding to proteins from different stages of the parasite also show an adequate level of cross-reactivity. CS protein, the major sporozoite stage antigen and a prominent blood stage protein (CRA) have been reported to cross-react at protein level but its cross-reactivity with other antigens has not been reported (22, 23).

In TFE, the CD spectra of peptides 1,2, and 3 show two negative CD bands with extrema at - 208 nm and - 223 nm. Exact band positions for these peptides are listed in Table 2. The positions and intensities of the bands suggest that the helical conformation is discern- ible for peptides 1, 2, and 3 in this solvent. In water two negative CD bands at - 206 nm and 222 nm were observed for peptides 1 and 2, suggesting the presence of helical conformation, whereas for peptide 3, a strong negative band at 201 nm and a weak negative band at 222 nm were observed in water, indicating largely disordered conformations in this solvent. However, in TFE the helical content is considerably higher than that in water for all the three peptides (Table 2).

In water, the CD spectra of peptide 4 showed two negative CD bands at 207 nm and 227 nm in addition to a strong positive CD band at 21 5 nm (Table 2). This spectrum is characteristic of peptide in random con- formation (24). However, in TFE, the positive band at

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P. Kaur ef al.

FIGURE 4 Cross-reactivity among synthetic malaria repeat peptides. Peptide 1

anti-RESA serum: Peptide 2 anti-RESA serum; Peptide 3 anti-FIRA serum; Peptide 4 anti-ABRA serum: Peptide 5 anti-CSP serum.

Antiserum to immunogen"

ELlSA A A,,, values using the peptides belowh to coat the wells'

Peptide 1 Peptide 2 Peptide 3 Peptide 4 Peptide 5 RESA RESA FIRA ABRA CSP

Peptide 1 RESA Peptide 2 RESA Peptide 3 FIRA Peptide 4 ABRA Peptide 5 CSP

3.21

I .67

0.18

1.28

0.55

I .29

I .28

0.54

1 . S l

0.35

2.33

2.47

I .34

0.54

0.64

I .72

1.59

0.15

2.33

0.64

I .05

0.83

0.13

1.43

1.64

'Each peptide was conjugated to Bovine Scruni Albumin (BSA) as carrier and then used as immunogen. hEach peptide was conjugated to tetanus toxoid (TT) and this conjugate was used to coat the wells. 'Each value represents a mean of values obtained for duplicate wells. Each serum was tested at 10-lold serial dilutions ranging from lo..' through 10 ', Values given in this table represent those obtained at 10 ' dilution of each serum. although in a large majority of titrations. antibody level remained detectable even at a dilution of 10 '.

TABLE 2 CD data !or malarirr repear unir peprides

Medium Peptide i. [el x lo-' M L [O] x 10 ' M

H, 0 I 2 3 4

5

TFE 1 2 3 4 5

206 208 20 1 207 215 200

207 208 208 2OX 205

- - 15.5 - - 11.0 -21.0 - 1.0 + 4.9

-- 22.0

-- 28.5 -- 30.5 -- 26.0 - 3.0

-- 22.5

222 223 220 227

225

220 223 224 225 222

- 6.25 - 4.25 - 6.0 - 1.3

- 6.75

- 15.0 - 17.5 - 10.0

- 12.75 - 0.975

2 15 nm completely disappears and only two negative bands at 208 nm and 225 nm are observed. These ne- gative bands are less intense than the corresponding CD bands for other repeat unit peptides.

The CD spectrum of peptide 5 in water showed a strong negative band at 200nm and a weak negative band at - 225 nm. This type of spectrum is character- istic of helical conformation in equilibrium with the random structure. Like other peptides, peptide 5 also showed a higher degree of ordered structure in TFE with two strong negative CD bands a t 205nm and 222 nm. Theoretical con formational predictions for peptides 1 to 5 (Table 3), based on Chou-l'asman calculations, were related to actual conformations measured by circular dichroism (25, 26). The spectra were fitted with the reference spectra of polylysine

51 8

Malaria antigens

TABLE 3 Chou-Fasmun calculutions on malaria repeat unit peptides

Residues 1-4 Residues 5-8 Residues 9-12 Residues 13-end

(pa) (Pb) (Pt) (pa) (Pb) (pt) (pa) (Pb) (pt) (pa) (Pb) (Pt)

Peptide 1 1.23 0.71 0.92 1.23 0.71 RESA Peptide 2 1.23 0.71 0.92 1.30 0.68 RESA Peptide 3 1.03 1.42 0.76 1.02 0.93 FIRA Peptide 4 1.09 0.84 1.07 1.02 0.73 ABRA Peptide 5 0.87 0.68 1.31 0.87 0.68 CSP

(27). The [ O ] , values and the peak extrema for pep- tides 1-5 in different media are summarized in Table 2.

DISCUSSION

All the malarial antigens described so far contain repeat structures (11, 14). Most of these antigens are conserved, highly immunogenic and have been pointed out as potential targets for a malaria vaccine (28). Synthetic peptides from the repeat region of such antigens are the subject of the present study. Poly- morphic antigens that are not targets of vaccine development, although containing repeat units, like S-antigen, were not included in this study (29).

It is clear from Table 3 that peptides 1 and 2 have a strong potential to adopt helical conformation. Calculations from CD data yield a helical content of 56% and 58% for peptides 1 and 2 in TFE, whereas in water the values are 29% and 26% respectively. It may be concluded that these two peptides have a tendency to acquire substantial helical character in solution. It is interesting to note that both these pep- tides are highly immunogenic and the antibodies raised against them show a high level of cross-reactiv- ity. However, their tendency to adopt helical structure may or may not be related to their high antigenicity.

It appears (Table 3) that both helical and 8-struc- ture are probable for peptide 3. CD calculations in- dicate that this peptide has a helical content of 52% and 25% in TFE and water respectively. These values compare well with those for peptides 1 and 2. Anti- bodies raised against peptides 1 and 2 recognised FIRA antigen peptide 3 quite efficiently. It may be pointed out that at the protein level these two antigens, RESA and FIRA, show remarkable cross- reactivity (1 1). However, when antibodies were raised against peptide 3 as an antigen, they reacted rather poorly with peptide 2, and even more so with peptide 1. The reason for this observation is not quite clear.

0.92 1.23 0.71 0.92 1.23 0.71 0.92

0.96 1.23 0.71 0.92 1.30 0.68 0.96

0.97 1.03 0.83 1.08 1.10 0.93 0.87

1.18 1.25 0.53 1.13 1.00 0.75 1.26

1.31 0.87 0.68 1.31 0.87 0.68 1.31

Whether antibodies against FIRA cross-react with RESA at protein antigen level or not, has not been reported so far.

CD spectra of peptide 4 showed different features when compared with other peptides. From the posi- tions and intensities of the bands (Table 2) it appears that in TFE this peptide begins to acquire some sort of organised structure, although compared to CD of other peptides in TFE, the extent of helical structure is much lower. Chou-Fasman calculations predict that helical and turn structures are almost equally prob- able for peptide 4. Antibodies against peptide 4 cross- reacted with peptides 1 and 2 appreciably, and also with sporozoite stage repeat structure peptide 5, but not so well with peptide 3. It is interesting to note that peptides 1, 2, and 4 share an overall high content of asparagine and glutamic acid and a high content of asparagine in peptide 5, whereas peptide 3 is com- pletely devoid of asparagines.

Peptide 5 corresponds to the most abundant and highly conserved antigen of sporozoite stage of malaria parasite ( 5 , 30). This antigen has been the basis for the sporozoite stage subunit vaccines and as such, a subject for extensive studies (7-9). The observa- tion that repeat sequences of CS proteins and some blood stage antigens frequently contain asparagine and/or proline led to speculations that their confor- mations are rich in P-bends (30). Gibson & Scheraga (3 1 ) have predicted two stable helical conformations for the tetradodecapeptide (NANP), , whereas Carson et al. (32) have predicted it to be an unusual structure, a 12,, right-handed helix. 'H NMR studies on (NANP), indicate a fast conformational averaging in water, whereas in water-methanol, mixed sequences of 8 I turns and half turns appear to exist.

Estimation of secondary structural parameters of peptide 5 yielded reasonable helical content (32% and 41% respectively in water and TFE). Antibodies raised against peptide 5 cross-reacted with all other

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P. Kaur et al.

peptides corresponding to blood-stage antigens, al- though the level of cross-reactivity is somewhat lower when compared with that of other peptides.

Repeat unit sequences have been included in syn- thetic vaccines (7-9). However, it appears that in malaria a multiple epitope polypeptide will be more suited to provide a protection than a vaccine based on a single antigen (10). High immunogenicity of the epitopes constituting such a polypeptide is crucial. In this context our finding that peptide 1 is the most immunogenic and cross-reactive of the repeat pep- tides, may be of importance in designing a synthetic polypeptide vaccine. Interestingly, this tetrapeptide repeat (peptide 1) has also been suggested to be a strong T-cell epitope, and of higher immunogenicity than the octapeptide repeat (peptide 2) (34, 35). It is also noteworthy, and perhaps important, too, that in most repeat units of malaria antigens there is an abundance of amino acids which are well accepted in helical motifs (25). Another prominent feature of these repeat units, a relative abundance of as- paragines, might also reflect on secondary structural preferences of these sequences. Asparagine, although recognised as a weak a-breaker, is known to be the most common amino acid found in fi-turns in protein structures (26, 30). Repeating turn motifs can be ex- pected to stabilise spiral or helical motifs, as has been predicted for (NANP)n repeat polypeptide (3 I) . It has also been proposed that, for a peptide chain consisting of multiple tandem repeats, cooperative interactions will ensure that the most suitable conformations are helical or near helical (31). Although structure deter- mination of small polypeptides by circular dichroism or theoretical predictions alone may not be entirely satisfactory, the CD studies of repeat sequence pep- tides suggest that helical or near helical structures may be the dominant ones. It is tempting to suggest that this similarity in secondary structural features may be in some way responsible for the cross-reactivity ob- served among peptides 1-5 (36).

A C K N O W L E D G E M E N T S

Efficient typing of this manuscript by Ms. Gita Srinivasan and Ms. R. Radha Is gratefully acknowledged.

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Address:

Dr. V.S. Chauhan International Centre for Genetic Engineering and Biotechnology NII Campus Shaheed Jeet Singh Marg New Delhi - 110 067 India

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