Immunogenicity of Plasmodium falciparum and Plasmodium vivax circumsporozoite protein repeat multiple antigen constructs (MAC)

PII: SO264-410X(97)00290-9

&cone, Vol. 16, No. 9110. pp. 982-988, 1998 C 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-41 OX/98 $19+0.00

ELSEVIER

Immunogenicity of Plasmodium falciparum and Plasmodium vivax circumsporozoite protein repeat multiple antigen constructs (MAC) Venkatachalam Udhayakumar*$, Ae Saekhou”, Sunan Fang?, Danny Jue?, Robert M. Wohlhueterf and A.A. Lal” In this study we characterized the immunogenic properties of three different multispecies multiple antigen constructs (WCs) carrying the circumsporozoite protein (CSP) repeats of human malaria parasites, Plasmodium falciparum and P. vivax We synthesized tetrameric MACs containing the antigenic repeats from the CSP of P. vivax-like parasite in two arms and CSP repeat sequences of either P. vivax type-l (vivax-likelvivax type-l MAC), P. vivax type-2 (vivax-likelvivax type-2 MAC), or P. falciparum (vivax-likelfalciparum MAC) in the other two arms. Mice of four different genetic backgrounds (H-z’, H-2”, H-2’: and H-2k) were immunized with these MACs in Freund’s adjuvant. All three MAC preparations were found to elicit antibodies to P. vivax-like CSP repeats in BlO.BR, BlO.A, and C57BLl6 mice. On the other hand, in BlO.D2 mice only vivax-likefvivax type-l MAC, but not the other two MAC’s induced antibodies to the P. vivax-like CSP repeats. In mice immunized with vivax-likelvivax type-l MAC, antibodies to P. vivax type-l CS repeat peptides were induced in BlO.BR, BlO.A, and C57BLl6 mice, but not in 810.02 mice. Antibody responses to P. vivax type-2 repeats were not induced in any of the four strains of mice that were immunized with vivax-likelvivax type-2 MAC. While BlO.BR, BlO.A, and C57BLl6 mice produced antibodies to NANP repeats of P. falciparum CSP following immunization with vivaxlikelfalciparum MAC, BlO.02 mice failed to elicit antibodies to this repeat. All the sera that showed positive reactivity to peptides in enzyme-linked immunosorbent assay were found to react with sporozoites by IFA. In conclusion, these results showed that naturally immunogenic epitopes from diflerent species of malaria parasites can be incorporated in a single vaccine construct to induce immune responses against multiple epitopes. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Malaria

vaccine:

circumsporozoite

protein;

peptide

vaccine

Plrrsrnodium,frrlcipatwn and I? vivax arc the most prevalent species of malaria parasites, and they account for 300-500 million cases of malaria and a death toll of l-2 million annually’. Although earlier studies have shown that immunization with irradiated sporozoites can provide protection against malaria in human volunteer?. large scale vaccination with either irradiated sporozoites or blood stage parasites are limited *Molecular Vaccine Section, Immunology Branch, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30084, USA. tBiotechnology Core Facility Branch, Scientific Resource Program, National center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30084, USA. $Author to whom all correspondence should be addressed. (Received 2 January 1997; revised version received 3 November 1997; accepted 3 November 1997)

982

Vaccine

1998 Volume

16 Number

9/10

because of practical limitations and biosafety considerations. Therefore attempts are being made to develop synthetic or recombinant vaccines as adjuncts to control malaria along with other vector control efforts. Circumsporozoite protein (CSP) has been most extensively studied as a vaccine candidate antigen in malaria. Although CSP based vaccines were found to induce complete protection in rodent models, their success in human vaccine trials have been limited (reviewed in ref. ‘). Earlier attempts to USC the P,fdciparum CSP repeats linked to heterologous T-helper epitopes were only partially successful in human vaccine trials against sporozoite challenge”.‘. Currently, attempts are being made to improve the efficacy of synthetic malaria vaccines by incorporating suitable protective epitopes in a configuration to induce appropriate immune responses.

lmmunogenicity

of MACs with dual peptide sequences:

antigen peptidc (MAP) system The multiple developed by Tam has become an important technological advancement in designing synthetic pcptide vaccines’. Unlike linear peptides, MAPS, which are synthesized as branched peptides. have some distinct advantages. Thcsc include: (i) high immunogenicity; (ii) rapid development of antibodies; (iii) long lasting memory; (iv) the fact that T-helper epitopes can be included without a ne’zd for conjugation to a carrier protein and therefore the ratio of T- and B-cell epitopes in the MAP vaccine can be controlled; (v) induction of antibodies highly specific to target antigens including conlformation dependent epitopes; and (vi) the fact that multiple epitopes can be incorporatcd”. The immunogenic properties of the MAP system have been evaluated using CSP based malarial epitopes”‘-‘5. The MAPS were found to induce higher antibody titrc than the linear peptides and these antiMAP antibodies were found to react with sporozoites. Mice that were vaccinated with MAPS were also found to be protected against a subsequent lethal challenge with rodent malaria parasites”‘.“. Recently. we have investigated the immunogenic potential of multiple antigen constructs (MAC), which are a new generation of MAP-like molecules designed to present heterologous epitopes in a branched tetravalent lys-Nlcu core. WC have found that MACs which incorporated protective cpitopes from the CSP of p berghei and /? yuefii induced protection against sporoSimilarly, MACs which contained zoite challenge’“. universal T-helper epitopes from tetanus toxoid and B epitopes from l? viva.~ type-l repeats induced antibodies against protective B cpitopes in saimiri monkcys’7. Since inclusion of T-helper epitopes from a hetcrologous source will not be suitable for inducing the memory response during the natural parasite exposure, in this study we investigated if presentation of two heterologous natural epitopes in a MAC can induce antibodies to both epitopes and whether these two epitopes can complement each other in terms of T-helper function. We developed three tetrameric MACs containing the antigenic repeats from the CSP of I! v&ax-like parasite in two arms and CSP repeat sequences of either I? vivu~ type- I, P vhav type-2, or l? ,fafciparum in the other two arms. The results of this study show that the MACs are highly immunogenic and antibodies to two different heterologous epitopes in alternate arms can be induced.

MATERIALS AND METHODS Synthetic MAC vaccine preparation Three different MACs, which contained CSP repeat sequences of different human malaria parasites, were synthesized and their schematic structure is given in Figure I. The CSP repeats [(APGANQEGGAA) x 21 of fl vivax-like parasite was incorporated in two alternative arms of all three tetra-branched MACs. In the other two arms CSP repeats of [(GDRAAGQPA) x 2]of I? vivax type-l (vivax-like/vivax type-l MAC), [(ANGAGNQPG) x 21 of p vivax type-2 (vivaxlike/vivax type-2 MAC), or [(NANP) x 51 of t? ,fakiparum (vivax-like/falciparum MAC) were included. The MACs were synthesrzed with the novel use of

VIVAX-LIKENIVAX

V. Udhayakumar

et al.

TYPE 1 MAC PGANQEGGAAAPGANQEGGA NLeu-Lys GDRAAGQPAGDRAAGQPA

1

1

NLeu PGANQEGGAAAPGANQEGGAA NLeu-Lys

VIVAX-LIKE-NIVAX

1

TYPE 2 MAC PGANQEGGAAAPGANQEGGA NLeu-Lys NGAGNQPGANGAGNQPGI

NLeu PGANQEGGAAAPGANQEGGAA NLeu-Lys NGAGNQPGANGAGNQPG

VlVAX-LIKE/ FALCIPARUM MAC PGANQEGGAAAPGANQEGGAA NLeu-Lys

1

NANPNANPNANPNANPNANP NLeu NLeu-Lys

PGANQEGGAAAPGANQEGGAA

1

NANPNANPNANPNANPNANP Figure

1

Schematic

structure of MAC

N-( Fmoc)-Lysine-E-( Dde)-OOH (Novabiochem, San Diego, CA). Lysines were added as the last amino acids to a norleucine-lysine-(norleucine)? core in order to facilitate tetrameric branch formation. Fmoc chemistry was used throughout the synthesis. After Fmoc deprotection, the first peptide was synthesized on the deprotected groups. t-Boc protected N-terminal amino acid was then incorporated into the peptide to prevent further addition. The Dde-protected groups are then removed with hydrizine and the second peptides sequentially synthesized. Standard Fmoc cleavage and de-protection protocols were then followed to make MACs that contain two copies of each of two different epitopes. Linear peptides The linear synthetic peptides PL35 [(APGAN[( GDRAAGQPA) x 31, PL2 QEGGAA) x 31, PLl repeats and NANP [(ANGAGNQPG) x 31, the CSP repeat sequences [(NANP) x 51 re P resenting of p ~ivawlike-parasite, F1 vivax type-l. and type-2, and p falciparnm respectively were made at the CDC-Biotechnology Core Facility as reported elsewhere’“. These peptides were found to be > YO% pure by HPLC analysis and were used in enzymelinked immunosorbent assay (ELISA) for antibody determinations. Mice and immunization Inbred strains of mice such as BIO.Br, BlO.A, BlO.D2, and C.57 BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and they were used at 8-12 weeks of their age. Groups of five mice were immunized intraperitoneally with 50 /(g of MACs emulsified in Frcund’s complete adjuvant. Then,

Vaccine

1998 Volume

16 Number

9/l 0

983

lmmunogenicity

of MACs with dual peptide sequences:

2 weeks later, the mice received booster injections in the same way with MACs emulsified in Frcund’s incomplete adjuvant. On day 241 after the primary immunization an additional booster was given by emulsifying MACs (50 /(g per mouse) in Freund’s incomplete adjuvant. The control group of mice received a similar amount of adjuvants only. Enzyme-linked

immunosorbent

Immunofluorescent assay Sporozoites from infected salivary glands were dissected into normal saline with 20% fetal bovine serum. The sporozoites were air dried and stored at -70°C until use. The MAbs were allowed to react with sporozoites for 30 min at room temperature, washed, and probed with fluorescein isothiocyanate (FITC)labelled goat anti-mouse IgG (Tago, Burlingame CA). The slides were read under a fluorescent microscope. The titre was defined as the highest dilution of sera that gave a positive reaction in the IFA (immunofluorescence) assay. In all the experiments monoclonal antibodies specific for each of the sporozoite species was included. Sporozoites of t! simiovuk were used as surrogates for t? vivax-like parasites, since both the parasites have identical CSP”. Sera from adjuvant control mice and mouse IgG were used as negative controls.

RESULTS Immunogenicity of MACs containing the CSP repeats Mice of four different genetic backgrounds (BlO.BR, BlO.A, BlO.D2, and C57BW6) were immunized with three different MACs emulsified in Freund’s complete adjuvant. Two of the arms in these three MACs contained CSP repeats from I? vivux-like parasites. The other two arms contained CSP repeats from either p vivux type I (vivax-like/vivax type-l MAC), p viva type 2 (vivax-like/vivax type-2 MAC), or F! julcipunrm (vivaxlike/falciparum MAC). The CSP repeats of p viva-like parasite contains natural T and B epitopes which wcrc recognized by mice of H-2;‘.k.h genetic backgrounds but not H-2” genetic background (V. Udhayakumar, unpublished results). The T-helper epitopes in the CSP

Vaccine

1998 Volume

16 Number

et al.

repeats of p vivux type I are H-2” restricted, FI “~IWX fv/>e II are H-2” restricted and NANP repeats of p @cipurum are H-2” rcstrictcd’3.“‘. Thus. WC wcrc intcrestcd in determining if presentation of CSP repeats from two different plasmodial species in a single MAC can induce antibodies to both scqucnces. Immunogenicity

assay

ELlSA was performed essentially as previously peptides (10 /lg ml ‘) were describedlX. Briefly, dissolved in PBS (0.01 M, PH 7.2) and 100 /II of this solution was added to lmmunolon II‘ (Dynatech Laboratories Inc., Alexandria VA) micro dilution plates and incubated overnight at 4°C. The unbound sites were blocked with PBS containing 5% of cascin and 0.5% of Tween 20. Test sera were serially diluted (log?) starting at 1:lOO dilution and incubated for I h at room temperature, and after washing, horse radish peroxidase conjugated goat anti-mouse IgG (Fisherbiotech) was added to detect antibody activity. The commercially available substrate solution (Kirkegaard and Perry Laboratories, Gaithersburg MD) was used to develop colour, and the reaction was arrested IO min later with 0.1 M sulphuric acid. The plates were read using a ELISA plate reader at 650 nm. The highest dilution which gave an absorbance (A) of > 0.2 was defined as titre.

984

V. Udhayakumar

9/l 0

of vivax-like/vivax

type-l MAC

Sera from mice that wcrc immunized with vivax-like/ vivax type-l MAC were tested for the prcscncc of antibodies to individual CSP repeats of FI vivux-like parasite and /? vivax type-I parasite. The data are shown in Figwe 2. Serum antibodies to P vivlcx-like parasite appeared as early as I5 days after first immunization in BIO.BR, and Bl0.A mice and it reached a peak level by day 38. In C57BL/h mice the onset of antibody production was slower than in BlO.BR and BIO.A mice and it reached peak lcvcl by day 73. The level of antibodies were about IO to l5-fold lower in this mouse strain when compared to the BIO.BR and BI0.A. The lowest level of antibodies were seen in Bl0.D2 mice, which is of H-2” background. It is important to point out here that mice of this H-2d background did not elicit antibodies when immunized with linear peptides representing the CSP repeats of P vivux-like parasite (data not shown); however, the same peptide sequence in MAC form could break the genetic barrier to mount the antibody response. In all of these mice the antibody levels persisted for at least 7 months after immunization and a booster immunization on day 241 after the primary immunization resulted in increase in antibody titres. The anti-vivax type-l antibody titres were higher in BIO.BR and Bl0.A mice; howcvcr, the magnitude of the antibody titre was considerably lower than that of anti-vivax-like CSP antibody titre. Earlier studies have shown that mice of H-2h genetic background wcrc non-responders to CSP repeats from p viva type-l. In this experiment, considerable anti-vivax type-l titres were found in the C57BW6 mice which were of H-2” background. In BlO.D2 mice, there was no antibody titres to vivax type-l repeats, which is consistent with the earlier tindings that the mice of H-2” background did not respond to these sequences’“. Immunogenicity

of vivax-like/vivax

type-2 MAC

Sera collected from mice immunized with vivax-like/ vivax type 2 MAC were tested against CSP repeat peptides of vivax-like and vivax type-2 to dcterminc individual antibody titrcs (Figure 3). Antibody titrcs against vivax-like repeats were highest in BIO.BR and BIO.A mice which were followed by C57BW6 mice. BlO.D2 mice, unlike those immunized with vivax/vivax type-l MAC, did not mount antibody response to vivax-like repeats. This finding suggests that the overall structure of MAC may have an important role in determining its immunogenecity in different mouse strains. In general the sera from most of the vivax-like/vivax type I immune mice showed no significant reactivity with vivax type-2 repeats. The only exception to this was that 2/5 Bl0.A (1:25600 titre in both mice) and l/5 BIO.BR (l:l2800) mice showed some titres to CSP repeats of /? vivux type-2. This finding suggests that the vivax type-2 CSP repeats may not have strong helper

lmmunogenicity

Anti-vixax-like 1,000,000,000

of MACs with dual peptide sequences:

V. Udhayakumar

et al.

Anti-vivax type 1 CSP repeat titers

CSP repeat titers

1,000,000,000

Fl

BlO.BR

BlO.BR

ii .e

100,000,000

t;i ti C57/BL6 E

10,000,000 C57BL6

L

E

1,ooo,ooo

8

r

$

‘,/,

z? ; ._

, , , , , ,j 15

38

73

100,000 10,000

?

1,000

103 224 251 266

100

15

Days post immunization

38

73

103 224 251 266

Days post immunization Figure 2 lmmunogenicity of vivax-like/vivax type-l MAC in four different inbred strains of mice. Sera were collected at indicated time intervals after the primary immunization (day 0) with this MAC as described in Section 2. Serum antibody titres were determined using linear peptides PL35 and PLl representing CSP repeat sequences of vivax-like and vivax type-l parasites respectively. Each point represent the geometric mean antibody titre. Each data point represents three to five mice.

at least in the genetic backgrounds that were used in this study. epitopes,

Immunogenicity

of vivax-like

falciparum

of mice

MAC

Sera obtained from mice immunized with vivax-like/ falciparum MAC were tested against CSP repeat

Anti-vixax-like

peptides of vivax-like and falciparum (F@u~ 4). As seen with the other two MACs used in this study, antibody titre to vivax-like repeat was higher in BlO.BR and Bl0.A mice. Interestingly, in C57BW6 mice the antibody titre to vivax-like repeat was 10 to 50-fold higher than was seen in the same strain of mice immunized with other two MAC preparations. It is

CSP repeat titer

Anti-vivax

10,000,000

type-2 CSP repeat titers

1 ,ooo.GQo,ooo

-I

BlO.BR

5

5 1,ooo,ooo

.$j

z 8.

f! n 8

1oo,Ixlo,ooo

SE

C57lBL6

q

100,000

;ij

$

1o,ooo,ooo

C57BL6 H

E

0)

s

i-4 ,g ‘;

10,000

,&

s

1,000

100

15

36

73

103

224

251

266

15

Days post immunization

36

73

103 224

251

266

Days post immunization

Figure 3 lmmunogenicity of vivax-like/vivax type-2 MAC in four different inbred strains of mice. Sera were collected at indicated time intervals after the primary immunization (day 0) with this MAC as described in Section 2. Serum antibody titres were determined using linear peptides PL35 and PL2 representing CSP repeat sequences of vivax-like and vivax type-2 parasites respectively. Each point represents the geometric mean antibody titre. Each data point represents four to five mice.

Vaccine

1998 Volume

16 Number

9/10

985

lmmunogenicity

of MACs with dual peptide sequences:

Anti-vixax-like

V. Udhayakumar

et al.

Anti-falciparum

CSP repeat titers

CSP repeat titers

BlO.BR

BlO.BR B

1o,ooo,ooo

5

.E

5

B

C57lBL6

1,ooo,ooo

C57BL6

W

W

&! 1,ooo,ooo

100,000

10,000

.s f

1,000

1,000

15

36

73

103

224

251

100

266

Days post immunization

Days post immunization

Figure 4 lmmunogenicity of vivax-like/falciparum MAC in four different inbred strains of mice. Sera were collected at indicated time intervals following primary immunization (day 0) with this MAC as described in Section 2. Serum antibody titres were determined using linear peptides PL35 and NANP repeat peptides representing CSP repeat sequences of vivax-like and P. falciparum parasites respectively. Each point represents the geometric mean antibody titre. Four to five mice were in each group.

known that NANP sequences induce T-helper responses in H-2h mice, and this increase may be due to the additional cognate help by NANP sequence to epitopes in the CSP repeats of l? r&~-like parasite. However, this hypothesis remains to be tested. The antibody titres persisted for at least 7 months in all of the responder strains. A booster immunization on day 241 did not increase antibody titres in all, but C57BWh mice. In BlO.D2 mice there was no response to vivaxlike repeats indicating that not all the MAC constructs can overcome genetic restriction. As expected, the antibody titres to the NANP sequence was highest in C57BW6 mice, which is a responder strain to NANP repcats. In B1O.A and BIO.BR mice, both of which are nonresponders to NANP repeats, there was a considerable antibody titre to this repeat sequence. While the antibody titres to NANP persisted until about 7 months in BI0.A mice, it declined faster in BIO.BR mice. A booster immunization on day 241 resulted in a slight increase in antibody titres in C57BL/6 and BlO.BR, but not in other mice. The antibody titre in BlO.D2 mice did not rise to any appreciable level. The sporozoite reactivity of anti-MAC sera The ability of anti-MAC sera to react with the sporozoites were tested by IFA assay and the results are presented in Figure 5. As shown in Figure 5A, antivivax-likeivivax type-l MAC sera from BlO.BR, BI0.A and C57BL/6 mice recognized sporozoites of I? simiovul~, a monkey parasite with CSP repeats identical to p viva.u-like parasites and used as a surrogate in this experiment. The IFA titres were proportional to the corresponding ELISA titre. Sera from B10.D2 mice, which showed the lowest ELISA titre against vivax-like

986

Vaccine

1998 Volume

16 Number

9/l 0

CSP repeats, were also able to react with II simiovul~ sporozoites (surrogates for I? vivLrx-like sporozoitcs). The anti-vivax-likeivivax type-l MAC sera from all strains except BlO.D2 recognized sporozoites of l? iivux-type 1 parasite. The antisera obtained from BIO.BR, BI0.A and C57BL/6 mice immunized with vivax-likeivivax type-2 MACs showed IFA reactivity to p simiovafr but not with F! vivux type-2 sporozoites (Figure 5B). Sera from BlO.D2 mice immunized with the same MAC did not recognize the sporozoites from these two species. Thus, the overall IFA reactivity was consistent with the respective ELISA titres. The sera from BlO.BR, BIO.A, and C57BW6 mice immunized with vivax-like-falciparum MAC showed IFA reactivity to sporozoites of I! simiovul~ (Figure SC). The sera from C57BL/6 mice immunized with vivax-likeifalciparum MAC showed a higher anti-/? sinziot& sporozoite titre than the same strain of mice immunized with the other two MACs. As cxpcctcd. BlO.D2 scra did not react with p simioval~ sporozoitcs. The highest IFA titre against I? fulci@rum sporozoites were seen with antisera from C57BW6 mice. Immune sera from BIO.A and BIO.BR mice also showed reactivity with p ,fukipurum sporozoites, but the titrc was about tenfold lower than in the C57BL/6 mice. Sera from non-responder BlO.D2 mice did not show reactivity with sporozoites from either t! simiovule or p ,ful~ipurum.

DISCUSSION This study shows that tetrameric MACs containing naturally immunogenic CSP repeats from two different species of malaria parasites are highly immunogenic

lmmunogenicity

of MACs with dual peptide sequences:

and they can generate antibodies recognizing the native CSP expressed on the surface of sporozoitcs. The previous stud& have shown that the MAPS were better immunogens than linear peptides, and that they were able to induce protective immunity against malaria in rodents”.“‘.“. However, the MACs used in this study were different from the MAP developed by Tam in three respects. First, the MACs were synthesizd on a nor-leucine con: instead of lysine to anchor peptide branches, and this allowed a better characterization of the product (D. Jut et uf. unpublished results). Second, WCintroduced two different peptide sequences in alternate arms while Tam used the homologous peptides in all arms consisting of T and B epitopes placed in tandem. Third, we used CSP repeat sequences of different human malaria parasites without any heterologous T-helper epitopes. The experimental results from this study showed that the MACs containing immunogenic T- and B-cell epitopes were highly immunogenic, as, demonstrated by their ability to induce high levels of antibody titre. The results of this study confirm recent studies showing that MACs containing heterologous peptide sequence in alternative arms can induce effective antibody response’“.“.“‘. It is important to point out that although in our MAC preparations the molar concentrations of a given peptide sequence arc reduced to 50% when compared to MAPS (due to incorporation of a different sequence in other arms) it did not compromise the immunogenicity of the product. This is consistent with the earlier observations

A

V. Udhayakumar

et al.

that MAPS with eight branches of the same sequences were no better than MAPS with four branches of the same sequence. It appears that the macromolecular configuration of MACs may be an important determinant in influencing the immunogenicity than the number of given peptide sequences in a given MAC. This hypothesis needs further veritication which can be tested by constructing MACs with different peptide sequence in all four arms. One of the potential concerns with our MAC design was that epitopes in one arm can compete with the heterologous epitopes in the other arm to induce antigenic competition. The results from this study using tetra-branched MACs rule out against such a possibility. For example, C57BLi6 mice which can recognize CSP repeats of both vivax-like and falciparum parasites responded more vigorously to both of these sequences after immunization with the MAC containing these two repeat sequences. A similar pattern was also seen in BIO.BR and BI0.A mice immunized with vivax-like/ vivax type-l MAC. In several instances immunization with MAC preparations induced antibodies to CSP repeats in non-responder genetic strains of mice. For example in the following cases the antibody responses were seen in mouse strains that were nonresponder to these cpitopes: (i) C57BL/6 mice that do not respond to I? ~~WXY type 1 repeats showed antibody titres to these repeats when immunized with vivax-likeivivax tvpe-I M’AC; (ii) B10. BR and BI0.A mice which’ are nonresponders to NANP repeats became responsive

C

B

J

n P. simiovale !IDP.vivax tvoe'I

100,000

BlO.BR

BIO.A

BlO.D2 C57BU6

-

-

B10l!D2

810 .D2

The sera collected on day 103 following the immunization were used to test their reactivity against the sporozoites of indicated species. Each sera were serially diluted (log,-fold) starting from 1:lOO dilution. The data points represent the geometric mean titre of three to five sera that were individually tested. (A) shows the reactivity of sera from vivax-like/vivax type-l MAC immunized mice, (B) shows the reactivity of sera from vivax-like/vivax type-2 MAC immunized mice, and (C) shows the reactivity of sera from vivax-like/falcip,arum MAC immunized mice. Figure

5

Th ie I.eactivity of anti-MAC sera with sporozoites.

Vaccine 1998 Volume 16 Number 9/l 0

987

lmmunogenicity

of MACs with dual peptide sequences:

after immunization with vivax-like/falciparum MAC; and (iii) antibodies to vivax-like CSP repeat scquencc in genetically nonresponder BlO.D2 mice occurred following immunization with vivax-like/vivax type-l MAC. At least two different explanations can hc offered to explain these results. First, it is possible that T-helper epitopes in one arm probably provide cognate help to B epitopes in the other arm, thereby enhancing the immunogenicity. Second, the presentation of peptides in MAC configuration could alter their immunogenic properties in such a way as to overcome some genetic restrictions. Further experiments are needed to test these hypotheses. It is also important to point out that the magnitude of antibody response to the B-cell epitopes in the nonresponder backgrounds were relatively lower than the response seen in the responder backgrounds. In addition, not all the MACs could break the genetic restriction of the immune response. Although vivaxlikeivivax type-2 and vivax-like/falciparum MACs had the same CSP repeat sequence of vivax-like parasites as that was present in vivax-like/vivax type-l MACs, they failed to mount an antibody response in BlO.D2 mice. We do not know whether this difference is due to some three-dimensional structural features imposed by the amino acid sequences in the parallel arms, or due to some other characteristics of the MAC. The previous studies with MAP preparations have also shown that only some but not all the preparations could overcome genetic restriction (reviewed in ref. “). We hypothesize that the three-dimensional conformation of MAC/MAP preparations can alter the processing and presentation of peptide sequences. In conclusion, we have shown that MACs containing CSP repeat sequences from different malaria parasites can be highly immunogenic and induce antibodies that recognize the native structure of the protein expressed on the parasites. This approach opens up ways to develop peptide based multi-species vaccines for malaria and other infectious agents.

V. Udhayakumar 4

5

6

7

8

9 10

11

12

13

14

15

ACKNOWLEDGEMENTS This work was supported by in part by USAID grant (DPE-5979-A-00-1036-00). The authors are grateful to Dr William Collins for providing sporozoites. Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the US Department of Health and Human Services.

16

17

REFERENCES Greenwood, B., Marsh, K. and Snow, R. Why do some African children develop severe malaria? farasitol. Today 1991, 7, 277-281. Clyde, D.F., McCarthy, V.C.. Miller, R.M. and Hornick, R.B. Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 1973, 266, 398-403. Rieckmann, K.H., Carson, P.E.. Beaudoin, R.L., Cassells, J.S. and Sell, K.W. Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans. R. Sot. Trop. Med. Hyg. 1974, 68, 258-259.

988

Vaccine

1998 Volume

16 Number

9/10

18

19

20

et al.

Rieckmann, K.H., Beaudoin, R.L., Cassells, J.S. and Sell, K.W. Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull. World Health Organ. 1979, 57, (SuppLl), 261-265. Nussenzweig. V. and Nussenzweig, R.S. Rationale for the development of an engineered sporozoite malaria vaccine. Adv. Immunol. 1989, 45,283-334. Ballou. W.R., Hoffman, S.L., Sherwood, J.A., Hollingdale, M.R., Neva, F.A., Hockmeyer, W.T., Gordon, D.M., Schneider, I., Wirtz. R.A., Young, J.F.. Wasserman, G.F.. Reeve, P., Diggs, CF. and Chulay, J.D. Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet 1987, 1,1277-1281. Herrington, D.A., Clyde, D.F., Losonsky, G., Cortesia, M., Murphy, J.R., Davis, J., Baqar, S., Felix, A.M., Heimer, E.P., Gillessen, D., Nardin, E., Nussenzweig, R.S., Nussenzweig, V., Hollingdale, M.R. and Levine, M.M. Safety and immunogenicity in man of synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 1987, 328, 257-259. Tam, J.P. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Nat/ Acad. Sci. USA 1988, 85, 5409 Tam, J.P. Recent advances in multiple antigen peptides. J. Immunol. Methods 1988, 196,17-32. Tam, J.P., Clavijo, P., Lu, Y.A., Nussenzweig, V., Nussenzweig, R.S. and Zavala, F. Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria. J. Exp. Med. 1990, 171, 299-306. Chai. SK., Clavijo, P., Tam, J.P. and Zavala, F. Immunogenic properties of multiple antigen peptide systems containing defined T and B epitopes. J. Immunol. 1992, 149, 2385-2390. Munesinghe, D.Y.. Clavijo, P., Calle, M.C., Nussenzweig, R.S. and Nardin, E. lmmunogenicity of multiple antigen peptides (MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein. fur. J. Immunol. 1991,21,3015-3020. Pessi, A., Valmori, D., Migliorini, P., Tougne, C., Bianchi. E., Lambert, P.H., Corradian, G. and Giudice, G.D. Lack of H-2 restriction of the Plasmodium falciparum (NANP) sequence as multiple antigen peptide. Eur. J. Immunol. 1991, 21, 2273-2276. Calvo-Cake, J.M., de Oloveira, G.A., Clavijo, P.. Maracic, M., Tam, J.P., Lu, Y., Nardin, E.H.. Nussenzweig, R.S. and Cochrane. A.H. lmmunogenicity of multiple antigen peptides containing B and non-repeat T cell epitopes of the circumsporozoite protein of Plasmodium falciparum. J. Immunol. 1993, 150,1403-1412. Wang, R., Charoenvit, Y., Corradin, G., Porrozzi, R., Hunter, R.L., Glenn, G., Alving, C.R., Church, P. and Hoffman, S.L. Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine. J. Immunol. 1995, 154, 2784-2793. Reed, R.C., Louis-Wileman, V., Fang, S.H., Jue, D., Wohlhueter, R.M., Hunter, R. and Lal, A. Multiple antigen constructs (MACs): Induction of sterile immunity against sporozoite stage of rodent malaria parasites Plasmodium berghei and P. Yoelii. Vaccine 1997, 15, 482-488. Yang, C., Collins, W.E., Xiao, L., Saekhou, A.M., Reed, R.C., Nelson, CO.. Hunter, R.L., Jue, D.L., Fang, S., Wohlhueter, R.M., Udhayakumar, V. and Lal, A. Induction of protective antibodies in Saimiri monkeys by immunization with a multiple antigen construct (MAC) containing the Plasmodium vivax circumsporozoite protein repeat region and a universal T-helper epitope of tetanus toxin. Vaccine 1997, 15, 377-386. Qari, S.H., Shi, Y.P., Goldman, I.F., Udhayakumar, V., Alpers, M.P., Collins, W.E. and Lal, A.A. Identification of Plasmodium v&x-like human malaria parasite. Lancet 1993, 341, 786783. Nardin, E.H., Barr, P.J., Heimer, E. and Etlinger, H.M. Genetic restriction of the murine humoral immune response to a recombinant Plasmodium vivax circumsporozoite protein. Eur. J. Immunol. 1988,18,1119-l 122. Ahlborg, N. Synthesis of a diepitope multiple antigen peptide containing sequences from two malaria antigens using Fmoc chemistry. J. Immunol. Methods 1995, 179, 269-275.