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The Journal of Immunology, 2004, 172: 6167-6174.
Copyright © 2004 by The American Association of Immunologists

Fusion of Two Malaria Vaccine Candidate Antigens Enhances Product Yield, Immunogenicity, and Antibody-Mediated Inhibition of Parasite Growth In Vitro 1

Weiqing Pan2, Daqing Huang, Qingfeng Zhang, Li Qu, Dongmei Zhang, Xiaoli Zhang, Xiangyang Xue and Feng Qian

Department of Etiologic Biology, Second Military Medical University, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and ~1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1–19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1–19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The emergence and rapid widespread occurrence of drug-resistant parasites and of mosquitoes resistant to insecticides demand a search for new tools to control the disease. Vaccination is one such tool that may control and even eradicate the disease from the world. Due to the complex life cycle and antigenic diversity of the malaria parasite, polyvalent subunit malaria vaccines containing multiple protective domains or epitopes from different Ags may be necessary, and may be constructed as chimeric proteins (1, 2). Such constructs may provide approaches to address the issue of parasite variation and to enhance the immunogenicity and protective efficacy of vaccination.

The ~200-kDa merozoite surface protein-1 (MSP1) 3 and the apical membrane Ag (AMA-1) of Plasmodium falciparum are attractive malaria vaccine candidates (3, 4). These two Ags are located on the parasite surface and undergo proteolytic processing before invasion of merozoite into the erythrocyte. It has been proposed that the proteins may play a role in the invasion process. Immunization of the Aotus monkey with native as well as recombinant MSP1 provided partial to complete protection against challenge of P. falciparum (3, 5). Similar immunization experiments in rodents also generated high levels of protection (6). A portion of MSP1 targeted by protective immunity has been mapped to the 19-kDa C-terminal region (MSP1–19) which contains two epidermal growth factor (EGF)-like domains (7, 8). This region is the target of some mAbs that have the ability to inhibit the growth of the parasite in vitro (9). In the Aotus monkey challenge model of P. falciparum, MSP1–19 has also proven to protect the animals from infection (10).

AMA-1 is an integral membrane protein of 83 kDa that contains 16 conserved cysteine residues forming eight intramolecular disulphide bonds (11). Results with rodent and monkey models demonstrated that immunization with native or recombinant AMA-1 can provide protection against parasite challenge (12, 13). The disulphide-bond stabilized molecular conformation is necessary for protection. A three-domain substructure to the AMA-1 ectodomain was recently suggested (14) and the most C-terminal of the disulphide-bonded domains in AMA-1 (AMA-1(III)), which has a "cysteine knot-like" structure consisting of ~40 residues, may also be carried in on the surface of the invading merozoite as occurs with MSP1–19 (15). The solution structure of AMA-1(III) has been recently determined and it has been demonstrated that this molecular domain is the target of inhibitory Abs isolated from humans in malaria-endemic regions (16).

Because both MSP1–19 and AMA-1(III) are targets of inhibitory Abs and their disulphide-bond based structures are essential for inhibition, we have constructed a P. falciparum chimeric protein 2.9 (PfCP-2.9) comprising the AMA-1(III) from the 3D7 line and MSP1–19 from the Wellcome/K1 line of P. falciparum, respectively. Consequently, fusion of the two malaria vaccine candidates greatly enhances the product yield and immunogenicity of the individual components. Moreover, sera from rabbits as well as rhesus monkeys immunized with the chimeric protein almost completely inhibit parasite growth in vitro that are mediated by a combination of inhibitory Abs generated by the individual components of PfCP-2.9.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis of the PfCP-2.9 gene and parts thereof

The amino acid sequence of PfCP-2.9 was translated into a DNA sequence using yeast Pichia pastoris codon usage (GenBank accession number AY496866). The sequence of the resulting synthetic gene was divided into eight oligonucleotide primers of ~80 nt in length. The oligonucleotides were synthesized using an Applied Biosystems 394 synthesizer (Foster City, CA) and were purified by electrophoresis in 10% polyacrylamide gel. The overlapping region between two primers varied from 15 to 20 nt in length. The eight primers were assembled by using the asymmetric PCR-based method as described previously (17) to generate 723 bp of the PfCP-2.9 gene. The product was isolated from the PCR by 1% agarose electrophoresis and inserted into the pBSK plasmid via XhoI and EcoRI restriction sites. The recombinant plasmid was transferred into Escherichia coli DH5{alpha}. Five clones containing the insert were sequenced to identify a clone with error-free sequence of the gene. To generate a DNA fragment of the individual components of the chimeric protein, two pairs of primers were designed and synthesized. Primers for the AMA-1(III) fragment were P1 (5'-CCG CTC GAG AAA AGA CAA CAA TCA TCT TAC ATT G-3') and P2 (5'-CG GAA TTC CTA TTA ATG GTG ATG GTG ATG GTG CAT TTT ATC ATA AGT TGG-3') while those for MSP1–19 were P3 (5'-CCG CTC GAG AAA AGA TTA CAA ATT TCT CAA CAT C-3') and P4 (5'-CG GAA TTC CTA TTA ATG ATG ATG ATG ATG ATG ATT AGA GGA AGA GCA GAA G-3'). Both DNA fragments were generated by PCR and confirmed by DNA sequencing. To facilitate purification, 6xHis tags were added to the C terminus of the two fragments.

Production of the recombinant proteins

The synthetic genes of PfCP-2.9, AMA-1(III) and MSP1–19, were first inserted into pPIC9 Pichia expression vector, respectively, and then each BamHI/SalI fragment was cut out from the plasmid and ligated into the pPIC9k vector which is identical to pPIC9 except the Kanr gene that confers resistance to G418. The resulting recombinant plasmids were linearized by BglII before they were transferred by electroporation into P. pastoris GS115. Selection of His+ transformants and G418-resistant clones were conducted according to the manufacturer’s instructions (Invitrogen, San Diego, CA). The selected clone was grown in minimal glycerol medium at 30°C overnight and then resuspended in buffered methanol-complex medium containing 5% methanol to induce expression of the target gene. Fermentation of the PfCP-2.9-expressing strain was conducted in a 15-L fermentor. The 250-ml culture of the clone grown at 30°C for 22 h was inoculated in the fermentor containing 6 L of minimal salt fermentation medium (per liter of the medium: MgSO4, 14.0 g; CaSO4.2H2O, 0.9 g; K2SO4, 18 g; KOH, 4 g; 26 ml of 85% H3PO4 and 4 ml of PTM1 trace metals solution). The cells were grown at 30°C and harvested at 72 h after methanol induction.

Purification of PfCP-2.9 protein from the supernatant was performed by using three steps including phenyl hydrophobic interaction, ion-exchange (DEAE) and gel filtration (Superdex 75; Pharmacia Biotech, Piscataway, NJ) chromatography. The fermentation supernatant filtered through a 2.0-µm filter was mixed with (NH4)2SO4 to a final concentration of 0.8 M and adjusted to pH 7.2. The material was applied to a l.5-L phenyl hydrophobic interaction column. The resin was equilibrated in the buffer A (10 mM PB, 0.8 M pH 7.2 (NH4)2SO4). The protein was eluted with 10 mM PB. The elution was then fractionated by ion-exchange chromatography on a 500-ml DEAE column. The protein was eluted with buffer B (10 mM PB, 0.35 M NaCl (pH 7.2)). The final step of the purification was performed on a 1.7-L Superdex 75 column and the PfCP-2.9 protein was eluted with buffer C (10 mM PB, 0.15 M NaCl (pH 7.2)). To purify the individual proteins, culture supernatants were dialyzed against buffer D (50 mM NaH2PO4, 300 mM NaCl (pH 8.0)) and then applied directly to Ni2+ chelate columns. The resin was extensively washed with 20 mM imidazole hydrochloride in buffer D and the proteins were eluted with 250 mM imidazole hydrochloride in buffer D.

Immunization studies

Monkey experiments were conducted at the Kunming Institute of Zoology (Chinese Academy of Sciences, Kunming, China). Ten rhesus monkeys (Macaca mulatta) with a history of no malaria exposure were screened for lack of Abs to P. falciparum by immunofluorescence assay (IFA) (see below). New Zealand White rabbits were purchased from Shanghai Laboratory Animal Center (Chinese Academy of Sciences, Shanghai, China). The immunogen emulsion was prepared by formulation of the purified PfCP-2.9 protein with the Montanide ISA720 adjuvant by mixing a 70% volume of the adjuvant with a 30% volume of the Ag solution using a homogenizer at 4000 rpm for 4 min. The Montanide ISA720 adjuvant (Seppic, Paris, France) is an oily adjuvant formulation containing a natural metabolizable oil of vegetable origin. The animals (rabbits or rhesus monkeys) were immunized i.m. four times at 4-wk intervals with 1 ml of the emulsion containing various concentration of the Ag. Sera obtained from each animal before immunization as well as after each immunization were analyzed for specific Abs by ELISA as well as IFA.

ELISA was performed in triplicate on diluted serum samples in a 96-well flat-bottom plate (Thermo Labsystems, Franklin, MA). The plates were coated overnight at 4°C with 100 µl of recombinant protein at a concentration of 1.0 µg/ml diluted in carbonate buffer (0.159% Na2CO3, 0.293% NaHCO3, pH 9.6) in each well. Plates were blocked with PBS containing 3% skim milk at 37°C for 1 h. Serum samples diluted in PBS containing 3% skim milk were added to the plates (100 µl), and incubated at 37°C for 1 h. HRP-conjugated goat anti-rhesus monkey, goat anti-rabbit, or goat anti-mouse IgG with 1/1000 dilutions in PBS containing 3% skim milk were added to the plates for a further 1-h incubation at 37°C. At every step, plates were washed three times with PBST (PBS containing 0.05% Tween 20) and one time with PBS using a plate washer. Bound secondary Abs were detected by adding 100 µl of TMB (3,3',5,5'-tetramethyl benzidine) substrate solution at room temperature for 10 min, and the reaction was stopped by 50 µl of 2 M H2SO4. The plates were read at an absorbance of 450 nm (ELx800 ELISA reader; Bio-Tek Instruments, Winooski, VT). Cut-off values were determined as the mean plus three SDs for the preimmunization sera. IFA was performed according to the procedure described previously (18) using cultured schizonts and merozoites of P. falciparum FCC1/HN isolate as Ag.

Growth inhibition assay

The function of immune sera or specific Abs was determined by detecting their ability to inhibit growth of the parasite in vitro. P. falciparum 3D7 and FCC1/HN lines were used for the assay. The FCC1/HN line was isolated from Hainan Island, China and its continuous cultivation in vitro was established in 1979. This line has been adapted to rabbit sera for cultivation. We analyzed the sequence of MSP1–19 of FCC1/HN and it belongs to the E-KNG variation. The sequence of the AMA1 gene of FCC1/HN can be found in GenBank (accession number AF277003). The FCC1/HN line was cultured using rabbit sera whereas the 3D7 line was cultured using human sera according to the method described previously (19). To isolate specific Abs for the inhibition assay, purified recombinant proteins were immobilized on CNBr-activated Sepharose 4B (Pharmacia Biotech) according to the manufacturer’s instructions. The Abs eluted from the columns were assayed for inhibition activity. The inhibition assay was performed with the starting culture containing 2% hematocrit and ~0.5% parasitemia with the majority of late trophozoites and schizonts. One hundred seventy microliters of the culture suspension and 30 µl of the test serum or various concentrations of Abs were added in triplicate wells to 96-well flat-bottom plates and incubated at 37°C for 72 h. During the period of cultivation, the medium was refreshed every 24 h. Thin blood smears were prepared to determine parasitemia. The inhibition rate was determined according to the following: % inhibition = (Pc – Ps) – (Pt – Ps)/(Pc – Ps) x 100%, where Pc is the parasitemia of preimmune sera, Pt is the parasitemia of immune sera, and Ps is the parasitemia of starting culture.

T cell proliferation assay

PBLs of rhesus monkeys immunized with either the purified PfCP-2.9 protein formulated with the adjuvant ISA720 or the adjuvant only were isolated from 5 to 8 ml of heparinized blood with Ficoll-Hypaque (Pharmacia Biotech) by centrifugation at 2500 rpm for 20 min at room temperature. The cells at the interphase were collected and washed twice with culture medium. The lymphocytes were resuspended in the medium and adjusted to 2 x 106 cells/ml. One hundred microliters of the cell suspension were added to each well of a 96-well plate and to this, 100 µl of culture medium containing various concentration of the Ag or PHA (as positive control) were added. The cells were incubated at 37°C in a 5% CO2 atmosphere for 60 h and then labeled with 1 µCi of [3H]thymidine (Shanghai Nuclear Institute, Shanghai, China) for 8 h. The cells were harvested, and the incorporation was measured by a liquid scintillation counter. The stimulation index (SI) was determined according to the following: SI = the geometric mean cpm for the Ag-stimulated culture/the geometric mean cpm for unstimulated medium culture.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design and production of the PfCP-2.9 recombinant protein

The sequence of MSP1–19 from the 3D7 line and of AMA-1(III) from the Wellcom/K1 line were fused via a hinge encoding a Gly-Pro-Gly motif repeat as well as a small peptide encoded by a multiple cloning site (mcs) to generate PfCP-2.9 protein (Fig. 1B). The amino acid sequence of PfCP-2.9 was translated into a DNA sequence in which the codon usage was designed to optimize expression of the gene in yeast. Three potential glycosylation sites on this chimeric protein were eliminated by changing codons from Asn to Gln. The DNA sequence of the PfCP-2.9 gene was synthesized using the asymmetric PCR-based method as described previously (17). In addition, DNA sequences of the individual AMA-1(III) and MSP1–19 were then generated by PCR using the PfCP-2.9 gene as a template (Fig. 1C). Yeast P. pastoris GS115 was transfected with three plasmids containing the genes for PfCP-2.9, AMA-1(III), and MSP1–19, respectively. These constructs were designed to secrete each of the proteins into the medium. As shown in Fig. 1D, the PfCP-2.9 protein was produced and secreted at an extremely high level in shaker flasks with a yield of 815 mg/L whereas both AMA-1(III) and MSP1–19 were produced at much lower levels, with yields <40 mg/L. The PfCP-2.9 protein constituted over 85% of the total supernatant proteins. Thus, the fusion of the two proteins produced ~20-fold greater expression compared with expression of the individual AMA-1(III) and MSP1–19 proteins in the same system and conditions. Moreover, 2600 mg/L of the expressed PfCP-2.9 was achieved in a 15-L fermentation run as measured by scanning densitometry of Coomassie brilliant blue staining gels when the conditions for the fermentation were optimized. Three batches of the protein were produced under good manufacture practice conditions and >40% of the protein was recovered from the purification process. Three batches of the recombinant protein were purified to near homogeneity from the supernatant with >98% purity determined by image analysis of Coomassie blue stained SDS-PAGE (Fig. 1E).



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  		FIGURE 1. Schematic representation of the PfCP-2.9 gene construct and recombinant protein. A, The localization of domain III of AMA-1 and MSP1–19 on the two molecules. B, Structure of PfCP-2.9. The AMA-1(III) and MSP1–19 fragments were fused via a hinge-mcs-hinge fragment. The hinge consists of Gly-Pro-Gly amino acid repeats. The main unique restriction sites in the mcs are KpnI, PstI, MluI, ClaI, and EcoR I to allow further insertion of sequences for other candidate Ags. C, DNA fragments encoding the individual AMA-1(III) and MSP1–19 proteins were generated by PCR using two pairs of primers and the PfCP-2.9 gene as template. D, Comparison of small-scale expression yields of the three constructs. Twenty microliters of the Pichia flask culture supernatant collected 72 h after induction from each construct were loaded on a 15% SDS-PAGE gel followed by Coomassie staining. Lane M: Prestained molecular mass marker; lane 1: AMA-1(III); lane 2: PfCP-2.9 and lane 3: MSP1–19. The expressed product in each lane is indicated by an arrow. E, Three batches of PfCP-2.9 protein were purified to near homogeneity by a combined three-step purification. The homogeneity and reproducibility of the recombinant protein were determined by analysis of Coomassie blue-stained SDS-PAGE gel. Ten micrograms of the purified protein were loaded on nonreducing SDS-PAGE gel. Lane M: Prestained molecular mass marker; lanes 1–3: the protein from batch nos. 1–3. F, Interaction of PfCP-2.9 with a panel of mAbs recognizing the conformational epitope. The same amount of PfCP-2.9 was subjected to each lane on SDS-PAGE under reducing (+) and nonreducing (–) conditions and was analyzed by Western blot using mAbs that recognize conformational epitopes on MSP1–19. mAb9.8 was used as negative control.

 
Characterization of the recombinant protein

The PfCP-2.9 contains 18 cysteine residues, of which six are located in AMA-1(III) and the rest in MSP1–19, to form nine intramolecular disulfide bonds. The protective immune responses induced by either AMA-1 or MSP1–19 have been shown to be dependent on protein conformation (15, 20). Thus, it was critical to retain both native conformations after fusion of the two proteins into a single molecule. To gain an insight into conformational properties of the chimeric protein, six specific anti-PfMSP1–19 murine mAbs including mAb 5.2, 12.8, 2F10, 2.2, 1E1, and 111.2 (18, 21, 22, 23) were studied for binding to PfCP-2.9. Murine mAb 9.8 that recognizes epitopes located in MSP1 but outside of MSP1–19 was used as negative control (22). Of these mAbs, 2.2 and 12.8 bind to the first EGF domain of MSP1–19 whereas 111.2 requires the presence of both EGF-like domains (20). mAb12.8 is an inhibitory Ab while mAbs 2.2 and 1E1 are blocking Abs (23, 24). All six specific anti-MSP1–19 mAbs recognize conformational epitopes on MSP1–19. The results showed that all mAbs interacted with the recombinant PfCP–2.9 protein. Moreover, the interaction was reduction-sensitive (Fig. 1F), indicating that these critical epitopes were in a conformation similar to that of the native molecule.

It is thought that N-glycosylation of Plasmodium proteins is a rare event (25). However, the native sequence of PfCP-2.9 contains three potential N-glycosylation sites. Considering that overglycosylation of proteins occurs in the yeast expression system, the potential glycosylation sites existing in the chimeric protein were eliminated by changing the amino acid Asn to Gln. To investigate whether the protein produced in Pichia was glycosylated, a Dig Glycan Differentiation kit (Roche Molecular Biochemistry, Basel, Switzerland) was used to detect the protein. Six kit reagents recognized their corresponding glycoproteins (positive controls), whereas none of the reagents interacted with PfCP-2.9, implying that the recombinant protein was probably not N-glycosylated.

The N-terminal sequence of the purified protein was determined by Edman degradation from the first residue to position 15. The determined sequence was found to be identical to the designed sequence.

Immunogenicity of the chimeric protein and its individual components

To evaluate the immunogenicity of PfCP-2.9, the >98% pure protein described above was prepared for immunization experiment. Rabbits were immunized with the Ag formulated with various adjuvants including ISA720, ISA51 (Seppic), alum, and Freund’s. ISA51, ISA720, and Freund’s adjuvant gave similar Ab responses but higher Ab response as compared with alum adjuvant (data not shown). Immunization of rabbits with increasing protein doses (50, 100, 200, 400, and 800 µg) indicated that a 200-µg dose gave the highest anti-PfCP-2.9 Ab response (data not shown).

To compare the immunogenicity of PfCP-2.9 with that of its individual components, MSP1–19 and AMA-1(III), rabbits were immunized (three animals by group). Rabbits in groups 1-4 received purified AMA-1(III), MSP1–19, a mixture of AMA-1 and MSP1–19, and PfCP-2.9, respectively. The proteins were all formulated with ISA720 and administrated at a dose of 200 µg. Sera from the animals were used to measure the Ab response to AMA-1(III), MSP1–19, and PfCP-2.9. As shown in Table I, PfCP-2.9 induced Abs recognizing both AMA-1(III) and MSP1–19. Moreover, the anti-AMA-1(III) Ab titer induced by PfCP-2.9 was ~18-fold higher than that induced by AMA-1(III) and 11-fold higher than the mixture of AMA-1(III) and MSP1–19. In addition, the anti-MSP1–19 Ab titer was 11-fold higher than that induced by MSP1–19 and ~4-fold higher than that induced by the mixture. These data indicate that the immunogenicity of both AMA-1(III) and MSP1–19 were significantly enhanced via fusion of the two candidate molecules.


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  		Table I. Comparison of ELISA Ab titers of rabbits immunized with AMA-1 (III), MSP1–19, mixture of AMA-I (III) and MSP1–19, and PfCP-2.9a

 
Two individual components of the PfCP-2.9 were fused via a hinge consisting of 28-aa residues. To detect any immune response to the hinge in various animals, a peptide (28 residues, designated as P28) of the hinge was synthesized and used as coated Ag to evaluate Abs of immune sera to the hinge by ELISA. As shown in Table II, no specific Abs to the hinge were induced in five strains of mice. Although anti-hinge Abs were detectable in both rabbits and monkeys, except that one monkey was negative, the Ab constitutes only a very little portion of total Abs to the chimeric protein. In addition, we have searched several databases including the nrdb (nonredundant database), remtrembl (remaining TrEMBL), gbest (GBr126-EST), and andgbhtc (GB123-high throughput cDNA) to identify any sequence homology between the hinge and human proteins. The result showed that the score is <35.0 with the expect value above 0.007 (significant difference), indicating there are no sequence homologues between the hinge and human proteins.


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  		Table II. Ab response to the hinge

 
In vitro parasite growth inhibition

Sera from immunized rabbits were used to determine the capability of Abs to PfCP-2.9 to inhibit the growth of P. falciparum in vitro. Fig. 2A showed that sera from rabbits immunized with the chimeric Ag formulated with either ISA720 or Freund’s adjuvant strongly inhibited parasite growth (98.3 and 96.3%, respectively). Moreover, inhibition was dependent on the presence of IgG as sera deleted of total IgG using protein A completely abolished inhibition of parasite growth. The Abs bound and eluted from the PfCP-2.9 affinity column inhibited parasite growth in vitro in a dose-dependent manner. The Abs completely inhibited parasite growth in vitro at a concentration of 1.5 mg/ml (Fig. 2B). To further confirm that the inhibition was mediated by specific Abs, we added various concentrations of purified PfCP-2.9 to the inhibitory sera to neutralize the specific Abs. Fig. 2C shows that addition of the PfCP-2.9 to the inhibitory sera affected inhibition in a dose-dependent manner. Addition of excess Ag (400 µg/ml) to the assay completely abolished the inhibition. Interestingly, addition of the Ag to preimmune sera (negative control) significantly inhibited parasite growth in a dose-dependent fashion. Indeed, when the culture contained 400 µg/ml PfCP-2.9, 68.0% of the parasite growth was inhibited (Fig. 2C).



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  		FIGURE 2. Inhibition of malaria parasite growth in vitro with rabbit sera. A, Parasites of the FCC1/HN isolate were cultured in RPMI 1640 medium containing preimmune sera or immune sera after the fourth immunization with ISA720 adjuvant (immunogen 1), PfCP-2.9 formulated with ISA720 (immunogen 2), reduced and alkylated PfCP-2.9 with ISA720 (immunogen 3), Freund’s adjuvant (immunogen 4), and PfCP-2.9 with Freund’s adjuvant (immunogen 5) to determine the efficacy of the immune sera to inhibit parasite growth (cross-hatched column). Inhibition of parasite growth using sera where the total IgGs were depleted by protein A binding is indicated by black columns. B, Inhibition of parasite growth by rabbit anti-PfCP-2.9 affinity-purified Abs and by total IgG from a pool of preimmune sera isolated using a protein A column. C, Purified recombinant PfCP-2.9 was added to the immune and preimmune sera to neutralize specific Abs to a final protein concentration of 0, 100, 200, and 400 µg/ml, respectively. The serum/protein mixtures were used in an inhibition assay.

 
We examined sera from rabbits immunized with individual AMA-1(III) or MSP1–19 and the mixture of the two Ags but these sera were shown to have no effect on inhibition of parasite growth (data not shown).

To investigate whether the inhibition was dependent on the conformation based on the disulfide bonds, rabbits were immunized with reduced and alkylated PfCP-2.9, and the resulting sera were found not to inhibit parasite growth (Fig. 2A, immunogen 3), indicating that induction of the growth-inhibitory Abs required a proper disulfide bond-based conformation.

To investigate whether inhibition mediated by anti-PfCP-2.9 Abs was a combination of both anti-AMA-1(III) and anti-MSP1–19 Abs, we prepared AMA-1(III) and MSP1–19 affinity columns to deplete either anti-AMA-1(III) or anti-MSP1–19 Abs from the inhibitory sera and assessed the flow-through for their ability to inhibit parasite growth. Quantification of Abs by ELISA showed that >95% of specific Abs were absent in the postcolumn sera. The flow-through depleted of either anti-AMA-1(III) or anti-MSP1–19 Abs were shown to partially inhibit parasite growth by 65 and 69%, respectively. Moreover, both the anti-AMA-1(III) and anti-MSP1–19 Abs bound and eluted from the affinity columns were shown to inhibit parasite growth in vitro in a dose-dependent manner (Fig. 3C). Interestingly, the anti-AMA-1(III) Abs and the anti-MSP1–19 Abs showed similar inhibition properties. At concentrations of 1.0 mg/ml and 1.5 mg/ml, the anti-AMA-1(III) Abs inhibited parasite growth by 72.9 and 100%, respectively, while the anti-MSP1–19 Abs inhibited growth by 80.4 and 94.4%, respectively. These data indicate that the individual components of PfCP-2.9 induced inhibitory Abs that combine to completely inhibit parasite growth.



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  		FIGURE 3. Specificity of the inhibitory Abs to the chimeric protein and its individual components. A, PfCP-2.9, AMA-1(III), and MSP1–19 affinity columns were used to deplete specific Abs from the sera and to purify the Abs as well. The flow-through and Abs eluted from each column were assayed for inhibition activity. B, Inhibition of parasite growth by immune sera (black column) and the flow-through from each affinity-column (white column). C, Inhibition of parasite growth by rabbit anti-AMA-1(III) and anti-MSP1–19 affinity-purified Abs.

 
Immunogenicity studies in nonhuman primates

We also investigated the immunogenicity of PfCP-2.9 in nonhuman primates. Ten rhesus monkeys were randomly divided into two groups (five per group). Monkeys in the immunized group (group I) received 200 µg of PfCP-2.9 formulated with ISA720 adjuvant, whereas the monkeys in the control group (group II) received the adjuvant only. The monkeys were immunized i.m. four times at 1-mo intervals. Sera obtained before immunization and 2 wk after each immunization were analyzed by ELISA and IFA for detection of PfCP-2.9-specific Abs and for measurement of in vitro growth inhibition of both the FCC1/HN and 3D7 lines of P. falciparum. As shown in Table III, all monkeys in group I showed strong immune response to the Ag with ELISA end titers of 1/867,000 to 1/5,700,000 and IFA end titers of 1/2,560 to 1/40,960, whereas no animal in group II had specific Abs. In an in vitro inhibition assay, sera from the monkeys in group I strongly inhibited the growth of the two lines of P. falciparum at a similar level with a 15% concentration of the sera in the culture. The inhibition was correlated with the Ab titers (Table III). Moreover, the level of inhibitory activity in monkey sera increased with the number of immunizations. After the fourth immunization, the sera of the five immunized monkeys almost completely inhibited parasite growth (Fig. 4). After a further two months, the inhibition activities present in the sera of two of the immunized monkeys were reduced from 99.7 to 75.3% (no. 2) and from 100 to 80.3% (no. 5), respectively.


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  		Table III. Evaluation of anti-PfCP-2.9 Abs and inhibition of in vitro parasite growth by sera from rhesus monkeys immunized with PfCP-2.9 formulated with ISA720.

 


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  		FIGURE 4. Kinetics of inhibitory activity on parasite growth (FCC1/HN). The sera from individual monkeys immunized with either 200 µg of PfCP-2.9 formulated by ISA720 adjuvant or ISA720 only were tested for the inhibition.

 
To investigate potential cellular response to the Ag, PBLs of all rhesus monkeys were isolated on day 48 (i.e., 20 days after second vaccination) and day 98 (i.e., 14 days after third vaccination). The isolated cells were used to determine peripheral lymphocyte proliferation. As shown in Table IV, various concentration of the PfCP-2.9 (i.e., 50, 100, and 200 µg/ml) can induce significant proliferation responses of lymphocytes recovered from monkeys immunized with the protein formulated with ISA720 (SI >3) whereas no significant proliferation response was observed in monkeys of the adjuvant control group (SI <3).


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  		Table IV. Proliferation response of lymphocytes recovered from immunized monkeys

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Poor immunogenicity of malaria Ags including MSP1–19 is one of the main scientific obstacles for malaria vaccine development. Strategies for optimizing immunogenicity of malaria Ags include use of different adjuvants, optimizing immunization protocols, and fusing exogenous T cell epitopes to the Ags. Fusion of MSP1–19 to carrier proteins such as tetanus toxoid can indeed enhance the IgG response to vaccine Ags, but it also induces a strong immune response to the carriers which is not required for the target disease. We have fused two leading malaria vaccine candidates into a single molecule and the resulting chimeric protein was shown to be highly immunogenic in both rabbits and monkeys. The highest titers of the PfCP-2.9-specific Abs in rabbits and monkeys were 1/5,130,000 and 1/5,700,000, respectively whereas the best titers to AMA-1(III) and MSP1–19 in rabbits were 1/40,200 and 1/35,100, respectively, using the individual components of PfCP-2.9 as immunogens. The enhancement of the immune response to the both individual components may be due to more T cell epitopes included in the construct. Our data also showed that positive proliferation response of lymphocytes was detected in the immunized monkeys.

The Abs to both AMA-1(III) and MSP1–19 isolated from malaria-exposed humans were shown to inhibit parasite growth in vitro (16, 26) and the levels of the Abs are associated with clinical immunity (27). The enhancement of the IgG response to the individual components of PfCP-2.9 may be responsible for improved inhibition of parasite growth in vitro because the sera from rabbits immunized with the individual components or a mixture of both individual proteins did not inhibit parasite growth in our experiment. Because in vitro inhibition has been shown to correlate with protection in vivo in the Aotus monkey model (28), it is very encouraging to find that the sera from four of five immunized monkeys provided complete inhibition of in vitro growth of two lines of P. falciparum.

The multivalent vaccine concept is an attractive strategy for the development of a malaria vaccine. Several attempts have been made to generate a multivalent vaccine by fusion of multiple "epitopes" into an immunogen. However, these epitopes were usually linear. We have constructed a chimeric protein named PfCP-2.9 consisting of two vaccine candidates that require disulfide bond-dependent conformation for which it has been shown that induction of protective immunity was completely dependent on conformation. Thus, it was critical that individual components of PfCP-2.9 closely resemble the native ones. Based on the fact that a panel of conformation-dependent mAbs reacted with PfCP-2.9 with reduction sensitivity and that the reduced and alkylated PfCP-2.9 did not induce any growth inhibitory Abs, the chimeric protein appears to correctly resemble its two components following appropriate design including a hinge GPG repeat and secretory expression. Moreover, rabbit anti-PfCP-2.9 Abs recognized nonreduced, but not reduced PfCP-2.9 (data not shown), indicating that a large proportion of Abs were induced by conformational epitopes on the molecule. We noted that the protein is quite stable. After being stored at 4°C for 18 mo or at 37°C for 4 wk, no degradation of the protein extracted from the emulsion was detected (data not shown). The high stability of the protein could be a result of resembling the protein correctly. Of particular interest is the fact that the inhibition of parasite growth in vitro was mediated by a combination of anti-AMA-1(III) and anti-MSP1–19 Abs. Furthermore, we were able to produce the chimeric protein at an extremely high yield and to use a simple method for its purification, providing the possibility for developing an affordable malaria vaccine that can be used widely in malaria-endemic developing countries.


   Acknowledgments
 
We thank Drs. Robin Anders, Tony Holder, and Howard Engers for invaluable help and advice, and also for mAbs. We also thank Aiguo Zhou and Jinhua Xing for technical assistance.


   Footnotes
 
1 This investigation received financial support from the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (TDR), the National 863 Project, and the National Outstanding Youths Fund in China. Back

2 Address correspondence and reprint requests to Dr. Weiqing Pan, Department of Etiologic Biology, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, China. E-mail address: malaria{at}guomai.sh.cn Back

3 Abbreviations used in this paper: MSP1, merozoite surface protein-1; AMA-1, apical membrane Ag-1; PfCP-2.9, P. falciparum chimeric protein 2.9; IFA, immunofluorescence assay; EGF, epidermal growth factor; SI, stimulation index; mcs, multiple cloning site. Back

Received for publication September 3, 2003. Accepted for publication March 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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