Production and Preclinical Evaluation of Plasmodium falciparum MSP-119 and MSP-311 Chimeric Protein, PfMSP-Fu24

ABSTRACT A Plasmodium falciparum chimeric protein, PfMSP-Fu24, was constructed by genetically coupling immunodominant, conserved regions of two merozoite surface proteins, the 19-kDa region C-terminal region of merozoite surface protein 1 (PfMSP-119) and an 11-kDa conserved region of merozoite surface protein 3 (PfMSP-311), to augment the immunogenicity potential of these blood-stage malaria vaccine candidates. Here we describe an improved, efficient, and scalable process to produce high-quality PfMSP-Fu24. The chimeric protein was produced in Escherichia coli SHuffle T7 Express lysY cells that express disulfide isomerase DsbC. A two-step purification process comprising metal affinity followed by cation exchange chromatography was developed, and we were able to obtain PfMSP-Fu24 with purity above 99% and with a considerable yield of 23 mg/liter. Immunogenicity of PfMSP-Fu24 formulated with several adjuvants, including Adjuplex, Alhydrogel, Adjuphos, Alhydrogel plus glucopyranosyl lipid adjuvant, aqueous (GLA-AF), Adjuphos+GLA-AF, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), and Freund's adjuvant, was evaluated. PfMSP-Fu24 formulated with GLA-SE and Freund's adjuvant in mice and with Alhydrogel and Freund's adjuvant in rabbits produced high titers of PfMSP-119 and PfMSP-311-specific functional antibodies. Some of the adjuvant formulations induced inhibitory antibody responses and inhibited in vitro growth of P. falciparum parasites in the presence as well as in the absence of human monocytes. These results suggest that PfMSP-Fu24 can form a constituent of a multistage malaria vaccine.

P lasmodium falciparum is responsible for causing over 2 million deaths annually, and 90% of these deaths are reported to occur in children under the age of 5 years. An effective vaccine represents a high-priority intervention strategy that could provide long-lasting protection from the disease (1)(2)(3)(4)(5). Several malaria vaccines, including the liver-stage vaccine, RTS,S/ASO1, have established that it is possible to interrupt the host-parasite interaction through vaccine-induced immune responses to antigenic targets of pre-erythrocytic, erythrocytic, and sexual stages of Plasmodium (6)(7)(8). However, the lower immune response and minimal to moderate protection seen with most of these vaccines suggest that there is an urgent need for more efficacious, second-generation vaccines (8)(9)(10). Development of highly efficacious second-generation blood-stage vaccines might be achieved with the use of a multiantigen recombinant protein and strong but safe immuneboosting adjuvants.
The important aspect of vaccine development against infectious diseases, including malaria, is the identification of an appropriate adjuvant which can improve the quality and magnitude of the immune response and is safe for use in humans. Aluminumcontaining adjuvants that are licensed for use in human vaccines are aluminum hydroxide (Alhydrogel) and aluminum phosphate (Adjuphos). Both these adjuvants are composed of loose aggregates of noncrystalline gel-like forms of aluminum salts and adsorb antigens via several mechanisms, among which electrostatic adsorption and ligand exchange appear to be important (11,12). Aluminum-containing adjuvants are associated with strong humoral and Th2 responses (13,14). Alhydrogel is a mainstay in current malaria vaccine formulations (15)(16)(17). Toll-like receptor (TLR) ligands are known as immune-potentiating adjuvants and exhibit high potential for prophylactic and therapeutic vaccines (18). The TLR4 agonist glucopyranosyl lipid A (GLA) is a new synthetic hexa-acylated lipid A molecule, which is a nontoxic derivative of lipopolysaccharide and an analog of the detoxified lipopolysaccharide (LPS) derivative monophosphoryl lipid A (MPL). GLA acts as a TLR4 agonist, induces maturation of dendritic cells, and stimulates production of several inflammatory cytokines and type I interferons (19,20). GLA adjuvant is formulated both as an aqueous solution (GLA-AF) and as a stable oilin-water emulsion (GLA-SE) consisting of squalene, glycerol, phosphatidylcholine, poloxamer surfactant, and ammonium phosphate buffer (Immune Design Corporation, Seattle, WA). Mechanistically, GLA-SE augments immunogenicity by inducing a Th1-biased immune response in terms of both antibody and T cell responses (20). Combinations of the TLR4 agonists with Alhydrogel increase the magnitude and avidity of the immune response in comparison with Alhydrogel alone. Whereas Alhydrogel alone induces a Th2 response, the inclusion of a TLR4 agonist can alter the quality of the immune response to a balanced Th1/Th2 response. Adjuplex is a novel adjuvant platform based on a proprietary combination of lecithin and a carbomer homopolymer, both of which are designated "generally regarded as safe" (GRAS) by the U.S. Food and Drug Administration. Nonclinical studies with HIV have indicated that it has potent immunostimulatory effects without reactogenicity and enhances protective immune responses via both cell-mediated and antibody-mediated mechanisms (21).
The approach using construction of fusion protein chimeras has been advocated for blood-stage vaccine development. Several studies reported that chimeric proteins based on merozoite surface antigens show significantly increased immune responses compared to single antigens (22,23). We had constructed a fusion chimera protein by fusing two merozoite antigens, the 19-kDa conserved carboxyl-terminal region of merozoite surface protein 1 (MSP-1 19 ) and an 11-kDa conserved region rich in B and Th epitopes of merozoite surface protein 3 (PfMSP-3 11 ) (24). PfMSP-1 and PfMSP-3 are two of the leading blood-stage vaccine candidates. Merozoite surface protein 1 (MSP-1) is synthesized as an ϳ195-kDa precursor protein that is proteolytically processed to form a multisubunit complex, expressed on the surface of merozoites (25). A 42-kDa glycosylphosphatidylinositol-anchored component, PfMSP-1 42 , is further cleaved at the time of invasion, leaving only a 19-kDa C-terminal domain (PfMSP-1 19 ) attached to the merozoite surface (25). The PfMSP-1 19 fragment has 12 cysteine residues which through disulfide linkage form two highly conserved epidermal growth factor (EGF)-like domains (26). PfMSP-1 19 antibodies have also been shown to correlate with naturally acquired immunity in several epidemiological studies (27). Several studies have shown that immunization with recombinant MSP-1 19 fails to produce high-titer antibodies (28)(29)(30)(31). However, inclusions of exogenous T cell epitopes have been shown to improve the immunogenicity of the PfMSP-1 19 fragment (32).
PfMSP-3 is another major merozoite surface protein that is being developed as a vaccine candidate on the basis of its involvement in antibody-mediated, monocyte-dependent protection acquired by humans upon natural exposure (33,34). Unlike PfMSP-1, PfMSP-3 lacks the characteristic transmembrane domain and is thought to associate loosely with the merozoite surface. PfMSP-3 protein contains three blocks of four tandem heptad repeats based on the AXXAXX motif at the N terminus, a glutamic acid-rich domain, and a putative leucine zipper sequence at the C terminus (35). It has been demonstrated that high levels of cytophilic antibodies to PfMSP-3 are directed toward the conserved N-terminal region of PfMSP-3, which also contains T-helper epitopes (36,37).
We had earlier produced PfMSP-Fu 24 in E. coli BLR(DE3) cells at the shake flask level and showed highly enhanced immunogenicity, including high levels of functional PfMSP-1 19 -and PfMSP-3-specific antibody responses in small animals (24). These findings warranted new studies to further develop PfMSP-Fu 24 as a vaccine candidate and to search for an adjuvant system that is appropriate for human use. In order to take this vaccine candidate to phase 1 clinical trials, we optimized the process for robust, scalable production of PfMSP-Fu 24 . In the present study, we report the production of PfMSP-Fu 24 in E. coli SHuffle 30 and optimization of a batch fermentation process on a 10-liter scale. We tested the preclinical immunogenicity of this clinical-grade recombinant PfMSP-Fu 24 in combination with the following adjuvants: (i) Freund's adjuvant, because of its known efficacy due to its high immunogenicity and widespread use in vaccine development in animals; (ii) Adjuplex, an experimental adjuvant that has been reported to increase vaccine-induced serum IgG antibody titers (21); (iii) Alhydrogel and Adjuphos, which stimulate a Th2type immune response characterized by increased antibody titers (38); and (iv) GLA-SE, a Th1-biased adjuvant that has recently been tested with few malaria antigens in mice (39)(40)(41). We found that both the magnitude and the type of the immune response were affected by the choice of adjuvant. PfMSP-Fu 24 formulated with Freund's adjuvant as well as with Adjuplex and GLA-SE induced high-titer antibodies against PfMSP-1 19 as well as the PfMSP-3 component, while PfMSP-Fu 24 formulated with Alhydrogel and Adjuphos induced low antibody titers in mice. However, in rabbits, Freund's adjuvant and Alhydrogel induced high antibody titers compared to GLA-SE adjuvant. Finally, PfMSP-Fu 24 -specific IgG from some the adjuvant formulations induced significant inhibitory antibody responses (i.e., Ͼ50% inhibition of parasite growth) by blocking erythrocyte invasion as well as by antibody-dependent cellular inhibition mechanisms.
Characterization of recombinant PfMSP-Fu 24 . Recombinant PfMSP-Fu 24 was analyzed for its molecular mass by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) at the International Centre of Genetic Engineering and Biotechnology, India, and N-terminal sequencing by Edman degradation followed by high-pressure liquid chromatography (HPLC) at the National Institute of Immunology, India. Recombinant PfMSP-Fu 24 was characterized for purity and homogeneity by the following methods. PfMSP-Fu 24 was evaluated for its purity on 15% SDS-PAGE gels and stained with Coomassie to observe shifts in mobility due to reduction of disulfide bonds by separating the antigen on SDS-PAGE gel before and after reduction with ␤-mercaptoethanol (␤-ME). The antigenicity of expressed proteins was confirmed by Western blotting using polyclonal antibodies against purified PfMSP-Fu 24 raised in mice to confirm the identity of full-length PfMSP-Fu 24 . HRP-conjugated goat anti-mouse immunoglobulin (Sigma) was used to detect the primary antibodies using diaminobenzidine (DAB). PfMSP-Fu 24 was analyzed for dimers and purity by analytical gel permeation chromatography with Superdex-75 (GE Healthcare). The conformation integrity of the PfMSP-1 19 fragment in recombinant PfMSP-Fu 24 was analyzed by enzyme-linked immunosorbent assay (ELISA) using conformation sensitive monoclonal antibodies (MAb) 2E10 and 1H4. MAb 2E10 and 1H4 were made commercially against bacterially expressed, highly pure PfMSP-1 19 (A&G Pharmaceuticals, Columbia, MD, USA) (42). For the assay, the ELISA plate was coated with PfMSP-Fu 24 and PfMSP-1 19 in coating buffer (50 mM carbonate buffer, pH 9.6) at a concentration of 0.5 mg/well in both the folded and denaturing forms. PfMSP-Fu 24 and PfMSP-1 19 were denatured by boiling the sample with 20% (vol/vol) ␤-ME for 5 min followed by incubation with 50 ml of 20% (wt/vol) iodoacetamide for 30 min at 37°C. The proteins used for coating were probed with MAbs 2E10 and 1H4 at a dilution of 1:2,000 in triplicate wells followed by HRP-conjugated goat anti-mouse immunoglobulin (Sigma) at a dilution of 1:1,000. In addition, recombinant PfMSP-Fu 24 was also probed with anti-PfMSP-119 anti-MSP3 antibody (43) (dilution, 1:1,000), followed by HRP-conjugated goat anti-mouse immunoglobulin (Sigma) at a dilution of 1:1,000 in parallel as another control. The ELISA was developed by the addition of 100 ml/well of O-phenylenediamine dihydrochloride (1 mg/ml; Sigma) and hydrogen peroxide (Merck) as the substrate. The reaction was stopped by adding 2 N sulfuric acid (Merck), and the absorbance was measured at 490 nm using an ELISA reader (Molecular Devices). Mean absorbance values and standard deviations for the triplicate wells of each antibody group were calculated and used for plotting graphs.
PfMSP-Fu 24 was tested for homogeneity by reverse-phase chromatography using a C 8 column (Supelco; Sigma-Aldrich). The gradient used for elution was developed using buffer A (0.05% trifluoroacetic acid in water) and buffer B (0.05% trifluoroacetic acid in acetonitrile). The column was initially washed in 10% buffer B, and the gradient reached 95% buffer B in 40 min. Residual host cell protein (HCP) content was determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting with commercially available kits (Cygnus Technologies, USA). The HCP standard recommended by the manufacturer was used. Endotoxin content in PfMSP-Fu 24 was estimated using a Limulus amoebocyte lysate (LAL) gel clot assay (Endosafe; Charles River, USA). The presence of free thiol groups, if any, was detected by using Ellman's reagent (5,5=-dithio-bis-3nitrobenzoic acid). Different concentrations of L-cysteine were used to plot the standard curve for estimation of free thiol groups in the protein.
Immunization of mice with PfMSP-Fu 24 . Six-to eight-week-old female BALB/c mice were obtained from the Jackson Laboratory and bred in the small-animal facilities of the International Centre for Genetic Engineering and Biotechnology (ICGEB) under pathogen-free conditions. New Zealand White female rabbits were obtained from National Center for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad, India. All animals were fed, housed, and looked after according to recommendations established by the Guide for the Care and Use of Laboratory Animals (ICGEB, India). ICGEB is licensed to conduct animal studies for research purposes under the registration number 18/1999/ CPCSEA (dated 1 October 1999). All the experimental protocols were approved by the ICGEB Institutional Animal Ethics Committee (IAEC).
Rabbits (n ϭ 3) were immunized subcutaneously with 100 g of PfMSP-Fu 24 formulated with complete Freund's adjuvant (CFA), Alhydrogel, and GLA-SE followed by 2 booster immunizations (100 g each) on days 28 and 56 and were bled 14 days after the last immunization.
Enzyme-linked immunosorbent assay (ELISA). The titers of PfMSP-Fu 24 -, PfMSP-1 19 -, and PfMSP-3 11 -specific antibodies in the serum samples were determined by ELISA. Ninety-six-well microplates (Nunc) were coated with 500 ng per well of PfMSP-1 19 or 200 ng per well of PfMSP3 11 or PfMSP-Fu 24 in 0.06 M carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Antigen-coated plates were washed and blocked with 2% low-fat milk in PBS (pH 7.2) for 2 h at 37°C. Two-fold serial dilutions of mouse or rabbit sera (100 l), were added to antigencoated wells in duplicate and incubated for 1 h at room temperature. Bound antibodies were detected using HRP-conjugated secondary antibodies specific for mouse or rabbit antibody immunoglobulin G (IgG), and IgG subclass-specific antibodies for mouse IgG (IgG1, IgG2a, IgG2b, and IgG3) were used for detection. Between incubations, the plates were washed with a 0.05% solution of Tween 20 in PBS by the ELISA plate washer AquaMax 2000 (Molecular Devices). Bound secondary Abs were detected by adding 100 l of O-phenylenediamine dihydrochloride and H 2 O 2 substrate solution at room temperature for 20 min, and the reaction was stopped with 50 l of 2 N H 2 SO 4 . The plates were read at an absorbance of 492 nm using a VersaMax ELISA reader (Molecular Devices). Cutoff values were determined as the mean plus three standard deviations (SDs) for the preimmunization sera.
Immunofluorescence assay (IFA) for PfMSP-Fu 24 . Anti-PfMSP-Fu 24 antibodies raised in mice immunized with different adjuvant formulations were tested for their abilities to recognize the native protein within the parasite by IFA as described earlier (44). Synchronized P. falciparum 3D7 parasites were air dried, fixed on 12-well immunofluorescence slides in methanol for 5 min, and stored at Ϫ80°C. The slides were brought to room temperature, blocked with 3% bovine serum albumin (BSA, fraction V; Sigma) in PBS for 30 min, and then washed twice for 5 min with PBS.
Anti-PfMSP-Fu 24 serum samples were diluted (1:100) in PBS with 3% BSA (Sigma) in PBS-Tween 20 and incubated for 1 h at room temperature in a humidified chamber. After extensive washings with PBS, the slides were incubated with anti-mouse IgG conjugated to Alexa 488-labeled secondary Ab at 1:300 with blocking buffer for 1 h at room temperature. Slides were washed, air dried, and mounted with an antifade agent with DAPI (Invitrogen). Fluorescence was examined under a Nikon SE300 microscope with a 100ϫ oil immersion objective.
Growth inhibition assays (GIA). IgGs were purified on protein G-Sepharose columns (GE Healthcare) from sera obtained from PfMSP-Fu 24 -as well as PfMSP-1 19 -and PfMSP-3 11 -immunized mice and rabbits as described in references 24 and 43, as per the manufacturer's instructions. Briefly, after dilution with the binding buffer (20 mM sodium phosphate buffer, pH 7.0), serum samples were loaded onto a protein G-Sepharose column equilibrated with the binding buffer. The column was washed with 10 column volumes of the binding buffer, and the bound IgG was eluted with 0.2 M glycine-HCl (pH 3.0). Eluted fractions were analyzed by SDS-PAGE, and fractions containing purified IgGs were pooled and dialyzed against incomplete RPMI 1640.
Purified IgG from mouse (pooled) and rabbit (individual) immune sera was tested for its ability to inhibit growth of the parasite in vitro. P. falciparum 3D7, Dd2, and 7G8 parasite lines were used in this assay. The inhibition assay was performed with the starting culture containing 2% hematocrit and approximately 0.3% parasitemia using the synchronized parasite with mostly late trophozoites and schizonts. Purified IgG from mouse and rabbit sera (preimmune and immune) was added to the parasite culture at final concentrations of 5.0 mg/ml. The assay was set up in duplicate and incubated for 44 h. Invasion inhibition efficiency was determined by scoring the percentage of infected erythrocytes by flow cytometry. For flow cytometry, 100 l of 10 g/ml ethidium bromide (Invitrogen Corporation) in PBS (pH 7.4) was mixed with 25 l of parasite culture and incubated for 30 min in darkness at room temperature. After staining, parasite culture was centrifuged and resuspended in 200 l/well of PBS, and the samples were processed using a FACSCalibur cytometer (BD Bioscience). The percentage of infected erythrocytes was evaluated using CellQuest software (BD Bioscience) by first gating for intact erythrocytes by side scatter and forward scatter parameters and subsequently determining the proportion of ethidium bromide-positive cells. The inhibition rate was determined as follows: % inhibition ϭ {1 Ϫ [(parasitemia of test IgG Ϫ parasitemia of starting culture)/(parasitemia of preimmune IgG Ϫ parasitemia of starting culture)]} ϫ 100.
Antibody-dependent cell inhibition (ADCI) assay. The ADCI assay was carried out by using purified IgG from rabbit sera according to procedures described previously (33). Briefly, human monocytes were purified from blood samples of healthy donors by using CD14 (monocyte/macrophage) microbeads (Miltenyi Biotech) according to the manufacturer's protocol. The study was approved by the Human Volunteers Research Ethical Committee of the International Centre for Genetic Engineering and Biotechnology prior to the study. Tightly synchronized P. falciparum 3D7 schizont-stage parasites (0.5% parasitemia and 2% hematocrit) were cocultured in a 96-well flat-bottom microculture plate with adherent human monocytes (2 ϫ 10 6 monocytes) in complete medium (RPMI medium containing 10% human serum). Purified antibodies at a concentration of 50 g/ml were added to these cultures and incubated at 37°C for 96 h in a mixed-gas environment (5% O 2 , 5% CO 2 , and 90% N 2 ). After 48 and 72 h of growth, 50 l of complete medium was added to each well. After 96 h of growth, parasitemia was estimated by flow cytometry. Control wells consisted of (i) parasite alone, (ii) parasite and control IgG (purified from naive mouse sera), (iii) parasites and monocytes, (iv) parasites and purified IgG without monocytes, and (v) parasites, control IgG, and monocytes. The specific growth inhibition index (SGI) was calculated as follows: {1 Ϫ [(percent parasitemia with monocytes and test antibodies/percent parasitemia with test antibodies)/ (percent parasitemia with monocytes and control IgG/percent parasitemia with control IgG)]} ϫ 100.
Statistical analysis. All data are presented as means Ϯ standard deviations; results were statistically analyzed. For analysis of the antibody responses, data were log transformed to conform to the normality and compared using a one-tailed paired t test. Data analyses were performed using GraphPad (GraphPad software, Inc., La Jolla, CA). A P value of Ͻ0.05 was considered statistically significant.

Expression, purification, and characterization of PfMSP-Fu 24 .
Our first goal was to work out fermentation conditions for scalable production of PfMSP-Fu 24 with high biomass and therefore high protein yield. We transformed E. coli BLR(DE3) cells using the plasmid (pET28a-MSP-Fu 24 ) construct used to produce the protein on a shake flask scale (24) (the PfMSP-Fu 24 construct is schematically represented in Fig. 1). From three 10-liter batch fermentations using semidefined media, we obtained a wet biomass of 25 g/liter. Plasmid stability tests showed that there was no  detectable loss of plasmid during fermentation. PfMSP-Fu 24 was purified from soluble fractions by a combination of metal affinity and cation-exchange chromatography to a purity less than or equal to 84%. The quantitative yields of purified PfMSP-Fu 24 produced were ϳ20 mg [ϳ2 mg/liter of E. coli BLR(DE3) fermentation culture] (Table 1 and Fig. 2).
To resolve the problem of low yield, we expressed PfMSP-Fu 24 in the E. coli SHuffle strain, which has disulfide isomerase (Dsbc) and allows improved cytoplasmic disulfide bond formation (45). We transformed E. coli SHuffle cells using the same plasmid (pET28a-MSP-Fu 24 ) construct. Each of the three different batches of 10-liter fermentation using semidefined media typically yielded a wet biomass of 24 g/liter. PfMSP-Fu 24 protein was then purified from the supernatant to Ͼ99% purity by a combination of metal affinity and cation-exchange chromatography (Table 1). Three different 10-liter batch fermentations gave a consistent yield of ϳ23 mg/liter of the protein from E. coli SHuffle fermentation. Analysis of PfMSP-Fu 24 by reverse-phase chromatography revealed the presence of a single symmetric peak, indicating that the protein was highly pure and homogeneous (Fig. 2B). The protein migrated as a single homogeneous band on SDS-PAGE with a molecular mass of ϳ19.2 kDa (Fig. 2C). Polyclonal antibodies against PfMSP-Fu 24 recognized both the native and denatured forms of recombinant PfMSP-Fu 24 (Fig. 3A). The endotoxin level in PfMSP-Fu 24 as determined by the Limulus amebocyte lysate assay was less than 0.25 endotoxin units (EU) per 25 g of protein.  The HCP content was determined by ELISA using an anti-E. coli antibody kit capable of quantifying the lowest concentration of HCP, and the purity of PfMSP-Fu 24 was tested with a Western blot HCP determination kit (Cygnus). No host cell protein contamination was observed in purified PfMSP-Fu 24 , as determined by an ELISA (0.3 ng/ml [0.03%]) and by Western blotting (data not shown). The protective immune responses induced by PfMSP-1 19 have been shown to be dependent on protein conformation (31). We tested the conformational integrity of the PfMSP-1 19 component in PfMSP-Fu 24 by two conformation-sensitive MAbs, 1H4 and 2E10 (42). ELISA showed that purified PfMSP-Fu 24 reacted strongly with both 1H4 and 2E10, confirming that PfMSP-Fu 24 retained conformational epitopes of native PfMSP-1 19 (Fig. 3B). Additionally, the reactivity of PfMSP-Fu 24 with these MAbs declined considerably under denatured conditions, revealing that the critical epitopes were present in the same conformation as the native protein. Ellman's test indicated that there were no free cysteines present in PfMSP-Fu 24 . Polyclonal antibodies against fulllength PfMSP-3 (43) and PfMSP-1 19 (42) recognized the native and denatured forms of PfMSP-Fu 24 (Fig. 3B).
As adjuvants are known to skew the immune response toward either a Th1-type antibody response (dominated in mice by IgG2a and IgG2b) or a Th2-type antibody response (dominated by IgG1) (40), IgG subclass distribution was assessed in mice immunized with PfMSP-Fu 24 formulated with different adjuvants. Mice immunized with PfMSP-Fu 24 exhibited high titers of PfMSP-Fu 24specific IgG1 antibodies (Fig. 5A). The presence of IgG1 isotypes was suggestive of a predominant Th2-type profile. In addition, we observed that PfMSP-Fu 24 -immunized mice showed significantly higher serum IgG1 responses to PfMSP-1 19 , whereas these mice showed a mixed IgG1 and IgG2a response against PfMSP-3 11 ( Fig.  5B and C).
The immunogenicity of PfMSP-Fu 24 formulated with Freund's adjuvant, Alhydrogel, and GLA-SE was also tested in rabbits with the same immunization regimen. Higher antibody titers for both PfMSP-1 19 and PfMSP-3 11 were induced with Freund's adjuvant and Alhydrogel formulation than with the GLA-SE formulation (Fig. 4B). The data indicate that the antibody response in rabbits was different from that in mice.
Anti-PfMSP-Fu 24 antibody recognizes the native parasite protein. Sera raised against PfMSP-Fu 24 formulated with different adjuvants were analyzed for their ability to recognize the native parasite protein in blood-stage P. falciparum 3D7 parasites by immunofluorescence. Antibodies to PfMSP-Fu 24 from all the adjuvant groups showed strong reactivity with the P. falciparum merozoite surface proteins MSP-1 and MSP-3 (Fig. 6).
Anti-PfMSP-Fu 24 antibodies inhibit parasite growth in an ADCI assay. Next, we tested the whether anti-PfMSP-Fu 24 antibodies mediate the monocyte-dependent antibody-mediated cellular inhibition (ADCI) of P. falciparum. PfMSP-3-specific antibodies have been reported to inhibit parasite growth in vitro in a monocyte-dependent manner (15). The specific growth inhibitory index (SGI) was calculated for all these ADCI assays. IgGs purified from sera from rabbits immunized with PfMSP-Fu 24 formulated with Freund's adjuvant and Alhydrogel showed potent effects against parasite strain 3D7 (SGI with Freund's adjuvant formulation ϭ 56%; SGI with Alhydrogel formulation ϭ 46%), and this was comparable to the ADCI activity of the IgGs purified from PfMSP-3 11 immune sera (SGI ϭ 52%). In contrast, no ADCI activity was seen with IgGs from the GLA-SE adjuvant group (Fig. 8).

DISCUSSION
It is necessary to be able to produce substantial quantities of stable and pure recombinant protein with a process that can be scaled up and is reproducible (46). The main aims of the present study were to (i) optimize the large-scale production of PfMSP-Fu 24 , (ii) assess the immunogenicity of PfMSP-Fu 24 in different adjuvant formulations, and (iii) investigate PfMSP-Fu 24 -specific antibodies for inhibition of erythrocyte invasion by three different strains of P. falciparum in vitro.
In our earlier work, we had expressed recombinant PfMSP-Fu 24 as a soluble protein at the shake flask level in E. coli BLR(DE3) and had gotten a yield of ϳ30 mg/liter (24). However, when the same PfMSP-Fu 24 protein was expressed in E. coli BLR(DE3) on a 10-liter fermentation scale, the yield of protein dropped signifi-cantly, to approximately ϳ20 mg of 10-liter fermentation culture. In order to improve the yield and purity of the chimera protein, an alternative E. coli expression system was used to express PfMSP-Fu 24 in a 10-liter fermentation. We were able to significantly scale up the production of PfMSP-Fu 24 using E. coli SHuffle cells that express a signal-truncated disulfide bond isomerase (DsbC) in the cytoplasm and rearrange incorrect disulfide bonds (45). A 10-liter fermentation batch yielded ϳ230 mg of PfMSP-Fu 24 which was highly pure and homogeneous, as indicated by SDS-PAGE, reverse-phase chromatography, and gel permeation chromatography. The purified PfMSP-Fu 24 had a low endotoxin content and was free of thiol and host cell proteins.
PfMSP-1 19 is highly structured and folds into two epidermal growth factor-like domains due to the presence of six disulfide formulated with different adjuvants were tested at 5 mg/ml for inhibition of erythrocyte invasion by three different P. falciparum lines, 3D7, Dd2, and 7G8. (B) IgG purified from rabbit sera immunized with PfMSP-Fu 24 formulated with Freund's adjuvant, Alhydrogel, and GLA-SE were tested at 5 mg/ml for inhibition of erythrocyte invasion by P. falciparum strain 3D7. Percent growth inhibition (shown as means and standard deviations) was calculated by using the parasitemia of the culture grown in the presence of test IgG with parasitemia of the culture grown in the presence of preimmune IgG. linkages, which provide it with conformational stability (47,48). Maintaining proper conformation of PfMSP-1 19 is critical for the generation of protective antibodies. The conformational integrity of the PfMSP-1 19 component of PfMSP-Fu 24 was confirmed with the conformation-specific monoclonal antibodies 1H4 and 2E10 (42). Several monoclonal antibodies, including 1H4 and 2E10, have been shown to be directed against conformation-specific epitopes of PfMSP-1 19 (42). Loss of reactivity of the conformational MAbs with PfMSP-Fu 24 under reducing conditions indicated that disulfide linkages stabilized the tertiary structure of the protein.
Adjuvants are known to boost the magnitude and duration of antibody responses to antigen. In the comparative assessment of different adjuvants used in this study, mice immunized with the adjuvants most commonly used in human vaccines, Alhydrogel and Adjuphos, were not potent enough to induce high levels of functional PfMSP-1 19 -specific and PfMSP-3 11 -specific antibodies. Therefore, adjuvants more potent than Alhydrogel were definitively required for the induction of functional antibody responses to blood-stage malaria antigens. The magnitude of the antibody response to both PfMSP-1 19 and PfMSP-3 11 was improved by the use of GLA-SE and Adjuplex, and these responses were comparable to those obtained with Freund's adjuvant. While Freund's adjuvant, GLA-SE, and Adjuplex produced very high antibody titers, other aspects of the antibody response, such as recognition of native antigen on parasite (by IFA) and subclass distribution (predominant IgG1 indicative of Th2-type response), were much less influenced by adjuvants. When aqueous GLA was added to the Alhydrogel or Adjuphos formulation, the combination of both adjuvants led to an increase in antigen-specific antibody response, although the response was still lower than that seen with GLA-SE. In rabbits, Freund's adjuvant and Alhydrogel formulations elicited higher antibody titers than GLA-SE.
Immunization of mice with PfMSP-Fu 24 resulted in an increased antibody response to PfMSP-1 19 , compared to immunization with either PfMSP-1 19 alone or a mixture of PfMSP-1 19 and PfMSP-3 11 , as we reported previously (24). MSP-1 19 is known to be poorly immunogenic, eliciting low levels of antibodies and poor T-cell activation and functional T helper cells in humans (29,31). A number of studies have shown that the addition or presence of exogenous helper T-cell epitopes provides help to B cells, thereby enhancing the immunogenicity of PfMSP-1 19 (32). The induction of high levels of antibodies and the evidence of IgG class switching in PfMSP-Fu 24 -immunized mice support the critical role of T helper cells in the PfMSP-Fu 24 -induced immune response Furthermore, the T cell response was restricted to the PfMSP-3 11 domain but clearly provided adequate help for the activation and differentiation of both PfMSP-1 19 -and MSP-3 11 -specific B cells (24). Thus, a significant increase in PfMSP-1 19 -specific antibody response may be due to T cell epitopes contributed by the PfMSP-3 11 portion of this chimeric protein (32,49,50).
One of the major concerns for any fusion chimera vaccine construct in general is the possibility of interference in eliciting desired immune responses against individual components. Our results showed that there was no evidence for antigenic competition between PfMSP-1 19 and PfMSP-3 11 . This is consistent with other chimeric malaria vaccine constructs. For example, no antigenic competition was observed with PfCP2.9, a fusion chimera of PfMSP-1 19 and PfAMA-1 (51), or when PfCP2.9 was administered in combination with another recombinant antigen, PfEBA-175 (F2) (52). In another study, a fusion construct of Plasmodium yoelii MSP-1 and MSP-8 induced an antibody response comparable to that of the two components (22). Similarly, immunizations with the GLURP-MSP3 hybrid molecule generated a stronger antibody response against the individual GLURP and MSP3 domains, thereby validating the value of the approach (53).
Having established that there was no interference with the immune response to individual components in PfMSP-Fu 24 , we examined the functional potential of the antibody response. PfMSP-Fu 24 -elicited potent strain-transcending invasion-inhibitory antibodies in mice from different adjuvant groups, except the Alhydrogel and Adjuphos groups, strongly inhibited parasite invasion, as measured by GIA against P. falciparum. In addition, rabbit sera from the Freund's adjuvant and Alhydrogel formulation groups were able to directly inhibit the in vitro growth of different strains of P. falciparum. Likewise, these antibodies inhibited the growth of blood-stage parasites in the ADCI assay, which has been shown to correlate with in vivo protection against infection (24). This provided evidence that PfMSP-Fu 24 -specific antibodies mimicked both the growth inhibition and the ADCI effect of naturally occurring human antibodies against PfMSP-1 19 (54) and PfMSP-3 (33,55), respectively.
In conclusion, we were able to establish a simple protocol to express PfMSP-Fu 24 as a soluble protein in a novel expression system with high yields, which can be easily further scaled up for large-scale production under current good manufacturing practice (cGMP) conditions. PfMSP-Fu 24 formulated in both preclinical and human-approved adjuvants was more immunogenic than the individual PfMSP-1 19 and PfMSP-3 11 in small animals. Additionally, PfMSP-Fu 24 formulated with some of these adjuvants induced inhibitory antibody responses and inhibited in vitro growth of P. falciparum parasites in the presence and in the ab- antibody-dependent cellular inhibition (ADCI). P. falciparum 3D7 parasites at the schizont stage were cocultured with human monocytes in the presence of 25 g of purified IgG from rabbits immunized with PfMSP-Fu 24 formulated with Freund's adjuvant, Alhydrogel, and GLA-SE and from rabbits immunized with PfMSP-1 19 and PfMSP-3 11 formulated with Freund's adjuvant, and parasite growth was monitored after 96 h. The SGI of ADCI of parasite growth (shown as means and standard deviations) was calculated by using the parasitemia of the culture grown in the presence of immune IgGs plus monocytes compared with parasitemia of the culture grown in the presence of monocytes alone.
sence of human monocytes. These results offer the possibility of investigating PfMSP-Fu 24 for development as a malaria vaccine candidate.