Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants

Vaccine 28 (2010) 4977–4983

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants Cheng Du a , Sandra Nilsson b , Huijun Lu a , Jigang Yin a , Ning Jiang a,∗ , Mats Wahlgren b , Qijun Chen a,b,c,∗ a b c

Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Xian Da Lu 5333, Changchun 130062, China Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet and Swedish Institute for Infectious Disease Control, SE-171 77, Stockholm, Sweden Institute of Pathogen Biology, Chinese Academy of Medical Sciences, Dong Dan San Tiao 9, Beijing 100730, China

a r t i c l e

i n f o

Article history: Received 24 November 2009 Received in revised form 7 May 2010 Accepted 11 May 2010 Available online 26 May 2010 Keywords: Plasmodium falciparum Pf332-DBL Immunogenicity Adjuvant

a b s t r a c t The Plasmodium falciparum antigen 332 (Pf332) is a conserved blood-stage antigen, which has been suggested to play a role in parasite invasion. In the present study, we have investigated the immunogenicity of the Duffy-binding like (DBL)-domain of the Pf332 molecule in combination with different adjuvants in four animal species. Three of the adjuvants are applicable for human use (Montanide ISA 720, alum and levamisole), whilst Freund’s adjuvant served as a positive control adjuvant. Montanide ISA 720 was able to generate a significant and Th2-biased IgG response in BALB/c and C57BL/6 mice. Alum was a strong inducer of a Th2-type immune response only in BALB/c mice, whereas it was a poor adjuvant together with Pf332-DBL in C57BL/6 mice, rabbits and rats. Levamisole did not show any obvious adjuvant effect in any of the immunized animals. Thus in the case with Pf332-DBL, Montanide ISA 720 may be an adjuvant to further explore in the development of a vaccine against malaria. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Plasmodium falciparum malaria is one of the most important infectious diseases. With as many as 500 million clinical cases annually, malaria accounts for the death of over one million people every year [1]. One of the main hurdles in the combat against malaria is the emergence of drug-resistant P. falciparum strains and insecticide-resistant mosquitoes, which dramatically reduces the efficiency of conventional treatment and mosquito prevention [2–6]. Therefore, a vaccine that averts or reduces infection and minimizes morbidity and mortality would be an efficient tool in malaria control and preventive programs. Blood-stage vaccines aim at reducing the overall parasite burden and the associated morbidity. The main targets of such a vaccine are P. falciparum antigens expressed on the surface of merozoites and infected red blood cells (iRBC). Adding complexity however, surface antigens often undergo antigenic variation or are highly polymorphic between different parasite strains [7–12]. The success of a vaccine directed against these highly variable antigens would therefore depend on its ability to elicit a broad range of cross-reactive antibodies.

∗ Corresponding authors at: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Xian Da Lu 5333, Changchun 130062, China. Tel.: +86 43187836701. E-mail addresses: [email protected] (N. Jiang), [email protected] (Q. Chen). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.05.027

In recent years, the availability of genomic sequences has provided a powerful platform for rational antigen identification and in particular the identification of antigens with low variability but functional importance [13]. One such antigen is Pf332, a blood-stage protein that can be found in association with the RBC membrane in late-stage trophozoites and schizonts [14–16]. Pf332 is the largest protein identified in P. falciparum and it is present in all parasite strains studied so far [15,16]. The Pf332 antigen contains a conserved Duffy-binding like (DBL)-domain homologous to the DBL-domains of the erythrocyte-binding ligand (EBL) family of invasion proteins, indicating that Pf332 may exert functions related to the EBLs [16]. Furthermore, this molecule is immunogenic and specific antibodies exist in malaria-exposed individuals [17–21]. More interestingly, antibodies targeting different regions of the Pf332 molecule have demonstrated growth and/or invasion inhibitory properties in vitro [16,22,23]. We have recently found that affinity-purified naturally acquired human antibodies targeting the N-terminally located DBL-domain of Pf332 display a prominent invasion inhibitory effect in various parasite strains (manuscript in preparation). The conservation and distinct sequence of Pf332-DBL [16] as well as the potential role the molecule may play in parasite invasion argue for the exploration of this antigen as a malaria vaccine candidate. In the present study, we have examined the immunogenicity of the DBL-domain of Pf332 in combination with different adjuvants in four animal species. The development of synthetic peptide or recombinant subunit vaccines is often hampered by limited intrinsic immunostimulatory properties and proper adjuvants are

4978

C. Du et al. / Vaccine 28 (2010) 4977–4983

therefore essential. In order to transfer data obtained in animal studies to clinical trials, we selected three adjuvants; Montanide ISA 720, alum and levamisole, which are currently being used in human vaccination studies. We here demonstrate that immunizations with Pf332-DBL in combination with Montanide ISA 720 can generate a significant immune response in BALB/c mice and C57BL/6 mice, with immunoglobulin (Ig) G1 as the predominant isotype induced. These data support the use of Montanide ISA 720 in future immunization studies with Pf332-DBL. 2. Material and methods 2.1. Antigen preparation His- and GST-tagged fusion proteins were generated by cloning the gene fragment encoding the N-terminal DBL-domain of Pf332 (amino acid residues 1–216) [16] into the pQE70 (Qiagen, Düsseldorf, Germany) and pGEX-4T-1 vector (GE Healthcare, Uppsala, Sweden). The recombinant proteins were expressed as previously described [24] and subsequently purified using His GraviTrapTM (GE Healthcare) and Glutathione SepharoseTM (GE Healthcare) according to the manufacturer’s instructions. The quality of the recombinant proteins was determined by 12% SDS-PAGE gels. The recombinant proteins were extensively dialyzed against phosphate buffered saline (PBS) and protein concentration was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher scientific, Rockford, IL, USA). His-tagged Pf332-DBL was subsequently used for immunizations and GST-tagged Pf332-DBL for detection of antigen-specific antibodies. 2.2. Animals Experimental animals including BALB/c and C57BL/6 mice, New Zealand white rabbits and Sprague-Dawley rats were selected for the study. The animals were purchased from and kept in the Laboratory Animal Facility Center of the Norman Bethune Medical College of Jilin University. An ethical permission for working with laboratory animals was obtained from the Ethical Committee of the Institute of Zoonosis, Jilin University, China (Permit number 2008IZ-20). 2.3. Immunization regimen and serum sampling Animals were immunized with the His-tagged Pf332-DBL antigen formulated with four different adjuvants including complete Freund’s adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) (Sigma–Aldrich, St. Louis, MO, USA), Montanide ISA 720 (SEPPIC, Paris, France), aluminum hydroxide (alum), levamisole (both from Sigma) or PBS as a control group. To formulate the antigen-adjuvant emulsion, the protein was mixed with an equal volume of adjuvant solution in two syringes connected with an adaptor (Sigma). To prepare the levamisole solution, levamisole was first dissolved in 0.9% NaCl and subsequently diluted to 2% and the His-tagged fusion protein was premixed with 1% levamisole plus 1% glycerin. Each adjuvant test group contained eight mice, four rats, four rabbits and a control group with the same number of animals receiving the recombinant protein in PBS alone. Animals were immunized intramuscularly with the antigenadjuvant emulsion on week 0, 3, 6 and 9. The amount of recombinant protein in the immunization was 10 ␮g/mouse, 50 ␮g/rat and 100 ␮g/rabbit. Animals primed with antigen-CFA were boosted with antigen mixed in IFA in the subsequent three immunizations. Serum samples were collected from all animals in each group prior to the first immunizations and 2 weeks after each immunization as well as on week 13, 15 and 17. Sera were separated by

centrifugation of blood at 1500 × g for 10 min and stored at −80 ◦ C until further analysis. 2.4. Detection of antibody responses by ELISA Antibody responses in immunized animals were measured by ELISA. All experimental steps except for coating and washing were performed at 37 ◦ C. Briefly, Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4 ◦ C with GST-Pf332-DBL fusion protein dissolved in coating buffer (15 mM Na2 CO3 , 35 mM NaHCO3 , pH 9.6) to a concentration of 5 ␮g/ml. A separate row was coated with GST protein as a negative control. Plates were washed four times with PBS containing 0.05% Tween 20 and blocked for 3–4 h with 1% bovine serum albumin (BSA) in PBS. After washing, animal sera diluted 1:1000, 1:8000 and 1:64,000 were added in triplicates and allowed to incubate for 1 h. Plates were washed and bound IgG was detected by incubation for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG, goat anti-rat IgG, or goat anti-rabbit IgG (1:20,000, Sigma). After washing of plates, the assay was developed by adding 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma) and 9.7% diethanolamine (pH 9.8) as a substrate. The optical density (OD) was read at 405 nm after 15 min in a Biotek micro-ELISA auto-reader 808 (Bio-TEK Instruments, Winooski, USA). For typing the IgG subclasses, GST-Pf332-DBL fusion protein was coated on ELISA plates as described above and incubated with sera (1:1000) from immunized mice or rats. Antigen-specific IgG subclasses were thereafter identified with commercial kits; Biotinylated rat anti-mouse antibody- or BioLegend mouse antirat antibody kits (BioLegend San Diego, CA, USA) according to the protocols provided by the manufacturer. 2.5. Immuno-recognition by Western blot The purity of the recombinant protein and the antibody specificity was confirmed by Western blot. Briefly, recombinant His-Pf332-DBL and GST-Pf332-DBL proteins were separated on a 12% SDS-PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The purity of the recombinant protein was determined by probing membranes with mouse-anti-His antibodies and mouse-anti-GST antibodies (1:500, Sigma) as well as a mouse monoclonal antibody (1:250) specific for a peptide within the DBL-region of Pf332. To study antibody specificity, membranes were probed with sera (1:500) from BALB/c mice immunized with different adjuvants. Visualization was performed by using Sigma Fast BCIP/NBT tablets as substrates after a secondary probe with alkaline-phosphatase coupled goat anti-mouse IgG (Sigma) was added. In order to study immuno-recognition of native Pf332 protein, purified FCR3S1.2 parasites (36–40 h post-infection) were separated on a 6% SDSPAGE gel (2.5 × 105 iRBC/lane) and transferred onto nitrocellulose membranes. Membranes were probed with pre-immune sera (1:250) and immune sera (1:250) from BALB/c mice immunized with different adjuvants as well as a rabbit antibody generated against a central repeat region of Pf332 (EB200) [17], which was used as a positive control (1:2000). Visualization was performed by using the ECL-detection system (GE Healthcare) after a secondary probe with HRP-coupled anti-mouse IgG and HRP-coupled anti-rabbit IgG (1:5000, GE Healthcare) was added. 2.6. Statistical analyses Statistical analyses were performed in Prism version 5.0 (Graphpad software, San Diego, USA). Student’s unpaired t-test or Mann–Whitney test (when samples were not normally distributed) were used when comparing total IgG levels between groups of ani-

C. Du et al. / Vaccine 28 (2010) 4977–4983

4979

Fig. 1. Purified Pf332-DBL recombinant proteins. His- and GST-tagged Pf332-DBL fusion proteins were purified and the quality of the recombinant proteins was determined by a Coomassie stained SDS-PAGE gel where two main bands at 26 kDa (His-Pf332-DBL) and 54 kDa (GST-Pf332-DBL) representing full-length Pf332-DBL can be detected (A). The quality of the recombinant proteins was further confirmed by Western blot, where antibodies specific for the tags as well as the Pf332-DBL protein were used (B). Slight degradation was observed with the GST-Pf332-DBL protein. Full-length recombinant Pf332-DBL is indicated (*).

mals immunized with different adjuvants at a p < 0.05 significance level. Data are presented as mean and standard error of mean. 3. Results 3.1. Expression of the DBL-domain of Pf332 in Escherichia coli His- and GST-tagged Pf332-DBL fusion proteins were expressed in E. coli and purified by using His GraviTrapTM and Glutathione SepharoseTM , respectively. The purity of the recombinant proteins was determined by a Coomassie stained SDS-PAGE gel where two main bands at 26 kDa (His-Pf332-DBL) and 54 kDa (GST-Pf332-DBL) representing full-length recombinant protein could be detected (Fig. 1A). Protein quality was further confirmed by Western blot where both tag- and antigen-specific monoclonal antibodies were used (Fig. 1B). The His-tagged Pf332-DBL protein was dialyzed extensively in PBS and subsequently used for immunizations. 3.2. Antibody responses to the DBL-domain of Pf332 in combination with different adjuvants In order to study both immunogenicity and adjuvant effect, BALB/c and C57BL/6 mice, New Zealand white rabbits and SpragueDawley rats were immunized with Pf332-DBL in combination with different adjuvants or protein alone and Pf332 specific antibodies were measured by ELISA. Three of the adjuvants (Montanide ISA 720, alum and levamisole) are applicable for human use, whilst Freund’s adjuvant served as a positive control adjuvant. Animals were immunized on week 0, 3, 6 and 9 and sera were collected prior to the first immunization and 2 weeks after each immunization and the collection was continued until week 17. The polyhistidine-tagged Pf332-DBL protein was found to be immunogenic in all immunized animals, although the antibody responses varied between different animal species. Antibody levels reached a peak on week 11, 2 weeks after the fourth and final immunization, and IgG levels were generally found to be higher in BALB/c mice and rats (Fig. 2A and D) than in C57BL/6 mice and rabbits (Fig. 2B and C).

Fig. 2. IgG responses in four different animal species after the final immunization. BALB/c mice (A), C57BL/6 mice (B), New Zealand white rabbits (C) and SpragueDawley rats (D) were immunized with Pf332-DBL in combination with different adjuvants (Freund’s adjuvant, Montanide ISA 720, alum and levamisole) or protein alone. The IgG response was determined on week 11 (2 weeks after the fourth and final immunization). IgG levels are presented as optical density (OD) values at 405 nm with sera diluted 1:1000. Bars represent the mean of all animals. Experiments are performed in triplicates and error bars indicate standard error of mean. Lines and asterisks indicate a significant difference between groups by Student’s unpaired t-test or Mann–Whitney test (*** and *; p < 0.001 and p < 0.05, respectively).

In BALB/c mice, formulations of Pf332-DBL with Freund’s adjuvant, ISA 720 and alum were more immunogenic and yielded significantly higher IgG levels than when Pf332-DBL was used alone (p < 0.001; Fig. 2A). Moreover, the ISA 720 and alum formulations produced even higher antibody titers than Freund’s adjuvant (p < 0.001 and p < 0.05). C57BL/6 mice immunized with Pf332-DBL combined with Freund’s adjuvant and ISA 720 generated similar antibody responses and IgG levels were significantly higher compared to when mice were immunized with antigen in combination with alum, levamisole or protein alone (p < 0.001; Fig. 2B). In contrast to what was observed in BALB/c mice, the adjuvant effect of alum was very low in C57BL/6 mice. There was no significant difference in adjuvant effect on week 11 within the rabbit or rat groups although there was a trend towards a higher IgG response in rats immunized with ISA 720. The adjuvant effect of levamisole in combination with Pf332-DBL was weak or absent in all animals tested (Fig. 2A–D), indicating that levamisole is not a suitable adjuvant when immunizing with this antigen. 3.3. The longevity of antibody responses after immunizations The dynamic changes and longevity of antibodies specific for the Pf332-DBL antigen were monitored for 17 weeks after the first immunization. Antibody titers were boosted in all animals following the immunizations (Fig. 3). All BALB/c mice, C57BL/6 mice and rats including those immunized with protein alone, still had a significant amount of antibodies after 17 weeks compared to pre-immune animals (p < 0.001, p < 0.001 and p < 0.05). However, in the rabbit group only those immunized with antigen in combination with Freund’s adjuvant still had a significant amount of

4980

C. Du et al. / Vaccine 28 (2010) 4977–4983

Fig. 3. Dynamics and longevity of Pf332-DBL specific antibodies after repeated immunizations. BALB/c mice (A), C57BL/6 mice (B), New Zealand white rabbits (C) and Sprague-Dawley rats (D) were immunized with Pf332-DBL in combination with Freund’s adjuvants (blue), Montanide ISA 720 (green), alum (red), levamisole (purple) or protein alone (black) on week 0, 3, 6, and 9 and monitored for 17 weeks after the first immunization. IgG levels are presented as OD values at 405 nm with sera diluted 1:1000. Graph represents the mean of all animals. Experiments are performed in triplicates and error bars indicate standard error of mean. (I; immunization and B; boost). Due to the low antibody level elicited in BALB/c mice and rabbits when immunized with levamisole formulations, serum sampling was discontinued after the final immunization and levamisole data is as a result of this not present in (A) and (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Pf332-DBL specific antibodies compared to pre-immune animals after 17 weeks (p < 0.05). BALB/c mice immunized with protein combined with Freund’s adjuvant, ISA 720 and alum had significantly higher IgG levels after 17 weeks compared to mice immunized with protein alone (p < 0.001, p < 0.001 and p < 0.05), demonstrating the effect of the adjuvants (Fig. 3A). Further, BALB/c mice immunized with Pf332DBL in combination with ISA 720 had significantly higher IgG levels at 17 weeks than mice immunized with Freund’s adjuvant (p < 0.05), and IgG levels were found to be more stable over time (Fig. 3A). In contrast, there was a gradual decline in Pf332DBL specific antibodies after week 11 in BALB/c mice immunized with antigen in combination with both Freund’s adjuvant and alum. In C57BL/6 mice, Freund’s adjuvant and ISA 720 showed similar effects over time (Fig. 3B), with significantly higher IgG levels compared to C57BL/6 mice immunized with protein alone also at 17 weeks (p < 0.001). However, C57BL/6 mice generated antibody responses that gradually declined after the final immunization with all adjuvants investigated. Rabbits immunized with Freund’s adjuvant demonstrated a more prominent IgG response after the last immunization (Fig. 3C). However, the response was not significant compared to the other adjuvant groups. Immunizations with Pf332-DBL in combination with ISA 720 and alum resulted in a weak antibody response in the rabbits and IgG levels declined gradually after the final immunization. Also in rats, IgG levels declined gradually after the last immunization. Rats immunized in combination with ISA 720 however had significantly higher antibody levels at 17 weeks compared to animals immunized with protein alone (p < 0.05; Fig. 3D). Nei-

ther Freund’s adjuvant, alum nor levamisole was able to induce a strong IgG response in rats. BALB/c mice and rabbits immunized with Pf332-DBL in combination with levamisole elicited very low antibody titers, and serum sampling was therefore discontinued after the final immunization. IgG levels in the levamisole groups are as a result of this not present in the graphs for BALB/c mice and rabbits. 3.4. IgG subclasses after immunizations The antibody subclass that is induced after immunization is an indirect measure of the relative contribution of Th2- versus Th1type immune responses. In mice, production of IgG1 versus IgG2a is widely interpreted as a reflection of Th2- or Th1 reactivity. To examine which type of immune response the different adjuvants elicited, ratios of IgG1/IgG2a levels were examined in sera collected from mice and rats after the final immunization. BALB/c mice and C57BL/6 mice immunized with antigen in combination with Freund’s adjuvant generated a mixed Th1/Th2 response with similar levels of IgG1 and IgG2a being expressed (Table 1). However, rats immunized with Freund’s adjuvant generated predominantly a Th1-type response as revealed by a lower IgG1/IgG2a ratio. BALB/c mice, C57BL/6 mice and rats immunized with either ISA 720 or alum predominantly generated IgG1, indicating that these two adjuvants primarily induced a Th2-biased immune response in the animal species investigated. Immunization with protein alone induced predominantly IgG2a in BALB/c mice and rats, suggesting a Th1-biased immune response. However, mainly IgG1 was elicited in C57BL/6 mice as revealed by a higher IgG1/IgG2a ratio.

C. Du et al. / Vaccine 28 (2010) 4977–4983 Table 1 Ratios of IgG1/IgG2a subclasses of antibodies specific for Pf332-DBL in different animal species. Adjuvant

BALB/c mouse C57BL/6 mouse Rat

Freund’s adjuvant

ISA 720

Alum

Protein alone

0.9 0.8 0.2

2.4 5.2 1.2

1.5 22.7 17.5

0.4 6.6 0.2

3.5. Recognition of Pf332 by immune sera To analyze the specificity of the Pf332-DBL antibodies detected in the ELISA assays, Western blot analyses were carried out with both recombinant GST-Pf332-DBL protein and native Pf332 antigen expressed by the P. falciparum FCR3S1.2 parasite strain. Individual BALB/c sera specifically recognized recombinant GST-tagged Pf332-DBL protein, migrating at approximately 54 kDa (Fig. 4A). Furthermore, all BALB/c sera except for a serum from a BALB/c mouse immunized with a levamisole formulation had antibodies that specifically recognized native Pf332 migrating well above the 250 kDa marker (Fig. 4B), demonstrating that the sere were capable of specifically recognizing also native Pf332 protein expressed in the parasite. Some smaller bands, which are likely processed protein or proteolytic degradation of full-length native Pf332, were observed with all sera. None of the pre-immune sera displayed reactivity to the parasite extract. These data demonstrate that the reactivity observed in the ELISA assays was antigen specific. Also sera from immunized C57BL/6 mice, rabbits and rats detected both recombinant Pf332-DBL and native Pf332 (data not shown). 4. Discussion In the present study, we have examined the immunogenicity of the DBL-domain of Pf332 (Pf332-DBL) in combination with different adjuvants in four rodent animal species. In an effort to down select adjuvants for further development, we have tested three different adjuvants (Montanide ISA 720, alum and levamisole) that are applicable for human use (see Coler et al. [25] for review), whereas Freund’s adjuvant was selected as a control adjuvant. In general, the data show that Pf332-DBL, which can be readily

4981

produced in E. coli (Fig. 1 and our unpublished data), was immunogenic as specific antibodies were boosted after each immunization (Fig. 3). Furthermore, it is interesting to note that different rodent species generated diverse antibody responses towards Pf332-DBL in combination with the adjuvants, demonstrating the complexity of adjuvant studies. The rational of this study was based on previous findings, which have demonstrated that antibodies against Pf332 are commonly found in semi-immune adults and specific antibodies targeting different regions of Pf332 inhibit parasite growth [16,17,23]. In addition, the conserved feature of the DBL-domain of Pf332 makes it a suitable target for vaccination studies. Montanides (including ISA 51 and ISA 720) are water-in-oil emulsions containing squalene and mannide-monooleate as an emulsifier. They are similar in physical characteristics to incomplete Freund’s adjuvant (IFA) but biodegradable. Montanides have been extensively used in several malaria, HIV, and cancer vaccine trials [26–28] and studies have demonstrated that ISA 720 elicits a strong Th2-biased antibody response with a predominant production of IgG1 [29,30]. Here, the Montanide ISA 720 adjuvant showed an effect similar to that generated with Freund’s adjuvant, and in BALB/c mice ISA 720 induced an even stronger antibody response than did Freund’s adjuvant (p < 0.001; Figs. 2 and 3). Furthermore, BALB/c mice immunized with ISA 720 as an adjuvant demonstrated the slowest decrease in IgG levels. Also C57BL/6 mice and rats generated a significant antibody response when ISA 720 was used as an adjuvant compared to when Pf332-DBL was used alone, however, antibody levels gradually declined after the last immunization. Since the type of immune response generated may be critical for the effectiveness of a potential vaccine, we further determined the IgG1/IgG2a ratio of Pf332-DBL specific antibodies in the immunized animals. Production of IgG1 is generally considered to reflect a Th2-biased response, whereas induction of IgG2a reflects a Th1type response. ISA 720 generated a Th2-biased immune response as revealed by higher IgG1/IgG2a ratios in rats and also in the Th2prone BALB/c mice as well as the Th1-prone C57BL/6 mice, which is consistent with previous observations of ISA 720 as a Th2-inducer (Table 1). Aluminum-derived adjuvants are the most commonly used adjuvants in clinical trials and they have the reputation of being safe, give high antibody titers and long lasting antibody responses [25]. Moreover, alum is a potent inducer of a Th2 response, charac-

Fig. 4. Immuno-recognition of recombinant Pf332-DBL protein and native Pf332 antigen by BALB/c mice immunized with Pf332-DBL in combination with Freund’s adjuvant, Montanide ISA 720, alum or levamisole. One representative serum from the immunized mice was selected and assayed from each adjuvant group. Immuno-recognition of recombinant GST-Pf332-DBL protein by sera from immunized BALB/c mice (A). All sera specifically recognized recombinant GST-Pf332-DBL migrating at approximately 54 kDa. Immuno-recognition of native Pf332 from FCR3S1.2 parasite extracts (36–40 h post-infection) (B). Membranes with iRBC were probed with sera from pre-immune BALB/c mice (−) and immunized BALB/c mice (+). A rabbit polyclonal antibody specific for a repeat region within Pf332 (EB200) was used as a positive Pf332 control. All sera specifically recognized native Pf332 protein migrating well above the 250 kDa marker, except for a serum from a BALB/c mouse immunized with protein in combination with levamisole. Some smaller bands, which are likely processed protein or proteolytic degradation of full-length native Pf332, were observed with all sera from the immunized animals.

4982

C. Du et al. / Vaccine 28 (2010) 4977–4983

terized by antigen-specific IgG1 antibodies in the absence of IgG2a [31–33]. The precise mechanism by which alum promotes immune responses is unclear, although absorption of antigen onto particulate alum and thereby creating a depot-effect of delayed antigen release is thought to be one of the main mechanisms. Furthermore, recent data have demonstrated that the enhancing effect of alum on both cellular and humoral immunity, is partly due to the promoted effect on antigen uptake and differentiation of dendritic cells [34,35]. In this study, we found the adjuvant effect of alum to be significant on total IgG levels only in BALB/c mice, where alum induced even higher IgG levels than did Freund’s adjuvant (p < 0.05). Alum was however a poor antibody inducer in C57BL/6 mice, rabbits and rats, as levels were comparable to those obtained in animals immunized with protein alone. Similar to what was observed with ISA 720, alum induced a Th2-biased response. However, antibody levels declined gradually after the final immunization in all adjuvant groups. Levamisole is an imidozothiazole-derived anti-helminthic drug, which has been used in a broad range of hosts including sheep, cattle, horses, pigs, dogs, chickens as well as in humans. Clinically, co-infection with helminthes and other pathogens is common in individuals living in developing countries [36,37]. Further, helminth infections have frequently resulted in a polarized immune response, which may affect host responses to the co-infected pathogen (see Hartgers and Yazdanbakhsh [38] for review). When used as an adjuvant in combination with DNA based vaccines or viral vaccines, levamisole enhances cell-mediated immunity and induces a Th1-biased immune response by stimulating an upregulation of antigen presentation and costimulation [39–41]. Thus, administration of an antigen in combination with levamisole as an adjuvant may be double-beneficial to the host. However, in our study immunization with Pf332-DBL in combination with levamisole elicited much lower antibody levels as compared to the other adjuvants. Levamisole might therefore not be a suitable adjuvant when immunizing with this recombinant protein. In conclusion, it is well known that immunity against malaria is slow to develop and that a successful subunit based malaria vaccine must contain an effective immune-facilitator or an adjuvant, which can both boost and direct the immune response. In this study, we demonstrate that immunization of BALB/c mice with the DBL-domain of Pf332 in combination with Montanide ISA 720 can generate a strong and significant IgG response, with IgG1 as the predominant isotype produced. The immune response in BALB/c mice was even stronger than the response generated by Freund’s adjuvant, an adjuvant universally used for antibody production. The effect of ISA 720 was significant also in C57BL/6 mice and rats, although antibody levels declined somewhat after the final immunization. Since ISA 720 has been included in malaria vaccine trials, a further vaccination study with the Pf332 antigen in combination with Montanide ISA 720 can be pursued. Acknowledgement We are very grateful to Professor Weiqing Pan at Tongji University, Shanghai, China, who provided the Montanide ISA 720 adjuvant used in this study. The anti-EB200 antibody was a kind gift from Professor Klavs Berzins at Stockholm University, Sweden. The study was conducted with support to QC from the National Basic Research Program of China (973 Program, No. 2007CB513100) and grant to Young Distinguished Scientist of NSFC, China and the National S&T Key Project (2008zc10004-011), China. References [1] Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Transactions of the Royal Society of Tropical Medicine and Hygiene 2000;94(April (Suppl. 1)):S1–90.

[2] White NJ. Antimalarial drug resistance. Journal of Clinical Investigation 2004;113(April (8)):1084–92. [3] N’Guessan R, Corbel V, Akogbeto M, Rowland M. Reduced efficacy of insecticidetreated nets and indoor residual spraying for malaria control in pyrethroid resistance area, Benin. Emerging Infectious Diseases 2007;13(February (2)):199–206. [4] Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH. Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Molecular Biology 2000;9(October (5)):491–7. [5] Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Berge JB, Devonshire AL, et al. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Molecular Biology 1998;7(May (2)):179–84. [6] Chandre F, Darrier F, Manga L, Akogbeto M, Faye O, Mouchet J, et al. Status of pyrethroid resistance in Anopheles gambiae sensu lato. Bulletin of the World Health Organization 1999;77(3):230–4. [7] Biggs BA, Gooze L, Wycherley K, Wollish W, Southwell B, Leech JH, et al. Antigenic variation in Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 1991;88(October (20)):9171– 4. [8] Frank M, Kirkman L, Costantini D, Sanyal S, Lavazec C, Templeton TJ, et al. Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum. International Journal for Parasitology 2008;38(August (10)):1099–109. [9] Roy SW, Weedall GD, da Silva RL, Polley SD, Ferreira MU. Sequence diversity and evolutionary dynamics of the dimorphic antigen merozoite surface protein6 and other Msp genes of Plasmodium falciparum. Gene 2009;443(August (1–2)):12–21. [10] Freitas-Junior LH, Bottius E, Pirrit LA, Deitsch KW, Scheidig C, Guinet F, et al. Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature 2000;407(October (6807)):1018–22. [11] Roberts DJ, Craig AG, Berendt AR, Pinches R, Nash G, Marsh K, et al. Rapid switching to multiple antigenic and adhesive phenotypes in malaria Nature 1992;357(June (6380)):689–92. [12] Ferreira MU, Ribeiro WL, Tonon AP, Kawamoto F, Rich SM. Sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-1 (MSP-1) of Plasmodium falciparum. Gene 2003;304(January):65–75. [13] Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002;419(October (6906)):498–511. [14] Haeggstrom M, Kironde F, Berzins K, Chen QJ, Wahlgren M, Fernandez V. Common trafficking pathway for variant antigens destined for the surface of the Plasmodium falciparum-infected erythrocyte. Molecular and Biochemical Parasitology 2004;133(January (1)):1–14. [15] Mattei D, Scherf A. The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes. Gene 1992;110(January (1)):71–9. [16] Moll K, Chene A, Ribacke U, Kaneko O, Nilsson S, Winter G, et al. A novel DBLdomain of the P. falciparum 332 molecule possibly involved in erythrocyte adhesion. PLoS ONE 2007;2(5):e477. [17] Ahlborg N, Haddad D, Siddique AB, Roussilhon C, Rogier C, Trape JF, et al. Antibody responses to the repetitive Plasmodium falciparum antigen Pf332 in humans naturally primed to the parasite. Clinical and Experimental Immunology 2002;129(August (2)):318–25. [18] Balogun HA, Vasconcelos NM, Lindberg R, Haeggstrom M, Moll K, Chen Q, et al. Immunogenicity and antigenic properties of Pf332-C231, a fragment of a non-repeat region of the Plasmodium falciparum antigen Pf332. Vaccine 2009;28(December (1)):90–7. [19] Iqbal J, Perlmann P, Greenwood BM, Berzins K. Seroreactivity with the Plasmodium falciparum blood-stage antigen-Pf332 in adults and children from malaria-endemic regions. Clinical and Experimental Immunology 1993;94(October (1)):68–74. [20] Israelsson E, Balogun H, Vasconcelos NM, Beser J, Roussilhon C, Rogier C, et al. Antibody responses to a C-terminal fragment of the Plasmodium falciparum blood-stage antigen Pf332 in Senegalese individuals naturally primed to the parasite. Clinical and Experimental Immunology 2008;152(April (1)):64–71. [21] Kulane A, Siddique AB, Sarthou JL, Tall A, Dieye A, Perlmann H, et al. Human immune responses to the highly repetitive Plasmodium falciparum antigen Pf332. American Journal of Tropical Medicine and Hygiene 1999;61(July (1)):141–8. [22] Ahlborg N, Flyg BW, Iqbal J, Perlmann P, Berzins K. Epitope specificity and capacity to inhibit parasite growth in-vitro of human-antibodies to repeat sequences of the Plasmodium falciparum antigen-Ag332. Parasite Immunology 1993;15(July (7)):391–400. [23] Ahlborg N, Iqbal J, Bjork L, Stahl S, Perlmann P, Berzins K. Plasmodium falciparum: differential parasite growth inhibition mediated by antibodies to the antigens Pf332 and Pf155/RESA. Experimental Parasitology 1996;82(March (2)):155–63. [24] Flick K, Ahuja S, Chene A, Bejarano MT, Chen QJ. Optimized expression of Plasmodium falciparum erythrocyte membrane protein I domains in Escherichia coli. Malaria Journal 2004;(December):3. [25] Coler RN, Carter D, Friede M, Reed SG. Adjuvants for malaria vaccines. Parasite Immunology 2009;31(September (9)):520–8. [26] Genton B, Betuela I, Felger I, Al-Yaman F, Anders RF, Saul A, et al. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts

C. Du et al. / Vaccine 28 (2010) 4977–4983

[27]

[28]

[29]

[30]

[31]

[32]

selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea. Journal of Infectious Diseases 2002;185(March (6)):820–7. Slingluff CL, Yamshchikov G, Neese P, Galavotti H, Eastham S, Engelhard VH, et al. Phase I trial of a melanoma vaccine with gp100 (280–288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes. Clinical Cancer Research 2001;7(October (10)):3012–24. Toledo H, Baly A, Castro O, Resik S, Laferte J, Rolo F, et al. A phase I clinical trial of a mufti-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers. Vaccine 2001;19(July (30)):4328– 36. Sachdeva S, Ahmad G, Malhotra P, Mukherjee P, Chauhan VS. Comparison of immunogenicities of recombinant Plasmodium vivax merozoite surface protein 1 19- and 42-kilodalton fragments expressed in Escherichia coli. Infection and Immunity 2004;72(October (10)):5775–82. Yazdani SS, Shakri AR, Mukherjee P, Baniwal SK, Chitnis CE. Evaluation of immune responses elicited in mice against a recombinant malaria vaccine based on Plasmodium vivax Duffy binding protein. Vaccine 2004;22(September (27–28)):3727–37. Brewer JM, Conacher M, Hunter CA, Mohrs M, Brombacher F, Alexander J. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4-or IL-13-mediated signaling. Journal of Immunology 1999;163(December (12)):6448–54. Brewer JM, Conacher M, Satoskar A, Bluethmann H, Alexander J. In interleukin4-deficient mice, alum not only generates T helper 1 responses equivalent to Freund’s complete adjuvant, but continues to induce T helper 2 cytokine production. European Journal of Immunology 1996;26(September (9)):2062– 6.

4983

[33] Grun JL, Maurer PH. Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses. Cellular Immunology 1989;121(June (1)):134–45. [34] Kool M, Soullie T, van Nimwegen M, Willart MAM, Muskens F, Jung S, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. Journal of Experimental Medicine 2008;205(April (4)):869–82. [35] Sokolovska A, Hem SL, HogenEsch H. Activation of dendritic cells and induction of CD4 (+) T cell differentiation by aluminum-containing adjuvants. Vaccine 2007;25(June (23)):4575–85. [36] Hartgers FC, Obeng BB, Kruize YC, Dijkhuis A, McCall M, Sauerwein RW, et al. Responses to malarial antigens are altered in helminth-infected children. Journal of Infectious Diseases 2009;199(May (10)):1528–35. [37] Yatich NJ, Yi J, Agbenyega T, Turpin A, Rayner JC, Stiles JK, et al. Malaria and intestinal helminth co-infection among pregnant women in Ghana: prevalence and risk factors. American Journal of Tropical Medicine and Hygiene 2009;80(June (6)):896–901. [38] Hartgers FC, Yazdanbakhsh M. Co-infection of helminths and malaria: modulation of the immune responses to malaria. Parasite Immunology 2006;28(October (10)):497–506. [39] Jin HL, Li YJ, Ma ZH, Zhang FC, Xie QG, Gu DF, et al. Effect of chemical adjuvants on DNA vaccination. Vaccine 2004;22(July (21–22)):2925–35. [40] Kang YM, Jin HL, Zheng GX, Xie QF, Yin JM, Yu Y, et al. The adjuvant effect of levamisole on killed viral vaccines. Vaccine 2005;23(December (48–49)):5543–50. [41] Zhang WJ, Du XG, Zhao G, Jin HL, Kang YM, Xiao C, et al. Levamisole is a potential facilitator for the activation of Th1 responses of the subunit HBV vaccination. Vaccine 2009;27(August (36)):4938–46.