Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

+ MODEL

Microbes and Infection xx (2014) 1e10www.elsevier.com/locate/micinf

Original article

Antigenicity studies in humans and immunogenicity studies in mice: anMSP1P subdomain as a candidate for malaria vaccine development

Yang Cheng a, Eun-Hee Shin b,c, Feng Lu a,1, Bo Wang a, Jongseon Choe d, Takafumi Tsuboi e,Eun-Taek Han a,*

aDepartment of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do 200-701,

Republic of KoreabDepartment of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Disease,

Seoul National University Medical Research Center, Seoul 110-799, Republic of Koreac Seoul National University Bundang Hospital, Seongnam 463-707, Republic of Korea

dDepartment of Microbiology and Immunology, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do 200-701, Republic of KoreaeDivision of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan

Received 5 June 2013; accepted 11 February 2014

Abstract

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus thatbinds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33)were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels weresignificantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P( p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels ofTh1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19( p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P.vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profilingstudy provides evidence that MSP1P may be a potential candidate for vivax vaccine.� 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Plasmodium vivax; MSP1P; IgG subclasses; Cytokines; Immunogenicity

1. Introduction

Vivax malaria threatens almost 40% of the world’s popula-tion, but only two Plasmodium vivax vaccines are in preliminary(phase _) clinical trials [1]. Meanwhile, resistance of P. vivax tofirst-line antimalarial drugs such as chloroquine is increasing,and the resistance of mosquitoes to insecticide has exacerbatedthe threat of malaria. To date, there have been no effective

* Corresponding author. Tel.: þ82 33 250 7941; fax: þ82 33 255 8809.

E-mail addresses: [email protected], [email protected] (E.-T. Han).1 Present address: Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu,

People’s Republic of China.

Please cite this article in press as: Cheng Y, et al., Antigenicity studies in humans a

malaria vaccine development, Microbes and Infection (2014), http://dx.doi.org/10

http://dx.doi.org/10.1016/j.micinf.2014.02.002

1286-4579/� 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights

malaria vaccines [2]. These issues emphasize the urgent needfor an effective malaria vaccine. The P. vivax merozoite surfaceprotein 1 paralog (MSP1P) is a glycosylphosphatidylinositol(GPI)-anchored blood-stage protein, but is not closely related toMSP1 (11% identity and 22% similarity) [3,4]. PvMSP1P islocated on the surface of merozoites, and the C-terminus ofPvMSP1P is highly antigenic and binds erythrocytes (RBCs)[5]. This study indicates that MSP1P has potential as a candi-date for developing a vivax malaria vaccine.

MSP1 shares some characteristics with MSP1P including:(i) a conserved antigenic C-terminal; (ii) location; (iii) anEGF-like domain involved in GPI-anchoring; (iv) ability to

nd immunogenicity studies in mice: an MSP1P subdomain as a candidate for

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reserved.

mailto:[email protected]

mailto:[email protected]




2 Y. Cheng et al. / Microbes and Infection xx (2014) 1e10

bind RBCs; and (v) similar molecular weight (about 200 kDa)[3,5,6]. EGF-like domains contain conserved cysteines thatcan potentially form disulfide bonds, and are predicted to bestructurally homologous to surface proteins of Plasmodiumfalciparum [7]. These regions are obvious candidates formediating proteineprotein interactions, including receptor-binding function. Some GPI-anchored merozoite surface pro-teins of P. falciparum are generally refractory to gene deletion,suggesting that they must play important roles in blood-stagedevelopment [8]. The C-terminus of MSP1, which includesthese common regions, is a leading vaccine candidate forerythrocytic-stage parasites [9]. Moreover, recombinantMSP1-19 can induce immunity in monkey and mouse models[10e12].

The PvMSP1P C-terminus possesses the highest antige-nicity and binding capability within MSP1P [5]. However, thequestion of whether MSP1P is a rational candidate for vivaxmalaria vaccine development should be addressed in moredetail. To characterize the immunogenicity of the two C-ter-minus regions of MSP1P, splenocytes of mice immunized withthese peptides, and the levels of Th1/Th2 cytokines in culturesupernatants of splenocytes from the immunized mice weredetermined. The C-terminus of MSP1P was selected based on:(i) the identification of critical highly antigenic fragments, (ii)the ability of these fragments to bind to RBCs, and (iii) thehighly conserved nature of these regions.

2. Materials and methods

2.1. Human sera samples

Samples of sera were collected from 30 patients positive forvivax malaria (mean age 27.5 years, range 18e52 years) withsymptoms and positivity (mean parasitemia 0.118%, range0.027%e0.477%) identified by microscopy at local healthcenters and clinics in the Gyeonggi and Gangwon Provinces inendemic areas of the Republic of Korea (ROK). Eight serumsamples of healthy individuals (mean age 25 years, range11e44 years) that were negative for vivax malaria by micro-scopy were collected in nonendemic areas of the ROK.Sixteen samples were selected randomly from those from the30 patients positive for vivax malaria, and four normal sam-ples were selected from the eight healthy individuals wereused for determination of IgG subclass level. Written informedconsent to participate in this study was obtained from allsubjects. Approval for the use of the blood samples for thisstudy was obtained from the Kangwon National UniversityHospital Institutional Review Board.

2.2. Expression of PvMSP1 and PvMSP1P proteinfragments

Previously, we divided PvMSP1P (PlasmoDBPVX_099975) into eight small fragments based on MSP1structure [4], and recombinantly expressed a tag of six histi-dines as described previously [5]. In brief, the two MSP1P C-terminus fragments, MSP1P-19 and -33 (Fig. 1A) were

Please cite this article in press as: Cheng Y, et al., Antigenicity studies in humans


expressed using a wheat germ cell-free (WGCF) system(CellFree Sciences, Matsuyama, Japan) and purified using Ni-affinity chromatography under nondenaturing conditions.Experimental protocols involving animals were approved bythe Institutional Animal Care and Use Committee of KangwonNational University, and the experiments were conducted ac-cording to the Kangwon National University Ethical Guide-lines for Animal Experiments.

2.3. Immunization of mice with MSP1-19, MSP1P-19,and -33

Six- to eight-week-old female BALB/c mice (DBL, Seoul,ROK) were injected intraperitoneally with about 20 mg ofrecombinant MSP1-19, MSP1P-19, or -33 proteins inphosphate-buffered saline (PBS) with complete Freund’sadjuvant (SigmaeAldrich, St. Louis, MO) in a final volume of100 ml. Three mice were used per group. Booster injectionswere given after 3 and 6 weeks using the same amount ofantigen in PBS with incomplete Freund’s adjuvant (Sigma-eAldrich). Mouse blood samples were taken 2 weeks after thefinal booster.

2.4. SDS-PAGE and Western blot analysis

Ten micrograms of each recombinant PvMSP1P proteinwas prepared in reducing sample buffer, separated by 12%SDS-PAGE, and then stained with Coomassie Brilliant Blue.For Western blot analysis, the proteins were transferred elec-trophoretically to PVDF membranes (Millipore, Bedford,MA), and incubated with blocking buffer (5% nonfat dry milkin PBS containing 0.2% Tween 20, PBS/T) for 1 h at 37 �C.The blots containing recombinant proteins were then incu-bated for 1 h at 37 �C with either anti-penta-His antibody(Qiagen, Hilden, Germany) or antibodies against each re-combinant PvMSP1P fragment diluted 1:1000 in PBS/T. Themembranes were washed with PBS/T and incubated with a1:2000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (MP Biomedicals, Solon, OH) for 1 h at 37 �C.The blots were washed with PBS/T and developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium(BCIP/NBT, SigmaeAldrich). After the primary antibody re-actions and washing, the membrane was incubated with sec-ondary IRDye goat anti-mouse (1:10,000 dilution) (LI-CORBioscience, Lincoln, NE) antibodies for detecting His-taggedrecombinant protein to determine immune serum specificity.Data were obtained using the Odyssey infrared imaging sys-tem (LI-COR Bioscience) and analyzed with Odyssey soft-ware (LI-COR Bioscience).

2.5. Vivax patient serum screening using protein arrays

Sera from 30 patients infected with vivax malaria weretested against the recombinant PvMSP1P-19 and -33 proteinsusing protein arrays as previously described [5]. Briefly, 1 mlof 50 ng/ml recombinant PvMSP1P-19 or -33 proteins werespotted on PVDF membranes. Then, nonspecific binding sites

and immunogenicity studies in mice: an MSP1P subdomain as a candidate for

.1016/j.micinf.2014.02.002

A

B

SP GPIEGF EGF

MSP1P-33(274 aa)

MSP1P-19(86 aa)

TR PR

NH COOH

25015010075

5037

2520

15

10

kDa 33 192501501007550

37

252015

10

kDa 33 19C α-His

2501501007550

37

252015

10

kDa33 19

α-MrProtein

Fig. 1. Schematic diagram showing expression of PvMSP1P. (A) Schematic representation of PvMSP1P, the two constructs of MSP1P C-terminus used for

serological and cellular analysis. (B) Expression and purification of recombinant PvMSP1P-33 and -19 fragments. Both PvMSP1P-33 (about 40 kDa) and -19

(about 14 kDa) tagged with His were synthesized using a wheat germ cell-free protein expression system, then purified using a Ni-Sepharose column. These two

purified proteins existed in soluble elution fractions and were resolved by 12.5% SDS-PAGE. (C) Recombinant PvMSP1P-33 and -19 proteins were probed with

anti-His antibody (a-His) and immune mouse sera (a-M) under reducing conditions. Arrows indicate the target bands for native and recombinant proteins. rProtein,

recombinant protein; aa, amino acid; kDa, kilodalton; TR, tandem repeat region of heptapeptide; PR, polymorphic region; SP, signal peptide; EGF, epidermal

growth factor-like domains; GPI, glycophosphatidylinositol.

3Y. Cheng et al. / Microbes and Infection xx (2014) 1e10

on the arrays and their matrix were blocked with 5% BSA inPBS/T for 1 h at 37 �C. After probing with human serum(1:10), antibodies were visualized with 10 ng/ml Alexa Fluor546 goat anti-human IgG (Invitrogen, Carlsbad, CA) in PBS/Tand scanned in a fluorescence scanner (ScanArray Express,PerkinElmer, Boston, MA). Fluorescence intensities of arrayspots were quantified using the fixed circle method withScanArray Express software (version 4.0, PerkinElmer).

2.6. Indirect immunofluorescence assay (IFA)

Blood rich in schizont-stage parasites of P. vivax wascollected from a malaria patient in Thailand. Slides smearedwith parasite-infected blood were fixed with ice-cold acetonefor 3 min, dried, and stored at �80 �C. Before use, the slideswere thawed on blue silica gel (Samchun Chemical, Pyeong-taek, Gyeonggi, ROK) and nonspecific binding sites blockedwith PBS containing 5% nonfat dry milk at 37 �C for 30 min.Then, the samples were incubated with primary antibodies(rabbit anti-MSP1-19 [1:100 dilution] and mouse anti-MSP1P-19 [1:50 dilution]) at 37 �C for 1 h. After the primary antibodyreactions, the samples were treated with Alexa 546-conjugatedgoat anti-mouse IgG secondary antibody or Alexa488-conjugated goat anti-rabbit IgG secondary antibody(Invitrogen), and 40,6-diamidino-2-phenylindole (DAPI, Invi-trogen) to stain nuclei, at 37 �C for 30 min. The slides weremounted with ProLong Gold antifade reagent (Invitrogen) andvisualized under oil immersion using a confocal laser scanningmicroscope (FV200; Olympus, Tokyo, Japan) equipped with�20 dry and �60 oil objectives. Images were captured using



FV10-ASW 3.0 viewer software and prepared for publicationwith Adobe Photoshop CS5 (Adobe Systems, San Jose, CA).

2.7. Enzyme-linked immunosorbent assay (ELISA)

To investigate the prevalence of various IgG subclassesagainst MSP1P-19 and -33, sera from 30 vivax-positive pa-tients and eight negative serum samples were selected. Briefly,2.5 mg/ml of MSP1-19, 5 mg/ml of MSP1P-19, and 5 mg/ml ofMSP1P-33 in coating buffer (50 mM bicarbonate buffer, pH9.5) were incubated in 96-well ELISA plates (Costar, Corning,NY) for 2 h at room temperature. Nonspecific binding sites ofthe plates were blocked with 5% nonfat dry milk in PBS/T(0.1% Tween-20) for 1 h at 37 �C, and then the plates incu-bated with 100 ml of individual sera diluted 1:400 in PBS/T.Horseradish peroxidase (HRP)-conjugated antihuman IgG1,IgG2, IgG3, and IgG4 antibodies (Invitrogen) diluted 1:1000in PBS/T were used for detection. Chromogenic reactionswere developed as described previously [13]. The cutoff valuewas the mean plus two standard deviations (SD) of the opticaldensity at 450 nm (OD450) of eight negative samples.

Sixteen serum samples from patients infected with P. vivaxwere randomly selected from the 30 patient samples; and foursamples from unexposed subjects were selected as controls.The IgG subclass distribution was determined using a humanIgG subclass profile kit (Invitrogen), according to the manu-facturer’s instructions. Briefly, a series of concentrations ofIgG1, IgG2, IgG3, and IgG4 standards was established. Theconcentrations used were IgG1 (13.72, 6.86, 3.43, 1.72, 0.86,and 0.43 mg/ml), IgG2 (5.32, 2.66, 1.33, 0.67, 0.33, and


.1016/j.micinf.2014.02.002

A

B

0.0 0.2 0.4 0.60

5000

10000

15000

R2 < 0.1

Parasitemia %

)IFM(

33-P1PSM

GgIna

muH

20000

25000

R2 < 0.1

)I


0.17 mg/ml), IgG3 (1.34, 0.67, 0.34, 0.17, 0.084, and0.042 mg/ml), and IgG4 (0.76, 0.38, 0.19, 0.095, 0.048, and0.024 mg/ml) in 96-well ELISA assay plates. Human subclass-specific antibodies (50 ml/well) were added to wells coatedwith each antibody and then incubated with 50 ml of a 1:2000-dilution of serum samples, standards, and the ready-to-usehuman serum controls. Following aspiration of the samples,the wells were washed with 200 ml of washing buffer, and100 ml of a 1:50 dilution of HRP-conjugated anti-human IgGantibody solution was added. Color was developed with100 ml of 3,30,5,50-tetramethylbenzidine solution (Invitrogen)and absorbance at 450 nm was measured within 1 h of addingthe stop solution. The results were calculated using a logelogcurve fit.

To detect mouse immune serum titers, MSP1-19 (2.5 mg/ml), MSP1P-19 and -33 (5 mg/ml/sample) were coated onELISA plates as previously described [14]. After blockingwith 5% nonfat dry milk in PBS/T, a twofold serial dilution ofanti-MSP1-19, -MSP1P-19, and -MSP1P-33 mouse sera wasadd to each well. HRP-conjugated anti-mouse IgG antibody(H þ L) (dilution 1:10,000) (Pierce Biotechnology, Rockford,IL) was added to each well as a secondary antibody for 1 h at37 �C. The reaction was developed by adding 100 ml ofdiluted TMB solution (Invitrogen) for 15 min at 37 �C andthen stopped with 100 ml 1 N HCl, before optical density(OD) was measured at 450 nm. All samples were tested induplicate, and the mean absorbance was calculated. TheELISA titer was the dilution at which the absorbance unit wasnearest to 1.0.

An ELISA was also developed for quantitative analysis ofthe mouse IgG subclass. Briefly, to construct a standard curve,100 ml of purified mouse IgG1, IgG2a, IgG2b, or IgG3(Invitrogen) was coated onto 96-well plates at 256, 128, 64,32, 16, 8, and 4 ng/ml, and incubated with immune mouse seradiluted 1:1000 in PBS/T. HRP-conjugated anti-mouse IgG1,IgG2a, IgG2b, and IgG3 antibodies (Invitrogen) at 1:1000,1:1000, 1:2000, and 1:1000 dilutions, respectively, were usedto detect reactions. The color intensity was measured andcalculated using a logelog curve fit.
F
M(91-
2.8. T-cell assays
0.0 0.2 0.4 0.60

5000

10000

15000

Parasitemia %

P1PSM

otGgI

namuH

Fig. 2. Correlation between patient peripheral blood parasitemia and mean

immunoreactive fluorescence intensity (MFI). Correlation between immuno-

reactivity of total IgG against PvMSP1P-33 (A) and -19 (B) and parasitemia of

each vivax patient sample was determined for 30 vivax patient samples. R2 was

calculated using a polynomial.

Spleens were removed from mice 2 weeks after their thirdimmunization. Splenocytes were resuspended at 5 � 105 cells/ml in RPMI (Gibco, Invitrogen) supplemented with 1� anti-bioticeantimycotic (Gibco), and 10% fetal bovine serum(Gibco). One-hundred-microliter aliquots of cell suspensionswere distributed into round-bottom 96-well microcultureplates (Costar), and 100 ml (5 mg/ml) of each antigen wasadded. Meantime, splenocytes were stimulated with 5 mg/mlof concanavalin A (SigmaeAldrich) or 10 mg/ml of lipo-polysaccharides (LPS, SigmaeAldrich), which functioned aspositive controls, whereas medium alone was a negativecontrol. Cultures were assayed in triplicate. After 72 h with thecells at 2.5 � 105/well (at 37 �C and 5% CO2), around 150 mlof supernatant/well was collected and stored at �70 �C forcytokine determination. Fifty microliters of enhanced cell



viability buffer (Daeil Lab Service, Seoul, ROK) was added to50 ml of splenocyte culture medium for the final 4 h of a 72 hincubation period. The OD at 450 nm of each well wasmeasured with reference to the OD at 650 nm using an ELISAplate reader. The stimulation index (SI ) was calculated asfollows: mean OD450 of triplicate test wells/meanOD450 þ two standard deviations of six control wells. Prolif-eration was considered positive when the SI was >1.

Cytokine concentrations in culture supernatants and serafrom immunized mice were assayed using the BD CBA FlexSet kit (BD Biosciences, San Jose, CA). The cytokines assayedincluded mouse gamma interferon (IFN-g), tumor necrosisfactor (TNF), interleukin-12p70 (IL-12p70), IL-2, IL-4, andIL-10. The results were acquired on a FACSAriaII Cell Sorter(BD Biosciences) according to the manufacturer’s


.1016/j.micinf.2014.02.002


instructions, and analyzed using FCAP array software (SoftFlow, Kedves, Hungary).

2.9. Statistical analysis

Data were analyzed using GraphPad Prism software (SanDiego, CA), SigmaPlot (Systat Software, San Jose, CA) andMicrosoft Excel 2007 (Microsoft, Redmond, WA). Man-neWhitney U tests were used to compare the differencesbetween the means of each group in terms of their statisticalsignificance. Differences of p < 0.05 were considered

Healthy Patients Healthy Patients0.00.51.01.52.0

5

10

15

20

25p < 0.01

IgG1 IgG2

)lm/g

m(.cnocs

GgI

p = 0.1

A

B

IgG1 IgG2 IgG3 IgG4 IgG1 IgG20.0

0.1

0.2

0.3

0.4

0.6

0.8

1.0

1.2

43%

56%

27%

23%

70%

10%

MSP1-19 MSP

Human IgG subclasses to P

.D.Omn054

Fig. 3. The levels of IgG subclasses in sera from patients from the Republic of Korea

response to PvMSP1P. (A) Immunoreactivity and levels of IgG subclasses against ea

of antibodies to PvMSP1-19, PvMSP1P-19, and -33 in sera from vivax malaria pat

range 18e52 years) and unexposed healthy controls (n ¼ 8; mean age 28.5 years, ra

for antibodies against MSP1-19, MSP1P-19, and -33. The cutoff value was the mean

eight negative samples. (B) IgG subclass levels were determined in 16 of the 30 viv

mean levels of IgG subclasses in vivax patient samples were IgG1 16.7 (range 11

IgG4 0.9 (0.1e1.9) mg/ml. IgG levels in the sera of healthy individuals were: IgG1

and IgG4 0.2 (0.1e0.3) mg/ml. Bars indicate the IgG subclass concentration (mg/ml

the negative and positive groups were analyzed using ManneWhitney U tests. P v

P < 0.05 was considered a significant difference.



significant. Simple scatter-regression was used to make astandard curve.

3. Results

3.1. Expression of C-terminus of PvMSP1P, andproduction of immune sera

Recombinant proteins were successfully purified undernondenaturing conditions as shown in Fig. 1B. The corre-sponding immunoblots were probed with an anti-His tag

Healthy Patients Healthy Patients

p < 0.05

p = 0.12

IgG3 IgG4

IgG3 IgG4 IgG1 IgG2 IgG3 IgG4

73%

20%

57%

0%

20%

23%

1P-19 MSP1P-33

vMSP1P sub-domain antigens

who were positive for P. vivax infection and in sera from healthy individuals in

ch antigen in the sera of malaria patients from ROK was determined. The level

ients was determined by ELISA. Vivax patients (n ¼ 30; mean age 27.5 years,

nge 23e45 years) were randomly selected for IgG subclass prevalence studies

plus two standard deviations (SD) of the optical density at 450 nm (OD450) of

ax patient samples and four of the samples from eight healthy individuals. The

.9e19.7) mg/ml, IgG2 6.7 (4.1e11.2) mg/ml, IgG3 1.7 (1.1e2.2) mg/ml, and

9.2 (8.2e12.0) mg/ml, IgG2 4.8 (3.2e7.5) mg/ml, IgG3 0.3 (0.3e0.6) mg/ml,

) and its percentage in each sample. Differences between IgG subclass levels in

alues for IgG1 and IgG3 were <0.05 and those for IgG2 or IgG4 were >0.05.


.1016/j.micinf.2014.02.002


monoclonal antibody, and anti-rPvMSP1P immune mouseserum revealed a similar and specific pattern of migration forPvMSP1P (Fig. 1C). Preimmune mouse serum samples wereused as a negative control (data not shown).

3.2. Correlation between anti-PvMSP1P antibody levelsand parasitemia

To clarify whether anti-PvMSP1P antibody levels correlatewith protection, antibody responses against two antigens werecompared with the individual parasitemia of the 30 patients(Fig. 2). Although there was no significant inverse correlationbetween parasitemia and antibody levels against eitherPvMSP1P-33 (R2 < 0.1) or �19 (R2 < 0.1) antigens, there wasno case with high antibody levels with high parasitemia(Fig. 2A and B, top right quadrant). These findings suggest

IgG1 IgG2a IgG2b IgG3 IgG1 IgG2a0

50

100

200300400500600

MSP1-19 MSP1

)lm/gu(.cnocsGgI

MSP1-19 MSP1P-19 MSP1P-33

latotesuo

MIgG

Ig

Ig

Ig

Ig

A

B

Fig. 4. IgG subclass levels in immune mouse samples, their percentages, and tot

PvMSP1P-33, or -19, respectively (A). Isotypic distribution of the IgG responses

lated for each of the four subclasses in each group of three mice and are present

responses against PvMSP1-19 and the C-terminal of the PvMSP1P in three BALB

specific antigens, respectively (C). IgG responses were analyzed by ELISA after t

expressed as mean titers � SD.



that the level of anti-PvMSP1P antibody may not stronglycontribute to the inhibition of parasite growth.

3.3. IgG subclass against PvMSP1P C-terminus in vivaxpatients

We analyzed the prevalence of each IgG subclass for an-tibodies against MSP1-19, MSP1P-19, and -33. We found thatIgG3 was the predominant IgG subclass for antibodies againstMSP1P-19 (73%) and MSP1-19 (70%) and IgG1 was thesubdominant IgG subclass for antibodies against MSP1P-33(prevalence 57%) and MSP1P-19 (43%) (Fig. 3A).Compared with MSP1-19, all the IgG subclasses were moreprevalent for antibodies against MSP1P-19, but only IgG1 andIgG4 antibodies were more prevalent against MSP1P-33.Nevertheless, the IgG subclass that predominated antibodies

MSP1-19 MSP1P-19 MSP1P-331

10

100

1000

10000

100000

1000000

GgIlatotfosreti T

IgG2b IgG3 IgG1 IgG2a IgG2b IgG3

P-19 MSP1P-33

G3

G2b

G1

G2a

C

al IgG titers. Three groups of three mice were immunized with PvMSP1-19,

to the MSP1-19, MSP1P-19 and -33 antigens. Geometric means were calcu-

ed as percentages of the total IgG responses in BALB/c mice (B). Total IgG

/c mice per group immunized with PvMSP1-19 and the PvMSP1P C-terminal-

he final immunizations against MSP1-19, MSP1P-19 and -33. The results are


.1016/j.micinf.2014.02.002


against MSP1P-19 and -33 differed, whereas MSP1P-19 andMSP1-19 shared a similar predominant subclass of antibodies.To determine the IgG subclass concentrations in the sera ofvivax patients, we evaluated 16 vivax patients and four serumsamples from healthy individuals. The mean concentrations ofIgG1, IgG2, IgG3, and IgG4 were 16.7, 6.7, 1.0, and 0.5 mg/ml, respectively, compared with 9.2 mg/ml, 4.8 mg/ml,0.4 mg/ml, and 0.25 mg/ml, respectively, in the four samplesfrom healthy individuals (Fig. 3B). Importantly, despite thehigh prevalence of IgG3 antibodies against MSP1-19 andMSP1P-19 in vivax patients, the concentration of IgG3 invivax patient sera was not high. Nevertheless, our data clearlyshow that the levels of IgG1 ( p < 0.01) and IgG3 ( p < 0.05)in patients with vivax infections were significantly greater thanthose in healthy controls.

3.4. IgG1 and IgG2b are the predominant class ofantibodies against the C-terminus of MSP1P inimmunized mice

We determined the levels of IgG subclasses for antibodiesin sera from mice immunized against three antigens. IgG1concentrations were highest in the three groups of immunizedmice, although IgG1 concentrations were similar to those ofIgG2b in mice immunized with MSP1P-19. The level ofIgG2b antibodies against MSP1P-19 (mean concentration447.0 mg/ml) was notably higher than those of IgG2b anti-bodies against MSP1-19 (39.3 mg/ml) and MSP1P-33(22.0 mg/ml) (Fig. 4A). Fig. 4B shows that noncytophilic an-tibodies, IgG1 plus IgG2b, were major components of theantibody response in mice immunized with MSP1-19 andMSP1P-33. There was no significant difference between the

Fig. 5. Localization of PvMSP1P in the mature schizont stage. Schizont-stage par

antisera against PvMSP1P-19 and PvMSP1-19 (merozoite surface marker) (B), or du

Nuclei are visualized with DAPI in merged images. PI, preimmune mouse serum;



levels of cytophilic and noncytophilic isotype from sera ofmice immunized with MSP1P-19.

3.5. Titers of IgG antibodies against the C-terminus ofMSP1P compared with those against MSP1-19

The titers of IgG antibodies against MSP1-19, MSP1P-19,and -33 were 130,000 � 46,000, 80,000 � 10,000 and90,000 � 50,000 (mean � SD), respectively (Fig. 4C). Thetwo C-terminal fragments of MSP1P (MSP1P-19 and -33),clearly induced an immune response as large as that seen inmice immunized with MSP1-19.

3.6. PvMSP1P is a merozoite surface protein

IFA was conducted using anti-PvMSP1 and anti-PvMSP1Psera. In parasites, PvMSP1P was localized to the merozoitesurface (Fig. 5A). To confirm the merozoite surface localiza-tion of PvMSP1P, we compared with merozoite surface pro-tein PvMSP1-19 in the schizont stage (Fig. 5B) usingpreimmune mouse serum as a negative control (Fig. 5C).

3.7. Effect of PvMSP1-19, PvMSP1P-19, and -33 on theTh1/Th2 ratio

We assessed the lymphoproliferation of splenocytes takenfrom immunized BALB/c mice in response to in vitro stimu-lation with 5 mg/ml of MSP1-19, MSP1P-19, or -33 (Fig. 6).Splenocytes from mice immunized with MSP1-19 showed lowproliferative responses (SI range 0.9e2.0), whereas higherlymphoproliferation was observed in response to stimulationwith MSP1P-19 (SI range 1.6e3.5), and MSP1P-33 (SI range

asites were labeled with antisera against PvMSP1P-19 (A), dual-labeled with

al-labeled with antisera against PvMSP1-19 and preimmune mouse serum (C).

DIC, differential interference contrast. Bar represents 5 mm.


.1016/j.micinf.2014.02.002

ConALPS

MSP1-19ConA

LPS

MSP1P-19ConA

LPS

MSP1P-33ConA

LPS

MSP1-19

MSP1P-19

MSP1P-330

1

2

3

4

5

MSP1-19 MSP1P-33MSP1P-19

Immunized mice

)IS(xedninoitalu

mitS

NI

A

MSP1-19

MSP1P-19

MSP1P-33

MSP1-19

MSP1P-19

MSP1P-33

MSP1-19

MSP1P-19

MSP1P-330

5

10

200

400

600

800

IFN-IL-2 TNF

p < 0.001

p < 0.001

p < 0.05

p < 0.01 p < 0.001

p < 0.05

)lm/gp(. cn ocenikoty C

MSP1-19

MSP1P-19

MSP1P-33

MSP1-19

MSP1P-19

MSP1P-330

5

10

100

200

300

400

IL-4 IL-10

p < 0.05

p < 0.01

)lm/gp(.cnoc

enikotyC

B

C

Fig. 6. Cytokine levels in 72-h culture supernatants of splenocytes from BALB/c mice immunized with either MSP1-19, MSP1P-19, or -33, stimulated in vitro with

each protein. (A) The stimulation index (SI ) of splenocytes from mice immunized with MSP1-19, MSP1P-19, or -33 following stimulation with culture medium

and 5 mg/ml of recombinant MSP1-19, MSP1P-19, or -33, respectively. Proliferation of splenocytes stimulated with concanavalin A or LPS as positive controls is

indicated. The SI was calculated as: (mean OD450 of triple test wells)/(mean OD450 þ two standard deviations of six control wells). Proliferation was considered

positive when the SI was >1. The results are expressed as mean SI � SD. Th1 cytokine profile (B), and Th2 cytokine profile (C). Shown are individual results for

each mouse tested in duplicate with samples taken after the third immunization; the black horizontal bars represent the geometric means of groups of mice. The IL-

4 levels against MSP1-19 were too low to detect. The p values, calculated using Student’s t test, indicate the number of sera analyzed; p values of <0.05 are shown.


1.4e3.3) with splenocytes isolated from mice immunized withMSP1P-19 and -33, respectively (Fig. 6A). By contrast, whensplenocytes from unimmunized mice were stimulated by thethree antigens, the levels of splenocyte stimulation by eachantigen were much lower than those of other groups (Fig. 6A).After antigen stimulation, the levels of cytokines in culture



supernatants were determined. Splenocytes from mice immu-nized with MSP1P-19 or MSP1P-33 showed a Th1-predominant response with high levels of secretion of TNF,IFN-g, and IL-2 (Fig. 6B). By contrast, levels of IL-10 and IL-4 cytokines indicating a Th2 profile were detectable as lowlevel of secretion (Fig. 6C). Although splenocytes from mice


.1016/j.micinf.2014.02.002


immunized with MSP1-19 secreted significantly low levels ofTNF, IL-2 and IFN-g cytokines compared with those ofPvMSP1P-19 and -33, it indicated a Th1-predominantresponse compared with Th2 cytokine profiles from low orunder detectable levels of IL-10 and IL-4 cytokines.

As a negative control, splenocytes from nonimmunizedmice (NI group) were also stimulated with MSP1-19, MSP1P-19 or -33, but the cytokine levels generated were borderlinenegative. Nevertheless, the data showed that splenocytesstimulated with MSP1P-19 and -33 were significantly differentfrom those stimulated with MSP1-19 groups ( p < 0.05). IL-12p70 secretion was detected at only a background level(data not shown).

4. Discussion

In light of the important role of PvMSP1P in binding RBCs,we tested our hypothesis that as a GPI-anchored protein,MSP1P may be a blood-stage candidate for vaccine develop-ment. Therefore, we characterized the immune responseinduced by two blood-stage candidates for vaccine develop-ment, the C-termini of MSP1 (MSP1-19) and MSP1P(MSP1P-19 and -33) in human patients in areas of the ROKwhere malaria is endemic. Furthermore, we also analyzed thehumoral and cellular immune responses induced by thesecandidates in mice.

The limited polymorphism seen in MSP1-19 may be theresult of its high immunogenicity [15e17], and that this frag-ment of MSP1 is incorporated on the merozoite surface througha GPI anchor that is taken into the erythrocyte [18]. The GPIsare potent agonists of the toll-like receptor, as are adjuvants thatfacilitate immune responses [19]. Previously, we determinedthe total IgG antibody response in sera of patients from an areain the ROKwhere malaria is endemic whowere infected with P.vivax and reported that about 80% of this population had anti-bodies against MSP1-19, and 68% and 60% had antibodiesagainst MSP1P-19 and -33, respectively [5]. PvMSP1P pos-sesses limited polymorphisms of the EGF-like domains amongglobal isolates [4], and its proteolytic processing mechanismsare still unclear. IgG1 was the most abundant IgG isotype, andhad the highest levels among the malaria patients [20]. Inhumans, both IgG1 and IgG3 antibodies against PvMSP1-19inhibit erythrocyte invasion [21,22]. The increased levels ofmalaria-specific IgG1 and IgG3 responses are associated withreduced risk of clinical malaria and lower parasitemia [23,24].In particular, IgG3 has the highest activity in terms of com-plement activation and binding to phagocytic cells [25].Another study on the N- and C-termini of MSP1 showed thatIgG subclass prevalence was dependent on the endemic regionwhere the isolate originated [26]. Here, our data indicate that inendemic regions of the ROK, IgG3 and IgG1 are the predomi-nant IgG subclasses in naturally acquired humoral immuneresponses against MSP1-19 and MSP1P-19. The antigenicprofiling of MSP1P-19 shows a similar humoral response inhumans as that to MSP1-19.

To date, IgG is the only immune effector known tocontribute to naturally acquired malaria immunity in humans



[27]. In the present study, IgG1 is the predominant IgG sub-class generated in mice by immunization with MSP1-19 orMSP1P-33. Relatively more IgG2b is produced by miceimmunized with MSP1P-19 than any other IgG subclass.Intriguingly, previous studies have reported that the role ofmouse IgG2b is similar to that of IgG3 in humans [28].Indeed, we found that the levels of IgG2b in mice immunizedwith MSP1P-19 paralleled levels of IgG3 in patients with P.vivax infections. These findings suggest that MSP1P-19 inmice is a valid model for understanding the immune response,and may compensate for the difficulty of culturing this parasitein vitro. As such, it warrants further study.

Th1 responses are important for clearance of the malariaparasite [29,30]. The secretion of IFN-g by both CD4þ andCD8þ T cells is associated with a protective immuneresponse, which appears to be critically linked to, or to actthrough, IFN-g production, thereby allowing an early andsustained Th1 response. Another Th1 cytokine, TNF, is pro-tective against the parasite [31]. Severe malarial anemia hasbeen associated with low IL-10 responses [32], whereas res-piratory distress is associated with abnormally large amountsof IL-10. We found both PvMSP1P C-terminal fragmentsinduce a predominant Th1 profile response, suggesting theirpotential for malaria vaccine development, especially frag-ment PvMSP1P-19.

In summary, our data show that PvMSP1P-19 can induce apotent immune response. To confirm whether this candidate isimportant, functional assays including a short-time growthinhibition assay will be developed.

Acknowledgments

We thank Young-Cheol Lim, Department of Molecular andCellular Biochemistry, School of Medicine, Kangwon Na-tional University, Republic of Korea, for his technical assis-tance. This work was supported by National ResearchFoundation of Korea Grant funded by Mid-Career ResearcherProgram MEST (20110016401).

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Documents

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development