INFECTION AND IMMUNITY, Feb. 2010, p. 595–602 Vol. 78, No. 20019-9567/10/$12.00 doi:10.1128/IAI.00877-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Baculovirus-Based Nasal Drop Vaccine Confers Complete Protection againstMalaria by Natural Boosting of Vaccine-Induced Antibodies in Mice�†‡

Shigeto Yoshida,* Hitomi Araki, and Takashi YokomineDivision of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Tochigi 329-0498, Japan

Received 4 August 2009/Returned for modification 1 September 2009/Accepted 30 October 2009

Blood-stage malaria parasites ablate memory B cells generated by vaccination in mice, resulting in dimin-ishing natural boosting of vaccine-induced antibody responses to infection. Here we show the development ofa new vaccine comprising a baculovirus-based Plasmodium yoelii 19-kDa carboxyl terminus of merozoite surfaceprotein 1 (PyMSP119) capable of circumventing the tactics of parasites in a murine model. The baculovirus-based vaccine displayed PyMSP119 on the surface of the virus envelope in its native three-dimensionalstructure. Needle-free intranasal immunization of mice with the baculovirus-based vaccine induced strongsystemic humoral immune responses with high titers of PyMSP119-specific antibodies. Most importantly, thisvaccine conferred complete protection by natural boosting of vaccine-induced PyMSP119-specific antibodyresponses shortly after challenge. The protective mechanism is a mixed Th1/Th2-type immunity, which isassociated with the Toll-like receptor 9 (TLR9)-dependent pathway. The present study offers a novel strategyfor the development of malaria blood-stage vaccines capable of naturally boosting vaccine-induced antibodyresponses to infection.

Malaria, which is transmitted by anopheline mosquitoes, isan enormous public health problem worldwide and every yearkills 1 to 2 million people, mostly children residing in Africa.Clearly, an effective vaccine for the control of malaria is ur-gently needed.

The 42-kDa carboxyl terminus of merozoite surface protein1 (MSP142) is a leading malaria vaccine candidate. In a murinemodel, vaccination with the 19-kDa carboxyl terminus of Plas-modium yoelii MSP1 (PyMSP119) confers protection againstchallenge, and the protective immunity correlates with the hightiter of PyMSP119-specific antibodies (6, 15). Despite its prom-ising potential, none of the MSP1-based Plasmodium falcipa-rum vaccine candidates have shown satisfactory outcomes inhuman clinical trials. With current antigen-adjuvant formula-tions, it has been difficult to induce robust antibody responsesin humans (18). Besides the poor immunogenicity, polymor-phisms in the P. falciparum msp1 gene are thought to representanother big obstacle for the development of vaccines based onthis molecule (24, 29).

Are poor immunogenicity and gene polymorphism really themain reasons why the MSP1-based vaccine candidates in hu-man phase II trials are much less effective than those in animalmodels? In a murine model, immunization with recombinantPyMSP119 vaccines in Freund’s adjuvant induced high titers ofPyMSP119-specific antibodies, leading to protection against le-thal challenge. Although the PyMSP119-specific antibodies atthe time of infection are consumed to impair P. yoelii growth,

no natural boosting of vaccine-induced PyMSP119-specific an-tibody responses is elicited during infection (31). Recent stud-ies demonstrated that the parasite induces apoptotic deletionof vaccine-specific memory B cells, long-lived plasma cells, andCD4� T cells, resulting in failure of the naturally boostingantibody response to malaria parasites during infection (13, 32,33). This is supported by sero-epidemiological studies showingthat a significant proportion of Africans do not possess IgGantibodies to P. falciparum MSP1 despite repeat exposure tomalaria (9–11). Thus, it is likely that malaria parasites manip-ulate the host’s apoptotic pathway to subvert the generationand/or maintenance of immunological memory (21). To date,however, little evidence has been documented on a host’s im-mune response to infection, specifically regarding the naturalboosting associated with vaccine-induced immune responses(26). Most malaria vaccine studies with animal models and inhuman clinical trials have focused mainly on the evaluation ofimmunization-induced immune responses present before chal-lenge. We hypothesize that the limited success of blood-stagevaccines in human clinical trials is mainly due to apoptosisinduction of vaccine-induced memory B cells by the parasite. Ifso, it is essential to develop a new vaccine vector capable notonly of inducing strong protective immune responses but alsoof circumventing the parasite-induced apoptosis of vaccine-specific immune cells.

The baculovirus Autographa californica nucleopolyhedrosisvirus (AcNPV) is an enveloped, double-stranded DNA virusthat naturally infects insects. AcNPV has long been used as abiopesticide and as a tool for efficient production of complexanimal, human, and viral proteins that require folding, subunitassembly, and extensive posttranslational modification in in-sect cells (22, 23). In recent years, AcNPV has been engineeredfor expression of complex eukaryotic proteins (e.g., vaccinecandidate antigens) on the surface of the viral envelope (12, 17,25, 34, 35) and has emerged as a new vaccine vector withseveral attractive attributes, including (i) low cytotoxicity, (ii)

* Corresponding author. Mailing address: Division of Medical Zo-ology, Department of Infection and Immunity, Jichi Medical Univer-sity, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. Phone:81-285-58-7339. Fax: 81-285-44-6489. E-mail: shigeto@jichi.ac.jp.

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 9 November 2009.‡ The authors have paid a fee to allow immediate free access to this

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an inability to replicate in mammalian cells, and (iii) an ab-sence of preexisting antibodies. AcNPV also possesses strongadjuvant properties which can activate dendritic cell (DC)-mediated innate immunity through MyD88/Toll-like receptor 9(TLR9)-dependent and -independent pathways (1), and intra-nasal (i.n.) immunization with AcNPV protects mice from alethal challenge of influenza virus through innate immune re-sponses (2). Therefore, nasal mucosal tissues, which are abun-dant in DCs and macrophages, may be attractive sites forimmunization with AcNPV-based vaccines to induce TLR9-mediated immune responses.

In the present study, we describe i.n. immunization with anAcNPV-based PyMSP119 vaccine (AcNPV-PyMSP119surf) as amodel of a blood-stage vaccine and evaluate the vaccine efficacyin a murine model. Needle-free nasal drop immunization withthis vaccine induced not only strong systemic humoral immuneresponses with high titers of PyMSP119-specific antibodies butalso natural boosting of PyMSP119-specific antibody responsesshortly after challenge, conferring complete protection. Theseresults suggest that the needle-free nasal drop malaria vaccinebased on the baculoviral vector could open a new avenue todeveloping a novel blood-stage malaria vaccine delivery platform.

MATERIALS AND METHODS

Mice and parasites. Female BALB/c mice, 7 to 8 weeks of age at the start ofthe experiment, were purchased from Nippon Clea (Saitama, Japan). TLR9-deficient mice on a BALB/c background were kindly provided by S. Akira(University of Osaka, Suita, Japan). P. yoelii 17XL, a lethal murine malariaparasite, was kindly provided by T. Tsuboi (Ehime University, Matsuyama, Ja-pan) and used for challenge infections.

Recombinant baculovirus. The DNA sequence corresponding to amino ac-ids Pro1659 to Gly1757 of P. yoelii 17XL MSP119 was amplified using theprimers pPyMSP119-F1 (5�-CTGCAGGACTACAAGGACGACGATGACAAGGAATTCGGTGTAGACCCTAAACATGTATGTGTTGATACAAGAGAT-3�) and pPyMSP119-R1 (5�-CCCGGGCTCCCATAAAGCTGGAAGAACTACAGAATACACCT-3�). The resulting PCR product was ligated into thePstI/SmaI sites of pBACsurf-1 (Novagen, Madison, WI) to construct a bac-ulovirus transfer vector, pBACsurf-PyMSP119. A recombinant baculovirus,AcNPV-PyMSP119surf, was generated according to the manufacturer’s (No-vagen) protocol. The purified baculovirus particles were free of endotoxin(�0.01 endotoxin unit/109 PFU), as determined by use of an Endospecyendotoxin measurement kit (Seikagaku Co., Tokyo, Japan).

Recombinant proteins. A recombinant PyMSP119 protein of P. yoelii 17XL,created as a fusion protein with glutathione S-transferase (GST-PyMSP119), wasexpressed in Escherichia coli, purified using a GST affinity column (GE Health-care) as described previously (5), and used as an immunogen for vaccination ofmice and as an enzyme-linked immunosorbent assay (ELISA) antigen. A recom-binant PyMSP119 protein of P. yoelii 17XL (yMSP119), produced as a His6-taggedprotein in Saccharomyces cerevisiae, was obtained from MR4 (Manassas, VA)and used as an ELISA antigen. We confirmed that GST-PyMSP119 and yMSP119

were equally recognized by P. yoelii-hyperimmune sera, which were obtainedfrom BALB/c mice that had recovered from repeated P. yoelii 17XL infection bytreatment with chloroquine as described previously (8). We also confirmed thatthe GST-PyMSP119 and yMSP119 proteins were antigenically equivalent in theELISA by using sera from AcNPV-PyMSP119surf-immunized mice.

Western blotting, IFA, and immuno-electron microscopy. Western blotting,indirect immunofluorescence assay (IFA), and immuno-electron microscopywere carried out as described previously (34). Full details of these methods areprovided in the supplemental material.

Immunization and challenge infections. Mice were immunized three times at3-week intervals with 5 � 107 PFU of baculovirus virions by either the intramus-cular (i.m.) or intranasal (i.n.) route. For i.n. immunization, a total of 50 �l,which was divided into three doses at 5-min intervals, was inoculated by nasaldrop with a Pipetman pipette. As a comparative control, mice were immunizedintraperitoneally (i.p.) with 50 �g of GST-PyMSP119 in 2 mg of aluminumhydroxide (Imject alum; Pierce) three times at 3-week intervals. For oral immu-nization, mice were deprived of food and water for 4 h and then orally immu-

nized four times at 2-week intervals with 0.2 ml of phosphate-buffered saline(PBS) containing AcNPV-PyMSP119surf (5 � 107 PFU), GST-PyMSP119 (50�g), or GST-PyMSP119 (50 �g) plus wild-type AcNPV (AcNPV-WT) (5 � 107

PFU) by intubation with an animal-feeding needle (Natsume, Tokyo, Japan). Foreach route of immunization, sera were collected and mice were challenged with1 � 103 live P. yoelii 17XL-parasitized red blood cells (pRBC) by intravenous(i.v.) injection 2 weeks after the final immunization. To examine the vaccineefficacy of AcNPV-PyMSP119surf, the baculovirus-produced PyMSP119 proteinmay be more suitable as a reference immunogen than E. coli-produced GST-PyMSP119. However, our conventional purification methods cannot removesmall traces of contamination of baculovirus virions and genomic DNA, whichhave strong adjuvant properties to induce innate immunity. Therefore, thepresent study used E. coli-produced GST-PyMSP119 as a standard recombinantPyMSP119 vaccine formulated with alum. The course of parasitemia was moni-tored by microscopic examination of Giemsa-stained thin blood smears obtainedfrom tail bleeds. All care and handling of the animals were in accordance withthe guidelines for animal care and use prepared by Jichi Medical University.

ELISA for antibody titers and isotypes. Sera obtained from immunized micewere collected by tail bleeds 2 weeks after the final immunization, prior tochallenge. For some mice, sera were also collected periodically after challenge.For PyMSP119-specific antibodies of mice immunized with AcNPV-based vac-cines, precoated ELISA plates with 100 ng/well GST-PyMSP119 were incubatedwith serial dilutions of sera. For PyMSP119-specific antibodies of mice immu-nized with GST-PyMSP119, precoated ELISA plates with 100 ng/well yPyMSP119

were incubated with serial dilutions of sera to avoid cross-reaction with GST. Weconfirmed that sera obtained from mice immunized with AcNPV-PyMSP119surfreacted equally with GST-PyMSP119 and yPyMSP119. For blood-stage parasite-specific antibodies, P. yoelii 17XL antigen was prepared from blood of P. yoelii17XL-infected mice as described previously (3). Specific IgGs were detectedusing horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H�L)(Bio-Rad, Hercules, CA). For isotype determination, HRP-conjugated rabbitanti-mouse IgM, IgG� (Southern Biotechnology Associates Inc., Birmingham,AL), IgG1, IgG2a, IgG2b, and IgG3 (Zymed Laboratories, San Francisco, CA)antibodies were used. The plates were developed with peroxidase substrate solution[H2O2 and 2,2�-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)]. The optical density(OD) at 414 nm of each well was measured using a plate reader. End-point titerswere expressed as the reciprocal of the highest sample dilution for which the OD wasequal to or greater than the mean OD for nonimmune control sera.

Cytokine detection. Sera were collected at various times after challenge andstored at �80°C until analysis. The presence of cytokines was analyzed using theBioPlex system (Bio-Rad) as described by de Jager et al. (7). The followingcytokines were analyzed: interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-12p70, gran-ulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon(IFN-�), and tumor necrosis factor alpha (TNF-�).

Statistical analyses. Statistical analysis was performed with Graphpad Prismsoftware (Graphpad Software Inc.). Fisher’s exact probability test was used tocompare the numbers of surviving animals in different groups, and the Mann-Whitney U test was used to compare peak levels of parasitemia between twogroups. Wilcoxon and Mann-Whitney U tests were used to compare antibodylevels between groups for paired and unpaired data, respectively. Spearman’srank correlation test was used to assess associations between antibody levels andmaximal parasitemia. A P value of �0.05 was considered statistically significantfor these analyses.

RESULTS

Construction of baculovirus-based PyMSP119 vaccine.AcNPV-PyMSP119surf harbored a gene cassette that consistedof the gp64 signal sequence and the Pymsp119 gene fused to theN terminus of the coding region for the AcNPV major enve-lope protein gp64 under the control of the polyhedrin pro-moter (Fig. 1A). Western blot analysis showed that bothanti-FLAG monoclonal antibody (MAb) and P. yoelii-hyper-immune serum recognized the PyMSP119-gp64 fusion protein,at a molecular mass of 75 kDa (Fig. 1B, lanes 2 and 4, respec-tively). AcNPV-PyMSP119surf was treated with 10-fold serialdilutions of 2-mercaptoethanol (2-ME). Reduction of theconcentration of 2-ME increased the reactivity of the 75-kDa band for P. yoelii-hyperimmune serum (Fig. 1C). In

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contrast, anti-FLAG MAb, which recognizes a linearepitope, retained the same reactivity of the 75-kDa bandirrespective of the 2-ME concentration (Fig. 1D). Immuno-electron microscopy showed that PyMSP119 was displayedon the viral envelope of AcNPV-PyMSP119surf (Fig. 1E).These results indicate that the PyMSP119-gp64 fusion pro-tein forms oligomers on the virus envelope and retains thethree-dimensional structure of the native PyMSP119 proteinwith correctly formed disulfide bonds.

i.n. immunization with AcNPV-PyMSP119surf induces highlevels of PyMSP119-specific antibody titer. i.n. immunizationwith AcNPV-PyMSP119surf induced significantly higherPyMSP119-specific antibody titer levels than did i.m. immuni-zation (2.7-fold; P � 0.01) (Fig. 2A). Immune sera obtainedafter immunization through the i.m. and i.n. routes containedpredominantly IgG1 and IgG2a (IgG1-to-IgG2a ratio � 0.63

and 0.54, respectively), indicating a mixed Th1/Th2-type im-mune response. The mucosal immunization-inducible IgG2bwas significantly increased in the group receiving i.n. immuni-zation. Immunization with GST-PyMSP119 in alum induced apredominantly Th2-type immune response characterized by ahigh IgG1/IgG2a ratio (�159). The total PyMSP119-specific

FIG. 1. Construction and analysis of recombinant AcNPV express-ing PyMSP119. (A) Schematic diagram of AcNPV-PyMSP119surf ge-nome. PyMSP119 was expressed as a PyMSP119-gp64 fusion proteinunder the control of the polyhedron promoter. Numbers indicate theamino acid positions of the PyMSP119-gp64 fusion protein and theendogenous gp64 protein. pPolh, polyhedrin promoter; SP, gp64 signalsequence; FLAG, FLAG epitope tag; pgp64, gp64 promoter.(B) Western blot analysis of AcNPV-PyMSP119surf. AcNPV-WT(lanes 1, 3, and 5) and AcNPV-PyMSP119surf (lanes 2, 4, and 6) weretreated with loading buffer containing 1% 2-ME and examined usinganti-FLAG MAb (lanes 1and 2), P. yoelii-hyperimmune serum (lanes 3and 4), and anti-gp64 MAb (lanes 5 and 6). Arrows indicate thepositions of PyMSP119-gp64 fusion protein and endogenous gp64. (Cand D) Structural analysis of PyMSP119-gp64 fusion protein. AcNPV-PyMSP119surf was treated with loading buffer containing various con-centrations of 2-ME. The reactivity of the PyMSP119-gp64 fusion pro-tein was examined using either P. yoelii-hyperimmune serum (C) oranti-FLAG MAb (D). The concentrations of 2-ME are shown at thetop. (E and F) Electron micrographs of AcNPV-PyMSP119surf dis-playing PyMSP119 on the viral envelope. AcNPV-PyMSP119surf wastreated with either mouse anti-GST-PyMSP119 antiserum (E) or nor-mal mouse serum (F) followed by labeling with anti-mouse–gold con-jugate. The surfaces of the virions were strongly labeled with goldparticles (arrows). Bars, 100 nm.

FIG. 2. PyMSP119-specific antibody responses. Sera were collectedfrom individual mice (10 mice/group) 3 weeks after the last immuni-zation. (A) The individual sera were tested for total IgG, IgG1, IgG2a,IgG2b, and IgG3 specific for PyMSP119 by ELISA. The data representone of two experiments, which had similar results. Data are presentedas means standard deviations (SD) for the groups. Significant dif-ferences in total IgG titers between different groups were evaluatedusing two-tailed Fisher’s exact probability test. �, P � 0.01. (B and C)Confocal fluorescence micrographs of sera obtained from mice immu-nized with AcNPV-PyMSP119surf. Free merozoites (B) and matureschizonts (C) were clearly stained (green) by serum (1:500 dilution)obtained from one mouse immunized i.n. with AcNPV-PyMSP119surf.Cell nuclei were visualized by DAPI (4�,6-diamidino-2-phenylindole)staining (blue). Similar results were observed for all sera obtained frommice immunized i.m. and i.n. with AcNPV-PyMSP119surf. Bar, 10 �m.(D) PyMSP119-specific antibody responses induced by mucosal immu-nization regimens. Groups of mice (n 10) were immunized either i.n.with GST-PyMSP119 plus AcNPV-WT, i.n. with GST-PyMSP119 alone,or orally with AcNPV-PyMSP119surf. Sera were collected from indi-vidual mice 3 weeks after the last immunization and tested forPyMSP119-specific antibodies and IgG isotypes by ELISA. Ig63 wasundetectable in all groups. Data are presented as means SD for thegroups. �, P � 0.01.

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antibody levels were not different between the GST-PyMSP119

and i.n. AcNPV-PyMSP119surf groups. The IgG3 antibody wasundetectable in the GST-PyMSP119 group (�1:100). Thus,each vaccination regimen induced different IgG class switch-ing. These immune sera strongly reacted with native PyMSP119

on the parasites, with circumferential staining on the surfacesof free merozoites (Fig. 2B) after schizont rupture in additionto the detection of PyMSP119 in mature schizonts (Fig. 2C).

Protection of mice immunized with AcNPV-PyMSP119surfagainst challenge infection. The groups of immunized micewere challenged i.v. with lethal P. yoelii 17XL, and the courseof infection was monitored (Table 1). All but one of the non-immunized control mice died with high parasitemia (76%) byday 8 postchallenge. Mice immunized either i.m. or i.n. withAcNPV-WT suffered severe courses of infection with highmaximum parasitemia levels (�70% parasitemia) and 100%mortality, indicating that AcNPV alone had no protective ef-fect. In the i.m. AcNPV-PyMSP119surf group, 7 of 15 micesurvived after challenge, with high maximum parasitemia(mean standard error of the mean [SEM], 51.1% 5.1%). All mice immunized with i.n. AcNPV-PyMSP119surfsurvived after challenge. Compared with the i.m. AcNPV-PyMSP119surf group, the i.n. AcNPV-PyMSP119surf groupexperienced significantly lower maximum parasitemia levels(7.56% 3.95%; P � 0.01) and earlier parasite clearance(14.7 1.4 days; P � 0.05). In the GST-PyMSP119 group, 7of 15 mice survived after challenge, with moderate maxi-mum parasitemia (37.8% 7.2%). PyMSP119-specific anti-body titers were inversely related to maximal parasitemia,and reduction of parasitemia and protection from deathdepended upon a high titer of PyMSP119-specific antibodiesat challenge (Fig. 3A). Antibody titers over 1:80,000 con-ferred 100% (22/22 mice) survival, while antibody titersunder 1:80,000 resulted in high parasitemia and 12.5% (1/8mice) survival. Thus, i.n. immunization with AcNPV-PyMSP119surf induced a solid level of protection against a

lethal challenge infection. Interestingly, the protection effi-cacy was only 40% (2/5 mice) in the i.n GST-PyMSP119-plus-AcNPV-WT immunization group (Table 1), despite hightiters of PyMSP119-specific antibodies (�4 � 105; predom-inantly IgG1 and IgG2b) (Fig. 2D). The two surviving micedid not induce natural boosting after challenge (data notshown). i.n. immunization with GST-PyMSP119 alone in-duced very low titers of PyMSP119-specific antibodies (�6 �103; predominantly IgG1) (Fig. 2D) and conferred no pro-tection. These results indicate that although AcNPV itselfcan act as a mucosal adjuvant and induce systemic antibodyresponses, display of PyMSP119 on the surface of the viralenvelope is essential to induce protective immunity. Unlikei.n. immunization, oral immunization with AcNPV-PyMSP119surf induced low levels of PyMSP119-specific an-tibodies (�4 � 104; predominantly IgG1 and IgG2b) (Fig.2D) and conferred no protection (Table 1).

Immunization with AcNPV-PyMSP119surf induces naturalboosting of vaccine-induced antibodies to infection. Determin-ing if PyMSP119-specific antibody responses induced by immu-nization were boosted in response to infection was critical toour study. When parasites were cleared, the PyMSP119-specificantibody levels of the i.m. and i.n. AcNPV-PyMSP119surfgroups were increased 8.0- and 8.5-fold, respectively (Fig. 3B).The PyMSP119-specific antibody titer of the i.n. AcNPV-PyMSP119surf group at day 29 was significantly higher (P 0.014) than that of the i.m. AcNPV-PyMSP119surf group, in-

FIG. 3. Antibody induction in response to infection. (A) Correlationbetween PyMSP119-specific antibody titer and parasitemia. Mice wereranked based on maximal parasitemia (Spearman’s rank correlation; r �0.616; P 0.0009). Crosses, death. (B) Comparison of antibody induc-tion between days 0 and 29 postchallenge in surviving mice. A total of 22surviving mice were from the i.m. (n 5) and i.n. (n 10) AcNPV-PyMSP119surf groups and the GST-PyMSP119-plus-alum (n 7) group.PyMSP119-specific antibody titers of each group were compared betweendays 0 and 29. Data are means standard errors (SE) for the groups.Significant differences between each group were evaluated using theMann-Whitney U test. �, P � 0.01.

TABLE 1. Protection of BALB/c mice immunized with AcNPV-PyMSP119surf against P. yoelii challengea

Vaccine (route) % Maximumparasitemia

Time toclearance

(days)

No. ofsurvivors/totalno. in group

(%)

No immunization 75.56 2.35 22 1/15 (10)b

AcNPV-WT (i.m.) 74.48 2.36 0/10 (0)AcNPV-WT (i.n.) 72.04 1.07 0/4 (0)AcNPV-PyMSP119surf

(i.m.)51.09 5.12 8.1 2.0 7/15 (47)b

AcNPV-PyMSP119surf(i.n.)

7.56 3.95* 14.7 1.4** 15/15 (100)**b

AcNPV-PyMSP119surf(oral)

62.39 2.51 0/8 (0)

GST-PyMSP119 �alum (i.p.)

37.76 7.16 17.9 3.3 7/15 (47)b

GST-PyMSP119 (i.n.) 59.12 7.27 0/5 (0)GST-PyMSP119 �

AcNPV-WT (i.n.)58.67 12.70 19.5 5.5 2/5 (40)

a Data represent means SE for total mice in each group. Significant differ-ences between the i.m. and i.n. AcNPV-PyMSP119surf groups were evaluatedusing Fisher’s exact probability test or the Mann-Whitney U test (*, P � 0.05; **,P � 0.01).

b Cumulative data from two independent experiments.

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dicating a stronger natural boosting effect by i.n. immuniza-tion. In contrast, there was no difference in the antibodylevels of the GST-PyMSP119 group before and after infec-tion. Interestingly, compared with the IgG1, -2a, and -2bisotypes, which increased 2.5- to 5-fold, IgG3 levels wereelevated 11- and 36-fold for i.m. and i.n. immunization,respectively (data not shown).

Natural boosting of PyMSP119-specific antibodies was in-duced during the course of infection. To examine when nat-ural boosting of PyMSP119-specific antibodies was induced,PyMSP119-specific antibody titers during the course of in-fection were determined. The kinetics of antibody titers wasplotted with the course of parasitemia (Fig. 4A). PyMSP119-specific antibodies induced by i.n. immunization with AcNPV-

PyMSP119surf drastically increased (eightfold) during days 10to 13 postchallenge, coinciding with the decreasing parasitemiaand parasite clearance, suggesting that the quick response ofnatural boosting may play critical roles in effective clearance ofthe parasites. In contrast, the antibody levels of the GST-PyMSP119 group gradually declined, to 10% of prechallengelevels, from days 7 to 20 postchallenge, as high levels of para-sitemia were developed. This finding also supports the dem-onstration that PyMSP119-specific antibodies alone can controlparasitemia postchallenge (31).

We further addressed the developed antiparasite antibodytiters on days 0 and 29 postchallenge. The P. yoelii-specificIgG titer was significantly increased on day 29 for bothgroups (Fig. 4B), while P. yoelii-specific IgM was signifi-

FIG. 4. Antibody and cytokine responses during the course of infection. Groups of mice were immunized either i.n. with AcNPV-PyMSP119surfor i.p. with GST-PyMSP119 in alum and then challenged i.v. with 103 P. yoelii-parasitized RBC. Parasitemia was monitored daily for 4 days afterchallenge, and sera were collected periodically postchallenge to measure antibody titers and cytokine production. (A) Kinetics of PyMSP119-specific antibody titers and parasitemia during the course of infection. Each point for antibody titers (solid lines) represents the mean SD (n 3). One representative parasitemia level for each group (dotted lines) is shown. i.n. AcNPV-PyMSP119surf data are shown in orange, andGST-PyMSP119-plus-alum data are shown in green. (B and C) IgG (B) and IgM (C) responses to blood-stage parasites at days 0 and 29. Data aremeans SE for the groups (n 3). Significant differences between different groups were evaluated using two-tailed Fisher’s exact probability test.��, P � 0.01; �, P � 0.05. (D to F) Kinetics of cytokine responses. Each point represents the mean SD (n 3). (D) TNF-�; (E) IL-5; (F) IL-10.i.n. AcNPV-PyMSP119surf data are shown in orange, and GST-PyMSP119-plus-alum data are shown in green. Data for nonimmunized mice areshown in blue.

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cantly elevated in the GST-PyMSP119 group but not in thei.n. AcNPV-PyMSP119surf group (Fig. 4C). These resultssuggest that the increasing P. yoelii-specific IgG in the i.n.AcNPV-PyMSP119surf group were mainly due to natural boost-ing of PyMSP119-specific antibodies against native PyMSP119 onmerozoites, resulting in effective parasite clearance. The increasein P. yoelii-specific IgM in the GST-PyMSP119 group during in-fection was likely due to primary immune responses to parasiteantigens other than PyMSP119.

For blood-stage parasites, the magnitudes, kinetics of pro-duction, and overall balance of proinflammatory and anti-in-flammatory cytokines are critically important to infection out-come. We monitored cytokine expression profiles withparasitemia in immunized mice during the course of infection(Fig. 4D to F). Much lower levels of proinflammatory (TNF-�)and anti-inflammatory (IL-5 and IL-10) cytokines were in-duced in the sera of the i.n. AcNPV-PyMSP119surf group thanin those of the GST-PyMSP119 group. Thus, i.n. immunizationwith AcNPV-PyMSP119surf circumvented cytokine dysregula-tion caused by high parasitemia.

TLR9 is involved in the mechanism of protection by i.n.immunization with AcNPV-PyMSP119surf. To determine therole of TLR9 in the AcNPV-PyMSP119surf-induced protec-tion, TLR9-deficient mice on a BALB/c background were im-munized and challenged with P. yoelii 17XL as describedabove. Significantly lower levels of PyMSP119-specific antibodytiters were induced in TLR9-deficient mice immunized withi.n. AcNPV-PyMSP119surf and GST-PyMSP119 in alum than in

the corresponding normal BALB/c mouse groups (Fig. 5A).The IgG isotype in the i.m. and i.n. AcNPV-PyMSP119surfgroups was mainly IgG1 (IgG1/IgG2a ratio � 4.36 and 5.39,respectively), and IgG3 antibody was undetectable (�1:100) inany group (Fig. 5B). Thus, Th2 immune responses are inducedin TLR9-deficient mice, completely contrasting with the mixedTh1/Th2-type immune responses of normal BALB/c miceshown in Fig. 2A.

During challenge, the protection efficacy for each immu-nized group was almost abolished in TLR9-deficient mice (Ta-ble 2). Even though the immunized mice had high antibodytiters (�8 � 104), 82% (14/17 animals) of mice died with highparasitemia (�60%). The three surviving mice from eachgroup did not induce natural boosting of PyMSP119-specificantibodies in response to infection.

DISCUSSION

In the present study, we developed a new PyMSP119 vaccinebased on a baculoviral vector and highlighted natural boostingof vaccine-induced humoral immune responses during thecourse of infection in mice. Both i.n. AcNPV-PyMSP119surfand GST-PyMSP119 (standard recombinant PyMSP119 vaccineformulated with alum) immunizations induced high titers ofPyMSP119-specific antibodies, to the same level. Duringthe course of infection, mice immunized with AcNPV-PyMSP119surf induced natural boosting of PyMSP119-specificantibody responses shortly after challenge, coinciding with de-creasing parasitemia and parasite clearance. In particular, i.n.immunization with AcNPV-PyMSP119surf conferred 100% sur-vival with low parasitemia. Although immunization with GST-PyMSP119 in alum conferred 47% survival against challenge, thesurviving mice exhibited no natural boosting of the PyMSP119-specific antibody response to infection. The results presentedherein offer a novel strategy for the development of baculoviralvector-based malaria blood-stage vaccines capable of naturallyboosting vaccine-induced antibody responses to infection.

The types of immune responses induced by immunization werequalitatively different between the i.n. AcNPV-PyMSP119surf andGST-PyMSP119 groups, with predominantly IgG1 and IgG2a

FIG. 5. PyMSP119-specific antibody response in TLR9-deficientmice. Sera were collected from individual TLR9-deficient mice (8mice/group) 3 weeks after the last immunization and tested forPyMSP119-specific antibodies and IgG isotypes by ELISA. (A) Com-parison of PyMSP119-specific antibody responses between TLR9-defi-cient mice and normal BALB/c mice. Data are means SE for thegroups. Significant differences between each group were evaluatedusing the Mann-Whitney U test. ��, P � 0.01; �, P � 0.05. (B) Isotypeprofiles of PyMSP119-specific antibodies. IgG3 was undetectable in allgroups. The data represent one of two experiments, which had similarresults. Data are means SD for the groups.

TABLE 2. Protection of TLR9-deficient mice immunized withAcNPV-PyMSP119surf against P. yoelii challenge

Vaccine (route) % Maximumparasitemiaa

Time toclearance

(days)

No. of survivors/totalno. in group (%)with PyMSP119-

specific antibody titer

�8 � 104 �8 � 104

No immunization 89.04 0.64 0/7 (0)AcNPV-WT (i.n.) 86.80 3.65 0/8 (0)AcNPV-PyMSP119surf

(i.m.)68.13 9.30 14 1/2 (50) 0/6 (0)

AcNPV-PyMSP119surf(i.n.)b

69.24 4.80 22 1/7 (7) 0/8 (0)

GST-PyMSP119 � alum(i.p.)b

64.16 5.18 21 1/8 (7) 0/6 (0)

a Data are means SE for total mice in each group. There was no signif-icant difference between the immunized groups and the nonimmunizedgroup, as evaluated using Fisher’s exact probability test and the Mann-Whitney U test.

b Cumulative data from two independent experiments.

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(mixed Th1/Th2 type) and predominantly IgG1 (Th2 type)responses, respectively. In the case of the Th2-type immuneresponse, when robust PyMSP119-specific IgG1 (noncytophilicIgG) was induced by immunization with GST-PyMSP119 inFreund’s adjuvant, sterile protection was achieved without de-tectable parasites (28, 30). However, once parasites develop toa detectable level (�0.1% parasitemia) in the blood, suppres-sion of parasitemia requires newly generated immune re-sponses to parasite antigens other than PyMSP119 responseselicited early during infection (31). This is consistent with ourresults showing that P. yoelii-specific IgM antibody titers, butnot PyMSP119-specific IgG antibody titers, were elevated dur-ing infection in the GST-PyMSP119 group. These results sug-gest that although PyMSP119-specific IgG1 may function byblocking parasite invasion or inhibiting MSP1 processing,which is required for erythrocyte entry, memory B cells pro-ducing PyMSP119-specific IgG1 would be deleted from thecirculation by parasite-induced apoptosis (33). The analyses ofthe kinetics of antibody and cytokine production during infec-tion suggested that days 10 to 13 after challenge would be acritical turning point for subsequent parasite clearance for thei.n. AcNPV-PyMSP119surf and GST-PyMSP119 groups. Thei.n. AcNPV-PyMSP119surf group induced natural boosting ofvaccine-induced antibodies, resulting in rapid parasite clear-ance. In contrast, the GST-PyMSP119 group suffered fromcytokine dysregulation caused by high parasitemia. This maytrigger parasite-induced apoptosis, resulting in diminished nat-ural boosting of PyMSP119-specific antibody responses to in-fection and further increasing parasitemia. In addition, induc-tion of IgG2a and IgG2b (cytophilic IgG) antibodies may alsobe necessary, but not a prerequisite, for parasite clearance bythe natural boosting of vaccine-induced antibody responses.Kumar et al. showed that even though immunization withrecombinant PyMSP119 formulated in CpG ODN1826 emulsi-fied in Montanide ISA 51, an adjuvant that favors Th1 differ-entiation, induced high levels of PyMSP119-specific IgG2a andIgG2b, no natural boosting was induced after challenge (19).These results suggest that in addition to the ability to induce amixed Th1/Th2-type immune response, memory B cells pro-duced by AcNPV-PyMSP119surf may possess qualitative andquantitative properties capable of resistance to parasite-in-duced apoptosis.

Mucosal immunization induces antigen-specific Th1- and/orTh2-type immune responses, depending on the nature of theantigen, adjuvant, and antigen delivery vehicle used. Althoughboth i.m. and i.n. immunizations with AcNPV-PyMSP119surfinduced a mixed Th1/Th2-type immune response, i.n. immu-nization led to higher PyMSP119-specific antibody titers andstronger natural boosting after challenge than did i.m. immu-nization. The i.n. immunization with GST-PyMSP119 plusAcNPV-WT induced high levels of PyMSP119-specific IgG1and IgG2b titers and protected 40% (2/5 mice) of mice againstchallenge, but no natural boosting was induced. When choleratoxin B was used as a mucosal adjuvant with rPyMSP119, aTh2-type immune response (predominantly IgG1 and IgG2b)was induced, and 38% (3/8 mice) of mice were protectedagainst challenge (14). Thus, the protection efficacy (100%) ofi.n. immunization with AcNPV-PyMSP119surf was superior tothose of other nasal immunization regimens. This suggests thati.n. immunization with AcNPV-PyMSP119surf displaying

PyMSP119 on the surface of the viral envelope would be es-sential for inducing Th1 cell-derived IFN-� production, whichdetermines switching to IgG2a as a consequence of the cognateinteraction of B cells with Th1 cells (4).

The source of Th1 cells and the precise mechanism of nat-ural boosting are not yet defined. It is possible that i.n. immu-nization with AcNPV-PyMSP119surf activated DCs and mac-rophages, which are abundant in nasal passage-associatedlymphoid tissue (NALT), a mucosal inductive site in the upperrespiratory tract for humoral and cellular immune responses(36), through a TLR9-dependent pathway. In fact, AcNPVitself (composed of viral genomic DNA containing abundantCpG motifs) possesses strong adjuvant properties which canactivate DC-mediated innate immunity through MyD88/TLR9-dependent and -independent pathways (1). The presentstudy provides important insights into the roles of TLR9 fol-lowing immunization with AcNPV-PyMSP119surf. In theTLR9-deficient mice immunized with AcNPV-PyMSP119surf,class switching to IgG2a was severely impaired, and naturalboosting and protective efficacy were completely abolished.Jegerlehner et al. showed that IFN-�-independent IgG2a classswitching is regulated by direct TLR9 signaling in B cells (16).It is likely that AcNPV-PyMSP119surf-induced natural boost-ing after challenge is associated with a TLR9-dependent path-way, and direct stimulation of B cells by AcNPV-PyMSP119surf(after the second and third immunizations) could be the driv-ing factor for class switch recombination to IgG2a in NALT.Nasal drop vaccines have several attractive features comparedwith parenteral vaccines (e.g., safety, cost-effectiveness, and easeof administration), but studies on their use have been limitedalmost exclusively to protection against mucosally transmittedpathogens. We have provided evidence that i.n. immunization is afeasible alternative for preventing malaria transmitted throughnonmucosal routes. Further studies are necessary to fully un-derstand the responses of antigen-presenting cells (APCs)in the NALT (mice) and Waldeyer’s ring (humans), consist-ing of the adenoids and tonsils (20), to AcNPV.

Although 8 of 14 TLR9-deficient mice immunized withGST-PyMSP119 in alum had high (�8 � 104) anti-PyMSP119

antibody titers (Table 2), which are sufficient for normal miceimmunized with GST-PyMSP119 to suppress parasite growth,all mice died with high parasitemia. The failure of protectionsuggests another role of TLR9. It has been reported thatblood-stage parasites activate DCs through a TLR9-dependentpathway (27). In addition, we observed that unlike normalBALB/c mice, TLR9-deficient mice passively immunized withanti-PyMSP119 antibody died with high parasitemia when chal-lenged with P. yoelii 17XL (data not shown). Thus, TLR9 mayenhance antigen presentation by DCs, resulting in induction ofantibody responses to parasite antigens during the course ofinfection. Further studies of TLR9-mediated memory B-cellresponses to baculovirus-based vaccines and natural humanmalaria infection are needed.

Needle-free nasal drop immunization with the baculovirus-based vaccine provides important insights into (i) enhance-ment of immunogenicity of PfMSP142, (ii) strong adjuvanteffects through TLR9, and (iii) subsequent natural boosting toimprove current PfMSP142-based vaccine candidates.

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ACKNOWLEDGMENTS

We thank C. Seki and H. Nagumo for excellent assistance with theELISAs and with handling of the mice. We also thank N. Takahashiand Y. Ishihara for help with the BioPlex system assay and withimmuno-electron microscopy, respectively, and H. Matsuoka for hos-pitality to H.A. and T.Y.

This work was supported by grants from the Ministry of Education,Culture, Sports and Science of Japan (21390126).

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Editor: J. H. Adams

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