Baculovirus-Based Nasal Drop Vaccine Confers Complete Protection against Malaria by Natural Boosting of Vaccine-Induced Antibodies in Mice

Abstract

Blood-stage malaria parasites ablate memory B cells generated by vaccination in mice, resulting in diminishing natural boosting of vaccine-induced antibody responses to infection. Here we show the development of a new vaccine comprising a baculovirus-based Plasmodium yoelii 19-kDa carboxyl terminus of merozoite surface protein 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-dimensional structure. Needle-free intranasal immunization of mice with the baculovirus-based vaccine induced strong systemic humoral immune responses with high titers of PyMSP1₁₉-specific antibodies. Most importantly, this vaccine conferred complete protection by natural boosting of vaccine-induced PyMSP1₁₉-specific antibody responses shortly after challenge. The protective mechanism is a mixed Th1/Th2-type immunity, which is associated with the Toll-like receptor 9 (TLR9)-dependent pathway. The present study offers a novel strategy for the development of malaria blood-stage vaccines capable of naturally boosting vaccine-induced antibody responses to infection.

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

The 42-kDa carboxyl terminus of merozoite surface protein 1 (MSP1₄₂) is a leading malaria vaccine candidate. In a murine model, vaccination with the 19-kDa carboxyl terminus of Plasmodium yoelii MSP1 (PyMSP1₁₉) confers protection against challenge, and the protective immunity correlates with the high titer of PyMSP1₁₉-specific antibodies (6, 15). Despite its promising potential, none of the MSP1-based Plasmodium falciparum vaccine candidates have shown satisfactory outcomes in human clinical trials. With current antigen-adjuvant formulations, it has been difficult to induce robust antibody responses in humans (18). Besides the poor immunogenicity, polymorphisms in the P. falciparum msp1 gene are thought to represent another big obstacle for the development of vaccines based on this molecule (24, 29).

Are poor immunogenicity and gene polymorphism really the main reasons why the MSP1-based vaccine candidates in human phase II trials are much less effective than those in animal models? In a murine model, immunization with recombinant PyMSP1₁₉ vaccines in Freund's adjuvant induced high titers of PyMSP1₁₉-specific antibodies, leading to protection against lethal challenge. Although the PyMSP1₁₉-specific antibodies at the time of infection are consumed to impair P. yoelii growth, no natural boosting of vaccine-induced PyMSP1₁₉-specific antibody responses is elicited during infection (31). Recent studies demonstrated that the parasite induces apoptotic deletion of vaccine-specific memory B cells, long-lived plasma cells, and CD4⁺ T cells, resulting in failure of the naturally boosting antibody response to malaria parasites during infection (13, 32, 33). This is supported by sero-epidemiological studies showing that a significant proportion of Africans do not possess IgG antibodies to P. falciparum MSP1 despite repeat exposure to malaria (9-11). Thus, it is likely that malaria parasites manipulate the host's apoptotic pathway to subvert the generation and/or maintenance of immunological memory (21). To date, however, little evidence has been documented on a host's immune response to infection, specifically regarding the natural boosting associated with vaccine-induced immune responses (26). Most malaria vaccine studies with animal models and in human clinical trials have focused mainly on the evaluation of immunization-induced immune responses present before challenge. We hypothesize that the limited success of blood-stage vaccines in human clinical trials is mainly due to apoptosis induction of vaccine-induced memory B cells by the parasite. If so, it is essential to develop a new vaccine vector capable not only of inducing strong protective immune responses but also of circumventing the parasite-induced apoptosis of vaccine-specific immune cells.

The baculovirus Autographa californica nucleopolyhedrosis virus (AcNPV) is an enveloped, double-stranded DNA virus that naturally infects insects. AcNPV has long been used as a biopesticide and as a tool for efficient production of complex animal, human, and viral proteins that require folding, subunit assembly, and extensive posttranslational modification in insect cells (22, 23). In recent years, AcNPV has been engineered for expression of complex eukaryotic proteins (e.g., vaccine candidate antigens) on the surface of the viral envelope (12, 17, 25, 34, 35) and has emerged as a new vaccine vector with several attractive attributes, including (i) low cytotoxicity, (ii) an inability to replicate in mammalian cells, and (iii) an absence of preexisting antibodies. AcNPV also possesses strong adjuvant properties which can activate dendritic cell (DC)-mediated innate immunity through MyD88/Toll-like receptor 9 (TLR9)-dependent and -independent pathways (1), and intranasal (i.n.) immunization with AcNPV protects mice from a lethal challenge of influenza virus through innate immune responses (2). Therefore, nasal mucosal tissues, which are abundant in DCs and macrophages, may be attractive sites for immunization with AcNPV-based vaccines to induce TLR9-mediated immune responses.

In the present study, we describe i.n. immunization with an AcNPV-based PyMSP1₁₉ vaccine (AcNPV-PyMSP1₁₉surf) as a model of a blood-stage vaccine and evaluate the vaccine efficacy in a murine model. Needle-free nasal drop immunization with this vaccine induced not only strong systemic humoral immune responses with high titers of PyMSP1₁₉-specific antibodies but also natural boosting of PyMSP1₁₉-specific antibody responses shortly after challenge, conferring complete protection. These results suggest that the needle-free nasal drop malaria vaccine based on the baculoviral vector could open a new avenue to developing 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 of the 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 malaria parasite, was kindly provided by T. Tsuboi (Ehime University, Matsuyama, Japan) and used for challenge infections.

Recombinant baculovirus.

The DNA sequence corresponding to amino acids Pro₁₆₅₉ to Gly₁₇₅₇ of P. yoelii 17XL MSP1₁₉ was amplified using the primers pPyMSP1₁₉-F1 (5′-CTGCAGGACTACAAGGACGACGATGACAAGGAATTCGGTGTAGACCCTAAACATGTATGTGTTGATACAAGAGAT-3′) and pPyMSP1₁₉-R1 (5′-CCCGGGCTCCCATAAAGCTGGAAGAACTACAGAATACACCT-3′). The resulting PCR product was ligated into the PstI/SmaI sites of pBACsurf-1 (Novagen, Madison, WI) to construct a baculovirus transfer vector, pBACsurf-PyMSP1₁₉. A recombinant baculovirus, AcNPV-PyMSP1₁₉surf, was generated according to the manufacturer's (Novagen) protocol. The purified baculovirus particles were free of endotoxin (<0.01 endotoxin unit/10⁹ PFU), as determined by use of an Endospecy endotoxin 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), was expressed in Escherichia coli, purified using a GST affinity column (GE Healthcare) as described previously (5), and used as an immunogen for vaccination of mice and as an enzyme-linked immunosorbent assay (ELISA) antigen. A recombinant PyMSP1₁₉ protein of P. yoelii 17XL (yMSP1₁₉), produced as a His₆-tagged protein 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 obtained from BALB/c mice that had recovered from repeated P. yoelii 17XL infection by treatment with chloroquine as described previously (8). We also confirmed that the GST-PyMSP1₁₉ and yMSP1₁₉ proteins were antigenically equivalent in the ELISA by using sera from AcNPV-PyMSP1₁₉surf-immunized mice.

Western blotting, IFA, and immuno-electron microscopy.

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

Immunization and challenge infections.

Mice were immunized three times at 3-week intervals with 5 × 10⁷ PFU of baculovirus virions by either the intramuscular (i.m.) or intranasal (i.n.) route. For i.n. immunization, a total of 50 μl, which was divided into three doses at 5-min intervals, was inoculated by nasal drop with a Pipetman pipette. As a comparative control, mice were immunized intraperitoneally (i.p.) with 50 μg of GST-PyMSP1₁₉ in 2 mg of aluminum hydroxide (Imject alum; Pierce) three times at 3-week intervals. For oral immunization, mice were deprived of food and water for 4 h and then orally immunized four times at 2-week intervals with 0.2 ml of phosphate-buffered saline (PBS) containing AcNPV-PyMSP1₁₉surf (5 × 10⁷ PFU), GST-PyMSP1₁₉ (50 μg), or GST-PyMSP1₁₉ (50 μg) plus wild-type AcNPV (AcNPV-WT) (5 × 10⁷ PFU) by intubation with an animal-feeding needle (Natsume, Tokyo, Japan). For each route of immunization, sera were collected and mice were challenged with 1 × 10³ live P. yoelii 17XL-parasitized red blood cells (pRBC) by intravenous (i.v.) injection 2 weeks after the final immunization. To examine the vaccine efficacy of AcNPV-PyMSP1₁₉surf, the baculovirus-produced PyMSP1₁₉ protein may be more suitable as a reference immunogen than E. coli-produced GST-PyMSP1₁₉. However, our conventional purification methods cannot remove small traces of contamination of baculovirus virions and genomic DNA, which have strong adjuvant properties to induce innate immunity. Therefore, the present study used E. coli-produced GST-PyMSP1₁₉ as a standard recombinant PyMSP1₁₉ vaccine formulated with alum. The course of parasitemia was monitored by microscopic examination of Giemsa-stained thin blood smears obtained from tail bleeds. All care and handling of the animals were in accordance with the guidelines for animal care and use prepared by Jichi Medical University.

ELISA for antibody titers and isotypes.

Sera obtained from immunized mice were collected by tail bleeds 2 weeks after the final immunization, prior to challenge. For some mice, sera were also collected periodically after challenge. For PyMSP1₁₉-specific antibodies of mice immunized with AcNPV-based vaccines, precoated ELISA plates with 100 ng/well GST-PyMSP1₁₉ were incubated with serial dilutions of sera. For PyMSP1₁₉-specific antibodies of mice immunized with GST-PyMSP1₁₉, precoated ELISA plates with 100 ng/well yPyMSP1₁₉ were incubated with serial dilutions of sera to avoid cross-reaction with GST. We confirmed that sera obtained from mice immunized with AcNPV-PyMSP1₁₉surf reacted equally with GST-PyMSP1₁₉ and yPyMSP1₁₉. For blood-stage parasite-specific antibodies, P. yoelii 17XL antigen was prepared from blood of P. yoelii 17XL-infected mice as described previously (3). Specific IgGs were detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (Bio-Rad, Hercules, CA). For isotype determination, HRP-conjugated rabbit anti-mouse IgM, IgGγ (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 titers were expressed as the reciprocal of the highest sample dilution for which the OD was equal to or greater than the mean OD for nonimmune control sera.

Cytokine detection.

Sera were collected at various times after challenge and stored at −80°C until analysis. The presence of cytokines was analyzed using the BioPlex system (Bio-Rad) as described by de Jager et al. (7). The following cytokines were analyzed: interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α).

Statistical analyses.

Statistical analysis was performed with Graphpad Prism software (Graphpad Software Inc.). Fisher's exact probability test was used to compare the numbers of surviving animals in different groups, and the Mann-Whitney U test was used to compare peak levels of parasitemia between two groups. Wilcoxon and Mann-Whitney U tests were used to compare antibody levels between groups for paired and unpaired data, respectively. Spearman's rank correlation test was used to assess associations between antibody levels and maximal parasitemia. A P value of <0.05 was considered statistically significant for these analyses.

RESULTS

Construction of baculovirus-based PyMSP1₁₉ vaccine.

AcNPV-PyMSP1₁₉surf harbored a gene cassette that consisted of the gp64 signal sequence and the Pymsp1₁₉ gene fused to the N terminus of the coding region for the AcNPV major envelope protein gp64 under the control of the polyhedrin promoter (Fig. 1A). Western blot analysis showed that both anti-FLAG monoclonal antibody (MAb) and P. yoelii-hyperimmune serum recognized the PyMSP1₁₉-gp64 fusion protein, at a molecular mass of 75 kDa (Fig. 1B, lanes 2 and 4, respectively). AcNPV-PyMSP1₁₉surf was treated with 10-fold serial dilutions of 2-mercaptoethanol (2-ME). Reduction of the concentration of 2-ME increased the reactivity of the 75-kDa band for P. yoelii-hyperimmune serum (Fig. 1C). In contrast, anti-FLAG MAb, which recognizes a linear epitope, retained the same reactivity of the 75-kDa band irrespective of the 2-ME concentration (Fig. 1D). Immuno-electron microscopy showed that PyMSP1₁₉ was displayed on the viral envelope of AcNPV-PyMSP1₁₉surf (Fig. 1E). These results indicate that the PyMSP1₁₉-gp64 fusion protein forms oligomers on the virus envelope and retains the three-dimensional structure of the native PyMSP1₁₉ protein with correctly formed disulfide bonds.

FIG. 1.

Construction and analysis of recombinant AcNPV expressing PyMSP1₁₉. (A) Schematic diagram of AcNPV-PyMSP1₁₉surf genome. PyMSP1₁₉ was expressed as a PyMSP1₁₉-gp64 fusion protein under the control of the polyhedron promoter. Numbers indicate the amino acid positions of the PyMSP1₁₉-gp64 fusion protein and the endogenous gp64 protein. pPolh, polyhedrin promoter; SP, gp64 signal sequence; 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) were treated with loading buffer containing 1% 2-ME and examined using anti-FLAG MAb (lanes 1and 2), P. yoelii-hyperimmune serum (lanes 3 and 4), and anti-gp64 MAb (lanes 5 and 6). Arrows indicate the positions of PyMSP1₁₉-gp64 fusion protein and endogenous gp64. (C and D) Structural analysis of PyMSP1₁₉-gp64 fusion protein. AcNPV-PyMSP1₁₉surf was treated with loading buffer containing various concentrations of 2-ME. The reactivity of the PyMSP1₁₉-gp64 fusion protein was examined using either P. yoelii-hyperimmune serum (C) or anti-FLAG MAb (D). The concentrations of 2-ME are shown at the top. (E and F) Electron micrographs of AcNPV-PyMSP1₁₉surf displaying PyMSP1₁₉ on the viral envelope. AcNPV-PyMSP1₁₉surf was treated with either mouse anti-GST-PyMSP1₁₉ antiserum (E) or normal mouse serum (F) followed by labeling with anti-mouse-gold conjugate. The surfaces of the virions were strongly labeled with gold particles (arrows). Bars, 100 nm.

i.n. immunization with AcNPV-PyMSP1₁₉surf induces high levels of PyMSP1₁₉-specific antibody titer.

i.n. immunization with AcNPV-PyMSP1₁₉surf induced significantly higher PyMSP1₁₉-specific antibody titer levels than did i.m. immunization (2.7-fold; P < 0.01) (Fig. 2A). Immune sera obtained after immunization through the i.m. and i.n. routes contained predominantly IgG1 and IgG2a (IgG1-to-IgG2a ratio ≅ 0.63 and 0.54, respectively), indicating a mixed Th1/Th2-type immune response. The mucosal immunization-inducible IgG2b was significantly increased in the group receiving i.n. immunization. Immunization with GST-PyMSP1₁₉ in alum induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio (≅159). The total PyMSP1₁₉-specific antibody levels were not different between the GST-PyMSP1₁₉ and i.n. AcNPV-PyMSP1₁₉surf groups. The IgG3 antibody was undetectable in the GST-PyMSP1₁₉ group (<1:100). Thus, each vaccination regimen induced different IgG class switching. These immune sera strongly reacted with native PyMSP1₁₉ on the parasites, with circumferential staining on the surfaces of free merozoites (Fig. 2B) after schizont rupture in addition to the detection of PyMSP1₁₉ in mature schizonts (Fig. 2C).

FIG. 2.

PyMSP1₁₉-specific antibody responses. Sera were collected from individual mice (10 mice/group) 3 weeks after the last immunization. (A) The individual sera were tested for total IgG, IgG1, IgG2a, IgG2b, and IgG3 specific for PyMSP1₁₉ by ELISA. The data represent one of two experiments, which had similar results. Data are presented as means ± standard deviations (SD) for the groups. Significant differences in total IgG titers between different groups were evaluated using two-tailed Fisher's exact probability test. *, P < 0.01. (B and C) Confocal fluorescence micrographs of sera obtained from mice immunized with AcNPV-PyMSP1₁₉surf. Free merozoites (B) and mature schizonts (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 from mice immunized i.m. and i.n. with AcNPV-PyMSP1₁₉surf. Bar, 10 μm. (D) PyMSP1₁₉-specific antibody responses induced by mucosal immunization 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 individual mice 3 weeks after the last immunization and tested for PyMSP1₁₉-specific antibodies and IgG isotypes by ELISA. Ig63 was undetectable in all groups. Data are presented as means ± SD for the groups. *, P < 0.01.

Protection of mice immunized with AcNPV-PyMSP1₁₉surf against challenge infection.

The groups of immunized mice were challenged i.v. with lethal P. yoelii 17XL, and the course of infection was monitored (Table 1). All but one of the nonimmunized control mice died with high parasitemia (76%) by day 8 postchallenge. Mice immunized either i.m. or i.n. with AcNPV-WT suffered severe courses of infection with high maximum parasitemia levels (>70% parasitemia) and 100% mortality, indicating that AcNPV alone had no protective effect. In the i.m. AcNPV-PyMSP1₁₉surf group, 7 of 15 mice survived after challenge, with high maximum parasitemia (mean ± standard error of the mean [SEM], 51.1% ± 5.1%). All mice immunized with i.n. AcNPV-PyMSP1₁₉surf survived after challenge. Compared with the i.m. AcNPV-PyMSP1₁₉surf group, the i.n. AcNPV-PyMSP1₁₉surf group experienced 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, 7 of 15 mice survived after challenge, with moderate maximum parasitemia (37.8% ± 7.2%). PyMSP1₁₉-specific antibody titers were inversely related to maximal parasitemia, and reduction of parasitemia and protection from death depended upon a high titer of PyMSP1₁₉-specific antibodies at challenge (Fig. 3A). Antibody titers over 1:80,000 conferred 100% (22/22 mice) survival, while antibody titers under 1:80,000 resulted in high parasitemia and 12.5% (1/8 mice) survival. Thus, i.n. immunization with AcNPV-PyMSP1₁₉surf induced a solid level of protection against a lethal challenge infection. Interestingly, the protection efficacy was only 40% (2/5 mice) in the i.n GST-PyMSP1₁₉-plus-AcNPV-WT immunization group (Table 1), despite high titers of PyMSP1₁₉-specific antibodies (≅4 × 10⁵; predominantly IgG1 and IgG2b) (Fig. 2D). The two surviving mice did not induce natural boosting after challenge (data not shown). i.n. immunization with GST-PyMSP1₁₉ alone induced very low titers of PyMSP1₁₉-specific antibodies (≅6 × 10³; predominantly IgG1) (Fig. 2D) and conferred no protection. These results indicate that although AcNPV itself can act as a mucosal adjuvant and induce systemic antibody responses, display of PyMSP1₁₉ on the surface of the viral envelope is essential to induce protective immunity. Unlike i.n. immunization, oral immunization with AcNPV-PyMSP1₁₉surf induced low levels of PyMSP1₁₉-specific antibodies (≅4 × 10⁴; predominantly IgG1 and IgG2b) (Fig. 2D) and conferred no protection (Table 1).

TABLE 1.

Protection of BALB/c mice immunized with AcNPV-PyMSP1₁₉surf against P. yoelii challenge^a

Vaccine (route)	% Maximum parasitemia	Time to clearance (days)	No. of survivors/total no. 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)

^aData represent means ± SE for total mice in each group. Significant differences between the i.m. and i.n. AcNPV-PyMSP1₁₉surf groups were evaluated using Fisher's exact probability test or the Mann-Whitney U test (*, P < 0.05; **, P < 0.01).

^bCumulative data from two independent experiments.

FIG. 3.

Antibody induction in response to infection. (A) Correlation between PyMSP1₁₉-specific antibody titer and parasitemia. Mice were ranked based on maximal parasitemia (Spearman's rank correlation; r = −0.616; P = 0.0009). Crosses, death. (B) Comparison of antibody induction between days 0 and 29 postchallenge in surviving mice. A total of 22 surviving 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 between days 0 and 29. Data are means ± standard errors (SE) for the groups. Significant differences between each group were evaluated using the Mann-Whitney U test. *, P < 0.01.

Immunization with AcNPV-PyMSP1₁₉surf induces natural boosting of vaccine-induced antibodies to infection.

Determining if PyMSP1₁₉-specific antibody responses induced by immunization were boosted in response to infection was critical to our study. When parasites were cleared, the PyMSP1₁₉-specific antibody levels of the i.m. and i.n. AcNPV-PyMSP1₁₉surf groups 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, indicating a stronger natural boosting effect by i.n. immunization. In contrast, there was no difference in the antibody levels of the GST-PyMSP1₁₉ group before and after infection. Interestingly, compared with the IgG1, -2a, and -2b isotypes, which increased 2.5- to 5-fold, IgG3 levels were elevated 11- and 36-fold for i.m. and i.n. immunization, respectively (data not shown).

Natural boosting of PyMSP1₁₉-specific antibodies was induced during the course of infection.

To examine when natural boosting of PyMSP1₁₉-specific antibodies was induced, PyMSP1₁₉-specific antibody titers during the course of infection were determined. The kinetics of antibody titers was plotted with the course of parasitemia (Fig. 4A). PyMSP1₁₉-specific antibodies induced by i.n. immunization with AcNPV-PyMSP1₁₉surf drastically increased (eightfold) during days 10 to 13 postchallenge, coinciding with the decreasing parasitemia and parasite clearance, suggesting that the quick response of natural boosting may play critical roles in effective clearance of the parasites. In contrast, the antibody levels of the GST-PyMSP1₁₉ group gradually declined, to 10% of prechallenge levels, from days 7 to 20 postchallenge, as high levels of parasitemia were developed. This finding also supports the demonstration that PyMSP1₁₉-specific antibodies alone can control parasitemia postchallenge (31).

FIG. 4.

Antibody and cytokine responses during the course of infection. Groups of mice were immunized either i.n. with AcNPV-PyMSP1₁₉surf or 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 after challenge, 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, and GST-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 are means ± 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-α; (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 are shown in blue.

We further addressed the developed antiparasite antibody titers on days 0 and 29 postchallenge. The P. yoelii-specific IgG titer was significantly increased on day 29 for both groups (Fig. 4B), while P. yoelii-specific IgM was significantly elevated in the GST-PyMSP1₁₉ group but not in the i.n. AcNPV-PyMSP1₁₉surf group (Fig. 4C). These results suggest that the increasing P. yoelii-specific IgG in the i.n. AcNPV-PyMSP1₁₉surf group were mainly due to natural boosting of PyMSP1₁₉-specific antibodies against native PyMSP1₁₉ on merozoites, resulting in effective parasite clearance. The increase in P. yoelii-specific IgM in the GST-PyMSP1₁₉ group during infection was likely due to primary immune responses to parasite antigens other than PyMSP1₁₉.

For blood-stage parasites, the magnitudes, kinetics of production, and overall balance of proinflammatory and anti-inflammatory cytokines are critically important to infection outcome. We monitored cytokine expression profiles with parasitemia in immunized mice during the course of infection (Fig. 4D to F). Much lower levels of proinflammatory (TNF-α) and anti-inflammatory (IL-5 and IL-10) cytokines were induced in the sera of the i.n. AcNPV-PyMSP1₁₉surf group than in those of the GST-PyMSP1₁₉ group. Thus, i.n. immunization with AcNPV-PyMSP1₁₉surf circumvented cytokine dysregulation caused by high parasitemia.

TLR9 is involved in the mechanism of protection by i.n. immunization with AcNPV-PyMSP1₁₉surf.

To determine the role of TLR9 in the AcNPV-PyMSP1₁₉surf-induced protection, TLR9-deficient mice on a BALB/c background were immunized and challenged with P. yoelii 17XL as described above. Significantly lower levels of PyMSP1₁₉-specific antibody titers were induced in TLR9-deficient mice immunized with i.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-PyMSP1₁₉surf groups was mainly IgG1 (IgG1/IgG2a ratio ≅ 4.36 and 5.39, respectively), and IgG3 antibody was undetectable (<1:100) in any group (Fig. 5B). Thus, Th2 immune responses are induced in TLR9-deficient mice, completely contrasting with the mixed Th1/Th2-type immune responses of normal BALB/c mice shown in Fig. 2A.

FIG. 5.

PyMSP1₁₉-specific antibody response in TLR9-deficient mice. Sera were collected from individual TLR9-deficient mice (8 mice/group) 3 weeks after the last immunization and tested for PyMSP1₁₉-specific antibodies and IgG isotypes by ELISA. (A) Comparison of PyMSP1₁₉-specific antibody responses between TLR9-deficient mice and normal BALB/c mice. Data are means ± SE for the groups. Significant differences between each group were evaluated using the Mann-Whitney U test. **, P < 0.01; *, P < 0.05. (B) Isotype profiles of PyMSP1₁₉-specific antibodies. IgG3 was undetectable in all groups. The data represent one of two experiments, which had similar results. Data are means ± SD for the groups.

During challenge, the protection efficacy for each immunized group was almost abolished in TLR9-deficient mice (Table 2). Even though the immunized mice had high antibody titers (≥8 × 10⁴), 82% (14/17 animals) of mice died with high parasitemia (>60%). The three surviving mice from each group did not induce natural boosting of PyMSP1₁₉-specific antibodies in response to infection.

TABLE 2.

Protection of TLR9-deficient mice immunized with AcNPV-PyMSP1₁₉surf against P. yoelii challenge

Vaccine (route)	% Maximum parasitemiaa	Time to clearance (days)	No. of survivors/total no. 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)

^aData are means ± SE for total mice in each group. There was no significant difference between the immunized groups and the nonimmunized group, as evaluated using Fisher's exact probability test and the Mann-Whitney U test.

^bCumulative data from two independent experiments.

DISCUSSION

In the present study, we developed a new PyMSP1₁₉ vaccine based on a baculoviral vector and highlighted natural boosting of vaccine-induced humoral immune responses during the course of infection in mice. Both i.n. AcNPV-PyMSP1₁₉surf and GST-PyMSP1₁₉ (standard recombinant PyMSP1₁₉ vaccine formulated with alum) immunizations induced high titers of PyMSP1₁₉-specific antibodies, to the same level. During the course of infection, mice immunized with AcNPV-PyMSP1₁₉surf induced natural boosting of PyMSP1₁₉-specific antibody responses shortly after challenge, coinciding with decreasing parasitemia and parasite clearance. In particular, i.n. immunization with AcNPV-PyMSP1₁₉surf conferred 100% survival with low parasitemia. Although immunization with GST-PyMSP1₁₉ in alum conferred 47% survival against challenge, the surviving mice exhibited no natural boosting of the PyMSP1₁₉-specific antibody response to infection. The results presented herein offer a novel strategy for the development of baculoviral vector-based malaria blood-stage vaccines capable of naturally boosting vaccine-induced antibody responses to infection.

The types of immune responses induced by immunization were qualitatively different between the i.n. AcNPV-PyMSP1₁₉surf and GST-PyMSP1₁₉ groups, with predominantly IgG1 and IgG2a (mixed Th1/Th2 type) and predominantly IgG1 (Th2 type) responses, respectively. In the case of the Th2-type immune response, when robust PyMSP1₁₉-specific IgG1 (noncytophilic IgG) was induced by immunization with GST-PyMSP1₁₉ in Freund's adjuvant, sterile protection was achieved without detectable parasites (28, 30). However, once parasites develop to a detectable level (∼0.1% parasitemia) in the blood, suppression of parasitemia requires newly generated immune responses to parasite antigens other than PyMSP1₁₉ responses elicited early during infection (31). This is consistent with our results showing that P. yoelii-specific IgM antibody titers, but not PyMSP1₁₉-specific IgG antibody titers, were elevated during infection in the GST-PyMSP1₁₉ group. These results suggest that although PyMSP1₁₉-specific IgG1 may function by blocking parasite invasion or inhibiting MSP1 processing, which is required for erythrocyte entry, memory B cells producing PyMSP1₁₉-specific IgG1 would be deleted from the circulation by parasite-induced apoptosis (33). The analyses of the kinetics of antibody and cytokine production during infection suggested that days 10 to 13 after challenge would be a critical turning point for subsequent parasite clearance for the i.n. AcNPV-PyMSP1₁₉surf and GST-PyMSP1₁₉ groups. The i.n. AcNPV-PyMSP1₁₉surf group induced natural boosting of vaccine-induced antibodies, resulting in rapid parasite clearance. In contrast, the GST-PyMSP1₁₉ group suffered from cytokine dysregulation caused by high parasitemia. This may trigger parasite-induced apoptosis, resulting in diminished natural boosting of PyMSP1₁₉-specific antibody responses to infection and further increasing parasitemia. In addition, induction of IgG2a and IgG2b (cytophilic IgG) antibodies may also be necessary, but not a prerequisite, for parasite clearance by the natural boosting of vaccine-induced antibody responses. Kumar et al. showed that even though immunization with recombinant PyMSP1₁₉ formulated in CpG ODN1826 emulsified in Montanide ISA 51, an adjuvant that favors Th1 differentiation, induced high levels of PyMSP1₁₉-specific IgG2a and IgG2b, no natural boosting was induced after challenge (19). These results suggest that in addition to the ability to induce a mixed Th1/Th2-type immune response, memory B cells produced by AcNPV-PyMSP1₁₉surf may possess qualitative and quantitative properties capable of resistance to parasite-induced apoptosis.

Mucosal immunization induces antigen-specific Th1- and/or Th2-type immune responses, depending on the nature of the antigen, adjuvant, and antigen delivery vehicle used. Although both i.m. and i.n. immunizations with AcNPV-PyMSP1₁₉surf induced a mixed Th1/Th2-type immune response, i.n. immunization led to higher PyMSP1₁₉-specific antibody titers and stronger natural boosting after challenge than did i.m. immunization. The i.n. immunization with GST-PyMSP1₁₉ plus AcNPV-WT induced high levels of PyMSP1₁₉-specific IgG1 and IgG2b titers and protected 40% (2/5 mice) of mice against challenge, but no natural boosting was induced. When cholera toxin B was used as a mucosal adjuvant with rPyMSP1₁₉, a Th2-type immune response (predominantly IgG1 and IgG2b) was induced, and 38% (3/8 mice) of mice were protected against challenge (14). Thus, the protection efficacy (100%) of i.n. immunization with AcNPV-PyMSP1₁₉surf was superior to those of other nasal immunization regimens. This suggests that i.n. immunization with AcNPV-PyMSP1₁₉surf displaying PyMSP1₁₉ on the surface of the viral envelope would be essential for inducing Th1 cell-derived IFN-γ production, which determines switching to IgG2a as a consequence of the cognate interaction of B cells with Th1 cells (4).

The source of Th1 cells and the precise mechanism of natural boosting are not yet defined. It is possible that i.n. immunization with AcNPV-PyMSP1₁₉surf activated DCs and macrophages, which are abundant in nasal passage-associated lymphoid tissue (NALT), a mucosal inductive site in the upper respiratory tract for humoral and cellular immune responses (36), through a TLR9-dependent pathway. In fact, AcNPV itself (composed of viral genomic DNA containing abundant CpG motifs) possesses strong adjuvant properties which can activate DC-mediated innate immunity through MyD88/TLR9-dependent and -independent pathways (1). The present study provides important insights into the roles of TLR9 following immunization with AcNPV-PyMSP1₁₉surf. In the TLR9-deficient mice immunized with AcNPV-PyMSP1₁₉surf, class switching to IgG2a was severely impaired, and natural boosting and protective efficacy were completely abolished. Jegerlehner et al. showed that IFN-γ-independent IgG2a class switching is regulated by direct TLR9 signaling in B cells (16). It is likely that AcNPV-PyMSP1₁₉surf-induced natural boosting after challenge is associated with a TLR9-dependent pathway, and direct stimulation of B cells by AcNPV-PyMSP1₁₉surf (after the second and third immunizations) could be the driving factor for class switch recombination to IgG2a in NALT. Nasal drop vaccines have several attractive features compared with parenteral vaccines (e.g., safety, cost-effectiveness, and ease of administration), but studies on their use have been limited almost exclusively to protection against mucosally transmitted pathogens. We have provided evidence that i.n. immunization is a feasible alternative for preventing malaria transmitted through nonmucosal routes. Further studies are necessary to fully understand the responses of antigen-presenting cells (APCs) in the NALT (mice) and Waldeyer's ring (humans), consisting of the adenoids and tonsils (20), to AcNPV.

Although 8 of 14 TLR9-deficient mice immunized with GST-PyMSP1₁₉ in alum had high (>8 × 10⁴) anti-PyMSP1₁₉ antibody titers (Table 2), which are sufficient for normal mice immunized with GST-PyMSP1₁₉ to suppress parasite growth, all mice died with high parasitemia. The failure of protection suggests another role of TLR9. It has been reported that blood-stage parasites activate DCs through a TLR9-dependent pathway (27). In addition, we observed that unlike normal BALB/c mice, TLR9-deficient mice passively immunized with anti-PyMSP1₁₉ antibody died with high parasitemia when challenged with P. yoelii 17XL (data not shown). Thus, TLR9 may enhance antigen presentation by DCs, resulting in induction of antibody responses to parasite antigens during the course of infection. Further studies of TLR9-mediated memory B-cell responses to baculovirus-based vaccines and natural human malaria infection are needed.

Needle-free nasal drop immunization with the baculovirus-based vaccine provides important insights into (i) enhancement of immunogenicity of PfMSP1₄₂, (ii) strong adjuvant effects through TLR9, and (iii) subsequent natural boosting to improve current PfMSP1₄₂-based vaccine candidates.