Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium

Malaria remains a severe public health problem and causes significant economic losses worldwide. In 2016, there were ∼216 million malaria cases and an estimated 445,000 malaria deaths, mainly in children under 5 y of age (1). Malaria infection is initiated following Plasmodium sporozoite injection into the skin during the taking of a blood meal by Anopheles mosquitoes. The sporozoites then migrate to the liver and invade hepatocytes. Before clinical symptoms of malaria occur during the blood stage of infection, the sporozoites of Plasmodium falciparum in the liver develop into exoerythrocytic schizonts for 5–6 d. P. vivax and P. ovale can develop dormant liver-stage forms, known as hypnozoites, which cause relapsing blood-stage infections months or years after the primary infection. Currently, the only licensed drug for the radical cure of P. vivax hypnozoites is primaquine (PQ), and artemisinin-based combination therapies are recommended by the World Health Organization as the first-line treatment for blood-stage P. falciparum malaria. However, PQ has a high associated risk of life-threatening hemolytic anemia in people with glucose-6-phosphate-dehydrogenase enzyme deficiency (2). For future malaria eradication strategies, safer radical curative compounds that efficiently kill hypnozoites are required.

A series of studies performed by Nussenzweig and colleagues (3–6) in 1986–1987 revealed that exogenously administered IFN-γ effectively inhibits the development of liver-stage parasites in vitro and in vivo. Recently, Boonhok et al. (7) reported that IFN-γ–mediated inhibition occurs at least partially in an autophagy-related, protein-dependent manner in infected hepatocytes. Additionally, Liehl et al. (8) reported that hepatocytes infected with wild type (WT) liver-stage parasites induce type I IFN secretion via host cell sensing of Plasmodium RNA, resulting in a reduction of the liver-stage burden. These findings suggest that IFN-mediated immunotherapy against liver-stage parasites might be effective. However, new antihypnozoite drugs (e.g., rIFNs or appropriate IFN inducers) have not been developed yet.

Autographa californica nucleopolyhedrosis virus, a type of baculovirus (BV), is an enveloped dsDNA virus that naturally infects insects. BVs possess unique characteristics that activate dendritic cell (DC)-mediated innate immunity through both MyD88/TLR9-dependent and -independent pathways (9). Takaku and colleagues (10, 11) reported that BV also directly activates murine NK cells through the TLR9 signaling pathway, which leads to the induction of NK cell–dependent antitumor immunity. Based on the unique adjuvant properties of BV that induce DC maturation and NK cell activation, which are prerequisites for generating robust and long-lasting adaptive immune responses, we have developed BV-based malaria vaccines effective for all three parasite stages: the pre-erythrocytic stage (12–14), asexual blood stage (15, 16), and sexual stage (17, 18).

P. berghei, a rodent malaria parasite, has been extensively used in vaccine development studies as part of a mouse model of malaria (19), and chimeric P. berghei–expressing P. falciparum or P. vivax Ags have been recently developed to evaluate a number of human malaria vaccines (20). Historically, P. berghei has proved to be analogous to the malarias of humans and other primates in most essential aspects of structure, physiology, and life cycle (21), despite staying in the murine liver for only up to 48 h, which is a much shorter period compared with human malaria parasites. In the current study, a challenge infection model based on the pre-erythrocytic stage of P. berghei has been developed, and BV-mediated innate immunity against the parasite was examined. Our results clearly demonstrate that BV i.m. administration not only elicits short-term sterile protection against Plasmodium sporozoite infection but also eliminates liver-stage parasites completely through the type I IFN signaling pathway. Because of its potent IFN-inducing characteristics, BV has the potential to be developed not only as a new malaria vaccine additive capable of protecting vaccine recipients for a short period before and after malaria infection but also as a new nonhemolytic, single-dose drug.

Female inbred BALB/c (H-2^d) mice were obtained from Japan SLC (Hamamatsu, Shizuoka, Japan) and used in all experiments at 7–8 wk of age. TLR9-deficient (TLR9^−/−) mice on a BALB/c background were kindly provided by S. Akira (University of Osaka, Suita, Japan). Spodoptera frugiperda and HepG2 cells were maintained as described previously (13). Three transgenic P. berghei ANKA parasites were used in this study: GFP P. berghei (Pb-conGFP) (22), luciferase P. berghei (Pb-Luc) (23), and P. falciparum circumsporozoite (PfCSP)/P. berghei (PfCSP-Tc/Pb) (24). These transgenic parasites were maintained by cyclical passaging through BALB/c mice and Anopheles stephensi (SDA 500 strain) at the Kanazawa University and Jichi Medical University according to a standard protocol (12, 25).

All animal care and handling procedures were approved by the Animal Care and Ethical Review Committee of Kanazawa University (no. 22118–1) and Jichi Medical University (no. 09193), Japan. For animal experiments, all efforts were made to minimize suffering in the animals. Mice were anesthetized with ketamine (100 mg/kg, i.m.; Daiichi Sankyo, Tokyo, Japan) and xylazine (10 mg/kg, i.m.; Bayer, Tokyo, Japan) when necessary.

The recombinant BVs BV expression system (BES)-GL3 and BV dual expression system (BDES)-sPfCSP2-WPRE-Spider have been described previously (26). The purified BV particles were free of endotoxin (<0.01 endotoxin units/10⁹ PFU), as determined by the Endospecy endotoxin measurement kit (Seikagaku, Tokyo, Japan). The recombinant adenoviruses human adenovirus serotype 5 (AdHu5)-expressing luciferase and AdHu5-sPfCSP2 have been described previously (14). In this manuscript, BDES-sPfCSP2-WPRE-Spider and AdHu5-sPfCSP2 are described as BDES-PfCSP and AdHu5-PfCSP, respectively.

A. stephensi mosquitoes were infected by feeding on infected mice using standard methods of mosquito infection. On days 21–24 postinfection, the salivary glands of the mosquitoes were collected by hand dissection. Salivary glands were collected in DMEM (Thermo Fisher Scientific, Tokyo, Japan) and homogenized in a plastic homogenizer. The free sporozoites were counted in a disposable hemocytometer-counting chamber using phase-contrast microscopy.

BALB/c mice were i.v., i.m., or intranasally administered 10⁴–10⁸ PFU of BES-GL3. Alternatively, instead of BES-GL3, BALB/c mice were i.m. injected with 50 μg of CpG oligodeoxynucleotides 1826 (5′-TCCATgACgTTCCTgACgTT-3′, Fasmac, Tokyo, Japan). The mice were i.v. challenged with 1000 Pb-conGFP sporozoites or 1000 parasitized RBCs at various time intervals (6 h–14 d). The mice were checked for P. berghei blood-stage infection by microscopic examination of Giemsa-stained thin smears of their tail blood, prepared on days 5, 6, 7, 8, 11, and 14 postchallenge. The time required to reach 1% parasitemia was determined as described previously (27). A minimum of 20 fields (original magnification ×1000) were examined before a mouse was deemed to be negative for infection. Protection was defined as the complete absence of blood-stage parasitemia on day 14 postchallenge.

BALB/c mice were i.v. injected with 1000 Pb-conGFP sporozoites and then i.v. (10⁷ PFU) or i.m. (10⁸ PFU) injected with BES-GL3 at various time intervals (6, 24, or 42 h postinfection). Alternatively, instead of BV, a single high (2 mg) or low (0.1 mg) dose of PQ (PQ diphosphate 98%; Sigma-Aldrich, St. Louis, MO), with corresponding concentrations of roughly 100 mg/kg of body weight and 5 mg/kg of body weight, respectively, was i.p. administered 24 h after the injection of 1000 Pb-conGFP sporozoites. The mice were checked for P. berghei blood-stage infection and evaluated for 1% parasitemia as described above.

BALB/c mice were i.v. or i.m. injected with BES-GL3 on day 0, and d-luciferin (15 mg/ml; OZ Biosciences, Marseille, France) was then i.p. administered (150 μl/mouse) to these mice at various timepoints. The animals were anesthetized with a ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture 10 min later, and the luciferase expression was detected with an in vivo imaging system (IVIS), IVIS Lumina LT (PerkinElmer, Waltham, MA). Alternatively, BALB/c mice were i.v. injected with 1000 Pb-Luc sporozoites, followed 24 or 42 h later by i.m. administration of BES-GL3 (10⁸ PFU) into the left thigh muscle. At 72 h after sporozoite injection, the luciferase expression was detected as described above. On days 5–14 postinfection, the same mice were analyzed for blood-stage infections by determination of the course of parasitemia in Giemsa-stained thin blood films of tail blood.

BALB/c mice were i.v. or i.m. injected with BV, and serum samples were subsequently harvested from whole blood obtained by cardiopuncture at various times and stored at −20°C until analysis. The concentrations of cytokines in the sera were determined by sandwich ELISA using a Mouse IFN-γ ELISA MAX Standard Kit (BioLegend, San Diego, CA), Mouse IL-12/IL-23 (p40) ELISA MAX Standard Kit (BioLegend), or Mouse TNF-α ELISA MAX deluxe kit (BioLegend) according to the manufacturer’s instructions. The IFN-α concentration was determined by sandwich ELISA as described previously (8). In brief, rat mAb against mouse IFN-α (clone RMMA-1; PBL Biomedical Laboratories, Piscataway, NJ) was used as the capture Ab (2 μg/ml for coating), rabbit polyclonal Ab against mouse IFN-α (PBL Biomedical Laboratories) was used at 80 neutralizing units/ml for detection, and HRP-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA) was used as the secondary reagent. Recombinant mouse IFN-α (PBL Biomedical Laboratories) was used as the standard. The lower detection limits for the IFN-γ and IFN-α immunoassays were each <20 pg/ml, whereas those for the IL-12 and TNF-α immunoassays were each <10 pg/ml. The concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) in the sera were determined by using a GPT/GOT assay kit (Transaminase CII-test; Wako Pure Chemical Industries, Tokyo, Japan) according to manufacturer’s instructions.

Pooled sera were obtained from blood harvested by cardiopuncture from five BALB/c mice that had been i.m. injected 6 h previously (−6 h) with BES-GL3, and the concentrations of IFN-α and IFN-γ were measured immediately. On the same day, the IFN-α and IFN-γ in 100-μl aliquots of the pooled sera were neutralized by incubation on ice for 6 h with anti–IFN-α (anti-mouse IFN-α, rabbit serum; PBL Biomedical Laboratories) and anti–IFN-γ (Ultra-LEAF Purified anti-mouse IFN-γ Ab; BioLegend) Abs, respectively, according to the manufacturer’s instructions. At 24 h, after being i.v. injected with 1000 Pb-conGFP sporozoites, BALB/c mice were subsequently i.v. injected with 100 μl of the sera that had been treated with either anti–IFN-α or anti–IFN-γ. For the IFN administration experiment, BALB/c mice that had been i.v. injected with 1000 Pb-conGFP sporozoites 24 h before were then i.v. administered either 8619 pg of IFN-α or 4705 pg of IFN-γ. For each experiment, the mice were checked for P. berghei blood-stage infection and evaluated for 1% parasitemia as described above.

The heat inactivation of BV (HI-BV) was prepared from BES-GL3 by heat inactivation at 56°C for 30 min. HepG2 cells were seeded in a 48-well cell culture plate (Sigma-Aldrich) at a density of 4 × 10⁴ cells per well for 24 h. Cells were transduced with either HI-BV (10⁸ PFU) or BES-GL3 (10⁸ PFU) at a multiplicity of infection of 100. After 24 h, the culture medium was removed, and cell extracts were prepared by the addition of cell culture lysis reagent (Promega, Madison, WI) according to the manufacturer’s instructions. The luminescence intensities of the samples were measured using a microplate reader (GloMax 96 Microplate Luminometer; Promega), and the light reaction of each well was measured for 5 s. Luciferase activity is expressed in relative luminescence units. BALB/c mice were i.m. injected with HI-BV or BES-GL3 (live BV) 24 h after being i.v. injected with 1000 Pb-conGFP sporozoites. The mice were then checked for P. berghei blood-stage infection and evaluated for 1% parasitemia as described above.

BALB/c (WT or TLR9^−/−) mice were i.m. injected with 10⁸ PFU of BES-GL3. Alternatively, 50 μg of CpG oligodeoxynucleotides 1826 were administered i.m. Six hours later, whole livers were obtained by dissection of the treated mice. Each whole liver was placed in a 5-ml plastic tube containing 4 ml of buffer RLT (Qiagen, Valencia, CA) containing 1% 2-ME. Two stainless steel beads (5-mm external diameter) were added to the mixture. Once the tube was capped, it was attached to a μT-12 Beads Crusher (TAITEC, Saitama, Japan) and vigorously shaken at 2500 rpm for 3.5 min. Total RNA was isolated from 100-μl aliquots of the homogenates by using an RNeasy Kit (Qiagen). cDNA was synthesized by using random hexamers and MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA). Quantitative analysis of RNA transcripts was performed by real-time PCR with SYBR Green Premix Ex Taq (Takara, Tokyo, Japan). All the oligonucleotide primers used for real-time PCR are detailed in Supplemental Table I. Amplification of gapdh was performed in each experiment. The cycle threshold (Ct) value of each sample was standardized based on the gapdh Ct value (∆Ct), and each ∆Ct value was normalized to that of the ∆Ct value from PBS-treated control WT mice (∆∆Ct). Results are shown as the relative expression (1/2^∆∆Ct).

Recombinant viruses expressing PfCSP, AdHu5-PfCSP, or BDES-PfCSP have been described previously (14, 26). BALB/c mice were first i.m. immunized with AdHu5-PfCSP (5 × 10⁷ PFU). After 3 wk, the mice were i.v. challenged with 1000 PfCSP-Tc/Pb sporozoites, and after 24 h, the mice were then i.m. immunized either with BDES-PfCSP (1 × 10⁸ PFU) or PBS. The mice were checked for P. berghei blood-stage infection and evaluated for 1% parasitemia as described above. Protected mice were i.v. rechallenged with 1000 PfCSP-Tc/Pb sporozoites, and protection was defined as described above.

For evaluation of the anti-PfCSP Ab response, sera from immunized mice were collected from tail blood samples taken 3 wk after the prime immunization and 2 wk after the boost immunization. PfCSP-specific Ab levels were quantified by ELISA using Escherichia coli–produced PfCSP as described previously (14).

Details concerning the study outline, sample size, and statistical analysis are shown in the main text, figures, and figure legends. A two-tailed Fisher exact probability test was performed to determine the significance of differences in the protective efficacies of the vaccines using Statistical Package for the Social Sciences software (version 19; Chicago, IL). In all other experiments, statistical differences between the experimental groups were analyzed by the methods described in the individual figure legends. The p values <0.05 were considered statistically significant. Statistical analyses were performed with either Prism version 7.0a (GraphPad Software, La Jolla, CA) or Microsoft Excel (Redmond, WA).

This study investigated the effects induced by BV on innate immune responses relating to malaria infection. We used the BV called BES-GL3, which harbors two gene cassettes consisting of the luciferase gene under the control of the CMV promoter and the decay accelerating factor (DAF) gene under the control of the p10 promoter. The DAF-shielded BES-GL3 has been shown to be complement resistant (26). BES-GL3 i.m. administration into the left thigh muscle of mice initially increased the luciferase expression levels robustly, but these levels gradually decreased to 2% of peak expression on day 28 (Fig. 1A), which is consistent with findings from previous studies (26, 28). Among the various cell types tested in vitro, hepatocytes were found to be the most effective at taking up BV (29), suggesting a potential use for BV as a vector for liver-directed gene transfer. However, direct evidence of in vivo liver-directed gene transfer has not been reported previously because BV-mediated gene transfer into hepatocytes via i.v. injection is severely hampered by serum complement (30). In this study, use of our complement-resistant, DAF-shielded BES-GL3 revealed for the first time, to our knowledge, that i.v. administered BV effectively transduces hepatocytes in vivo (Fig. 1B).

We next examined the serum kinetics of ALT, AST, and proinflammatory cytokines following BES-GL3 i.v. administration. IFN-γ and TNF-α levels rapidly peaked at 6 h and decreased to baseline by 24 h (Fig. 1C, 1D). Similarly, the ALT and AST levels each rapidly reached their peaks at 12 h and decreased to baseline by 48 h (Fig. 1E, 1F). Compared with i.v. administration, i.m. administration did not affect the ALT levels; although the AST level trended higher following i.m. administration, this difference did not reach statistical significance (Fig. 1G, 1H). ALT is a sensitive indicator of liver damage, so these results suggest that, for BV, i.m. administration may be less destructive than i.v. administration.

The experimental designs for animal trials are illustrated in Supplemental Fig. 2A, and Table I summarizes the protective efficacy results for BV administration against sporozoite challenge. First, to examine the effects of BV i.v. administration, mice were i.v. administered 10⁷ PFU of BES-GL3. At 6 h after BV injection, which coincides with peak IFN-γ production, the mice were i.v. challenged by 1000 Pb-conGFP sporozoites, which are transgenic P. berghei constitutively expressing GFP. All BV-injected mice were protected, whereas all PBS- and AdHu5-PfCSP–injected mice that were treated similarly became infected. Next, we investigated the effects of BES-GL3 i.m. administration (10⁸ PFU) followed by sporozoite challenge at various intervals after BV injection. After i.m. administration of BES-GL3, all mice were protected for at least 7 d. However, there was a complete loss of protection by 14 d after BES-GL3 i.m. administration, and no delay of parasitemia was observed in these mice. Additionally, no protection was observed in mice treated intranasally with BES-GL3.

BES-GL3 i.v. administration failed to provide protection against challenge with 1000 parasitized RBCs at 6 h after BV injection, indicating that BV has no residual effect on blood-stage parasites. CpG i.m. administration at 6 or 24 h prior to challenge conferred protection against sporozoite challenge in 90 or 80% of mice, respectively. This is consistent with previous work showing short-term (2-d) protection induced by CpG i.m. administration (50 μg) against challenge with 100 P. yoelii sporozoites (31), although only partial protection (50%) was observed when the challenge occurred at 7 d after CpG i.m. injection. Thus, the protective efficacy induced by BES-GL3 i.m. administration is more effective and longer lasting (7 d) compared with that induced by CpG. All PBS-treated control mice developed blood-stage infection within 6 d following an i.v. injection of 1000 Pb-conGFP sporozoites.

Pathways stimulated by type I and II IFNs can lead to the killing of hepatocytes infected with liver-stage parasites (3–8). Because BV is a potent inducer of type I and II IFNs (32, 33), and we observed BV-mediated protection as described above, we next investigated whether BV-induced IFNs could kill liver-stage parasites in vivo. To examine the elimination effects on the trophozoite and exoerythrocytic (mature) schizont stages, we administered BES-GL3 i.v. or i.m. at two different intervals (24 and 42 h) following sporozoite challenge (Supplemental Fig. 2B). Table II summarizes these results on the elimination efficacy of BES-GL3 administration against liver-stage parasites. Blood-stage parasites were completely prevented in all mice that had been i.v. injected with BES-GL3 at 24 h postinfection; in contrast, the protective effectiveness of BES-GL3 i.v. administration was diminished when mice received it at 42 h postinfection instead. The same results were obtained when mice were i.m. injected with BES-GL3.

To visualize parasite elimination by BV, mice were infected with Pb-Luc, which are transgenic P. berghei constitutively expressing luciferase, and then examined via IVIS; this is a highly sensitive method for detecting liver- and blood-stage parasites. Parasites were observed in the liver at both 24 and 42 h postinfection (Fig. 2A, 2B, respectively; left panels). BES-GL3 i.m. administration into the left thigh muscle at 24 h postinfection eliminated the liver-stage parasites completely at 72 h postinfection, whereas the PBS control treatment failed to prevent the development of blood-stage parasites (Fig. 2A; right panel). Although BES-GL3 i.m. administration into the right thigh muscle at 42 h postinfection also failed to prevent the development of blood-stage parasites (Fig. 2B; right panel), it caused a significant delay of parasitemia (Fig. 2C). The exoerythrocytic merozoites of P. berghei are released from infected hepatocytes into the blood stream at 44–48 h after the liver stage (34). Therefore, this result indicates that even for exoerythrocytic schizonts (42 h postinfection), the elimination effect of BV i.m. administration was invoked in the liver within 2–6 h. Lower doses (10⁴ and 10⁶ PFU) of BES-GL3 administered at 24 h postinfection failed to prevent blood-stage parasites. However, a significant delay of parasitemia was observed for the dose of 10⁶ PFU of BES-GL3 (Fig. 2D), indicating that the elimination effect is dependent on the amount of BV that is i.m. administered.

As PQ is the only licensed drug for the radical cure of P. vivax hypnozoites, we compared the elimination effects of BV with those of PQ. Two different doses of PQ, high dose (2 mg/mouse) and low dose (0.1 mg/mouse), were i.p. administered. A single administration of high dose PQ eliminated the liver-stage parasites completely (Table II), whereas a single low dose of PQ was suboptimal, producing only a reduction in parasite burden in the liver and a significant delay of parasitemia (Fig. 2C). The World Health Organization–recommended treatment schedule for PQ is 15 mg/d for 14 d, but because high doses of PQ often cause side effects like nausea, vomiting, and stomach cramps, these side effects can limit patient compliance, potentially resulting in PQ resistance (35, 36). Thus, a single dose of BV i.m. administration might have important advantages of over PQ.

CpG i.m. administration eliminated early liver-stage parasites completely at 6 h postinfection (Table III); however, although this treatment caused a significant delay of parasitemia, it had little effect on mature schizonts (24 h postinfection) (Fig. 2C). BV possesses unique characteristics that activate DC-mediated innate immunity through MyD88/TLR9-dependent and -independent pathways (9). Therefore, we next investigated whether TLR9 plays an important role in BV-mediated parasite killing in the liver. A single dose of i.m. administered BES-GL3 (10⁸ PFU) completely prevented blood-stage parasites in all TLR9^−/− mice that had been previously infected with liver-stage parasites. In contrast, no elimination effect or parasitemia delay was observed in TLR9^−/− mice following i.m. administration of CpG (50 μg) (Table III). These results clearly demonstrate that BV-mediated parasite killing occurs via TLR9-independent pathways.

BV i.v. administration was reported to produce type I IFNs through TLR-independent and IRF3-dependent pathways in mice (9). To further investigate IFN production following BV i.m. administration, the IFN serum levels were measured in WT and TLR9^−/− mice at 6 h after BES-GL3 i.m. administration. As with i.v. administration, i.m. administration of BES-GL3 produced IFN-α in not only WT mice (6311 ± 2363 pg/ml) but also TLR9^−/− mice (1590 ± 737 pg/ml), whereas mice i.m. injected with PBS or CpG did not produce detectable IFN-α (<20.0 pg/ml) (Fig. 3A). IFN-γ, a type II IFN, was also produced in both WT mice (1367 ± 1303 pg/ml) and TLR9^−/− mice (488 ± 132 pg/ml) following i.m. administration of BES-GL3 (Fig. 3B). Compared with BV, CpG i.m. administration in WT mice induced much less IFN-γ but much more IL-12 (Fig. 3B, 3C). This result indicates that IL-12 and/or IFN-γ induced by CpG administration induced a partial protective efficacy (80–90%) against the sporozoite infection (Table I) but was insufficient to completely eliminate liver-stage parasites (Table III). Notably, CpG i.v. administration induces a high level of IFN-γ with considerable systemic side effects (37, 38). These results indicate that BES-GL3 i.m. administration induces production of both type I and II IFN via TLR9-independent pathways.

To determine whether serum cytokines act as effectors against liver-stage parasites, a serum transfer assay was performed (Supplemental Fig. 2C). Pooled sera were collected from donor mice at 6 h after they had been i.m. injected with BES-GL3 or PBS. An aliquot of the pooled sera (100 μl/animal) was transferred to each recipient mouse at 24 h after their i.v. injection with 1000 sporozoites. One of the five recipient mice effectively eliminated the liver-stage parasites, and the other four infected recipient mice showed a significant delay in the time to 1% parasitemia (mean delay of 3.54 d; p = 0.0008, compared with the PBS sera group) (Fig. 3D). The elimination efficacy in the passive serum-transferred mice was lower than that in mice directly injected by BV. This is likely due to the relatively low amount of effector molecules, such as IFN-α and IFN-γ, that are contained in only 100 μl of serum.

We next examined whether neutralization of IFN-α or IFN-γ in the sera altered the effect of the sera on liver-stage parasites. Either anti–IFN-α or anti–IFN-γ Ab was incubated with 100 μl of the sera, which contained 8619 pg/ml of IFN-α and 4705 pg/ml of IFN-γ. Complete neutralization of IFN-α was confirmed by ELISA. The IFN-α– or IFN-γ–neutralized sera (100 μl) was i.v. administered to recipient mice that had been i.v. injected 24 h previously with 1000 sporozoites. The anti–IFN-α Ab treatment completely abrogated the serum-induced delay of parasitemia, whereas the anti–IFN-γ Ab treatment only partially impaired the serum-induced elimination effect (Fig. 3D). To assess the effects of exogenous IFN-α and IFN-γ on the elimination of liver-stage parasites, rIFN-α (8619 pg/mouse) or rIFN-γ (4705 pg/mouse) was i.v. administered to mice that had been i.v. injected 24 h before with 1000 sporozoites (Supplemental Fig. 2D). IFN-α administration eliminated the liver-stage parasites completely, whereas IFN-γ administration only partially eliminated them but caused a significant delay in the time to 1% parasitemia (mean delay of 3.82 d; p = 0.0082, compared with the PBS group) (Fig. 3E). The IFN-α–mediated parasite elimination may occur via an effector mechanism distinct from or in addition to that activated by IFN-γ. It is also possible that the effector mechanisms induced by IFN-α and IFN-γ may still be synergistically operative but that an alternate protective mechanism may be activated by BV. Miller et al. (39) similarly showed that IFN-γ produced by NKT cells following type I IFN signaling from infected hepatocytes plays an important role in the elimination of liver-stage parasites.

BV inactivation was previously reported to completely abrogate its immunopotentiation effect, partly because of the abolished induction of type I IFN production (40). To address the influence of BV inactivation on the elimination of liver-stage parasites, BES-GL3 that had been inactivated by heat treatment at 56°C for 30 min (HI-BV) was i.m. administered to mice that had been i.v. injected with 1000 sporozoites 24 h previously. The successful inactivation of live BES-GL3 by the heat treatment was confirmed by the substantially complete lack of luciferase activity observed following its transduction into HepG2 cells (Fig. 3F). Unexpectedly, HI-BV induced a significant delay in the time to 1% parasitemia (mean delay of 2.17 d; p = 0.0079, compared with the PBS group); although all the HI-BV–treated mice eventually developed parasitemia in contrast with the BV-treated mice (Fig. 3G). These results suggest that heat-labile viral components in BV play an important role in cell transduction, resulting in type I IFN production by innate immunity via DNA sensing, whereas heat-resistant components of BV also contribute to parasite elimination through an IFN-independent mechanism. Supplemental Table II summarizes the results on the elimination efficacy of serum transfer, IFN administration, and HI-BV administration against liver-stage parasites.

Signal transduction of type I IFNs results in the induction of numerous IFN-stimulated genes (ISGs) (41). Some ISGs participate in direct antimicrobial activities, such as apoptosis induction and posttranscriptional event regulation of microbial killing, mainly acting as antiviral responses. Gene-targeting studies have distinguished four effector pathways of the IFN-mediated antiviral response: the Mx GTPase pathway, 2′-5′ oligoadenylate synthetase (OAS)-directed RNase L pathway, protein kinase R (PKR) pathway, and ISG15 ubiquitin-like pathway (42). Additionally, several ISGs such as IFN-induced proteins with tetratricopeptide repeats (IFITs), as well as the transcription factors IRF3 and IRF7, are responsible for sensing liver infection by Plasmodium sporozoites (8). To confirm the involvement of ISGs, the gene expression levels in the livers of mice that had been i.m. injected with BES-GL3 were measured by quantitative RT-PCR. BES-GL3 significantly induced the gene expression of several antiviral proteins (Isg15, Mx1, Oas1a/b, Oasl1, and Pkr) in WT mice (Fig. 4A). All these genes, except Oas1a/b, possibly because of the gene locus, were also upregulated by BV in TLR9^−/− mice. Gene expression of IFIT proteins, such as Ifit1, Ifit3, and Ifit44, were markedly and significantly enhanced by BV in both WT and TLR9^−/− mice (Fig. 4B). Gene expression of the transcription factors Irf3 and Irf7 were also induced by BV in the same manner (Fig. 4C). These results indicate that systemic type I IFN secretion following BV i.m. administration in the thigh muscle strongly induced ISGs in the liver.

To evaluate our newly developed malaria vaccine in an AdHu5-PfCSP–prime/BDES-PfCSP–boost heterologous immunization regimen (14), mice were challenged twice, once before and once after the BDES-PfCSP boost (see Supplemental Fig. 2F). All mice immunized with AdHu5-PfCSP–prime/BDES-PfCSP–boost heterologous regimen were protected against both the first and second challenges (Table IV, group 2). In contrast, AdHu5-PfCSP–prime immunization alone did not confer protection (Table IV, group 1). All control mice i.m. injected with PBS became infected (Table IV, groups 3 and 4). Thus, BDES-PfCSP boosting was able to exert not only a therapeutic effect on liver-stage parasites, but also a prophylactic effect on sporozoites. High levels of anti-PfCSP Ab titers (>1:10⁵) were induced by the AdHu5-PfCSP–prime/BDES-PfCSP–boost heterologous immunization regimen (Supplemental Fig. 1), a finding that is consistent with our previous study (14). The experimental designs for these animal trials are illustrated in Supplemental Fig. 2E, 2F.

In this study, we show that BV i.m. administration not only elicits short-term sterile protection against sporozoite infection, but also eliminates liver-stage parasites completely. For liver-stage parasites proliferating vigorously at 24 h postinfection, the BV-induced, fast-acting innate immune responses completely killed them within the following 20 h and prevented blood-stage parasite development. A major challenge for strategies combating the human malaria parasite P. vivax is the presence of hypnozoites in the liver. PQ is currently the only available drug that kills the dormant hypnozoites of P. vivax, but its severe side effects in G6PD-deficient people prevent the widespread use of this drug (43). To date, the closely related malaria parasite P. cynomolgi, which infects nonhuman primates, has been the gold-standard in vivo model for studying hypnozoites (44). Although P. berghei does not have a dormant hypnozoite stage, it is possible that the therapeutic effect of BV against liver-stage infection may provide clues on how to eliminate hypnozoites. Further experiments in the P. cynomolgi model are needed to evaluate the potential of BV as a new, nonhemolytic, single-dose alternative to PQ.

BV possesses attractive attributes as a new vaccine vector [e.g., its low cytotoxicity (45), inability to replicate in mammalian cells (46, 47), and absence of pre-existing Abs against it (48)]. This study suggests a further unique advantage of BV as a vaccine additive with short-term protection against malaria via its intrinsic potent immunostimulatory property. In phase II–III malaria vaccine trials, all volunteers are presumptively treated with three daily doses of antimalaria drug for 1 wk before the final vaccination and rechecked for asexual P. falciparum parasitemia at 1 wk after the final vaccination. Any subject who tests parasite positive is treated with a second-line drug or excluded from the trial (49). Thus, clinical trials aim to test vaccine efficacy after all vaccine schedules are completed to assess the maximum effect. For clinical application, however, vaccine recipients remain in danger of infection until the full vaccination schedule is completed. If BV was coadministered as an additive with a newly developed malaria vaccine like RTS,S, the joint vaccine would be expected to not only minimize the risk of infection for vaccine recipients during the vaccination schedule, but also generate robust and long-lasting adaptive immune responses via innate immune system stimulation by BV acting as an adjuvant. Our previous study showed that repeated administration of BV reduces its transduction efficacy by Ab-mediated neutralization (23). Further studies are needed to address whether the induction of innate immune responses by BV is linked to its transduction efficacy.

This study showed that IFN-α and IFN-γ were rapidly and robustly produced in serum at 6 h after BV administration. Interestingly, the prophylactic effect against sporozoite infection lasted for at least 7 d, even though IFN-γ and TNF-α serum levels returned to baseline by 24 h. We speculate that the prophylactic effect on day 7 after BV administration may be because of the killing of parasites in the liver rather than to an invasion blockade mediated by serum components. Our results are consistent with recent data from Liehl et al. (8) reporting that the ISGs induced in infected hepatocytes by a high dose of sporozoites reduced the liver-stage burden. However, the parasite-induced IFN-α responses failed to eliminate every parasite. Collectively, these data suggest that the innate immune responses induced by i.m. injection of BV comprise not only IFN production, but also activation of an unknown pathway in the liver, and that BV injection induces these more rapidly and effectively than does sporozoite infection. Further experiments using transgenic mice incapable of responding via the type I and II IFN pathways will be needed to clarify the dependency of BV-mediated Plasmodium elimination on IFN-mediated killing. A better understanding of the molecular mechanisms by which BV administration confers both protection against and elimination of pre-erythrocytic parasites will provide new strategies for malaria drug and vaccine development.

IFN-α has been extensively explored for its efficacy in various disease conditions and is currently used as a standard treatment in several illnesses. However, its use is accompanied by a wide variety of possible side effects (50), such as autoimmune thyroiditis. This study found that BV i.m. administration, which induced 8619 pg/ml of IFN-α in mouse sera while maintaining normal ALT levels, completely killed liver-stage parasites. The manufacturing cost of BV would be much lower than that of rIFN-α. Thus, BV i.m. administration also has great potential for use as an alternative IFN-α–based immunotherapy; its high biological activity, cost-effectiveness, noninvasive nature, and minimal adverse effects make it superior to the current IFN-α therapy using rIFN-α via i.v. administration.

In conclusion, BV effectively induces fast-acting innate immune responses that provide powerful first lines of both defensive and offensive attacks against pre-erythrocytic parasites. Our results illustrate the potential of BV as a new potent prophylactic and therapeutic immunostimulatory agent against pre-erythrocytic–stage parasites. Even though the first-in-human trials of BV have not yet been conducted, our previous study showed that the BV-based vaccine vector is safe and well tolerated with acceptable reactogenicity and systemic toxicity in a primate model (13). In addition, the BV system itself has proven to be clinically suitable and has been approved for the purposes of vaccination. BV-based vaccines include Cervarix (GlaxoSmithKline, Rixensart, Belgium), a human papillomavirus (strain 16 and 18) viral-like particle vaccine against cervical cancer, and Provenge (Dendreon, Seattle), which is an immune-therapeutic vaccine against prostate cancer. Although further studies are needed to clarify the prophylactic and therapeutic effects of BV in a nonhuman primate malaria model (e.g., doses and repeated treatment), our results highlight useful characteristics of BV that may pave the way for developing new malaria drug and vaccine strategies.

We thank K. Takagi, K. Genshi, M. Tokutake, and C. Seki for A. stephensi production and animal care and T. Yoshii and T. Amano for IVIS and cytokine ELISA experiments. We are indebted to S. Akira (Osaka University) for providing TLR9^−/− mice and to BioLegend for providing antisera. We also thank S. Shida for critical reading of the manuscript and Katie Oakley (Edanz Group) for editing the English text of a draft of the manuscript.

S.Y. and M.I. are named inventors on a patent pending related to BV as a new agent against pre-erythrocytic malaria parasite (2018-57311). Neither of the products in this patent have been commercialized. None of the authors have undertaken any consultancies relevant to this study. The authors have no financial conflicts of interest.