Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines

Background Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP119) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic. Methodology and Results In this study, we assessed the protective efficacies of a series of MSP119- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP119 induced high titers of PfMSP119-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP119 in place of native PbMSP119. Similarly, neither P. berghei MSP119- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP119- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa. Conclusion This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP119- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP119 and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.


Introduction
Malaria is an enormous public health problem worldwide and kills one to two million people every year, mostly children residing in Africa. Clearly, an effective vaccine for the control of malaria is urgently needed. The selection of protein antigens for malaria vaccine development has been hampered by the lack of a reliable and readily accessible challenge system for Plasmodium falciparum. Accordingly, much attention has focused on the study of laboratory rodents infected by murine malaria parasite species, most notably P. yoelii and P. berghei. Although not perfect models for human infection, these systems have proved useful, and important advances in our understanding of the principles of vaccine design have followed their use. For blood-stage vaccine development, in particular, the P. yoelii-murine model has greatly contributed to the evaluation of protective efficacies of blood-stage antigens prior to human clinical trials. Based on the P. yoelii model, many asexual blood-stage candidate antigens have been identified for malaria vaccine development. Of these, two leading malaria blood-stage vaccine candidates, merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1), have been intensively studied as promising vaccine candidates. These two antigens are well conserved across all species of Plasmodium and play important roles in erythrocyte invasion and blood-stage growth. Several passive and active immunization studies have indicated that both antigens elicit protective immune responses and serve as targets for invasion-blocking antibodies [1,2,3].
MSP1 is synthesized as an approximately 200-kDa precursor protein at the schizont stage and is further proteolytically cleaved into a number of discrete products residing on the surface of the merozoite that invades the erythrocyte [4]. After processing, the C-terminal 19-kDa fragment (MSP1 19 ) remains on the merozoite surface during erythrocyte invasion and therefore is an ideal target for blocking parasite invasion into the erythrocyte [5]. Several studies have shown that immunization with the bacteriallyproduced recombinant MSP1 19 with an adjuvant completely protects mice against P. yoelii challenge [6,7,8]. The P. falciparum MSP1 19 has been implicated as a target for protective immunity in a large number of studies, including seroepidemiological studies of naturally-acquired immunity, vaccination studies in non-human primates and in vitro cultures [9]. In particular, antibodies to MSP1 19 , either affinity purified from immune human sera or monoclonal or polyclonal experimental sera, are capable of inhibiting parasite growth in vitro [10,11,12]. Recently, however, the value of these in vitro assays has come into question because cytophilic MSP1 19 -specific antibodies appear to be more important for controlling infection than previously thought [13,14] and the protective efficacy of MSP1 19 -based vaccines do not correlate with anti-MSP1 19 antibody titers or in vitro parasite-inhibitory activity in animal models [15].
AMA1, synthesized as a 60-80-kDa protein during schizogony, is a microneme protein involved in merozoite invasion of erythrocytes. AMA1 possesses a large N-terminal cysteine-rich ectodomain, followed by a single transmembrane domain and a short C-terminal cytoplasmic tail. The ectodomain has been divided into three domains (I, II, and III) based on the disulfide bond position [16,17] and the recent crystal structure [18]. Domain III binds to human erythrocytes [19] and serves as a target for growth-inhibitory antibodies [20]. Immunization with parasite-derived AMA1 and recombinant AMA1 induced significant levels of protection against P. yoelii challenge in mice [21] and against P. falciparum challenge in Aotus monkeys [22], respectively. Despite its promising potential, neither PfMSP1 19 -nor PfAMA1based vaccine candidates have yet shown satisfactory outcomes in human clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic, although poor immunogenicity and genetic polymorphisms are thought to be major obstacles for vaccine development using these molecules [23,24,25,26].
We have recently developed a baculoviral-based vaccine (BBV) expressing PyMSP1 19 on the surface of the viral envelope [27]. Adjuvant-free intranasal immunization with this vaccine induced not only strong systemic humoral immune responses with high titers of PyMSP1 19 -specific antibody but also natural boosting of PyMSP1 19 -specific antibody responses shortly after challenge, and conferred complete protection. As a next step, we have generated a PfMSP1 19 -BBV vaccine to address the possibility of its use in a clinical setting. In the present study, we evaluated the protective efficacies of a series of MSP1 19 -and AMA1-BBVs against challenge with transgenic P. berghei expressing PfMSP1 19 as well as P. berghei in mice. Our results show that although immunization with these BBVs induced high levels of antigen-specific antibody titers, none of the immunized mice were protected against challenge. In contrast, immunization with PyMSP1 19 -and PyAMA1-BBVs provided 100% and 40% protection against lethal challenge with P. yoelii, respectively. These data suggest that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1 19 and AMA1. The present study provides important insights for malaria bloodstage vaccine development using P. yoelii and P. berghei.

Ethics Statement
All care and handling of the animals was in accordance with the Guidelines for Animal Care and Use prepared by Jichi Medical University, following approval (ID: 09193) by the Jichi Medical University Ethical Review Board.

Mice and parasites
Female BALB/c and C57/BL6 mice, 7 to 8 weeks of age at the start of the experiments, were purchased from Nippon Clea (Tokyo, Japan). P. berghei ANKA were used for challenge infection. P. yoelii 17XL, a lethal murine malaria parasite, was kindly provided by T. Tsuboi (Ehime University, Matsuyama, Japan). P. falciparum 3D7 was kindly provided by K. Kita (The University of Tokyo, Tokyo, Japan). Pb-PfM19 [28] [27] to construct baculovirus transfer vectors.
For IFA, erythrocytes infected with parasites were washed, aliquoted onto multiwell slides, and fixed in 4% paraformaldehyde or methanol/acetone (4:6) for 30 min. Sera were diluted 1:1,000 and incubated on the slide at room temperature for 1 h following permeabilization with 1% Triton X in PBS. After washing, the slides were incubated with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse IgG for 1 h, washed, and covered with a drop of VECTASHIELD TM with DAPI (49 6-diamidion-2phenylindole) (Vector Laboratories). Bound antibodies were detected using a BZ 9000 fluorescence microscope (Keyence, Tokyo, Japan).

Immunization and challenge infections
Mice were immunized three times at 3-week intervals with 5610 7 pfu of BBV either by an intramuscular (i.m.) or intranasal (i.n.) route as described previously [27]. As a comparative control, mice were immunized intraperitoneally (i.p.) with 50 mg of GST-PbMSP1 19 , GST-PfMSP1 19 or GST-PyMSP1 19 in 2 mg of aluminum hydroxide (ImjectH Alum, Pierce) three times at 3week intervals. For each route of immunization, 2 weeks after the final immunization, sera were collected and mice were challenged with 1,000 live parasite-infected red blood cells (pRBC) by intravenous injection. The course of parasitemia was monitored by microscopic examination of Giemsa-stained thin smears of tail blood.

Enzyme-linked immunosorbent assay (ELISA) for antibody titers
Sera obtained from immunized mice were collected by tail bleeds 2 weeks after the final immunization prior to challenge. For some mice, serum was also collected periodically after challenge. For MSP1 19 -specific antibody detection, pre-coated ELISA plates with 100 ng/well GST-PfMSP1 19 , GST-PbMSP1 19 , GST-PyMSP1 19 , PyAMA1-D3, PbAMA1-D3, and yPyMSP1 19 were incubated with serial dilutions of sera obtained from immunized and control mice. MSP1 19 -or AMA1D3-specific antibodies were detected using HRP-conjugated goat anti-mouse IgG (H+L) (Bio-Rad). The plates were developed with peroxidase substrate solution [H 2 O 2 and 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonate)]. The optical density (OD) at 414 nm of each well was measured using a plate reader. Endpoint titers were expressed as the reciprocal of the highest sample dilution for which the OD was equal or greater than the mean OD of non-immune control sera.

Infection and drug treatment
Groups of five mice were infected with P. yoelii XL or P. berghei ANKA pRBC. When the parasitemia had reached 1-3%, mice were treated i.m. on 3 consecutive days with 100 ml of 10 mg/ml Artemether InjectionH (Kunming Pharmaceutical Corp., Kunming, China) dissolved in olive oil (Yoshida Pharmaceutical Corp., Tokyo, Japan). Four weeks after the completion of the infection and drug cure regimen, mice were re-infected three times with 1,000 live pRBC of homologous parasite at 4-week intervals. Selfcured mice were challenged with 1,000 live pRBC of heterologous parasites (P. yoelii XL or P. berghei ANKA). The same experiment was repeated. The course of parasitemia was monitored by microscopic examination of Giemsa-stained thin smears of tail blood.

Construction of MSP1 19 -BBV
Recently, we have developed a new PyMSP1 19 -BBV (AcNPV-PyMSP1 19 surf) that displays PyMSP1 19 on the surface of the baculoviral envelope. Adjuvant-free intranasal immunization with this vaccine induced strong systemic humoral immune responses with high titers of PyMSP1 19 -specific antibody, naturally boosted the PyMSP1 19 -specific antibody response a short time after infection, and allowed 100% of mice to self-cure with very low parasitemia [27]. To apply this baculoviral vaccine system to P. falciparum MSP1 19 vaccine development, we generated AcNPV-PfMSP1 19 surf and AcNPV-PbMSP1 19 surf ( Figure 1A). Each construct harbored a gene cassette that consisted of the gp64 signal sequence and the MSP1 19 gene fused to the N-terminus of the AcNPV major envelope protein gp64. Expression of these gene cassettes was driven by the polyhedrin promoter. Thus these BBVs were designed to express MSP1 19 on the viral envelope as a gp64 fusion protein.
tion-dependent epitope [31], reacted with very faint doublet bands with relative molecular masses (M r ) of 75 and 85 kDa in the presence of 2-ME ( Figure 1B, lanes 1-2). Much stronger smear bands with high M r were seen in the absence of 2-ME, (lane 3), indicating formation of oligomer complexes. P. berghei-hyperimmune serum reacted with a 75-kDa band corresponding to the PbMSP1 19 -gp64 fusion protein in the presence of 5% 2-ME. Similar to the PfMSP1 19 -gp64 fusion protein complex, strong smear bands of PbMSP1 19 -gp64 fusion protein with high M r were seen in the absence of 2-ME (lane 6). These results are consistent with previous results showing that the PyMSP1 19 -gp64 fusion protein was susceptible to treatment with 2-ME as detected using P. yoelii-hyperimmune serum (lanes 7-9) [27]. The anti-gp64 mAb reacted with three MSP1 19 -gp64 fusion proteins and endogenous gp64 (lanes 10-12). The total intensity of endogenous gp64 plus each MSP1 19 -gp64 fusion protein band seems to be similar to that of each smear band (lanes 3, 6 and 9) under the non-reducing conditions. These results indicate that these three MSP1 19 -gp64 fusion proteins form oligomer complexes not only with MSP1 19 -gp64 fusion protein but also endogenous gp64 on the virus envelope and retain the three-dimensional structures of the native MSP1 19 with correctly formed disulfide bonds. AcNPV-PyMSP1 19 surf (lanes 7, 8, 9 and 12) were treated with the loading buffer with 5% 2-ME (lanes 1, 4, 7, 10, 11 and 12), 0.5% 2-ME (lanes 2, 5 and 8) or without 2-ME (lanes 3, 6 and 9) and examined using the 5.2 mAb (lanes 1-3), P. berghei-hyperimmune serum (lanes 4-6), P. yoelii-hyperimmune serum (lanes 7-9) and anti-gp64 mAb (lanes 10-12). Positions of MSP1 19  High level PfMSP1 19 -specifc antibody titers induced by AcNPV-PfMSP1 19 surf did not confer protection against Pb-PfMSP1 parasites Both i.m. and i.n. immunization of BALB/c mice with AcNPV-PfMSP1 19 surf induced high titers of PfMSP1 19 -specifc antibodies (72,600619,300 and 167,000647,300, respectively) ( Table 1,  EXP1). These immune sera strongly reacted with native PfMSP1 19 on P. falciparum schizonts with circumferential staining, characteristic of an antigen present on the parasite surface ( Figure 1C). However, none of the immunized mice survived following challenge with Pb-PfM19 parasites (transgenic P. berghei expressing PfMSP1 19 in place of native PbMSP1 19 ). In accordance with our previous study [27], the i.m. and i.n. immunization with AcNPV-PyMSP1 19 surf conferred 50% and 100% protection, respectively, against P. yoelii challenge infection ( Table 1, EXP2,G4-5). Moreover, we and others have demonstrated that mice immunized with E. coli-producing GST-PyMSP1 19 formulated in Freund's or alum adjuvant were protected against P. yoelii challenge infection. While immunization with GST-PyMSP1 19 plus alum provided 70% protection against P. yoelii challenge infection, mice similarly immunized with the same preparation of GST-PfMSP1 19 did not survive following Pb-PfMSP1 parasite challenge, although the immunization induced high titers of PfMSP1 19 -specifc antibodies (134,000628,600). Interestingly, one of 10 naïve mice self-cured from high parasitemia following P. yoelii 17XL infection (Table 1, EXP2 G1). We observed several monocytes actively phagocytosing the parasites in the blood of the self-cured mouse (Supplementary Figure S1). This is a very rare case because P. yoelii 17XL infection of BALB/c mice was 100% lethal in our previous experiments. The parasite clearance by phagocytosis may be due to the activation of innate immunity during infection. It would be interesting to address the triggers behind the induction of protective immunity in a naïve mouse during infection.
To examine whether natural boosting of PfMSP-1 19 -specific antibodies was induced, the kinetics of the PfMSP1 19 -specific antibody titers and parasitemia during the course of infection were determined. PfMSP1 19 -specific antibodies induced by i.m. and i.n. immunization with AcNPV-PfMSP1 19 surf increased 2.7-and 4.2fold 11 days after challenge infection (Figure 2), indicating natural boosting by challenge infection. However, the immunized groups died with high levels of parasitemia and anemia but without signs of cerebral malaria, which is similar to the non-immunized group.
AcNPV-PbMSP1 19 surf was ineffective against P. berghei To address the possibility that P. berghei is resistant to immune responses to PbMSP1 19 , AcNPV-PbMSP1 19 surf was generated with a construct similar to AcNPV-PyMSP1 19 surf and AcNPV-PfMSP1 19 surf. As for the Pb-PfMSP1 parasites, none of the BALB/c mice immunized i.m or i.n. with AcNPV-PbMSP1 19 surf survived following P. berghei challenge ( Table 1, EXP3 G4-5), although immunization induced high titers of PbMSP1 19 -specifc antibodies (103,000631,700 and 97,800649,800, respectively) with strong reactivity against P. berghei mature schizonts ( Figure 1E). In addition, none of the BALB/c mice immunized with GST-  Figure S2). Since P. berghei ANKA infection of C57BL/6 mice, but not BALB/c mice, has been shown to lead to ''cerebral malaria'' [32], it is important to examine whether the vaccine efficacy and the course of infection are different between BALB/c and C57BL/6 mice. All groups of C57BL/6 mice infected with P. berghei ANKA (Table 1, EXP 3 G6-8) died exhibiting low parasitemia (,15%) 8-10 days after challenge, which may be due to cerebral malaria (Supplementary Figure S2). Similar to BALB/c mice, there is no difference in the course of infection and survival time between AcNPV-PbMSP1 19 and nonimmunized B57BL/6 groups (Supplementary Figure S2). Thus AcNPV-PbMSP1 19 did not contribute to any protective effect or reduction of symptoms either in BALB/c or C57BL/6 mice, indicating that P. berghei and Pb-PfM19 parasites circumvent immune responses to MSP1 19 -BBVs, which are effective for P. yoelii.
Naturally acquired protective immunity to P. berghei confers resistance to P. yoelii, but not vice versa While many studies have consistently shown that the ELISA titer of MSP1 19 -and AMA1-immunized mice correlates with protective immunity against P. yoelii challenge, the corresponding P. berghei vaccines failed to protect against P. berghei. Therefore, it was of interest to determine the degree of heterologous immunity occurring between P. yoelii and P. berghei. BALB/c mice infected either with P. yoelii or P. berghei were drug-cured by three doses of Artemether. This drug treatment regimen completely cleared parasitemia so that no recurrence or recrudescence parasite appeared. The self-cured mice were re-infected three times with the homologous parasite. At the first challenge after drug treatment, some mice developed low levels of parasitemia (,10%) in both groups, and all self-cured within 7 days (data not shown). No parasitemia appeared following the second and third homologous challenges, indicating that mice naturally acquired sterile protective immunity against homologous infection. Subsequently, these mice were challenged with the heterologous parasite. All of the self-cured mice from P. berghei completely protected against P. yoelii with undetectable levels of parasitemia (Table 3, EXP6 G1), indicating the acquisition of cross-resistance to P. yoelii infection. In contrast, all the self-cured mice from P. yoelii suffered from severe courses of P. berghei infection with high maximum parasitemia levels (.70% parasitemia) and 100% mortality (G2). Thus naturally acquired sterile immunity induced by drug treatment and repeated P. berghei infection can persistently protect mice against P. yoelii infection, but not vice versa.

Discussion
In the present study, we demonstrate that the two leading malaria blood-stage vaccine candidate antigens, MSP1 19 and AMA1, failed to protect against P. berghei and its transgenic parasite challenge infection, although immunization with these vaccines induced high levels of antigen-specific antibody titers, and the immune sera strongly reacted with the blood-stage parasites.
Our previous study showed that immunization with AcNPV-PyMSP1 19 surf completely clears P. yoelii shortly after challenge by a quick natural boosting response [27]. On the other hand, immunization with the bacterially-produced GST-PyMSP1 19 plus alum impairs P. yoelii growth at the time of infection by induced PyMSP1 19 -specific antibodies, resulting in a delay in the onset of a patent parasitemia and protracted period of parasite inhibition [6,33,34,35]. Therefore, these two vaccine formulas inducing different protective immune responses are suitable immunogens to investigate whether MSP1 19 -specific immune responses could confer protection or affect the course of infection in P. berghei ANKA and its transgenic models. Since MSP1 19 is highly structured on the surface of merozoites, folding into two epidermal growth factor-like domains [36], development of MSP1 19 -based vaccines with its native three-dimensional structure would be necessary to induce protective immune responses. This is similar to were treated with the loading buffer containing 1% 2-ME and examined using P. yoelii-hyperimmune serum (lanes 1 and 2), or P. berghei-hyperimmune serum (lanes 3 and 4). (C-J) Immunofluorescence patterns of sera obtained from mice immunized with four AMA1-BBVs on methanol-acetone fixed smears of erythrocytes infected with P. yoelii (C and E) and P. berghei (G and I). The smears were incubated with serum obtained from an individual mouse immunized either with AcNPV-PyAMA1-D123surf (C), AcNPV-PyAMA1-D3surf (E), AcNPV-PbAMA1-D123surf (G) or AcNPV-PbAMA1-D3surf (I), and antibody binding was detected with a secondary FITC-labeled antibody. Cell nuclei were visualized by DAPI staining on the corresponding smears (D, F, H and J). Scale bar, 10 mm. doi:10.1371/journal.pone.0013727.g003 PyMSP1 19 , PfMSP1 19 and PbMSP1 19 displayed on BBVs, which are stabilized by disulfide bonds, as evidenced by the loss of conformational mAb and hyperimmune IgG binding, respectively, in immunoblots with reduced MSP1 19 -BBVs ( Figure 1B). Additional evidence of the correct structure includes the induction of IFA-reactive antibodies ( Figures 2C-H). In the P. berghei transgenic parasite model, although the AcNPV-PfMSP1 19 surf group elicited natural boosting of vaccine-induced PfMSP1 19 -specific antibody responses during infection, there was no significant reduction of parasitemia or prolonging of survival time compared to nonimmunized groups. Similarly, neither PbMSP1 19 -nor PbAMA1-BBV was effective against P. berghei. In addition, the bacteriallyproduced GST-PbMSP1 19 protein with alum adjuvant conferred no protection. Thus P. berghei and its transgenic parasites completely circumvent immune responses induced by MSP1 19and AMA1-based vaccines.
One possible mechanism by which the growth of P. berghei cannot be controlled by high levels of PbMSP1 19 -or PbAMA1specific antibodies is that P. berghei possesses additional molecules and/or mechanisms other than MSP1 19 and AMA1 for erythrocyte invasion that are lacking in P. yoelii. In support of this, we found that when mice self-cured from P. berghei infection by drug treatment and subsequent P. berghei re-infections, they naturally acquired sterile immunity against P. yoelii as well as P. berghei, but not vice versa. This is consistent with a previous report that protective cross-immunity operates only in one direction (P. bergheiRP. yoelii) since mice immunized with a formalin-fixed blood-stage P. yoelii were fully susceptible to P. berghei [37]. P. falciparum has been shown to have a number of invasion pathways and to be capable of entering erythrocytes by sialic acid-dependent and -independent pathways [38]. While no such parallel invasion pathways have been described for rodent malaria parasites, it is possible that the multiplicity of merozoite surface proteins in P. berghei may reflect involvement in alternative pathways of invasion. Although a transgenic P. berghei line expressing PyMSP1 19 in place of native PbMSP1 19 would be a good model to address this possibility, we have failed to generate the transgenic line using similar methodology for the construction of Pb-PfM19 [28], suggesting that PbMSP1 19 and PfMSP1 19 share similar functions essential for growth and/or invasion, but not with PyMSP1 19 .
Another possible mechanism is that P. berghei infection may impair antibody-dependent cellular inhibition of parasite growth. We have previously demonstrated that a genetically engineered bispecific single-chain antibody targeted to human CD3 and PfMSP1 19 promotes merozoite phagocytosis and growth inhibition in in vitro P. falciparum culture through cooperation with T cells and monocytes [39]. Evidence has been accumulated to suggest that antibody action via Fc interaction with monocytes/macrophages plays an important role in protective immunity against blood-stage parasites [14,40,41]. In the case of the P. yoelii model, MSP1 19 and AMA1-specific antibodies may effectively function in the parasite killing by cooperation with monocytes/macrophages. However, P. berghei infection may strongly suppress monocyte/  macrophage activation through Fc receptors with MSP1 19 -or AMA1-specific antibodies. If P. falciparum uses mechanisms similar to those utilized by P. berghei to circumvent MSP1 19 -and AMA1based vaccine properties, P. berghei rather than P. yoelii would be a more useful model to identify and evaluate new blood-stage vaccine candidate antigens for P. falciparum. Recently, P. berghei has been genetically engineered to express representative vaccine candidate antigens (e.g., PfMSP1 19 , PfAMA1, PfCSP, Pfs25 and Pvs25) from the human malaria parasites, P. falciparum and P. vivax [28,42,43,44,45]. These transgenic P. berghei parasites provide great potential to investigate the protective efficacies of vaccine candidates against the human malaria parasites in vivo. Very recently, we have shown that transmission blocking vaccines using the BBV system can induce a remarkable reduction of malaria transmission to mosquitoes, directly evaluated by immunization of mice following challenge with Pfs25-or Pvs25-expressing P. berghei [46,47]. Although P. berghei transgenic parasites expressing PfMSP1 19 and PfAMA1 have been used to evaluate the inhibitory effects on parasite growth in vitro and through passive immunization [15,28,48], in vivo challenge experiments following active immunization have not been reported. Unlike P. yoelii, P. berghei has not been used for the evaluation of vaccine efficacy against blood-stage parasites, although the parasite has been well-studied not only for preerythrocytic vaccine development but also a cerebral malaria model [49]. To date, the usefulness of the transgenic P. berghei model for evaluating protective efficacies of blood-stage antigens remains unclear. In the present study, we were unable to show the usefulness of PfMSP1 19 -BBV as a promising malaria vaccine candidate using the transgenic P. berghei, although a similar construct of PyMSP1 19 -BBV provided 100% protection in the P. yoelii model [27]. We still cannot exclude the possibility that the absence of protection induced by the PfMSP1 19 vaccine formulations is due to qualitative differences between the PyMSP1 19 and PfMSP1 19 vaccine formulations used here, rather than differences in parasite susceptibility to anti-MSP1 19 responses, although western blot and immunologiocal analyses show that the three MSP1 19 -gp64 fusion proteins as well as GST-MSP1 19 proteins were expressed at quantitatively similar levels and immunization with these proteins induced high titers of MSP1 19specific antibodies with strong reactivity against mature schizonts of the corresponding parasites. Further experiments using PfMSP1-based vaccines with GMP level, which have been evaluated in human clinical trials, would be needed to investigate the susceptibility of Pb-PfMSP1 19 to anti-PfMSP1 19 responses.
Our results provide important insights for malaria blood-stage vaccine development using P. yoelii and P. berghei and highlight the need to deeply investigate the relationship between rodent and human parasites. Obviously, it is important to elucidate the discrepancy in the vaccine efficacies observed between the P. yoelii model and human clinical trials. A better understanding of the precise relationship between rodent and human parasites should lead to a transfer of information from transgenic P. berghei to human vaccine development prior to human clinical settings. Figure S1 Photomicrographs of Giemsa-stained thin blood smears of the self-cured mouse. Ten non-immunized mice were infected with P. yoelii 17XL-pRBC by i.v. injection. The course of parasitemia was monitored daily from 4 days post-challenge by microscopic examination of Giemsa-stained thin blood smears obtained from tail bleeds. One of these mice self-cured from high parasitemia of P. yoelii infection. The mouse cleared the parasites 21 days after challenge. The photomicrographs of the self-cured mouse were taken at 19, 21 and 24 days after challenge. Arrows indicate malaria pigment in monocytes phagocytosing the parasites. Original magnification, 61,000. Found at: doi:10.1371/journal.pone.0013727.s001 (6.18 MB TIF) Figure S2 The course of parasitemia. BALB/c and C57BL/6 mice were immunized i.m. with AcNPV-PbMSP1 19 surf or AcNPV-WT and challenged i.v. with 10 3 P. berghei-pRBC. Parasitemia was monitored daily from 5 days post-challenge. All groups of BALB/c and C57BL/6 mice died 18 and 10 days after challenge, respectively. Data (mean Â 6SD) are from the BALB/c (EXP3 G 1, 3 and 4) and C57BL/6 (EXP3 G6-8) shown in Table 1. closed triangle, non-immunized; open square, AcNPV-WT; closed circle, AcNPV-PbMSP1 19 surf. Found at: doi:10.1371/journal.pone.0013727.s002 (6.11 MB TIF)