Baculovirus-Vectored Multistage Plasmodium vivax Vaccine InducesBoth Protective and Transmission-Blocking Immunities againstTransgenic Rodent Malaria Parasites

Masanori Mizutani,a Mitsuhiro Iyori,a Andrew M. Blagborough,b Shinya Fukumoto,c Tomohiro Funatsu,a Robert E. Sinden,b

Shigeto Yoshidaa

Laboratory of Vaccinology and Applied Immunology, Kanazawa University School of Pharmacy, Kanazawa, Japana; Division of Cell and Molecular Biology, Department ofLife Sciences, Imperial College London, London, United Kingdomb; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and VeterinaryMedicine, Hokkaido, Japanc

A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individualsagainst infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite,respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmis-sion in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simul-taneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein,thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculo-virus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vac-cine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies againsttransgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, whichfunctions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalar-ial pre-erythrocytic and TB response via a single immunization regimen.

Plasmodium vivax is currently the most widely distributed humanmalaria parasite, with an “at risk” population in 2010 of almost 3

billion people (a third of the global population) and approximately100 to 300 million clinical cases each year (1, 2). Several factors, in-cluding (i) the recent appearance of chloroquine-resistant P. vivax,(ii) the lack of alternatives to primaquine for attacking the dor-mant liver-stage hypnozoites, and (iii) increasing global temper-atures caused by climate change, raise concerns about increases inthe risk of severe P. vivax disease (3–6). Although the importanceof P. vivax vaccines is recognized, the lack of long-term in vitroculture systems in red blood cells and suitable animal models aswell as the complex life cycle of this parasite has hindered advancesin the development of a potent vaccine (7, 8).

The development of malaria vaccines has been focused mostlyon single antigens from different stages of the parasite life cycle: (i)the pre-erythrocytic stages (including the liver stages), (ii) theasexual blood stages, and (iii) the mosquito sexual stages, whereantigens expressed on the gametocyte, gamete, zygote, or ookineteare targeted to prevent transmission from the human hosts tothe mosquito vectors (9). There are concerns that the single-stagevaccine may not be effective because of sequence variabilityamong different parasite isolates, host genetic restriction of im-mune responses to specific epitopes, and short-lived protectiveimmunity induced by some single-antigen vaccines (10). There-fore, a multistage vaccine, which targets several antigens expressedin different stages of the parasite’s development, should logicallyprovide more efficacious protection than immunization with avaccine against a single stage.

Among single-stage vaccines, transmission-blocking vaccines(TBVs) targeting the sexual-stage antigens have great potential tobe used as a component of a multistage vaccine in combination

with vaccines from other parasite stages. A TBV combined withother stage antigens as a multistage vaccine should deliver pro-nounced benefits by preventing infections in individuals and re-ducing parasite transmission in communities. Recently, Theisenet al. reported that a multistage vaccine expressing P. falciparumsexual- and blood-stage antigens induced strong transmission-blocking (TB) activity and functional activity against the bloodstage, as evaluated by a membrane feeding assay and an in vitrogrowth inhibition assay, respectively, using immune sera (11). Toour knowledge, however, no published study has directly assessed,in parallel, the in vivo effectiveness of protection and the TB activ-ity of multistage malaria vaccines by the use of a parasite challengetest. The development of both a new vaccine system and a suitablesmall-animal model is required to evaluate the effectiveness ofprotection and TB activity before proceeding to expensive andethically complex human clinical trials.

We have recently developed a new vaccine platform systembased on the baculovirus Autographa californica nucleopolyhe-drosis virus (AcNPV) called the baculovirus dual-expression sys-

Received 12 May 2014 Returned for modification 31 May 2014Accepted 26 July 2014

Published ahead of print 4 August 2014

Editor: J. H. Adams

Address correspondence to Shigeto Yoshida, shigeto@p.kanazawa-u.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02040-14.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02040-14

4348 iai.asm.org Infection and Immunity p. 4348 – 4357 October 2014 Volume 82 Number 10

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

tem (BDES). BDES is capable of displaying an antigen on the viralenvelope by the use of a baculovirus-derived polyhedrin and ex-pressing it upon transduction of mammalian cells by cytomegalo-virus (CMV) promoters and so can function as both a vaccinecomponent and a DNA vaccine, respectively (12). For a suitablesmall-animal model, our strategy for evaluating BDES malariavaccines is to use transgenic rodent malaria parasites to evaluateprotective and TB efficacies against target antigens from a humanmalaria parasite. Such transgenic parasites provide a safe, cheap,and more practical alternative to using nonhuman primate mod-els for preclinical challenge studies of malaria vaccine efficacy. Ourprevious studies have shown that BDES was an effective malariavaccine platform for all three stages of the parasites, including thepre-erythrocytic stage (12, 13), asexual blood stage (14, 15), andsexual stage (16, 17), when transgenic P. berghei parasites express-ing human Plasmodium antigens were used for their evaluation. Ifoptimized, BDES vaccines could be adopted for developing potentmultistage malaria vaccines.

In the present study, we chose the circumsporozoite protein(CSP) and P25 protein as multistage target antigens for pre-eryth-rocytic- and sexual-stage antigens, respectively. CSP is expressedon the surface of sporozoites and liver-stage parasites. The RTS,Svaccine, which targets P. falciparum CSP, is the most advancedprotective malaria vaccine candidate to date (18). The sexual-stage antigen P. vivax Pvs25 (a homolog of P25 in other Plasmo-dium spp.), located on the surface of the unfertilized macrogameteand ookinete, has been exceptionally well characterized andshown to confer TB immunity in experimental animals (19–21).

We have generated a BDES multistage vaccine expressingPvs25 and PvCSP for a TBV and a pre-erythrocytic vaccine, respec-tively. This vaccine elicited both protective and TB efficacies againsttransgenic P. berghei parasites expressing the above-described an-tigens in mice. These results indicate that the BDES multistagevaccine has potential for preventing malarial infections in individ-uals while also reducing parasite transmission within populationsin regions of malaria endemicity. Hence, in terms of an effectivemalaria vaccine, the BDES multistage vaccine could represent anew alternative to the current vaccine delivery platforms.

MATERIALS AND METHODSEthics statement. Experimental protocols and all care and handling of theanimals were in accordance with the guidelines for animal care and useprepared by Kanazawa University (reference no. 22118-1) and ImperialCollege London (reference no. PPL 70/7185).

Cell lines and Abs. Spodoptera frugiperda (Sf9) cells and COS-7 cellswere maintained as described previously (13). The monoclonal antibodies(MAbs) used were as follows: 2F2, MAb specific for the repeat sequence(DRAD/AGQPAG) of PvCSP of the Salvador-I VK210 strain [PvCSP-(Sal)] (MRA-184; Malaria Research and Reference Reagent ResourceCenter [MR4], Manassas, VA); 2E10E9, MAb specific for the repeat se-quence (ANGAGNQPG) of PvCSP of the Papua New Guinea VK247strain [PvCSP(PNG)] (MRA-185; MR4); N1-1H10, MAb specific forPvs25 (18); and 28B8, MAb specific for anopheline antiplatelet protein(AAPP) (13). A rabbit polyclonal Ab against the baculovirus capsid VP39protein was used as described previously (13).

Plasmid construction and parasite transfection. To construct thetransfer plasmid for generating the PvCSP(Sal)/P. berghei [PvCSP(Sal)/Pb] transgenic parasite line in place of native PbCSP, the DNA sequencecorresponding to amino acids His24 to Asp351 of the entire PvCSP(Sal) gene(GenBank accession number M11926) minus its signal sequence and glyco-sylphosphatidylinositol (GPI) anchor was amplified from pEU3-PvCSP(Sal)using pPvCSP(SalI)-F4 and pPvCSP(Sal)-R2 primers (see Table S1 in the

supplemental material). The DNA sequence corresponding to amino acidsLys372 to Asn395 of the GPI anchor of P. falciparum CSP (GenBank accessionnumber AAN87615) was amplified from pBS-5=UTR-PfCSP-Tcell (13)using pPvCSP(SalI)-F5 and PfCSP-R3 primers. Our previous study con-firmed that the GPI anchor moieties derived from P. falciparum couldfunction in the transgenic rodent parasites (13). The DNA sequence en-coding PvCSP(Sal) and the GPI anchor of PfCSP were linked by overlap-ping PCR using pPvCSP(SalI)-F4 and pPfCSP-R3 primers and then li-gated into the XmaI-XhoI site of pBS-5=UTR-PfCSP-Tcell to generatepBS-5=UTR-PvCSP(Sal)-DHFR-3=. The transgenic parasite [PvCSP(Sal)/Pb] was generated by transfection of wild-type P. berghei ANKA 2.34(WT-Pb) with the linearized pBS-5=UTR-PvCSP(Sal)-DHFR-3= plasmid,as described previously (22). For genotype analysis, genomic PvCSP-(Sal)/Pb DNA was extracted from PvCSP(Sal)/Pb-infected mouse bloodusing a QIAamp DNA blood minikit (Qiagen, Hilden, Germany). Re-placement of the PbCSP gene with the PvCSP gene was confirmed by PCRusing a range of forward and reverse gene-specific primers (see Table S1).Transgenic Pvs25DR3 parasites were used to investigate TB efficacy asdescribed previously (23).

Infectivity and fitness of PvCSP(Sal)/Pb parasites. Anopheles ste-phensi mosquitoes (SDA 500 strain) were allowed to feed on PvCSP(Sal)/Pb-infected mice. The infectivity of PvCSP(Sal)/Pb parasites transmittedfrom mice to mosquitoes was assessed at day 10 postfeeding. Mosquitomidguts were dissected, and oocyst prevalence and intensity were re-corded. The infectivity of PvCSP(Sal)/Pb transmitted from mosquitoes tomice was assessed at day 14 postfeeding. Mice were challenged by the bitesof three to seven infected mosquitoes and checked for the development ofblood-stage parasites by microscopic examination of Giemsa-stained thinblood smears.

Recombinant baculoviruses. To generate BDES-Pvs25-PvCSP-G andBDES-PvCSP-G constructs, the DNA sequence corresponding to His24-Lys353 of PvCSP(Sal) was amplified from pEU3-PvCSP (VK210) usingpPvCSP-F1 and pPvCSP-R1 primers and then cloned into the ApaI andXmaI sites of pGL3 (Promega, Madison, WI) to construct pGL3-PvCSP-(Sal). The DNA sequence corresponding to Pro84-Asp295 of the PvCSP-(PNG) repeat (GenBank accession number M69059) with the G6S1 hingesequence was amplified from pEU3-PvCSP (VK247) using pPvCSP-PNG-F1 and pPvCSP-PNG-R1 primers and then cloned into the AgeI andXmaI sites of pGL3 to construct pGL3-PvCSP(PNG). A 0.6-kb fragmentof the repeat region of PvCSP(PNG) was excised from pGL3-PvCSP-(PNG) by digestion with AgeI and XbaI and then inserted into the XmaIand XbaI sites of pGL3-PvCSP(Sal) to construct pGL3-PvCSP. ThePvCSP DNA sequence corresponding to PvCSP(Sal) linked to the repeatregion of PvCSP(PNG) was amplified from pGL3-PvCSP using pPvC-SP(B)-F3 and pPvCSP-PNG-R1 primers and then ligated into the EcoRIand XmaI sites of pTriEx-PfCSP-gp64 (13) to construct the pTriEx-PvCSP-gp64 baculovirus transfer vector. The DNA sequence correspond-ing to Phe421 and Arg511 of the vesicular stomatitis virus envelope G(VSV-G) protein (GenBank accession number X03633) was amplifiedfrom pCMV-VSV-G (RIKEN Bioresource Center, Tsukuba, Japan) usingpVSV-G-F1 and pVSV-G-R1 primers and then cloned into the XmaI andXhoI sites of pTriEx-PvCSP-gp64 to construct the pTriEx-PvCSP-G bac-ulovirus transfer vector. To construct the Pvs25-PvCSP-expressing bacu-lovirus transfer vector, a 0.6-kb fragment of the Pvs25 gene was excisedfrom pTriEx-Pvs25-gp64 (17) and inserted into the EcoRI and BglII sitesof pTriEx-PvCSP-G to construct the pTriEx-Pvs25-PvCSP-G baculovirustransfer vector. Three recombinant baculoviruses were generated in Sf9cells by cotransfection of the corresponding plasmid vector with BacVec-tor-2000 DNA (Merck Millipore, Guyancourt, France) following themanufacturer’s protocol. Purification of viral particles was performed asdescribed previously (12).

Western blotting. Protein samples from BDES particles and salivaryglands containing sporozoites were separated by 8% sodium dodecyl sul-fate-polyacrylamide gel electrophoresis, transferred to an Immobilon-Ptransfer membrane (Merck Millipore), and then probed with multiple

Baculovirus-Based Multistage Plasmodium vivax Vaccine

October 2014 Volume 82 Number 10 iai.asm.org 4349

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

MAbs and anti-VP39 polyclonal Abs (13). Bound Abs were subsequentlydetected by electrochemiluminescence (GE Healthcare, Waukesha, WI)as described previously (13).

Transduction and indirect immunofluorescence assay (IFA). COS-7cells (1 �104/well) were transduced with BDES particles at a multiplicityof infection (MOI) of 500. After 48 h of incubation with BDES particles,the cells were incubated with an Alexa Fluor 594-conjugated anti-PvCSP-(Sal) MAb or an Alexa Fluor 488-conjugated anti-PvCSP(PNG) MAb tostain the two allelic forms of PvCSP. To stain Pvs25, cells were incubatedwith an anti-Pvs25 MAb followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Biosource International, Camarillo,CA). Hoechst 33342 (Invitrogen, Carlsbad, CA), a permeative DNA dye,was used for live-cell staining.

For IFAs, sporozoites isolated from mosquito salivary glands wereloaded onto glass slides and fixed as described previously (13). Slides werestained with anti-PvCSP(Sal) MAb-conjugated Alexa Fluor 594 andmounted with a drop of Vectashield containing 4=,6=-diamidino-2-phe-nylindole (Vector Laboratories, Burlingame, CA). An LSM710 invertedlaser scanning microscope (Carl Zeiss, Tokyo, Japan) was used for imageacquisition.

Immunizations and challenge infections. Female BALB/c mice (H-2Kd) were immunized intramuscularly (i.m.) three times at 3-week inter-vals with 1 � 108 PFU of BDES particles. PvCSP(Sal)/Pb-infected mos-quitoes were prepared and used for challenge infection as describedpreviously (12). Briefly, 2 weeks after their third immunization, the micewere challenged by the bites of PvCSP(Sal)/Pb-infected mosquitoes. Mos-quitoes were allowed to feed on the abdomen of each mouse for 15 min.To ensure a high probability of infection (in the control mice), all micereceived a minimum of three bites and a maximum of seven bites. Salivaryglands from all blood-engorged mosquitoes in the control group weredissected to confirm the presence of sporozoites. Where necessary, mos-quito feeds were performed repeatedly until three to seven infected mos-quitoes had bitten each mouse.

Recombinant proteins. The DNA sequence corresponding to aminoacids His24 to Lys353 of the PvCSP(Sal) gene and the DNA sequence corre-sponding to amino acids Pro84 to Asp295 of the PvCSP(PNG) repeat wereexcised from pGL3-PvCSP(Sal) and pGL3-PvCSP(PNG), respectively, by di-gestion with EcoRI and XmaI. Each of the PvCSP(Sal) and PvCSP(PNG)DNA fragments was cloned into the EcoRI and XmaI sites of the pET32 (�)vector (Merck Millipore). Recombinant PvCSP(Sal) and PvCSP(PNG) re-peat regions, created as thioredoxin fusion proteins, were expressed in Esch-erichia coli and were purified using a nickel-nitrilotriacetic acid (Ni-NTA)column (Qiagen) followed by dialysis against phosphate-buffered saline(PBS), as described previously. These proteins were used as antigens forenzyme-linked immunosorbent assays (ELISAs).

ELISA Ab titers. Sera from immunized mice were collected by tailbleeds 2 weeks after the final immunization and prior to sporozoite chal-lenge. Ab titers specific for PvCSP(Sal), PvCSP(PNG) repeat, and Pvs25were quantified by ELISA. Ninety-six-well plates (Corning Life Sciences,Tokyo, Japan) precoated with rPvCSP(Sal) (80 ng/well), rPvCSP(PNG)repeat (150 ng/well), or rPvs25 (112 ng/well) (17) were incubated withserially diluted sera from the immunized and control mice. Antigen-spe-cific IgG and subclasses (IgG1, IgG2a, IgG2b, and IgG3) were measured byELISA as described previously (13).

TB assays. In three separate experiments, mice were immunized i.m.three times with BDES-Pvs25-PvCSP-G or BDES-Pvs25-gp64 at threeweekly intervals. Two weeks later, mice were divided into two groups(three per group) and infected intraperitoneally with 1 � 106 live WT-Pb-or Pvs25DR3-parasitized red blood cells (pRBCs). Similarly, mice wereimmunized i.m. with AcNPV-WT, divided into two groups, and infectedas described above. At 3 days postinfection, �50 A. stephensi mosquitoeswere allowed to feed per infected mouse. At 24 h postfeeding, mosquitoeswere briefly anesthetized with CO2, and any unfed mosquitoes were re-moved. The blood-fed mosquitoes were then maintained on fructose (8%[wt/vol] fructose, 0.05% [wt/vol] p-aminobenzoic acid) at 19 to 22°C and

50% to 80% relative humidity. At day 10 postfeeding, mosquito midgutswere dissected, and the oocyst prevalence and intensity were recorded.

Statistical analyses. Statistical differences between the experimentalgroups were analyzed using Student’s t test. The two-tailed Fisher’s exactprobability test was used to determine statistical differences in the protec-tive efficacy of BDES. For active immunizations, significance was assessedusing the Mann-Whitney U test to examine differences in oocyst countsper mosquito. Fisher’s exact probability test was used to examine differ-ences in the infection prevalence rates. P values that were �0.05 wereconsidered statistically significant. Statistical analyses were performed us-ing Microsoft Excel, Prism version 5 (GraphPad Software Inc., La Jolla,CA), and SPSS Statistics (version 19; IBM, Chicago, IL).

RESULTSBDES vaccine constructs. We have previously developed a BDES-Pvs25-gp64 vaccine that displays Pvs25 on the surface of the viralenvelope (17). Immunization with this vaccine induced stronghumoral immune responses with high anti-Pvs25 titers and highTB activity (17). To use this system to generate a novel multistageP. vivax vaccine, the gene encoding PvCSP was assembled in framewith the gene encoding Pvs25 and then introduced into the BDESconstruct. There are different allelic forms of PvCSP, and the twomost common PvCSP alleles are derived from the Salvador-I(VK210; Sal) strain and the Papua New Guinea (VK247; PNG)strain (24). The main variation between these allelic forms is in thecentral repeat region of CSP, which is a possible target for neutral-izing Abs. To encompass the two genetic variants of PvCSP, thegene encoding full-length PvCSP(Sal) was fused with that of therepeat region of PvCSP(PNG), here called “PvCSP(Sal-PNG)”(Fig. 1A). BDES-Pvs25-PvCSP-G harbors the gene encoding PvCSP(Sal-PNG) fused to Pvs25 through the G6S1 hinge sequence andthe C-terminal portion of VSV-G under the control of a dualpromoter consisting of the CMV immediate-early enhancer-pro-moter and the polyhedrin promoter (Fig. 1B). The C-terminalportion of VSV-G comprises the ectodomain plus the transmem-brane and cytoplasmic tail domains of VSV-G, which effectivelydisplay foreign proteins on the baculovirus envelope (25). BDES-PvCSP-G, BDES-PvCSP-gp64, and BDES-Pvs25-gp64 were usedas control groups for monovalent vaccines.

Pvs25-PvCSP fusion protein expression on the surface of theviral envelope. Western blotting shows that the anti-PvCSP(Sal)MAb (2F2), which is specific for the PvCSP(Sal) repeat, reactedwith Pvs25-PvCSP-G as a multiple ladder of bands with relativemolecular masses (Mr) of 140,110 and 90 kDa (Fig. 2A; see alsoFig. S1A in the supplemental material), whereas the anti-PvCSP-(PNG) MAb (2E10E9), which is specific for the PvCSP(PNG) re-peat, reacted with multiple bands of 140, 110, 90, 60 and 40 kDa(Fig. 2A; see also Fig. S1A). The predicted Mr of the Pvs25-PvCSP-G fusion protein is 110 kDa. Multiple ladder banding wasalso seen in BDES-PvCSP-G and BDES-PvCSP-gp64, when re-acted with the same MAbs (Fig. 2A; see also S1B). We hypothesizethat these multiple bands may be due to protein degradation in theinsect cells rather than to post-translational modifications, be-cause there is no potential N-linked glycosylation site in the pre-dicted amino acid sequence of PvCSP. The level of PvCSP(Sal)displayed by BDES-Pvs25-PvCSP-G was much higher than that ofBDES-PvCSP-G and BDES-PvCSP-gp64, when normalized usingthe viral capsid protein VP39 as an internal control (Fig. 2A; seealso S1A and B in the supplemental material). Both BDES-Pvs25-gp64 and BDES-Pvs25-PvCSP-G displayed antigens with the pre-dicted Mr, and the amount of Pvs25-PvCSP fusion protein dis-

Mizutani et al.

4350 iai.asm.org Infection and Immunity

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

played by BDES-Pvs25-PvCSP-G was 3-fold higher than that ofthe Pvs25 protein displayed by BDES-Pvs25-gp64 (Fig. 2B). Sincethe protein degradation in BDES-Pvs25-PvCSP-G was much lessthan that in other monovalent vaccines (see Fig. S1 in the supple-mental material), it is possible that Pvs25 can increase the proteinstability on the surface of viral envelope, resulting in the increaseof protein display.

To examine expression of conformation-dependent epitopes,the viral display profiles of BDES-Pvs25-PvCSP-G were analyzed

by Western blotting using anti-Pvs25 and anti-FLAG MAbs in theabsence or presence of 0.001% to 1% 2-mercaptoethanol (2-ME).A FLAG MAb, which recognizes a linear epitope within the N-ter-minal tag, reacted with a single band of some 110 kDa at all 2-MEconcentrations (Fig. 2C). The 110-kDa band corresponds to thepredicted Mr of the Pvs25-PvCSP fusion protein, whereas theband around 250 kDa is likely a dimer of the Pvs25-PvCSP fusionprotein. When 2-ME was added at concentrations above 1%, noband was detected by the anti-Pvs25 MAb (N1-1H10), which rec-

FIG 1 Schematic representation of BDES vaccine constructs. (A) Map of the sequence encoding the PvCSP(Sal-PNG) tandem protein. The full-lengthPvCSP(Sal)and PvCSP(PNG) repeat genes are connected by a G6S1 linker (Linker) gene. The sequences of each repeat region are as follows: a, DRADGQPAG;b, DRAAGQPAG; c, DRAAGQAAG; d, QPGANGAGN; e, GNQPGANGA; and f, QPGANGAGG. (B) Construction of BDES vaccines. BDES-Pvs25-PvCSP-Gharbors the gene encoding Pvs25 fused to PvCSP(Sal-PNG) by a G6S1 linker. Expression of the gene cassettes was driven by a dual promoter comprising the CMVimmediate-early enhancer-promoter (pCMV) and the polyhedrin promoter (pPolh). SP, the gp64 signal sequence; F, FLAG epitope tag; NT, Sal-NT, N terminusof PvCSP(Sal); Sal-CT, C terminus of PvCSP(Sal); G, transmembrane region of VSV-G protein; H, His epitope tag.

FIG 2 Antigen expression of BDES vaccines incorporated into viral particles. (A) The BDES products indicated were treated with loading buffer containing 1%2-mercaptoethanol, boiled for 5 min, and then subjected to SDS-PAGE as described in Materials and Methods. Antigen expression was detected with anti-PvCSP(Sal) MAb, anti-PvCSP(PNG) MAb, or anti-VP39 polyclonal Ab, as indicated on the left. (B) Antigen expression of BEDS-Pvs25-gp64 and BDES-Pvs25-PvCSP-G was detected with anti-FLAG MAb and anti-VP39 polyclonal Ab. (C and D) BDES-Pvs25-PvCSP-G virions were treated with various concentrations(0% to 1%) of 2-ME and then boiled for 5 min. Anti-FLAG MAb (C) and anti-Pvs25 MAb (D) were used. The 2-ME concentrations are shown above each gel.

Baculovirus-Based Multistage Plasmodium vivax Vaccine

October 2014 Volume 82 Number 10 iai.asm.org 4351

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ognizes a conformation-dependent epitope of Pvs25 (19). In con-trast, reduced concentrations of 2-ME (below 0.1%) increased thereactivity of the Pvs25-PvCSP fusion protein to the anti-Pvs25MAb (Fig. 2D). These results suggest that the Pvs25-PvCSP fusionprotein on the viral envelope might retain the three-dimensionalstructure of the native Pvs25 protein, which is essential for induc-tion of Abs with TB activities (19).

Pvs25-PvCSP fusion protein expression in mammalian cells.To investigate the potential of BDES as a DNA-based vaccine,COS-7 cells were transduced with a series of BDES vaccines.Strong immunofluorescence signals were detected with MAbsagainst PvCSP(Sal), PvCSP(PNG), and Pvs25 in COS-7 cellstransduced with BDES-Pvs25-PvCSP-G 48 h after transduction(Fig. 3). The expression levels of the PvCSP(Sal) repeat in BDES-

FIG 3 In vitro expression of PvCSP and Pvs25 in mammalian cells by transduction of BDES vaccines. (A) COS-7 cells were transduced with AcNPV-WT or BDESvaccines at an MOI of 500 for each. Forty-eight hours later, these cells were incubated with the Alexa Fluor 594-conjugated anti-PvCSP(Sal) (red) or the AlexaFluor 488-conjugated anti-PvCSP(PNG) (green). (B) Bound anti-Pvs25 MAbs were detected with fluorescein isothiocyanate-labeled anti-mouse IgG by fluo-rescence microscopy (green). Cell nuclei were visualized by Hoechst (blue). Original magnification, �400. Bars � 50 �m.

Mizutani et al.

4352 iai.asm.org Infection and Immunity

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

Pvs25-PvCSP-G were much higher than those of the monovalentBDES-PvCSP-G and BDES-PvCSP-gp64 constructs. No signalwas detected from cells transduced with AcNPV-WT (Fig. 3).Treatment with the transcriptional inhibitor actinomycin Dmarkedly reduced the protein expression, indicating that de novoantigen expression was induced in the mammalian cells (see Fig.S2 in the supplemental material). The expression level of Pvs25 inBDES-Pvs25-PvCSP-G was high and equivalent with that of mon-ovalent BDES-Pvs25-gp64 (Fig. 3B). Thus, BDES-Pvs25-PvCSP-G had a capability of transducing to the mammalian cellswith high expression levels of PvCSP(Sal-PNG) and a moderateexpression level of Pvs25, which may provide not only humoralbut also cellular immune responses to both Pvs25 and PvCSP.

Ab responses in mice immunized with BDES vaccines. Thetiters and isotype profiles of PvCSP(Sal)-, PvCSP(PNG) repeat-,and Pvs25-specific IgGs induced by the BDES vaccines were de-termined by ELISA. The PvCSP(Sal)-specific IgG titer was in-duced in the mice immunized with BDES-Pvs25-PvCSP-G(116,030 � 94,708); this value range is much higher than those ofBDES-PvCSP-gp64 (456 � 925) and BDES-PvCSP-G (24,388 �20,927) (Fig. 4A). The PvCSP(PNG) repeat- specific IgG titer wasalso induced by BDES-PvCSP-gp64 (23,293 � 16,720), BDES-PvCSP-G (10,797 � 12,350), and BDES-Pvs25-PvCSP-G (18,736� 16,136) (Fig. 4A). Anti-Pvs25 titers were more strongly inducedby immunization with BDES-Pvs25-gp64 (396,429 � 233,426)and BDES-Pvs25-PvCSP-G (500,571 � 236,705) (Fig. 4B). Theresults indicate that higher expression levels of the Pvs25-PvCSPfusion protein both in viral particles and host cells could enhancehumoral responses. Analysis of the IgG subclasses revealed thatBDES-Pvs25-PvCSP-G immune sera contained predominantlyIgG1 and IgG2a against both PvCSP(Sal) and Pvs25 (Fig. 5). The

IgG1/IgG2a ratios for PvCSP(Sal) and Pvs25 were approximately0.8 and 0.6, respectively, indicating that activation of Th1- andTh2-type cells occurred during antigen presentation.

Evaluating the protective and TB efficacies of BDES-Pvs25-PvCSP-G using transgenic parasites. Fig. 6 shows an evaluationscheme for determining the protective and TB efficacies of BDES-Pvs25-PvCSP-G using transgenic parasites in a murine model. Toevaluate the protective efficacy of the vaccine, a transgenic P. ber-ghei line expressing the full-length PvCSP(Sal) in place of nativePbCSP [PvCSP(Sal)/Pb] was generated. Genomic integration ofthe PvCSP(Sal) gene was confirmed by PCR using gene-specificprimers (see Fig. S3A in the supplemental material). The Westernblotting and IFA data showed that PvCSP is expressed on thesurface of the sporozoites isolated from the salivary glands ofPvCSP(Sal)/Pb-infected mosquitoes (see Fig. S3B and C). In themosquitoes, the levels of infectivity (numbers of oocysts per mos-quito) and the rates of infectivity (percentages of infected mosqui-toes) of the WT-Pb and PvCSP(Sal)/Pb lines were comparable{58.5 � 6.8 oocysts/midgut [WT] versus 60.4 � 6.6 [PvCSP(Sal)/Pb]; 95% [WT-Pb] versus 95% [PvCSP(Sal)/Pb] infectivity; 54and 60 mosquitoes were infected with WT-Pb and PvCSP(Sal)/Pb, respectively} (see Fig. S3D). Additionally, Table 1 shows that34 of 35 mice in the control groups (PBS and AcNPV-WT) be-came infected following the bites of three to seven PvCSP(Sal)/Pb-infected mosquitoes, indicating that the infectivity of PvCSP-(Sal)/Pb sporozoites was comparable to that of the WT-Pb line inmice. To evaluate the TB efficacy of the vaccine, a transgenic P.berghei line expressing Pvs25 in place of native Pbs25 and Pbs28(Pvs25DR3) that we generated previously was used (17).

Evaluation of protective efficacy. We examined the ability ofBDES vaccines to protect mice against challenge with PvCSP-

FIG 4 Ab responses to PvCSP and Pvs25. Sera were collected from mice immunized with BDES-Pvs25-PvCSP-G (n � 36), BDES-PvCSP-G (n � 30),BDES-PvCSP-gp64 (n � 30), and BDES-Pvs25-gp64 (n � 6) after the third immunization. Ab titers specific for PvCSP(Sal) (A), PvCSP(PNG) repeat (A), andPvs25 (B) were determined by ELISA. Data are the means � standard deviations (SD) of the results. Significant differences in each immunization group wereevaluated using Student’s t test. *, P � 0.01 (BDES-Pvs25-PvCSP-G versus BDES-PvCSP-G or BDES-PvCSP-gp64); **, P � 0.05 (BDES-Pvs25-PvCSP versusBDES-PvCSP-G and BDES-Pvs25-PvCSP-G versus BDES-Pvs25-gp64); NS, not significant (BDES-Pvs25-PvCSP-G versus BDES-PvCSP-gp64).

FIG 5 Ab isotype responses induced by BDES-Pvs25-PvCSP-G. Mice were immunized with BDES-Pvs25-PvCSP-G (n � 17). Individual sera after the thirdimmunization were tested for total IgG, IgG1, IgG2a, IgG2b, and IgG3 specific for PvCSP(Sal) (A) or Pvs25 (B) using ELISA. Data are means � SD of the results.

Baculovirus-Based Multistage Plasmodium vivax Vaccine

October 2014 Volume 82 Number 10 iai.asm.org 4353

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

(Sal)/Pb (Fig. 6A). Immunized mice were challenged by the bitesof PvCSP(Sal)/Pb-infected mosquitoes, and the prevalence ofsubsequent blood-stage infections was monitored for a 2-weekperiod. Cumulative data from independent experiments areshown in Table 1. Significant protection was observed in the miceimmunized with BDES-Pvs25-PvCSP-G (43%), but not BDES-PvCSP-G (12%) or BDES-PvCSP-gp64 (3%), compared to thecontrol mice. BDES-Pvs25-PvCSP-G vaccination provided a sig-nificant reduction in parasitemia on days 5 and 7 postchallengewhen mice were infected with the transgenic parasites (Table 1).BDES-Pvs25-PvCSP-G improved the protective efficacy againstPvCSP(Sal)/Pb compared to the results seen with other monova-lent vaccines. On day 7 postinfection, there was no significantcorrelation between anti-PvCSP(Sal) Ab titers and parasitemia orbetween anti-PvCSP(Sal) titers and protection (see Fig. S4 in thesupplemental material).

Evaluation of in vivo TB activity. In the active-immunizationstudies, groups of mice (n � 3) immunized with BDES vaccineswere infected with Pvs25DR3-pRBCs or WT-Pb-pRBCs, follow-ing which direct-feeding assays were performed on individualmice (Fig. 6B). Groups of approximately 50 mosquitoes were al-lowed to feed on each mouse, and at day 10 postfeeding, the mos-quito midguts were dissected, and the oocyst numbers werecounted. There was no significant difference between vaccinegroups and the AcNPV-WT group with respect to oocyst meanintensity and prevalence in the mosquitoes infected with WT-Pb,indicating that anti-Pvs25 Abs had no effect on WT-Pb (Fig. 7Aand Table 2). In contrast, BDES-Pvs25-PvCSP-G and BDES-Pvs25-gp64 each caused significant reductions in oocyst intensity(Fig. 7B and Table 2), achieving an 84% (BDES-Pvs25-PvCSP-G)and a 56% (BDES-Pvs25-gp64) inhibition of infection intensity(Fig. 7A and Table 2). Oocyst prevalences were similarly reduced(82% and 60%, respectively). Pvs25DR3 has (previously reported)reduced infectivity compared with WT-Pb (23), although theoocyst numbers produced by Pvs25DR3 were sufficient to mea-sure significant TB efficacy (17). BDES-Pvs25-PvCSP-G had TBefficacies (mean TB intensity and mean TB prevalence) signifi-cantly higher than those of BDES-Pvs25-gp64 vaccine. There wasno significant correlation between anti-Pvs25 Ab titers and TBefficacies (mean TB intensity and mean TB prevalence) (see Fig. S5in the supplemental material).

DISCUSSION

Malaria multistage vaccines that include sexual-stage antigens forblocking parasite transmission are of particular importance forthe control of P. vivax, as there is great difficulty in reducing par-asite transmission because hypnozoite development in the liverand the early formation of gametocytes in this species allow trans-mission to occur before the onset of clinical symptoms in the host(26). The present study showed for the first time that the P. vivaxmultistage vaccine based on BDES can elicit both protective andTB efficacies in a murine model.

FIG 6 Scheme for evaluation of BDES-Pvs25-PvCSP-G efficacy using transgenic parasites. (A) Evaluation of protective efficacy. Groups of mice were immunizedwith BDES-Pvs25-PvCSP-G (bivalent), BDES-PvCSP-G (monovalent), or BDES-PvCSP-gp64 (monovalent) three times and then challenged by bites fromPvCSP(Sal)/Pb-infected mosquitoes (3 � mosquito bites � 7). The protective efficacy of vaccination was evaluated by the levels of sterile protection andparasitemia. (B) Evaluation of TB efficacy. Groups of mice were immunized with BDES-Pvs25-PvCSP-G (bivalent) or BDES-Pvs25-gp64 (monovalent) threetimes and then infected with Pvs25DR3-pRBCs or WT-Pb-pRBCs. Approximate 50 mosquitoes were allowed to feed on each mouse. At day 10 postfeeding,mosquito midguts were dissected, and the oocyst numbers were counted. TB efficacy was evaluated by oocyst intensity and prevalence. The detailed proceduresare described in Materials and Methods.

TABLE 1 Protection of mice immunized with BDES vaccines againstchallenge by the bites of PvCSP(Sal)/Pb parasite-infected mosquitoesa

Vaccine

No. of protectedmice/total no. ofmice (%)b

% of parasitemia � SDc

Day 5 Day 7

PBS 0/10 (0) 0.12 � 0.13 1.69 � 1.23AcNPV-WT 1/25 (4) 0.05 � 0.08 1.55 � 0.08BDES-PvCSP-gp64 1/29 (3) 0.11 � 0.16 1.76 � 0.96BDES-PvCSP-G 3/26 (12) 0.03 � 0.05 1.05 � 1.04BDES-Pvs25-PvCSP-G 13/30 (43)d 0.01 � 0.02e 0.77 � 0.71f

a Cumulative data from three independent experiments.b “Protected” was defined as the complete absence of blood-stage parasitemia on day 14postchallenge.c Giemsa-stained thin smears of tail blood were prepared after challenge. Data are themeans � standard deviations (SD) of the results.d P � 0.05 (Fisher’s exact probability test) compared to the other vaccine/controlgroups.e P � 0.05 (Student’s t test) compared to the other vaccine/control groups.f P � 0.05 (Student’s t test) compared to the control group (PBS and AcNPV-WT) orBDES-PvCSP-gp64 results.

Mizutani et al.

4354 iai.asm.org Infection and Immunity

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

Our strategy for evaluation of the BDES multistage vaccineused appropriately designed and phenotyped transgenic rodentmalaria parasites for assessing murine immune responses to indi-vidual antigenic targets from a human malaria parasite. We andother groups have developed several transgenic rodent malariaparasites expressing human malaria antigens (e.g., PfCSP, PfMSP-119, Pfs25, and Pvs25) to evaluate the immunogenicity and/orprotective efficacy of vaccines (13, 15–17, 27–30). Here, we de-scribe the successful development of PvCSP [PvCSP(Sal)/Pb]transgenic P. berghei parasite expressing the full-length PvCSP-(Sal) in place of the natural P. berghei counterpart. The infectivitylevels of PvCSP(Sal)/Pb from mosquitoes to mice and vice versaare comparable to those of WT-Pb. The availability of transgenicparasites expressing vaccine target antigens from the three major

life stages of the parasite (sporozoite stage, blood stage, and sexualstage) would provide invaluable tools for evaluating the func-tional in vivo efficacy of vaccine-induced immune responses andidentifying protective epitopes.

BDES-Pvs25-PvCSP-G conferred more protective efficacy(43%) than BDES-PvCSP-G (12%), as evaluated by natural infec-tion from the bites of PvCSP(Sal)/Pb-infected mosquitoes (Table1). Recently, two groups have reported that passive immunizationof the MAb-specific CSP repeat region was able to reduce liver-stage burden when mice were challenged by the bites of infectedmosquitoes using transgenic parasites (30, 31), indicating that Absprovide effective protection against a natural infection. One po-tential reason why there was no significant correlation betweenthe anti-PvCSP Ab titers and the protective efficacy among indi-

FIG 7 TB efficacy against Pvs25DR3 by active immunization. Individual mice were immunized three times with BDES-Pvs25-gp64 (n � 6) or BDES-Pvs25-PvCSP-G (n � 6). Control mice were administered AcNPV-WT (n � 6). Three groups of immunized mice were subdivided into two groups, each containingthree mice. Each group of mice was infected with WT-Pb (n � 3) (A) or with Pvs25DR3 (n � 3) (B). Mosquitoes were allowed to feed on the infected mice bya direct-feeding assay. At day 10 postfeeding, mosquito midguts were dissected, and oocyst intensity and prevalence were determined. Data points representmosquitoes that fed on individual mice. x axis points represent individual mice, immunized with the corresponding regimen. Mouse 1 in the WT-Pb-infectedcontrol group died prior to challenge. Horizontal lines indicate the mean numbers of oocysts observed (� standard errors of the means [SEM]). Statisticalsignificance was determined by the Mann-Whitney U test (oocyst intensity) or the Fisher’s exact probability test (infection prevalence) (Table 2).

TABLE 2 Evaluation of transmission-blocking activity by active immunizationa

VaccineChallengeparasite

Mean intensity(oocysts permidgut) (�SEM)

Mean prevalence(% mosquitoesinfected) (�SEM)

Mean TBintensity (%)b

Mean TBprevalence (%)c

BDES-Pvs25-PvCSP-G WT-Pb 44.83 (3.20) 92.44 (0.52)Pvs25DR3 0.16 (0.07) 7.79 (2.65) 84*,** 82†,††

BDES-Pvs25-gp64 WT-Pb 40.14 (3.08) 90.19 (3.91)Pvs25DR3 0.48 (0.16) 17.27 (3.23) 56* 60†

AcNPV-WT WT-Pb 44.19 (4.65) 87.80 (0.30)Pvs25DR3 1.00 (0.13) 43.63 (1.84)

a Significance was assessed using the Mann-Whitney U test (to examine the difference in oocyst counts per mosquito) and the Fisher’s exact probability test (to examine thedifference in infection prevalences). *, P � 0.05 (Mann-Whitney U test) compared to AcNPV-WT; **, P � 0.05 (Mann-Whitney U test) compared to BDES-Pvs25-gp64; †, P �0.05 (Fisher’s exact probability test) compared to AcNPV-WT; ††, P � 0.05 (Fisher’s exact probability test) compared to BDES-Pvs25-gp64.b Percent mean TB intensity was calculated by comparison with AcNPV-WT.c Percent mean TB prevalence was calculated by comparison with AcNPV-WT.

Baculovirus-Based Multistage Plasmodium vivax Vaccine

October 2014 Volume 82 Number 10 iai.asm.org 4355

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

vidual mice may be the highly differing numbers of sporozoites(ranging from 0 to 1,297 per bite) injected into the mice followingthe bites of infected mosquitoes (32). Technologies to examine thenumber of sporozoites injected into an individual by mosquitobite are not currently at the level of throughput that would allowutilization in a study of this scope.

Another reason is that cellular immune responses to PvCSPmay play a critical role in the protective efficacy. It is known thatboth humoral immunity and cellular immunity to the pre-eryth-rocytic parasite stages can provide protection against sporozoiteinfection (33). In the case of PvCSP, however, there is no cytotoxicT lymphocyte (CTL) epitope on the PvCSP sequence capable ofrecognition by C57BL/6 (H-2Kb) mice (34). Similarly, the BALB/c(H-2Kd) mice we used here may not be able to recognize the CTLepitope on PvCSP. Therefore, anti-PvCSP Abs induced by BDES-Pvs25-PvCSP-G could theoretically make a significant contribu-tion to protection against infections naturally transmitted bysporozoites, although we cannot fully discount the possibility of arole for cellular immune responses in immune protection, asbaculoviral vectors have the potential to induce CTLs via stimu-lation by innate immunity (12).

Compared with a single-stage antigen-based vaccine, a multi-stage and/or multiantigen malaria vaccine should provide moreefficacious protection for individuals and a TB effect that de-creases the burden of infection on individual communities. How-ever, a major concern for the development of multivalent vaccinesis the potential for vaccine interference which would be associatedwith poor immune responses (35). In fact, it has been reportedthat immune responses to the individual components of multian-tigen malaria vaccines can potentially be suppressed by immuneinterference (36–43). The BDES-Pvs25-PvCSP-G vaccine success-fully induced high Pvs25-, PvCSP(Sal)-, and PvCSP(PNG) repeat-specific Ab titers, suggesting an absence of immune interference.Interestingly, the bivalent BDES-Pvs25-PvCSP-G induced signif-icantly higher anti-Pvs25 Ab titers and TB activity than the mon-ovalent BDES-Pvs25-gp64, suggesting the possibility that Pvs25can stabilize PvCSP to facilitate the development of P. vivax mul-tistage vaccines.

In summary, the present report is the first step toward devel-oping malaria multistage vaccines capable of preventing malariainfections in individuals and the spread of parasites in communi-ties, while further studies are needed to elucidate the mechanismof the vaccine effects and subsequently improve their effects bysimultaneously expressing multiple antigens from different stagesof the parasite life cycle. Our recent study showed that the BDESvaccine is safe and well tolerated with acceptable reactogenicityand systemic toxicity in a primate model (13). BDES vaccines offera promising new alternative to current human malaria vaccinedelivery platforms for first-in-human clinical trials.

ACKNOWLEDGMENTS

We thank Mami Koketsu (Obihiro University of Agriculture and Veteri-nary Medicine) for excellent assistance with Giemsa-stained thin bloodsmears and mouse handling and Mark Tunnicliff (Imperial College, Lon-don) for mosquito rearing. We also thank Takafumi Tusboi for providingpEU3-PvCSP(Sal) and pEU3-PvCSP(PNG) plasmids and the MR4 forproviding Abs against P. vivax proteins.

This work was supported in part by Grants-in-Aid for Scientific Re-search (B) 21390126 from the Japan Society for the Promotion of Science(JSPS) and Japan-UK Joint Research Projects of JSPS Bilateral Programs(7301001475) to S.Y. and awards from the PATH Malaria Vaccine Initia-

tive, Medicines for Malaria Venture, and the Fraunhofer Institute forA.M.B.

REFERENCES1. Guerra CA, Howes RE, Patil AP, Gething PW, Van Boeckel TP, Tem-

perley WH, Kabaria CW, Tatem AJ, Manh BH, Elyazar IR, Baird JK,Snow RW, Hay SI. 2010. The international limits and population at riskof Plasmodium vivax transmission in 2009. PLoS Negl. Trop. Dis. 4:e774.http://dx.doi.org/10.1371/journal.pntd.0000774.

2. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, SnowRW. 2010. Estimating the global clinical burden of Plasmodium falcipa-rum malaria in 2007. PLoS Med. 7:e1000290. http://dx.doi.org/10.1371/journal.pmed.1000290.

3. Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM. 2007.Vivax malaria: neglected and not benign. Am. J. Trop. Med. Hyg.77(Suppl):79 – 87.

4. Kochar DK, Das A, Kochar SK, Saxena V, Sirohi P, Garg S, Kochar A,Khatri MP, Gupta V. 2009. Severe Plasmodium vivax malaria: a report onserial cases from Bikaner in northwestern India. Am. J. Trop. Med. Hyg.80:194 –198.

5. Sina B. 2002. Focus on Plasmodium vivax. Trends Parasitol. 18:287–289.http://dx.doi.org/10.1016/S1471-4922(02)02329-2.

6. Picot S. 2006. Is Plasmodium vivax still a paradigm for uncomplicatedmalaria? Med. Mal. Infect. 36:406 – 413. (In French.) http://dx.doi.org/10.1016/j.medmal.2006.06.001.

7. Herrera S, Corradin G, Arevalo-Herrera M. 2007. An update on thesearch for a Plasmodium vivax vaccine. Trends Parasitol. 23:122–128. http://dx.doi.org/10.1016/j.pt.2007.01.008.

8. Carlton JM, Sina BJ, Adams JH. 2011. Why is Plasmodium vivax aneglected tropical disease? PLoS Negl. Trop. Dis. 5:e1160. http://dx.doi.org/10.1371/journal.pntd.0001160.

9. Hoffman SL, Miller LH. 1996. Perspectives on malaria vaccine develop-ment, p 1–12. In Hoffman SL (ed), Malaria vaccine development: a multi-immune response approach. American Society for Microbiology, Wash-ington, DC.

10. Shi YP, Das P, Holloway B, Udhayakumar V, Tongren JE, Candal F,Biswas S, Ahmad R, Hasnain SE, Lal AA. 2000. Development, expression,and murine testing of a multistage Plasmodium falciparum malaria vaccinecandidate. Vaccine 18:2902–2914. http://dx.doi.org/10.1016/S0264-410X(00)00045-1.

11. Theisen M, Roeffen W, Singh SK, Andersen G, Amoah L, van deVegte-Bolmer M, Arens T, Tiendrebeogo RW, Jones S, Bousema T, AduB, Dziegiel MH, Christiansen M, Sauerwein R. 2014. A multi-stagemalaria vaccine candidate targeting both transmission and asexual para-site life-cycle stages. Vaccine 32:2623–2630. http://dx.doi.org/10.1016/j.vaccine.2014.03.020.

12. Yoshida S, Kawasaki M, Hariguchi N, Hirota K, Matsumoto M. 2009.A baculovirus dual expression system-based malaria vaccine inducesstrong protection against Plasmodium berghei sporozoite challenge inmice. Infect. Immun. 77:1782–1789. http://dx.doi.org/10.1128/IAI.01226-08.

13. Iyori M, Nakaya H, Inagaki K, Pichyangkul S, Yamamoto DS, KawasakiM, Kwak K, Mizukoshi M, Goto Y, Matsuoka H, Matsumoto M,Yoshida S. 2013. Protective efficacy of baculovirus dual expression systemvaccine expressing Plasmodium falciparum circumsporozoite protein.PLoS One 8:e70819. http://dx.doi.org/10.1371/journal.pone.0070819.

14. Yoshida S, Araki H, Yokomine T. 2010. Baculovirus-based nasal dropvaccine confers complete protection against malaria by natural boostingof vaccine-induced antibodies in mice. Infect. Immun. 78:595– 602. http://dx.doi.org/10.1128/IAI.00877-09.

15. Yoshida S, Nagumo H, Yokomine T, Araki H, Suzuki A, Matsuoka H.2010. Plasmodium berghei circumvents immune responses induced bymerozoite surface protein 1- and apical membrane antigen 1-basedvaccines. PLoS One 5:e13727. http://dx.doi.org/10.1371/journal.pone.0013727.

16. Mlambo G, Kumar N, Yoshida S. 2010. Functional immunogenicity ofbaculovirus expressing Pfs25, a human malaria transmission-blockingvaccine candidate antigen. Vaccine 28:7025–7029. http://dx.doi.org/10.1016/j.vaccine.2010.08.022.

17. Blagborough AM, Yoshida S, Sattabongkot J, Tsuboi T, Sinden RE.2010. Intranasal and intramuscular immunization with Baculovirus DualExpression System-based Pvs25 vaccine substantially blocks Plasmodium

Mizutani et al.

4356 iai.asm.org Infection and Immunity

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

vivax transmission. Vaccine 28:6014 – 6020. http://dx.doi.org/10.1016/j.vaccine.2010.06.100.

18. Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Methogo BG, Kab-wende AL, Adegnika AA, Mordmuller B, Issifou S, Kremsner PG,Sacarlal J, Aide P, Lanaspa M, Aponte JJ, Machevo S, Acacio S, Bulo H,Sigauque B, Macete E, Alonso P, Abdulla S, Salim N, Minja R, MpinaM, Ahmed S, Ali AM, Mtoro AT, Hamad AS, Mutani P, Tanner M,Tinto H, D’Alessandro U, Sorgho H, Valea I, Bihoun B, Guiraud I,Kabore B, Sombie O, Guiguemde RT, Ouedraogo JB, Hamel MJ,Kariuki S, Oneko M, Odero C, Otieno K, Awino N, McMorrow M,Muturi-Kioi V, Laserson KF, Slutsker L, et al. 2012. A phase 3 trial ofRTS,S/AS01 malaria vaccine in African infants. N. Engl. J. Med. 367:2284 –2295. http://dx.doi.org/10.1056/NEJMoa1208394.

19. Hisaeda H, Collins WE, Saul A, Stowers AW. 2001. Antibodies toPlasmodium vivax transmission-blocking vaccine candidate antigensPvs25 and Pvs28 do not show synergism. Vaccine 20:763–770. http://dx.doi.org/10.1016/S0264-410X(01)00402-9.

20. Peiris JS, Premawansa S, Ranawaka MB, Udagama PV, MunasingheYD, Nanayakkara MV, Gamage CP, Carter R, David PH, Mendis KN.1988. Monoclonal and polyclonal antibodies both block and enhancetransmission of human Plasmodium vivax malaria. Am. J. Trop. Med.Hyg. 39:26 –32.

21. Carter R. 2001. Transmission blocking malaria vaccines. Vaccine 19:2309 –2314. http://dx.doi.org/10.1016/S0264-410X(00)00521-1.

22. Sumitani M, Kasashima K, Yamamoto DS, Yagi K, Yuda M, MatsuokaH, Yoshida S. 2013. Reduction of malaria transmission by transgenicmosquitoes expressing an antisporozoite antibody in their salivary glands.Insect Mol. Biol. 22:41–51. http://dx.doi.org/10.1111/j.1365-2583.2012.01168.x.

23. Ramjanee S, Robertson JS, Franke-Fayard B, Sinha R, Waters AP, JanseCJ, Wu Y, Blagborough AM, Saul A, Sinden RE. 2007. The use oftransgenic Plasmodium berghei expressing the Plasmodium vivax antigenP25 to determine the transmission-blocking activity of sera from malariavaccine trials. Vaccine 25:886 – 894. http://dx.doi.org/10.1016/j.vaccine.2006.09.035.

24. Lim CS, Tazi L, Ayala FJ. 2005. Plasmodium vivax: recent world expan-sion and genetic identity to Plasmodium simium. Proc. Natl. Acad. Sci.U. S. A. 102:15523–15528. http://dx.doi.org/10.1073/pnas.0507413102.

25. Zhou J, Blissard GW. 2008. Display of heterologous proteins on gp64nullbaculovirus virions and enhanced budding mediated by a vesicular sto-matitis virus G-stem construct. J. Virol. 82:1368 –1377. http://dx.doi.org/10.1128/JVI.02007-07.

26. malERA Consultative Group on Vaccines. 2011. A research agenda formalaria eradication: vaccines. PLoS Med. 8:e1000398. http://dx.doi.org/10.1371/journal.pmed.1000398.

27. Goodman AL, Blagborough AM, Biswas S, Wu Y, Hill AV, Sinden RE,Draper SJ. 2011. A viral vectored prime-boost immunization regime tar-geting the malaria pfs25 antigen induces transmission-blocking activity.PLoS One 6:e29428. http://dx.doi.org/10.1371/journal.pone.0029428.

28. Bauza K, Malinauskas T, Pfander C, Anar B, Jones EY, Billker O, HillAV, Reyes-Sandoval A. 2014. Efficacy of a Plasmodium vivax malariavaccine using ChAd63 and modified vaccinia Ankara expressing throm-bospondin-related anonymous protein as assessed with transgenic Plas-modium berghei parasites. Infect. Immun. 82:1277–1286. http://dx.doi.org/10.1128/IAI.01187-13.

29. Persson C, Oliveira GA, Sultan AA, Bhanot P, Nussenzweig V, NardinE. 2002. Cutting edge: a new tool to evaluate human pre-erythrocyticmalaria vaccines: rodent parasites bearing a hybrid Plasmodium falcipa-rum circumsporozoite protein. J. Immunol. 169:6681– 6685. http://dx.doi.org/10.4049/jimmunol.169.12.6681.

30. Espinosa DA, Yadava A, Angov E, Maurizio PL, Ockenhouse CF, ZavalaF. 2013. Development of a chimeric Plasmodium berghei strain expressingthe repeat region of the P. vivax circumsporozoite protein for in vivoevaluation of vaccine efficacy. Infect. Immun. 81:2882–2887. http://dx.doi.org/10.1128/IAI.00461-13.

31. Sack BK, Miller JL, Vaughan AM, Douglass A, Kaushansky A, Miko-

lajczak S, Coppi A, Gonzalez-Aseguinolaza G, Tsuji M, Zavala F, SinnisP, Kappe SH. 2014. Model for in vivo assessment of humoral protectionagainst malaria sporozoite challenge by passive transfer of monoclonalantibodies and immune serum. Infect. Immun. 82:808 – 817. http://dx.doi.org/10.1128/IAI.01249-13.

32. Medica DL, Sinnis P. 2005. Quantitative dynamics of Plasmodium yoeliisporozoite transmission by infected anopheline mosquitoes. Infect. Im-mun. 73:4363– 4369. http://dx.doi.org/10.1128/IAI.73.7.4363-4369.2005.

33. Vaughan AM, Kappe SH. 2012. Malaria vaccine development: persistentchallenges. Curr. Opin. Immunol. 24:324 –331. http://dx.doi.org/10.1016/j.coi.2012.03.009.

34. Teixeira LH, Tararam CA, Lasaro MO, Camacho AG, Ersching J,Leal MT, Herrera S, Bruna-Romero O, Soares IS, Nussenzweig RS,Ertl HC, Nussenzweig V, Rodrigues MM. 2014. Immunogenicity of aprime-boost vaccine containing the circumsporozoite proteins of Plas-modium vivax in rodents. Infect. Immun. 82:793– 807. http://dx.doi.org/10.1128/IAI.01410-13.

35. Vidor E. 2007. The nature and consequences of intra- and inter-vaccineinterference. J. Comp. Pathol. 137(Suppl 1):S62–S66. http://dx.doi.org/10.1016/j.jcpa.2007.04.014.

36. Rogers WO, Weiss WR, Kumar A, Aguiar JC, Tine JA, Gwadz R, HarreJG, Gowda K, Rathore D, Kumar S, Hoffman SL. 2002. Protection ofrhesus macaques against lethal Plasmodium knowlesi malaria by a heterol-ogous DNA priming and poxvirus boosting immunization regimen. In-fect. Immun. 70:4329 – 4335. http://dx.doi.org/10.1128/IAI.70.8.4329-4335.2002.

37. Zhou Z, Todd CW, Wohlhueter RM, Price A, Xiao L, Schnake P,Bonner PC, Martin AM, Goldman IF, De La Vega P, Udhayakumar V,Lal AA. 2006. Development, characterization and immunogenicity of amulti-stage, multi-valent Plasmodium falciparum vaccine antigen (FAL-VAC-1A) expressed in Escherichia coli. Hum. Vaccin. 2:14 –23. http://dx.doi.org/10.4161/hv.2.1.2437.

38. Sedegah M, Charoenvit Y, Minh L, Belmonte M, Majam VF, Abot S,Ganeshan H, Kumar S, Bacon DJ, Stowers A, Narum DL, Carucci DJ,Rogers WO. 2004. Reduced immunogenicity of DNA vaccine plasmids inmixtures. Gene Ther. 11:448–456. http://dx.doi.org/10.1038/sj.gt.3302139.

39. Jiang G, Charoenvit Y, Moreno A, Baraceros MF, Banania G, Richie N,Abot S, Ganeshan H, Fallarme V, Patterson NB, Geall A, Weiss WR,Strobert E, Caro-Aquilar I, Lanar DE, Saul A, Martin LB, Gowda K,Morrissette CR, Kaslow DC, Carucci DJ, Galinski MR, Doolan DL.2007. Induction of multi-antigen multi-stage immune responses againstPlasmodium falciparum in rhesus monkeys, in the absence of antigen in-terference, with heterologous DNA prime/poxvirus boost immunization.Malar. J. 6:135. http://dx.doi.org/10.1186/1475-2875-6-135.

40. Jiang G, Shi M, Conteh S, Richie N, Banania G, Geneshan H, ValenciaA, Singh P, Aguiar J, Limbach K, Kamrud KI, Rayner J, Smith J, BruderJT, King CR, Tsuboi T, Takeo S, Endo Y, Doolan DL, Richie TL, WeissWR. 2009. Sterile protection against Plasmodium knowlesi in rhesus mon-keys from a malaria vaccine: comparison of heterologous prime booststrategies. PLoS One 4:e6559. http://dx.doi.org/10.1371/journal.pone.0006559.

41. Wang R, Richie TL, Baraceros MF, Rahardjo N, Gay T, Banania JG,Charoenvit Y, Epstein JE, Luke T, Freilich DA, Norman J, Hoffman SL.2005. Boosting of DNA vaccine-elicited gamma interferon responses inhumans by exposure to malaria parasites. Infect. Immun. 73:2863–2872.http://dx.doi.org/10.1128/IAI.73.5.2863-2872.2005.

42. Sedegah M, Charoenvit Y, Aguiar J, Sacci J, Hedstrom R, Kumar S,Belmonte A, Lanar DE, Jones TR, Abot E, Druilhe P, Corradin G,Epstein JE, Richie TL, Carucci DJ, Hoffman SL. 2004. Effect on antibodyand T-cell responses of mixing five GMP-produced DNA plasmids andadministration with plasmid expressing GM-CSF. Genes Immun. 5:553–561. http://dx.doi.org/10.1038/sj.gene.6364125.

43. Forbes EK, Biswas S, Collins KA, Gilbert SC, Hill AV, Draper SJ. 2011.Combining liver- and blood-stage malaria viral-vectored vaccines: inves-tigating mechanisms of CD8� T cell interference. J. Immunol. 187:3738 –3750. http://dx.doi.org/10.4049/jimmunol.1003783.

Baculovirus-Based Multistage Plasmodium vivax Vaccine

October 2014 Volume 82 Number 10 iai.asm.org 4357

on June 22, 2020 by guesthttp://iai.asm

.org/D

ownloaded from