Protective Humoral Immunity Elicited by a Needle-Free MalariaVaccine Comprised of a Chimeric Plasmodium falciparumCircumsporozoite Protein and a Toll-Like Receptor 5 Agonist,Flagellin

Daniel Carapau,a* Robert Mitchell,a Adéla Nacer,a* Alan Shaw,b* Caroline Othoro,a Ute Frevert,a Elizabeth Nardina

Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine, New York, New York, USAa; Vaxinnate Corporation, Cranbury, NewJersey, USAb

Immunization with Plasmodium sporozoites can elicit high levels of sterile immunity, and neutralizing antibodies from pro-tected hosts are known to target the repeat region of the circumsporozoite (CS) protein on the parasite surface. CS-based subunitvaccines have been hampered by suboptimal immunogenicity and the requirement for strong adjuvants to elicit effective hu-moral immunity. Pathogen-associated molecular patterns (PAMPs) that signal through Toll-like receptors (TLRs) can functionas potent adjuvants for innate and adaptive immunity. We examined the immunogenicity of recombinant proteins containing aTLR5 agonist, flagellin, and either full-length or selected epitopes of the Plasmodium falciparum CS protein. Mice immunizedwith either of the flagellin-modified CS constructs, administered intranasally (i.n.) or subcutaneously (s.c.), developed similarlevels of malaria-specific IgG1 antibody and interleukin-5 (IL-5)-producing T cells. Importantly, immunization via the i.n. butnot the s.c. route elicited sporozoite neutralizing antibodies capable of inhibiting >90% of sporozoite invasion in vitro and invivo, as measured using a transgenic rodent parasite expressing P. falciparum CS repeats. These findings demonstrate that func-tional sporozoite neutralizing antibody can be elicited by i.n. immunization with a flagellin-modified P. falciparum CS proteinand raise the potential of a scalable, safe, needle-free vaccine for the 40% of the world’s population at risk of malaria.

Plasmodium sporozoite vaccine studies in the 1960s and ’70sprovided the first demonstration that sterile immunity for ma-

laria could be elicited in murine models and in human volunteers(1–4). Immune sera from the protected hosts identified the cir-cumsporozoite (CS) protein covering the sporozoite surface as thetarget of antibody-mediated immunity (5, 6), and more recentstudies have confirmed the CS protein as one of the major targetsof protective immunity elicited by sporozoite immunization (7).A recent phase III clinical trial of a Plasmodium falciparum CSsubunit vaccine, termed RTS,S, represents the first malaria vac-cine to reach testing for commercial licensure (8, 9). The resultsdemonstrate a reduction of clinical disease in 31% of neonatesaged 6 to 12 weeks and in 56% of infants aged 5 to 17 months.While the endpoint in these trials was clinical disease, these resultssupport efforts to develop more efficacious second-generationmalaria vaccines to elicit sterile immunity for individuals living inareas where malaria is endemic, as well as for travelers to thoseregions.

A critical factor in the development of subunit vaccines is theneed for strong adjuvants to stimulate innate immune responsesrequired to initiate adaptive cellular and humoral immunity. Im-munogenicity of the RTS,S vaccine is adjuvant dependent, and aformulation in alum, the aluminum adjuvant present in most li-censed vaccines, elicited suboptimal protection against sporozoitechallenge (10, 11). The current RTS,S adjuvant formulation wasderived empirically and is comprised of a mixture of monophos-phoryl lipid A (MPL) and QS21, a purified fraction of the deter-gent saponin, in an oil-in-water emulsion (AS02) or a liposome(AS01) formulation (12, 13).

In contrast to empirically defined adjuvants which lack amechanistic rationale, pathogen-associated molecular patterns

(PAMPs) provide well-defined protein, lipid, or other molecularmoieties that are known to stimulate cytokine/chemokine pro-duction by innate immune cells (14). A number of natural andchemically synthesized Toll-like receptor (TLR) agonists, such asbacterial cell wall lipopeptides and CpG motifs of bacterial DNA,have been used as adjuvants (15, 16). Bacterial flagellin, an agonistof TLR5, is one of the limited number of protein PAMPs (17) andthus can be expressed readily in Escherichia coli, using standardscale-up and quality control methods, for production of low-costvaccines for developing countries. Multiple studies have demon-strated the adjuvant properties of flagellin for enhancing T and Bcell responses to bacterial, viral, and parasite antigens (18–23).Recent phase I trials demonstrated safety and immunogenicity ofan influenza vaccine produced by VaxInnate, Inc., which is com-prised of a recombinant fusion protein of flagellin and the globu-

Received 1 March 2013 Returned for modification 21 March 2013Accepted 5 September 2013

Published ahead of print 16 September 2013

Editor: J. H. Adams

Address correspondence to Elizabeth Nardin, elizabeth.nardin@nyumc.org.

* Present address: Daniel Carapau, Instituto de Medicina Molecular, Unidade deMalária, Lisbon, Portugal; Adéla Nacer, Unité de Biologie des Interactions Hôte-Parasite, Institut Pasteur, Paris, France; Alan Shaw, Vedantra Pharmaceuticals, Inc.,Cambridge, Massachusetts, USA.

D.C. and R.M. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00263-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00263-13

4350 iai.asm.org Infection and Immunity p. 4350 – 4362 December 2013 Volume 81 Number 12

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

lar head of hemagglutinin (HA) (24–26). The clinical trials dem-onstrated induction of virus-neutralizing antibodies in young (18to 49 years) and elderly (�65 years) volunteers following intra-muscular (i.m.) immunization with the flagellin-modified HAvaccine.

In the present study, we examined the immunogenicity andprotective efficacy of a recombinant P. falciparum CS proteinmodified with either a full-length flagellin of Salmonella entericaserovar Typhimurium, i.e., FljB (STF2), or a truncated flagellinprotein (STF2�) in which the hinge region containing immuno-dominant flagellin B cell epitopes was removed. The P. falciparumCS in these constructs was either a nearly full-length CS protein ora triepitope sequence, T1BT*, containing well-defined T and B cellepitopes of P. falciparum CS that are recognized by human andmurine immune cells. Immunization of mice with either flagellin-modified CS construct, administered via the intranasal (i.n.) orsubcutaneous (s.c.) route, elicited systemic malaria-specific im-mune responses of similar magnitudes and specificities. However,significant levels of sporozoite-neutralizing antibodies were elic-ited only by i.n. immunization, with �90% reductions in parasitelevels in sporozoite invasion assays in vitro. Mice immunized withflagellin-modified CS administered i.n., but not s.c., had �90%reductions in liver parasite burdens in vivo following challengewith Plasmodium-infected mosquitoes.

This is the first description of a needle-free flagellin-modifiedP. falciparum CS protein vaccine that can elicit functional sporo-zoite-neutralizing antibodies. Optimization of an i.n. malaria vac-cine would provide significant cost and safety advantages in re-source-poor areas of the world, which experience the majority ofthe 250 to 500 million infections and �1 million deaths each yearcaused by the Plasmodium parasite.

MATERIALS AND METHODSFlagellin-modified P. falciparum circumsporozoite protein. Two typesof flagellin-modified fusion proteins containing the P. falciparum CS pro-tein were expressed in E. coli (Fig. 1). The STF2.(T1BT*) constructs con-tained full-length flagellin from Salmonella enterica serovar Typhimu-rium FljB (STF2) fused to T1BT*, a triepitope malaria sequencerepresenting multiple immunodominant T and B cell epitopes of P. fal-ciparum CS protein that were identified using sera and cells from P. falcipa-rum sporozoite-immunized volunteers (27–29) (Fig. 1B). The T1 and Bepitopes are located in the repeat region of the P. falciparum CS protein (NF54isolate), while the universal T cell epitope, T*, is located in C-terminal aminoacids (aa) 326 to 345 (Fig. 1A). Constructs containing T1BT* as either a singlecopy [STF2.(T1BT*)1X] or four copies [STF2.(T1BT*)4X] were compared forimmunogenicity.

A second type of flagellin-modified protein, STF2�.CS, contained anearly full-length P. falciparum CS that lacked only the N-terminal 23-aasignal sequence and the C-terminal 14 aa, containing the putative glyco-sylphosphatidylinositol (GPI) anchor sequence, fused to the C terminusof a truncated form of the S. enterica serovar Typhimurium flagellin(STF2�) from which the hypervariable hinge region (aa 170 to 415) wasdeleted to reduce the size of the chimeric fusion protein (Fig. 1B). Aflexible linker sequence (GAPVDPASPW) was inserted into the hingeregion of the truncated flagellin protein to facilitate interactions betweenthe N- and C-terminal regions required for binding to TLR5 (30). TheSTF2� truncated flagellin was previously shown to function as a potentTLR5 agonist and adjuvant in murine studies of a West Nile virus vaccine(20).

The E. coli-expressed recombinant proteins were purified by columnchromatography or by use of nickel chelating columns, as previously de-scribed (19–21). Endotoxin levels were quantified using a Limulus amoe-

bocyte lysate (LAL) assay, and all constructs contained �0.01 endotoxinunit (EU)/�g. Western blots and an enzyme-linked immunosorbent assay(ELISA) using monoclonal antibody (MAb) 2A10, specific for P. falcipa-rum CS repeats, were used to confirm the purity and antigenicity of theflagellin-modified recombinant proteins (see Fig. S1 in the supplementalmaterial).

TLR5 agonist activity of the flagellin-modified CS proteins was as-sessed using a murine cell line, RAW 264.7, transfected with human TLR5(hTLR5), as previously described (19). Briefly, hTLR5-transfected RAW264.7 and untransfected RAW 264.7 cells were cultured in 96-well plates at3 � 104 to 5 � 104 cells/well in Dulbecco’s modified Eagle’s medium(DMEM) (Cellgro, Fisher Scientific, Pittsburgh, PA) supplemented with10% fetal calf serum (FCS) (HyClone, Logan, UT). Cells were treated for5 h with serial dilutions of flagellin-modified CS or flagellin only, andlevels of tumor necrosis factor alpha (TNF-�) in the supernatant weremeasured by ELISA (Invitrogen, Carlsbad, CA).

Dendritic cells (DC). (i) Murine DC. Bone marrow-derived dendriticcells (BMDC) were isolated from femurs and tibias of naive BALB/c orC57BL/6 mice by flushing with complete high-glucose DMEM with 10%FCS, penicillin-streptomycin, and -mercaptoethanol. Cells were centri-fuged at 300 � g for 5 min, and the pellet was resuspended in red blood celllysis buffer at room temperature (RT) for 5 min. Washed cells were cul-tured in complete DMEM supplemented with 30% conditioned mediumobtained from an Ag8.653 cell line transfected with the murine granulo-cyte-macrophage colony-stimulating factor (GM-CSF) gene (31), kindlyprovided by A. Rodriguez, New York University. Cells were cultured at37°C and 5% CO2 in complete medium containing GM-CSF, which waschanged every other day.

An immature myeloid DC line, D1, derived from spleens of C57BL/6mice (32, 33), was provided by A. Rodriguez, New York University. Cellswere grown in high-glucose DMEM supplemented with 10% FCS, peni-cillin-streptomycin, and 30% conditioned medium from transfectedAg8.653 cells expressing murine GM-CSF.

(ii) hDC. Human DC (hDC) were derived from monocytes purifiedfrom peripheral blood mononuclear cells (PBMC) following differentia-tion in vitro to myeloid CD11c CD14� DC by stimulation with humaninterleukin-4 (IL-4) and GM-CSF cytokines (R&D Systems, Minneapolis,

FIG 1 (A) Schematic diagram of P. falciparum CS protein showing locationsof the T1 and B cell epitopes within the central repeat region and the universalTh cell epitope, T*, in the C terminus. (B) Schematic diagrams of E. coli-expressed recombinant proteins comprised of full-length flagellin (STF2)combined with P. falciparum T1BT* epitopes as either a single copy[STF2.(T1BT*)1X] or four copies [STF2.(T1BT*)4X]. A second type of con-struct, STF2�.CS, was comprised of a truncated flagellin protein (STF2�) andnearly full-length P. falciparum CS protein.

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4351

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

MN), as previously described (34). The immature DC were cryopreservedand stored at �80°C until used. To obtain hDC for phenotypic assays,thawed cells were incubated overnight and stimulated the following daywith IL-4 and GM-CSF (R&D Systems).

(iii) DC phenotypes. Subset and maturation markers were measuredin a FACSCalibur flow cytometer (Becton, Dickinson, Franklin Lakes,NJ). Cells were preincubated on ice for 30 min with Fc block (CD16/32;BD Biosciences, San Diego, CA) diluted 1:200 in fluorescence-activatedcell sorter (FACS) buffer (3% FCS in phosphate-buffered saline [PBS]).Cells were then incubated on ice for 30 min with a 1:100 dilution offluorophore-conjugated antibodies: CD40-fluorescein isothiocyanate(CD40-FITC) (BD Biosciences), IAd-phycoerythrin (IAd-PE) or IAb-PE(BD Biosciences), CD86-peridinin chlorophyll protein/Cy5.5 (CD86-PerCP/Cy5.5), and CD11c-allophycocyanin (CD11c-APC) (both fromBiolegend, San Diego, CA). For FACS analysis, cells were gated on CD11c,and levels of CD40, major histocompatibility complex class II (MHC II),and CD86 were analyzed with FlowJo software. Controls included cellsstimulated with E. coli K-12-derived lipopolysaccharide (LPS) (InVivo-gen, San Diego, CA) or a maturation-inducing cytokine cocktail, in thecase of hDC.

Uptake of flagellin-modified CS protein in vitro. Murine BMDC orhuman DC were plated into 8-well Permanox Lab-Tek chamber slides at2 � 105 cells per well and incubated with 10 or 100 �g/ml STF2�.CS orSTF2.(T1BT*)4X at 4°C for 30 min to allow synchronized binding, fol-lowed by 30 min or 24 h at 37°C to allow endocytic uptake. Cells were fixedin situ with 4% paraformaldehyde in PBS for 15 min at RT, washed with afixation/permeabilization solution (Biolegend), and incubated in thesame solution for 10 min at RT. Cells were permeabilized with 0.25%Triton X-100 in PBS. The presence of internalized CS protein in perme-abilized, fixed DC was determined by labeling with biotinylated MAb2A10, specific for P. falciparum CS repeats, followed by washing with 6%bovine serum albumin (BSA) in PBS and incubation for 30 min on icewith streptavidin-conjugated 585-nm Qdots (Invitrogen, Life Technolo-gies, Grand Island, NY). Cells were then washed three times with FACSbuffer and acquired in a FACSCalibur flow cytometer, as described above.

Immunization and challenge. Immunogenicity and protective effi-cacy of the flagellin-modified CS proteins were tested in C57BL/6 andBALB/c mice (3 to 6 mice/group) (Jackson Laboratories, Bar Harbor,ME). TLR5 knockout (KO) mice were kindly provided by AndrewGewirtz, Emory University, Atlanta, GA. Mice were housed in AAALAC-approved animal facilities according to NYU School of Medicine IACUC-approved protocols. Mice were immunized s.c. or i.n. with 4 to 6 doses offlagellin-modified CS (10 to 50 �g/dose) at 2-week intervals. For i.n. im-munization, 10 �l was placed in each naris of anesthetized mice. Micewere used at �8 weeks of age, as nasopharynx-associated lymphoid tissue(NALT) organogenesis does not begin until after birth and reaches mat-uration at �8 weeks of age (35). Controls included mice immunized withfull-length or truncated flagellin without CS or with unmodified T1BT*peptide. Sera were collected 14 days after each immunization and stored at�20°C until used.

To test protective efficacy, mice were challenged by exposure to thebites of 5 to 15 mosquitoes infected with PfPb, a transgenic P. bergheirodent parasite expressing P. falciparum CS repeats (36, 37). The mecha-nism of immunity was investigated by depletion of T cells by injection of100 �g anti-CD4 (GK 1.5) and anti-CD8 (2.43) MAbs on three consecu-tive days prior to challenge. Mice were dissected at 40 h postchallenge, andparasite 18S rRNA levels in liver extracts were quantified by real-timequantitative PCR (qPCR) (38). Briefly, livers were placed in TRI reagent(Molecular Research Center, Inc., Cincinnati, OH) and homogenizedwith a PowerGen 125 tissue homogenizer (Fisher Scientific, Pittsburgh,PA). Total RNA was extracted from the homogenate by use of chloro-form-isoamyl alcohol (24:1), the aqueous phase was separated, and RNAwas precipitated with isopropyl alcohol. RNA pellets were washed with70% ethyl alcohol, dried, resuspended in diethyl pyrocarbonate (DEPC)H2O, and dissolved by heating at 44°C for up to 1 h. Total RNA was

converted into cDNA by using reverse transcriptase (Applied Biosystems,Carlsbad, CA), and qPCR was performed using primers specific for the P.berghei 18S rRNA gene and SyBr green (Qiagen, Valencia, CA). The num-ber of 18S rRNA copies in each sample was based on a standard curvegenerated with known amounts of plasmid 18S cDNA. Inhibition of�90% of 18S rRNA copy numbers compared to that in naive challengedmice was considered significant, based on previous studies using intrave-nous injection of known numbers of sporozoites which demonstratedthat a log reduction in sporozoite numbers is required to give a 1-daydelay in the prepatent period (1, 39–41).

Serologic assays. (i) ELISA. IgG titers were determined using 96-wellplates coated with P. falciparum CS repeat peptide (T1B)4, recombinantfull-length (STF2) or truncated (STF2�) flagellin protein, or flagellin-modified CS protein STF2.(T1BT*)4X or STF2�.CS. Results for individualsera are expressed as geometric mean titers (GMT) with upper and lower95% confidence intervals (CI). A �4-fold difference in GMT was consid-ered significant. IgG subtypes were determined using CS repeat peptide-coated wells and an ELISA kit containing an enzyme-labeled MAb specificfor murine IgG subtypes (Southern Biotech, Birmingham, AL).

(ii) Sporozoite assays. Reactivity with native CS on P. falciparumsporozoites was measured by an indirect immunofluorescence assay (IFA)using 2-fold serum dilutions starting at 1:80. The endpoint titer was thelast dilution giving unequivocal positive fluorescence. Reactivity with vi-able sporozoites was measured by the circumsporozoite precipitin (CSP)reaction (42), in which 2-fold dilutions of sera were incubated with viablePfPb sporozoites expressing P. falciparum CS repeats. Formation of aterminal CSP reaction was determined by phase microscopy, and the end-point taken was the last serum dilution giving positive CSP reactions on 2sporozoites among a total of 20 sporozoites counted.

(iii) TSNA. Functional anti-repeat antibodies were measured in an invitro transgenic sporozoite neutralization assay (TSNA) as previously de-scribed (37, 43). Briefly, 2 � 104 transgenic PfPb sporozoites, in which theP. berghei CS repeats have been replaced with P. falciparum CS repeats,were incubated for 40 min on ice with a 1:5 dilution of immune or naiveserum. The preincubated PfPb sporozoites were then added to confluenthuman HepG2 hepatoma cells and cultured at 37°C and 5% CO2. After 48h, cells were lysed and homogenized using QIAshredder columns (Qia-gen), and total RNA was extracted using a PureLink RNA minikit (LifeTechnologies, Grand Island, NY) according to the manufacturer’s in-structions. The level of parasites in the cultures was measured by real-timeqPCR, as described above. Results were expressed as numbers of copies ofP. berghei 18S rRNA contained in the cell extracts. A �90% reduction in18S rRNA copy number compared to cultures receiving PfPb sporozoitesin medium or naive serum was considered significant.

Cellular assays. (i) ELISPOT assay. Th1- and Th2-type cell responseswere measured by gamma interferon (IFN-�) and IL-5 ex vivo enzyme-linked immunosorbent spot (ELISPOT) assays using splenocytes ob-tained 7 to 14 days after the last immunization. For expanded ELISPOTassays, spleen cells were cultured in vitro for 7 days with T1BT* peptide (10�g/ml) prior to assay. Cellular responses measured in the expandedELISPOT assay reflect the presence of malaria-specific memory T cells(44).

For ELISPOT assays, washed cells were added at 4 � 105/well inELISPOT plates coated with either anti-mouse IFN-� or anti-mouse IL-5(BD Biosciences). Malaria peptides were added at 10 �g/ml, and flagellin-modified CS or flagellin recombinant proteins were used at 1 �g/ml. Ir-radiated naive splenocytes were added as a source of antigen-presentingcells. The plates were incubated at 37°C and 5% CO2 for 40 h and devel-oped according to the manufacturer’s instructions. The number of spot-forming cells (SFC) was determined using an automated reader (CTLTechnologies, Amarillo, TX), and results are expressed as mean numbersof SFC/106 cells standard deviations (SD).

(ii) Cytometric bead arrays. Supernatants from ELISPOT cultureswere collected and stored at �20°C. Cytokines in the supernatants weremeasured using cytometric bead arrays (CBAs) according to the manu-

Carapau et al.

4352 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

facturer’s instructions (BD Biosciences). Bead fluorescence was measuredin a FACSCalibur flow cytometer (Becton, Dickinson) and converted tocytokine concentrations by using CBA Analysis (version 1.4.2) or FCAPsoftware from BD Biosciences.

Confocal microscopy. DC cultures were analyzed with an invertedLeica TCS SP2 AOBS confocal system equipped with a Ludin chamber forcontrol of temperature and CO2 concentration. Data were acquired withLeica confocal software; Imaris 7.4 (Bitplane, Saint Paul, MN), Image-ProPlus (Media Cybernetics, Bethesda, MD), AutoDeBlur (Media Cybernet-ics, Bethesda, MD), and NIH ImageJ were used for further image process-ing and deconvolution. Confocal figure panels were assembled in AdobePhotoshop.

Statistics. Statistical analyses used GraphPad Prism, version 6.00, forWindows (GraphPad Software, La Jolla, CA). Pairwise comparisons usedStudent’s t test or the nonparametric Mann-Whitney rank sum test, whilemultiple comparisons used analysis of variance (ANOVA) or the Kruskal-Wallis test with the Dunn procedure. Reductions of �90% of parasiterRNA copy numbers were considered significant in both the in vitro and invivo assays. The chi-square test (2 � 2 contingency tables) was used tocompare the numbers of mice in groups with �90% inhibition. In alltests, P values of �0.05 were considered statistically significant.

RESULTSFlagellin-modified P. falciparum CS functions as a TLR5 ago-nist. Two types of flagellin-modified P. falciparum CS recombi-nant proteins (Fig. 1), comprised of either full-length flagellin andimmunodominant CS epitopes [STF2.(T1BT*)n] or truncatedflagellin and a nearly full-length CS protein [STF2�.CS], wereanalyzed for TLR agonist activity, immunogenicity, and protec-tive efficacy. Both STF2.(T1BT*)n containing full-length flagellin(Fig. 2A) and STF�.CS containing truncated flagellin (Fig. 2B)effectively stimulated RAW 264.7 cells transfected with humanTLR5. The cytokine levels elicited by STF2�.CS stimulation ofhTLR5-transfected cells were comparable to levels obtained fol-lowing stimulation with STF2� only, indicating that the presenceof the �25-kDa CS protein at the C terminus of the truncatedflagellin did not interfere with flagellin-mediated TLR5 signaling(Fig. 2B). The lack of stimulation of untransfected RAW 264.7cells (Fig. 2A and B, open symbols), which express TLR4, is con-sistent with the low endotoxin levels (�0.01 EU/�g) and highlevels of purity of the flagellin-modified CS constructs (see Fig. S1in the supplemental material).

Flagellin-modified P. falciparum CS enhances murine andhuman DC maturation. Given the ability to trigger cytokine pro-duction in hTLR5-transfected cells (Fig. 2A and B), the flagellin-modified CS constructs were examined for the ability to stimulatemurine and human DC. TLR5 was detected by flow cytometry ona subset of human monocyte-derived DC (hDC), with approxi-mately 18% of CD11c hDC reacting with anti-TLR5 antibody(Fig. 2C). The percentage of TLR5-positive cells did not changewhen the hDC cultures were stimulated with flagellin or with mat-uration medium, suggesting that the levels of TLR5 expressed onthe hDC were not modified during DC maturation and may rep-resent a subset of the CD11c DC population.

In contrast to the human DC, murine BMDC stained onlyweakly with anti-TLR5 antibody (data not shown), as noted inprevious studies (45). However, TLR5 was detected on a murineimmature DC line, D1, with approximately 40% of the cells stain-ing positive (Fig. 2D).

Signaling through TLR5 leads to upregulation of costimulatorymolecules required for DC-T cell interactions that initiate adap-tive immune responses (23, 46). To determine if flagellin interac-

tion with TLR5 induced maturation of human DC, CD86 expres-sion on monocyte-derived hDC stimulated with STF2�.CS, orwith unmodified CS without flagellin, was measured by confocalmicroscopy and flow cytometry. Increased CD86 expression wasdetected on hDC incubated with STF2�.CS compared to cellsincubated with PBS (Fig. 3A). Recombinant CS protein alone,without flagellin, elicited only low levels of CD86 in hDC, indicat-ing that the flagellin moiety of the modified CS was required forupregulation of CD86.

The expression of CD86 on hDC increased with time of incuba-tion. After 6 h of incubation with STF2�.CS or STF2.(T1BT*)4X, 10to 20% of hDC were CD86, with levels increasing to approxi-mately 40% in 24-h cultures (Fig. 3B). The truncated flagellin

FIG 2 TLR5 signaling by flagellin-modified CS constructs. Human TLR5-transfected RAW 264.7 cells (closed symbols) or untransfected cells (opensymbols) were stimulated with 10-fold dilutions of (A) STF2.(T1BT*)1X or apositive-control protein, flagellin-modified ovalbumin (STF2.OVA), or (B)STF2�.CS, and levels of TNF-� in culture supernatants were measured byELISA. (C) Flow cytometry of TLR5 expression in human monocyte-derivedDC that were immature, matured with a cytokine cocktail, or stimulated withSTF2 flagellin (10 �g/ml). (D) Flow cytometry of TLR5 expression in murineD1 cells, an immature DC line.

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4353

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

protein (STF2�) was as effective as full-length flagellin (STF2) instimulating upregulation of costimulatory molecules on the hDC.The similar percentages of CD86-positive cells and similar meanfluorescence intensities (MFI) (data not shown) in cultures stim-ulated with flagellin-modified CS constructs or flagellin only in-dicate that the presence of the large CS protein or multiple T1BT*copies did not alter the interaction with TLR5 on hDC.

The flagellin-modified CS constructs also increased murineDC expression of costimulatory molecules. Incubation of D1 cellswith STF2�.CS or STF2.(T1BT*)4X stimulated upregulation ofCD40 and CD86, as well as increasing the percentage of cells ex-pressing MHC II molecules (Fig. 3C). Incubation with either full-length flagellin (STF2) or truncated flagellin (STF2�) elicited sim-ilar increases, indicating that upregulation of costimulatorymolecules on D1 cells was induced by the flagellin moiety, asfound with the hDC.

DC uptake of flagellin-modified CS proteins. In addition toexpression of costimulatory molecules, DC must also effi-ciently internalize antigens to process and present peptides to

MHC II-restricted CD4 T helper cells. To assay uptake offlagellin-modified CS, murine BMDC were incubated with eitherSTF2.(T1BT*)4X or STF2�.CS, followed by permeabilization andstaining with MAb 2A10, specific for P. falciparum CS repeats.Intracellular CS was detected in murine BMDC incubated witheither of the flagellin-modified CS constructs (Fig. 4A). Cells in-cubated with STF2 flagellin only did not stain with MAb 2A10,confirming the specificity of antibody staining for malaria CS pro-tein.

Uptake of flagellin-modified CS by murine BMDC was timeand dose dependent. After incubation with 10 �g/ml STF2�.CS,intracellular CS could first be detected in BMDC at 0.5 to 6 h ofincubation, with the intensity of staining increasing after 24 h (Fig.4B). Cells incubated with a log lower concentration of STF2�.CS(1 �g/ml) gave lower levels of intracellular CS (data not shown).Only low levels of MAb 2A10 stain were detected in cells incubatedwith T1BT* peptide without flagellin (data not shown), suggestingthat the presence of flagellin in the fusion protein enhanced anti-gen uptake, as reported for other flagellin fusion proteins (45).

Human DC also internalized the flagellin-modified CS after 24h of incubation with 10 �g/ml of STF2�.CS (Fig. 4C). Lower levelsof intracellular CS were detected following incubation of cells withSTF2.(T1BT*)4X, which may reflect enhanced detection by theMAb due to the presence of a larger number of repeats in thefull-length CS protein (41 repeats) than in the (T1BT*)4X con-struct (24 repeats).

Humoral immunity elicited by flagellin-modified P. falcipa-rum CS constructs. To assess immunogenicity, antibody re-sponses were measured in C57BL/6 and BALB/c mice injected s.c.with the flagellin-modified CS constructs. In preliminary studies,a flagellin-CS fusion protein containing four copies of the T1BT*sequence, STF2.(T1BT*)4X, induced higher anti-CS repeat anti-body titers with faster kinetics than constructs containing only asingle copy of the malaria epitopes (see Fig. S2 in the supplementalmaterial). Further studies therefore compared the immunogenic-ity of STF2.(T1BT*)4X to that of STF2�.CS, which contains theentire repeat region (41 tetramer repeats) as well as multipleCD4 and CD8 T cell epitopes in the C terminus of CS.

C57BL/6 and BALB/c mice immunized s.c. with STF2�.CS orSTF2.(T1BT*)4X developed antibodies with similar fine specifici-ties and peak GMT in ELISA (Table 1). IgG titers in both strains ofmice were highest against the respective immunogens, with peakGMT of �105 following s.c. immunization with eitherSTF2.(T1BT*)4X or STF2�.CS. High titers of anti-flagellin anti-bodies (GMT � 104), were also detected following immunizationwith either of the flagellin-modified CS proteins (data not shown).In contrast, antibody to the CS repeat peptide was a log lower, withpeak titers of 2 � 103 to 3 � 103 in both strains of mice. Overall,there was no significant difference (�4-fold) in ELISA titers elic-ited by s.c. immunization with the different flagellin-modified CSconstructs, indicating that both full-length and truncated flagellinprovided similar adjuvant properties for induction of antibody tomalaria CS repeats.

The antibodies elicited in BALB/c and C57BL/6 mice immu-nized with either of the flagellin-modified CS constructs also re-acted with native CS protein on P. falciparum sporozoites. A max-imum IFA titer of 103 to 104 was detected in pooled immune sera,consistent with the magnitude of the anti-repeat antibodies mea-sured by ELISA. These antibodies also reacted with viable sporo-zoites and elicited circumsporozoite precipitin (CSP) titers when

FIG 3 DC maturation following stimulation with flagellin-modified CS con-structs. (A) Confocal microscopy of monocyte-derived human DC expressionof costimulatory molecule CD86 (green) following incubation with unmod-ified CS protein or STF2�.CS. The DC nuclei were stained with Hoechststain (blue). (B) Percentages of CD86 human DC after 6 or 24 h of stim-ulation with the flagellin-modified CS construct STF2.(T1BT*)4X or STF�.CSor with full-length or truncated flagellin (STF2 and STF2�). Means with SDfor replicates are shown. Data are representative of two independent experi-ments. (C) Percentages of murine D1 cells expressing costimulatory and MHCII molecules following stimulation with 10 �g/ml of flagellin or flagellin-mod-ified CS proteins. Means with SD for replicates are shown. Data are represen-tative of three independent experiments.

Carapau et al.

4354 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

immune sera were incubated with PfPb sporozoites (data notshown). The CSP reaction reflects the shedding of antibody-cross-linked surface CS protein (42, 47), demonstrating that the anti-bodies elicited with flagellin-modified CS proteins effectively rec-ognized native CS protein on the viable sporozoite.

Route of immunization. S. enterica serovar Typhimurium is abacterial pathogen that invades the host through the gut mucosa.To determine if flagellin-modified CS was immunogenic whendelivered via a mucosal route, C57BL/6 mice were immunized i.n.,and levels of antibody were compared to those in s.c. immunizedmice. Mice immunized i.n. developed a systemic IgG responsecomparable in magnitude and fine specificity to that with s.c. im-munization (Table 1). Antibody to the STF2�.CS immunogenincreased with increasing i.n. doses, with a peak GMT of 105 ob-tained post-third dose, similar to the magnitude and kinetics ofthe antibody response following s.c. immunization (Fig. 5A). Nosignificant increase (�4-fold) was obtained following additionali.n. or s.c. booster immunizations. A large proportion of antibod-ies elicited by either the i.n. or s.c. route were specific for flagellin,with peak GMT of �104. All mice also developed antibodies to CSrepeats following i.n. immunization, although, as found with s.c.immunization, the peak GMT was 103, a log lower than that ofanti-flagellin antibodies.

To confirm that the immunogenicity of the flagellin-modifiedCS constructs was mediated through TLR signaling, mice lackingTLR5 were immunized i.n. or s.c. with STF2.(T1BT*)4X. Follow-ing priming and boosting by either the i.n. or s.c. route, only asingle tlr5�/� mouse had antibody to the immunogen, and nonehad antibody to flagellin, while all wild-type (WT) mice had sero-converted to both the immunogen and flagellin (data not shown).Additional doses of STF2.(T1BT*)4X did not compensate for thelack of TLR5 in the tlr5�/� mice (Fig. 5B). After a total of fourimmunizations, delivered either i.n. or s.c., the GMT of antibodyspecific for immunogen, flagellin, or CS repeats in the tlr5�/�

mice was 2 logs lower than corresponding titers in WT mice, in-dicating that development of malaria-specific antibodies as well asflagellin-specific antibodies was TLR5 dependent. The presence oflow levels of antibody in the hyperimmunized TLR5 KO mice mayreflect inefficient signaling through cytosolic inflammasomes orother innate immune pathways (48).

IgG subtypes in mice immunized with flagellin-modified CS.Mice immunized either i.n. or s.c. with STF2�.CS developedmixed Th1/Th2-type antibody responses. At a 1:80 serum dilu-tion, high levels of IgG1 anti-repeat antibodies were detected, withlower levels of Th1-associated IgG2a/c antibodies (Fig. 6A). A pre-dominance of the IgG1 subtype was also observed in the anti-flagellin antibody response, along with IgG2a/c and IgG2b, inboth s.c. and i.n. immunized mice (Fig. 6B). Few or no IgG3 an-tibodies were observed to either CS repeats or flagellin.

Similarly, mice immunized with STF2.(T1BT*)4X had onlyIgG1 and IgG2b subtype antibodies, with no detectable Th1-typeIgG2a/c antibody (see Fig. S3 in the supplemental material). Onlylow levels of IgA to flagellin, but not to CS repeats, were detected insera of i.n. immunized mice (data not shown).

Cellular immunity elicited by flagellin-modified P. falcipa-rum CS constructs. To assess T cell responses, Th1-type (IFN-�)and Th2-type (IL-5) cytokine-producing cells were enumeratedby ELISPOT assays using spleen cells of mice immunized s.c. ori.n. with STF2�.CS. In ex vivo ELISPOT assays, the majority ofIL-5- and IFN-�-specific spot-forming cells (SFC) in the immune

FIG 4 DC uptake of flagellin-modified CS constructs. (A) FACS histogram ofmurine BMDC following 24 h of incubation with flagellin or flagellin-modi-fied CS (10 �g/ml). Intracellular CS was detected by labeling permeabilizedcells with biotinylated MAb 2A10 followed by streptavidin-conjugated Qdots.(B) MAb 2A10-positive murine BMDC measured after incubation for varioustimes with flagellin-modified CS constructs or flagellin-only controls (STF2 orSTF2�). Means with SD for replicates are shown. Data are representative ofthree independent experiments. (C) Dose-dependent increase in MAb 2A10staining of human DC following 24 h of incubation with flagellin-modified CSconstructs or flagellin-only controls. Means with SD for replicates are shown.Data are representative of three independent experiments.

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4355

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

spleens were specific for the STF2�.CS immunogen or for theSTF2� flagellin protein (see Fig. S4 in the supplemental material).Stimulation with the malaria T1BT* peptide elicited minimal orno IL-5- and IFN-�-secreting T cells.

To enhance detection of malaria-specific T cells, immunespleen cells were cultured with the malaria peptide for 7 days priorto testing in an expanded ELISPOT assay. In peptide-expandedcultures, IL-5 SFC were detected upon restimulation with theT1BT* malaria peptide, with higher levels in mice immunized i.n.than in those immunized s.c. (Student’s t test; P � 0.006) (Fig.7A). The i.n. immunized mice also had higher levels of IL-5 SFCspecific for the STF2�.CS immunogen (Student’s t test; P �0.002). Similar levels of malaria-specific IL-5 SFC were observedin mice immunized i.n. with STF2.(T1BT*)4x or STF2�.CS (Stu-

dent’s t test; P � 0.10) (Fig. 7B). IFN-�-secreting SFC could not beevaluated due to high background levels in the expandedELISPOT assay.

To further explore T cell responses in the i.n. immunized mice,Th1-type (IL-2, IFN-�, and TNF-�) or Th2-type (IL-4, IL-5 orIL-6, and IL-10) cytokines were measured in supernatants of thestimulated cells by using a cytometric bead array (CBA). Consis-tent with results obtained in the expanded ELISPOT assay, IL-5was detected in supernatants following T1BT* restimulation ofcells from mice immunized i.n. with STF2.(T1BT*)4X orSTF2�.CS (Fig. 7C). Conversely, IL-6 levels were higher followingstimulation with STF2.(T1BT*)4X or STF2�.CS than with the ma-laria T1BT* peptide (Fig. 7D). Th1-type cytokines were either notdetected in the culture supernatants (IL-2 and IFN-�) or not dif-

TABLE 1 Immunogenicity of flagellin-modified CS proteins

Immunogena

Route ofadministration Mouse strain

ELISA GMT (range) for IgGb

IFA titercImmunogen CS repeats

STF2.(T1BT*)4X s.c. BALB/c 188,203 (163,840–327,680) 2,941 (1,280–20,480) 1,280s.c. C57BL/6 142,631 (81,920–327,680) 2,941 (1,280–10,240) 1,280i.n. C57BL/6 520,160 (327,680–655,360) 8,127 (640–40,960) 2,560

STF2�.CS s.c. BALB/c 463,410 (327,680–655,360) 3,620 (2,560–5,120) 5,120s.c. C57BL/6 412,851 (327,680–655,360) 2,032 (1,280–2,560) 10,240i.n. C57BL/6 655,360 (655,360) 2,032 (1,280–5,120) 20,480

a Mice (3 to 5/group) were immunized by the s.c. or i.n. route with 4 or 5 doses of either STF2.(T1BT*)4X or STF2�.CS (10 to 50 �g/dose). Sera were obtained 14 to 21 days afterthe final immunization.b Individual sera were tested by ELISA, using plates coated with homologous immunogen or P. falciparum CS repeat peptide as antigen.c Pooled immune sera were tested by IFA using P. falciparum sporozoites.

FIG 5 Kinetics and fine specificities of antibody responses in mice immunized with flagellin-modified CS constructs. (A) Kinetics of IgG response of C57BL/6mice (3 mice/group) immunized s.c. or i.n. with STF2�.CS (50 �g/dose), as measured by ELISA using CS repeat peptide, flagellin (STF2�), or STF2�.CS as thecoating antigen. Results shown are geometric mean titers (GMT) with 95% confidence intervals. (B) IgG GMT in tlr5�/� mice and WT controls (3 mice/group)immunized s.c. or i.n. with four doses of STF2.(T1BT*)4X (10 �g/dose). Following four immunizations, all WT controls seroconverted. In contrast, none of thetlr5�/� mice immunized s.c. had detectable antibodies to CS repeats or flagellin, with one mouse having detectable antibodies to the immunogen. Two of threetlr5�/� mice immunized i.n. had detectable antibodies to CS repeats, with one mouse having detectable antibodies to flagellin and immunogen. The tlr5�/� micethat did not seroconvert were assigned a value of 20 for calculation of GMT.

Carapau et al.

4356 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

ferent from levels in medium controls (TNF-�), consistent with apredominantly Th2-type T cell response detected by ELISPOTassay.

Protective efficacy of flagellin-modified P. falciparum CSconstructs. To determine whether immunization with the flagel-lin-modified CS constructs elicited functional antibodies, sera

from immunized mice were tested for the ability to inhibit in vitroinvasion of hepatoma cells by PfPb sporozoites, which aretransgenic P. berghei parasites that express the P. falciparum CSrepeat region (37, 43). The sporozoite-neutralizing activity inthe immune sera was dose dependent. Serum obtained follow-ing three i.n. immunizations with either STF2.(T1BT*)4X orSTF2�.CS was not inhibitory (0% or 42% inhibition, respectively)(Fig. 8A). Additional boosters increased sporozoite-neutraliz-ing activity, with sera of mice immunized i.n. with 5 doses ofSTF2.(T1BT*)4X and STF2�.CS giving 98% and 96% inhibi-tion, respectively, compared with naive serum. An additional6th dose of immunogen did not significantly increase the levelof inhibition, with STF2.(T1BT*)4X immune serum giving 95%inhibition and STF2�.CS immune serum giving 99% inhibition.Inhibition of �90% is considered significant, as a 90% reductionin sporozoite numbers is required to give a 1-day delay in theprepatent period following sporozoite challenge (39–41).

The level of sporozoite-neutralizing activity in immune sera ob-tained after the 5th or 6th dose of STF2.(T1BT*)4X or STF2�.CS wascomparable to that obtained with 25 �g/ml of MAb 2A10, aninhibitory antibody specific for P. falciparum CS repeats (6). Noinhibition was found when sporozoites were preincubated withMAb 3D11, specific for P. berghei CS repeats, indicating that neu-tralizing activity was specific for P. falciparum CS repeats.

To determine whether the route of immunization was a factorin the induction of neutralizing antibodies, immune sera of miceimmunized i.n. were compared to those of s.c. immunized mice byTSNA. Following immunization with five doses of STF2�.CS, thesera of mice immunized i.n. gave �90% inhibition, while immunesera from mice immunized s.c. gave a mean 69% reduction inparasite burden (Fig. 8B).

FIG 6 IgG subtypes in mice immunized with flagellin-modified CS. Antibod-ies specific for CS repeats (A) or flagellin (B) were measured in pooled immunesera (1:80 dilution) from mice immunized i.n. or s.c. with four doses ofSTF2�.CS or flagellin (50 �g/dose). OD, optical density.

FIG 7 Flagellin-modified CS constructs elicit Th2-type cytokine responses. Spleen cells were obtained from mice (A) immunized s.c. or i.n. with four doses ofSTF2�-CS (50 �g/dose) or (B) immunized i.n. with five doses of either STF2.(T1BT*)4X or STF2�.CS (10 �g/dose). Malaria-specific cells were expanded in vitrowith T1BT* peptide for 7 days prior to ELISPOT assay. Results are shown as numbers of spot-forming cells (SFC)/106 cells following restimulation with T1BT*peptide, flagellin (STF�), or the respective immunogen [STF2�-CS or STF2.(T1BT*)4X]. IL-5 (C) or IL-6 (D) in pooled cell culture supernatants were measuredusing multiplex CBA. Cytokine concentrations are shown in pg/ml, after subtraction (�) of levels in medium-only cultures.

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4357

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

Although the transgenic PfPb parasites used in the TSNA ex-press P. falciparum repeats, they remain rodent parasites that canefficiently infect mice and develop into normal liver- and blood-stage infections (36), thus allowing the measurement of in vivoprotective immunity following challenge. Consistent with levels ofinhibition observed in the in vitro TSNA, the rRNA copy numberwas significantly lower in the i.n. immunized mice than in naivemice, while the difference was not significant in s.c. immunizedmice 40 h after challenge by the bites of PfPb-infected mosquitoescompared to naive challenge controls (Fig. 8C). All of the i.n.immunized mice (3/3 mice) had �90% reductions in liver-stageburdens (mean, 98%), while in the s.c. group, 0/3 mice had �90%inhibition (mean, 61%) (�2; P � 0.014).

The correlation of in vitro and in vivo results suggested that themechanism of immunity in the flagellin-CS-immunized mice isantibody mediated. The sporozoite-neutralizing activity in thesera of the i.n. immunized mice was antibody concentration de-pendent. In TSNA of immune sera obtained from mice immu-nized i.n. with five doses of STF2�.CS, there was a significantdifference in 18S rRNA copy number observed at a 1:5 dilution(90% inhibition) compared to a 1:10 serum dilution (77% inhibi-tion) (Fig. 9A). The level of inhibition at a 1:5 dilution correlatedwith the level of protection following challenge, with a mean 93%reduction of parasite burdens in the livers of immunized mice(Fig. 9B). Protection in vivo was confirmed to be antibody medi-

ated, as depletion of CD4 and CD8 T cells prior to challenge didnot abrogate immune resistance (Fig. 9B, hatched bars).

DISCUSSION

Subunit vaccines produced in standard bacterial expression sys-tems provide numerous safety and cost advantages over attenu-ated or viral vector-based vaccines. A needle-free vaccine deliv-ered by i.n. immunization would further reduce costs byeliminating the need for sterile syringes, trained medical person-nel, and biohazard waste disposal. While frequently used to in-duce strong mucosal immunity, i.n. immunization also elicits sys-temic antibody responses comparable to those obtained by s.c.injection (Table 1) and therefore has the potential to target blood-borne pathogens such as Plasmodium parasites.

The TLR agonist properties of S. enterica serovar Typhimu-rium flagellin have been developed as adjuvants for bacterial, viral,and protozoan subunit vaccines which lack endogenous PAMPsrequired to stimulate innate immune responses that initiate adap-tive humoral and cellular immunity (reviewed in reference 23).Previous studies demonstrated that S. enterica serovar Typhimu-rium type 1 (FLiC) or type 2 (FljB) flagellin, as well as flagellinfrom other Gram-negative bacteria, can be used as a vaccine ad-juvant. The enhanced immunogenicity of STF2 flagellin fusionproteins compared to mixtures of flagellin and antigen (19, 20) isconsistent with the requirement for colocalization of both the TLR

FIG 8 Sporozoite-neutralizing antibody and protection in mice immunized with flagellin-modified CS constructs. (A) TSNA was carried out using a 1:5 dilutionof pooled sera (3 mice/group) obtained after the 3rd to 6th dose of STF2�-CS, STF2.(T1BT*)4X, or unmodified T1BT* peptide (10 �g/dose). Results measuredby qPCR analysis of hepatoma cell extracts obtained at 48 h postinfection are shown as mean P. berghei 18S rRNA copy numbers. MAb 2A10, specific for P.falciparum CS repeats, and MAb 3D11, specific for P. berghei CS repeats, served as positive and negative controls, respectively. (B) TSNA was carried out usingindividual sera (1:5 dilution) from 3 mice/group immunized i.n. or s.c. with five doses of STF2�.CS (50 �g/dose). 18S rRNA copy numbers in cultures with i.n.immune sera, but not s.c. immune sera, were significantly reduced compared to naive control levels. (C) In vivo protection was measured in the mice used forpanel B following challenge by exposure to the bites of 5 to 15 PfPb-infected mosquitoes. Results shown are mean parasite 18S rRNA levels in livers harvested at40 h postinfection, as measured by qPCR. Consistent with in vitro TSNA, the liver parasite burden was reduced in i.n. immunized but not s.c. immunized micecompared to naive controls following challenge. *, P � 0.05 by the Kruskal-Wallis test; ns, nonsignificant.

Carapau et al.

4358 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

agonist and antigen in the same phagosome for optimal immuno-genicity (49, 50).

The construction of the flagellin-modified malaria constructswas based on flagellin-modified viral and bacterial vaccines whichhave been shown to elicit neutralizing antibodies to West Nile,dengue, and influenza viruses, as well as CD8 T cell-mediatedimmunity to Listeria, in murine models (19–21). In recent phaseI/II studies, an influenza vaccine comprised of the HA globularhead inserted at both the hinge region and C terminus of flagellinwas shown to be safe and efficacious when low doses were admin-istered i.m. to elderly (�65 years) and young (18 to 49 years)volunteers (25, 26).

In the present study, CS protein modified with either full-length (STF2) or truncated (STF2�) flagellin was found to stim-ulate cytokine production by RAW cells transfected with humanTLR5. The lack of response of untransfected RAW cells, whichexpress TLR4, demonstrated that the purified constructs were freeof bacterial LPS contaminants, consistent with the low levels ofendotoxin detected by LAL assay. In additional studies, we foundthat C3H/HeJ mice, which are hyporesponsive to LPS due to a

mutation in TLR4, developed peak antibody titers to CS repeats(GMT, 7,241) and flagellin (GMT, 97,420) after immunizationwith flagellin-modified CS protein (data not shown) that werecomparable to those of BALB/c and C57BL/6 mice (Table 1), in-dicating that adjuvanticity was mediated by flagellin and not byminor bacterial contaminants from the E. coli expression system.Immunogenicity was dependent on flagellin stimulation throughTLR5 signaling, as tlr5�/� mice developed minimal or no anti-body responses following immunization with the flagellin-modi-fied CS protein (Fig. 5B). The adjuvant activity of flagellin hasbeen shown to require direct engagement of flagellin with TLR5on CD11c DC (45). The flagellin-modified CS constructs in-creased expression of costimulatory molecules and MHC IImolecules on a TLR5 murine DC line (D1) and on monocyte-derived hDC. TLR5 staining of only a subpopulation of cellsmay reflect the presence of different DC subsets or activationstates (51, 52).

The CS repeat region is known to contain a protective B cellepitope (5, 6). The magnitudes of the repeat-specific antibodieselicited by the flagellin-modified CS constructs, containing eitherfull-length or minimal epitopes of CS, were similar (Table 1),indicating that the presence of only a small number of CS repeatsand a T helper epitope was sufficient to elicit sporozoite-neutral-izing antibodies. The systemic anti-repeat IgG antibody responsefollowing i.n. immunization was not significantly different frompeak titers obtained by s.c. immunization, and the hierarchy ofantibody responses remained the same: immunogen � flagellin �CS repeats (Fig. 5A). However, despite the similar kinetics, speci-ficities, and magnitudes of antibody responses, immune sera ofmice immunized i.n. had higher levels of sporozoite-neutralizingantibodies in vitro than those of s.c. immunized mice, and thiscorrelated with higher levels of protection in vivo in i.n. immu-nized mice than in s.c. immunized mice (Fig. 8). Protection in vivowas mediated by antibody, as depletion of T cells from immu-nized mice prior to challenge did not reduce levels of inhibition(Fig. 9B).

The mechanism of the enhanced neutralizing activity of an-tibodies elicited by i.n. immunization compared to s.c. immu-nization remains to be defined. The affinities of anti-repeatantibodies elicited by i.n. or s.c. immunization were similar inelution assays with chaotropic NH4SCN (see Fig. S3 in the supple-mental material). Both of the flagellin-modified CS constructs,STF2.(T1BT*)4X and STF2�.CS, administered either i.n. or s.c.,elicited predominantly IgG1 (Th2-type) anti-repeat antibodies.Consistent with predominant IgG1 antibody responses, the ma-jority of T cells elicited by the flagellin-modified CS constructswere of the Th2 type, based on production of IL-5 and IL-6, withminimal IFN-�, as measured by ELISPOT assay and CBA (Fig. 7).Vaccines containing flagellin as adjuvant frequently elicit a mixedTh1/Th2-type response skewed toward Th2-type antibody sub-types in murine studies (19, 22, 53) and in volunteers immunizedwith a flagellin-modified flu vaccine (25, 26). Thus far, a role for Fcor immunoglobulin (Ig) subtypes in antibody-mediated sporozo-ite neutralization has not been demonstrated, as Fab of anti-repeatMAbs as well as MAbs of various IgG subtypes can neutralizesporozoite infectivity in vivo (54, 55). A requirement for high con-centrations of anti-repeat antibody rather than a specific IgG sub-type would be consistent with the immobilization of sporozoites,thereby preventing motility required for invasion of cells in theliver (56). Recent studies have also shown that anti-repeat anti-

FIG 9 Antibody-mediated protection in mice immunized i.n. with flagellin-modified CS protein. Mice (n � 6 mice/group) were immunized i.n. withSTF2�.CS or STF2 only (50 �g/dose). (A) TSNA was carried out using a 1:5 or1:10 dilution of individual sera obtained after the fifth dose. Results shown aremean 18S rRNA copy numbers in 48-h extracts of HepG2 cell cultures asmeasured by qPCR. (B) In vivo protection measured in immunized mice thatwere nondepleted (3 mice/group; solid bars) or depleted of T cells (3 mice/group; hatched bars) by injection of anti-CD4 and anti-CD8 MAbs (100 �g ofeach MAb) for 3 days prior to challenge by exposure to the bites of PfPb-infected mosquitoes. Results shown are mean 18S rRNA copy numbers in liverextracts obtained at 40 h postchallenge, as measured by qPCR. *, P � 0.05; **,P � 0.01 by the Kruskal-Wallis test; ns, nonsignificant.

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4359

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

bodies immobilize sporozoites in the skin at the site of the mos-quito bite, thereby preventing their egress into the circulation andtransit to the liver (57).

The differences in the antibodies elicited by i.n. and s.c. immu-nizations may be related to the unique structure and cell popula-tions found in the murine NALT (58, 59). We recently carried outconfocal imaging of the NALT of mice immunized with flagellin-modified CS proteins (60). In contrast to lymph nodes, B cellspredominate in the NALT, as found also in Peyer’s patches of thegut-associated lymphoid tissue (GALT). The high levels of B cellspersisted following a �2-fold increase in the overall size of theNALT in the flagellin-CS-immunized mice. A similar increase inNALT size was noted in mice immunized with flagellin alone,suggesting a role for TLR5 signaling in stimulating cell influx ormultiplication within the NALT. Moreover, CD11c DC withtransepithelial dendrites, as well as microfold (M) cells, werefound in the NALT, which could potentially play a role in antigencapture and/or presentation, as found in GALT (61, 62). In hu-mans, the lympho-epithelial structures of the Waldeyer’s ring,considered the equivalent of the murine NALT (63), are enlargedin infants, the primary target population for an intranasal malariavaccine. Although most standard childhood vaccines are giveni.m. or s.c., an i.n. pediatric flu vaccine was recently approved bythe FDA (64).

Our analysis of the immunogenicity of flagellin-modified CSrecombinant proteins demonstrates that antibodies elicited by i.n.immunization, although of relatively modest titer, can neutralizesporozoite infectivity in vitro and in vivo. These results suggest thatfunctional assays based on transgenic parasites expressing P.falciparum epitopes, rather than static serologic assays such asELISA, may provide a more biologically relevant measurement ofthe sporozoite-neutralizing potential of vaccine-induced re-sponses. These studies provide the first demonstration that an i.n.administered TLR5 agonist-modified P. falciparum CS proteincan elicit protective antibody-mediated immune responses andsupport further efforts to investigate needle-free malaria vaccines.While the data are encouraging, the level of parasite inhibitionobserved in the flagellin-CS-immunized mice would not be ex-pected to provide high levels of sterile immunity, as TSNA titers of1:40 and higher have been obtained in previous murine studiesusing parenterally administered CS peptide-protein conjugates(65), while TSNA activities of immune sera from STF2�.CS-im-munized mice did not exceed a 1:5 dilution (Fig. 9A). Based onanalysis of NALT, optimization of adjuvant formulations to im-prove mucoadhesion and to specifically target NALT antigen-pre-senting cells (66–68) would be expected to improve the protectiveefficacy of i.n. delivered flagellin-CS vaccines. Delivery of opti-mized vaccine formulations i.n. would facilitate large-scale vacci-nation programs for the control, elimination, and possible eradi-cation of P. falciparum, the most lethal of all human Plasmodiumparasites, as well as the other human-infective Plasmodium species(69).

ACKNOWLEDGMENTS

VaxInnate is acknowledged as a source of flagellin and flagellin-modifiedCS proteins, and we thank A. Price and V. Nakaar for production andpurification of the recombinant proteins. We thank Rita Altszuler forexcellent technical assistance on all phases of the research and LeydaCabrera for technical assistance on serological assays. We thank David

O’Neill and Nina Bhardwaj for providing cryopreserved human dendriticcells.

This research was supported by NIH/NIAID grant R56 AI 083655(E.N.) and NIH/NCRR grant S10 RR019288 (U.F.).

REFERENCES1. Nussenzweig RS, Vanderberg J, Most H, Orton C. 1967. Protective

immunity produced by the injection of X-irradiated sporozoites of Plas-modium berghei. Nature 216:160 –162.

2. Clyde DF. 1975. Immunization of man against falciparum and vivaxmalaria by use of attenuated sporozoites. Am. J. Trop. Med. Hyg. 24:397–401.

3. Clyde DF. 1990. Immunity to falciparum and vivax malaria induced byirradiated sporozoites: a review of the University of Maryland studies,1971–75. Bull. World Health Organ. 68(Suppl):9 –12.

4. Rieckmann KH. 1990. Human immunization with attenuated sporozo-ites. Bull. World Health Organ. 68(Suppl):13–16.

5. Yoshida N, Nussenzweig RS, Potocnjak P, Nussenzweig V, Aikawa M.1980. Hybridoma produces protective antibodies directed against thesporozoite stage of malaria parasite. Science 207:71–73.

6. Nardin EH, Nussenzweig V, Nussenzweig RS, Collins WE, HarinasutaKT, Tapchaisri P, Chomcharn Y. 1982. Circumsporozoite proteins ofhuman malaria parasites Plasmodium falciparum and Plasmodium vivax.J. Exp. Med. 156:20 –30.

7. Kumar KA, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC,Zavala F, Nussenzweig V. 2006. The circumsporozoite protein is animmunodominant protective antigen in irradiated sporozoites. Nature444:937–940.

8. Agnandji ST, Lell B, Soulanoudjingar SS, Fernandes JF, Abossolo BP,Conzelmann C, Methogo BG, Doucka Y, Flamen A, Mordmuller B,Issifou S, Kremsner PG, Sacarlal J, Aide P, Lanaspa M, Aponte JJ,Nhamuave A, Quelhas D, Bassat Q, Mandjate S, Macete E, Alonso P,Abdulla S, Salim N, Juma O, Shomari M, Shubis K, Machera F, HamadAS, Minja R, Mtoro A, Sykes A, Ahmed S, Urassa AM, Ali AM,Mwangoka G, Tanner M, Tinto H, D’Alessandro U, Sorgho H, Valea I,Tahita MC, Kabore W, Ouedraogo S, Sandrine Y, Guiguemde RT,Ouedraogo JB, Hamel MJ, Kariuki S, Odero C, Oneko M, Otieno K,Awino N, Omoto J, Williamson J, Muturi-Kioi V, Laserson KF, SlutskerL, Otieno W, Otieno L, Nekoye O, Gondi S, Otieno A, Ogutu B,Wasuna R, Owira V, Jones D, Onyango AA, Njuguna P, Chilengi R,Akoo P, Kerubo C, Gitaka J, Maingi C, Lang T, Olotu A, Tsofa B, BejonP, Peshu N, Marsh K, Owusu-Agyei S, Asante KP, Osei-Kwakye K,Boahen O, Ayamba S, Kayan K, Owusu-Ofori R, Dosoo D, Asante I,Adjei G, Chandramohan D, Greenwood B, Lusingu J, Gesase S, Mala-beja A, Abdul O, Kilavo H, Mahende C, Liheluka E, Lemnge M,Theander T, Drakeley C, Ansong D, Agbenyega T, Adjei S, BoatengHO, Rettig T, Bawa J, Sylverken J, Sambian D, Agyekum A, Owusu L,Martinson F, Hoffman I, Mvalo T, Kamthunzi P, Nkomo R, Msika A,Jumbe A, Chome N, Nyakuipa D, Chintedza J, Ballou WR, Bruls M,Cohen J, Guerra Y, Jongert E, Lapierre D, Leach A, Lievens M, Ofori-Anyinam O, Vekemans J, Carter T, Leboulleux D, Loucq C, Radford A,Savarese B, Schellenberg D, Sillman M, Vansadia P. 2011. First results ofphase 3 trial of RTS,S/AS01 malaria vaccine in African children. N. Engl. J.Med. 365:1863–1875.

9. 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, Otieno W, Otieno L, OtsyulaN, Gondi S, Otieno A, Owira V, Oguk E, Odongo G, Woods JB, OgutuB, Njuguna P, Chilengi R, Akoo P, Kerubo C, Maingi C, Lang T, OlotuA, Bejon P, Marsh K, Mwambingu G, Owusu-Agyei S, Asante KP,Osei-Kwakye K, Boahen O, Dosoo D, Asante I, Adjei G, Kwara E,Chandramohan D, Greenwood B, Lusingu J, Gesase S, Malabeja A,Abdul O, Mahende C, Liheluka E, Malle L, Lemnge M, Theander TG,Drakeley C, Ansong D, Agbenyega T, Adjei S, Boateng HO, Rettig T,Bawa J, Sylverken J, Sambian D, Sarfo A, Agyekum A, Martinson F,Hoffman I, Mvalo T, Kamthunzi P, Nkomo R, Tembo T, Tegha G,

Carapau et al.

4360 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

Tsidya M, Kilembe J, Chawinga C, Ballou WR, Cohen J, Guerra Y,Jongert E, Lapierre D, Leach A, Lievens M, Ofori-Anyinam O, OlivierA, Vekemans J, Carter T, Kaslow D, Leboulleux D, Loucq C, RadfordA, Savarese B, Schellenberg D, Sillman M, Vansadia P. 2012. A phase 3trial of RTS,S/AS01 malaria vaccine in African infants. N. Engl. J. Med.367:2284 –2295.

10. Stoute JA, Heppner DG, Jr, Mason CJ, Siangla J, Opollo MO, KesterKE, Vigneron L, Voss G, Walter MJ, Tornieporth N, Cohen JD, BallouWR. 2006. Phase 1 safety and immunogenicity trial of malaria vaccineRTS,S/AS02A in adults in a hyperendemic region of western Kenya. Am. J.Trop. Med. Hyg. 75:166 –170.

11. Gordon DM, McGovern TW, Krzych U, Cohen JC, Schneider I, La-Chance R, Heppner DG, Yuan G, Hollingdale M, Slaoui M, Hauser P,Voet P, Sadoff JC, Ballou WR. 1995. Safety, immunogenicity, and effi-cacy of a recombinantly produced Plasmodium falciparum circumsporo-zoite protein-hepatitis B surface antigen subunit vaccine. J. Infect. Dis.171:1576 –1585.

12. Owusu-Agyei S, Ansong D, Asante K, Kwarteng Owusu S, Owusu R,Wireko Brobby NA, Dosoo D, Osei Akoto A, Osei-Kwakye K, Adjei EA,Boahen KO, Sylverken J, Adjei G, Sambian D, Apanga S, Kayan K,Vekemans J, Ofori-Anyinam O, Leach A, Lievens M, Demoitie MA,Dubois MC, Cohen J, Ballou WR, Savarese B, Chandramohan D,Gyapong JO, Milligan P, Antwi S, Agbenyega T, Greenwood B, Evans J.2009. Randomized controlled trial of RTS,S/AS02D and RTS,S/AS01Emalaria candidate vaccines given according to different schedules in Gha-naian children. PLoS One 4:e7302. doi:10.1371/journal.pone.0007302.

13. Kester KE, Cummings JF, Ofori-Anyinam O, Ockenhouse CF, KrzychU, Moris P, Schwenk R, Nielsen RA, Debebe Z, Pinelis E, Juompan L,Williams J, Dowler M, Stewart VA, Wirtz RA, Dubois MC, Lievens M,Cohen J, Ballou WR, Heppner DG, Jr. 2009. Randomized, double-blind,phase 2a trial of falciparum malaria vaccines RTS,S/AS01B and RTS,S/AS02A in malaria-naive adults: safety, efficacy, and immunologic associ-ates of protection. J. Infect. Dis. 200:337–346.

14. Pulendran B, Ahmed R. 2006. Translating innate immunity into immu-nological memory: implications for vaccine development. Cell 124:849 –863.

15. Coffman RL, Sher A, Seder RA. 2010. Vaccine adjuvants: putting innateimmunity to work. Immunity 33:492–503.

16. Pulendran B, Ahmed R. 2011. Immunological mechanisms of vaccina-tion. Nat. Immunol. 12:509 –517.

17. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, EngJK, Akira S, Underhill DM, Aderem A. 2001. The innate immune re-sponse to bacterial flagellin is mediated by Toll-like receptor 5. Nature410:1099 –1103.

18. McEwen J, Levi R, Horwitz RJ, Arnon R. 1992. Synthetic recombinantvaccine expressing influenza haemagglutinin epitope in Salmonella flagel-lin leads to partial protection in mice. Vaccine 10:405– 411.

19. Huleatt JW, Jacobs AR, Tang J, Desai P, Kopp EB, Huang Y, Song L,Nakaar V, Powell TJ. 2007. Vaccination with recombinant fusion pro-teins incorporating Toll-like receptor ligands induces rapid cellular andhumoral immunity. Vaccine 25:763–775.

20. McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P,Price A, Jacobs A, Takahashi VN, Huang Y, Nakaar V, Alexopoulou L,Fikrig E, Powell TJ. 2007. A West Nile virus recombinant protein vaccinethat coactivates innate and adaptive immunity. J. Infect. Dis. 195:1607–1617.

21. Huleatt JW, Nakaar V, Desai P, Huang Y, Hewitt D, Jacobs A, Tang J,McDonald W, Song L, Evans RK, Umlauf S, Tussey L, Powell TJ. 2008.Potent immunogenicity and efficacy of a universal influenza vaccine can-didate comprising a recombinant fusion protein linking influenza M2e tothe TLR5 ligand flagellin. Vaccine 26:201–214.

22. Bargieri DY, Rosa DS, Braga CJ, Carvalho BO, Costa FT, EspindolaNM, Vaz AJ, Soares IS, Ferreira LC, Rodrigues MM. 2008. New malariavaccine candidates based on the Plasmodium vivax merozoite surface pro-tein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin.Vaccine 26:6132– 6142.

23. Mizel SB, Bates JT. 2010. Flagellin as an adjuvant: cellular mechanismsand potential. J. Immunol. 185:5677–5682.

24. Talbot HK, Rock MT, Johnson C, Tussey L, Kavita U, Shanker A, ShawAR, Taylor DN. 2010. Immunopotentiation of trivalent influenza vaccinewhen given with VAX102, a recombinant influenza M2e vaccine fused tothe TLR5 ligand flagellin. PLoS One 5:e14442. doi:10.1371/journal.pone.0014442.

25. Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, Liu G,Kavita U, Song L, Dark I, Shaw A. 2010. Safety and immunogenicity ofa recombinant hemagglutinin influenza-flagellin fusion vaccine(VAX125) in healthy young adults. Vaccine 28:8268 – 8274.

26. Taylor DN, Treanor JJ, Strout C, Johnson C, Fitzgerald T, Kavita U,Ozer K, Tussey L, Shaw A. 2011. Induction of a potent immune responsein the elderly using the TLR-5 agonist, flagellin, with a recombinant hem-agglutinin influenza-flagellin fusion vaccine (VAX125, STF2.HA1 SI).Vaccine 29:4897– 4902.

27. Nardin EH, Herrington DA, Davis J, Levine M, Stuber D, Takacs B,Caspers P, Barr P, Altszuler R, Clavijo P, Nussenzweig RS. 1989.Conserved repetitive epitope recognized by CD4 clones from a malaria-immunized volunteer. Science 246:1603–1606.

28. Moreno A, Clavijo P, Edelman R, Davis J, Sztein M, Sinigaglia F,Nardin E. 1993. CD4 T cell clones obtained from Plasmodium falcipa-rum sporozoite-immunized volunteers recognize polymorphic sequencesof the circumsporozoite protein. J. Immunol. 151:489 – 499.

29. Calvo-Calle JM, Hammer J, Sinigaglia F, Clavijo P, Moya-Castro ZR,Nardin EH. 1997. Binding of malaria T cell epitopes to DR and DQmolecules in vitro correlates with immunogenicity in vivo: identificationof a universal T cell epitope in the Plasmodium falciparum circumsporo-zoite protein. J. Immunol. 159:1362–1373.

30. Eaves-Pyles TD, Wong HR, Odoms K, Pyles RB. 2001. Salmonellaflagellin-dependent proinflammatory responses are localized to the con-served amino and carboxyl regions of the protein. J. Immunol. 167:7009 –7016.

31. Stockinger B, Zal T, Zal A, Gray D. 1996. B cells solicit their own helpfrom T cells. J. Exp. Med. 183:891– 899.

32. Pierre P, Turley SJ, Gatti E, Hull M, Meltzer J, Mirza A, Inaba K,Steinman RM, Mellman I. 1997. Developmental regulation of MHC classII transport in mouse dendritic cells. Nature 388:787–792.

33. Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, Adorini L,Zimmermann VS, Davoust J, Ricciardi-Castagnoli P. 1997. Maturationstages of mouse dendritic cells in growth factor-dependent long-term cul-tures. J. Exp. Med. 185:317–328.

34. O’Neill DW, Bhardwaj N. 2005. Differentiation of peripheral bloodmonocytes into dendritic cells. Curr. Protoc. Immunol. Chapter 22:Unit22F.4. doi:10.1002/0471142735.im22f04s67.

35. Kiyono H, Fukuyama S. 2004. NALT- versus Peyer’s-patch-mediatedmucosal immunity. Nat. Rev. Immunol. 4:699 –710.

36. 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.

37. Othoro C, Johnston D, Lee R, Soverow J, Bystryn JC, Nardin E. 2009.Enhanced immunogenicity of Plasmodium falciparum peptide vaccinesusing a topical adjuvant containing a potent synthetic Toll-like receptor 7agonist, imiquimod. Infect. Immun. 77:739 –748.

38. Bruna-Romero O, Hafalla JC, Gonzalez-Aseguinolaza G, Sano G, TsujiM, Zavala F. 2001. Detection of malaria liver-stages in mice infectedthrough the bite of a single Anopheles mosquito using a highly sensitivereal-time PCR. Int. J. Parasitol. 31:1499 –1502.

39. Nussenzweig RS. 1967. Increased nonspecific resistance to malaria pro-duced by administration of killed Corynebacterium parvum. Exp. Parasi-tol. 21:224 –231.

40. Vanderberg JP, Nussenzweig RS, Most H. 1968. Further studies on thePlasmodium berghei-Anopheles stephensi-rodent system of mammalianmalaria. J. Parasitol. 54:1009 –1016.

41. Schmidt LH, Fradkin R, Genther CS, Rossan RN, Squires W. 1982. Thecharacteristics of untreated sporozoite-induced and trophozoite-inducedinfections. Am. J. Trop. Med. Hyg. 31:612–645.

42. Vanderberg J, Nussenzweig R, Most H. 1969. Protective immunity pro-duced by the injection of X-irradiated sporozoites of Plasmodium berghei.V. In vitro effects of immune serum on sporozoites. Mil. Med. 134:1183–1190.

43. Kumar KA, Oliveira GA, Edelman R, Nardin E, Nussenzweig V. 2004.Quantitative Plasmodium sporozoite neutralization assay (TSNA). J. Im-munol. Methods 292:157–164.

44. Flanagan KL, Lee EA, Gravenor MB, Reece WH, Urban BC, Doherty T,Bojang KA, Pinder M, Hill AV, Plebanski M. 2001. Unique T cell effectorfunctions elicited by Plasmodium falciparum epitopes in malaria-exposedAfricans tested by three T cell assays. J. Immunol. 167:4729 – 4737.

45. Bates JT, Uematsu S, Akira S, Mizel SB. 2009. Direct stimulation of

Needle-Free Flagellin-CS Protein Malaria Vaccine

December 2013 Volume 81 Number 12 iai.asm.org 4361

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

Tlr5/ CD11c cells is necessary for the adjuvant activity of flagellin. J.Immunol. 182:7539 –7547.

46. Means TK, Hayashi F, Smith KD, Aderem A, Luster AD. 2003. TheToll-like receptor 5 stimulus bacterial flagellin induces maturation andchemokine production in human dendritic cells. J. Immunol. 170:5165–5175.

47. Cochrane AH, Aikawa M, Jeng M, Nussenzweig RS. 1976. Antibody-induced ultrastructural changes of malarial sporozoites. J. Immunol. 116:859 – 867.

48. Vijay-Kumar M, Carvalho FA, Aitken JD, Fifadara NH, Gewirtz AT.2010. TLR5 or NLRC4 is necessary and sufficient for promotion of hu-moral immunity by flagellin. Eur. J. Immunol. 40:3528 –3534.

49. Blander JM, Medzhitov R. 2004. Regulation of phagosome maturation bysignals from Toll-like receptors. Science 304:1014 –1018.

50. Blander JM, Medzhitov R. 2006. Toll-dependent selection of microbialantigens for presentation by dendritic cells. Nature 440:808 – 812.

51. Ciacci-Woolwine F, McDermott PF, Mizel SB. 1999. Induction of cyto-kine synthesis by flagella from gram-negative bacteria may be dependenton the activation or differentiation state of human monocytes. Infect.Immun. 67:5176 –5185.

52. Honko AN, Mizel SB. 2005. Effects of flagellin on innate and adaptiveimmunity. Immunol. Res. 33:83–101.

53. Agrawal S, Agrawal A, Doughty B, Gerwitz A, Blenis J, Van Dyke T,Pulendran B. 2003. Cutting edge: different Toll-like receptor agonistsinstruct dendritic cells to induce distinct Th responses via differentialmodulation of extracellular signal-regulated kinase-mitogen-activatedprotein kinase and c-Fos. J. Immunol. 171:4984 – 4989.

54. Potocnjak P, Yoshida N, Nussenzweig RS, Nussenzweig V. 1980. Mon-ovalent fragments (Fab) of monoclonal antibodies to a sporozoite surfaceantigen (Pb44) protect mice against malarial infection. J. Exp. Med. 151:1504 –1513.

55. Ak M, Bower JH, Hoffman SL, Sedegah M, Lees A, Carter M, BeaudoinRL, Charoenvit Y. 1993. Monoclonal antibodies of three different immu-noglobulin G isotypes produced by immunization with a synthetic peptideor native protein protect mice against challenge with Plasmodium yoeliisporozoites. Infect. Immun. 61:2493–2497.

56. Stewart MJ, Nawrot RJ, Schulman S, Vanderberg JP. 1986. Plasmodiumberghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect. Immun. 51:859 – 864.

57. Vanderberg JP, Frevert U. 2004. Intravital microscopy demonstratingantibody-mediated immobilisation of Plasmodium berghei sporozoitesinjected into skin by mosquitoes. Int. J. Parasitol. 34:991–996.

58. Kuper CF, Koornstra PJ, Hameleers DM, Biewenga J, Spit BJ, Dui-

jvestijn AM, van Breda Vriesman PJ, Sminia T. 1992. The role ofnasopharyngeal lymphoid tissue. Immunol. Today 13:219 –224.

59. van der Ven I, Sminia T. 1993. The development and structure of mousenasal-associated lymphoid tissue: an immuno- and enzyme-histochemical study. Reg. Immunol. 5:69 –75.

60. Nacer A, Carapau D, Mitchell R, Meltzer A, Shaw A, Frevert U, NardinEH. 3 July 2013. Imaging murine NALT following intranasal immuniza-tion with flagellin-modified circumsporozoite protein malaria vaccines.Mucosal Immunol. doi:10.1038/mi.2013.48.

61. Chieppa M, Rescigno M, Huang AY, Germain RN. 2006. Dynamicimaging of dendritic cell extension into the small bowel lumen in responseto epithelial cell TLR engagement. J. Exp. Med. 203:2841–2852.

62. Neutra MR, Mantis NJ, Kraehenbuhl JP. 2001. Collaboration of epithe-lial cells with organized mucosal lymphoid tissues. Nat. Immunol.2:1004 –1009.

63. Davis SS. 2001. Nasal vaccines. Adv. Drug Deliv. Rev. 51:21– 42.64. Heinonen S, Silvennoinen H, Lehtinen P, Vainionpaa R, Ziegler T,

Heikkinen T. 2011. Effectiveness of inactivated influenza vaccine in chil-dren aged 9 months to 3 years: an observational cohort study. LancetInfect. Dis. 11:23–29.

65. Przysiecki C, Lucas B, Mitchell R, Carapau D, Wen Z, Xu H, Wang XM,Nahas D, Wu C, Hepler R, Ottinger E, Ter Meulen J, Kaslow D, ShiverJ, Nardin E. 2012. Sporozoite neutralizing antibodies elicited in mice andrhesus macaques immunized with a Plasmodium falciparum repeat pep-tide conjugated to meningococcal outer membrane protein complex.Front. Cell. Infect. Microbiol. 2:146. doi:10.3389/fcimb.2012.00146.

66. Kobayashi T, Fukushima K, Sannan T, Saito N, Takiguchi Y, Sato Y,Hasegawa H, Ishikawa K. 2013. Evaluation of the effectiveness and safetyof chitosan derivatives as adjuvants for intranasal vaccines. Viral Immu-nol. 26:133–142.

67. Manocha M, Pal PC, Chitralekha KT, Thomas BE, Tripathi V, GuptaSD, Paranjape R, Kulkarni S, Rao DN. 2005. Enhanced mucosal andsystemic immune response with intranasal immunization of mice withHIV peptides entrapped in PLG microparticles in combination with UlexEuropaeus-I lectin as M cell target. Vaccine 23:5599 –5617.

68. Kataoka K, Fujihashi K, Oma K, Fukuyama Y, Hollingshead SK, SekineS, Kawabata S, Ito HO, Briles DE, Oishi K. 2011. The nasal dendriticcell-targeting Flt3 ligand as a safe adjuvant elicits effective protectionagainst fatal pneumococcal pneumonia. Infect. Immun. 79:2819 –2828.

69. Alonso PL, Brown G, Arevalo-Herrera M, Binka F, Chitnis C, Collins F,Doumbo OK, Greenwood B, Hall BF, Levine MM, Mendis K, NewmanRD, Plowe CV, Rodriguez MH, Sinden R, Slutsker L, Tanner M. 2011.A research agenda to underpin malaria eradication. PLoS Med.8:e1000406. doi:10.1371/journal.pmed.1000406.

Carapau et al.

4362 iai.asm.org Infection and Immunity

on February 19, 2021 by guest

http://iai.asm.org/

Dow

nloaded from