Polymeric Linear Peptide Chimeric Vaccine-Induced Antimalaria Immunity Is Associated with Enhanced In Vitro Antigen Loading

Abstract

Immunization of mice with Plasmodium berghei or Plasmodium yoelii synthetic linear peptide chimeras (LPCs) based on the circumsporozoite protein protects against experimental challenge with viable sporozoites. The immunogenicity of LPCs is significantly enhanced by spontaneous polymerization. To better understand the antigenic properties of polymeric antimalarial peptides, we studied the immune responses elicited in mice immunized with a polymer or a monomer of a linear peptide construct specific for P. yoelii and compared the responses of antigen-presenting cells following incubation with both peptide species. Efficient uptake of the polymeric peptide in vitro resulted in higher expression of the coactivation markers CD80, CD40, and CD70 on dendritic cells and higher proinflammatory cytokine production than with the monomeric peptide. Macropinocytosis seems to be the main route used by polymeric peptides internalized by antigen-presenting cells. Spontaneous polymerization of synthetic antimalarial-peptide constructs to target professional antigen-presenting cells shows promise for simple delivery of subunit malaria vaccines.

Malaria is globally the most devastating vector-borne disease, responsible for more than 1 million fatalities annually (37). Malaria is also one of the most deadly transmissible diseases (6). The development of a safe and effective malaria vaccine remains an urgent, yet unmet, medical need for vast populations living in areas of endemicity (6, 39). Although revived interest in malaria research and vaccine development has recently inspired a wave of clinical trials with some promising vaccine candidates, an ideal formulation remains a distant prospect (15, 39). The lack of surrogate markers to predict protection and the complexity of the parasite life cycle have hindered the development of effective vaccines. Epidemiological and clinical evidence from vaccine trials suggest that an ideal malaria vaccine should include several antigens (14, 16). However, the inclusion of multiple antigens in a single vaccine formulation is logistically challenging and requires the use of efficient delivery systems. We have used polymeric linear peptide chimeras (LPCs) as a simple and efficient alternative to deliver subunit vaccines (8, 36).

Proof-of-principle studies confirmed that immunization with LPCs containing circumsporozoite protein sequences elicited protection against malaria challenge using Plasmodium berghei and Plasmodium yoelii rodent malaria models (8, 36). LPCs are synthetic linear peptides that contain a Plasmodium promiscuous CD4⁺ T-cell epitope synthesized in tandem with B-cell and cytotoxic T-lymphocyte (CTL) epitopes. A distinguishing feature of LPCs is the inclusion of amino- and carboxyl-terminal cysteine residues that form cross-linkages for spontaneous polymerization. This results in an array of homopolymeric peptide species, easily confirmed using mass spectroscopy (8). Peptide homopolymerization is critical for antigenicity and immunogenicity (8).

To better understand the protective effects of polymeric linear peptides, we compared the immune responses of mice immunized with peptide polymer or monomeric forms of the same sequence and the antigen-presenting-cell processing of both peptide species. The monomeric form contained the same three P. yoelii-specific epitopes as the polymeric form but lacked cysteine residues necessary for polymerization to occur. The results suggested that polymerization of antimalarial-peptide constructs plays a role in peptide processing by antigen-presenting cells, peptide immunogenicity, and protection against parasite challenge.

MATERIALS AND METHODS

Parasites, animals, and cell lines.

P. yoelii 17X/MR4-267 parasites were obtained from the Malaria Research and Reference Reagent Resource Center. The complete parasite life cycle was maintained using Anopheles stephensi and sporozoites isolated from salivary glands (8). BALB/c (H-2^d) mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were females and were 8 to 10 weeks of age at the time of the first immunization. The mice were housed in microisolation cages, and all procedures were approved by Emory University's Institutional Animal Care and Use Committee.

The mouse macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (TIB-71; ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and supplemented with penicillin/streptomycin. To harvest the cells, culture flasks containing a monolayer of RAW264.7 cells were scraped using a cell scraper and washed prior to the experiments.

Polymeric and monomeric LPCs.

The topology of the LPCs has been described previously (8, 36). The sequences and molecular masses of the synthetic peptides used in this study are summarized in Table 1. All peptides were assembled using the standard 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy. LPCs and the 5(6)-carboxyfluorescein (FAM)-tagged derivatives were synthesized at the Emory Microchemical Facility. The extent of peptide homopolymerization was evaluated by surface-enhanced laser desorption/ionization mass spectrometry as previously described (8). The fluorescent derivatives were synthesized as a new batch synthesis, and FAM was introduced into the resin after assembly of all amino acids. This procedure allowed the attachment of the fluorochrome to the N terminus. The peptides were purified by reverse-phase high-performance liquid chromatography and characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

TABLE 1.

Terminology, nomenclature, amino acid sequences, and molecular masses of synthetic peptides used in the study

Terminology	Nomenclaturea	Amino acid sequenceb	Molecular mass (Da)
Polymer (LPCcys+)	cys-PyT*-PyB-PyCTL-cys	C-KQISSQLTEEWS-QGPGAPQGPGAPQGPGAP-SYVPSAEQI-C	4,139
FAM polymer (FAM-LPCcys+)	FAM-cys-PyT*-PyB-PyCTL-cys	FAM-C-KQISSQLTEEWS-QGPGAPQGPGAPQGPGAP-SYVPSAEQI-C	4,494
Monomer (LPCcys−)	PyT*-PyB-PyCTL	KQISSQLTEEWS-QGPGAPQGPGAPQGPGAP-SYVPSAEQI	3,933
FAM monomer (FAM-LPCcys−)	FAM-PyT*-PyB-PyCTL	FAM-KQISSQLTEEWS-QGPGAPQGPGAPQGPGAP-SYVPSAEQI	4,289
FAM PyT53 epitope	FAM-PyT53	FAM-TRNQIRDLSILKARLLKRK	2,681
FAM-fibronectin	FN-C/H-V	WQPPRARIGK-FAM	1,783
P. yoelii CTL epitope	PyCTL	SYVPSAEQI	993
PvT53PfB control	PvT53-PfB	SKDQIKKLTSLKNKLERRQN-NANPNANPNANP	3,617
Human immunodeficiency virus V3 loop control	V3	TRPNNNTRRRLSIGPGRAFYARR	2,729

^aT* and T53, T-cell epitope; B, B-cell epitopes; CTL, CD8⁺ CTL epitope; Py, P. yoelii; Pv, Plasmodium vivax; Pf, Plasmodium falciparum.

^bIndividual epitopes are separated by hyphens. FAM indicates the FAM-tagged peptide.

Vaccination and challenge infection.

Groups of 10 female BALB/c mice were subcutaneously immunized with 50 μg of P. yoelii-specific cys-PyT*-PyB-PyCTL-cys polymer (LPC_cys+) or PyT*-PyB-PyCTL monomer (LPC_cys−) emulsified in Montanide ISA51 (Seppic Inc., Fairfield, NJ). The immunizations were conducted on days 0, 20, and 40. Placebo groups received phosphate-buffered saline (PBS) formulated with the same adjuvant. Four mice from each group were euthanized 20 days after the first immunization, and their pooled spleen cells were used for determining peptide-specific cytokine-secreting cells using enzyme-linked immunospot (ELISPOT) assays. Twenty days after the last immunization, the remaining mice were challenged by intravenous inoculation of 100 P. yoelii 17X sporozoites, dissected from the salivary glands of A. stephensi mosquitoes and resuspended in 200 μl of sterile RPMI. This parasite inoculum, in our hands, infected 100% of naïve BALB/c mice. To control for sporozoite viability, naïve mice were also included in the challenge.

The inhibition of liver stage development was determined 40 h after challenge by quantification of the parasite burden using real-time PCR as described previously (7, 40). Briefly, total RNA from the livers was treated with RQ1 DNase (Promega) and reverse transcribed with a High Capacity cDNA Reverse Transcription Kit and random hexamers (Applied Biosystems, Foster City, CA). Five microliters of cDNA, corresponding to 250 ng of total RNA, was used as the template for the real-time PCR amplifying P. yoelii 18S rRNA (iCycler; Bio-Rad Laboratories, Hercules, CA). The following primers were used for specific amplification: 5′-GCTCGTAGTTGAACTTCAAGGGTA-3′ and 5′-GCAACGAGGCATGAGATATCGA-3′. A TaqMan MGB (minor groove binding) probe linked to a 6-FAM reporter dye at the 5′ end and a nonfluorescent quencher at the 3′ end (5′-CTTGGCTAGATTCTTGGCTCC-3′) was incorporated into the PCR mixture to detect the generated PCR product. We included reactions amplified with β-actin primers and probe (Applied Biosystems) as positive controls and normalizing references. The profile of the reaction was 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min.

Peptide priming and P. yoelii parasite boosting.

P. yoelii-infected mosquitoes taken 12 days after an infectious blood meal were irradiated with 10,000 rads of gamma radiation from a ¹³⁷Ce source (Gammacell 3001; MDS Nordion, Vancouver, Canada). For isolation of salivary gland sporozoites, infected mosquitoes were sequentially washed in 70% ethanol, medium 199 medium containing amphotericin B (Fungizone) (5%), and medium 199 containing penicillin and streptomycin (5%) and placed on a glass slide. The salivary glands were dissected and homogenized on ice. The irradiated sporozoites were diluted in medium containing penicillin and streptomycin without serum. Mice were primed with 50 μg of LPC_cys+ or with adjuvant alone, followed by three boosting immunizations of 20,000 irradiated sporozoites 1 week apart.

Antibody reactivity.

The specificities of the antibodies elicited by immunization with peptides were determined by enzyme-linked immunosorbent assay using Immulon 4HB plates (Thermo Labs Systems, Franklin, MA) coated with 1 μg/ml of the corresponding synthetic peptide diluted in PBS. Wells were blocked with 5% bovine serum albumin in PBS and 0.05% Tween 20 for 2 h at 37°C. Serum dilutions in PBS with 2.5% bovine serum albumin and 0.05% Tween 20 were added in duplicate. The plates were incubated for 1 h at 37°C, and bound antibodies were detected using peroxidase-labeled goat anti-mouse total immunoglobulin G and H₂O₂/2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as a substrate (KPL, Gaithersburg, MD). Optical densities were determined using a Versamax enzyme-linked immunosorbent assay reader (Molecular Device Corporation, Sunnyvale, CA) with a 405-nm filter. Endpoint titers, defined as the last serum dilution having an optical density greater than 3 standard deviations above the mean optical density of preimmune sera, were determined using sera obtained from six mice in each group 20 days after the first immunization or 40 h after challenge, when the animals that received three immunizations were euthanized for parasite load quantification.

Sera obtained from BALB/c mice after the third immunization with 50 μg of LPC_cys+ or LPC_cys− were pooled, and the antibody reactivity against native protein was evaluated by indirect immunofluorescence using air-dried P. yoelii sporozoites. Several dilutions were tested, and the reactivity was evaluated using fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (KPL, Gaithersburg, MD) diluted in PBS-0.4% Evans Blue dye.

Quantification of cytokine production.

The frequency of peptide-specific T cells was determined by gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-specific ELISPOT assays. Spleen cells obtained from groups of mice 20 days after first or third immunizations were tested. ELISPOT assays were performed ex vivo in nitrocellulose microplates (Millipore, Bedford, MA) coated with rat capture anti-mouse IFN-γ or rat capture anti-mouse IL-4 (BD Biosciences Pharmingen, San Diego, CA) following the instructions from the manufacturer. Freshly isolated spleen cells from four mice were pooled, and duplicate aliquots of different concentrations (1 × 10⁶ and 5 × 10⁵ cells/ml) were plated in RPMI containing 10% fetal calf serum. T helper cells were activated by addition of LPCs (10 μg/ml) or synthetic peptides representing P. yoelii-specific B-cell, universal T-cell, or CTL epitopes (8). After 24 h for IFN-γ or 48 h for IL-4, the plates were washed and incubated with biotinylated rat anti-mouse IFN-γ or biotinylated rat anti-mouse IL-4 (BD Biosciences Pharmingen), followed by incubation with streptavidin-horseradish peroxidase. The reaction was developed using 3-amino-9-ethylcarbazole (BD Biosciences Pharmingen) and evaluated using an immunospot analyzer (Cellular Technology-Becton Dickinson, San Diego, CA). Concanavalin A (Sigma Aldrich Corp., St. Louis, MO) was included as a positive control for cellular activation. The results were expressed as the total number of spot-forming cells (SFC) per 10⁶ splenocytes. IL-2, IL-5, and IL-10 were also determined in 48-h supernatants using cytokine multiplex analysis (Pierce SearchLight technology; Pierce Boston Technology, Woburn, MA).

Bone marrow-derived DCs.

Bone marrow-derived dendritic cells (DCs) were generated as described by Inaba et al. (18). In brief, following erythrocyte lysis, bone marrow cells from femur and tibia were cultured at 10⁶ cells/ml in serum-free Cellgro medium (Cellgenix, Freiburg, Germany) supplemented with 2 mM l-glutamine, 10 mM HEPES, 100 U/ml penicillin, 10 μg/ml streptomycin, and 20 ng/ml murine granulocyte-macrophage colony-stimulating factor (R&D Systems). The medium was replaced on days 2 and 4, and the cells (immature DCs) were harvested on day 6. DCs were purified using bead-conjugated anti-CD11c monoclonal antibody (clone N418), followed by positive selection through a paramagnetic column (MACS system; Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's protocol. Using this immunomagnetic cell-sorting approach, we consistently obtained over 95% pure populations of DCs as determined by flow cytometry.

DCs were incubated at 37°C for 48 h with LPC_cys− or LPC_cys+ (20 μg/ml) in serum-free medium. The cell culture supernatant was collected for quantification of cytokine production as described above for T lymphocytes. To evaluate the expression of maturation markers, DCs were washed, incubated with Fc-block for 30 min, washed again, and incubated for 25 min at 4°C with FITC-anti-mouse CD40 (clone HM40-3; eBioscience, San Diego, CA); allophycocyanin-anti-mouse CD86 (clone GL1; eBioscience, San Diego, CA); FITC-anti-mouse CD80 (clone 16-10A1; eBioscience, San Diego, CA); allophycocyanin-anti-mouse I-A/I-E (clone M5/114.15.2; eBioscience, San Diego, CA); and phycoerythrin (PE)-anti-mouse CD11c (clone N418; eBiosceince, San Diego, CA). The cells were washed three times and analyzed on a FACScalibur flow cytometer (Becton Dickinson).

Peptide uptake and cell transduction.

Splenocytes (5 × 10⁵ cells per well) derived from naïve BALB/c mice were incubated for 2 h at 37°C in RPMI medium containing fluorochrome-tagged LPCs (10 μg/ml) in the presence or absence of anti-major histocompatibility complex (MHC) class I at 1 μg/10⁶ cells (clone A5A1-Do4; Abcam, Cambridge, MA) and/or anti-MHC class II monoclonal antibodies at 0.5 μg/10⁶ cells (clone M5/114.15.2; Abcam Cambridge, MA). Anti-MHC class I and class II blocking monoclonal antibodies were used to determine the abilities of anti-MHC antibodies to modify peptide internalization. After extensive washing using RPMI supplemented with 10% FBS, the cells were incubated with either PE-labeled anti-mouse CD11c (clone HL3; Pharmingen, San Jose, CA), PE-CD14 (clone rmC5-3; Pharmingen, San Jose, CA), PE-CD19 (clone 1D3; Pharmingen, San Jose, CA), or PE-CD3 (clone 17A2; Pharmingen, San Jose, CA) at 4°C for 30 min. Peptide uptake was evaluated by flow cytometry and expressed as a percentage of viable FAM-labeled cells.

To characterize the effects of temperature on cell peptide uptake, RAW 264.7 cells (0.5 × 10⁶ cells/well) were washed and incubated at 4°C or 37°C for 2 h with FAM-labeled LPC_cys− or FAM-labeled LPC_cys+ (10 μg/ml). After three washes in Dulbecco's modified Eagle's medium (with 10% FBS), the cells were trypsinized (0.25% in EDTA) for 6 min at 37°C, washed once with PBS, and analyzed by fluorescence-activated cell sorter (FACS). To evaluate the effects of specific treatments, RAW264.7 cells were pretreated for 30 min at 37°C with chloroquine (200 μM; Sigma), heparinase III (1.0 U; Sigma), cytochalasin D (10 μM; Sigma), or nystatin (50 μg/ml; Sigma) or for 60 min with amiloride (0.2 mM; Sigma). FAM-labeled LPC_cys− or LPC_cys+ was added at a concentration of 10 μg/ml and incubated for an additional 2 h. After incubation, RAW264.7 cells were treated with trypsin to remove peptides bound to the cell surface (32). The vital dye propidium iodide was added to the cell suspension before FACS analysis.

For confocal studies, DCs were plated at a density of 5 × 10⁵ cells/well in eight-well chamber glass slides (LAB-Tek; Nalge Nunc International, Rochester, NY), maintained at 37°C for 40 min to allow cell adhesion, and cooled on ice for 40 min. The cells were incubated with FAM-labeled LPC_cys− or LPC_cys+ (10 μg/well) for 40 min at 37°C and washed twice in warm PBS and once in PBS with trypan blue (0.001%) to quench the fluorescence of the noninternalized peptides (24). The cells were fixed for 10 min at room temperature in PBS with 4% paraformaldehyde, pH 7.2. After fixation, cholera toxin B labeled with Alexa Fluor 594 (2 μg/ml; Molecular Probes-Invitrogen, Carlsbad, CA), rhodamine-phalloidin (3 μl/ml), or Syto 62 red fluorescence (6 μl/ml; Molecular Probes-Invitrogen, Carlsbad, CA) was used to label plasma membranes or nuclei. The cells were mounted in Vectashield (Vector, Burlingame, CA) and examined under a 100× oil immersion objective on a Zeiss LSM 510 confocal microscope. Images were analyzed using Zeiss LSM Browser. For studies of endosomal colocalization, DCs (1 × 10⁶ cells/well) were incubated for 2 h with labeled LPC_cys− or LPC_cys+ (10 μg/well), washed extensively, and exposed for 30 min to LysoTracker Red (50 nM; Molecular Probes-Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For live cells, confocal images were acquired using a Zeiss LMS 510 inverted confocal microscope.

Statistics.

Student t tests were used to compare values for cytokine production, cellular uptake, and expression of costimulatory molecules for cells stimulated in vitro with LPC_cys− or LPC_cys+. Parasite loads in the livers of mice immunized with LPC_cys− or LPC_cys+ were compared with those of nonimmunized control mice by analysis of variance. P values of <0.05 were considered significant. To measure the effects of specific treatments on peptide uptake, data were normalized to calculate a relative mean fluorescence channel (MFC) as follows: MFC = (MFC for cells incubated with LPCs + inhibitor/MFC for cells incubated with LPC at 37°C) × 100.

RESULTS

Comparative immunogenicities of monomeric and polymeric LPCs.

In mice immunized with polymeric LPC_cys+, antibody titers were fourfold higher than in mice immunized with monomeric LPC_cys− after priming and more than 1,200-fold higher following parasite challenge (Table 2). A similar trend was observed when serum samples were tested with LPC_cys− (Table 2). By immunofluorescence assay, mice immunized with polymeric LPC_cys+ had antibody titers of 1:3,200 versus <1:100 in mice immunized with monomeric LPC_cys− (data not shown).

TABLE 2.

Immune responses elicited in BALB/c mice after immunization with monomeric or polymeric peptides

Immunogen	Stimulation	After first immunization				After challenge
		Antibody titera	ELISPOTb		Cytokine (Δpg/ml)c (IL-2)	Antibody titera	Protection (%)d	ELISPOTb		Cytokine (Δpg/ml)c
			IFN-γ	IL-4				IFN-γ	IL-4	IL-2	IL-5	IL-10
LPCcys−	LPCcys+	508 ± 165	15 ± 4	12 ± 9	0	1,280 ± 0	0	1,289 ± 769	191 ± 38	52.2 ± 2	0	0
	LPCcys−	452 ± 175	47 ± 18	23 ± 12	22.4 ± 6	1,280 ± 0	0	3 ± 4	16 ± 9	21.5 ± 1	0	2 ± 3
	PyCTL				0		0			0	0	0
LPCcys+	LPCcys+	2,032 ± 1,537	14 ± 3	16 ± 3	20.4 ± 16	1,558,718 ± 655,360	53 ± 15	2,106 ± 104	192 ± 77	98.2 ± 2	296.2 ± 1	69.8 ± 1
	LPCcys−	806 ± 303	22 ± 5	18 ± 5	4 ± 4	183,904 ± 10,031	53 ± 15	0	10 ± 6	0	82.7 ± 5	0
	PyCTL				9.7 ± 7		53 ± 15			69 ± 46	2.2 ± 1	0

^aTotal immunoglobulin G antibody titers determined by ELISA using serum samples obtained 20 days after priming or 40 h after challenge. Comparative results of anti-peptide reactivity were evaluated using LPC_cys+ or LPC_cys−. The data are presented as the median reciprocal final antibody titer obtained from six mice per group tested individually ± standard deviation (differences in antibody titers after priming, P < 0.01; differences after challenge, P < 0.0001).

^bNumber of IFN-γ− or IL-4-secreting cells as determined by ELISPOT assay after priming or 40 h after challenge using ex vivo stimulation of spleen cells with LPC_cys+ or LPC_cys− peptide. The data are presented as mean numbers of SFC per 10⁶ spleen cells pooled from five mice and tested in quadruplicate ± standard deviations (differences in IFN-γ-secreting cells after challenge, P = 0.03).

^cCytokine production was measured after 48 h of peptide stimulation in culture supernatants from a pool of splenocytes obtained from five mice. The supernatants were tested in duplicate using ex vivo stimulation with LPC_cys+, LPC_cys−, or PyCTL. The data are expressed in Δpg/ml ± standard deviation and represent the cytokine levels obtained after peptide stimulation subtracted from baseline levels obtained with medium alone (differences in cytokine production after challenge, P = 0.013).

^dComparative evaluation of protective efficacy in mice immunized with LPC_cys− or LPC_cys+ after challenge with P. yoelii sporozoites. The numbers represent percentages of parasite load inhibition calculated by comparison of P. yoelii liver stage parasite rRNA copies obtained in peptide-immunized mice with copy numbers in mice immunized with adjuvant alone ± standard deviations. The data summarize the results of one out of two replicate experiments using groups of five mice, which showed similar outcomes (P = 0.016).

Cellular immune responses induced by a single immunization were modest, with low numbers of LPC-specific IFN-γ- and IL-4-secreting cells in both groups (Table 2). IL-2 was identified in 48-h culture supernatants of splenocytes obtained from mice immunized with LPC_cys+ in response to both LPC peptide species and PyCTL. This is in contrast to production of IL-2 only after ex vivo stimulation with LPC_cys− in animals immunized with the monomer. After parasite challenge, cytokine production by splenocytes obtained from mice immunized with LPC_cys+ showed a mixed Th1/Th2 response, with production of IFN-γ, IL-4, IL-2, IL-5, and IL-10 when the cells were stimulated with LPC_cys+ but not with the monomeric form. Consistent with differences in antigenic properties between LPC_cys+ and LPC_cys−, splenocytes derived from mice immunized with the monomeric peptide produced IFN-γ, IL-4, and IL-2 after ex vivo stimulation with LPC_cys+ but not with the autologous immunogen. Interestingly, IL-4, IL-2, and IL-5 recall responses to PyCTL were also detected in mice immunized with the polymeric peptide. Using intracellular staining for flow cytometry, we confirmed that LPC_cys+-specific IFN-γ-secreting cells in the spleens of LPC_cys+-immunized mice were CD4⁺ (data not shown). More relevantly, after experimental challenge with viable sporozoites, mice immunized with LPC_cys+ had significantly lower parasite loads in the liver than mice immunized with LPC_cys− (P < 0.05) (Table 2).

The high prevalence of peptide-specific IFN-γ-secreting cells induced by immunization with the polymeric peptide after sporozoite challenge suggested proliferation and/or compartmentalization in response to the parasite exposure. To evaluate the magnitude of the recall response elicited by P. yoelii sporozoites, we quantified the peptide-specific IFN-γ T cells in the livers or spleens of mice primed with LPC_cys+ and subsequently exposed to irradiated sporozoites. Boosting of LPC_cys+-immunized mice with irradiated sporozoites resulted in a T-cell-mediated recall response. Following the unique exposure to irradiated sporozoites, the prevalence of LPC-specific or CTL-specific T cells that secreted IFN-γ was higher in the livers and spleens of mice primed with LPC_cys+ than in those exposed to irradiated sporozoites without peptide priming (Table 3) (P = 0.032). The number of LPC-specific IFN-γ-secreting cells was three times higher in the liver than in the spleen.

TABLE 3.

Compartmentalization of the T-cell recall response to irradiated P. yoelii sporozoites

Compartment	Peptide priminga		Montanidea
	LPCcys+	PyCTL	LPCcys+	PyCTL
Spleen	387 ± 0.5	481 ± 51	108 ± 0.5	215 ± 4
Liver	1037 ± 31	1750 ± 658	150 ± 5	560 ± 80

^aTotal number of IFN-γ-secreting cells ± standard deviation in liver or spleen compartments of BALB/c mice as determined by ELISPOT assay. The mice were primed with LPC_cys+ and subsequently boosted with irradiated P. yoelii sporozoites as described in Materials and Methods. The results are compared with data obtained with mice that received adjuvant alone before the exposure to irradiated P. yoelii sporozoites. The data are presented as mean numbers of SFC per 10⁶ spleen cells or liver mononuclear cells pooled from four mice and tested in quadruplicate (P = 0.032).

LPC uptake by DCs.

The two main populations of splenocytes involved in peptide uptake were CD14⁺ or CD11c⁺ (Fig. 1). After peptide incubation in the presence of anti-MHC class I and anti-MHC class II monoclonal antibodies, 35.1% of CD14⁺ splenocytes took up polymeric LPC_cys+ versus 29.3% for monomeric LPC_cys−. For CD11c⁺ splenocytes, 25.3% took up polymeric LPC_cys+ versus 18.8% for monomeric LPC_cys−. CD3⁺ and CD19⁺ splenocytes were able to preferentially take up polymeric LPC_cys+ in comparison with the monomeric LPC_cys− (CD3⁺, 6.89% versus 1.59%, P = 0.008; CD19⁺, 5.23% versus 2.61%, P = 0.004). Peptide uptake was independent of association with MHC molecules, since the addition of blocking monoclonal antibodies did not abolish this activity. The FAM-tagged synthetic peptide FN-C/H-V, derived from the heparin-binding domain of fibronectin, which has been reported to interact with cell surface proteoglycans (21, 27, 29), was used as a positive control.

FIG. 1.

Flow cytometry analysis of the cellular internalization of LPC_cys+ or LPC_cys− synthetic peptides. Splenocytes derived from naïve mice were incubated with FAM-labeled LPC_cys+ polymer or FAM-labeled LPC_cys− monomer at 10 μg/ml for 2 h in the presence of anti-MHC class I and anti-MHC class II blocking antibodies. Cells were then incubated with either anti-mouse PE-CD3, PE-CD11c, PE-CD14, or PE-CD19. Peptide uptake was evaluated by flow cytometry and expressed as a percentage of FAM-labeled cells plus standard deviation of two independent experiments. The cells were incubated with LPC_cys− (filled bars), LPC_cys+ (hatched bars), or fibronectin-derived positive control peptide (open bars).

LPC_cys+ induced upregulation of cell surface markers of DC maturation (CD40, CD80, and CD70) on CD11c⁺ cells (Fig. 2) (P < 0.05). Consistent with these results, secretion of monocyte chemoattractant protein 1 (MCP-1), IL-6, IL-12p70, and tumor necrosis factor alpha (TNF-α) by CD11c⁺ DCs was higher following incubation with polymeric LPC_cys+ than with LPC_cys− (Fig. 3) (P = 0.02).

FIG. 2.

Expression of maturation surface molecules on DCs after incubation with LPC_cys− (filled bars) or LPC_cys+ (hatched bars). The monomeric peptides PvT53PfB (open bars) and V3 (shaded bars) were included as negative controls. The synthetic peptide Pv53PfB, which contains a promiscuous Plasmodium vivax T-cell epitope and a Plasmodium falciparum B-cell epitope, has been previously described (9). The V3 peptide represents a B-cell epitope derived from the gp120 surface glycoprotein of human immunodeficiency virus (23). Bone marrow-derived DCs from BALB/c mice were incubated for 48 h with the corresponding peptide at 20 μg/ml, using serum-free medium. After incubation, cells were collected; stained for CD80, CD40, I-A/I-E, CD70, or CD86; and analyzed by FACS. The data represent mean relative fluorescence units plus standard deviations from two independent experiments. *, P < 0.05.

FIG. 3.

DC cytokine production after incubation with LPC_cys− (filled bars) or LPC_cys+ (hatched bars) synthetic peptides. MCP-1, IL-6, IL12p70, and TNF-α were determined in cell-free medium 48-h supernatants using multiplex protein assays. The results are plotted as the mean cytokine concentration plus standard deviation. The values obtained with medium alone were included for comparison (open bars). The mean cytokine concentrations determined in supernatants from cells stimulated with lipopolysaccharide, as a positive control, were as follows: MCP-1, 58.9 ± 24; IL-6, 36,667 ± 5,248; IL12p70, 31 ± 6; TNF-α, 6,504 ± 599.

Incubation with fluorescence-labeled polymeric LPC_cys+ resulted in a punctuated fluorescence pattern in CD11c⁺ DCs (Fig. 4A and B). After membrane translocation, the peptide was localized in vesicular compartments in the cytoplasm, suggesting an endocytic pathway of internalization. Only polymeric LPC_cys+ colocalized with lysosomes (Fig. 4C and D). Polymeric LPC_cys+ (Fig. 4B), but not LPC_cys− (data not shown), colocalized completely with cholera toxin B, suggesting that the peptide is localized in lipid rafts.

FIG. 4.

Cytoplasmic localization in DCs of FAM-labeled peptides after internalization as determined by confocal microscopy. Highly purified and viable DCs were treated with FAM-LPC_cys+ or FAM-LPC_cys− derivatives for 40 min at 37°C. The cells were then washed and fixed. After fixation, the cells were labeled with rhodamine-phalloidin (F-actin) (A) or cholera toxin B labeled with Alexa Fluor 594, which binds the GM1 glycosphingolipid (B), and Syto 62 (blue nuclear staining in panels labeled Merge [A and B]). Live DCs loaded with FAM-LPC_cys+ (C) or FAM-LPC_cys− (D) were washed and incubated with LysoTracker Red (endosomal marker).

Peptide uptake at 4°C versus 37°C was inhibited for monomeric LPC_cys− and polymeric LPC_cys+ (P < 0.05) (Fig. 5). LPC_cys+ uptake by RAW264.7 cells was decreased by following pretreatment with chloroquine, an inhibitor of endosomal acidification, indicating that phagocytosis is involved in LPC uptake (Fig. 5). The effect of pretreatment with cytochalasin D suggests that transduction of LPC is F-actin dependent (Fig. 5). Pretreatment with heparinase III, nystatin, and amiloride had no effect. We hypothesized that lipid rafts mediate the transduction of LPC.

FIG. 5.

Uptake and endocytosis inhibition of FAM-LPC_cys− or FAM-LPC_cys+ peptides in RAW264.7 cells as determined by FACS analysis. RAW264.7 cells were incubated for 2 h at 4°C with FAM-LPC_cys− (filled bars) or FAM-LPC_cys+ (hatched bars). Endocytic inhibition was performed by pretreating the cells with 200 μM of chloroquine, 1.0 U of heparinase III, 10 μM of cytochalasin D, 50 μg/ml of nystatin, and 0.2 mM of amiloride. Untreated cells incubated at 37°C were used as control cells. The data represent relative mean fluorescence units from three representative experiments. *, P < 0.05.

DISCUSSION

Polymeric synthetic peptide constructs show promise for efficient delivery of subunit malaria vaccine antigens (9). Spontaneous polymerization and inclusion of Plasmodium universal T-cell epitopes are distinguishing features of the peptides that confer protection against parasite challenge in the rodent malaria model. Enhanced uptake of the polymerized peptide by antigen-presenting cells accounts for the improved immunogenicity of polymeric peptide compared to the homologous monomer. While both the polymer and the monomer translocate in vitro to the cytoplasm of antigen-presenting cells, only the polymer colocalized with lysosomal structures. Synthetic polymerized peptide upregulated cytokine production and expression of cell surface maturation markers by DCs. Polymerization of linear peptides improved peptide presentation and could be used to enhance vaccine potency.

Although it is difficult to demonstrate in vivo, the ability of P. yoelii polymeric peptide to be efficiently internalized by professional antigen-presenting cells may be responsible for the enhanced immune response to this form. Targeting of antigen-presenting cells is a desired characteristic in viral and cancer vaccine candidates (12). Processes involved in the translocation of the polymeric versus monomeric forms of the P. yoelii-specific peptide through cellular membranes of antigen-presenting cells could explain the significant differences observed in vivo regarding protection from parasite challenge. The kinetics of polymeric-peptide uptake contrast markedly with those of the homologous monomer lacking cysteine residues required for spontaneous polymerization. Low concentrations of polymeric peptide required for efficient uptake are consistent with receptor-mediated endocytosis, rather than pinocytosis, which would require higher protein concentrations (31, 33). The polymeric peptide is rapidly accumulated in perinuclear vesicles that can be compartmentalized with cholera toxin B, suggesting interaction with lipid raft proteins (30). Receptor-mediated endocytosis and recruitment of proteins to lipid rafts in DCs are mechanisms associated with cell activation (1). One hypothesis is that monomeric peptides are presented mainly by nonprofessional antigen-presenting cells, in contrast to the targeting of these cells by polymeric peptides. Similar mechanisms have been associated with immune tolerance observed with short synthetic peptides in a variety of vaccination models (5, 38). To test the hypothesis that enhanced loading of antigen-presenting cells explains improved antimalarial immune responses in vivo, it will be important to compare protective immune responses in naïve mice injected with DCs pulsed with polymeric or monomeric peptides.

Protection using irradiated sporozoites, genetically attenuated sporozoites, or long synthetic peptides has been correlated with efficient induction of a heterogeneous pool of IFN-γ-secreting T cells (4, 19, 26). Immunization with a P. yoelii-specific preerythrocytic peptide polymer, followed by experimental challenge, resulted in massive expansion of IFN-γ-secreting cells in the spleens of immunized mice and reduction of the parasite burden in the livers of LPC_cys+-immunized mice. Immunization with monomer did not result in expansion of peptide-specific IFN-γ-secreting cells and was not protective. In addition, priming with P. yoelii-specific polymeric peptide, followed by exposure to irradiated sporozoites, stimulated the expansion of peptide-specific T cells in the livers of immunized mice. This indicates that exposure to parasites initiated a compartmentalized recall response, a critical aspect of antimalarial immunity, in animals immunized with synthetic peptide constructs (17).

Synthetic peptides have traditionally been considered poor immunogens that require complex stoichiometry or potent adjuvant formulations to induce a robust immune response. We have used spontaneous polymerization to enhance the immunogenicity of subunit vaccines. The P. yoelii-specific peptide shares features with a family of compounds known as cell-penetrating peptides (CPPs) (32) that can deliver a diverse repertoire of bioactive molecules (25). CPPs are linear sequences ranging between 10 and 30 amino acids that contain positively charged residues (tryptophan, arginine, or phenylalanine). Peptide uptake is enhanced by the interaction of positively charged residues with negatively charged membranes, resulting in endocytosis (3). In contrast with CPPs, the P. yoelii peptides tested here contain a single tryptophan with an isoelectric point (pI) of 3.96. This is in sharp contrast with the representative CPP penetratin, with a pI of 12.7. P. yoelii-specific polymeric peptides do not share the cationic characteristics described for CPPs, suggesting a different mechanism for membrane translocation. Prediction of secondary structure suggests that P. yoelii-specific polymeric peptides have amino- and carboxyl-terminal helices separated by a random-coil section, compared with a single amino-terminal helix for the monomeric form. Similar structural features have recently been reported for a compound with CPP-like function (28). The P. yoelii-specific polymeric peptide may be useful as a carrier to deliver different bioactive products to the cytoplasm.

Recent studies have shown that metabolic inhibitors, low temperature, and known endocytic inhibitors could inhibit the transduction of cationic CPPs (11, 20). Our data indicate that uptake for both compounds was substantially reduced at low temperatures, suggesting a mechanism of endocytosis, a polyfunctional mechanism that allows cells to internalize macromolecules into transport vesicles derived from the plasma membrane (10). Endocytosis involves several pathways and can be mediated by several membrane receptors that trigger dramatic changes in the plasma membrane morphology and composition (13, 34). Heparinase III treatment did not reduce the translocation rate, suggesting that the synthetic peptides do not use sulfated proteoglycans. Furthermore, the cell translocation process of monomeric and polymeric peptides was not modified by cholesterol depletion using nystatin, suggesting that the process is not mediated by caveola internalization. In contrast with the effect on monomeric-peptide internalization, both cytochalasin D and chloroquine inhibit the internalization of the polymeric peptide. These experiments suggest that the polymeric peptide uses a lipid raft-dependent entry. Interestingly, the use of amiloride, a lipid raft disruption agent, improved cell uptake for both peptides. Overall, our data indicate that an endocytic pathway independent of Na⁺/H⁺ exchange depletion is functional. Confocal-microscopy studies of colocalization of the polymeric peptide with cholera toxin B support a process of internalization mediated by lipid raft macropinocytosis (2, 22, 35).

In conclusion, the P. yoelii polymeric LPC characterized in this report has CPP capability. This feature would explain the significant improvement in immunogenicity compared with the monomeric peptide species. Our study provides a framework for future development of peptide chimeras for use as delivery systems for subunit vaccines.