1 Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA
2 Immunology Group, Institute of Chemistry and Biochemistry, University of Salzburg, Salzburg, Austria
3 Dermatology Branch, NCI/NIH, Bethesda, MD 20892, USA
4 Department of Entomology and 5 Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD, 20910 USA
Correspondence to: G. C. Tsokos; E-mail: gtsokos{at}usuhs.mil
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Abstract |
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Keywords: complement, host defense, malaria, T lymphocytes
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Introduction |
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The need to enhance the efficacy of DNA vaccination has led to the development of a variety of strategies, most notably the addition of molecular adjuvants designed to enhance the function and activation of antigen-presenting cells (APC). B cells are powerful APC because they can capture and accumulate antigen by using their antigen-specific surface Ig. A variety of factors and signals initiate and enhance B cell APC function and thus represent potential adjuvants. Among these is C3d, which is generated from C3 during complement activation, and binds to complement receptor 2 (CR2, CD21), on mature B cells. C3d-mediated B cell activation occurs as a consequence of simultaneous engagement of the antigen-specific surface-bound Ig and CD21 by antigen/C3d complexes (47). The crucial role of this signaling process is reflected by impaired B cell responses in mice lacking either C3 (8) or CR2 (9, 10). Recently, it has been demonstrated, however, that as an adjuvant, C3d can also function independently of CD21/CD35 (11).
Fusion of two or three tandem copies of C3d to recombinant hen egg lysozyme (HEL) has been shown to enhance dramatically the immune response to HEL (12). This approach was subsequently used successfully to enhance the efficacy of DNA vaccines. Indeed, constructs encoding genes for pathogen-derived antigens linked in tandem to sequences encoding for C3d delivered more protective immune response than constructs without the C3d (1316).
In the present study, we investigated the potential adjuvant effect of C3d in a DNA vaccine against P. berghei malaria. Using a previously established CSP-based DNA vaccine known as CSP(-A) (3) we asked whether the use of C3d as molecular adjuvant would accelerate the onset of the immune response and induce protective immunity after a single immunization. However, in contrast to previous reports for other infection models (1316), we observed a significant reduction of protection against a challenge with sporozoites when two copies of C3d were attached to the CSP(-A) gene, even after three immunizations. Our experiments show that C3d associates with the C-terminus of CSP, shifts the immune response to a Th1-type response, limits the humoral response against C-terminal epitopes and significantly reduces protection against P. berghei infection. In summary, this study (i) demonstrates that the fusion of antigens to C3d is not a generally applicable adjuvant strategy, (ii) clearly shows that CSP-based protective immunity against P. berghei is Th2 and not Th1 mediated and (iii) provides compelling evidence for a novel escape mechanism of the malaria parasite, involving components of the complement system.
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Methods |
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The murine C3d DNA fragment was amplified by PCR from a plasmid containing the C3 gene, which was a gift from John Lambris (University of Pennsylvania). The forward primer (gggagatctacccccgcaggctctggg) added a unique BglII site at the 5' end and inactivated the thioesterification site by replacing the TGT (Cys) with TCT (Ser). The reverse primer (ggggatccggggaggtggaaggacacatcc) added a unique BamHI site at the 3' end. A precursor plasmid WR65/00/C3d was prepared, ligating the C3d PCR product that was digested with BglII and BamHI, into the WRG6518 vector (1), and the insert was sequenced. A precursor plasmid [pCI-TPA/1C3d(pre)] containing a single copy of C3d was prepared by excising the C3d fragment from WR65/00/1C3d with BamHI and BglII and inserting it into pCI-TPA(-EEF motif) digested with BglII. Adding a second copy of C3d required first inserting a (G4S)2 linker (12) into WR65/00/1C3d digested with HindIII and BamHI, generating WR65/00/1C3d+(G4S)2. The two complementary oligonucleotides used to make the linker were gatctggaggaggaggctccggaggaggaggctccggatccgaattca and agcttgaattcggatccggagcctcctcctccggagcctcctcctcca; they added unique 5' BglII and 3' HindIII sites. The pCI-TPA/3C3d(pre) plasmid, which contains three copies of C3d, was prepared by excising the C3d+(G4S)2 fragment from WR65/00/1C3d+(G4S)2 by digesting with BamHI and BglII and inserting it into pCI-TPA/C3d(pre) digested with BglII, making pCI-TPA/2C3d(pre). This process was repeated from pCI-TPA/2C3d(pre), making pCI-TPA/3C3d(pre). The CS/3C3d chimeric plasmid was created by excising the CSP fragment from the CSP(-A) plasmid with BamHI and inserting it into pCI-TPA/3C3d(pre) digested with BglII. The plasmid used for expressing three tandem copies of C3d (p3C3d) was prepared by cutting pCI-TPA/3C3d(pre) with BamHI and BglII and re-ligating. Plasmids for immunization studies were prepared by Genoquest Inc. (Gaithersburg, MD, USA) using Endo Free Plasmid Giga Kits (Qiagen, Valencia, CA, USA). Purified plasmid DNA was stored in TE(Tris-EDTA[Ethynelo-Diamine-Tetraacetate]) buffer at 20°C.
Expression of recombinant CSP in Escherichia coli and protein purification
The glutathione (GSH)-S-transferase (GST)-fusion expression vector, pGEX-6P, was obtained from Pharmacia. A 48-mer oligonucleotide that codes for six consecutive histidines for nickel chelate affinity chromatography was synthesized containing BamHI and HindIII termini. The pGEX-6P vector was digested with BamHI and HindIII, the oligonucleotide linker was ligated into the expression vector and the reconstructed His-tag-modified pGEX vector was sequenced. The P. berghei CSP gene was sub-cloned from the expression vector pCI-TPA/PbCSP using BamHI into the pGEX-His6 vector (3). The PbCSP expression vector was identified as pGST/PbCSP-His. Escherichia coli BL21 (DE3) cells were transformed and expression was tested.
Protein was purified using a two-step affinity chromatography method based on GSHSepharose 4 Fast Flow (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and Ni+2-nitrilo-tri-acetic acid Superflow (Qiagen). Frozen bacterial cell paste was re-suspended in ice-cold PBS/10 mM dithiothreitol (DTT)/1 mM phenylmethylsulfonylfluoride/0.2 mg/ml lysozyme. The bacterial homogenate was lysed by microfluidization. Following lysis, Triton X-100 was added to 1.0% (v/v final). The lysate was incubated on ice with gentle stirring for 30 min. The Triton X-100-soluble supernatant was collected by centrifugation for 1 h at 15 000 rpm. The GSHSepharose resin was prepared by pre-equilibration with PBS/10 mM DTT/1% Triton X-100. Following the application of the soluble supernatant to the column by gravity flow method, the column was washed extensively with equilibration buffer. Protein was eluted with 10 mM GSH, reduced form (Aldrich Chemical, St Louis, MO, USA), in 50 mM Tris, pH 8.0. The elution peak was dialyzed against PBS/10% glycerol/10 mM imidazole/0.3% Empigen BB (v/v final) (Albright and Wilson, Calbiochem, La Jolla, CA, USA) at 4°C overnight. For metal chelate chromatography, Ni+2-NTA resin was pre-equilibrated with PBS/10 mM imidazole/0.3% Empigen BB and the sample was loaded under gravity flow conditions. Following the application of the sample, the column was first washed with PBS/10 mM imidazole/0.3% Empigen followed by a second wash with PBS/0.1% Empigen BB/30 mM imidazole. The recombinant protein was eluted with PBS/500 mM imidazole. The peak fractions were collected and dialyzed against PBS/10% glycerol and stored frozen at 80°C in aliquots.
Expression of recombinant CSP N-terminal and C-terminal flanking regions in E. coli
The N-terminal and C-terminal flanking sequences were obtained by PCR amplification from the expression vector, pCI-TPA/PbCSP (2). The amplification primers for the N-terminal fragment were 5'CSP N-T 1131 (5'gtcagcggatccgatggac) and 3'CSP N-T 1370 (5'tggtggtggggatccttgtttcaatttattattacgctc) and for the C-terminal 5'CSP C-T 1809 5'caaccaggatccggtggtaataacaataacaaaaataataataatgacgattc) and 3'CSP C-T 2072 ( 5'tcttcaggatccacttgaac). Internal BamHI sites are underlined. After amplification, fragments were gel purified, digested with BamHI and ligated to the linearized/dephosphorylated pGex-6P1 vector. The bacterial strain BL21de3 was transformed with each of the vectors, inserts were sequenced and plasmids screened for expression. The clones NT-pGex-6P1 and CT-pGex-6P1 expressed the N-terminal and C-terminal of products of PbCSP, respectively. Recombinant proteins were purified over GSHSepharose as described for the full-length recombinant CSP.
Detection of CSP in transfected cells
BHK cells American Type Tissue Collection (Rockville, MD, USA) were transfected with 2 µg of an equal mixture of expression plasmid [CSP(-A), CS/2C3d, CS/3C3d, 2C3d or 3C3d] and pcDNA, by using lipofectamine plus (Invitrogen/Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions. For flow cytometric assays, secretion was blocked by adding 2 µM brefeldin A (Pharmingen, San Diego, CA, USA) to the culture for 8 h beginning 18 h after transfection. For western blotting, cells were not treated with brefeldin A and instead, culture supernates containing secreted protein were analyzed. For flow cytometric analysis of protein expression, cells were trypsinized and prepared for intracellular staining by using the Cytofix/cytoperm kit following the manufacturer's instructions (Pharmingen). CSP expression was detected with pooled hyperimmune serum from animals immunized with CSP(-A) by gene gun [isotype control is pooled serum from mice immunized with pcDNA using the same regimen as with CSP(-A)]. C3d expression was determined using a commercially available polyclonal rabbit anti-human C3d antibody (DAKO, Fort Collins, CO, USA), and normal rabbit IgG was used as isotype control. Affinity-purified, PE-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL, USA) and Spectrared-conjugated goat anti-rabbit IgG were used as second-step reagents. Data were expressed as percent specific positive cells and mean fluorescence intensity of the gated positive cells. For analysis of protein expression by western blot, culture supernatants containing secreted antigen were harvested 24 h after transfection and centrifuged at 100 000g for 1 h to pellet membrane vesicles. Samples equivalent to 2 x 105 cells per lane were applied to 420% polyacrylamide gel (Invitrogen) and size fractionated by SDS gel electrophoresis (120 V, 90 min). Subsequently, proteins were transferred onto nitrocellulose membranes (25 V, 1 h). The membranes were blocked in PBS + 0.3% Tween-20 for 30 min at room temperature (RT), then incubated with 1 : 20 000 diluted mAb 4B10 (directly conjugated to HRP, gift from Robert Wirtz, CDC, Atlanta, GA, USA) for 1 h at RT. After three washes with PBS + 0.05% Tween, reactive bands were visualized using enhanced chemiluminescence (ECL) plus and exposing the membranes to photographic film.
Detection of C3d binding to CSP
BHK cells were transfected with various plasmids (pcDNA, 2C3d or 3C3d) as described above. Secretion was blocked by adding 2 µM brefeldin A to the culture for 8 h beginning 24 h after transfection. Cells were harvested by trypsinization and washed and cell lysates prepared by freeze thawing and brief sonication. To assay for CSP and C3d binding, microtiter wells were treated overnight with 50 ng per well of (i) the C-terminal flanking sequence of CSP (CSP-CT), (ii) the N-terminal flanking sequence of CSP (CSP-NT), (iii) 24-mer peptide representing the repeat region of CSP (repeat region, 50 ng/ml), (iv) recombinant GST/CSP or (v) GST protein (GST, 50 ng/ml) as negative control. Plates were then blocked with PBS + 1% BSA for 1 h at RT, then cell lysates from transfected cells were added and incubated overnight at 4°C. Binding of C3d was detected by the anti-C3d antibody (1 µg/ml), followed by goat anti-rabbit antiserum conjugated with alkaline phosphatase (dilution 1 : 500). After adding the substrate BluePhos (Kirkegaard Perry, Gaithersburg, MD, USA), plates were read at 570 nm.
Immunization
Mice used for immunizations were 6- to 8-week old BALB/c females from the Jackson Laboratory (Bar Harbor, ME, USA). Sera were collected 1 day prior to each immunization and 2 weeks after the last immunization, which was prior to challenge with live P. berghei sporozoites. Sera were preserved by adding sodium azide (final concentration, 0.2%) and stored at 4°C. Mice were vaccinated three times at 4-week intervals epidermally with a Helios gene gun (Bio-Rad, Hercules, CA, USA). Vaccine was prepared by precipitating plasmid DNA onto gold beads (1.6-µm diameter) with CaCl2 in the presence of spermidine at a loading rate of 2 µg DNA mg1 of gold (1). Mice received a dose of 3 µg of DNA divided between three non-overlapping areas on the shaved abdomen at a helium pressure of 400 psi.
Determination of antibody titers by immunofluorescence assay
End point antibody titers were determined as described previously by indirect immunofluorescence assay (IFA) on air-dried, methanol-fixed P. berghei ANKA strain sporozoites (1).
ELISA
Ninety-six-well plates (Immunolon 2 HB, Thermo Labsystems, Franklin, MA, USA) were coated by overnight incubation with recombinant GST/CSP, the N-terminal part of CSP (CSP-NT), the C-terminal part of CSP (CSP-CT) or a 24-mer peptide (DPPPPNPN)3 representing a CSP repeat epitope at a concentration of 1 µg/ml in PBS at 4°C. Plates were washed with PBS/0.1% Tween-20 using the 96-well plate automatic ELISA-plate washer (Skatron, Sterling, VA, USA) and blocked with blocking buffer (PBS, 1% BSA, pH 7.5) for 1 h at 37°C. Sera were serially diluted in blocking buffer, incubated for 2 h at 37°C and then washed. Alkaline phosphatase-conjugated goat anti-mouse IgG, IgG1 and IgG2a (all from Southern Biotechnology) detection antibodies were added in blocking buffer (1 : 1000) and incubated for 1 h at 37°C. The assay was developed with BluePhos substrate (Kirkegaard Perry) for 30 min at RT, then stopped with stopping solution (Kirkegaard Perry) and read at 570 nm. Antibody concentration was determined by establishing a standard curve (run in parallel with each assay) with purified mouse IgG, IgG1 and IgG2a. For each serum, we determined a concentration that was within the linear portion of the reaction curve and used this dilution to extrapolate the actual antibody concentration in the assay wells.
Antibody avidity determination by thiocyanate elution
Based on their respective antibody titers, serum samples were diluted in blocking buffer to obtain comparable concentrations of IgG, incubated for 2 h at 37°C and then washed. Antigenantibody interactions were disrupted by the addition of increasing concentrations of the chaotropic agent sodium thiocyanate (NaSCN) in PBS (0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 M NaSCN) for 15 min (17). Plates were then washed to remove the NaSCN and processed following the standard ELISA protocol. The effective concentration of NaSCN required to release 50% of antiserum (ED50) was determined and used to compare the affinity of the antibody response induced by the vaccines.
ELISA-SPOT analysis
ELISA-SPOT plates (Millipore, Bedford, MA, USA) were coated with capturing mAb (Pharmingen, clone R4-6A2 for IFN-, clone 11B11 for IL-4, 4 µg/ml) overnight at 4°C in sterile PBS. Plates were then blocked with PBS + 1% BSA and later cells were seeded at 105 lymphocytes per well in HL-1 medium (GIBCO/Invitrogen, Carlsbad, CA, USA). Lymphocytes were obtained by lysing RBCs by osmotic shock (ACK Ammonium Chlorid Potassium (K) lysis buffer, GIBCO/Invitrogen). Cells were stimulated with 20 µg/ml recombinant CSP, peptides CS5770 (18), CS5865 (19), CS252260 (20) and CS252260 (21) or a mix of these peptides. Plates were incubated for 24 h (IFN-
) or 48 h (IL-4) before cells were washed out and the biotinylated, detecting mAb was added (Pharmingen, clone XMG1.2 for IFN-
, clone BVD6-24G2 for IL-4, 2 µg/ml). Detecting mAb was incubated overnight at 4°C. Finally, wells were treated with streptavidinalkaline phosphatase (Southern Biotechnology; 1 : 1000 diluted) for 2 h and the assay was developed using nitro-blue tetrazolium chloride/5-ßromo-4-chloro-3'-indoly-phosphate-p-toluidine salt substrate (Pierce). Analysis of the ELISA-SPOT assay plates was done using the C.T.L. imaging system (ImmunoSpot Analyzer, Cellular Technology Ltd, Cleveland, OH, USA). The number of spots in wells containing only unstimulated lymphocytes was used as background control. Spots reported from cultures with stimulated lymphocytes were corrected using their corresponding media control.
Magnetic bead separation
Single-cell suspensions were prepared and erythrocytes were lysed by using ACK lysis buffer. CD4+ or CD8+ splenocytes were purified using magnetic bead-labeled antibodies (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer's instructions. Separation was performed by using the Miltenyi OctoMACS system, which produces a >95% pure population. Cells were then washed with HL-1 medium (Invitrogen), suspended at 1 x 106 cells/ml and plated at 1 x 105 cells per well along with 2 x 105 irradiated APC per well (splenocytes from naive mice, irradiated with 3000 rad). ELISA-SPOT analysis was performed as described above. Wells containing only irradiated APC and antigen were set up to ensure that the APC did not produce any cytokines.
Parasite challenge
Fourteen days after the final immunization, mice were challenged by subcutaneous inoculation of 30005000 P. berghei sporozoites dissected from infected mosquito glands. Infection was determined by the presence of blood-stage parasites in Giemsa-stained thin blood smears 1 week after challenge. Animals that were not infected at that time were tested again 1 week later. We used this analysis schedule because animals that are infected with P. berghei ANKA strain malaria parasites do not self-cure.
Statistical analysis
A group size of 10 mice was chosen because, in efficacy studies, this number gives a 75% probability of observing a difference between the control group and the vaccinated group at the 95% confidence level (one tail) if 90% of the control animals were infected and 50% of the vaccinated animals were not infected. Chi-square analysis (two tails) was used to determine if a vaccine had a protective effect by comparing the proportion of infected animals observed in a vaccinated group with the proportion in pcDNA control group. Chi-square analysis (two tails) was also used to compare the protective effect of the CSP(-A) and CS/C3d chimeric vaccines. Comparisons of IFA results from vaccine groups were made by KruskalWallis followed by MannWhitney tests, and comparison of ELISA results were made by ANOVA on log-transformed data followed by Dunnet's tests with the CSP(-A) vaccine group as the control comparator. The binding of 2C3d and 3C3d to GST/CS or GST/CS C-terminal flanking sequence, GST/CS N-terminal flanking sequence or CS repeat peptide was compared by using ANOVA with binding to a GST control serving as the comparator for Dunnet's test (22).
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Results |
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Discussion |
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This study has revealed a number of novel and unexpected findings. First, C3d limits the efficacy of a CSP-based DNA vaccine against P. berghei (Table 1); second, the number of copies of C3d affects the magnitude and the balance of CSP-specific Ig isotypes (Figs 2 and 3) induced as well as the IL-4 response to CSP-derived CD4 and CD8 T cell epitopes measured ex vivo (Fig. 4); third, it limits the anti-CSP C-terminal flanking sequence-specific antibody response by masking the epitopes in this region of the molecule (Table 3) and fourth, it decreases the production of IgG1 anti-sporozoite and anti-CSP antibody response (Figs 2 and 3). The C3d binding to the CSP C-terminus may be a mechanism for exploitation of the innate immune response by the parasite to suppress the development of an acquired immune response.
The presence of C3d limits the induction of protection against malaria infection. Whether given one or three times, both CS/2C3d and CS3C3d were more protective than pcDNA (challenge control). The proportion of animals that were not infected after three immunizations with CSP(-A) was significantly greater than that observed for either CS/2C3d or CS/3C3d.
Cognizance of the importance of antigen-specific IL-4-producing CD4+ T cells in protection against malaria infection is increasing. Protection induced after immunization either with radiation-attenuated sporozoites or with the CSP-specific CD8+ T cell epitope SYVPSAEQI depends on the induction of IL-4-producing CD4+ T cells that are specific for liver-stage antigens (25). Immunity in humans vaccinated with radiation-attenuated sporozoites also correlates with the induction of IL-4-producing CD4+ CD45RO+ T cells, but not IFN--producing T cells specific for erythrocytic-stage malaria parasites (26). Gene gun-mediated vaccination with the CSP gene induces strong type 2 humoral immune responses (13). Typically, this type of immunity is difficult to deviate toward type 1 (27). Our results demonstrate that the presence of C3d either as a gene chimera with CSP or co-injected with CSP(-A) indeed diverted the induction of the expected type 2 humoral immune response. This diversion can be attributed to C3d, which is pro-inflammatory and immunoregulatory (28, 29) and activates murine CD4+ T cells bearing the CR1/2 complement receptor (30, 31), causing them to express a Th1 phenotype (30). Despite inducing a strong CD8+ IFN-
response, the CS/2C3d vaccine failed to protect mice against malaria infection, which can be explained by its failure to induce a strong IL-4 response. Although both CSP(-A) and CS/3C3d induced a significant protective effect, the protection induced by the CSP(-A) vaccine was the stronger of the two. Both vaccines induced large quantities of repeat-specific antibodies (Fig. 3), high-titer IFA-reactive antibodies (Fig. 2) and large numbers of CSP-specific IL-4- and IFN-
-producing splenocytes (Fig. 4). The stronger protective effect of the CSP(-A) vaccine is most readily explained by its ability to induce antibodies specific for the C-terminal flanking sequence (Fig. 3), which has been shown to be protective in other studies (32, 36).
The observation that C3d interacts with the C-terminus of CSP (Table 3) and may mask epitopes that are important for protection has interesting implications for hostparasite interactions. This region of the CSP is known to contain several adhesion motifs including those for properdin and thrombospondin. This properdin motif, found in Plasmodia spp. and other micro-organisms, may modulate susceptibility to infection or disease (3436). The adhesion motifs in the C-terminus of CSP have been implicated with the initial entry of the sporozoites into the liver cells (37) and protection (32, 33). Our experiments suggest that these motifs might also play an effective role in immune deviation. Hepatocytes, the target cells of sporozoites, are the major producers of C3 (30). Thus, binding of the C-terminus of CSP to C3d could represent an elegant immune escape mechanism of the parasite. We can only speculate as to how this mechanism might work in vivo. Although sporozoites can fix heterologous complement, they do not appear to fix homologous complement (38). The CSP-based properdin motifs on sporozoites might inhibit complement fixing by binding C3b such that it is oriented in the wrong direction. This type of C3b binding without fixation could then induce the CSP reaction and shedding of multimeric immunoregulatory complexes. Protection in the irradiated sporozoite vaccine model does not argue against this mechanism because this approach induces immunity that is strongly T cell dependent (39). It involves CS as well as non-CSPs (40, 41) and it is characterized by minimal induction of sporozoite-specific antibodies (40).
In this study, we have shown that C3d decreased the protective effect and deviated immunity induced by what has proven to be a highly effective CSP-based DNA vaccine against malaria infection. Therefore, the vaccine-enhancing effects of C3d reported previously against some viral pathogens cannot be extended readily to parasite and probably other pathogens. The potential for C3d-mediated non-responsiveness or immunosuppression has been shown previously (12, 42). One of the studies report the failure to induce HEL-specific antibodies when mice were immunized with one copy, but not two or three copies of C3d (12). Another study using bovine rotavirus and bovine herpesvirus showed inhibition of humoral and cellular immune responses after intradermal injection of chimeric plasmids containing either one or two copies of C3d (42). Differences in the nature of the immunogen (chimeric HEL-C3d protein) or in the modality (needle immunization rather than gene gun injection of a DNA vaccine) and route (intradermal instead of epidermal injection of a DNA vaccine) do not allow a direct comparison with our results. Other potential vaccines contrived similarly to the ones presented in this communication will probably also require detailed characterization. Finally, the possibility that plasmodia may bind elements of the innate immune system in order to evade a protective adaptive host immune response presents additional strategic considerations for the development of malaria vaccines.
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Acknowledgements |
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Abbreviations |
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APC | antigen-presenting cell |
CR2 | complement receptor 2 |
CSP | circumsporozoite protein |
DOC | day-of-challenge |
DTT | dithiothreitol |
GPI | glycosyl-phosphatidylinositol |
GSH | glutathione |
GST | glutathione-S-transferase |
HEL | hen egg lysozyme |
IFA | immunofluorescence assay |
RT | room temperature |
TPA | tissue plasminogen activator |
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Notes |
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Transmitting editor: T. F. Tedder
The authors' views are personal and are not to be construed as official policy of the Department of Defense or the U.S. Army. Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. All procedures were reviewed and approved by the Institute's Animal Care and Use Committee, and performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.
Received 18 May 2004, accepted 6 December 2004.
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References |
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