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Infection and Immunity, July 2002, p. 3479-3492, Vol. 70, No. 7
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.7.3479-3492.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Alexandra Rodríguez,1,
J. Mauricio Calvo-Calle,2 Fanny Guzmán,1 Patricia De la Vega,3 Manuel Elkin Patarroyo,1 Mary R. Galinski,4 and Alberto Moreno4*
Fundación Instituto de Inmunología de Colombia (FIDIC), Santafé de Bogotá, Colombia,1 Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York,2 Naval Medical Research Center, Silver Spring, and Department of Microbiology, School of Medicine, University of Maryland, Baltimore, Maryland,3 Emory Vaccine Research Center at Yerkes Regional Primate Research Center of Emory University, Atlanta, Georgia4
Received 20 November 2001/ Returned for modification 18 January 2002/ Accepted 25 March 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Separate malaria vaccines must be produced to target both P. falciparum and P. vivax, as the two species are quite distinct phylogenetically and antigenically (13). Malaria vaccines must also be able to target the exoerythocytic and blood stage forms of the parasite, generate both humoral and cellular immune responses, overcome genetic restriction, and stimulate memory cells.
Individuals living in areas where malaria is endemic develop an effective immunity capable of controlling parasitemia only after numerous infections, and repeated exposure is required to maintain this immunity (23). Experiments performed almost 40 years ago showed that complete protection could be provided by immunization with radiation-attenuated sporozoites (52). The focus since has been to determine which molecules or combination of molecules would be able to stimulate a similar protective immune response. Studies with rodent and nonhuman primate malaria models have shown that cellular as well as humoral immunity mechanisms have an important role in establishing a protective immune response (24). One widely studied target of this immunity is the circumsporozoite (CS) protein (54), which covers the surfaces of sporozoites, the liver-infective stage of the parasite. Proteins expressed by blood stage forms of the parasite can also confer protective immunity. One of the most extensively tested vaccine candidates from this stage is the merozoite surface protein-1 (MSP-1) (33). Investigations of the effector mechanisms involved in protection induced by immunization with MSP-1 suggest the involvement primarily of antibodies (7).
For these and other malaria vaccine candidate antigens, recombinant protein, DNA, and peptide immunogen approaches have been evaluated (32, 66). While there has been progress in generating immune responses with each approach, most immunogens tested have been limited in the ability to induce significant responses in a large number of genetically diverse individuals. Peptide immunogens offer several advantages (64), including the possibility of designing chimeras containing selected universal T-cell epitopes that can induce such responses. Qualitative and quantitative approaches have been used to identify universal T-cell epitopes. Allele-specific motifs were first demonstrated by isolating and subsequently sequencing class I self-peptides (15). The introduction of M13 phage display permitted the simultaneous characterization of several peptide-binding motifs (27). The introduction of methodology to isolate HLA class II molecules from Epstein-Barr virus (EBV)-transformed human B-cell lines, allowed peptide-binding interactions to be assayed by peptide competition experiments using biotinylated or radiolabeled indicator peptides (29). Using this approach, several universal epitopes have been defined for different pathogens, allergens, and autoantigens (38).
A malarial universal T-helper (Th) cell epitope described in the P. falciparum CS protein (45, 46) was recently shown to induce a significant antibody response in 7 of 10 individuals as part of a synthetic polyoxime-multiple-antigen peptide (MAP) immunogen (50). The prediction of this universal Th cell epitope through in vitro peptide-binding assays was validated in mice (5). These studies demonstrate that the abilities of synthetic peptides to interact with major histocompatibility complex (MHC) class II molecules can be directly correlated with their immunogenicity in vivo and that universal epitopes can induce a broad immune response that overcomes MHC restriction. Several HLA-DR promiscuous epitopes were subsequently described in P. falciparum preerythrocytic-stage proteins, but their in vivo relevance with regard to the development of immunity has not been reported (12).
Promiscuous T-cell epitopes have the inherent potential for functioning as universal epitopes (28). Based on the structural features of several DR alleles, pocket 4 interactions are known to determine the physical characteristics of amino acid residues in the amino-terminal portion of the binding peptides (4, 37). Peptides able to be adjusted at pocket 4 contain position 1 (P1) anchor residues, which are aromatic or hydrophobic residues (F, W, Y, L, I, V, or M). Secondary anchor residues have also been attributed to the sixth position (P6) in the linear peptide sequence and correspond to short or hydrophobic residues (S, T, C, A, P, V, I, L, or M). This structural pattern has been described as the P1-P6 motif and is a characteristic of a major group of DR alleles (65) that allows the promiscuous and potentially universal binding of epitopes.
In an effort to identify novel universal Plasmodium Th epitopes and to test their immunogenic functionality to overcome genetic restriction, we chose the P. vivax MSP-1 (PvMSP-1) molecule as our model. In comparison to P. falciparum MSP-1, the T-cell reactivity to PvMSP-1 has not been extensively characterized (10, 56). We constructed 86 PvMSP-1 peptides spanning this 210-kDa protein and used peptide competition assays to evaluate their binding to purified human class II alleles. The potential for their binding promiscuity was also analyzed based on their P1-P6 sequence motif patterns. The Th functions of eight selected peptide sequences were then studied by lymphoproliferation tests using cells from individuals with previous contact with P. vivax malaria and by analyzing the antibody and cytokine responses induced in mice immunized with Cys-T-B-Cys linear-peptide chimeras (LPCs) that include in tandem potential PvMSP-1 universal T-cell epitopes and the well-characterized immunodominant P. falciparum CS protein B-cell epitope, (NANP)3. This B-cell epitope is known to induce a significant antibody response in H-2b mice, while H-2d (BALB/c) mice have been considered nonresponders. Our data indicate that this genetic restriction to the (NANP)3 B-cell epitope can be overcome in H-2d (BALB/c) mice by including in the chimeric immunogens the PvMSP-1 promiscuous Th epitopes we identified. The Cys-T-B-Cys LPCs were exceptionally immunogenic in BALB/c mice and were able to elicit significant levels of neutralizing antibodies to the (NANP)3 B-cell epitope regardless of their H-2d genetic background. The potential and practicality of utilizing such LPCs for vaccine development is discussed.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice and immunizations. BALB/c (H-2d) mice were obtained from Charles River (Wilmington, Mass.). Groups of four female mice were immunized with 50 µg of the individual peptides tested emulsified in Freund's adjuvant (FA) (GIBCO Invitrogen Corp.); complete FA was used for the first immunization, followed by incomplete FA for subsequent immunizations. The mice received three intraperitoneal inoculations at 30-day intervals and were bled via the tail vein for antibody analyses 30 days after each immunization and at various time points following the final inoculation to reach a total of 300 days. The sera of each group were pooled for all antibody analyses. Two mice from each group were euthanized at day 200 to recover spleen cells for use in enzyme-linked immunospot (ELISPOT) assays. All animal protocols were approved by Emory University's Institutional Animal Care and Use Committee and followed accordingly.
HLA-DR heterodimer purification. The following EBV-transformed homozygous B-cell lines were used as sources of human soluble class II molecules: DR1, WT100BIS [International Histocompatibility Workshop number 9006 (DRB1*0101)]; DR3, COX [WSNO 9022 (DRB1*0301)]; DR4, BSM [WSNO 9032 (DRB1*0401)]; and DR11, BM21 [WSNO 9043 (DRB1*1101)]. The B-cell lines were maintained in culture using RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 50 µg of streptomycin/ml, 50 U of of penicillin/ml, and 10% heat-inactivated fetal calf serum (FCS). Membrane proteins were extracted from 109 EBV- transformed B cells using 1% (vol/vol) Nonidet P-40 and a mixture of protease inhibitors, and the DRB1* heterodimers were purified by immunoaffinity procedures as described previously (29).
Synthetic peptides. (i) PvMSP-1. Eighty-six 20-mer peptides spanning the complete PvMSP-1 protein from the Belem strain of P. vivax (GenBank accession number M60807 [9]) were synthesized by the multiple-solid-phase technique (Fig. 1). A series of amino- and carboxyl-terminally truncated peptides were also produced based on the sequences of peptides 19 and 53 (Table 1). All peptides were assembled using standard tert-butoxycarbonyl solid-phase peptide synthesis strategy on a p-methylbenzhydrylamine resin (Bachem California, Torrance, Calif.) as previously described (34). Briefly, tert-butoxycarbonyl N-protected amino acids were employed for peptide synthesis (Bachem California) and deblocked by trifluoroacetic acid treatment. Didyclohexilcarbodiimide was employed as the carboxy-activating reagent. Residues with low coupling efficacy required hydroxybenzotriazole activation (Aldrich Chemical, Milwaukee, Wis.). The optimum coupling reaction time was set to 1 h and monitored by the qualitative Kaiser test (61). The coupling step was repeated when necessary. Final peptide deprotection and cleavage was carried out by low-high hydrogen fluoride treatment and then the peptides were extracted using 5% acetic acid. Peptide purity was assessed by reverse-phase high-performance liquid chromatography, and the relative mass was characterized by spectroscopy.
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(iii) LPCs. Six promiscuous peptide sequences selected for binding to purified DRB1* molecules were chosen to design 34-mer Cys-T-B-Cys LPCs. Based on our evaluation of published results concerning the immunogenicity induced with linear peptides containing T- and B-cell epitopes, a polarity of T-B was chosen (16-18, 55). The synthetic peptides thus contain the 20-mer T-cell epitope at the N terminus of the construct, immediately followed by the minimal P. falciparum NANP repeat, (NANP)3. Cysteine residues were added to the amino- and carboxyl-terminal ends of the peptides to allow polymerization of the peptides by oxidation (43). As controls, two peptides containing three (PfB3) or six (PfB6) NANP repeats were also synthesized with terminal cysteine residues (see Fig. 3). In addition, a peptide containing the genetically restricted P. falciparum T1 epitope from the CS protein was produced (PfT1-PfB3) (47, 48).
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(ii) Peptide-binding competition assays. Peptide-binding competition assays were conducted to measure the abilities of unlabeled peptides to compete with biotinylated indicator peptides for binding to purified HLA-DR molecules. For these assays, biotinylated polyalanine 9- or 10-mer peptides containing allele-specific binding motifs were used (5). For DRB1*0101, DRB1*0401, and DRB1*1101 assays, biotinylated GFK(A)7 was used as the indicator peptide. Similarly, the appropriate biotinylated GYR(A)6L peptide was used as the indicator peptide for DRB1*0301. These indicator peptides were implemented previously to define HLA-binding peptides, and their affinity for HLA molecules is well characterized (5). According to this assay, a good competitor is a peptide capable of inhibiting over 50% of the binding of the indicator peptide to the HLA molecule being tested.
The biotinylated indicator peptide and class II molecules were incubated with 5- to 10-fold dilutions (0.001 to 100 µM) of the unlabeled PvMSP-1 competitor peptides. Binding was determined by measuring the OD in the presence versus the absence of competitor peptide. Inhibition was calculated as a percentage using the following formula: 100 x [1 - (
OD with competitor peptide/OD without competitor)].
Antibody assays. (i) Standard ELISAs.
The fine specificity of the antibodies elicited by immunization with individual chimeras was determined by ELISA using Immulon-2 plates (Dynatech Laboratories, Chantilly, Va.) coated with 1 µg of peptides/ml representing the T-cell epitopes, the polymeric B-cell epitope, or the Cys-T-B-Cys LPCs. The abilities of the peptides to bind the ELISA plates were determined by in situ biotinylation as described previously (25). After being blocked, the plates were incubated with sera diluted in PBS with 2.5% bovine serum albumin for 1 h at 37°C, and the bound antibodies were then detected using peroxidase-labeled goat anti-mouse immunoglobulin G (IgG) and H2O2-2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (KPL) as a substrate. ODs were determined using a VERSAmax ELISA reader (Molecular Device Corp., Sunnyvale, Calif.) with a 405-nm filter. The endpoint was measured as the highest dilution of sera having a OD greater than the mean plus two standard deviations obtained using nonimmune sera. Individual serum, analyzed at different time points, showed no significant differences among the groups. Final titers were then determined using pools of sera.
(ii) Subtype determinations. Isotype profiles of the antibodies elicited by immunization with the LPCs were also determined by ELISA. After serum incubation, the plates were washed and incubated with biotinylated rat anti-mouse MAbs IgG1, IgG2a, IgG2b, IgG3, IgE, or IgM (BD PharMingen, Franklin Lakes, N.J.) or biotin-labeled goat anti-mouse IgA (KPL) for 90 min. After the plates were washed, the bound antibodies were detected using horseradish peroxidase-streptavidin (BD PharMingen) and H2O2-2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) as a substrate.
The affinities of anti-peptide antibodies were assessed by a thiocyanate elution-based ELISA (44). The procedure was similar to that described above for the standard ELISAs with the inclusion of an extra step. After the plates were washed following incubation of the pooled serum dilutions, ammonium thiocyanate in PBS was added to the wells in duplicate in concentrations ranging from 0 to 8 M. The plates were allowed to stand for 15 min at room temperature before they were washed and the assay proceeded as described above. The concentration of ammonium thiocyanate required to dissociate 50% of the bound antibody was determined by linear regression analysis.
(iii) ISI assays. Inhibition of sporozoite invasion (ISI) assays were performed as described previously (42). Briefly, human hepatoma cell line HepG2 A16 cells were collected after trypsinization and plated at 50,000 per well in glass Lab-Tek chamber slides (Nunc, Rochester, N.Y.). Pools of sera were added in triplicate in a volume of 50 µl at 1:50 dilution. P. falciparum sporozoites (NF54 strain) were obtained from infected Aedes stephensi mosquitoes and adjusted to 2 x 104/50 µl of medium. The sporozoites were added to the hepatoma cells to a final serum dilution of 1:100 and incubated for 3 h at 37°C. The culture was subsequently washed and fixed with cold methanol. The parasites were visualized by immunocytochemistry using the P. falciparum-specific MAb NFS1 (42). This MAb was also used as a positive control of inhibition, and cultures with normal sera served as negative controls. The average numbers of exoerythrocytic stage forms (EEF) in triplicate cultures were recorded, and percent inhibition was calculated with the following formula: [1 - (mean number of EEF in cultures with immune serum/mean number of EEF in cultures with normal serum)] x 100.
(iv) Indirect IFAs. Sera obtained from mice immunized with the same synthetic peptide constructs were pooled, and the antibody reactivity was evaluated by indirect immunofluorescence assays (IFAs) using air-dried P. falciparum (NF54 strain) sporozoites and acetone-fixed P. vivax schizonts, kindly provided by John W. Barnwell. Several serum dilutions were tested, and the reactivity was evaluated using fluorescein isothiocyanate-labeled goat anti-mouse IgG (KPL) diluted in PBS-0.4% Evans Blue.
T-cell lines and lymphoproliferation assays. Terminal T-cell lines were established from the volunteer participants' PBMCs immediately after isolation by cultivation in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% nonessential amino acids (GIBCO Invitrogen Corp.), 1 mM sodium pyruvate, 25 U of penicillin/ml, 50 µg of streptomycin/ml, and 5 x 10-5 M 2-mercaptoethanol (complete RPMI), with 10% heat-inactivated AB+ serum (ICN Biomedicals, Costa Mesa, Calif.). The cells were incubated with a peptide pool containing a total 10-µg/ml final concentration of individual 20-mer synthetic peptides representing eight different regions of the PvMSP-1 protein selected by their abilities to bind four different MHC alleles. After 3, 7, and 10 days of culture at 37°C in 5% CO2, the cells were supplemented with complete RPMI-10% FCS containing 100 U of recombinant interleukin-2 (IL-2) (BD PharMingen)/ml.
Lymphoproliferation assays were performed using 2 x 104 T cells from the T-cell lines described above and 5 x 104 autologous PBMCs inactivated with mytomicin C (Sigma, St. Louis, Mo.) as antigen-presenting cells. The cells were incubated with complete RPMI supplemented with 10% FCS in 96-well plates in the presence or absence of individual peptides at 1 and 10 µg/ml. Phytohemagglutinin P and staphylococcal enterotoxin B were included as positive controls of proliferation. After 48 h, the cultures were pulsed overnight with 1 µCi of [3H]thymidine (Amersham-Pharmacia, Little Chalfont, Buckinghamshire, United Kingdom)/well and harvested on glass filters, and the specific incorporation was evaluated by liquid scintillation counting. The results are expressed as the stimulation index (SI), calculated as the mean counts per minute in triplicate peptide-stimulated wells/mean counts per minute in medium-treated control wells. T-cell responses are considered positive for cases in which the SI is higher than 2.
ELISPOT assays.
The frequency of murine peptide-specific T lymphocytes was determined in two mice with gamma interferon (IFN-
)- and IL-5-specific ELISPOT assays conducted 200 days after the first immunization, when the antibody responses reached a plateau. The assays were performed in nitrocellulose microplates (Millipore, Bedford, Mass.) coated with rat capture anti-mouse IFN- or rat anti-mouse IL-5 (PharMingen) (10 µg/ml) in PBS. Duplicate aliquots of different concentrations (1 x 106 and 5 x 105) of freshly isolated spleen cells from immunized mice were plated in RPMI containing 10% FCS. Th cells were activated by the addition of 10 µg of LPCs/ml or peptides representing B- or T-cell epitopes alone. After 24 h for IFN- and 48 h for IL-5, the plates were washed and incubated with 2 µg of biotinylated rat anti-mouse IFN-/ml and 5 µg of biotinylated rat anti-mouse IL-5 (PharMingen)/ml and followed by incubation with streptavidin-horseradish peroxidase. The reaction was developed using 3,3-diaminobezidine-tetra-hydrochloridedehydrate (Sigma) and evaluated in an Immunospot analyzer (Cellular Technology-Becton Dickinson, San Diego, Calif.).
Statistical analysis. Stimulation indexes were analyzed using the Mann-Whitney U test. Linear correlation was calculated to evaluate the relationship among ISI and IFA titers, antibody isotype profiles, and affinity assays. For affinity of anti-peptide antibody assays, the concentration of ammonium thiocyanate required to dissociate 50% of bound antibodies was determined by linear regression. The significance of differences in cytokine production between cells stimulated in vitro with and without synthetic peptides was analyzed with Student's t test following log10 transformation to normalize the distribution of data; P values of 0.05 or less were considered to be significant.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recognition of the PvMSP-1 class II promiscuous-binding peptides by human T cells. To determine whether the eight promiscuous PvMSP-1 peptides that bound all four DR molecules in the peptide-binding assays are recognized by T cells of individuals with a history of P. vivax malaria infections, T-cell proliferation responses to these peptides were assessed from samples acquired from 33 volunteers living in a region of Colombia, South America, where P. vivax is endemic. Each individual had previously documented P. vivax infections and, in some cases, P. falciparum malaria. Control samples were also obtained from nonexposed volunteers living in areas where P. vivax is not endemic. Terminal T-cell lines were tested for proliferation, after 10 days of specific PvMSP-1 peptide stimulation, with autologous mitomycin-treated PBMCs as antigen-presenting cells. Although the DRB1* allele prevalence could not be determined in this cohort of individuals, the alleles tested for binding represent over 70% of the Hispanic populations, assuming a representative main subtype (12, 65).
Consistent with the broad pattern of HLA-DR binding we observed, most of the DRB1*-selected synthetic peptides elicited a proliferative response in the cells from volunteers with previous contact with P. vivax malaria but not in the cells from nonexposed individuals (Fig. 2B and C). The frequency of responders among exposed individuals ranged from 18 to 48%, in contrast to 3 to 23% for nonexposed volunteers. Differences between groups were statistically significant for peptides 4, 6, 8, 19, 25, and 45 (P < 0.05). Most of the responders showed reactivity to more than one peptide; however, none of the volunteers responded to all of the peptides. All of the donor cells showed positive responses to both phytohemagglutinin-P and staphylococcal enterotoxin B, with SIs ranging from 2.8 to 164.
Design of LPCs to study the Th activity of the six most conserved PvMSP-1 potential universal Th epitopes. The six promiscuous PvMSP-1 peptides displaying the highest conservation, as noted above, were selected for development of Cys-T-B-Cys LPC chimeras and were renamed PvT4, PvT6, PvT8, PvT19, PvT45, and PvT53, corresponding to their original peptide numbers (Fig. 3). Interestingly, five of these six linear peptides contain at least one or several overlapping P1-P6 DR allele structural binding motifs (65), which are noted in boldface as follows: PvT4, NFVGKFLELQIPGHTDLLHL; PvT6, FNQLMHVINFHYDLLRANVH; PvT8, LDMLKKVVLGLWKPLDNIKD; PvT19, LEYYLREKAKMAGTLIIPES; and PvT53, SKDQIKKLTSLKNKLERRQN. The B-cell epitope selected for inclusion in the LPCs is the repeat tetramer (NANP)n of the P. falciparum CS protein. Antibody responses to this epitope have been extensively characterized in several models (42, 47, 57), and they are known to be able to neutralize sporozoites and inhibit their invasion of hepatocytes (42). An additional chimeric peptide, which includes a genetically restricted Th epitope of the CS protein (PfT1) that is unable to provide help for antibody production against the (NANP)n sequence in H-2d mice, was used as a control (47).
The accessibility of the PfCS (NANP)3 repeat B-cell epitope in these constructs was determined by ELISA using the specific PfCS protein MAb 2A10. Figure 4A shows the profiles of recognition of the different Cys-T-B-Cys LPCs. Most of the peptides were recognized by MAb 2A10 at concentrations as low as 0.048 ng/ml. Nonetheless, the PvT4-PfB3, PvT6-PfB3, and PvT8-PfB3 constructs were not recognized as efficiently as the others, suggesting the presence of structural constraints associated with polymerization which could prevent or limit exposure of the B-cell epitope. The results also demonstrate better reactivity with the PfB6 construct than with the PfB3 peptide. Based on these observations, the peptide containing six repeats (PfB6) rather than three was selected for the subsequent fine characterization of the anti-NANP antibodies elicited by immunization with the Cys-T-B-Cys LPCs.
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Immunogenicity of LPC constructs in mice. The immunogenicity of the peptide chimeras were tested in BALB/c mice. It is well known that these mice do not produce significant levels of antibodies upon (NANP)n immunization, regardless of whether the repeats are included in recombinant protein or peptide constructs (47, 57). Groups of four mice were immunized three times intraperitoneally with 50 µg of individual Cys-T-B-Cys chimeras or the control peptides PfB3 and PfB6 emulsified in complete or incomplete FA, and the antibody responses were followed for 300 days by ELISA using PfB6 as an antigen (Fig. 4B). In mice immunized with PfB3, or an allele-specific T-cell epitope (PfT1), the antibody responses to the PfB6 (NANP) sequence were very low and boosting did not change the pattern of the responses. In fact, these antibody titers mimic those obtained upon immunization with saline (Fig. 4B). However, the PfB6 construct induced significant levels of antibodies, which were boosted by each immunization. The inclusion of P. vivax promiscuous T-cell epitopes in the LPCs dramatically increased the responses to the (NANP)3 sequence, with titers reaching a peak at day 120 and antibody titers ranging between 1:2 x 105 and 1:3.3 x 106. Single immunizations with PvT4-PfB3, PvT6-PfB3, PvT8-PfB3, and PvT19-PfB3 chimeras were able to induce antibody responses with titers over (1:2) x 104. Lower but substantial antibody titers were obtained after boosting with the chimeras PvT4-PfB3, PvT6-PfB3, PvT8-PfB3, and PvT45-PfB3, which all consistently had lower recognition by MAb 2A10. Strikingly, the antibody levels remained essentially the same for the 300-day period evaluated.
Antibodies induced by the immunogens were also evaluated by ELISA using individual chimeras as antigens. The magnitudes and kinetics of the antibody titers followed patterns of recognition similar to those obtained with PfB6 as the antigen (data not shown). The antibody responses peaked 60 days after the last immunizations, with titers ranging between (1:1) x 105 and (1:1.3) x 107. Importantly, the PvT19-PfB3 and PvT53-PfB3 immunogens induced an antibody peak followed by a plateau at day 90 with ELISA titers up to (1:1.3) x 107 against the corresponding LPCs. This contrasts with the final titers of (1:3.2) x 106 and (1:1.6) x 106 obtained with the PfB6 construct. These results suggest that PvT19-PfB3 and PvT53-PfB3 contain additional B-cell epitopes. To characterize the potential recognition of the T-cell epitope-containing peptides by antibodies elicited by the PvT19-PfB3 and PvT53-PfB3 immunogens, a new set of synthetic peptides containing amino- and carboxyl-terminal truncations were synthesized and tested by ELISA. Table 1 summarizes the responses using pools of serum obtained at day 120. These results suggest that the T-cell epitope-containing peptide PvT53-PfB3 contains two different B-cell epitopes. In contrast, the minimal B-cell epitope recognized by immunization with the chimera PvT19-PfB3 seems to be present in a 16-mer core region. The presence of B-cell determinants in the Th sequences could explain the higher immunogenicities of both peptides in comparison with other chimeras.
Several reports have indicated that different antibody isotypes are involved in protection elicited by natural contact with malaria parasites. We therefore determined the IgG subtypes in the sera from the mice immunized with the individual Cys-T-B-Cys LPCs. High antibody responses mediated by IgG1, IgG2a, and IgG2b were obtained with all of the chimeras. However, mice immunized with PvT4-PfB3, PvT8-PfB3, and PvT19-PfB3 more consistently elicited anti-repeat antibodies of all IgG subclasses. In contrast, the antibody responses elicited with PvT6-PfB3, PvT45-PfB3, and PvT53-PfB3 were predominantly of the IgG1 and/or IgG2b subtype (Table 2). This suggests that different populations of Th cells are induced after immunization with chimeras containing the P. vivax promiscuous T-cell epitopes. Only constructs based on peptides PvT6 and PvT19 induced significant levels of IgG3.
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- and IL-5-secreting cells (Table 3). The cultures were pulsed with peptides representing T-B-, T-, or B-cell epitopes. A negative control using only cells cultured in medium was included, and a combination of phorbol 12-myristate 13-acetate and ionomycin served as a positive control. Spleen cells from mice immunized with different LPCs showed T-cell epitope-specific IFN- production. There were no significant differences in IFN- secretion when the cells were tested with PfB6 or the corresponding LPCs. In contrast, we determined significant differences in IL-5 production after in vitro stimulation with PfB6, LPCs, or peptides representing the T-cell epitopes (Table 3).
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Antibodies recognizing the P. falciparum sporozoites were more complex and differed depending upon the construct used (Table 2). T-B chimeras containing the P. vivax Th epitopes produced a range of anti-sporozoite responses, with IFA antibody titers as low as 1:800 for the PvT8-PfB3 construct and as high as 1:409,600 for the PvT19-PfB3 construct. These constructs could be grouped as high responders (PvT6-PfB3, PvT19-PfB3, and PvT53PfB3), intermediate responder (PvT4-PfB3), and low responders (PvT8-PfB3 and PvT45-PfB3). Further, constructs PfB3 and PfB6 and the PfT1-PfB3 constructs induced much greater anti-sporozoite antibody titers than we had expected, suggesting that the polymerization of the peptides due to the presence of the terminal Cys residues had in fact increased the representation and immunogenicity of the B-cell epitopes in these constructs.
The biological relevance of these antibodies was also evaluated by testing their abilities to inhibit the invasion of target cells. As expected, the greatest inhibition was observed with the antisera that showed the highest reactivities to sporozoites. An interesting observation, however, is that construct PfB6, in spite of its lower anti-sporozoite titer, produced a higher inhibition than T-B chimeras with intermediate or low levels of anti-sporozoite responses. The ISI assays were correlated with anti-peptide antibody titers (total IgG, IgG isotypes, IgM, IgE, and IgA) (Table 2), IFA analyses, and antibody affinity assays. There were positive correlations between ISI and titers evaluated by IFA against P. falciparum sporozoites (r = 0.905; P = 0.0025), total IgG (r = 0.909; P = 0.0023), IgG1 (r = 0.836; P = 0.009), IgG2a (r = 0.752; P = 0.025), IgG2b (r = 0.782; P = 0.018), IgA (r = 0.811; P = 0.013), and the total IgG antibody affinity (r = 0.814; P = 0.012), but not with IgG1, IgE, or IgM.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several factors contributed to our decision to initiate this study with PvMSP-1 as a primary model: (i) epidemiological evidence of broad PvMSP-1 recognition by lymphocytes from people living in areas of endemicity (63), (ii) evidence in clinical and preclinical trials with different Plasmodium species that partial protection could be induced in susceptible hosts by immunization with vaccine candidates based on MSP-1 (33), (iii) indications that proliferation and production of cytokines could be elicited in vivo and in vitro using recombinant MSP-1 constructs (10, 21), and (iv) our objective to increase the knowledge base on immunity to P. vivax antigens. Further, genetic diversity of MSP-1 has been suggested to contribute to the parasite's immune evasion strategies. T-cell epitopes described previously in polymorphic regions of MSP-1 from P. falciparum have been reported to exhibit genetic restriction, inducing responses in a limited number of individuals (56).
Our analysis of PvMSP-1 is the most comprehensive assessment to date of promiscuous Th cell epitopes contained in the Plasmodium MSP-1 malaria vaccine candidate antigen. In fact, to our knowledge, this is the most thorough analysis of the presence of Th cell epitopes for any Plasmodium antigen. Moreover, we show that the promiscuous epitopes defined for PvMSP-1 can provide significant T-cell help for the P. falciparum (NANP)3 B-cell epitope. This observation suggests that the data reported here have potentially much larger implications. If in fact certain T-cell epitopes defined for this or other Plasmodium antigens can provide significant T-cell help for various Plasmodium antigens, or even other types of antigens, their potential utility could be wide ranging. Previous experiments designed to evaluate the in vivo significance of the association of B- and T-cell epitopes in immunogens have shown that the magnitude of the response is limited by low T-cell epitope density (2). Peptide dendrimers, including MAP constructs, which consist of linear peptides attached to a core matrix, have been considered promising approaches to develop peptide vaccines with a high density of B- and T-cell epitopes (68). The combination of B- and T-cell epitopes in Plasmodium-specific traditional MAP structures led to a sustained protective immune response in murine models (68) and a strong antibody response, lymphoproliferation, and cytokine production in human volunteers (49, 50). Nevertheless, the production of these MAPs requires very precise stoichiometry yet results in heterogeneous final products and very poor yields (2, 36). The addition of cysteine residues to anchor peptides to the core matrix is a recent improvement that has facilitated the inclusion of several epitopes in MAPs, as well as the characterization of the in-process product (2, 39). However, the process of developing and analyzing MAP constructs remains complex and costly compared to linear peptides.
We sought a simpler and more economical approach for presenting a high density of Plasmodium epitopes, with the goal of designing constructs that were easy to produce, easy to characterize, stable, and highly immunogenic. Consequently, a high density of B- and T-cell epitopes was obtained using the straightforward approach of adding cysteine residues at the amino- and carboxyl-terminal ends of linear-peptide T-B chimeras (43, 59) (Fig. 3). The cysteine residues enabled the LPCs to spontaneously polymerize (43). The antigenicity of the constructs was confirmed by ELISA using a MAb specific to the (NANP)3 B-cell epitope, and recall T-cell responses were assessed by conducting lymphoproliferation assays. The immunogenicity of the polymers was evaluated in mice in the absence of carrier molecules, as we had also hoped to be able to bypass the need for carriers and ultimately strong adjuvants. The resulting LPC immunogens were highly effective, with the resulting humoral immune response far surpassing the antibody levels previously observed for the extensively studied (NANP)3 B-cell epitope (8, 39, 42, 47, 57). Furthermore, the ability of the LPCs to overcome genetic restriction to the (NANP)3 B-cell epitope in BALB/c mice and the observed potential for isotype class switching and cellular recall responses add strong support for further basic and preclinical evaluation of the LPC approach for malaria vaccine development.
The rationale behind developing chimeras containing a P. falciparum B-cell epitope, and not a P. vivax B-cell epitope(s), was based on the fact that the immune response against the P. falciparum NANP repeat is particularly well characterized (5, 47). We were also testing the hypothesis that P. vivax Th epitope sequences could provide help to an unrelated antigen. In spite of the fact that the phylogenetic distance of P. vivax and P. falciparum has led to them being largely antigenically distinct species, our data suggest that there may be ways to combine effective epitopes from multiple species to produce a multispecies malaria vaccine. Whereas the concept of multistage epitope vaccines was adopted about 20 years ago (53), with cocktail approaches being investigated since to develop multicomponent P. falciparum vaccines, the concept of a multispecies malaria vaccine has not yet been pursued. Our finding that the P. vivax Th epitopes can provide help to generate a significant antibody response against the P. falciparum (NANP)3 B-cell epitope is the first demonstration of a multispecies malaria vaccine immunogen.
Our results show that eight out of 86 20-mer PvMSP-1 peptides bind competitively to human DRB1* alleles, which have been shown to represent over 70% of individuals in Hispanic populations (12, 65). In contrast to the definitive class I binding of 8- to 10-mer peptides, class II molecules show promiscuity in their association with peptides 10 to 20 amino acids long (31). Six out of the eight promiscuously binding peptides induced a spectrum of lymphoproliferation reactivities in 24 to 48% of individuals with prior contact with P. vivax, consistent with a recall response. This is in sharp contrast with the reactivity in 3 to 23% of normal volunteers, results that have been previously reported in malaria, and is consistent with the broad promiscuity of MHC class II association or cross-reactivity (23, 62). This percentage of reactivity is very close to the frequency found in individuals exposed to P. vivax malaria in areas of Brazil with similarly low endemicity, where the prevalence of recall lymphoproliferative responses to PvMSP-1 N-terminal recombinant proteins was reported to be 30 to 47.2% (63). Nevertheless, the prevalence of recognition observed for the PvMSP-1 peptide sequences is lower than that reported in other systems (11). This could be attributed to the fact that the PvMSP-1 studies were conducted without CD4 enrichment and using PBMCs derived from volunteers living in an area of low malaria endemicity. Although all volunteers had previous P. vivax infections, a comparatively low prevalence of memory cells would be expected in an area of low endemicity versus an area of high endemicity (63). Hence, the positive recognition of the PvMSP-1 peptide sequences by recall lymphocytes that we observed is in fact striking. Typically, T-cell epitope recognition by nonfractionated cells is obtained only with the use of antigen-presenting cells, such as dendritic cells, with strong costimulatory activity and a high MHC-peptide complex density (41).
Importantly, we identified significant IFN- production after in vitro stimulation with peptides representing the promiscuous T-cell epitopes. IFN- has been involved as a key effector molecule in the clearance of malaria parasites (67). The PvT45 peptide (KKIKAFLETSNNKAAAPAQS) induced a particularly high level of IFN--producing cells and deserves additional characterization. Interestingly, while DNA immunizations with plasmids containing Pvmsp-1 sequences are immunogenic in mice (10), with DNA representing the amino-terminal (682-aa) and 19-kDa carboxyl-terminal regions generating significant antibody responses, only the plasmid representing the amino-terminal portion consistently induced IFN- production (10). Four out of the six promiscuous peptide sequences we identified correspond to this region. In contrast, no IFN- production was observed in a recent study of the immunogenicity of a series of MAP constructs representing P. falciparum MSP-1, CS, and liver stage antigen-1 epitopes (39). This study also demonstrated strong genetic restriction of these MAP immunogens in several strains of mice, including BALB/c.
Another recent study using peptide-binding assays on filter papers with soluble DRB1*0101 and DRB1*0405 biotinylated molecules identified several P. falciparum MSP-1 T-cell epitopes, with a high percentage (8.3 to 25.8%) of promiscuity noted (19). However, this study did not entail a complete survey of peptides representing the entire protein and did not utilize competition peptide-binding assays. While the present binding analyses of 86 PvMSP-1 peptides, which resulted in the identification of eight unique promiscuous T-cell epitopes that bind DRB1*0301, DRB1*0401, DRB1*1101, and DRB1*0101 alleles, is certainly more thorough, it is important to realize that since these are not overlapping peptides, additional potential PvMSP-1 T-cell epitopes could have escaped detection. Thus, it remains uncertain how many are actually present across this protein. In any case, the dramatic Th cell response attributed to several of our PvMSP-1 Th epitopes brings to light the question of how many such epitopes will be satisfactory for the development of an effective malaria vaccine. If epitopes like the ones defined here can show a broad Th activity for CS protein antibody expression, it is reasonable to predict that they could have a similar potential for other B-cell epitopes as well. It is now critical to determine the fullest potential of these sequences. If they can provide essential Th activity to various B-cell epitopes, we must question the relevance of further extensive searching for novel promiscuous Th cell epitopes in various Plasmodium vaccine candidate antigens.
Analysis of the antibody response to the chimeras containing the PvMSP-1 peptide sequences Pv19 and Pv53, which elicited the highest antibody titers against the T-B peptides, suggests the presence of novel B-cell epitopes within the PvMSP-1 Th epitope sequences. The results are quite remarkable for the persistence of the immune response, with anti-peptide antibody titers between 1:2.5 x 104 and 1:1.6 x 106 detected up to 300 days after the first immunization. Importantly, antibodies elicited against these LPCs were also found to recognize P. vivax merozoites, showing a classic MSP immunofluorescence staining pattern for the surface of the merozoites (33). On the other hand, all constructs with the (NANP)3 sequence elicited antibodies that reacted by IFA with the surfaces of P. falciparum sporozoites and were able to inhibit invasion of target hepatocytes in a classical ISI assay.
When antibody isotypes relating to the individual LPCs were assessed, distinct patterns were observed. Similar titers of IgG1, IG2a, and IgG2b were induced in mice immunized with the chimeras PvT4-PfB3, PvT8-PfB3, and PvT19-PfB3, suggesting that the Th activity includes both Th1- and Th2-type responses. In contrast, the predominant IgG subtypes in mice immunized with chimeras PvT6-PfB3, PvT45-PfB3, and PvT53-PfB3 were IgG1 and IgG2b, suggesting a predominant Th2 activity. The production of anti-peptide IgA and a low level of IgE antibodies is also noteworthy; so far, though, the IgE titers have been very low and would not seem to pose safety concerns. Consistent with the Th2 and IgA antibody profiles, a significant number of IL-5-secreting cells were detected 200 days after the first immunization. The cytokine profile was obtained by in vitro boosting with individual peptides in the absence of in vitro selection or CD4 enrichment.
Differences in cytokine and isotype profiles for different immunogens have been reported to be influenced by the selection of adjuvants (69). While a deeper analysis is beyond the scope of this report, the fact that all of the mice in this study received the LPCs emulsified in the same adjuvant suggests a real participation of the peptide constructs in the modulation of the specific cytokine and isotype profiles observed here. FA was chosen for the present study in order to be able to compare the humoral immune response with that obtained for the (NANP)3 epitope in numerous previous studies. Importantly, even more dramatic results were generated more recently in BALB/c mice with one of the LPCs by using clinical-grade adjuvants and subcutaneous immunization procedures (unpublished data).
Ongoing research must now investigate (i) the immune response to these and other LPCs in different strains of mice, (ii) the molecular mechanisms involved in the preferential induction of a particular Th1 or Th2 immunogenic profile, (iii) the potential synergic effect obtained by the combination of several promiscuous T-cell epitopes, (iv) the potential of including the reported Th epitopes with nonmalarial antigens, and (v) the magnitude and functional significance of LPC-specific memory cells. Several factors may influence the commitment of the immune response to Th1 or Th2 phenotypes in vivo (60). In particular, altered peptide ligands can lead to changes in the interaction between a T-cell receptor and an MHC-peptide complex. Altered peptide ligands have been shown to modify the kinetics of the immune response to well-defined T-cell epitopes (14). Recently, Ise and colleagues demonstrated a preferential Th1 or Th2 cytokine secretion profile in vitro using peptide analogues (35). In general, it has been suggested that increasing the affinity of the wild-type peptides for MHC class II alleles above baseline affinity leads to enhanced priming for Th1. In contrast, decreasing the affinity leads to the Th2 phenotype. It may be relevant in this regard that the PvT19-PfB3 chimera, which induced the highest antibody response, also had the highest affinity to two human alleles (50% inhibitory concentrations for DRB1*0401 and DRB1*1101, 11.45 and 19.14 µM), in contrast to 50% inhibitory concentrations ranging between 23 and 250 µM for the other LPCs (data not shown).
In summary, we have demonstrated the presence of eight promiscuous T-cell epitopes in the PvMSP-1 molecule. Novel synthetic LPC Cys-T-B-Cys constructs including these epitopes proved to be highly immunogenic in BALB/c mice. Functional (NANP)3 antibodies measured by ISI assays and cytokine profiles consistent with long-lasting Th activity were obtained after immunization in all animals tested, suggesting that the genetic restriction to B-cell epitopes is not an insurmountable obstacle to developing effective malaria vaccines. The remarkable immunogenicity of the linear peptides also suggests that complex synthetic structures or adjuvant-built peptides are not required to induce a vigorous immune response. The LPC approach described here expands the possibilities for rapidly producing and testing novel multimeric vaccines. Finally, we have shown that epitopes derived from different malaria parasite species and life cycle stages can be combined in a single linear peptide to form a very effective immunogen.
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ACKNOWLEDGMENTS |
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I.C.-A. and A.R. contributed equally to this work.
We thank Soraya Angel, Juan Carlos Gómez, and the personnel at the Battalion 21 Vargas, Granada, Meta, Colombia, for their assistance in obtaining blood samples. We are also grateful to the volunteers who willingly participated in these studies. We thank Elizabeth Nardin for kindly providing the Plasmodium CS protein anti-repeat MAbs, Joseli de Oliveira-Ferreira for her advice with ELISPOT assays and critical reading of the manuscript, John W. Barnwell for providing P. falciparum sporozoites and P. vivax schizonts for IFA analyses and critical review of the manuscript, and Vladimir Corredor for critical review of the manuscript.
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FOOTNOTES |
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Present address: Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, N.Y.
Present address: Departamento de Biologia Molecular, Instituto de Investigaciones Biomedicas, UNAM, Mexico, D.F. Mexico.
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), PvT6-PfB3 (
), PvT8-PfB3 (
), PvT19-PfB3 (
), PvT45-PfB3 (
), and PvT53-PfB3 (•) are the individual LPCs containing PvMSP-1 promiscuous T-cell epitopes. PfT1-PfB3 (+) represents the LPC containing the P. falciparum genetically restricted T1 epitope. PfB3 (
) and PfB6 (
) represent the polymeric peptides containing only the B-cell epitope (NANP)n with three and six repeats, respectively. Mice immunized with saline emulsified in FA represent the placebo group (
). The data are presented as geometric mean titers obtained with a pool of sera obtained at different time points. The immunizations were conducted at 0, 30, and 60 days as indicated by the syringes.