Berghei Circumsporozoite Protein Research Articles

Immunization with a Circumsporozoite Epitope Fused to Bordetella pertussis Adenylate Cyclase in Conjunction with Cytotoxic T-Lymphocyte-Associated Antigen 4 Blockade Confers Protection against Plasmodium berghei Liver-Stage Malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

T cell receptor-ligand interactions: a conformational preequilibrium or an induced fit.

Kinetic parameters of T cell receptor (TCR) interactions with its ligand have been proposed to control T cell activation. Analysis of kinetic data obtained has so far produced conflicting insights; here, we offer a consideration of this problem. As a model system, association and dissociation of a soluble TCR (sT1) and its specific ligand, an azidobenzoic acid derivative of the peptide SYIPSAEK-(ABA)I (residues 252-260 from Plasmodium berghei circumsporozoite protein), bound to class I MHC H-2K(d)-encoded molecule (MHCp) were studied by surface plasmon resonance. The association time courses exhibited biphasic patterns. The fast and dominant phase was assigned to ligand association with the major fraction of TCR molecules, whereas the slow component was attributed to the presence of traces of TCR dimers. The association rate constant derived for the fast phase, assuming a reversible, single-step reaction mechanism, was relatively slow and markedly temperature-dependent, decreasing from 7.0 x 10(3) at 25 degrees C to 1.8 x 10(2) M(-1).s(-1) at 4 degrees C. Hence, it is suggested that these observed slow rate constants are the result of unresolved elementary steps of the process. Indeed, our analysis of the kinetic data shows that the time courses of TCR-MHCp interaction fit well to two different, yet closely related mechanisms, where an induced fit or a preequilibrium of two unbound TCR conformers are operational. These mechanisms may provide a rationale for the reported conformational flexibility of the TCR and its unusual ligand recognition properties, which combine high specificity with considerable crossreactivity.

Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein–protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle.

Predominant cell-mediated immunity in the oral mucosa: gene gun-based vaccination against infectious diseases

Background: Direct immunization via epithelial surfaces has been considered for many vaccine approaches, including DNA vaccines. It remains to be determined, however, which body site is suitable for genetic vaccination. Objective: To characterize the effects of the oral mucosa-mediated genetic vaccination, we compared antigen-specific immune responses of the oral mucosal DNA vaccine to the flank skin vaccination against influenza virus and malaria parasite. Methods: DNA vaccines against the influenza A/WSN/33 (H1N1) hemagglutinin and the malaria Plasmodium berghei circumsporozoite protein were administered respectively three times at 3-week intervals into the oral mucosa, skin, or liver of hamsters. The effects of their vaccine were evaluated by antigen-specific antibody production and cell-mediated killing activity. Furthermore, the in vivo malaria challenge test was also performed after the vaccination. Results: Significant specific antibody production was not observed in each case, but interferon-gamma production and cell-mediated killing activity were strongly induced in splenic lymphocytes from hamsters with the oral vaccination. The in vivo malaria challenge after the oral mucosal vaccination significantly delayed the blood-appearance day of the parasites in comparison with other immunization sites ( P<0.05). Conclusion: These results suggest that gene immunization via the oral mucosa may induce cell-mediated immunity more efficiently than via the skin or liver, and that the oral mucosa may be one of the most suitable tissues for gene gun-based DNA vaccination against infectious diseases.

Immuno-electron microscopic observation of Plasmodium berghei CTRP localization in the midgut of the vector mosquito Anopheles stephensi.

The subcellular localization of Plasmodium berghei circumsporozoite protein and thrombospondin-related adhesive protein (PbCTRP) in the invasive stage ookinete of P. berghei was studied in the midgut of Anopheles stephensi by immuno-electron microscopic observations using polyclonal antibodies and immuno-gold labeling. PbCTRP was found to be associated with the micronemes of a mature ookinete throughout the movement from the endoperitrophic space to the basal lamina of the midgut epithelium. PbCTRP was also observed in the electron-dense area outside the ookinete, which might have been secreted from the apical pore. PbCTRP is found most abundantly at the site of contact between the apical end of an ookinete and the basal lamina of an epithelial cell. These results suggest that PbCTRP functions as an adhesion molecule for ookinete movement into the midgut lumen and epithelial cell and for ookinete association with the midgut basal lamina and transformation into an oocyst.

Potentiation by a novel alkaloid glycoside adjuvant of a protective cytotoxic T cell immune response specific for a preerythrocytic malaria vaccine candidate antigen

We have recently demonstrated that the novel glycoalkaloid tomatine, derived from leaves of the wild tomato Lycopersicon pimpinellifolium, can act as a powerful adjuvant for the elicitation of antigen-specific CD8 + T cell responses. Here, we have extended our previous investigation with the model antigen ovalbumin to an established malaria infection system in mice and evaluated the cellular immune response to a major preerythrocytic stage malaria vaccine candidate antigen when administered with tomatine. The defined MHC H-2k d class I-binding 9-mer peptide (amino acids 252–260) from Plasmodium berghei circumsporozoite (CS) protein was prepared with tomatine to form a molecular aggregate formulation and this used to immunise BALB/c (H-2k d) mice. Antigen-specific IFN-γ secretion and cytotoxic T lymphocyte activity in vitro were both significantly enhanced compared to responses detected from similarly stimulated splenocytes from naive and tomatine-saline-immunised control mice. Moreover, when challenged with P. berghei sporozoites, mice immunised with the CS 9-mer-tomatine preparation had a significantly delayed onset of erythrocytic infection compared to controls. The data presented validate the use of tomatine to potentiate a cellular immune response to antigenic stimulus by testing in an important biologically relevant system. Specifically, the processing of the P. berghei CS 9-mer as an exogenous antigen and its presentation via MHC class I molecules to CD8 + T cells led to an immune response that is an in vitro correlate of protection against preerythrocytic malaria. This was confirmed by the protective capacity of the 9-mer-tomatine combination upon in vivo immunisation. These findings merit further work to optimise the use of tomatine as an adjuvant in malaria vaccine development.

The synthetic, oxidized C-terminal fragment of thePlasmodium. berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.

Direct Immunization of Malaria DNA Vaccine into the Liver by Gene Gun Protects against Lethal Challenge of Plasmodium berghei Sporozoite

The liver is the first target organ for malaria parasites immediately after the bite of an infected mosquito. We studied local immunization of malaria DNA vaccines at the site of the liver using a gene gun as a useful tool for in vivo transfection of foreign genes. A malaria DNA vaccine consisting of the Plasmodium berghei circumsporozoite protein (PbCSP) gene plus the mouse IL-12 gene was bombarded directly by a gene gun into mouse liver once or into the skin twice. A marked protective effect was induced by gene bombardment into the liver (more than 71%) compared with that into the skin (less than 33%). A Th1-type immune response and high production of iNOS were observed in the hepatic lymphocytes from mice bombarded into the liver, resulting in more effective protection compared with those bombarded into the skin. These results provide an important implication on the development of efficient malaria vaccine strategies.

Extracellular processing and presentation of a 69-mer synthetic polypeptide to MHC class I-restricted T cells

The classical pathway for MHC class-I-restricted Ag presentation processes cytosolic Ag synthesized in or delivered into the cytosol for binding to MHC class I molecules in the ER. Alternatively, Ag may be processed and bind class I molecules in endocytic compartments or at the cell surface after regurgitation of processed peptides. We show that a 69-mer synthetic polypeptide that carries the optimal 9-mer Kd-restricted epitope from the Plasmodium berghei circumsporozoite protein, PbCS 245-253, is presented to CD8 + T cells after a short incubation (1–2 h) with target cells. The presentation kinetics correlate with the length of the peptides when shorter peptide analogues are used. This presentation is independent of the transporters associated with antigen processing and presentation (TAP), does not require newly synthesized proteins and does not proceed via regurgitation of intracellularly processed peptides. In contrast, it is substantially decreased in the absence of β2 microglobulin or serum. Taken together, these data suggest that serum components, such as proteases and β2 microglobulin, allow the processing and loading of exogenous polypeptides onto empty cell surface class I molecules for presentation to CTL.

Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites.

The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.

Cytotoxic T cell induction with ratchet peptide libraries

Immunization with synthetic peptides are used to induce cytotoxic T cell (CTL) responses in vivo. However, CTL peptide vaccines require the use of multiple peptides to overcome genetic diversity associated with MHC restriction, and proir epitope identification from the chosen protein template. We describe here a method whereby all nonamer sequences from a longer template can be synthesized simultaneously in a ratchet peptide library (RPL) covering all potential epitopes within a protein. We synthesized an RPL based on a template sequence from the Plasmodium berghei circumsporozoite (CS) protein (CSRPL). Using a lipopeptide formulation we immunized mice i.p. with the CSRPL and elicited CS specific CTL, which recognized the CS252-260 H-2K d restricted CTL epitope. Published by Elsevier Science Ltd.

Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity

Peptides from Plasmodium berghei circumsporozoite protein (CS) and influenza A virus nucleoprotein (NP) were entrapped in microparticles prepared from poly (lactide-co-glycolide) polymers, and the microparticles were administered parenterally to mice. After immunization with single or multiple doses, splenocytes were tested for a cytotoxic T cell (CTL) response and high levels of CTL activity were detected. The CTL induced were CD8 +, MHC class I restricted, and could recognize virus infected cells. Peptide entrapped in microparticles of mean size <500nm were better inducers of CTL than larger microparticles (mean > 2 μm and above). Microparticles could also be used to deliver lipid modified peptides (lipopeptides) and elicited higher levels of cytolytic activity than either free peptide in microparticles or lipopeptide alone. Microparticles provide a novel way of inducing a CTL response using synthetic peptides.

Re-investigation of the circumsporozoite protein-based induction of sterile immunity against Plasmodium berghei infection

Although the circumsporozoite protein (CSP) of the malaria parasite is the most immunologically characterized protein, the goal of using this protein in an effective vaccine has not yet been realized. Monoclonal antibody against the repetitive immunodominant B-epitope of the CSP can protect mice from malaria, but vaccines that induce antibody against this epitope do not consistently induce protection. Toward developing a rationale for a CSP-based effective vaccine, we have re-investigated the ability of anti-CSP repeat antibodies, as induced by different CSP vaccine formulations with several adjuvants, to confer sterile immunity against sporozoite challenge. Using Plasmodium berghei rodent malaria model and several CSP subunit vaccine constructs, we found that a formulation consisting of the P. berghei CSP repetitive epitope, (DPPPPNPN) 2 (CS), conjugated to BSA by carbodiimide, formulated in a block copolymer and detoxified lipopolysaccharide (RaLPS) adjuvant, was particularly promising. Mice were immunized and boosted with vaccines that contain varying malarial peptide-carrier ratios of 6:1 (CS 6-BSA), 55:1 (CS 55-BSA) and 170:1 (CS 170-BSA). Following immunization, the animals were challenged with live sporozoites. Two types of effects were observed in vaccinated mice. First, sterile immunity was induced in 100%, 50% and 29% of mice that were immunized with the CS 170-BSA, CS 55-BSA, and CS 6-BSA vaccine conjugates, respectively. The second effect of immunization was observed with the CS 170-BSA conjugate vaccine primed mice; a boost in IFA titers followed sporozoite challenge. In addition, we observed that IgG1 isotype titer against the surface of the sporozoite, as measured by IFA, and antibody avidity parallel sterile immunity. These findings reiterate the potential of the CSP as a malaria vaccine candidate antigen, and suggest that the induction of sterile immune responses depends on inducing antibody of the appropriate isotype, avidity and specificity.

Efficient binding to the MHC class I K d molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif

Peptides eluted from the MHC class I K d molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Len in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr 2 was substituted by other amino acids, i.e. Len, Ile or Met. These peptides were variants of the 252–260 K d-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K d molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.

CD8+ cell activation to a major mastocytoma rejection antigen, P815AB: requirement for tum- or helper peptides in priming for skin test reactivity to a P815AB-related peptide.

Delayed-type hypersensitivity (DTH) responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum-) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum- antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum- peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8+ cell-mediated DTH to P815AB was unaffected by the ablation of CD4+ cells, the same treatment, or neutralization of IFN-gamma, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4+ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum- cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.

Monopalmitic acid-peptide conjugates induce cytotoxic T cell responses against malarial epitopes: importance of spacer amino acids

Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2- RS)-propyl]- N-palmitoyl-( R)-cysteine (tripam-C). Inbred mice [( BALB c × C57BL 6 )F1 ] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freund's complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates.

Malaria vaccine: Immunization of mice with a synthetic T cell helper epitope alone leads to protective immunity

The immunogenicity of the non-repetitive sequences of the Plasmodium berghei circumsporozoite (CS) protein was studied using synthetic peptides. Two CS sequences (residues 20-39 and 57-70) exhibiting T cell helper activity were identified. Immunization of BALB/c mice with a branched peptide containing either the 20-39 or the 57-70 sequence and two repeats (B epitope) in a linear sequence induced high titers of anti-repeat and anti-sporozoite antibodies. Mice immunized with the T-B construct (high antibody titers) or with the 57-70 epitope alone (no serum anti-repeat or anti-peptide antibodies) were protected to a similar degree after challenge with infective sporozoites. No protection was obtained in mice immunized with the 20-39 epitope. These results indicate that BALB/c mice can be protected either by effector T cells or by high levels of anti-repeat antibodies. Thus, in the same strain, a double mechanism of protection can be obtained by a synthetic peptide vaccine.

Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Monoclonal antibodies recognize a processing dependent epitope present in the mature CS protein of various plasmodial species.

In the present paper, we have characterized the specificity of a series of monoclonal antibodies (MoAbs) against Plasmodium berghei sporozoites, selected for their lack of reactivity with the repeat domain of the circumsporozoite (CS) protein. We found that these MoAbs recognize Pb44, the mature membrane form of the CS protein, but they do not react with Pb54, its precursor. Furthermore, these MoAbs do not react with any of the synthetic peptides representing the linear sequence of the P. berghei CS protein nor do they react with a recombinant CS (rCS) protein. These observations indicate that the epitope recognized by these antibodies is expressed only after the processing of the CS protein has occurred. This 'processing dependent epitope' is present in sporozoites of P. berghei, and also in those of P. falciparum, P. yoelii and P. brasilianum. It is not present in sporozoites of the P. cynomolgi-P. vivax complex and of P. gallinaceum. These anti-sporozoite MoAbs strongly inhibit sporozoite invasion of hepatoma cells, in vitro, however, they displayed no protective effect in an in vivo assay.