Plasmodium Berghei Circumsporozoite Protein Research Articles

C3d-defined complement receptor-binding peptide p28 conjugated to circumsporozoite protein provides protection against Plasmodium berghei

Immune response against circumsporozoite protein (CSP) of Plasmodium berghei, a major surface protein on the sporozoite, confers protection in various murine malaria models. Engineered DNA vaccine encoding CSP and 3 copies of C3d caused an unexpected loss in protection attributed to the binding of C3d to the C-terminal region of CSP. Because the C3d region known as p28 represents the complement receptor (CR) 2-binding motif, we developed a CSP-3 copies of p28 DNA construct (CSP-3p28). CSP-3p28-immunized mice were better protected against P. berghei sporozoites than CSP-immunized mice 6 weeks after the 2nd boost, produced sufficient IgG1 anti-CSP and CSP C-terminus antibody and failed to produce IgG2a. CSP-3C3d-immunized mice were not protected, failed to produce IgG1 and produced high amounts of IgG2a. We conclude that use of the CR2-binding motif of C3d as molecular adjuvant to CSP results in anti-malaria protective immune response probably by targeting the chimeric protein to CR2.

Baculovirus-based Vaccination Vectors Allow for Efficient Induction of Immune Responses Against Plasmodium falciparum Circumsporozoite Protein

Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

Effective Targeting of Liposomes to Liver and Hepatocytes In Vivo by Incorporation of a Plasmodium Amino Acid Sequence

Several species of the protozoan Plasmodium effectively target mammalian liver during the initial phase of host invasion. The purpose of this study was to demonstrate that a Plasmodium targeting amino acid sequence can be engineered into therapeutic nanoparticle delivery systems. A 19-amino peptide from the circumsporozoite protein of Plasmodium berghei was prepared containing the conserved region I as well as a consensus heparan sulfate proteoglycan binding sequence. This peptide was attached to the distal end of a lipid-polyethylene glycol bioconjugate. The bioconjugate was incorporated into phosphatidylcholine liposomes containing fluorescently labeled lipids to follow blood clearance and organ distribution in vivo. When administered intravenously into mice, the peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver. Fluorescence and electron microscopy demonstrated that the liposomes were accumulated both by nonparenchymal cells and hepatocytes, with the majority of the liposomal material associated with hepatocytes. Accumulation of liposomes in the liver was several hundredfold higher compared to heart, lung, and kidney, and more than 10-fold higher compared to spleen. In liver slice experiments, liposome binding was specific to sites sensitive to heparinase. Incorporation of amino acid sequences that recognize glycosaminoglycans is an effective strategy for the development of targeted drug delivery systems.

Direct processing and presentation of antigen from malaria sporozoites by professional antigen‐presenting cells in the induction of CD8+ T‐cell responses

Irradiated malaria sporozoites induce better protection than viable untreated sporozoites. We observed early differences between irradiated and viable untreated sporozoites in priming responses in vivo to a protective CD8 T-cell epitope, pb9, of the circumsporozoite protein of Plasmodium berghei. Sporozoites were processed for MHC class I presentation by dendritic cells (DC) to prime pb9-specific IFN-gamma-producing CD8 T cells. DC pulsed with untreated and irradiated sporozoites were similarly capable of priming central memory T-cell responses, detectable by the IFN-gamma cultured ELISPOT assay. However, irradiation significantly enhanced sporozoites' ability to prime effector T-cell responses detectable by the IFN-gammaex vivo ELISPOT assay. Irradiation also enhanced the ability of splenic APC to process and present sporozoites in order to re-stimulate pb9-specific polyclonal and clonal T-cell responses. Sporozoites did not stimulate T cells in the absence of APC. Over-irradiation decreased the sporozoites' T-cell stimulating capacity in vitro at high parasite doses, which may indicate that an optimal irradiation dose is necessary to induce protective immunity by sporozoite inoculation. The induction of sporozoite-specific CD8 T-cell responses without the need for liver stage infection identifies a potentially important mechanism in the development of pre-erythrocytic immunity.

A strong CD8 + T cell response is elicited using the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei together with the adjuvant QS-21: quantitative and phenotypic comparison with the vaccine model of irradiated sporozoites

Stable protective immunity can be achieved against malaria by the injection of radiation-attenuated sporozoites (γ-spz) and is mediated by IFN-γ producing CD8 + T cells targeting the pre-erythrocytic stages. An efficient malaria vaccine should mimic this immunity. We compared the immune response specific for the circumsporozoite protein (CSP) of Plasmodium berghei ( P. berghei), an important target of this protective response, elicited in mice immunized with the long synthetic polypeptide (LSP) PbCS 242–310, representing the C-terminus of the CSP of P. berghei, with the adjuvant QS-21 or injected with γ-spz. The ex vivo evaluation of the CD8 + T cell response by IFN-γ ELISPOT assay revealed that the injection of LSP with QS-21 induced, compared to γ-spz, a similar frequency of peptide-specific lymphocytes in the spleen but a eight-fold increase in the draining lymph-nodes. A very high frequency of CD8 + T cells, specific for the sequence PbCS 245–253, a H-2K d-restricted CTL epitope, was obtained in the liver and spleen of mice immunized with the two regimens. Even though the frequency of H-2K d PbCS 245–253 multimer +, CD8 + T cells was higher in γ-spz immunized mice, the frequency of IFN-γ producing CD8 + T cells was comparable. The phenotype of the CD8 + T cell responses was characterized with the help H-2K d PbCS 245–253 multimer and most of the CSP-specific CD8 + T cells represented an intermediate subset between effector and central memory with CD44 high, CD45RB high, CD62L low and CD122 high. The number of memory CD8 + T cells decreased after the last LSP immunization but could be boosted to higher level with live spz. The unique combination of LSP PbCS 242–310 and the adjuvant QS-21 induced an immune response that was comparable in terms of quality to the one generated with γ-spz. This confirmed the potential of LSP as malaria vaccine candidates as well as for the study of the repertoire of targets of protective immunity in the γ-spz vaccine model.

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.

OM-174, a new adjuvant with a potential for human use, induces a protective response when administered with the synthetic C-terminal fragment 242–310 from the circumsporozoite protein of Plasmodium berghei

The goal of this project was the evaluation of a novel immunomodulatory adjuvant for human use, OM-174, which is a soluble adjuvant derived from Escherichia coli lipid A. For this study, we used a synthetic peptide, known for its safety and reproducibility and the murine model of BALB/c mice. The long peptide ( PbCS 242–310) used corresponds to the C-terminal region of the circumsporozoite protein (CSP) that is the major protein on the surface of Plasmodium sporozoites. Subcutaneous injections of PbCS 242–310 in combination with soluble adjuvant OM-174 induced long lasting peptide-specific antibody titres comparable to those obtained by immunization with incomplete Freund’s adjuvant (IFA). The ex vivo evaluation of the CD8 + T cell response by IFN-γ ELISPOT assay revealed that the injection of polypeptide with OM-174 adjuvant induced, compared to IFA, a similar and an eight-fold increased frequency of peptide-specific lymphocytes in the draining lymph-nodes and in the spleen, respectively. The CD8 + T-cells are specific for the sequence PbCS 245–253, a well-known H-2K d-restricted CTL epitope, and are cytotoxic as shown in a chromium release assay. Immunization of BALB/c mice with this polypeptide in combination with adjuvant OM-174 conferred a protection after challenge with live Plasmodium berghei sporozoites. The strong antibody and CTL responses observed to a synthetic peptide in mice, the safety profile of the adjuvant and its extensive physico-chemical characterization suggest that OM-174 has a potential use in vaccine formulations for humans.

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.

The synthetic, oxidized C-terminal fragment of the Plasmodium 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.

Induction of a cytotoxic T lymphocyte response by immunization with a malaria specific CTL peptide entrapped in biodegradable polymer microspheres

We have previously reported that biodegradable polymer microspheres (MS) are capable of eliciting strong and long-lasting antibody and T cell proliferative responses for either natural protein antigens or synthetic peptides. In this study, we investigated the possibility of inducing antigen-specific cytotoxic T lymphocyte (CTL) responses in vivo with a short synthetic peptide from the circumsporozoite (CS) protein of Plasmodium berghei (Pb) 252–260 by using different MS formulations. We show that injection of mice with a short CTL epitope microencapsulated in MS or adsorbed on empty MS enhanced a specific CTL response comparable to that obtained with the incomplete Freund's adjuvant (IFA) formulation, indicating that MS are a potent antigen delivery system/immunostimulant for CTL response. These results might be of practical interest for MS preparation and development of subunit vaccines.

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.

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.