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To determine the optimum combination, concentration, and formulation of synthetic peptides and adjuvants to induce protective CTL responses against the Plasmodium yoelii circumsporozoite protein (PyCSP), BALB/c mice were immunized with linear and multiple antigen peptides (MAP) including PyCSP CTL and Th epitopes in Montanide's ISA51, Lipofectin, and Lipofectamine. An H-2K(d)-restricted PyCSP CTL epitope, SYVPSAEQI (amino acids (aa) 280-288), recognized by protective CTL clones, was included in the following peptides: a 9-aa linear peptide (SYVPSAEQI; PyCSP9), a 20-aa linear peptide (aa 280-299; SYVPSAEQILEFVKQISSL; PyCSP20), a MAP containing four branches of PyCSP20 (MAP(280-299)), and a linear peptide and a MAP(MAP(280-299)p2p30) in which PyCSP20 was colinearly synthesized with two universal Th epitopes from tetanus toxin (p2p30). A MAP containing the PyCSP Th epitope (aa 57-70; KIYNRNIVNRLLGD) was included in some experiments. The highest specific lytic activity against peptide-pulsed target cells was obtained with splenocytes from mice immunized with three doses at 3-wk intervals of MAP(280-299)p2p30 in Lipofectin or Lipofectamine. Forty percent of the mice immunized with MAP(280-299)p2p30 and Lipofectin were protected against sporozoite challenge. Immunization with CTL and Th epitopes co-linearly synthesized in a MAP induced significantly better CTL than did immunization with the same sequence as a linear peptide, or immunization with a mixture of two individual MAPs, one with the CTL epitope and the second with the Th epitope.

Plasmodium falciparum sporozoite surface protein 2 (SSP2), also known as TRAP, is included in experimental human malaria vaccines because Plasmodium yoelii SSP2 is the target of protective CD8+ CTL that eliminate P. yoelii-infected hepatocytes in mice. We now report that immunization with a synthetic branched-chain peptide including four copies of a PySSP2 sequence, NPNEPS, and two tetanus toxin T helper epitopes in the adjuvant TiterMax, or with an 18 amino acid peptide (NPNEPS)3 in the adjuvant protects A/J, but not BALB/c or C57BL/6 mice. Transfer of T lymphocyte-enriched immune splenocytes protects naive mice; in vivo depletion of CD4+ T cells eliminates vaccine-induced protection; and in vivo treatment with anti-IFN-gamma reverses vaccine-induced activity against infected hepatocytes. Lymph node cells from immunized A/J, BALB/c, and C57BL/6 mice recognize the (NPNEPS)3 peptide in vitro. However, the protected A/J mice respond with a predominantly Th1 pattern of lymphocyte response, and the non-protected strains of mice respond with a Th2 pattern. There are many examples of CD4+ T cells transferring protection against infectious organisms. However, to our knowledge, this is the first formal demonstration that immunization with a linear synthetic peptide induces CD4+ T cell-dependent, IFN-gamma dependent, genetically restricted sterile protective immunity against an infectious agent.

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

In this study we have investigated the interaction between two DR11 alleles (DB*1101 and DB*1104) and two previously described tetanus toxin (tt) universal T cell epitopes P2(tt830-843) and P30(tt947-967) by means of a functional cytotoxic competition assay. Both truncation analysis and single alanine substitution analysis were performed. In addition, the capacity of truncated and single alanine substituted peptides to be recognized by human T cell clones from donors bearing the DR1101 or DR1104 alleles was assessed. In the case of truncated peptides the same binding and recognition pattern was observed with both alleles. Longer peptides were better competitors and more potent stimulators, a result that should be taken into account when these peptides are used as immunogens. None of the single alanine substitutions could abrogate or strongly diminish the inhibitory capacity of the analogues tested indicating the lack of strong "anchor residues" present in P2 and P30 and implicated in DR binding. In addition, although the original peptide sequences were presented to specific T cell clones with comparable efficiency, some of the alanine single substituted peptides were better recognized in association with one of the alleles by clones derived from individuals bearing the homologous allele. The only exception was the tt951-967 analogue ttW955A, which was preferentially recognized in association with the DR1104 allele regardless of the clone tested. This suggests that, although it binds to both alleles with comparable efficiency, the MHC-peptide complex so formed is conformationally distinguishable by specific T cell clones.

Synthetic constructs were assembled as multiple Ag peptide systems containing repetitive sequences of Plasmodium falciparum and Plasmodium berghei, the causative agents of human and murine malaria respectively, and two universal human tetanus toxin T cell epitopes 830-843 and 947-967. These constructs were tested for antibody production in mice and for their capacity to stimulate human PBL and tetanus toxin-specific T cell clones. A high antibody titer can be obtained in mice when multiple Ag peptide systems are injected in various adjuvants or in PBS alone. Furthermore, all constructs can activate PBL from every donor tested. However, a variable response was obtained when different clones specific for the two tetanus toxin universal epitopes were used. These constructs may represent possible candidates for a malaria vaccine.

Murine models of hypersensitivity to allergens are useful tools for the evaluation of preclinical strategies to down-regulate the IgE response.To monitor the long-term kinetics of T and B cell responses to allergen as a function of allergen dosage and to investigate the effect of parallel immunization with a second antigen; to correlate B cell response with anaphylaxis.CBA/J mice were sensitized every other week by subcutaneous injections of phospholipase A2 (PLA2) and/or ovalbumin (OVA) adsorbed to alum. Specific antibody isotype responses, T cell proliferation, T cell cytokine production and anaphylaxis were assessed throughout the sensitization phase.Low-dose immunization with PLA2 (0.1 microg) favoured a long-term, specific T helper (Th)2 response with high IgE and IL-4 production in contrast to high-dose PLA2 (10 microg) immunization, which biased the immune response towards a Th1 response with high IgG2a and low IL-4 production. Parallel immunization with an unrelated antigen (ovalbumin) had a significant bystander effect on the immunization with PLA2, which was also dose-dependent. Finally, although anaphylaxis as measured by rectal temperature drop was allergen-specific, it could be induced in the high- and low-dose immunization groups, and was not solely dependent on IgE levels.Though low-dose allergen immunization appears to induce an efficient IgE response, the intensity and quality of this response may be modulated by bystander effects of parallel immunization and does not correlate strictly with anaphylaxis. This observation has relevance to the design of clinical immunotherapy protocols using murine model-based data.

We report the first synthetic peptide vaccine eliciting strong CD8(+) and CD4(+) T lymphocyte responses in humans. The vaccine, representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282-383) was well tolerated and strong sporozoite-specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN-gamma, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8(+) T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8(+) T cell response is needed.

We synthesized 17 long synthetic peptides (LSP) spanning the whole 200-kDa Plasmodium falciparum liver stage antigen-3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper-endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3-LSP develop in malaria-exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non-repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3-LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3-LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN-gamma-specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44-119, 1026-1095, 1601-1712) also contained epitopes recognized by H-2(d) class I-restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

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.

To evaluate a long peptide-based allergy vaccine in a murine model, CBA/J mice were sensitized with low dose alum-adsorbed phospholipase A2 (PLA2), a major bee venom allergen. Presensitized mice were treated by daily i.p. injections of a mixture of three long overlapping peptides (44- to 60-mer) spanning the entire PLA2 molecule (100 microg/peptide) for 6 consecutive days. This therapeutic approach induced a sharp drop in PLA2-specific IgE, an increase in specific IgG2a, and a marked T cell hyporesponsiveness. T cell cytokine secretion was characterized by a shift from a Th2 to a Th1 profile. Prophylactic treatment of naive mice with long peptides prior to sensitization with PLA2 induced a comparable modulation of B and T cell responses. Upon i.p. challenge with native PLA2, presensitized mice treated with the long peptide mixture were fully protected from anaphylaxis. This indicated that allergen-derived long overlapping peptides were safe and able to modulate an established Th2 response or to prevent its development. Furthermore, long peptide-based immunotherapy provided clinical protection against anaphylaxis, thus appearing as a promising approach of the therapy of allergic diseases.

Preclinical evaluation of synthetic peptides corresponding to the C-terminal regions of the circumsporozoite (CS) protein in various Plasmodia showed that these preparations were immunogenic and safe upon injection in various animal models. Additionally, the corresponding peptide from Plasmodium falciparum was widely recognized by sera and PBL obtained from semi-immune adults living in malaria endemic areas. Moreover, the CS C-terminal peptide derived from P. berghei conferred protection upon challenge with live sporozoites in mice. A GLP preparation of the synthetic peptide corresponding to residues 282-383 of the Pf CS, NF-54 strain is currently evaluated in a open, non-randomized, Phase I human trial. Data obtained after the second antigen injection show that the malaria vaccine Pf CS 282-383 is safe, well tolerated and gives rise to high antibody titre, CD4+ and CD8+ lymphocyte responses.

Plasma samples of individuals from two malaria-endemic villages on the Colombian Pacific coast and synthetic peptides representing different fragments of the central and flanking regions of the Plasmodium vivax circumsporozoite protein (CSP) were used to perform a fine mapping of the B-cell epitopes on the whole CSP. In addition, the immunogenicity of long polypeptides corresponding to the amino (N) and carboxyl (C) regions was evaluated in Aotus monkeys. The epitopes recognized after natural infection of humans and after immunization of Aotus with these synthetic peptides were compared. Human samples more frequently contained specific antibodies to the central region. The type-I repeat region of the CSP was predominantly recognized by the human sera (by 68% of those from the village of Zacarías and 75% of those from Bajo Calima), a significantly smaller population reacting with the type-II repeat (20% and 11%, respectively). Most of the sera reacting with the type-I repeat recognized the minimal epitope AGDR. Although the N- and C-terminal polypeptides were both highly immunogenic in Aotus and induced long-lasting antibodies, titres of antibodies to the C-terminal polypeptide were higher than those of antibodies to the N-terminal. Competitive inhibition assays performed using human and monkey plasma allowed the identification of dominant B-cell epitopes on sequence 71-90 (p8) from the amino region and sequence 332-361 (p24/p25) from the carboxyl region. The high prevalence of naturally induced antibodies to the three epitopes, the possible functional role of the corresponding sequences, and the high immunogenicity of these epitopes in Aotus could be of great importance in the development of a malaria vaccine based on P. vivax CSP.

Tetanus toxoid (Ttxd) encapsulated in polyester microspheres (MS) for single injection immunization have so far given pulsatile in vitro release and strong immune response in animals, but no boosting effect. This has been ascribed to insufficient toxoid stability within the MS exposed to in vivo conditions over a prolonged time period. This study examined the effect of co-encapsulated putative stabilizing additives.Two different Ttxd were encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA 50:50) and poly(D,L-lactic acid) (PLA) MS by spray-drying. The influence of co-encapsulated additives on toxoid stability, loading in and release from the MS, was studied by fluorimetry and ELISA.Co-encapsulated albumin, trehalose and gamma-hydroxypropyl cyclodextrin all improved the toxoid encapsulation efficiency in PLGA 50:50 MS. Albumin increased the encapsulation efficiency of antigenic Ttxd by one to two orders of magnitude. Further, with albumin or a mixture of albumin and trehalose ELISA responsive Ttxd was released over 1-2 months following a pulsatile pattern.Optimized Ttxd containing MS may be valuable for a single-dose vaccine delivery system.

Antigenic proteins encapsulated in biodegradable polyester microspheres (MS) can slowly denature or aggregate, which results in decreased antigenicity. In this study, we have evaluated the ability of co-encapsulated additives to protect against the loss of tetanus toxoid (TT) antigenicity.Antibody responses were analyzed after immunization of mice with TT microencapsulated in the presence of additives (TT-MS-additive).Immunization with TT-MS-additives gave rise to higher responses than those obtained in the absence of additive. BSA, trehalose. Gamma-hydroxypropylcyclodextrin and calcium salts preserved the immunogenicity of the incorporated antigen with the highest efficacy. Sustained responses were obtained with mixtures of fast and slowly releasing TT-MS containing BSA plus trehalose or calcium salts.The selected additives may stabilize the antigen in MS during storage and rehydration in body fluids. Regulated antigen release from MS-based vaccines permits a reduction of the antigen dose and optimization of single-dose vaccine formulations.

International audience; Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen-loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen-presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation-purified monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The two APC were prepared in parallel from the same donors and their phenotype and antigen-presenting capacity were compared. DC differed from MPH by a higher expression of HLA-DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti-CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I-restricted T-cell responses by exogenous peptide presentation.

Antigen degradation and peptide loading to major histocompatibility complex class I and class II molecules are described with special emphasis on "noncanonical" pathways. Examples of specific peptide loading for measles proteins are provided. In addition, characterization of defined epitopes presented to T cells can lead to the design of products of special interest in medicine and, in particular, in development of vaccines.

Synthetic polypeptides encompassing the non-repeated regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are very immunogenic in mice and are recognized by sera from donors living in regions where malaria is endemic, both in Africa and South America. Long polypeptides, encompassing the N- or C-terminal regions, have now been used to demonstrate peptide-specific T cells in donors living in an endemic area of Colombia. Although the N-terminal peptide (22-125) was recognized almost exclusively by donors from the endemic area, the patterns of recognition of the C-terminal peptide (289-390) in donors from endemic and non-endemic areas were similar and like the pattern with smaller peptides. The availability of the long polypeptides made it possible to compare T-cell responses to the non-repeated regions of the CSP with the presence of peptide-specific antibodies. No correlation was found and no antibodies were detected in donors from non-endemic regions. The long polypeptides also elicited strong antibody and T-cell responses in owl monkeys (Aotus lemurinus). The antibodies generated against the synthetic peptides in such monkeys also recognized sporozoites, the natural infective form of the parasite. The results emphasise the potential of the peptides tested as malaria-vaccine candidates. Not only are they recognized by humans at both the B- and T-cell level but they also elicit strong responses in monkeys and encompass several distinct T-cell epitopes, thus overcoming the limitations of specific, major-histocompatibility-complex restriction.

We investigated the Ag fine specificity of four TCRs that shared the same V beta segment but used V alpha s of three different subfamilies and displayed highly heterogeneous alpha and beta CDR3. The TCRs recognized the tetanus toxin tt830-843 (QYIKANSKFIGITE) epitope presented by DRB1*1302. By using a large panel of monosubstituted peptide analogues, we first defined the requirements for tt830-843 binding to DRB1*1302. We found that three residues, I832, N835, and G840, were critical for the interaction with DRB1*1302. Residues potentially contacted by the four TCRs were functionally defined by measuring the IL-2 response to the analogues. Except for the first and the last three residues, as well as I832 and G340, all of the others appeared to provide contacts with the four TCRs, indicating a considerable overlapping in the way these TCRs interact with the peptide. More importantly, and contrary to expectations, the two TCRs expressing the same V alpha/V beta germ-line segments showed a strikingly similar reactivity toward nearly all substitutions; moreover, more pronounced differences were observed when comparing TCRs using different V alpha segments. These results indicate that TCRs with entirely distinct CDR3s in the context of conserved V segments may not differ substantially in the way they recognize the ligand, and may provide new insights into understanding the formation of TCR/peptide/MHC ternary complexes. During these studies, we noticed that analogues with nonconservative substitutions at I832, which bound very unstably to DRB1*1302, could effectively stimulate T cells, suggesting a role of the TCR in contributing toward stabilization of peptide binding.

We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of the P. falciparum 7G8 isolate CS protein expressed on the sporozoite surface. They comprise what is believed to be the mature protein, except for the central repetitive B cell domain. BALB/c (H-2d) mice were immunized s.c. with 50 micrograms soluble CS polypeptides emulsified in IFA. After a single immunization, CS-specific helper and cytotoxic T lymphocytes (CTLs) could be obtained. The resultant CTLs obtained by in vitro restimulation of primed lymph node (LN) cells recognized H-2Kd target cells in the presence of short synthetic peptides defined in the present study. These epitopes are contained within the N- and C-terminal regions of the CS protein, and correspond to sequences 39-47 and 333-342. In addition, these CTLs can specifically lyse H-2d target cells transfected with the CS gene. These results suggest that, by immunization of mice with large soluble CS synthetic polypeptides in IFA, it is possible to obtain MHC class I-restricted T cell responses specific for the CS protein. This approach might be advantageous in the formulation of efficient malaria subunit vaccines.

Multiple-antigen peptide (MAP) constructs containing different T- and B-cell epitopes were assessed for their ability to be specifically recognized by murine and human T-cell clones. The different synthetic MAP constructs consisted of a malaria T-cell epitope or of a human universal tetanus toxin helper T-cell epitope collinearly synthesized with B-cell epitopes from the circumsporozoite proteins of different malaria parasites. All constructs were able to stimulate specifically T-cell clones. Interestingly, T-cell epitopes assembled as MAP constructs did not require processing for the specific stimulation of murine and human T-cell clones, as shown by retention of their stimulatory effect in the presence of glutaraldehyde-fixed antigen-presenting cells. However, processing was required for most of the synthetic constructs containing both T- and B-cell epitopes. Thus, the requirement for processing of these constructs seems to be dictated by the nature of the B-cell epitope present.

Previously, CD4+ T cell lines and clones were isolated after immunization of BALB/c and C57BL/6 mice with the Py1 peptide, a 21-mer synthetic peptide corresponding to a N-terminal segment of the circumsporozoite protein of Plasmodium yoelii. The clones were separated into the Th1 and Th2 subsets on the basis of lymphokine production. It was observed that immunization with the Py1 peptide induced preferentially Th1 cells in BALB/c and Th2 cells in C57BL/6 mice. These clones were then tested for their cytolytic ability in vitro. Some of the clones from BALB/c and C57BL/6 mice eliminated liver stage parasites from cultured hepatocytes in a MHC restricted manner. Nevertheless, none of these clones was able to lyse Py1 peptide-pulsed target cells. It was also found that two clones could protect BALB/c mice against a sporozoite challenge. These results provide evidence that CD4+ T cells, induced after priming with a defined peptide, could participate in the effector mechanisms against malaria liver stages.

The design, total chemical synthesis, and immunological properties of a four-alpha-helix bundle template-assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58-74 of the alpha 1 heavy chain domain of HLA-A2 was modeled in order to increase helix stability and amphiphilicity of the 17-mer peptide, preserving the residues for potential T-cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four-helix-bundle topology of the assembled peptide blocks. After extensive HPLC purification, stepwise solid-phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial alpha-helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four-alpha-helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA-A2-positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.

In order to identify T cell epitopes recognized by human in the Plasmodium vivax circumsporozoite protein, 28 overlapping synthetic peptides spanning the entire circumsporozoite protein were tested for their ability to stimulate proliferation of PBMC from 22 adults living in a malaria-endemic area of the Colombian Pacific Coast and from four individuals who never had a history of malaria infection. In addition, BALB/c mice were immunized with pools of peptides, and their lymph node cells were stimulated in vitro with individual peptides. Four epitopes were recognized by human lymphocytes but not all of them by mice. One of the epitopes was located inside the central repetitive B cell immunodominant domain. Several of the variants of the repeats were recognized by about one-third of the studied individuals. Another T cell epitope was located in the amino terminus and the other two in the carboxyl region. Peptides were recognized by both immune and nonimmune donors. Some of them were frequently recognized suggesting a lack of genetic restriction, whereas some others were recognized by only a few individuals but induced strong proliferation. These epitopes may be of potential value for a malaria subunit vaccine.

T lymphocytes expressing variable cell surface antigen receptors recognize "processed" forms of antigen, presented on the surface of other cells by molecules of the major histocompatibility complex (MHC). Naturally processed antigenic peptides can be replaced by synthetic ones. The synthetic peptide AYPPPPPTLA (P5) is an active competitor to the antigenic peptide HLA A24 170-182 (sequence RYLENGKETLQRA) that is recognized by A24 specific T cells in association with the H-2Kd class I MHC molecule. In P5 the five prolines were designed to play the role of a rigid spacer between the residue Y and the T-L unit, so as to mimic the role of Y171, T178, and L179 in the HLA A24 antigenic peptide, since these residues have proven to be the most important with respect to the binding of the HLA A24 peptide with the H-2Kd MHC molecule. Nuclear magnetic resonance studies allow us to demonstrate that in aqueous solution P5 adopts at least three long-lived conformations that can be classified with respect to the Y2-P3-P4 amide bonds as trans-trans, cis-trans, and cis-cis. Among these, the trans-trans form is present in 67% of the molecules while the two others share the remaining 33%. [on SciFinder (R)]

We have defined structural features that are apparently important for the binding of four different, unrelated antigenic epitopes to the same major histocompatibility complex (MHC) class I molecule, H-2Kd. The four epitopes are recognized in the form of synthetic peptides by cytotoxic T lymphocytes of the appropriate specificity. By analysis of the relative potency of truncated peptides, we demonstrated that for each of the four epitopes, optimal antigenic activity was present in a peptide of 9 or 10 amino acid residues. A comparison of the relative competitor activity of the different-length peptides in a functional competition assay, as well as in a direct binding assay based on photoaffinity labeling of the Kd molecule, indicated that the enhanced potency of the peptides upon reduction in length was most likely due to a higher affinity of the shorter peptides for the Kd molecule. A remarkably simple motif that appears to be important for the specific binding of Kd-restricted peptides was identified by the analysis of peptides containing amino acid substitutions or deletions. The motif consists of two elements, a Tyr in the second position relative to the NH2 terminus and a hydrophobic residue with a large aliphatic side chain (Leu, Ile, or Val) at the COOH-terminal end of the optimal 9- or 10-mer peptides. We demonstrated that a simple peptide analogue (AYP6L) that incorporates the motif can effectively and specifically interact with the Kd molecule. Moreover, all of the additional Kd-restricted epitopes defined thus far in the literature contain the motif, and it may thus be useful for the prediction of new epitopes recognized by T cells in the context of this MHC class I molecule.

With the use of flow cytometry, we recorded changes in intracellular ionized calcium [Ca2+]i of Indo-1 loaded T cells that were triggered by contact with APC. This rapid readout of TCR perturbation enabled us to monitor the formation of stimulatory Ag-MHC complexes on EBV-transformed B cells that were either pulsed with native tetanus toxoid (TT) or with a 12-amino-acid fragment of this protein. Neither unpulsed APC nor Ag-specific APC that were pulsed with native Ag and kept at +4 degrees C were able to induce changes in basal T cell [Ca2+]i in TT-specific T cell clones. After 1 h at 37 degrees C, however, the Ag-pulsed APC were able to induce a three-to-fourfold increase in [Ca2+]i. This length of time appeared to be almost independent of the concentration of Ag with which the APC were pulsed, suggesting that the lag time was due more to intracellular transit than to association of the processed Ag with the MHC molecule. Furthermore, the same lag time and independence of Ag concentration were found when the EBV-transformed B cells were pulsed with a mouse-anti-transferrin receptor mAb and tested for their capacity to trigger a T cell clone specific for processed mouse Ig. This indicates that, in addition to surface Ig, other receptors that are internalized can function in the same fashion in the uptake and processing of a soluble Ag. In contrast to what was found with intact native Ag, no lag time was observed when the APC were pulsed with high concentrations of a 12-amino-acid peptide, containing the amino acid sequence recognized by a TT-specific T cell clone, suggesting that the formation of MHC-peptide complexes occurs instantly. Pulsing with a lower peptide concentration, however, caused the appearance of a time-dependent increase in efficacy of Ag presentation, suggesting a slow accumulation of MHC-peptide complexes on the B cell membrane.

Immunization of DBA/2 (H-2d) mice with syngeneic P815 tumor cell transfectants that express HLA class I genes elicits CTL that recognize HLA in the context of H-2Kd molecules. Anti-HLA-CW3 CTL cross-react to a variable extent on the related alleles A3 and A24. Using a panel of target cells expressing native or recombinant HLA genes, we could map the epitope recognized by a CTL clone specific for CW3 to the second external (alpha 2) domain of CW3. Moreover, the epitope recognized by this clone could be mimicked by incubating P815 (HLA negative) target cells with a synthetic peptide corresponding to the C-terminal 12 amino acids of the CW3 alpha 2 domain (residues 171 to 182). Other independent anti-CW3 CTL clones with different fine specificities recognized the same CW3 peptide. In contrast, CTL clones specific for HLA-A24 or HLA-A3 that did not lyse P815-CW3 transfectants did not recognize this peptide. The CW3 peptide could be recognized on other tumor cell targets that were also of H-2d origin, but not on those of H-2b or H-2k origin. The requirement for the expression of H-2Kd by the target cells was directly demonstrated using L cell Kd transfectants. Our results suggest that the CTL response of DBA/2 mice immunized with P815-CW3 transfectants is predominantly Kd restricted and focused on epitopes contained within the 12 C-terminal amino acids of the alpha 2 domain.

Murine T cell clones that proliferated specifically in response to the protein antigen apo cytochrome c were derived and maintained in continuous culture. Two distinct clonotypes were observed with respect to the proliferative responses observed when a variety of peptides prepared from several species of cytochrome c were tested. These 2 clonotypes appeared to recognize 2 different regions in the cytochrome c molecule. Only 1 of the 2 clonotypes tested demonstrated helper cell activity for antibody formation in vitro.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

The specificity of peptide recognition by a number of Kd-restricted CTL clones specific for HLA-CW3 or HLA-A24 was investigated. The CTL clones were derived from DBA/2 (H-2d) mice immunized with syngeneic P815 mouse cells transfected with genes encoding HLA-CW3 or HLA-A24 class I molecules. We had previously shown that CTL clones that lysed P815-CW3 transfectant target cells could lyse P815 (HLA-) target cells incubated with synthetic CW3 peptides corresponding to the COOH-terminal end of the alpha 2 domain. In the present study, we found that Kd-restricted CTL clones that lysed P815-A24 transfectant target cells recognized a synthetic peptide from the same region (residues 170-182) of the A24 molecule. CW3 and A24 differ by only one amino acid within this region. Recognition of CW3 or A24 peptides corresponded exactly with lysis of P815-HLA transfectants both for clones that mutually exclusively lysed CW3 or A24 transfectant target cells and for CW3/A24 crossreactive CTL clones. The latter CTL clones that lysed both CW3 and A24 transfectant target cells showed a clear preference for the peptide corresponding to the immunizing HLA allele. The homologous CW3 and A24 peptides could compete with each other for recognition, in contrast to a peptide from the same region of HLA-B7. Peptides from the corresponding region of the endogenous Kd and Dd/Ld molecules could also inhibit recognition of CW3 and A24 peptides. Competition with peptides apparently occurred at the level of the target cell. These results are consistent with a model whereby MHC class I molecules position protein fragments or peptides for specific recognition by T cells.

Murine T-lymphocyte specificity was determined in a system of antigen driven in vitro T-cell proliferation using cytochrome c molecules from different species, their derived peptides and reconstituted hybrid proteins. It was observed that primed T cells could discriminate between peptide fragments which differed from each other at a single amino acid residue. These conclusions were substantiated by the pattern of cross-reactivity noted in the response of closely related cytochrome c proteins as well as when artificial hybrid molecules reconstituted by the covalent linkage of peptide fragments were analyzed. The pattern of specificity observed appeared to be haplotype (BDF1) dependent although similar conclusions about the fine specificity of T cells in the response to cytochrome c have been obtained in other strains but associated with different residues.

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.

In these studies, we have shown that the heme moiety of cyt c is a dominant T cell epitope that induces a large proliferative response in lymph node T cells derived from SJL and B10.A mice when presented on either unfixed or fixed syngeneic APCs. Not only is this vigorous response observed for cyt c-primed T cell populations but also for populations obtained from naive SJL or B10.A mice. The reactivity to the heme moiety falls under strict MHC restriction, in that it is present only in murine strains bearing either the I-Ak or I-As molecule and can be blocked by antibodies specific for these class II molecules. Therefore, these findings require that the current models describing the nature of T cell epitopes be extended to include nonpeptide molecules. Furthermore, as the heme moiety is ubiquitous throughout the organism, although sequestered within proteins, the existence of heme-reactive T cell populations in unprimed animals provides another example of the existence of self-reactive T cell clones.

Human T cell clones specific for epitopes 830-843 and 947-967 of tetanus toxin can be differentially activated in vitro when APC (PBL or LCL) from different donors are pulsed with tetanus toxin. Although PBL tested do not seem to exhibit substantial differences in the number of precursor T cells specific for these epitopes, APC from the same donors activate clone KT-2 specific for peptide 830-843 but not clone KT-30 specific for peptide 947-967. These APC express the proper restriction element because they can present the corresponding synthetic peptides. The failure to present a particular epitope might, however, be explained by the absence or presence of a protease(s) required for Ag presentation that may vary for different epitopes. Indeed, the protease inhibitor leupeptin was found to inhibit activation of KT-2 but not KT-30 T cell clone by the KK.35 B cell line normally capable of presenting either epitope. In summary, these data suggest that tetanus toxin processing and epitope formation by APC is distinct in different donors and for different epitopes.

The region of the horse cytochrome c molecule recognized by Mab SJL2-4 specific for the denatured form of the protein was located around residues 22-28. Binding studies on antigen pulsed macrophages were also performed. Surprisingly, heme peptide 1-65 was not recognised by Mab when bound on macrophages. This correlates with the incapacity of the same peptide to activate the T-cell clone 2-16. Binding sites on antigen pulsed macrophages varied between 0.5-2 x 10(6) per cell depending on the conditions used. The expression of the antigenic determinant as detected by Mab was also followed under different conditions (chloroquine, trypsin treatment) and time. Kinetics parameters of the antigen-antibody reaction in solution and on antigen bound macrophages were also determined and are dramatically different. This is correlated with a different structure of the peptide in solution and on macrophage cell surface.

The repetitive epitope (Asn-Ala-Asn-Pro = NANP) of the Plasmodium falciparum circumsporozoite protein is considered as the basis for the development of a recombinant or synthetic subunit vaccine against malaria. Vaccines consisting of (NANP)n molecules coupled to carrier proteins have already been tested in trials in human volunteers with partial success. In this paper we show that C57BL/6 mice, genetically responsive to carrier-free (NANP)n molecules, exhibit a secondary antibody response to (NANP) if they are primed with carrier-free (NANP)40 synthetic peptide, and then challenged with P. falciparum sporozoites. However, such a sporozoite-mediated boosting effect is not observed if C57BL/6 and BALB/c mice were previously primed with (NANP)40 peptide conjugated to carrier proteins. The genetic restriction of the murine antibody response to (NANP)n is overcome when mice bearing seven different H-2 haplotypes are immunized with entire P. falciparum sporozoites. These results may have implications for the understanding of natural or induced anti-sporozoite immunity, and show that the use of T-cell epitopes from the plasmodial antigenic repertoire would be very likely to represent an efficient approach for the development of a subunit malaria vaccine.

Surface- or biosynthetically labeled Lyt-2/3 antigens were isolated from cell lysates by immunoprecipitation and affinity chromatography with a monoclonal antibody. Tryptic digests of the individual subunits of 37,000, 32,000 and 28,000 apparent mol. wts were analysed by reverse-phase high-performance liquid chromatography and by two-dimensional peptide mapping. The results indicate that the 37,000 and 32,000 mol. wt components are structurally very similar whereas the 28,000 mol. wt component appears as a different molecule.

The ability of synthetic P. falciparum (NANP)n circumsporozoite peptides to elicit murine T cell proliferative responses was studied. When C57BL/6, C3H, and DBA/2 mice were injected with (NANP)40, only C57BL/6 (H-2b)-immune lymph node cells proliferated on restimulation in vitro with the same peptide. By using anti-I-A monoclonal antibodies or spleen cells from congenic H-2b mice as a source of antigen-presenting cells, the T cell proliferative response was shown to be restricted to the I-Ab region of the C57BL/6 haplotype. These results are in agreement with previous experiments which demonstrated that the anti-(NANP)40 antibody response was uniquely restricted to C57BL/6 (H-2b) mice. Several C57BL/6 long-term (NANP)n-specific T cell lines and clones were derived. All of the clones exhibited the L3T4 helper T cell phenotype. A considerable heterogeneity of T cell responses was observed when the lines and clones were stimulated with different concentrations of the various peptides studied. The results, together with the observed genetic restriction for both antibody and T cell responses, suggest that perhaps not all individuals who receive a similar repetitive tetrapeptide sporozoite malaria vaccine will develop T cell and or antibody responses.

An in vitro assay which measures antigen-induced proliferation of primed murine lymph node cells is described. The response is mediated by T eclls since it can be obtained by using nylon wool-passed lymphocytes (less than 1% Ig+ cells) and it can be abolished by treatment with anti-Thy 1.2 and C. Furthermore, LN cells from nu/nu mice injected with antigen do not demonstrate antigen-induced proliferation in contrast to the response observed in euthymic littermate controls. Other relevant parameters of this proliferative assay include the observations that the response is highly antigen specific, can be seen as early as 4 days and as late as 60 days after in vivo priming, is restricted to the use of certain sets of LN when animals are injected subcutaneously at the base of the tail, and can be seen with LN cells from mice primed with antigen in either CFA or ICFA. The ease of the assay coupled with its specificity and quantitative dimensions provides a direct and simple method to evaluate processes involved in antigen-induced murine T lymphocyte activation.