Marc Bossus

High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules.

We have investigated the immunogenicity in chimpanzees of twelve synthetic peptides derived from four new Plasmodium falciparum molecules expressed at pre-erythrocytic stages of the human malaria parasite. These parasite molecules were initially selected through their ability to be recognized by stage restricted human antibodies. Twelve 20- to 41-mer peptides representing potential human B- or T-cell epitopes were selected from these proteins, and synthesized. Six of these were modified by a C-terminal lipidic chain in order to re-inforce their immunogenicity. Strong B- and T-helper cell responses were induced in chimpanzees by lipopeptides injected without adjuvant and by peptides in Montanide. All twelve peptides induced CD4(+) T-cell proliferative responses, as well as the secretion of IFN-gamma (some of them at very high levels) and eleven peptides induced antibody responses. The immune responses elicited in this way were reactive with native parasite proteins, as shown by recall studies with sporozoite stage proteins, and proved to be long-lasting (up to 10 months after immunization). Our results support the strategy employed to select these four new malarial antigens and the corresponding peptides, and suggest that the immunizing formulations are both efficient and clinically acceptable.

Antigenicity and immunogenicity of P30-derived peptides in experimental models of toxoplasmosis

P30, also referred to as SAG-1, is now recognized as a major Toxoplasma gondii antigen potentially important for both diagnosis and immunoprophylaxis of toxoplasmosis. By using predictive algorithms, five synthetic peptides (48-67, 82-102, 213-230, 238-256 and 279-285) derived from P30, were investigated for B- and T-cell determinants in mouse and rat experimental models. Antibody recognition appeared more broadly distributed along the P30 sequence, whereas T-cell recognition was mainly targeted on the 238-256 peptide. In the absence of any carrier protein, this peptide induced a B- and T-cell immune response independent of the route of immunization (oral route or subcutaneous injection). This peptide (238-256) induced multiple antibody isotypes. In contrast with the 238-256 peptide, the 48-67 peptide, either free or in the form of a multiple antigenic peptide (MAP) construct or the 279-295 peptide, elicited antibodies associated with a TH2 response. This study reports for the first time the analysis of the antigenic and immunogenic properties of P30-derived peptides and are potentially useful for vaccinal strategies incorporating the P30 Toxoplasma gondii antigen.

Diagnostic value of a synthetic peptide derived from Echinococcus granulosus recombinant protein.

A specific monoclonal antibody (MAb; EG 02 154/12) directed against a protein epitope of Echinococcus granulosus antigen 5 was used to screen a cDNA library constructed from E. granulosus protoscoleces RNA. One clone designated Eg14 was selected and shown to code for an amino acid sequence partially homologous to that of the clone Eg6 previously identified with the same MAb. Hydrophobic cluster analysis showed that both recombinant antigens may adopt a similar alpha-helical organization and share a common conformational epitope. A synthetic peptide (89-122) mimicking the conformational site of Eg6 and Eg14 was constructed and demonstrated to be able to inhibit binding of the MAb and human hydatid sera to the Eg6 fusion protein (FP6) or to native hydatid antigens. To assess the diagnostic value of the peptide 89-122, we tested sera from patients infected with different parasites for their antibody reactivity with this peptide in ELISA. A high binding sensitivity and specificity of IgG-A-M antibodies were obtained with E. granulosus-infected patient sera. Moreover, the peptide 89-122 was found to be specifically recognized by IgE antibodies from patients with hydatid disease. These results indicate the particular interest of this synthetic peptide as a standardized antigen in diagnosis and treatment surveillance of hydatidosis.

Analysis of antigenicity and immunogenicity of five different chemically defined constructs of a peptide.

The aim of this study was to compare the antigenicity and the immunogenicity of five constructs of a peptide, including the peptide in single copy, a tandem repeat containing three copies, a copolymer with glutaraldehyde and two constructs based on the MAP (Multiple Antigenic Peptide) model, one containing two copies (MAP-2) and the other, eight copies of the peptide (MAP-8). The peptide used in this test was the 115-131 sequence derived from the rSm28-GST antigen of Schistosoma mansoni. All constructs were recognized by rSm28-GST specific antibodies in solid phase immunoassays. However, the binding was higher when the MAP-8 was used as antigen at least partly because of its better coating on the microtiter plates. In vitro lymphoproliferative assays showed that polymer was mitogenic, repeat and MAP-2 did not stimulate rSm28-GST specific T cells while MAP-8 induced a slight response. The injection of MAP-8 to rats led to important antibody and T cell responses higher than those obtained with the other constructs. The IgG2a (cytotoxic antibody in schistosomiasis)/IgG2c (blocking antibody) ratio was independent of the immunogen. Taken together these results demonstrate that both the antigenicity and the immunogenicity of a peptide containing T and B cell epitope(s) are strongly related to the molecular form whereby it is presented and that the MAP-8 construct can be useful in serodiagnosis or in vaccination trials using synthetic peptides.

Lipopeptide-based melanoma cancer vaccine induced a strong MART-27-35-cytotoxic T lymphocyte response in a preclinal study.

Identification of tumor antigens and their optimal antigenic peptides raised hopes for the development of peptide‐based immunotherapeutic vaccine strategies for human melanoma, however. Synthetic peptides alone are not immunogenic enough, and adequate formulation is critical for elaboration of peptide vaccines. To improve formulation, we evaluated 2 lipopeptide constructs, both including HLA‐A2‐restricted MART 27‐35‐CD8+ T lymphocyte (CTL) epitope covalently linked to universal tetanus toxoid (TT) 830‐843 helper T lymphocyte (HTL) epitope, in HLA‐A2 transgenic mouse models that mimic human CTL responses in vivo. These 2 constructs only differed in the formulation of their lipid tail. We showed that lipopeptide constructs were strongly recognized, in vitro, by human MART 27‐35 cytotoxic T cells derived from tumor‐infiltrating lymphocytes. The transgenic Mice immunized with these 2 MART lipopeptide formulations containing covalently linked HTL‐CTL epitopes induced strong MART 27‐35 cytotoxic T cells. This CTL induction was critically dependant on the presence of the helper T lymphocyte epitope. These results also showed that a single palmitoyl‐lysine chain is enough to assure immunogenicity of a given peptide and that the presence of a lipid tail bypass the need for adjuvant. These results support the selection of MART‐lipopeptide melanoma vaccine for evaluation in a clinical trial.

Analysis of the immune response elicited by a multiple antigen peptide (MAP) composed of two distinct protective antigens derived from the parasite Schistosoma mansoni.

In this report we analyse the immune response elicited by a Multiple Antigen Peptide (MAP), containing three peptide sequences derived from two distinct vaccine candidates against schistosomiasis; the Schistosoma mansoni 28 kDa Gluthatione‐S‐Transferase (Sm28GST) and the Schistosoma mansoni Triose‐Phosphate‐Isomerase (sTPI). We examined the immunogenicity of this construct, named MAP ‘DA’, in three distinct mouse strains. The B‐cell response, studied by measuring the production of different IgG isotypes, was mainly directed against the peptide derived from the Sm28GST, but also against the whole Sm28GST protein. In contrast, the T‐cell response, as assessed by proliferation assay and cytokine mRNA expression, was directed against the MAP construct, the peptides derived from the sTPI protein and the whole sTPI protein. Significantly, T‐cells from all MAP ‘DA’‐immunized mice, restimulated in vitro was the sTPI antigen, expressed IFN‐γ specific messengers. This cytokine has been described to play a major role in the reduction of the Schistosoma mansoni pathology. We thus demonstrate that a single MAP construct, composed of peptides from distinct antigens of Schistosoma mansoni, induced a B‐ and T‐cell response, including production of potentially protective IFN‐γ, irrespective of the MHC background.

Characterization of a monoclonal antibody directed against the NH2 terminal area of interleukin-2 (IL-2) and inhibiting specifically the binding of IL-2 to IL-2 receptor β chain (IL-2Rβ)

An anti-human IL-2 mAb (19B11β) was found to selectively block the binding of IL-2 to TS1β cells expressing the interleukin-2 receptor β (IL-2Rβ) without affecting binding to TS1α cells expressing the IL-2Rα receptor. It also specifically inhibits the IL-2 driven cell proliferation in TS1β cells. These observations have lead to the hypothesis that its epitope is related to an IL-2 area involved in binding with IL-2Rβ chain. This epitope was identified using various peptides covering the N-terminal half (including α helix A) of the 133 amino acids of IL-2. MAb 19B11gb does not recognize peptides 30–54 and 44–54 but recognizes peptides 1–22 and 1–30 with a good affinity. Furthermore, threonine in position no. 3 was found to be critical for the binding of mAb 19B11β. A relationship between the epitope of mAb 19B11β and the glycosylation of the IL-2 molecule was observed. This further demonstrates that the NH2 terminal area of IL-2 is critical for IL-2IL-2Rβ interactions. Two other mAbs were studied during the course of this work. They served as control for the study of mAb 19B11β and provide some additional insight concerning the question of IL-2IL-2R structure-function. MAb 16F11α selectively blocks the IL-2 binding to TS1α cells. The epitope of mAb 16F11 is conformational and it was not possible to study the corresponding IL-2IL-2Rα region of interaction. Epitope of mAb 3H9 is localized between residues 30 and 54 and does not affect the binding of IL-2 to IL-2Rα.

Evaluation of the effect of Sm28GST-derived peptides in murine hepatosplenic schistosomiasis: Interest of the lipopeptidic form of the C-terminal peptide

Among the synthetic peptides derived from the 28-kDa Schistosoma mansoni glutathione S-transferase (Sm28GST), immunization with the C-terminal peptide comprising amino acid residues 190-211 induced a reduction in splenomegaly, in the number of hepatic eggs and in hepatic fibrosis in mice infected by Schistosoma mansoni. The absence of antibodies specific for the Sm28GST or for the 190-211 peptide observed in our conditions of immunization with this peptide argued in favour of the involvement of cellular-dependent mechanisms in the reduction in hepatic pathology. This was confirmed by the passive transfer of 190-211 peptide-specific T-cell enriched spleen cells which reproduced the protective effect conferred by immunization with the 190-211 peptide. These 190-211 peptide-specific cells produced little IL4 and high levels of IFN-gamma, a potent inhibitor of collagen synthesis. Furthermore, the use of a lipopeptidic form of the 190-211 peptide significantly improved the reduction in hepatic pathology obtained with the uncoupled peptide and induced a durable protective response. These results provide encouraging information for the possible use of synthetic peptides in the immunoprophylaxis of Schistosomiasis.

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen

The P126 protein, a parasitophorus vacuole antigen of Plasmodium falciparum has been shown to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and the antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in terms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced antibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

Improved detection of human antibodies to a Plasmodium antigen using a peptide modified with Aib residues.

A 17‐mer sequence was selected as a model to study the influence of modifications of terminal ends both on the conformation of a peptide and on its antigenicity towards naturally developing antibodies. This sequence corresponded to a tandemly repeated motif, found in a long repetitive region, with high helical propensity, of a Plasmodium falciparum liver‐stage antigen (LSA‐1), immunogenic in man. Our model peptide was synthesized with ionizable or non‐ionizable ends, or modified in both extremities by introduction of the helix‐promoting residue α‐aminoisobutyric acid (Aib). Helical contribution, absent in the 17 amino‐acid sequence possessing ionizable ends, was detectable when non‐ionizable ends were introduced, and dramatically increased in the Aib‐modified analogue. The presence of ionizable ends totally abolished reactivity towards human sera, otherwise detectable with the peptide possessing non‐ionizable ends. While modification by Aib residues was neither detrimental nor beneficial to antigenicity in solution, it clearly resulted in an improved sensitivity of the specific antibody detection when used as solid‐phase antigen in ELISA.