Biomedical subjects

G Corradin

Publications and source records attributed to G Corradin.

At least 91 records · Page 5Linked to original sources

PubMed1994

Induction of a cytotoxic T cell response by co-injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund's adjuvant (IFA): further enhancement by pre-injection of IFA alone.

We have previously demonstrated that it is possible to induce a consistent and strong cytolytic T lymphocyte (CTL) response to synthetic peptides, corresponding to poorly immunogenic malaria CTL epitopes, by co-injecting them with peptides representing defined T helper (Th) epitopes in incomplete Freund's adjuvant (IFA). In this study we have tested different immunization protocols to improve further the elicitation of the CTL response. We show that the CTL response to a mixture of Th + CTL peptides administered in IFA was further enhanced by a previous injection of the Th epitope peptide in IFA. Moreover, we found that the response could be significantly augmented by a pre-injection of IFA alone. This enhancement was observed only if the Th epitope was also present in the second injection. The number of lymph node cells recovered was 2-3-fold higher in mice pre-injected with IFA, but the increase in specific CTL activity, expressed as lytic units per animal, by pre-injection of IFA was at least 10-20-fold. Thus, pre-injection of IFA clearly increases the magnitude of a subsequent CTL response.

Amino Acid Sequence↗

PubMed1994

Biologically addressable monolayer structures formed by templates of sulfur-bearing molecules.

We demonstrate that the combined application of Langmuir-Blodgett and self-assembly techniques allows the fabrication of patterns with contrasting surface properties on gold substrates. The process is monitored using fluorescence microscopy and surface plasmon spectroscopy and microscopy. These structures are suitable for the investigation of biochemical processes at surfaces and in ultrathin films. Two examples of such processes are shown. In the first example, the structures are addressed through the binding of a monoclonal antibody to a peptide. This demonstrates the formation of self-assembled monolayers by cysteine-bearing peptides on gold, and the directed binding of proteins to the structured layers. A high contrast between specific and unspecific binding of proteins is observed by the patterned presentation of antigens. Such films possess considerable potential for the design of multichannel sensor devices. In the second example, a structured phospholipid layer is produced by controlled self-assembly from vesicle solution. The structures created--areas of phospholipid bilayer, surrounded by a matrix of phospholipid monolayer--allow formation of a supported bilayer which is robust and strongly bound to the gold support, with small areas of free-standing bilayer which very closely resemble a phospholipid cell membrane.

Amino Acid Sequence↗

PubMed1994

Processing and presentation of tetanus toxin by antigen-presenting cells from patients with chronic granulomatous disease (CGD) to human specific T cell clones are not impaired.

The capacity of peripheral blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed B cell lines from CGD patients to process and present tetanus toxin (tt)-specific epitopes was assessed using various tt preparations and human tt-specific T cell clones. PBL from all of the donors were able to process and present either native tt and/or denatured tt to human T cell clones specific for various tt epitopes. Furthermore, no difference was found in the antigen requirement when normal or CGD EBV-B cell lines were used as antigen-presenting cells (APC). These results suggest that the deficiency in oxygen metabolism in CGD cells does not affect tt processing and presentation.

Amino Acid Sequence↗

PubMed1994

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice.

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

Animals↗

PubMed1993

Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages.

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.

Animals↗

PubMed1993

The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86.

The response to tetanus toxoid (TT) was studied in immune donors that carry two alleles of DR5 that differ only at DR beta residue 86: DRB1*1101 (G86, abbreviated 1101) and DRB1*1104 (V86, abbreviated 1104). A large number of TT-specific T cell clones was isolated and the epitopes recognized in association with 1101 and 1104 were mapped. We found that two epitopes (p2 and p32) can be recognized in association with both 1101 and 1104 while three epitopes (p23, p27 and p30) are recognized in association with 1101, but never in association with 1104. The sets of naturally processed self peptides displayed by 1101 and 1104 were characterized using alloreactive T cell clones. We found that all 1104 alloreactive clones recognize both 1104 and 1101, while approximately 30% of the alloreactive 1101 clones fail to recognize 1104. Thus it is apparent that both naturally processed TT and self peptides displayed on 1104 molecules represent a fraction of those displayed on 1101 molecules. The mechanism responsible for this differential presentation was investigated by comparing the capacity of 1101 and 1104 antigen-presenting cells to present TT or synthetic peptides to specific T cells and by measuring the binding of these peptides to DR molecules. Three sets of results suggest that V86 acts as a constraint to the binding of naturally processed peptides: (i) all 1104-restricted or alloreactive T cell clones recognize TT- or allo-epitopes presented by 1101 as well, thus ruling out a major effect of V86 as a residue determining T cell restriction specificity; (ii) presentation of naturally processed peptides correlates in general with the capacity of long synthetic peptides to bind to 1101 or 1104 and (iii) while the naturally processed p30 epitope discriminates between 1101 and 1104, a short synthetic peptide binds equally well to and is comparably recognized in association with both 1101 and 1104. Taken together these results suggest that the natural polymorphism at residue 86 might be a molecular switch that finely tunes the complexity of the peptide collection presented on DR molecules.

Alleles↗

PubMed1993

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

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

Amino Acid Sequence↗

PubMed1993

The interaction of antigenic peptides with the H-2Kd MHC class I molecule.

An important event in the recognition of antigen by T cells is the selective interaction of peptides with major histocompatibility complex (MHC) molecules. We have defined several critical structural features that promote the efficient interaction of antigenic peptides with the MHC class I molecule, H-2Kd. For four unrelated antigens, we found that optimal synthetic peptides were short, only 9 or 10 amino acids long. These and other H-2Kd-restricted peptides were found to share a distinct 2-residue binding motif. Two regions in the H-2Kd antigen binding site that might accommodate the motif residues were identified by analysis of Ala-substituted H-2Kd molecules. A molecular model showing the possible interaction of one antigenic peptide with the H-2Kd molecule is presented.

Amino Acid Sequence↗

PubMed1993

MHC class I H-2Kd-restricted antigenic peptides: additional constraints for the binding motif.

The previously defined binding motif of MHC class I H-2Kd-restricted antigenic peptides consists of a Y residue in position P2 and a hydrophobic residue with a large aliphatic side chain (L, I, or V) in position P9/P10 of optimal 9- or 10-mer peptides. We show now that the presence of a charged or a F residue in position P5 reduces the Kd-restricted competitor activity of several cytotoxic T lymphocyte (CTL) epitopes and model peptides, at a degree comparable to A substitutions for the P2 or the P9/P10 anchor residues. Various charged, polar, aromatic, and aliphatic amino acids were substituted for S256 in the CTL epitope Plasmodium berghei circumsporozoite (CS) 253-260 8-mer and in its CS 252-260 9-mer form, whereas a more restricted panel of substitutions was tested in the CTL epitopes influenza nucleoprotein 147-155 9-mer and HLA-CW3 170-179 10-mer. Analysis of all the Kd-restricted epitopes so far defined also revealed an uncharged residue at this position. These additional structural constraints present in the Kd binding motif may thus improve the prediction of new epitopes recognized by T cells in the context of this MHC molecule.

Amino Acid Sequence↗

PubMed1993

Presentation of T-cell epitopes assembled as multiple-antigen peptides to murine and human T lymphocytes.

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.

Amino Acid Sequence↗

PubMed1992

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides.

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freund's adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.

Amino Acid Sequence↗

PubMed1992

Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination.

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.

Amino Acid Sequence↗

PubMed1992

Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein.

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.

Amino Acid Sequence↗

PubMed1992

Immunization with synthetic peptides containing a defined malaria epitope induces a highly diverse cytotoxic T lymphocyte response. Evidence that two peptide residues are buried in the MHC molecule.

In the present study, we have explored ways of inducing a CTL response to a previously defined H-2Kd MHC class I restricted epitope in the circumsporozoite (CS) protein of Plasmodium berghei, and studied in detail the fine specificity of the response. We found that the s.c. injection of a variety of synthetic peptides emulsified in Freund's adjuvant efficiently induced a specific CTL response in (BALB/c x C57BL/6)F1 (H-2d x H-2b) mice. In contrast, BALB/c mice responded only marginally, consistent with the possible requirement for a concomitant Th response that would be provided by the C57BL/6 strain. Similar to our previous observations in analyzing CTL clones from sporozoite-immunized mice, the CTL response induced by peptide immunization was in part cross-reactive with an epitope from the Plasmodium yoelii species. The minimal P. berghei CS epitope, the octapeptide PbCS 253-260, was studied in detail by the analysis of a series of variant CS peptides containing single Ala substitutions. The relative antigenic activity for each variant peptide was calculated for 28 different CTL clones. Overall, the response to this P. berghei CTL epitope appeared to be extremely diverse in terms of fine specificity. This was evident among the CTL derived from sporozoite-immunized mice, as well as among those from peptide-immunized animals. The heterogeneity found at the functional level correlates with the highly diverse TCR repertoire that we have found for the same series of CTL clones in a study that is reported separately. The relative competitor activity for each Ala-substituted peptide was also determined in a quantitative functional competition assay. For the residues (Tyr253 and Ile260) within the 8-mer CS peptide, substitution with Ala reduced competitor activity by at least 40-fold, and for two others the reduction was 5- to 10-fold. When the relative antigenic activity for each CTL/peptide combination was normalized to the relative competitor activity of the peptide, a striking pattern emerged. The two residues that most affected competitor activity showed no additional effect on recognition beyond that observed for competition. In marked contrast, Ala substitutions at the other five positions tested varied widely, depending on the CTL/peptide combination. This pattern not only supports a model whereby the Tyr253 and Ile260 residues anchor the peptide to the Kd molecule, but also implies that they are virtually inaccessible to the TCR.

Amino Acid Sequence↗

PubMed1992

Dictyostelium discoideum as an expression host for the circumsporozoite protein of Plasmodium falciparum.

We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.

Amino Acid Sequence↗

PubMed1992

Mouse tumor rejection antigens P815A and P815B: two epitopes carried by a single peptide.

Mouse mastocytoma P815 expresses several distinct tumor rejection antigens recognized by syngeneic cytolytic T lymphocytes (CTL). Two of these tumor rejection antigens, P815A and P815B, are encoded by gene P1A, the sequence of which was reported previously. Tumor cell variants having lost one or both of these antigens were isolated by in vitro selection with CTL and also by collecting tumor cells that progressed in vivo after escaping a nearly complete immune rejection process. The structure of gene P1A in these antigen-loss variants was examined. Several A-B- variants presented a complete or partial deletion of the gene. One variant that had lost only antigen A displayed a point mutation in the first exon. Peptides were synthesized that corresponded to the normal sequence located in the region of this point mutation. They sensitized target cells to both anti-A and anti-B CTL. The homologous peptide encoded by the mutated gene of the P815 A-B+ variant sensitized cells only to anti-B CTL. We conclude that anti-A and anti-B CTL recognize on the same peptide two distinct epitopes that are affected differently by the mutation.

Amino Acid Sequence↗

PubMed1992

Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design.

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.

Amino Acid Sequence↗

PubMed1992

In vitro immune recognition of synthetic peptides from the Plasmodium falciparum CS protein by individuals naturally exposed to different sporozoite challenge.

The impact of duration and intensity of sporozoite challenge on the in vitro cell immune response to synthetic peptides of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated in residents of a malaria endemic area in Burkina Faso (West Africa). Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used to assess immune recognition of synthetic peptides corresponding to the polymorphic Th2R and Th3R regions, to the conserved CS.T3 sequence and to NANP and degenerate NVDP repeats. Immune responses were measured in adults and children from a village where they received more than 100 sporozoite inoculations per year and in adults living in a town, exposed to a 10-100 times lower challenge. A lifetime intense exposure apparently increased the ability to proliferate in response to most peptides in the rural adults, who all produced antibodies to NANP repeats. Surprisingly, cell cultures from these subjects seldom contained appreciable levels of IFN-gamma. In the urban adults, possibly due to the moderate challenge they are exposed to, significant differences in the proliferative potentials of the peptides could be detected. The highest stimulation indices were obtained with the genetically unrestricted CS.T3 peptide. Remarkably, proliferative responses to Th2R and Th3R appeared to be correlated with the humoral response to the CS protein, indicating a T helper significance of the epitopes. The differing proliferative potential of the polymorphic epitopes in the urban adults suggests that polymorphism might delay the development of immune responsiveness under conditions of sporadic transmission. The children from the highly malarious village displayed the lowest proliferative scores, accompanied by a high prevalence of antibodies to NANP repeats. On the basis of these findings, the hypothesis is proposed that a pure B cell reactivity to NANP repeats could ontogenetically precede the mounting of a conventional T-B cooperative immune response.

Adult↗