Biomedical subjects

G Corradin

Publications and source records attributed to G Corradin.

At least 19 recordsLinked to original sources

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↗

PubMed1992

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

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

Animals↗

PubMed1991

Differential stability of antigenic MHC class I-restricted synthetic peptides.

Various synthetic peptides recognized as Ag by CTL in the context of MHC class I molecules were tested for stability in vitro and in vivo. Peptide inactivation in vitro was quantitated by titrating the amount of peptide required to sensitize target cells for lysis by specific CTL clones. The degree of inactivation after overnight incubation at 37 degrees C varied widely among a series of antigenic peptides. Some were nearly unaffected, whereas others lost activity by more than 100-fold or even 10,000-fold. However, no correlation was found between susceptibility to serum inactivation and antigenic potency as measured in short term cytolytic assays. No inactivation occurred at 4 degrees C, or at 37 degrees C in the absence of serum, under the conditions used. Serum inactivation most likely involved proteolysis because it could be inhibited by protease inhibitors. Moreover, presumed cleavage products of a radiolabeled susceptible peptide could be visualized by TLC. In vivo, the persistence of the antigenic activity of the injected peptides, either in extracellular fluids or on tumor target cells growing in an ascites form, correlated with the degree of stability found for the peptides in vitro. The differential stability of synthetic peptides may have important consequences for attempts to manipulate the development of an immune response in vivo.

Amino Acid Sequence↗

PubMed1991

NMR studies of an oligoproline-containing peptide analogue that binds specifically to the H-2Kd histocompatibility molecule.

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%.

Amino Acid Sequence↗

PubMed1991

In vitro activity of CD4+ and CD8+ T lymphocytes from mice immunized with a synthetic malaria peptide.

In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoelii. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Py1) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region, (Gln-Gly-Pro-Gly-Ala-Pro)n, of the P. yoelii circumsporozoite protein when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Experiments were then designed to study the in vitro antiparasite efficacy of T cells elicited in vivo by peptide immunization. T-cell activity was evaluated on cultured hepatic stages of P. yoelii. Peptide immunizations led to the preferential activation of CD8+ T cells in BALB/c mice and of both CD4+ and CD8+ T cells in C57BL/6 mice. Parasite elimination was mediated directly by these cells and did not seem to be dependent on lymphokine secretion. These data suggest that peptide-primed CD4+ T cells as well as CD8+ T cells could be cytolytic for the hepatic phase of malaria parasites. The fact that the same peptide could activate different lymphocyte populations, depending on the strain of mouse, highlights the importance of a better understanding of the fine mechanisms behind the immune responses to synthetic peptides being tested for malaria vaccine development.

Animals↗

PubMed1991

H-2Kd-restricted antigenic peptides share a simple binding motif.

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.

Amino Acid Sequence↗

PubMed1991

Recognition by class II alloreactive T cells of processed determinants from human serum proteins.

Alloreactive T cells recognize a complex composed of an allogeneic major histocompatibility complex (MHC) molecule and a peptide derived from the processing of nonpolymorphic proteins. A sizable fraction of MHC class II alloreactive T cells is shown to recognize peptides derived from constitutive processing of human serum proteins. One such epitope is a fragment of human serum albumin. This epitope bound selectively to the human class II molecule DRw11 and was constitutively present on antigen-presenting cells in vivo. These data indicate that, in the case of MHC class II, peptides involved in allorecognition may originate from exogenous proteins.

Antigen-Presenting Cells↗

PubMed1991

Macrophage NO2- production as a sensitive and rapid assay for the quantitation of murine IFN-gamma.

Macrophage production of arginine-derived NO2- provides a simple method for detection and quantitation of interferon-gamma (IFN-gamma) in murine cell culture fluids. When the macrophage cell line RAW 264 is cultured overnight with IFN-gamma in the presence of 10 ng/ml LPS, NO2- release, as determined by a simple colorimetric assay, is proportional to the concentration of IFN-gamma and is inhibited by monoclonal antibody to IFN-gamma. A high degree of correlation was obtained when antigen stimulated T lymphocyte supernatants were tested for IFN-gamma by ELISA and the NO2- production assay.

Animals↗

PubMed1991

Lack of H-2 restriction of the Plasmodium falciparum (NANP) sequence as multiple antigen peptide.

The major surface antigen of malaria sporozoites, the circumsporozoite protein, contains a region of tandem amino acid repeats, which in the case of the human malaria parasite Plasmodium falciparum, consist of four amino acids Asn-Ala-Asn-Pro (NANP) repeated up to about 40 times. This repetitive sequence has been considered as the basis for the development of subunit vaccines against P. falciparum malaria. We and others had previously shown that synthetic and recombinant NANP peptides were immunogenic only in H-2b mice. In the present report we show that, when mice with different H-2 haplotypes are immunized with the repetitive NANP sequence incorporated in a synthetic branching multiple antigen peptide (MAP), all except one of the mouse strains tested mounted an anti-peptide antibody response. Such a response does not appear to be due to the peculiar assembly of the NANP sequence. In fact, MAP containing repetitive sequences from circumsporozoite proteins of other malaria parasites did not overcome the genetic restriction of the immune response to the linear peptides. These data show that in the case of the P. falciparum NANP repeats, their immunogenicity can be dramatically changed and increased when these peptides are assembled as MAP. This unexpected finding may be of interest in the design of synthetic candidate malaria vaccines.

Amino Acid Sequence↗

PubMed1991

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum.

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.

Amino Acid Sequence↗

PubMed1991

The identification of tyrosine as a common key residue in unrelated H-2Kd restricted antigenic peptides.

We have compared the activity of several Kd- or Ld-restricted antigenic peptides as competitors in a functional competition assay using cytolytic T lymphocyte (CTL) clones. All of four unrelated Kd-restricted peptides tested could compete with each other but not with the Ld-restricted peptide P91A-. 12-24 (P91A). Moreover, the P91A peptide failed to compete with the four Kd-restricted peptides. In contrast, another Ld-restricted peptide [mouse cytomegalovirus (MCMV) pp89 167-176] could clearly compete with both Kd- and Ld-restricted peptides. The comparison of a series of modified MCMV pp89 peptides suggested that distinct structural features allow the interaction of the peptide with the two different MHC class I molecules. We showed previously that the competitor activity of two different Kd-restricted antigenic peptides was reduced substantially upon Ala substitution of the single Tyr residues present in these peptides. We now show a similar effect for two additional Kd-restricted peptides. Our results thus suggest that Tyr may function as an 'anchor' residue for many antigenic peptides that bind to the Kd molecule. Molecular modeling of the presumed antigen-binding site of the Kd molecule revealed the presence of two deep cavities that may be involved in binding peptide amino acid side chains. A model illustrating one possible interaction of a Tyr-containing peptide with the Kd molecule is presented.

Alanine↗