Induction of antibodies against the Plasmodium falciparum p126 antigen in non-responder H-2b and partial-responder H-2d mice using synthetic peptides.

The p126 Plasmodium falciparum antigen is processed into two fragments, p50 and p73, the latter one containing the subfragments p47 and p18 when the schizonts rupture. An absence of antibody response against the p126 antigen has been reported recently in H-2b mice and limited to the p73 processed fragment in H-2d mice. Synthetic peptides corresponding to various domains of the molecule have been used to immunize mice in order to overcome the absence of an immune response. Synthetic peptides corresponding to the N-terminus of p50 or p18 as well as to the C-terminus of p47 were unable to induce anti-peptide antibodies when injected carrier-free or coupled to ovalbumin. Synthetic peptides corresponding to the C-terminus of p18 or composed of 6 or 9 serines were able to induce anti-peptide antibodies when injected coupled to a carrier protein. However, none of these antibodies was able to recognize the native p126 molecule. Various synthetic peptides corresponding to the 6-octapeptide [Nt47 (6 x 8)] or the 4-octapeptide [Nt47(4 x 8)] repeat sequence localized at the N-terminus of the p47 have also been used to immunize mice. No antibodies were generated using a carrier-free [Nt47(6 x 8)-Cys]2 or [Nt47 (4 x 8)-Cys]2 peptide, an octameric multiple antigen peptide construct [Nt47(6 x 8)]-MAP or the [Nt47(6 x 8)] coupled to one or two palmitic acids. In contrast, [Nt47(6 x 8)]-Cys coupled to either tetanus toxoid (TT) or ovalbumin (OVA) and [Nt47(4 x 8)]-Cys coupled to OVA induced antibodies against the synthetic peptide and the native p126 molecule in both H-2d and H-2b mice. A multiple antigen peptide construct [Nt47(4 x 8)-MSP-3b]-MAP containing 4 [Nt47(4 x 8)] and 4 [MSP-3b] also induced antibodies against the synthetic peptide [Nt47(4 x 8)-Cys]2 and the native p126 molecule in both H-2d and H-2b mice.

Improvement of the T-cell response to a non immunogenic peptide by its tandem association with a highly efficient T-helper peptide.

The 45-69 peptide, an helper T-cell epitope derived from the HIV nef protein is strongly immunogenic. A T-cell proliferative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein of the parasite Schistosoma mansoni, requires the presence of a carrier of the use of peptidic constructs. We demonstrate here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic constructs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent association of both peptides is necessary, since the coinjection of 45-69 and 115-131 peptides is not sufficient to induce a detectable anti-115-131 T-cell response. The mutual orientation between the respective tandem peptides (45-115 and 115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and immunogenicity but both allowed to reveal the existence of a specific T-cell response normally undetectable using 115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction of vaccines.

Effect of a lipopeptidic formulation on macrophage activation and peptide presentation to T cells.

We studied a 45-69 lipopeptide obtained by N-terminal modification with a N epsilon-palmitoyl lysine residue of the 45-69 peptide derived from the nef protein of HIV. T cells from animals immunized intraperitoneally with 45-69 lipopeptide proliferated in vitro in the presence of 45-69 peptide while no response was obtained after intraperitoneal immunization with 45-69 peptide. The efficiency of the 45-69 lipopeptide is supported by the covalent association to the N epsilon-palmitoyl lysine moiety. The immunogenicity of the 45-69 lipopeptide or of the unmodified peptide is dependent on the route of immunization but is not related to a mitogenic effect on cells or to an increase of the peptide antigenicity. Moreover, only 45-69 lipopeptide induces the secretion of cytokines such as IL-1, IL-6 and TNF-alpha by peritoneal macrophages. Finally, the use of 45-69 lipopeptide permits the activation of highly purified T cells without the addition of antigen-presenting cells. These results have implications for the formulation of synthetic vaccines.

Improvement of the T-cell response to a non-immunogenic peptide by its tandem association with a highly efficient T-helper peptide.

The 45-69 peptide, an helper T-cell epitope derived from HIV nef protein, is strongly immunogenic. A T-cell proliferative response was observed following immunization of Lou/M rats with 45-69 peptide administered in low dose and without any adjuvant. It is already known that the T-cell response to the 115-131 peptide of Sm28GST antigen, a protein of the parasite Schistosoma mansoni, requires the presence of a carrier or the use of peptidic constructs. We demonstrate here that a T-cell response against the 115-131 peptide can be obtained in the absence of adjuvant using peptidic constructs (115-45 and 45-115 peptides) resulting from tandem synthesis of 115-131 and 45-69 peptides. A covalent association of both peptides is necessary, since the co-injection of 45-69 and 115-131 peptides is not sufficient to induce a detectable anti-115-131 T-cell response. The mutual orientation between the respective tandem peptides (45-115 and 115-45) is critical for the T-cell response. These peptidic constructs possess distinct properties of antigenicity and immunogenicity but both allowed to reveal the existence of a 115-131 specific T-cell response normally undetectable using 115-131 peptide alone. This immunopharmacological approach should be useful in the rational design and construction of vaccines.

H-2b restriction of the immune response to the p126 Plasmodium falciparum antigen.

Inbred BALB/c (H-2d), CBA (H-2k) and C57B1/6 (H-2b) mice immunized with Plasmodium falciparum schizonts or culture supernates develop antibodies of different antigenic specificities. It has been observed that C57B1/6 mice were unable to produce detectable antibodies against the p126 antigen (native molecule and p73 or p50 processed fragments) compared with other inbred mice. Similar results were obtained using BALB congenic mice with a lack of p126 antibody response in H-2b mice, while H-2d and H-2k mice produced antibodies against the p126. Lymphocyte proliferation assays performed by incubation of spleen cells with immunopurified p126 were positive for immunized BALB/c (H-2d) and congenic H-2d or H-2k mice. On the other hand, no lymphocyte stimulation was observed with either C57B1/6 (H-2b) or congenic H-2b mice. These results suggest an MHC restriction of the immune response against the entire p126 (found in schizonts) and its p73 and p50 naturally processed fragments (found in culture supernates).

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.

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.

Functional analysis of a T cell line specific for antiidiotypic antibodies to a Schistosoma mansoni protective epitope. I. Role in the anti-S. mansoni antibody response.

Previous data have shown that from an antiparasitic IgE mAb (mAb1), antianti-Id IgG and IgE antibodies (Ab3) could be prepared. These Ab3 demonstrated the same functional properties as the Ab1, such as in vitro cytotoxic activity toward schistosomula and in vivo a protective effect against Schistosoma mansoni infection. To study the possible interactions between the idiotypic network and the regulation of isotypic expression, we focused on Id-specific T cells obtained by immunization with Ab2. Both Ab2 idiotopes and native schistosomula Ag were able to stimulate the proliferation of anti-Ab2 T cells in vitro. The activation of anti-Ab2 T cells by Ab2 shared the classic characteristics of Th cells, namely, it was MHC-restricted and required APC. A T cell line could be maintained in long term culture by stimulation with schistosomula Ag. The adoptive transfer of cells from this line to 26-kDa Ag-immunized or S. mansoni-infected rats led to a dramatic increase in the specific humoral response. This effect was restricted to antibodies specific for 26- and 56-kDa Ag (the targets of the mAb1) and was observed for the two isotypes tested, i.e., IgG and IgE. Finally, the helper effect on the antibody response could be further amplified by cooperation of anti-Ab2 T cells with Id-specific cells of the first generation (anti-Ab1 cells). Together with Ag-specific Th cells, the Id-specific T cells may, due to their specificity and their functional properties, play a major role in the induction and more importantly, in the maintenance of the immune response.

Obtention of a human primary humoral response against schistosome protective antigens in severe combined immunodeficiency mice after the transfer of human peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMC) from healthy donors were injected into C.B.-17 severe combined immunodeficiency (scid) mice which were subsequently immunized with crude Schistosoma mansoni adult worm antigenic preparation (SWAP) or with recombinant S. mansoni 28-kDa glutathione transferase (r-Sm-28-GST) antigen. PBMC from a S. mansoni-infected patient were also transferred. The specific human anti-SWAP and anti-Sm-28-GST antibody responses were monitored. The presence in both cases of human specific antibodies in scid mouse sera was determined by enzyme-linked immunosorbent assay and Western blotting techniques using anti-human immunoglobulin reagents. No antibodies were detected in these sera using anti-mouse immunoglobulin antisera. These antibodies were functional since a cytotoxic activity against schistosomula was observed when monocytes were incubated with scid mouse sera positive for anti-Sm-28-GST antibodies.

Antigenicity and immunogenicity of a multiple peptidic construction of the Schistosoma mansoni Sm-28 GST antigen in rat, mouse, and monkey. 1. Partial protection of Fischer rat after active immunization.

Among the schistosome proteins characterized as vaccine candidates, an Ag of 28 kDa (Sm-28-GST) has received considerable attention. It was shown to be antigenic in humans and protective in mice, rats, hamsters, and baboons. Synthetic peptides derived from its sequence have been used to characterize the immune response to the molecule and one of these, comprising aminoacids 115-131 has been shown to incorporate both T and B cell recognition sites in a variety of experimental models. An octameric ("octopus") construction of the 115-131 peptide has been synthesized and its antigenicity and immunogenicity have been examined. The octopus construct is immunogenic in rats, mice and baboons in the presence of CFA (for rodents) and Bacille-Calmette-Guérin vaccine (for primates) as adjuvants. This clearly indicates that the construction allowed the conservation of the immune sites of the cognate protein. Moreover, anti-octopus sera from immunized Fischer rats were able to mediate platelet-, macrophage-, and eosinophil-dependent cytotoxicity toward schistosomula. Rats immunized with the 115-131 octopus were partially protected against a challenge infection with Schistosoma mansoni cercariae and this was paralleled by an increased level of IgG and more importantly, of IgE Sm-28-GST-specific antibodies.

T-cell responsiveness towards various synthetic peptides of the P28 antigen in rat and mouse models during Schistosoma mansoni infection.

It has recently been demonstrated that the Schistosoma mansoni P28 antigen can induce a strong protective immunity after direct immunization in various experimental models. T lymphocytes from Fischer rats immunized with the recombinant P28 antigen were cultured in vitro in the presence of seven synthetic peptides derived from the amino acid sequence of the P28. The most significant and reproducible proliferation was obtained with the 24-43 and 115-131 synthetic peptides. In order to analyze whether these located determinants were also exposed to the host's immune system during the natural S. mansoni infection or after immunization with crude antigenic extracts of various development stages of the parasite, the T-cell responsiveness of infected or immunized Fischer rats and BALB/c mice was tested towards these synthetic peptides. The results showed that, in both permissive (mouse) and non-permissive (rat) hosts, 24-43 and 115-131 synthetic peptides are recognized during the course of infection and that there is a dynamic variation of this recognition. These peptides are also recognized by T cells educated against crude antigenic extracts of different developmental stages of the parasite which contained the native form of the P28 molecule. Taken together, the results indicated that these synthetic peptides derived from the recombinant P28 antigen can activate T lymphocytes educated against the native P28 molecule during the development and maturation of the parasite in their hosts. Therefore, they might be useful for the construction of synthetic vaccines against schistosomiasis.

In vitro and in vivo effects of interleukin 2 on the protozoan parasite leishmania.

T cells can have either resistance-promoting or disease-promoting effects in murine cutaneous leishmaniasis. It is known that the adoptive transfer of parasite-specific helper T cells led to an exacerbation of Leishmania-induced lesions. This work presents evidence that lymphokines produced by activated T cells could be involved in this exacerbating process by directly stimulating the parasite growth. In the presence of activated T cell supernatants, the in vitro growth of Leishmania mexicana amazonensis promastigotes was greatly enhanced. This effect was reproduced by addition of recombinant interleukin 2 (IL2). An anti-IL2 antibody partially reversed the stimulatory effect of IL. An in vivo in situ treatment of infected mice with IL 2 led to an exacerbation of the lesions. The increase in footpad swelling after IL2 treatment was correlated with a higher number of parasites per lesion. The protective effect of cyclosporin A against the development of Leishmania infection was abolished by IL2 treatment. As we observed that IL2 has a stimulatory effect on the in vitro Leishmania growth, we speculate that exacerbation of the lesions observed in vivo after IL2 treatment could be partially related with a direct effect of IL2 on the parasite growth.

Induction of platelet cytotoxic functions by lymphokines: role of interferon-gamma.

Antigen- or mitogen-stimulated CD4+/CD- lymphocytes produced factors able to induce normal human platelets into cytotoxic effectors toward the young larvae of Schistosoma mansoni. The neutralization by monoclonal anti-IFN-gamma antibody of the induction of the platelet killer effect, the presence of IFN-gamma in the CD4+/CD8- lymphocyte supernatant, and, finally, the direct inducer effect of recombinant IFN-gamma clearly demonstrated that IFN-gamma was one of the factors responsible for the induction of platelet cytotoxic functions.

Regulation of IgE synthesis by macrophages expressing FcE-receptors: role of interleukin 1.

Triggering rat macrophages with IgE complexes induced the production of interleukin 1-like activity (IL-1). The signal is delivered through the macrophage FcE receptor since stimulating macrophages with IgE bound to spleen cells (to avoid endocytosis) or with an anti-FcE receptor antibody linked to nonphagocytizable cells also led to IL-1 production. The molecular weight of IL-1 produced after IgE triggering is in the same range (30 kD) as previously described for rat IL-1. A positive feed-back effect of IL-1 on IgE response was suggested, as purified IL-1 was able to enhance IgE synthesis in vitro by lymphocytes from immunized animals.

Effect of schistosome-derived inhibitory factor on the cell cycle of T lymphocytes.

A schistosome-derived inhibitory factor (SDIF) with immunosuppressive properties has been investigated for its effect on human T cell proliferation. We show here that SDIF has no effect on the process of lymphocyte activation because peripheral blood leukocytes (PBL) stimulated with lectin in the presence of SDIF increased normally their RNA content and showed normal acquisition of interleukin 2 (IL-2) and transferrin receptors. IL-2 production was not altered by SDIF but utilization of IL-2 was decreased, suggesting that SDIF blocked cells before or in the early s phase. Jurkat T cell line cells physically enriched for G1 cells were also more susceptible to SDIF inhibition. On the contrary, normal PBL or Jurkat cells which were already in the s phase were no more inhibited by SDIF. While SDIF has no effect on T lymphocyte activation and on production of regulatory lymphokines it selectively blocks T cell proliferation at G1 transition of the cell cycle.

Schistosome-derived inhibitory factor: an immunosuppressive agent preferentially active on T lymphocytes.

We have previously shown that schistosome-derived inhibitory factors (SDIF) inhibited lymphocyte proliferation and induced immunosuppression. Crude SDIF was purified by successive gel filtration and reverse-phase high-performance liquid chromatography. Purified SDIF preparations strongly inhibited the proliferation of different T cell line cells, while other cell lines (B cells, macrophages and fibroblasts) were almost not affected by SDIF. The inhibition of T cell proliferation by SDIF was not mediated through an Interleukin-2-dependent mechanism since both Interleukin-2-dependent and -independent T cells were inhibited. SDIF-activity was absorbed by cells in a time- and cell-number-dependent fashion at 4 degrees C, suggesting the existence of a possible receptor for SDIF. However, the difference in sensitivity to SDIF proliferation inhibition could not be attributed to the presence or absence of this receptor since cells from SDIF-sensitive and SDIF-resistant cell lines absorbed SDIF activity in the same way.

Non-specific potentiation of T- and B-lymphocyte proliferation at the early stage of infection by Schistosoma mansoni: role of factors secreted by the larvae.

The response of rat lymphocytes to schistosomula released products (SRP) was examined. SRP non-specifically activated lymphocytes by potentiating their proliferative response to PHA, Con A or LPS. The parasite factor involved was dialysable and heat stable. The addition of SRP to cultures containing nylon-wool non-adherent lymph node cells resulted in a significant enhancement of cell proliferation. The effect of SRP on athymic nude (Nu/Nu) and litter mate (Nu/+) control rat cells indicated an effect on the proliferation of both B and T lymphocytes. SRP acted in a dose-dependent manner and its action was observed as early as the beginning of cell division. This corresponds to the in vivo situation, since at the early stage of infection increased proliferative responses of the lymph node cells to mitogens were observed. The adjuvant effect of SRP could partly explain the regulation of the cellular immune response observed during S. mansoni infection by the parasite itself and could represent one of the mechanisms involved in immunity to reinfection that is under the control of the parasite.

Inhibition of primary and secondary IgE-response by a schistosome-derived inhibitory factor.

Schistosome-derived inhibitory factor (SDIF) previously shown to inhibit lymphocyte proliferation, markedly decreased the primary IgE response of rats immunized with dinitrophenylated ovalbumin (DNP-OVA) when injected either simultaneously or shortly after antigen administration. No effect however was observed when SDIF was injected before the immunization. An inhibition of non-IgE anti-DNP antibodies was also found in SDIF-treated rats although the decrease was lower than with IgE antibody. IgE responses of both low and high IgE responder rats were reduced but a lower dose of SDIF was required in the case of high IgE responder Brown Norway rats. When SDIF was only given at the time of priming, the secondary IgE response was no longer modified. However, the administration of SDIF together with the second injection of the antigen induced marked decrease in the secondary IgE response. The effects of SDIF on primary and secondary IgE responses could be attributed to the inhibitory activity of the parasite-derived factor on lymphocyte proliferation. The observed inhibition of secondary IgE antibody responses confers to SDIF a pharmacological interest in allergic diseases.

Inhibition of cytotoxic T lymphocytes by a schistosome-derived inhibitory factor is independent of an inhibition of the production of interleukin 2.

Schistosome-derived inhibitory factor (SDIF) was shown to inhibit lymphocyte proliferation. While SDIF is not toxic to lymphoid cells, the generation of cytotoxic effector cells was inhibited by SDIF in a mixed lymphocyte culture. This inhibitory effect was not attributable to the induction of suppressor cells, as SDIF also inhibited the development of nonspecific suppressor cell activity in 7-day cultures of unstimulated spleen cells. The interleukine 2-dependent proliferation of cytotoxic T lymphocytes of or of blast cells was markedly reduced by the addition of SDIF. In contrast, the production of interleukine 2 itself was not impaired by SDIF. These results support the hypothesis of an inhibition by SDIF of a particular step of the mitotic cycle, probably posterior to the G0-G1 transition. An inhibitory activity of SDIF on the expression of the cytolytic activity of cytotoxic T lymphocytes was also observed.

Immunoregulation by Schistosoma mansoni.

Schistosoma mansoni is known to release an inhibitory factor of lymphocyte proliferation elicited in vitro. The effect of this dialyzable schistosome incubation product (DSIP) was tested in vivo on different aspects of the cell-mediated immune response. First, the DSIP injected into C57B1/6 mice markedly inhibited the delayed type hypersensitivity to sheep red blood cells (SRBC). Furthermore, the DSIP injected into S. mansoni infected Fisher rats at the beginning of the infection induced an inhibition of the specific lymphocyte response to S. mansoni antigen and of the spleen cell response to concanavalin A (Con A). The DSIP injected into uninfected rats also inhibited the spleen cell response to Con A. In uninfected as in infected rats injected with the DSIP, the lymphocyte response to Con A was restored after purification of the spleen cells on a nylon wool column. Moreover spleen cells from rats injected wtih the DSIP reduced the proliferative response of normal syngeneic spleen cells induced by Con A. This inhibition was not observed when cells from DSIP-injected rats were previously passed through a nylon wool column. In contrast, nylon wool depletion of spleen cells from infected rats injected with the DSIP did not restore the lymphocyte response to S. mansoni antigen. It seems tht DSIP could partly explain the modulation of the cellular immune responses observed during S. mansoni infection and could represent one of the mechanisms of this parasite's survival in the immunized host.