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G Del Giudice

Publications and source records attributed to G Del Giudice.

At least 73 records · Page 4Linked to original sources
PubMed1992

Plasmodium falciparum synthesizes O-glycosylated glycoproteins containing O-linked N-acetylglucosamine.

Asexual blood forms of the human malaria parasite, Plasmodium falciparum, synthesize a major glycosylated 195 kDa protein that has been considered for the development of a vaccine. beta-Elimination-borohydride reduction of the 195 kDa glycoprotein and its 16 kDa processed product after metabolic labeling of their carbohydrates, showed the presence of derived, labeled glucosaminitol and alanine. This suggests that the 195 and 16 kDa glycoproteins contain distinct O-glycosyl linkages and that N-acetylglucosamine and serine residues are involved in the attachment of carbohydrate moieties to the protein core. Endo-O-glycanase treatment of total glycoproteins shows that O-glycosidycally-linked sugars represent a major carbohydrate moiety in P. falciparum glycoproteins.

Acetylglucosamine↗
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

Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential.

Malaria infection crisis, at which the parasitemia drops precipitously and the parasite loses infectivity to the mosquito vector, occurs in many natural malaria systems, and has not been explained. We demonstrate that in a simian malaria parasite (Plasmodium cynomolgi in its natural host, the toque monkey), the loss of infectivity during crisis is due to the death of circulating intraerythrocytic gametocytes mediated by crisis serum. These parasite-killing effects in crisis serum are due to the presence in the serum of cytokines tumor necrosis factor and interferon gamma, which are produced by the host as a result of the malaria infection. The killing activity of each cytokine is absolutely dependent upon the presence of additional, as yet unidentified factor(s) in the crisis serum.

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

Mycobacterial heat-shock proteins as carrier molecules.

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guérin, BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies, overcoming the requirement of adjuvants and the genetic restriction of the antibody response to the peptide (Lussow et al., Proc. Natl. Acad. Sci. USA 1990. 87:2960). This initial work led us to the following observations. BCG had to be live for priming to lead to the induction of anti-peptide antibodies. Surprisingly, priming with other living microorganisms which chronically infect the macrophage (e.g. Salmonella typhimurium and Leishmania major) also induced anti-peptide antibodies in mice immunized with PPD-(NANP)40 conjugate. It was, thus, hypothesized that molecules expressed during active infection and also known to be highly conserved between species, namely the heat-shock proteins (hsp), could mediate the T cell sensitization required for the production of anti-peptide antibodies. In fact, when the PPD protion of the conjugate was replaced by a highly purified recombinant protein corresponding to the 65-kDa (GroEL-type) hsp of M. bovis, this resulted in the production of anti-(NANP) IgG antibodies in BCG-primed mice, irrespective of the major histocompatibility complex-controlled responsiveness to the (NANP) sequence itself. Further, similar induction of anti-peptide antibody response was also obtained with a recombinant 70-kDa (DnaK-type) hsp of M. tuberculosis, but not with a small molecular mass (18 kDa) of M. leprae. Finally, an adjuvant-free carrier effect for anti-peptide IgG antibody production in BCG-primed mice, was also exerted by the GroEL hsp of Escherichia coli. This finding that hsp can act as carrier molecules without requiring conventional adjuvants is of potential importance in the development of vaccine strategies.

Adjuvants, Immunologic↗
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

Multiple antigen peptides (MAPs) as candidate vaccines against malaria.

Multiple Antigen Peptides (MAPs), branched molecules where multiple copies of a desired antigenic sequence are assembled on a small peptide core, have been recently described as an alternative approach to the synthesis of high molecular weight immunogens. In comparison with conventional peptide-carrier conjugates, the MAPs show several advantages, including chemical unambiguity and ease of synthesis. A MAP based on the sequence of the repetitive domain of P. malariae sporozoites was immunogenic in a large number of mouse strains. When covalently linked to the corresponding sequence of the P. falciparum circumsporozoite protein, [NANP]40, the resulting conjugate showed the properties of a multivalent vaccine, overcoming the severe genetic restriction of the [NANP] sequence. A second generation of MAPs including both sequences, with more desirable chemical properties, was equally effective. These compounds represent a promising step towards the development of synthetic, multivalent peptide vaccines against human malaria.

Amino Acid Sequence↗
PubMed1990

Human T cell proliferative responses to Plasmodium vivax antigens: evidence of immunosuppression following prolonged exposure to endemic malaria.

Human T cell proliferative responses, of 33 adult Sri Lankans convalescing from Plasmodium vivax infections, to several P. vivax antigens (i.e. a soluble extract of asexual erythrocytic stage parasites and two cloned antigens that are potential vaccine candidates PV200 and GAM-1) were assessed. The peripheral blood mononuclear cell proliferative responses to the soluble extract of P. vivax, as assessed by studying both the proportion of responders and the degree of the response, were significantly lower in a group of individuals resident in a malaria endemic area in Sri Lanka than in another group that did not have a life-long exposure to malaria but had acquired the disease on a visit to an endemic region. Individuals of both groups responded equally well to mitogen. The responses to a non-malarial antigen such as purified protein derivative of tuberculin were only marginally lower in residents of the malaria-endemic region. These findings suggest that exposure to endemic P. vivax malaria leads to a specific immunosuppression to P. vivax antigens. Immunosuppression of a much lower degree was evident to a non-malarial antigen.

Adolescent↗
PubMed1990

A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes.

The major repetitive epitopes of the surface circumsporozoite (CS) protein of malaria sporozoites represent candidates for the development of subunit vaccines against malaria. However, previous experimental work has shown that repetitive peptides from the CS proteins of Plasmodium falciparum, P. vivax, P. yoelii and P. berghei are immunogenic only in mice with the H-2b or H-2k haplotype. This led to the conclusion that strong T helper epitopes from the non-repetitive CS sequences were required in the design of sporozoite vaccines. In the present study, we investigated the immunogenicity in mice of a octa-branched multiple antigen peptide (MAP) containing repeats of the CS protein of the human malaria parasite, P. malariae, [MAP8(NAAG)6], and found that mice with an H-2b, H-2d, H-2k, H-2f, H-2q, and H-2s haplotype produced anti-peptide antibodies after immunization and that only H-2r mice were nonresponsive. This antibody response, not induced in athymic H-2b nu/nu mice, was directed against the (NAAG) sequence, but not against the lysine core of the MAP construct. Finally, when covalently linked to a synthetic polymer of the repetitive (NANP) sequence of the P. falciparum CS protein, [MAP8(NAAG)6] behaved as a carrier molecule for the production of anti-(NANP)n antibodies in H-2d and H-2k mice, genetically nonresponder to the (NANP)n sequence. Should this wide immunogenicity of the P. malariae CS (NAAG) repetitive sequence also apply to humans, it might be considered for the design of multivalent subunit malaria vaccines.

Adjuvants, Immunologic↗
PubMed1990

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms.

The immunogenicity of the carrier-free synthetic peptide, (NANP)40, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)40 IgG antibodies. This decrease in the anti-(NANP)40 antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)40 peptide, and on an already established anti-(NANP)40 antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.

Amino Acid Sequence↗
PubMed1990

Peptide-primed CD4+ cells and malaria sporozoites.

We have mapped a T cell epitope in the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-mer synthetic peptide corresponding to the amino acid positions 59-79 (referred to as Py1), induced specific proliferation in BALB/c and C57BL/6 mice, and provided help for the production of antibodies to peptides from the repetitive region, (QGPGAP)n, of the same CS protein, when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Long-term CD3+CD4+CD8-TCR alpha beta+ T cell lines and clones were derived from both strains of mice. These lines and clones, that proliferated in an MHC-restricted fashion, did not recognize peptides from the homologous region of another murine malaria parasite, P. berghei. About 50% of these clones produced detectable amounts of IFN-gamma and IL-2, whereas the remaining produced IL-4, IL-5, and IL-6. In preliminary experiments, some of these clones specifically inhibited P. yoelii sporozoite development in vitro and conferred protection in vivo in passive transfer experiments. These findings show that heterogenous T cell populations are activated in mice upon immunization with a short peptide from the P. yoelii CS protein and that some of these cells could be active in the effector arm of the immune response against malaria sporozoites.

Amino Acid Sequence↗
PubMed1990

Anti-parasite effects of cytokines in malaria.

Cytokines induced during natural malaria infections, e.g., at crisis of a blood infection of Plasmodium cynomolgi, and during clinical paroxysms in human Plasmodium vivax infections, mediate killing of intra-erythrocytic blood stage malaria parasites. These cytokines, TNF and IFN-gamma, require additional, yet unidentified complementary factors that are present in "crisis" and "paroxysm" serum to kill intra-erythrocytic blood stage parasites. In contrast, cytokines, (mainly IFN-gamma) are able to effect killing of intra-hepatic stages of the parasite by themselves independent of serum complementary factors, suggesting that the mechanisms of killing may be different with respect to the two parasite stages. Cytokines also appear to be critical intermediates in mechanisms of clinical disease in malaria. Serum cytokine (TNF) levels and killing effects on blood stage malaria parasites were lower in patients who were exposed to endemic P. vivax malaria who had partial clinical immunity, than in non-immune patients. Evidence suggest that individuals acquire natural immunity to the disease by avoiding the induction of high levels of cytokines and complementary factors.

Animals↗
PubMed1990

Towards vaccine optimisation.

New molecular technologies have accelerated the search for sub-unit candidate vaccines. However, once identified the use of a candidate antigen must be optimised to reap the maximum benefit from the eventual vaccine. This optimisation should take into account both the needs of the target population, and the various ways of potentiating the protective immune response induced. One must be sure that the final product will be used. Hence, vaccine optimisation should strive toward meeting the needs of a specific epidemiological problem within the economic constraints of a given situation. This may be possible using novel delivery systems designed to limit the number of doses needed, improve the stability or facilitate the delivery of a particular vaccine. In meeting the needs of a target population in a field situation, one must also keep in mind certain safety factors that go beyond the usual regulatory constraints. The immune response to vaccine candidates can be potentiated in many ways. The ability to preferentially induce specific protective effector mechanisms: i.e., antibody isotypes, T-cell subsets, and T-cell sub-subsets, is becoming a reality. Carrier molecules designed to avoid the problems of epitope suppression and competition, and perhaps an eventual "carrier jam," are being developed. Adjuvants and immunostimulants may also help, but the critical issue here remains their acceptability for use in man. Finally novel strategies for the induction of the immune response may also potentiate the immune response in the optimisation of vaccines.

Adjuvants, Immunologic↗
PubMed1990

Use of a tuberculin purified protein derivative--Asn-Ala-Asn-Pro conjugate in bacillus Calmette-Guérin primed mice overcomes H-2 restriction of the antibody response and avoids the need for adjuvants.

Because of its immunodominancy, and because it is conserved in different geographical isolates of Plasmodium falciparum, the repetitive sequence of the circumsporozoite protein, (Asn-Ala-Asn-Pro)n [(NANP)n], has been envisaged for the development of an anti-falciparum malaria subunit vaccine. However, the murine immune response to (NANP)n peptides, either carrier-free or coupled to carrier proteins, was shown to be inducible only by using strong (e.g., Freund's) adjuvants. Furthermore, response to the carrier-free peptide, administered in adjuvant, is genetically restricted to I-Ab mice. In the present paper, we report that high titers of antibodies against the NANP repetitive epitope were obtained in responder C57BL/6 (H-2b) mice when they were primed with live BCG (bacillus Calmette-Guérin Mycobacterium tuberculosis var. bovis) and immunized once with the synthetic peptide (NANP)40 coupled to tuberculin purified protein derivative (PPD) without the use of any adjuvant. This approach also led to the production of high titers of anti-NANP antibodies in ASW (H-2s), B10.RIII (H-2r), BALB/c (H-2d), C3H/He (H-2k), and DBA/1 (H-2q) nonresponder mice after two injections of the conjugate. In both cases, BCG priming was obligatory for the induction of antibodies reacting with the synthetic peptide. The levels of anti-NANP antibodies in nonresponder BALB/c mice were demonstrated to be comparable to the levels induced after PPD-(NANP)40 immunization in Freund's complete or incomplete adjuvant. The antibodies induced were also capable of recognizing P. falciparum sporozoites in immunofluorescence assays and, furthermore, these antibodies inhibited the penetration of live sporozoites into human hepatocytes in vitro. This system functioned independently of the subjects' resistance or susceptibility to BCG infection. Given the widespread natural exposure to mycobacterial antigens and the extensive use of BCG and PPD in the human population, this approach might be envisaged for vaccination with malaria peptides.

Adjuvants, Immunologic↗
PubMed1990

Production of specific monoclonal antibodies to Salmonella typhi flagellin and possible application to immunodiagnosis of typhoid fever.

Four murine monoclonal antibodies (MAbs) to Salmonella typhi flagellin were produced. These MAbs did not react with eight other enterobacterial strains tested: Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi A, Escherichia coli, Shigella flexneri, Shigella sonnei, Yersinia enterocolitica, and Campylobacter jejuni. All four MAbs cross-reacted with Salmonella muenchen flagellin indicating specificity for d antigenic flagellar epitope. One MAb (C4) was selected to develop a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect S. typhi flagellin in serum samples. By use of this assay S. typhi flagellar antigen was detected in 95.5% of serum samples from patients with positive hemoculture for S. typhi, in 93.6% of samples from patients with positive serodiagnosis of typhoid fever, in 26% of samples collected from patients who were initially hemoculture-positive for S. typhi and who had undergone 7-8 d of chemotherapy, in 8.5% of samples from healthy persons from an endemic area, and in no samples from healthy persons from a nonendemic area. The presence of high levels of flagellin antibody titers did not interfere with the antigen detection. The detection of S. typhi flagellar antigen in patient serum may have practical value for rapid diagnosis of typhoid fever.

Animals↗
PubMed1990

Use of synthetic peptides in the study of the antibody response to Plasmodium vivax sporozoites.

Synthetic peptides reproducing 4 DRADGQPAG (D4) and a sequential array of DRADGQPAG and DRAAGQPAG repeats (DDAAD) of the Plasmodium vivax circumsporozoite (CS) protein were investigated for their potential use in the detection of P. vivax sporozoite antibodies in human sera. These peptides specifically inhibited the binding of monoclonal antibodies to the P. vivax CS protein in Western blots. However, when D4 and DDAAD peptides were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of human antibodies, more sera bound to the DDAAD (61%) than to the D4 peptide (22%). This binding was specific, and suggested that the DDAAD peptide contained epitopes constituted by the sequential array of DRADGQPAG and DRAAGQPAG repeat variants and absent in the D4 peptide. The ELISA using the DDAAD peptide was applied to the detection of P. vivax CS protein antibodies in a large number of sera from Kataragama, an endemic area in Sri Lanka. The prevalence of these antibodies increased with age, reaching 40% in adults greater than 50 years old. The ELISA employing the DDAAD peptide represents a simple and useful tool for the analysis of the antibody response to P. vivax sporozoites in naturally exposed individuals.

Age Factors↗
PubMed1990

Antibody responses to Plasmodium falciparum and P. vivax sporozoites in areas with stable and unstable malaria.

Availability of synthetic and recombinant peptides reproducing the repetitive regions of the circumsporozoite (CS) proteins of Plasmodium falciparum and P. vivax has allowed the development of assays for the detection of specific antibodies and of potential subunit vaccines. Knowledge of the immune responses to malaria sporozoites is a prerequisite for the optimal design of a sporozoite antigen-based vaccine. Studies carried out in areas with stable P. falciparum malaria (United Republic of Tanzania) have shown that antibodies against the synthetic peptide (NANP)40 increase as a function of age. Cluster analysis revealed marked inter-household variation of the anti-sporozoite antibody response, despite comparable risks of exposure to infectious bites. An age-related prevalence of anti-P. vivax sporozoite antibodies has been observed in an area of Sri Lanka with unstable malaria, using a 45-mer synthetic peptide reproducing a defined sequential array of the two main 9-mer variants of the P. vivax CS protein. In this area, anti-(NANP)40 antibodies became detectable after the first epidemic of P. falciparum malaria. Interestingly, their prevalence also increased with age. Since this population had not been exposed to P. falciparum malaria for at least 10 years previously, one can suggest that anti-sporozoite antibodies reflect the relative exposure to infectious bites in the different age groups, and, in turn, the transmission of the disease. This can be particularly useful in areas where entomological indices of transmission tend to be unreliable because of the low vectorial capacity and wide fluctuations in vector densities.

Age Factors↗