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1.
Vaccine ; 26(48): 6143-50, 2008 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-18804135

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25-545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials.


Assuntos
Antígenos de Protozoários/biossíntese , Indústria Farmacêutica/normas , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/normas , Proteínas de Membrana/biossíntese , Proteínas de Membrana/normas , Pichia/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/normas , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/toxicidade , Western Blotting , Clonagem Molecular , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Fermentação , Liofilização , Cobaias , Vacinas Antimaláricas/toxicidade , Masculino , Espectrometria de Massas , Proteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Pichia/metabolismo , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/toxicidade , Controle de Qualidade , Coelhos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/normas , Vacinas Sintéticas/toxicidade
2.
PLoS One ; 3(8): e2940, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698359

RESUMO

BACKGROUND: Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. METHODS: A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 15), 80 microg of AMA1-C1/Alhydrogel (n = 30), or 80 microg of AMA1-C1/Alhydrogel+564 microg CPG 7909 (n = 30). RESULTS: Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 microg or 80 microg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 microg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. CONCLUSION/SIGNIFICANCE: The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00344539.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/toxicidade , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Hidróxido de Alumínio/toxicidade , Animais , Antígenos de Protozoários/toxicidade , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/toxicidade , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/toxicidade , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/toxicidade , Segurança
3.
Parasitol Res ; 102(6): 1375-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18369662

RESUMO

Human Trichomonas vaginalis infection ranges from asymptomatic to mild, moderate or severe clinical manifestations. The reasons for diverse symptomatology have been found to vary in several attributes and both parasite and host factors appear to play a role in the pathogenesis. The present study reports the in vitro haemolytic and cytotoxic activity of crude soluble extract (CSE) antigen of T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. The haemolytic activity following the interaction of CSE antigen with human erythrocytes and cytotoxic activity by adding CSE antigen to normal human vaginal epithelial cells was significantly higher with the use of CSE antigen of isolates from symptomatic women as compared to those from asymptomatic women. Furthermore, cytotoxic effect was found to be pH dependent. The study demonstrates, for the first time, the significant effect of parasite antigen of isolates from symptomatic women as compared to those from asymptomatic women in inducing haemolytic and cytotoxic effect and supports the earlier report that contact-independent mechanism(s) may be playing a role in establishing symptomatic trichomoniasis.


Assuntos
Antígenos de Protozoários/toxicidade , Portador Sadio/parasitologia , Extratos Celulares/toxicidade , Células Epiteliais/efeitos dos fármacos , Hemólise , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/química , Animais , Antígenos de Protozoários/isolamento & purificação , Extratos Celulares/isolamento & purificação , Feminino , Humanos , Concentração de Íons de Hidrogênio , Trichomonas vaginalis/isolamento & purificação
4.
Parasite ; 11(2): 225-30, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15224585

RESUMO

In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM). Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA), associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion-of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.


Assuntos
Antígenos de Protozoários/imunologia , Cryptosporidium parvum/imunologia , Cryptosporidium parvum/ultraestrutura , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/toxicidade , Bovinos , Doenças dos Bovinos/parasitologia , Criptosporidiose/parasitologia , Fezes/parasitologia , Glicoproteínas/imunologia , Humanos , Microscopia Imunoeletrônica , Organelas/imunologia
5.
J Egypt Soc Parasitol ; 29(1): 281-9, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-12561907

RESUMO

An 18-20 kDa Cryptosporidum coproantigen (18-20 CCA), had been detected in the stool of infected humans and calves. A purified, electroeluted and concentrated 18-20 kDa antigen was tested in Ussing chamber, and electric parameters were tested before and after the addition of this antigen. A significant increase in the short circuit current (Isc) was detected. The enterotoxic effectof 18-20 kDa CCA, was time and dose dependent, heat labile, and Cl- dependent. The detected change in the short circuit current was not detected when the 18-20 kDa antigen wa incubated with its monospecific antibodies. These results indicate an enterotoxic activity of the 18-20 kDa antigen secreted from the released parasite and detected in stool of infected humans and calves. Additionally, they may help in developing an appropriate anti-secretory therapy for the intractable diarrhoea caused by cryptosporidiosis.


Assuntos
Antígenos de Protozoários/toxicidade , Cryptosporidium parvum/patogenicidade , Enterotoxinas/toxicidade , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Pré-Escolar , Criptosporidiose/parasitologia , Criptosporidiose/veterinária , Cryptosporidium parvum/imunologia , Fezes/parasitologia , Humanos , Íleo/parasitologia , Íleo/patologia , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Coelhos
6.
Infect Immun ; 60(5): 1894-901, 1992 May.
Artigo em Inglês | MEDLINE | ID: mdl-1563780

RESUMO

We have previously shown that malaria parasites liberate exoantigens which, through a phospholipid component, stimulate mouse macrophages to secrete tumor necrosis factor (TNF), which are toxic to D-galactosamine-sensitized mice, and which therefore might be involved in pathology. Plasmodium yoelii exoantigens detoxified by dephosphorylation or digestion with lipases do not induce TNF production. However, these partial structures inhibited its production in response to the exoantigens, although not to bacterial lipopolysaccharide (LPS). When pure phospholipids were tested in a macrophage assay, none stimulated the production of TNF, but phosphatidylinositol (PI) inhibited TNF induction by P. yoelii exoantigens. Moreover, inositol monophosphate (IMP) was the only one of a number of monophosphate saccharides tested which was inhibitory; inositol was not. Macrophages pretreated with PI, IMP, or detoxified exoantigens and then incubated with parasite exoantigens also yielded much less TNF. PI, IMP, and lipase-digested exoantigens of P. yoelii similarly inhibited the TNF-inducing activity of exoantigens of the human parasites Plasmodium falciparum and Plasmodium vivax. Neither PI nor IMP diminished TNF production in response to LPS, in contrast to a platelet-activating factor antagonist [1-O-hexadecyl-2-acetyl- sn-glycero-3-phospho(N,N,N-trimethyl hexanolamine)] which inhibited both exoantigen- and LPS-induced production of TNF. We conclude that at least two different parts of the molecule are involved in the induction of TNF secretion by parasite exoantigens: one requires the presence of a phosphate bound to inositol, and, since dephosphorylated exoantigens were also inhibitory, one does not. It would seem that both affect interactions between parasite-derived exoantigens and the macrophage receptors.


Assuntos
Antígenos de Protozoários/toxicidade , Fosfatidilinositóis/farmacologia , Plasmodium/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Feminino , Lipopolissacarídeos , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Plasmodium/patogenicidade , Fator de Ativação de Plaquetas/antagonistas & inibidores , Relação Estrutura-Atividade
7.
Immunology ; 75(1): 129-35, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1537589

RESUMO

In patients with malaria, the clinical manifestations of the disease are associated with the presence of high concentrations of tumour necrosis factor (TNF) in the serum. Blood-stage parasites of human and rodent malarial parasites release serologically related exoantigens which induce the production of TNF in vitro and in vivo and which can kill mice made hypersensitive to TNF by pretreatment with D-galactosamine. They also elicit the production of T-independent antibody, which blocks these effects. The capacity of the exoantigens to stimulate macrophages to secrete TNF does not require the presence of protein or carbohydrate, but is associated with a lipid whose activity can be abolished by treatment with phospholipase C. Treatments of the exoantigens which destroyed their activity in vitro also abrogated their immunogenicity and their toxicity for mice. No TNF-inducing activity could be detected in preparations of parasitized erythrocytes that was not associated with phospholipid, and the TNF-inducing properties of the malarial phospholipids are quite distinct from those of bacterial lipopolysaccharide. We conclude that release of potentially toxic phospholipids by parasites may be responsible for some of the pathology of malaria.


Assuntos
Antígenos de Protozoários/imunologia , Fosfolipídeos/imunologia , Plasmodium yoelii/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Antígenos de Protozoários/toxicidade , Feminino , Soros Imunes/imunologia , Malária/imunologia , Camundongos
8.
Immunol Lett ; 25(1-3): 207-12, 1990 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2283151

RESUMO

The production of cytokines, including tumour necrosis factor (TNF), may be involved in the pathology of malaria, as well as in protection against the parasite. We have shown that parasite exoantigens induce the secretion of TNF in vitro and in vivo and kill mice made hypersensitive to TNF. They elicit T-independent antibody that inhibits their capacity to stimulate TNF production and protects against toxicity in vivo, and those of human and rodent parasites are serologically related. Their active component does not appear to be protein. Here we review their properties and consider the epidemiological significance of our findings and their possible contribution to the development of an "anti-disease" vaccine.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Plasmodium berghei/imunologia , Plasmodium yoelii/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/química , Antígenos de Protozoários/toxicidade , Antígenos T-Independentes/imunologia , Reações Cruzadas , Feminino , Galactosamina/farmacologia , Técnicas In Vitro , Lipopolissacarídeos/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Vacinas Protozoárias , Fator de Necrose Tumoral alfa/biossíntese
9.
Immunology ; 66(4): 600-5, 1989 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2654012

RESUMO

Heat-stable soluble products of rodent malarial parasites induce activated peritoneal macrophages to secrete tumour necrosis factor (TNF) in vitro. Since heat-stable parasite antigens are known to be present in the circulation of patients with malaria and it has been suggested that much of the pathology of malaria is due to TNF, we investigated the ability of such antigens to induce the production of TNF in vivo and to be toxic to mice. Injection of antigens obtained from Plasmodium yoelii or from P. berghei into mice which had previously received the macrophage-activating agent Propionibacterium acnes induced the release of TNF into the serum in amounts equivalent to the maximum release induced by bacterial lipopolysaccharide (LPS). Specific antiserum blocked the ability to the boiled soluble antigens, but not of LPS, to induce release of TNF. Similarly, vaccination specifically inhibited the release of TNF into the serum in response to subsequent stimulation with the antigens, but not with LPS. Mice made hypersensitive to the lethal action of TNF by pretreatment with D-galactosamine were killed in a dose-related fashion by administration of antigen preparations; addition of specific antiserum or prior vaccination with the antigens protected such mice, but not those given LPS, from death. We conclude that, in malaria, soluble antigens derived from the parasites may act like a toxin by stimulating the production of TNF, an important mediator of endotoxic shock, and that immunization with such antigens may diminish TNF secretion and consequently many of the clinical manifestations of the disease.


Assuntos
Antígenos de Protozoários/toxicidade , Plasmodium berghei/imunologia , Plasmodium yoelii/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antígenos de Protozoários/imunologia , Feminino , Galactosamina , Ativação de Macrófagos , Propionibacterium acnes , Ratos , Solubilidade
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