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1.
Bioorg Med Chem ; 26(9): 2401-2409, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29650461

RESUMO

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease's most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides' secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals' sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Membrana/imunologia , Mycobacterium tuberculosis/fisiologia , Fragmentos de Peptídeos/imunologia , Vacinas contra a Tuberculose/imunologia , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/toxicidade , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Linhagem Celular Tumoral , Biologia Computacional , Desenho de Fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Membrana/síntese química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/toxicidade , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Vacinas contra a Tuberculose/síntese química , Vacinas contra a Tuberculose/metabolismo , Vacinas contra a Tuberculose/toxicidade , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/toxicidade
2.
Regul Toxicol Pharmacol ; 71(2): 269-78, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545314

RESUMO

RTS,S malaria antigen is weakly immunogenic as such and needs to be formulated with an adjuvant to improve the magnitude and duration of the immune responses to RTS,S. Two Adjuvant Systems, AS01 and AS02 were evaluated during the development of the RTS,S vaccine. The evaluation included non-clinical studies in rabbits to evaluate the local intramuscular tolerance following administration on a single occasion, and the local and systemic effects following repeated administrations of RTS,S/AS01 or RTS,S/AS02 formulations. In the first study, rabbits were injected on one occasion with RTS,S/AS01, RTS,S/AS02 or controls, and the local intramuscular tolerance was evaluated up to 3 days after injection. In the second study, the different formulations were injected on Days 0, 14, 28 and 42. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Macroscopic and microscopic evaluations were made after termination of the study. No sign of toxicity was detected following single or repeated administrations of the adjuvanted RTS,S formulations. Changes in haematology or clinical chemistry parameters were indicative of a developing immune response in the groups receiving either RTS,S formulation. All examined parameters returned to normal within 28 days after the last injection. The absence of toxicological effects following the injection of RTS,S/AS01 or RTS,S/AS02 in rabbits was supportive of further clinical evaluation of these two formulations.


Assuntos
Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/toxicidade , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/toxicidade , Animais , Análise Química do Sangue/métodos , Química Farmacêutica , Feminino , Injeções Intramusculares , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Coelhos
3.
Int J Pharm ; 465(1-2): 275-83, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24491528

RESUMO

A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E. coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, zeta potential, electrophoretic mobility and atom force microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity suggesting its employment as potential vaccine against Parietaria-induced allergies.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Portadores de Fármacos , Hipersensibilidade/prevenção & controle , Nanopartículas , Peptídeos/química , Proteínas de Plantas/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Alérgenos/química , Alérgenos/toxicidade , Animais , Antígenos de Plantas/química , Antígenos de Plantas/toxicidade , Basófilos/efeitos dos fármacos , Basófilos/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Estabilidade de Medicamentos , Eletroforese , Hemólise/efeitos dos fármacos , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Luz , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Microscopia de Força Atômica , Nanotecnologia , Proteínas de Plantas/química , Proteínas de Plantas/toxicidade , Espalhamento de Radiação , Tecnologia Farmacêutica/métodos , Vacinas Sintéticas/química , Vacinas Sintéticas/toxicidade
4.
J Neurochem ; 129(4): 696-703, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24471474

RESUMO

The most commonly used immunogen to induce experimental autoimmune encephalomyelitis is MOG35-55 , a 21-residue peptide derived from myelin oligodendrocyte glycoprotein (MOG). In most studies, mice exhibit a chronic disease; however, in some studies mice show a transient disease. One variable that is not often controlled for is the peptide fraction of the purified MOG material, which can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification. We compared the development of clinical signs in female C57Bl6 mice immunized with two commercially available MOG35-55 peptides of similar purity but different peptide fraction (MOG-A being 45%; MOG-B being 72%). A single immunization with MOG-A induced a chronic disease course with some recovery at later stages, whereas immunization with MOG-B induced a similar course of disease but with significantly lower average clinical scores despite a higher peptide content. The addition of a booster immunization significantly increased clinical severity with both preparations, and significantly reduced the average day of onset using MOG-A. To determine if the counter ion could influence disease, we compared MOG-B-containing trifluoroacetate with MOG-B-containing acetate. Although disease incidence and severity were similar, the average day of disease onset occurred approximately 5 days earlier with the use of MOG-B-containing trifluoroacetate. These results demonstrate that differences in peptide fraction influence the course of encephalomyelitis disease, which may be due in part to the levels of counter ions present in the purified material. These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources.


Assuntos
Acetatos/farmacologia , Autoantígenos/isolamento & purificação , Fracionamento Químico/métodos , Encefalomielite Autoimune Experimental/induzido quimicamente , Glicoproteína Mielina-Oligodendrócito/isolamento & purificação , Ácido Trifluoracético/farmacologia , Vacinas Sintéticas/isolamento & purificação , Acetatos/administração & dosagem , Acetatos/análise , Acetatos/toxicidade , Sequência de Aminoácidos , Animais , Autoantígenos/administração & dosagem , Autoantígenos/química , Autoantígenos/toxicidade , Progressão da Doença , Relação Dose-Resposta Imunológica , Contaminação de Medicamentos , Encefalomielite Autoimune Experimental/imunologia , Feminino , Imunização/métodos , Imunização Secundária , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Glicoproteína Mielina-Oligodendrócito/química , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/toxicidade , Índice de Gravidade de Doença , Fatores de Tempo , Ácido Trifluoracético/análise , Ácido Trifluoracético/toxicidade , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/toxicidade
5.
Anaerobe ; 18(3): 363-5, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22306065

RESUMO

In this work, we produced and evaluated a vaccine based on a ß toxoid of Clostridium perfringens type C produced in Escherichia coli (rBT). The non-toxic rBT was innocuous for mice and induced 14 IU mL(-1) of ß antitoxin in rabbits, complying with the European Pharmacopeia and CFR9 - USDA guidelines.


Assuntos
Vacinas Bacterianas/biossíntese , Clostridium perfringens/imunologia , Toxoides/biossíntese , Vacinas Sintéticas/biossíntese , Animais , Toxinas Bacterianas/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/toxicidade , Escherichia coli , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Toxoides/genética , Toxoides/toxicidade , Vacinação , Vacinas Sintéticas/genética , Vacinas Sintéticas/toxicidade
6.
Vaccine ; 30(11): 1917-26, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22269871

RESUMO

A recombinant botulinum vaccine (rBV A/B) is being developed to protect adults 18-55 years of age from fatal botulism caused by inhalational intoxication with botulinum neurotoxin complex (BoNT) serotype A, subtype A1 (BoNT/A1) and BoNT serotype B, subtype B1 (BoNT/B1). Fundamental to the advanced development process is an initial demonstration of product safety in animals. A comprehensive series of studies was conducted to evaluate the general toxicity, neurobehavioral toxicity and local reactogenicity of the rBV A/B vaccine prior to first use in humans. Toxicity was evaluated in CD-1 mice vaccinated with control material and three dosages of rBV A/B with or without Alhydrogel(®) by intramuscular (IM) injection on Study Days 0, 28, 56 and 70 in a volume of 100µL. Total immunizing protein given in each dose was either 0, 2, 4 or 8 µg/animal. Local reactogenicity was evaluated in mice at the dosages given and in New Zealand white (NZW) rabbits using the same injection volume (0.5 mL) and formulations (10, 20 and 40 g/mL total antigen with 0.2% (w/v) Alhydrogel(®)) intended for human use. The rBV A/B vaccine produced no apparent systemic or neurobehavioral toxicity and only transient mild inflammation at the injection site. Together these results indicated a favorable safety profile for rBV A/B and supported its use in a Phase 1 clinical trial.


Assuntos
Vacinas Bacterianas/toxicidade , Toxinas Botulínicas/toxicidade , Botulismo/prevenção & controle , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Camundongos , Coelhos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
7.
Immunol Invest ; 41(4): 356-66, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21864115

RESUMO

It is of fundamental importance to use an appropriate adjuvant to generate a potent immune response for immunotherapy. In this study, we had a comparative investigation on the effectiveness of two adjuvant formulations, liposome-polycation-DNA (LPD) and monophosphoryl lipid A(MPL) in combination with a truncated peptide of bFGF(tbFGF) as antigen. LPD/tbFGF induced continuously increasing antibodies expression during the whole immunization period. In contrast, the level of antibodies was variable in MPL/tbFGF-immunized mice, MPL/tbFGF elicited potent antibodies response in the early-phase of immunization (during the first 3 immunizations), but the later immunizations did not produce a significant increase in the level of antibodies. Evaluation of IFN-γ and IL-4 responses revealed that both LPD/tbFGF and MPL/tbFGF demonstrated generation of higher level of IFN-γ, whereas no significant increase in IL-4 levels was detected in the two groups. In addition, histological analysis exhibited obvious germinal centers in the spleen tissues of LPD/tbFGF mice. The data suggested that LPD would be a promising long-effective adjuvant due to its potent and persistent immunostimulation and MPL could play an appropriate role in short-acting immunization.


Assuntos
Adjuvantes Imunológicos/química , Lipídeo A/análogos & derivados , Oligodesoxirribonucleotídeos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Sequência de Aminoácidos , Animais , Anticorpos/sangue , Anticorpos/imunologia , Citocinas/metabolismo , DNA/química , DNA/imunologia , Feminino , Fator 2 de Crescimento de Fibroblastos/química , Lipídeo A/química , Lipídeo A/imunologia , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Poliaminas/química , Poliaminas/imunologia , Baço/imunologia , Baço/metabolismo , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
8.
J Immunol ; 187(3): 1176-83, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21705619

RESUMO

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.


Assuntos
Autoantígenos/administração & dosagem , Tolerância Imunológica , Colágenos não Fibrilares/administração & dosagem , Penfigoide Bolhoso/imunologia , Vacinas Virossomais/toxicidade , Animais , Autoantígenos/genética , Autoantígenos/imunologia , Sítios de Ligação de Anticorpos , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Epiderme/imunologia , Epiderme/metabolismo , Epiderme/patologia , Feminino , Tolerância Imunológica/genética , Imunização Secundária/efeitos adversos , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Neutrófilos/imunologia , Neutrófilos/patologia , Colágenos não Fibrilares/genética , Colágenos não Fibrilares/imunologia , Penfigoide Bolhoso/genética , Penfigoide Bolhoso/patologia , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/toxicidade , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/imunologia
9.
Clin Vaccine Immunol ; 17(8): 1163-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20554806

RESUMO

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.


Assuntos
Anticorpos Antibacterianos/sangue , Antitoxinas/sangue , Enterotoxinas/administração & dosagem , Enterotoxinas/imunologia , Imunidade nas Mucosas , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Substituição de Aminoácidos/genética , Animais , Animais Recém-Nascidos , Toxina da Cólera/administração & dosagem , Enterotoxinas/toxicidade , Fezes/química , Feminino , Humanos , Imunização Secundária/métodos , Imunoglobulina A/análise , Imunoglobulina G/sangue , Masculino , Proteínas Mutantes/administração & dosagem , Proteínas Mutantes/imunologia , Proteínas Mutantes/toxicidade , Mutação de Sentido Incorreto , Vacinas Antiestafilocócicas/efeitos adversos , Vacinas Antiestafilocócicas/toxicidade , Superantígenos/administração & dosagem , Superantígenos/imunologia , Superantígenos/toxicidade , Suínos , Fatores de Tempo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
10.
Transplantation ; 88(8): 1025-9, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19855249

RESUMO

BACKGROUND.: Vaccination against Epstein-Barr virus (EBV), inducing an antibody response to the envelope glycoprotein gp350, might protect EBV-negative children with chronic kidney disease from lymphoproliferative disease after transplantation. METHODS.: A phase I trial recruited children with chronic kidney disease to two successive cohorts given three injections of 12.5 microg (n=6) and 25 microg (n=10) recombinant gp350/alhydrogel vaccine over 6 to 8 weeks. RESULTS.: One in each cohort acquired wild EBV before the week 28 evaluation. Both doses were similarly immunogenic, inducing an IgG response in all 13 evaluable patients. Neutralizing antibodies were detected in four recipients (1/4 in the 12.5 microg and 3/9 in the 25 microg cohort). Median time from first vaccination to transplantation was 24 weeks. Immune responses declined rapidly and were unlikely to affect posttransplant events. DISCUSSION.: The vaccine was immunogenic but a prolonged vaccine schedule up to time of transplantation or improved adjuvants are required in future trials to reduce posttransplant EBV load and risk of lymphoproliferative disease.


Assuntos
Herpesvirus Humano 4/imunologia , Falência Renal Crônica/imunologia , Transplante de Rim/imunologia , Glicoproteínas de Membrana/imunologia , Vacinas Sintéticas/toxicidade , Proteínas da Matriz Viral/imunologia , Vacinas Virais/toxicidade , Adolescente , Animais , Células CHO/imunologia , Criança , Pré-Escolar , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/efeitos dos fármacos , Lactente , Glicoproteínas de Membrana/genética , Proteínas da Matriz Viral/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-19718687

RESUMO

BACKGROUND: An increasing number of women are being vaccinated during child-bearing years, including vaccination with BioThrax (Anthrax Vaccine Adsorbed, or AVA). As only a limited number of studies exist in humans that have examined the effects of AVA on reproductive health, this study was conducted in order to evaluate the impact AVA vaccination may have on pregnant female rabbits and their offspring. METHODS: Two hundred female rabbits were vaccinated with saline, adjuvant, or AVA twice prior to mating and on one of two occasions during gestation, in order to have exposure to the antigen during organogenesis. Blood samples were collected from does and fetuses/kits to assess the development and in utero transfer of antibodies to Bacillus anthracis protective antigen (anti-PA IgG). Half of the does underwent Caesarean-sectioning on gestation day 29 and a gross necropsy was performed on both the does and their fetuses. The other half were allowed to naturally deliver and gross necropsy of the does and their kits was performed on lactation day 29. RESULTS: ELISA results showed that anti-PA IgG was generated by the does and passed to the fetuses/kits at detectable levels. CONCLUSIONS: AVA directly, or indirectly through the production of anti-PA IgG, did not appear to have an adverse effect on the pregnant females or their offspring, as measured by mating and fertility indices, natural delivery observations, clinical signs, gross lesions, in utero growth and survival, morphological development, or kit viability.


Assuntos
Vacinas contra Antraz/toxicidade , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Embrião de Mamíferos/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Vacinas Sintéticas/toxicidade , Adjuvantes Imunológicos/toxicidade , Hidróxido de Alumínio/toxicidade , Animais , Vacinas contra Antraz/imunologia , Embrião de Mamíferos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Exposição Materna , Troca Materno-Fetal/imunologia , Gravidez , Coelhos , Reprodução/imunologia , Vacinas Sintéticas/imunologia
12.
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
13.
Vaccine ; 25(42): 7441-9, 2007 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-17870214

RESUMO

We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.


Assuntos
Alphavirus/genética , Vacinas contra Citomegalovirus/genética , Citomegalovirus/genética , Citomegalovirus/imunologia , Animais , Anticorpos Antivirais/sangue , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/toxicidade , Feminino , Vetores Genéticos , Humanos , Imunidade Celular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Coelhos , Replicon , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
14.
Artigo em Russo | MEDLINE | ID: mdl-17672125

RESUMO

Data on influence of Francisella tularensis C-complex preparations on formation of immunity against tularemia are presented. Study of cellular immunity characteristics as well as dynamics of antibody response was carried out on white mice and guinea pigs models. Absence of toxicity, pyrogenicity, and negative effects on immunocompetent cells in combination with protective activity points to possibility of use the C-complex as a component of a subunit vaccine.


Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Tularemia/imunologia , Tularemia/prevenção & controle , Vacinação , Animais , Anticorpos Antibacterianos/sangue , Apoptose , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/toxicidade , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Cobaias , Ativação Linfocitária , Proteínas de Membrana/imunologia , Camundongos , Baço/fisiologia , Timo/fisiologia , Tularemia/sangue , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
15.
Scand J Immunol ; 66(1): 43-51, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17587345

RESUMO

In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.


Assuntos
Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/prevenção & controle , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Western Blotting , Vacinas Anticâncer/administração & dosagem , Antígeno Carcinoembrionário/genética , Neoplasias Colorretais/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Guanosina Monofosfato/imunologia , Células HeLa , Humanos , Injeções a Jato , Camundongos , Plasmídeos , Proteínas Recombinantes , Linfócitos T/imunologia , Transfecção , Transgenes , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade
16.
Dig Liver Dis ; 38(8): 578-87, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16777500

RESUMO

BACKGROUND: Anti-angiogenesis therapy has showed a promising future in tumour treatment. More and more evidence suggest that endoglin is a powerful marker of angiogenesis in solid malignancies, including liver cancer. AIM: To explore whether a plasmid DNA encoding the porcine endoglin has the ability of breaking immune tolerance against endoglin-related tumour angiogenesis in mice. METHODS: A eukaryotic plasmid encoding the extracellular domain of porcine endoglin was constructed, and then used it as a xenogeneic DNA vaccine. Hepa1-6 and H22 hepatoma models were established to observe the anti-tumour activities. Western blot, enzyme-linked immunoadsorbent assay and enzyme-linked immunospot assay were used to determine the antibody characters. Immunohistochemistry and alginate-encapsulated tumour cell assay were used to observe the anti-angiogenesis effects. RESULTS: Immunotherapy with recombinant plasmid encoding extracellular domain of porcine endoglin was effective at both protective and therapeutic anti-tumour immunity in two hepatoma models. Autoantibodies against murine endoglin were identified. IgG1 and IgG2b were the major subclasses in response to recombinant plasmid encoding extracellular domain of porcine endoglin vaccination. Anti-endoglin antibody-producing B cells were significantly increased in the spleens of mice immunised with recombinant plasmid encoding extracellular domain of porcine endoglin. In addition, mouse self-immunoglobulins were found deposited on the blood vessels of recombinant plasmid encoding extracellular domain of porcine endoglin-immunised tumour tissues. The similar anti-tumour activity was induced by the adoptive transfer of the purified immunoglobulins from the sera of mice immunised with recombinant plasmid encoding extracellular domain of porcine endoglin. Furthermore, angiogenesis was apparently inhibited within the tumour tissues from the recombinant plasmid encoding extracellular domain of porcine endoglin-immunised mice, and the vascularisation of alginate balls was also reduced in recombinant plasmid encoding extracellular domain of porcine endoglin-immunised mice. Most importantly, recombinant plasmid encoding extracellular domain of porcine endoglin could really induce cytotoxic T lymphocyte-mediated cytotoxicity and inhibit cell proliferation against endothelial cells. In addition, both CD4+ and CD8+ T lymphocytes took part in the function of inhibiting tumour growth and were synergistically responsible for induction of the anti-tumour activities. CONCLUSIONS: This approach may provide an alternative strategy for liver cancer immunotherapy.


Assuntos
Vacinas Anticâncer/farmacologia , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas Experimentais/terapia , Neovascularização Patológica/terapia , Plasmídeos/farmacologia , Vacinas de DNA/farmacologia , Transferência Adotiva , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Autoanticorpos/efeitos dos fármacos , Autoanticorpos/imunologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/toxicidade , Carcinoma Hepatocelular/imunologia , Proliferação de Células/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Endoglina , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Ensaio de Imunoadsorção Enzimática , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina G/imunologia , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Fígado/irrigação sanguínea , Neoplasias Hepáticas Experimentais/imunologia , Camundongos , Neovascularização Patológica/imunologia , Plasmídeos/toxicidade , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Vacinas de DNA/toxicidade , Vacinas Sintéticas/farmacologia , Vacinas Sintéticas/toxicidade
17.
Proc Natl Acad Sci U S A ; 103(17): 6694-9, 2006 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-16617103

RESUMO

West Nile (WN) virus is an important cause of febrile exanthem and encephalitis. Since it invaded the U.S. in 1999, >19,000 human cases have been reported. The threat of continued epidemics has spurred efforts to develop vaccines. ChimeriVax-WN02 is a live, attenuated recombinant vaccine constructed from an infectious clone of yellow fever (YF) 17D virus in which the premembrane and envelope genes of 17D have been replaced by the corresponding genes of WN virus. Preclinical tests in monkeys defined sites of vaccine virus replication in vivo. ChimeriVax-WN02 and YF 17D had similar biodistribution but different multiplication kinetics. Prominent sites of replication were skin and lymphoid tissues, generally sparing vital organs. Viruses were cleared from blood by day 7 and from tissues around day 14. In a clinical study, healthy adults were inoculated with 5.0 log(10) plaque-forming units (PFU) (n = 30) or 3.0 log10 PFU (n = 15) of ChimeriVax-WN02, commercial YF vaccine (YF-VAX, n = 5), or placebo (n = 30). The incidence of adverse events in subjects receiving the vaccine was similar to that in the placebo group. Transient viremia was detected in 42 of 45 (93%) of ChimeriVax-WN02 subjects, and four of five (80%) of YF-VAX subjects. All subjects developed neutralizing antibodies to WN or YF, respectively, and the majority developed specific T cell responses. ChimeriVax-WN02 rapidly elicits strong immune responses after a single dose, and is a promising candidate warranting further evaluation for prevention of WN disease.


Assuntos
Vacinas Virais/farmacologia , Vírus do Nilo Ocidental/imunologia , Adolescente , Adulto , Animais , Método Duplo-Cego , Feminino , Humanos , Macaca fascicularis , Masculino , Dados de Sequência Molecular , Testes de Neutralização , Segurança , Linfócitos T/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacocinética , Vacinas Atenuadas/farmacologia , Vacinas Atenuadas/toxicidade , Vacinas Sintéticas/genética , Vacinas Sintéticas/farmacologia , Vacinas Sintéticas/toxicidade , Vacinas Virais/genética , Vacinas Virais/farmacocinética , Vacinas Virais/toxicidade , Replicação Viral , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/prevenção & controle , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/fisiologia , Vírus da Febre Amarela/genética
18.
Vaccine ; 24(15): 2790-8, 2006 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-16448727

RESUMO

The absence of standard guidelines from National and International regulatory agencies for the safety evaluation of biotechnology products challenges the ingenuity of toxicologists. At present, the development of standard pre-clinical toxicology protocols for such products is on an individual case basis. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed DNA based anti-rabies vaccine in India. The test compounds were DNA rabies vaccine [DRV (100 microg)] and combination rabies vaccine (CRV (100 microg DRV and 1/50 dose of cell culture vaccine)), intended for clinical use by intramuscular route on 1, 7, 14 and 28 day. As per the regular mandatory requirements, the study has been designed to undertake acute (single dose--10 days), sub-chronic (repeat dose--28 days) and chronic (intended clinical dose--120 days) toxicity tests using three dose levels viz. therapeutic, average (2 x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in Swiss Albino mice. The selection of the rodent model viz. Swiss Albino mice is based on affinity and rapid higher antibody response during the efficacy studies. Apart from physical, physiological, clinical, hematological and histopathology profiles of all target organs, the tier-I immunotoxicity parameters have also been monitored. There were no observational adverse effects even at levels of 10x therapeutic dose administration of DRV and CRV. The procedure also emphasizes on the designing of protocols for the products developed by recombinant technique.


Assuntos
Vacinas Antirrábicas/toxicidade , Vacinas de DNA/toxicidade , Animais , Feminino , Masculino , Camundongos , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/efeitos adversos , Testes de Toxicidade Aguda , Testes de Toxicidade Crônica , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/toxicidade
19.
Vet Immunol Immunopathol ; 107(3-4): 281-90, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16002149

RESUMO

The synthetic anti-Boophilus microplus vaccine SBm7462 derived from the tick intestinal protein, Bm86, induced a protective immune response when emulsified in saponin and used in cattle. Using a mice model, and with the objective of improving the vaccine by continual peptide release, it was encapsulated in PLGA 50:50 microspheres and inoculated in BALB/c mice to assess the immunological response by detection of anti-peptide IgGs. Comparative studies were made with the peptide emulsified in saponin and with another synthetic vaccine, and the microsphere/peptide was characterized for efficiency of encapsulation, in vitro release profile, morphology, size, peptide integrity after encapsulation and stability in different pHs. The findings showed that saponin enhances a better immune response from SBm7462 and that the PLGA 50:50 microspheres are suitable for use with this peptide.


Assuntos
Ixodidae/imunologia , Vacinas Sintéticas/administração & dosagem , Animais , Biodegradação Ambiental , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Preparações de Ação Retardada , Feminino , Imunoglobulina G/biossíntese , Ácido Láctico , Camundongos , Camundongos Endogâmicos BALB C , Microesferas , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Controle de Ácaros e Carrapatos , Infestações por Carrapato/imunologia , Infestações por Carrapato/prevenção & controle , Infestações por Carrapato/veterinária , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/toxicidade
20.
Vaccine ; 23(39): 4775-84, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15961194

RESUMO

Ricin toxin is a plant-derived ribosome inactivating protein (RIP) of extraordinary toxicity. Vaccination using ricin toxoid or its A chain (RTA) is protective in animals but both vaccines have two potential toxicities, RIP and vascular leak syndrome (VLS). Previously we described three recombinant RTA constructs from which both toxicities were eliminated by site-specific mutations. One mutant, V76M/Y80A, RiVax, has now been further characterized for immunogenicity and toxicity in animals. We have found that RiVax is safe at doses of at least 8 mg in mice, 800-fold higher than the protective dose, and induces neutralizing antibodies in both mice and rabbits.


Assuntos
Ricina/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos/sangue , Anticorpos Monoclonais/imunologia , Feminino , Soros Imunes/imunologia , Masculino , Camundongos , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/toxicidade , Vacinas , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/toxicidade
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