RESUMO
Porphyromonas gingivalis is a key periodontal pathogen that has been implicated in the aetiology of chronic adult periodontitis. The aim of this study was to characterize two potential vaccine candidates (PG32 and PG33) identified from a previous genomic sequence analysis. Gene knockout studies suggested that these proteins play an important role in bacterial growth and are transcriptionally linked. Analysis of 14 laboratory and clinical isolates of P. gingivalis found that in all strains, both genes were present with a high level of conservation and that the two proteins were also expressed in vitro. Truncated recombinant PG32 and PG33 proteins were produced in Escherichia coli in an attempt to increase the solubility of the proteins while retaining their native conformation. While most of the truncated proteins remained insoluble, two truncated proteins showed good solubility and high levels of protection in the P. gingivalis murine lesion model and may be considered as potential vaccine candidates for further testing in models of human periodontal disease.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Porphyromonas gingivalis/imunologia , Substâncias Protetoras/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Sequência Conservada , Modelos Animais de Doenças , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Vetores Genéticos , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Porinas/imunologia , Porinas/isolamento & purificação , Porphyromonas gingivalis/genética , Proteínas Recombinantes , Solubilidade , Transformação Genética/genética , Vacinas Acelulares/imunologiaRESUMO
Various formulations of the Plasmodium falciparum merozoite surface antigen, MSA-2, were made and tested in animals in order to select one for use in human vaccine trials. Recombinant constructs representing both major allelic forms of MSA-2 were formulated with a range of adjuvants and used to immunize rabbits, mice and sheep. After immunization, antibody responses obtained with the most potent adjuvants were at least tenfold greater than responses obtained with the least potent adjuvant Alhydrogel, which was used as the reference standard, although its lower potency indicated against its further use in clinical trials. Based on broadly similar results obtained with the three animal species, several adjuvants, including the water-in-oil adjuvant Montanide ISA 720, the oil-in-water adjuvant SAF-1, and liposomes containing lipid A formulated with Alhydrogel were demonstrated to be potent and potentially suitable for the clinical evaluation of MSA-2 as a candidate malaria vaccine antigen. Of these, ISA 720 was selected for further trial.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antígenos de Protozoários , Vacinas Antimaláricas/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Antígenos de Superfície/imunologia , Avaliação Pré-Clínica de Medicamentos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Plasmodium falciparum/imunologia , Coelhos , Ovinos , Especificidade da EspécieRESUMO
Saimiri sciureus boliviensis monkeys were immunized with the Plasmodium fragile form of the merozoite apical membrane antigen-1 produced using the baculovirus expression system and combined with Montanide ISA 720 adjuvant. Following three immunizations, monkeys were challenged with 10,000 P. fragile trophozoite parasites. Antibody titers determined by fluorescence microscopy indicated an enhanced response following the second immunization. Four of five control animals had parasite counts > 5% 18-26 days following challenge. Four of five immunized monkeys had reduced levels of maximum parasitemia or delays in accumulated parasite counts, suggestive of protection. Rechallenge of the animals with P. falciparum resulted in three of four adjuvant control animals developing patent parasitemia whereas none of five immunized animals were infected, suggesting some level of heterologous protection.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas , Malária/prevenção & controle , Proteínas de Membrana/imunologia , Plasmodium/imunologia , Proteínas de Protozoários/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , DNA de Protozoário/sangue , Modelos Animais de Doenças , Imunofluorescência , Imunização , Imunização Secundária , Vacinas Antimaláricas/efeitos adversos , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Proteínas de Membrana/genética , Parasitemia/prevenção & controle , Plasmodium/genética , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saimiri , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genéticaRESUMO
Saimiri sciurus monkeys were immunized at multiple sites with recombinant vaccinia viruses expressing Plasmodium falciparum antigen genes and boosted 4 weeks later. Control monkeys were immunized with a thymidine kinase-negative vaccinia virus mutant. Two weeks later, all of the monkeys were challenged by intravenous inoculation of P. falciparum (Indochina strain) parasites. A group of unimmunized monkeys was challenged in parallel. All of the monkeys that received vaccinia virus recombinants or the control virus produced good anti-vaccinia virus antibody responses. However, those that received a single construct containing ring-infected erythrocyte surface antigen (RESA) given at eight sites did not produce significant antibody to any of the three major RESA repeat epitopes after immunization but were primed for an enhanced antibody response after challenge infection with P. falciparum. Most of the monkeys produced detectable antibodies to the RESA epitopes after challenge infection. One group of monkeys was immunized with four constructs (expressing RESA, two merozoite surface antigens [MSA-1 and MSA-2], and a rhoptry protein [AMA-1]), each given at two sites. While these monkeys failed to produce significant antibody against MSA-2 or AMA-1 after immunization, they produced enhanced responses against these antigens after challenge infection. Immunization involved an allelic form of MSA-2 different from that present in the parasite challenge strain, so that the enhanced responses seen after challenge infection indicated the presence of T-cell epitopes common to both allelic forms. No groups of monkeys showed any evidence of protection against challenge, as determined by examination of the resulting parasitemias.