RESUMO
In this work, we assessed the efficacy of an experimental intranasal vaccine against urinary-tract infections. The vaccine contained a recombinant truncated FimH (rFimHt) adhesin plus CpG oligodeoxynucleotides. The efficacy of the vaccine was compared with that of an intramuscular vaccine that was formulated with the same immunogen plus Freund's adjuvant. Our results show that serum immunoglobulin G titers of vaccinated animals were similarly enhanced in both cases. However, the intranasal vaccine elicited higher vaginal-wash-specific immunoglobulin A titers against rFimHt than the intramuscular route. Both vaccines reduced the in vivo colonization of the bladder by uropathogenic Escherichia coli more than 100-fold in a murine cystitis model. Our results indicate that a recombinant truncated FimH adhesin plus CpG oligodeoxynucleotides is a suitable immunogenic combination that can contribute to the development of a highly efficacious urinary tract infection vaccine.
Assuntos
Adesinas de Escherichia coli/imunologia , Adjuvantes Imunológicos/genética , Cistite/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Fímbrias/imunologia , Vacinação/métodos , Adesinas de Escherichia coli/genética , Administração Intranasal , Animais , Cistite/prevenção & controle , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Infecções por Escherichia coli/prevenção & controle , Feminino , Proteínas de Fímbrias/genética , Imunidade nas Mucosas/imunologia , Imunoglobulina A/análise , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vagina/imunologiaRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/síntese química , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Imuno-Histoquímica , Camundongos , Oligonucleotídeos/imunologia , Oligonucleotídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Testes de Precipitina , CoelhosRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
Assuntos
Animais , Coelhos , Ratos , Oligonucleotídeos/síntese química , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/imunologia , Testes de Precipitina , Imuno-Histoquímica , Western Blotting , Reação em Cadeia da Polimerase , Anticorpos Monoclonais/imunologiaRESUMO
Using synthetic peptides and a combinatorial library of 56 mer random oligonucleotides, we have developed reagents that behave as "synthetic antibodies". The results obtained with the protein phosphatase 2A as a model system are shown here. The specificity of these reagents, named "oligobodies", has been demonstrated by Western blot analysis and immunohistochemistry. The oligobodies have enormous advantages compared to antibodies: their production is independent of the immune system, they can be prepared in a few days and there is no need for a purified target protein. These reagents can be produced even if the corresponding protein was never isolated or purified, since only a partial DNA sequence from a database provides enough information to make them.