RESUMO
The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.
Assuntos
Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Febres Hemorrágicas Virais/prevenção & controle , Vacinas Virais/imunologia , Animais , Antígenos Virais/genética , Proteção Cruzada , Células Dendríticas/imunologia , Células Dendríticas/virologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/virologia , Febres Hemorrágicas Virais/imunologia , Febres Hemorrágicas Virais/virologia , Cavalos , Humanos , Replicon , Vacinação , Vacinas Atenuadas , Vacinas Sintéticas , Vacinas Virais/administração & dosagem , Vacinas Virais/biossínteseRESUMO
AIM: To obtain human recombinant 70 kDa heat shock protein (Hsp70) in baculovirus expression system and to study its antiviral activity. MATERIALS AND METHODS: Baculovirus expression system was used to obtain recombinant HSP70. Plasmid pFastBacHTb-Hsp70 containing sequence coding HSP70 gene with insertion of 6 histidine residues in protein reading frame was constructed. Competent cells MAX Efficiency DH 10 Bac were transfected with pFastBacHTb-Hsp70 plasmid with following extraction of recombinant bacmid Bac-Hsp70. In order to obtain baculovirus expressing HSP70, Sf-9 cells were transfected with Bac-Hsp70 bacmid. Hsp70 extraction and purification was performed with column metal-chelating affinity chromatography using Ni2+ ions. Protective efficacy of recombinant human HSP70 was estimated using model of Venezuelan equine encephalitis (VEE) in mice. RESULTS: Recombinant bacmid Bac-Hsp70 was constructed based on Bac-to-Bac expression system. Baculovirus expressing human HSP70 have been produced after transfection of Sf-9 cells with Bac-Hsp70 bacmid. Cultivation of recombinant baculovirus in Sf-9 cells and application of metal-chelating affinity chromatography allowed to extract purified fraction of HSP70. Experiments on mice infected with VEE virus demonstrated significant protection from death after administration of HSP70 in dose 15 mcg/mice. CONCLUSION: Application of baculovirus expression system and insect cell line for accumulation of recombinant baculoviruses in combination with Ni(2+)-mediated metal-chelating affinity chromatography allowed to obtain highly purified human recombinant HSP70 with marked antiviral activity.
Assuntos
Antivirais/farmacologia , Baculoviridae , Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina Venezuelana/prevenção & controle , Proteínas de Choque Térmico HSP70/farmacologia , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Perspectives of using reverse genetics methods for constructing of recombinant influenza virus strains acceptable for use as live attenuated vaccines are discussed. Using of attenuated NS-vectors of influenza virus opens possibilities for the development of recombinant vaccines with optimal ratio of immunogenicity and safety. Reverse genetics is applicable for development of effective vaccines against new pathogens such as highly pathogenic avian influenza A/H5N1.
Assuntos
Clonagem Molecular/métodos , Vírus da Influenza A/genética , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Proteínas não Estruturais Virais/genética , Animais , Vetores Genéticos/genética , Humanos , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas não Estruturais Virais/imunologiaRESUMO
As a result of long-standing screening of chemopreparations of anti-rickettsiosis action in-vitro and in-vivo experiments and their clinical evaluation in the treatment of R. of any etiology, it is established that the most effective ones are those of the 4 groups: tetracyclines (doxicycline, minocyclines), ansamicines (rifampicine), ftorchinolones (cyprofloxacine, ofloxacine, pefloxacine) and the new generation of macrolides (asitromicine, klaritromicine). Since all the studied preparations have only rickettsiosis-static effect, a promising way to finding a rickettsiocidic preparation capable of preventing a possible persistence of a relevant agent in a human body is a combined application of highly effective chemopreparations (doxicycline, in particular) or pefloxacine with lysosomal alkalinizing drugs (chlorochin, ammonium chloride or amantadine). Antibiotics of prolonged anti-rickettsiosis action give a real chance for prevention even with a one-time dose.
Assuntos
Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/prevenção & controle , Animais , Antibacterianos/uso terapêutico , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , HumanosRESUMO
The informative indices of antibiotic efficacy in the treatment of typhus were determined in experiments on guinea pigs and used for the design of an integral index of a drug prophylactic activity against Rickettsia prowazekii. A procedure for a comprehensive estimation of an antibiotic prophylactic efficacy in experimental typhus by the whole complex of clinical, immunological and rickettsiological properties is described.