RESUMO
The highly immunogenic glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) is a very important element for entry of this virus into host cells. These characteristics have made this protein a very interesting HSV-2 subunit vaccine candidate. Despite efforts to prevent genital herpes using gD-based subunit vaccines, to date, clinical trials using this antigen have failed. Therefore, using a small animal model, we sought to determine if a tetramerized truncated form of gD subunit vaccine, produced by recombinant baculovirus infected insect larvae, would elicit better protection against genital herpes than a monomeric gD-2 subunit vaccine. Three out of 5 mice immunized with the tetramerized antigen produced in a baculovirus expression vector system, survived a lethal challenge with a wild type HSV-2 strain (for more than 3 weeks after challenge). In contrast, all the mice (5) immunized with the truncated protein, produced by the same methodology, died within 2 weeks after challenge. These results suggest that multimerization (increasing the structural complexity) of the truncated gD antigen might be more likely protective than the monomer form. Also the use of an alternative cost-efficient eukaryotic expression system is described.
Assuntos
Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor p53/genética , Proteínas do Envelope Viral/genética , Animais , Baculoviridae/genética , Escherichia coli , Feminino , Larva , Camundongos , Camundongos Endogâmicos BALB C , Mariposas , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Cultura de VírusRESUMO
Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreich's ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green fluorescent protein expression driven from the same vector but by the strong HSV-1 IE4/5 promoter was transient. Our data demonstrate for the first time sustained transgene expression in vivo by infectious delivery of a genomic DNA locus >100 kb in size. Such an approach may be suitable for gene rescue strategies in neurological disease, such as FRDA.
Assuntos
Cerebelo , Ataxia de Friedreich/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Proteínas de Ligação ao Ferro/administração & dosagem , Animais , Células Cultivadas , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/genética , Escherichia coli , Ataxia de Friedreich/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 1 , Imuno-Histoquímica , Proteínas de Ligação ao Ferro/genética , Óperon Lac/genética , Camundongos , Microscopia de Fluorescência , Transgenes/genética , FrataxinaRESUMO
Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.