Partial complementation between the immediate early proteins ICP4 of herpes simplex virus type 1 and IE180 of pseudorabies virus.

We previously described that the immediate early (IE) IE180 protein of PRV can down-regulate the transactivation of the ICP4 promoter of HSV-1, and that the d120 virus (an ICP4-deficient HSV-1 strain) can partially replicate its viral DNA in the presence of the IE180 protein. Herein, we demonstrate that this partial complementation of d120 by IE180 is sufficient for transcription of ß, γ1 and γ2 products such as DNA pol, VP16 and gC, respectively. However, expression levels are low for VP16 and even lower for the gC, such that IE180 is unable to fully substitute for ICP4 functionally. Viral progeny was not detected in PK15 cells expressing PRV IE180.

Expression and characterization of the gD protein of HSV-2 fused to the tetramerization domain of the transcription factor p53.

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.

Infectious delivery and long-term persistence of transgene expression in the brain by a 135-kb iBAC-FXN genomic DNA expression vector.

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.

Insect larvae biofactories as a platform for influenza vaccine production.

Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-insect larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect larvae produced four-fold more HA protein than insect cells per biomass unit (1 g of fresh larvae weight). A single infected Trichoplusia ni larva produced up to 113 µg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2×10(8) Sf21 insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on insect larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E.

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.

Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus.

Recombinant pseudorabies viruses (PRVs) gIS8 and N1aHTK were constructed by the insertion of a chimeric gene (alpha4-TK) from herpes simplex virus type 1 (HSV-1) into wild-type PRV. HSV-1 TK expression by these recombinant viruses resulted in enhanced sensitivity to ganciclovir, compared to that of the wild-type PRV, and was similar to the sensitivity shown by HSV-1. Infection with gIS8 or N1aHTK recombinant viruses led to expression of HSV-1 TK mRNA as an immediate-early (IE) gene, observed by downregulation of the HSV-1 alpha4 promoter. This negative regulation was due to a PRV IE protein, IE180. IE180, however, does not have all the regulatory functions of the infected-cell protein ICP4, as it does not restore the growth of ICP4-deficient HSV-1 mutants.