High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle.

Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.

Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens.

Two recombinant herpesviruses of turkey (HVT) expressing the VP2 protein of infectious bursal disease virus (IBDV or Gumboro disease virus) have been constructed: vHVT001 and vHVT002. The VP2 open reading frame was inserted at the locus of the small subunit of ribonucleotide reductase gene (HSV-1 UL40 homolog) without any exogenous promoter in vHVT001 and at the locus of gl gene (HSV-1 US7 homolog) under the control of the human cytomegalovirus immediate-early promoter in vHVT002. The isolation of these recombinant viruses indicated that the deleted genes were not required for replication of HVT in chicken embryo fibroblasts. Efficacy of these recombinant viruses against IBDV strain 52/70 and Marek's disease virus (MDV strain RB1B) virulent challenges was evaluated in chickens vaccinated at 1 day of age. In the IBDV challenge, a good protection against mortality and bursal gross lesion was observed in vHVT002-vaccinated chickens: 100% with 10(5) PFU dose and 60% with 10(4) PFU dose; in contrast, only a weak level of protection was achieved after vaccination with vHVT001. Protection levels against MDV challenge obtained with vHVT001 and vHVT002 were low (around 10%) compared to that induced by the parental HVT (84%). In spite of the low protection level against MDV, this is the first report which describes induction of full protection against IBDV with a single inoculation of a recombinant virus.

Localization of antigenic sites of the S glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement.

The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement. To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation. Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs). A region spanning residues 509 to 673 reacted with most MAbs tested. Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs. To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced. Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus. Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus. Site A2 contains residues 643, 649, and 656. Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing. The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement.

The complete sequence and gene organization of the short unique region of herpesvirus of turkeys.

The DNA sequence of the whole of the short unique region (U(s)) and that of part of the short terminal repeat (TRs) of herpesvirus of turkeys (HVT) were determined. HVT U(s) is 8.6 kbp long and contains eight potential open reading frames (ORFs). Seven of these have counterparts in the U(s) of herpes simplex virus type 1 (HSV-1). The homologous proteins include US1, US2, US10, protein kinase (US3) and the glycoproteins gD, gI and gE. In addition, HVT contains one ORF which has a counterpart in the U(s) of Marek's disease virus (MDV) but is not homologous to any other known herpesvirus gene. Although HVT and MDV proteins encoded by U(s) genes have evident similarities with proteins encoded by alphaherpesviruses, multiple alignment analysis of predicted amino acid sequences show that HVT proteins are more closely related to MDV proteins than to homologous proteins of mammalian alphaherpesviruses. The percentage amino acid identity between HVT and MDV U(s)-encoded proteins ranges from 35 to 65, the most conserved protein being encoded by the homologues of the HSV-1 US2 gene. Most of the genes are colinear with those of HSV-1 except US10 which is transposed in HVT and MDV. A characteristic feature of HVT is the fact that approximately two-thirds of the gE gene is located in the inverted repeats flanking U(s).