Protective immunity against herpes simplex virus generated by DNA vaccination compared to natural infection.

To evaluate the utility of plasmid DNA vaccination against disease caused by herpes simplex virus (HSV), we compared the strength of protection against lethal challenge following natural virus infection with that following vaccination with a plasmid encoding HSV glycoprotein gD (gD-DNA). We further determined the cellular basis of each type of protection using lymphocyte deficient knockout mice. Establishment of immunity to HSV using live virus immunization required CD8+ T cells and B cells, but not CD4+ or gamma/delta+ T cells, and was related to specific antibody levels; surprisingly, CD4 knockout mice had large quantities of IgG anti-HSV serum antibodies. Establishment of immunity to HSV using gD-DNA immunization approached the strength of that generated following sublethal infection, but was dependent on alpha/beta+ CD4+ T cells, CD8+ T cells, B cells, and even partially on gamma/delta+ T cells, and not strictly correlated with antibody levels.

Success and limitations of a naked plasmid transfection protocol for keratinocyte growth factor-1 to enhance cutaneous wound healing.

Our group and others have previously reported enhancement of cutaneous wound healing following the transfection of tissue with plasmid vectors expressing the DNA for growth factors. In these experiments, growth factor treated animals were usually compared to animals treated with control plasmid vector. To achieve consistent transfection, high DNA plasmid load and repeated penetrations of the wound by needle or gene gun were required. In the current experiments, we assessed the effect of the plasmid load and repeated tissue penetrations on wound healing of excisional wounds in diabetic C57 mice. Animals received 5 mm excisional wounds, and were assigned to the following groups, no treatment, phosphate buffered saline solution injections, and plasmid vector injection with and without the keratinocyte growth factor-1 gene. Intradermal injections of 100 microg plasmid were given adjacent to the wounds at days 1-5, 7 and 11. At day 9, wound closure was more advanced in keratinocyte growth factor-1 treated animals compared to those treated with control plasmid. But a detrimental effect of the DNA plasmid injection was evident from a comparison of the DNA control group versus the non-injected group. Therefore, the challenge for developing an effective system for the enhancement of wound healing lies in improving transfection efficiency.

Antibody response and protective capacity of plasmid vaccines expressing three different herpes simplex virus glycoproteins.

Plasmid expression vectors were constructed that contained the genes encoding herpes simplex virus 1 (HSV-1) glycoproteins C (gC), D (gD), and E (gE). Mice receiving two intramuscular injections of expression plasmid (50 microg) produced a specific HSV-1 antibody response. Mice receiving the gD plasmid were protected against a lethal intraperitoneal challenge of HSV-1 (5 x 10(4) pfu) but not against more demanding challenge doses. Protection with gC or gE plasmid vaccination could be demonstrated only if the inoculating dose of DNA was increased to 250 microg. In contrast, all mice immunized with vaccinia recombinants expressing either gC or gE survived HSV-1 challenge. Analysis of the HSV-1 antibody isotype produced by plasmid immunization revealed a response dominated by IgG2a. Combination delivery of all three glycoprotein expression plasmids provided better protection against lethal challenge, but mice receiving the combination were still not able to withstand increased challenge doses of virus.

In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats.

ABSTRACT Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.