ABSTRACT
We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 +/- 8 and 86 +/- 6% at 7 and 21 days, respectively, after monocrotaline (P < 0. 05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.
Subject(s)
Plasmids , Pulmonary Circulation/physiology , Transfection/methods , Vasculitis/physiopathology , Animals , Blotting, Southern , Chloramphenicol O-Acetyltransferase/genetics , Female , Gene Expression , Genes, Reporter , Liposomes , Mice , Mice, Inbred ICR , Mice, Transgenic , Monocrotaline , Pulmonary Circulation/drug effects , RNA, Messenger/analysis , Transgenes/genetics , Vasculitis/chemically inducedABSTRACT
Feline Infectious Peritonitis Virus (FIPV) is a coronavirus that induces an often fatal, systemic infection in cats. Various vaccines designed to prevent FIPV infection have been shown to exacerbate the disease, probably due to immune enhancement mediated by virus-specific immunoglobulins against the outer envelope (S) protein. An effective vaccine would be one that induces cell-mediated immunity without disease enhancing antibodies. In this report, we describe the use of a recombinant raccoon poxvirus that expresses the gene encoding the nucleocapsid protein of FIPV (rRCNV-FIPV N) as an effective vaccine against FIPV-induced disease. Cats were parenterally or orally vaccinated twice, three weeks apart. Cats were then orally challenged with Feline Enteric Coronavirus (FECV), which induces a subclinical infection that can cause enhancement of subsequent FIPV infection. Three weeks later, cats were orally challenged with FIPV. The FIPV challenge induced a fatal infection in 4/5 (80%) of the controls. On the other hand, all five cats vaccinated subcutaneously with rRCNV-FIPV N showed no signs of disease after challenge with FIPV. Four of the five subcutaneous vaccinates survived an additional FIPV challenge. Vaccination with rRCNV-FIPV N induced serum IgG antibody responses to FIPV nucleocapsid protein, but few, if any, FIPV neutralizing antibodies. In contrast to the controls, protected vaccinates maintained low FIPV serum neutralizing antibody titers after FIPV challenge. This suggests that the protective immune response involves a mechanism other than humoral immunity consisting of FIPV neutralizing antibodies.
