Safety and efficacy of an E2 glycoprotein subunit vaccine produced in mammalian cells to prevent experimental infection with bovine viral diarrhoea virus in cattle.

Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12 months were vaccinated, twice with a 4 week interval, with either a tE2 subunit vaccine (n = 8), a whole virus inactivated vaccine (n = 8) or left untreated as negative control group (n = 8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.

The fractal structure of polycation-DNA complexes.

We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.

Prediction of size distribution of lipid-peptide-DNA vector particles using Monte Carlo simulation techniques.

Concerns with insertional mutagenesis for retrovirus and immunogenicity for adenovirus have motivated research into development of non-viral vectors that can safely deliver desired gene constructs to target cells in tissues and organs. Many non-viral vectors suffer from unacceptably poor in vivo cell transfection and low transgene expression. Evidence suggests that cell transfection is linked to particle size - vector particles below about 200 nm are considered desirable. Experimental measurements indicate, however, that vector particles are susceptible to significant aggregation under most conditions of pH and ionic strength, including physiological conditions, although there are currently no means of predicting the kinetics of aggregation. The present paper addresses this challenge by presenting a mathematical framework based on the Monte Carlo simulation techniques for modelling the dynamics of aggregation. The approach is used to simulate the evolution of particle-size distribution for an integrin-targeting lipid-peptide-DNA vector system in buffers of different pH and ionic strength. The simulations required two input parameters, including the initial-size distribution of the particles and a fitting parameter (alpha). Comparison of simulations with experimental data showed that alpha was closely related to the zeta potential of the particles in the buffer medium, making simulations fully predictive. The modelling approach may be used in other vector systems.