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Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(d,l-lactic-co-glycolic acid) microparticles of less than 10 μm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01+ rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals.

Orthogonal subspace signal processing algorithms (OSPA) have been developed to extract the optical thickness of a porous silicon layer to within one part in 10 5 from its reflectivity spectrum. This is equivalent to a limit of detection (LOD) of -40 pm change in optical thickness for a 3 μm thick layer, or an LOD of 1/2000 of a monolayer coverage with antibodies, of molecular weight 160 k Daltons, within a layer with pores of 100 nm diameter. A large molecule {horseradish peroxidase (HRP), MWt 40 kDa} has been detected at a concentration of 1 μg/ml by measuring its direct binding to anti-HRP antibodies immobilised within a porous silicon layer. A competitive assay has been demonstrated for the detection of a small molecule {2, 4, 6 trinitrotoluene (TNT), MWt 227 Da} at 10 μg/ml. The projected LODs for HRP and TNT by these assays are 50 ng/ml and 1 μg/ml respectively.

The sensitivity of the optical reflectivity of porous silicon structures to the refractive index of liquid within the pores is studied for a single layer, a Bragg mirror and a microcavity. Sucrose solutions of concentration in the range 0.05 to 1.0% by weight are introduced into the pores within a flow cell in order to change the refractive index of the liquid in the pores from 1.3330 to 1.3344. Optimum wavelengths for detection via reflectivity changes are determined based on a signal to noise analysis. The optical thickness of the single layer is also monitored by measuring the fringe spacing via a Fourier transform technique. It is just possible to detect the effect of a change in refractive index of liquid in the pores of 0.00007 for both the reflectivity and optical thickness approaches.

To use porous silicon as an optical interferometric biosensor, the pores must be sufficiently large to allow easy ingress of reagents and the layer must also display Fabry-Perot optical cavity modes. Here the detection antibody is rabbit IgG and the analyte is α-rabbit IgG conjugated to horseradish peroxidase (HRP). For this model system, the pores should be >50 nm in diameter. Such diameters have been obtained in 0.05 Ω cm n-type silicon using anodisation followed by chemical etching in ethanolic KOH and also by anodising 0.005 Ω cm p-type material. The latter also displays optical cavity modes. The silicon surface is oxidised in ozone, silanised using aminopropylmethoxysilanes with one, two or three methoxy groups, and cross linked to IgG using glutaraldehyde. High specific binding is found for mono-, di- and tri-methoxy silanes, but the lowest non-specific binding is found for silanisation with the tri-methoxy silane.

An optimised water-in-oil-in-water double emulsion process for the microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic acid) (PLGA) was used to prepare microparticles from a range of different PLGA formulations. This process has been developed using pharmaceutically accepted solvents and is potentially scaleable. Incorporation of plasmid DNA in the microparticles of up to 11 microg/mg was obtained and the retention of plasmid DNA integrity was considerably greater than previously reported. Microparticle structure was determined, by scanning electron microscopy, to be hollow and size distribution characteristics were found to be independent of polymer formulation. The ability to vary the plasmid DNA release profile by changing the PLGA formulation and polymer concentration used in the encapsulation process was also demonstrated. This ability to control the release profile of the microparticles was shown to be especially important as the physical integrity of the encapsulated plasmid DNA was found to deteriorate with extended release times in vitro.

Protective immune responses in mice were obtained after oral immunization with rotavirus DNA vaccines encapsulated in poly(lactide-co-glycolide) (PLG) microparticles. The DNA vaccines used encoded outer capsid proteins VP4 and VP7; proteins that are the basis for rotavirus serotyping and the generation of virus neutralizing antibodies. One dose of vaccine was given to BALB/c mice by oral gavage (75 μg DNA/mouse). Rotavirus-specific serum antibodies and intestinal IgA antibodies were detectable by 6 weeks postimmunization. After challenge with homologous murine rotavirus at 12 weeks postimmunization, fecal rotavirus antigen was reduced significantly in immunized mice compared with controls. Protective immunity also was generated by oral delivery of unencapsulated VP 7 DNA vaccine but to a lesser degree. These results demonstrate that the oral route is effective for generating protective immune responses with rotavirus DNA vaccines targeting neutralization antigens.