DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy.

A generic approach to inducing high level CD8+ T cell responses would be of value for prophylactic and therapeutic immunisation against several infectious diseases. However, it has been very difficult to achieve such immune responses using available vaccination strategies. Malaria is one of several diseases against which a new generation of better CD8+ T cell-inducing vaccines might be useful and is unusual in that it allows assessment of vaccine efficacy in small numbers of volunteers in carefully controlled challenge studies. Here we review the identification of a heterologous prime-boost regime using DNA priming and recombinant modified vaccinia Ankara (MVA) boosting that induces high level T cell responses in both mice and non-human primates. Clinical trials to determine whether this prime-boost approach is immunogenic in humans are in progress.

Induction of CD8+ T cells using heterologous prime-boost immunisation strategies.

One of the current challenges in vaccine design is the development of antigen delivery systems or vaccination strategies that induce high protective levels of CD8+ T cells. These cells are crucial for protection against certain tumours and intracellular pathogens such as the liver-stage parasite of malaria. A liver-stage malaria vaccine should therefore include CD8+ T-cell-inducing components. This review provides an overview of prime-boost immunisation strategies that result in protective CD8+ T-cell responses against malaria with an emphasis on work from our laboratory. Possible mechanisms explaining why heterologous prime-boost strategies, in particular boosting with replication-impaired recombinant poxviruses, are so effective are discussed.

Percolation phenomenon of calcium bis(2-ethylhexyl) sulfosuccinate water-in-oil microemulsions by conductivity and dielectric spectroscopy measurements.

The sodium counterion (Na+) of the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) surfactant was exchanged with calcium Ca2+ to investigate the counterion charge effect on the structure of water in normal decane microemulsions. Ohmic conductivity and dielectric permittivity measurements were performed on samples at constant water to surfactant mole ratio [water]/[Ca(AOT)(2)]=26.6. Increasing the volume fraction of the dispersed phase phi, a percolation phenomenon was observed at the constant temperature of 25 degrees C. The percolation threshold was found at phi approximately 15% by Ohmic conductivity and static dielectric permittivity studied as a function of phi, and by the frequency dependence of the complex permittivity. Critical exponents typical of the static percolation mechanism (formation of bicontinuous microemulsions) were found below and above threshold. The comparison of these results obtained for the two different counterions, Ca2+ and Na+, in AOT surfactant water in normal decane microemulsions allows detection of an important difference. The percolation below threshold is dynamic for the sodium-based microemulsions, accounting for the formation of clusters of droplets, whereas calcium-based microemulsions show a static percolation. For this system, the coalescence of droplets begins to occur below threshold at phi approximately 12%.

Interaction of a synthetic peptide based on the neutrophil-derived antimicrobial protein CAP37 with dipalmitoyl-phosphatidylcholine membranes.

CAP37, a cationic antimicrobial protein of Mr 37 kDa is constitutively expressed in human neutrophils. A synthetic peptide, CAP37 P20-44, corresponding to amino acid residues 20 through 44 of the native CAP37 molecule has been shown to mimic the antimicrobial activity of the native protein. An analog of peptide CAP37 P20-44 was synthesized in which the cysteine residues at positions 26 and 42 were replaced with serine residues (CAP37 P20-44Ser). This resulted in a peptide that no longer exhibited bactericidal activity. The effect of different concentrations of the active CAP37 peptide, CAP37 P20-44, and its inactive analog, CAP37 P20-44Ser, on artificial lipid membranes composed of dipalmitoyl phosphatidylcholine (DPPC) was studied using small-angle X-ray scattering and differential scanning calorimetry. The results indicated that CAP37 P20-44 perturbs the periodicity of the lamellar structure as shown by small angle X-ray diffraction, while the effect of the inactive peptide is not as strong. Differential scanning calorimetry further confirms that CAP37 P20-44 interacts with lipid membranes as indicated by increased width of the transition and decreased peak height. Moreover, it completely abolishes the pretransition temperature of the DPPC membranes. The effect of the inactive peptide, CAP37 P20-44Ser on the thermotropic properties of DPPC was small. These studies suggest that CAP37 perturbs the lamellar structure of lipid bilayers and further suggests that the antibiotic action of the molecule may be through its interactions with the lipid components of the Gram negative bacterial membrane.

The binding site of NK receptors on HLA-C molecules.

The protection of cells expressing class I HLA molecules from NK lysis is mediated by natural killer cell inhibitory receptors (NKIR). Using site-directed mutagenesis, residues on HLA-C that determine the locus specificity (alphaVal-76), allotype group specificity (a dimorphism alphaAsn-80/Lys-80), and affinity of NKIR binding (a second pair of dimorphisms, alphaAla-73, Asp-90 or alphaThr-73, Ala-90) have been identified. Thus the "footprint" of the NKIR on the alpha1 helix of the class I MHC molecule HLA-C and its associated beta strands are similar in position to the site occupied by superantigens on and behind the alpha1 helix of the class II MHC molecule HLA-DR1, but further toward its C-terminus. The intermediate affinity binding of NKIR to HLA-C, determined by alpha73 and alpha90, has an essential role in preventing cross-reactivity and ensuring the availability of NK cells for immunosurveillance; low affinity and high affinity mutants are both physiologically impaired.

Human NK cells: their ligands, receptors and functions.

The expression, or lack thereof, of class I MHC glycoproteins has a marked influence on natural killer cell function. Cells which do not express class I MHC molecules are susceptible to lysis by NK cells, and transfection of these targets with class I MHC genes can render these cells resistant to NK attack. This inhibition of NK-killing is mediated by a novel family of receptors expressed mainly on NK cells, but also found on some T-cells. The function of these class I MHC binding receptors when expressed on T-cells is discussed also and a novel co-stimulatory activity of NKAR described. Lastly, a novel mechanism by which human cytomegalovirus evades immune surveillance by NK cells is documented.

Enhancement of class II-restricted T cell responses by costimulatory NK receptors for class I MHC proteins.

An important feature of the human immune system is the ability of T cells to respond to small quantities of antigen. Class II major histocompatibility complex (MHC)-restricted T cells that expressed a costimulatory natural killer (NK) cell receptor for class I MHC proteins were cloned. In the presence of low doses of superantigen, the proliferative response of these T cell clones was three- to ninefold greater when the T cells were costimulated by way of the NK receptor. Thus, the action of costimulatory NK receptors on T cells may play a significant role in initiating and sustaining immune responses.

Small-angle neutron scattering studies of protein-reversed micelle complexes.

Enzymes solubilized in organic solvents, hosted within the polar cores of surfactant aggregates, known as reversed micelles, provide many unique opportunities for new biocatalytic synthesis and protein separation processes. Small-angle neutron scattering (SANS) studies have shown that insertion of the protein cytochrome-c in the reversed micelle polar core causes a significant reapportioning of the surfactants and water between the filled and unfilled micelles, leading to an increase in overall size of the filled micelles relative to their empty counterparts. The simple shell and core model assuming single occupancy of the reversed micelles has significant limitations in interpreting data for high protein loadings, and points to the need for more detailed characterization of the protein-reversed micelle interactions.