Heterogeneity in the capsid protein of bovine enteric caliciviruses belonging to a new genus.

Some bovine enteric caliciviruses form a new genus in the family Caliciviridae. In this study, Bayesian phylogenetic analysis of 31 full length capsid sequences from Europe, North America and Asia revealed that this new genus had four currently circulating lineages that showed both temporal and geographical distribution. These groupings were supported by the distribution of the frequency of pair-wise distances. However, the nucleotide and amino acid heterogeneity was low, with a maximum nucleotide and amino acid divergence of 16.7% and 8.4%, respectively. Most variability was found between amino acid residues 288 and 420 of the capsid protein and the sequence motifs observed in this region supported the division of the four lineages. Homology modelling using the structure of the San Miguel sea lion capsid indicated that most variation occurred in the predicted P2 domain and thus, may affect antigenic sites on the surface of the capsid of this newly described genus.

Envelope protein variability among HBV-Infected asymptomatic carriers and immunized children with breakthrough infections.

A detailed study of hepatitis B virus (HBV) surface variants and their role in breakthrough infections has been conducted in The Gambia, West Africa. Samples from 1856 vaccinated subjects were tested for hepatitis B surface antigen (HBsAg). Evidence of infection was found in 11% (22/192) of subjects with breakthrough infections and 18 (81.8%) were also positive for HBV DNA following PCR analysis. A cohort of 58 unvaccinated carriers which also included 11 patients with hepatocellular carcinoma was also investigated in order to establish the prevalence of surface variants in the unvaccinated population. Analysis of the S gene from HBV PCR-positive subjects (n = 64) revealed little variation in the S gene of these subjects. Twenty-four S protein sequences (37.5%) were identical and a further 22 sequences differed by only a single amino acid. The K141E variant found in previous work was not detected and little variation was observed in the immunodominant "a" determinant; a single change was found in one vaccinated patient (Q129H) and nine changes detected among six unvaccinated carriers. This study showed that breakthrough HBV infection in vaccinated Gambians is mainly caused by the wild type genoytype E strain and that immune escape mutants are uncommon. However, HBV mutants may play a role in establishing infection later in life when anti-HBs antibodies have begun to decline. Further investigation is required to determine the cause of these breakthrough infections and whether they contribute to the establishment of the carrier state.

DNA vaccination can protect Cyprinus Carpio against spring viraemia of carp virus.

Several DNA constructs containing the spring viraemia of carp virus (SVCV) glycoprotein (G) gene were investigated for their ability to induce protection against SVCV following injection into myofibres. The constructs were pooled into four groups and co-injected with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor. Group 1 contained one full-length and two truncated G constructs under the control of the cytomegalovirus (CMV) promoter. Group 2 contained full-length constructs with the CMV promoter, the simian virus 40 promoter and a muscle-specific promoter. Group 3 contained constructs in which the G-gene was fused with a second gene in order to improve secretion of the G-protein or to enhance destruction of transfected myocytes by T cells. Group 4 contained constructs with the CMV-Intron A promoter in plasmids with or without CpG motifs. A small-scale trial in goldfish showed that antibody responses in at least half the fish were induced by three injections of plasmids from Groups 1 and 3 whereas T-cell like responses with stimulation indices of above 3 were induced in at least half the fish by Groups 2 and 4. A single-dose of each plasmid mix was then used to protect carp in a large-scale trial. Following challenge with a heterologous strain of SVCV that killed 64% of fish, the strongest protection was observed in carp that received the full length G-gene expressed by two plasmids driven by the CMV-Intron A promoter (Group 4), with a relative percentage survival of 48% (p=0.00008).

Modulating immune responses with dendritic cells: an attainable goal in veterinary medicine?

Dendritic cells (DCs) are antigen presenting cells that potently modulate immune responses with varying outcomes depending on the DC sub-population involved. To understand how DC sub-types arise, it is necessary to determine which factors influence their differentiation. At least three major sub-populations of DCs have been described in mice: CD4+/CD8- "myeloid" DCs, CD4-/CD8+ "lymphoid" DCs and Langerhans cell-derived DCs. Whilst somewhat comparable populations have been described in man, in most other species very little is known. The identification of cytokines which stimulate proliferation of DC precursors, and the observation that the cytokine environment influences the phenotype and the function of the DCs that subsequently develop, has provided a useful tool for evaluating these rare cells. We describe the influence of cytokines on the phenotype of DCs generated in the rat. Using bone marrow cells as the source of precursors we generated "myeloid-type" DCs from the adherent population using granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4 and Flt-3L or "lymphoid-type" DCs from the non-adherent population using cytokines which included IL-7, IL-3, SCF and TNFalpha. In order to facilitate similar approaches to the study of equine DCs we have identified the nucleotide sequence encoding GM-CSF from the m-RNA of equine PBMC stimulated with Concanavalin A, amplified the cDNA by PCR and cloned it in eukaryotic and prokaryotic expression vectors. We report on the structure and function of this molecule.

Enhanced immunogenicity of a hepatitis B virus peptide vaccine using oligosaccharide ester derivative microparticles.

Controlled release microspheres can overcome many of the disadvantages of multiple vaccine delivery such as rate of uptake and cost of administration. Proteins and peptides are difficult to administer using conventional polymers owing to protein degradation, premature release and stability. Here we report the successful development of room temperature stable, controlled release formulations using oligosaccharide ester derivatives (OEDs) of trehalose and a synthetic peptide analogue of hepatitis B surface antigen. Employing a range of different OED preparations, we have optimised the immunogenicity of the peptide formulation such that mice injected with a single preparation of microspheres consisting of trehalose octaacetate (TR101; Group G) produce high titre anti-hepatitis B (anti-HBs) surface antigen antibodies. The kinetics of the immune response could be manipulated with different peptide/OED formulations and correlated with the OED composition of the microspheres. Our data demonstrate the considerable potential of OED microspheres as novel delivery systems for vaccines. The ability to induce strong immune responses, without the requirement for multiple doses or cold-chain storage, could radically improve vaccination programmes in developing countries.