- Volume 89, Issue 5, 2008

This study used a duck hepatitis B virus (DHBV) model to evaluate whether a novel DNA vaccination protocol alone or associated with antiviral (lamivudine) treatment was able to clear the intrahepatic covalently closed, circular viral DNA (cccDNA) pool responsible for persistence of infection. DHBV carriers received DNA vaccine (on weeks 6, 10, 13, 14, 28 and 35) targeting the large envelope and/or core proteins alone or combined with lamivudine treatment (on weeks 1–8) or lamivudine monotherapy. After 10 months of follow-up, a dramatic decrease in viraemia and liver DHBV cccDNA (below 0.08 cccDNA copies per cell) was observed in 9/30 ducks (30 %) receiving DNA mono- or combination therapy, compared with 0/12 (0 %) from lamivudine monotherapy or the control groups, suggesting a significant antiviral effect of DNA immunization. However, association with the drug did not significantly improve DHBV DNA vaccine efficacy (33 % cccDNA clearance for the combination vs 27 % for DNA monotherapy), probably due to the low antiviral potency of lamivudine in the duck model. Seroconversion to anti-preS was observed in 6/9 (67 %) ducks showing cccDNA clearance, compared with 1/28 (3.6 %) without clearance, suggesting a significant correlation (P<0.001) between humoral response restoration and cccDNA elimination. Importantly, an early (weeks 10–12) drop in viraemia was observed in seroconverted animals, and virus replication did not rebound following the cessation of immunotherapy, indicating a sustained effect. This study provides the first evidence that therapeutic DNA vaccination is able to enhance hepadnaviral cccDNA clearance, which is tightly associated with a break in humoral immune tolerance. These results also highlight the importance of antiviral drug potency and an effective DNA immunization protocol for the design of therapeutic vaccines against chronic hepatitis B.

The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

Variation in PrP prion gene sequence appears to modulate susceptibility to chronic wasting disease (CWD), a naturally occurring prion disease affecting four North American species of the family Cervidae. Wapiti (Cervus elaphus nelsoni) PrP is polymorphic at codon 132 [methionine (M) or leucine (L)]. We genotyped 171 samples, collected between 2002 and 2005 from CWD-infected and uninfected wapiti from three free-ranging populations in Colorado, USA, to study influences of PrP polymorphisms on CWD susceptibility further. Overall genotype frequencies for 124 apparently uninfected animals were 65.3 % MM132, 32.3 % ML132 and 2.4 % LL132; for 47 CWD-infected animals, these frequencies were 70.2 % MM132, 27.7 % ML132 and 2.1 % LL132. Surprisingly, our data revealed that, among recent (approx. 2002–2005) CWD cases detected in free-ranging Colorado wapiti, the three PrP codon 132 genotypes were represented in proportion to their abundance in sampled populations (P≥0.24) and all three genotypes showed equivalent susceptibility to infection.

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.