Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies.

The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata.

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.