Present recommendations for the management of alpha interferon-based treatment in patients with chronic hepatitis C virus (HCV) infection are based on HCV RNA measurements before, during, and after antiviral therapy (6,22). Changes in HCV RNA serum concentrations during the early phase of interferon-based therapy have been analyzed based on complex models of viral kinetics and applied to the prediction of treatment outcomes (13,16,31). In different studies, a high predictive value for virologic nonresponse (98 to 100%) was observed for HCV genotype 1-and genotype 4-to 6-infected patients, with a decline in the HCV RNA serum concentration of less than 2 log steps between baseline and week 12 of (pegylated) alpha interferon-ribavirin combination therapy (3,5,8). Alternatively, an absolute HCV RNA concentration above 30,000 IU/ml may be used for decisions about early treatment discontinuation at week 12 (3). In addition, for genotype 1-and genotype 4-to 6-infected patients at week 24 of treatment, it is recommended that therapy be discontinued on the basis of detectable HCV RNA in serum by qualitative PCR-based assays (3,5,19). Recently, for patients infected with genotype 2 or 3, the HCV RNA concentration at baseline and viral decline at week 4 have been described as highly predictive for virologic response to pegylated alpha interferonribavirin combination therapy (4,18,30,32). Furthermore, for assessment of virologic response to currently developed direct antiviral drugs (i.e., protease and polymerase inhibitors), proper HCV RNA quantification for the different HCV genotypes is critical (1,14,25,26). WHO HCV international standard, 96/790 (2,7,11,17,23,24,27). However, continuing limitations are the lack of complete automation (22), the necessity for dilutions for quantification by standard PCR-based assays (6), the relatively low sensitivity of quantitative HCV RNA assays (31), and the need for different test systems for qualitative and quantitative HCV RNA measurements (31). Furthermore, standardization of results to IU are mainly based on HCV genotype 1 panels, and little is known about the variability of commercially available HCV RNA assays for quantification of different HCV genotypes.Real-time PCR methods for the quantification of HCV RNA have the advantage of linear amplification over a broad dynamic range, together with an integrated, automated detection system. With efficient HCV RNA extraction, they have the * Corresponding author. Mailing address:

The nucleotide sequence of the gene (pac) coding for penicillin G acylase from E. coli ATCC 11105 was determined and correlated with the primary structure of the two constituent subunits of this enzyme. The pac gene open reading frame consists of four structural domains: Nucleotide positions 1-78 coding for a signal peptide, positions 79-705 coding for the alpha subunit, positions 706-867 coding for a spacer peptide, and positions 868-2538 coding for the beta subunit. Plasmids were constructed which direct the synthesis of a pac gene product lacking the signal peptide, and the synthesis of the alpha subunit or the beta subunit. The following results were obtained: The two dissimilar subunits are processing products of a single precursor polypeptide; the spacer peptide is removed during processing; the precursor polypeptide lacking the signal sequence is accumulated in the cytoplasm; it is not processed proteolytically in the cytoplasm and it does not display enzyme activity. Processing, therefore, requires translocation through the cytoplasmic membrane; processing follows a distinct sequential pathway in vitro.

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The two constituent subunits of the enzyme penicillin acylase from Escherichia coli strain ATCC 11 105 are derived from a single precursor polypeptide by post-translational processing. Mutant penicillin acylase precursors were constructed carrying insertions and deletions in various domains and they were analysed for their processing behaviour. It was found that an endopeptide region of appropriate size and an intact C-terminus were absolutely necessary for the maturation process. Internal deletions within the b-subunit domain also prevented post-translational cleavage. Processing competence, therefore, was not merely determined by the amino acid sequence in the vicinity of the processing sites but relied on a correct overall conformation of the protein. The processing pathway in vivo proceeds via an intermediate comprising the a subunits plus endopeptide and is thus identical to the pathway which has been determined previously by in vitro analysis. The post-translational modification of the precursor is probably not carried out by a specific processing enzyme(s) as the heterologous expression of the penicillin acylase (pac) structural gene yielded processed and active enzyme in different enterobacteria and in a Pseudomonas speciesThe derivation of two polypeptides from a single gene product can be achieved by proteolytic processing of a precursor polypeptide. Whereas this mechanism is commonly involved in the formation of viral proteins or eucaryotic polypeptide hormones [l], it is rarely found in bacteria. The synthesis of penicillin acylase (penicillin G amidohydrolase EC 3.5.1.11) from Escherichia coli strain ATCC 11105 is one of the few examples known which occurs via the maturation of a precursor polypeptide [2, 31. In this case, the enzymatically inactive precursor protein comprises (a) a signal peptide; (b) the CI subunit; (c) a peptide of 54 amino acids which is absent in the mature enzyme (endopeptide); (d) the p subunit (see Fig. 1) [4]. The active enzyme is an aP heterodimer which is localized in the periplasmic space and which hydrolyzes penicillin G to the b-lactam moiety and the phenylacetic acid side-chain. Using a precursor lacking the signal peptide, it has been demonstrated by in vitro processing experiments that the precursor is first cleaved at the N-terminus of the fl subunit resulting in mature fl subunit and a protein consisting of the M subunit plus endopeptide. The removal of the endopeptide from the CI subunit involves a further intermediate composed of a subunit and of only part of the endopeptide (see Fig. 1)In order to define the structural requirements for processing, we have studied the processing characteristics of mutant precursor polypeptides carrying insertions or deletions in different domains of the penicillin acylase structural gene (pac). Evidence is presented that an endopeptide of adequate size and an appropriate folding state of the precursor polypeptide [41.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.

We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.

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