Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR).

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces. INTRODUCTIONThe 51 serotypes of human adenoviruses (Ads) are classified into six species, A-F (Benkő et al., 2000;Horwitz, 2001), and species B Ads are divided further into B1 and B2 serotypes, for which tropism differs. The B1 group comprises Ad3, 7, 16, 21 and 50 and predominantly infects the upper respiratory tract, whereas the B2 group serotypes Ad11, 14, 34 and 35 are associated with kidney and urinary-tract infections, with fatal outcome in immunocompromised patients (Leen & Rooney, 2005;Schmitz et al., 1983;Wadell, 2000). Species B Ads bind a different cell-surface receptor from most of the species A, C, D, E and F Ad serotypes, which bind to the coxsackievirus and adenovirus receptor (CAR) (Bergelson et al., 1997;Defer et al., 1990;Roelvink et al., 1998;Stevenson et al., 1995). Several groups have identified the membrane cofactor CD46 as an attachment receptor for species B serotypes, including Ad11 (Segerman et al., 2003b), Ad35 (Gaggar et al., 2003) and Ad3 (Sirena et al., 2004). CD46 belongs to a family of regulators of complement activation, whose biological role is to prevent complement activation on autologous tissue (Liszewski et al., 1991). Based on virus competition experiments and antibody-mediated blocking of various Ads on human cells, it has been suggested that more than one species B receptor exist (Segerman et al., 2003a;Sirena et al., 2004;Tuve et al., 2006), and it remains controversial whether CD46 functions as attachment receptor for all species B serotypes (Gaggar et al., 2003;Gustafsson et al., 2006;Marttila et al., 2005;Segerman et al., 2003b;Tuve et al., 2006). Additional Ads that bind to CD46 include species D Ad37 and 49 (Lemcke...

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BackgroundGlycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering.ResultsIn this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.ConclusionThe present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.