P.I.T. (d) 5d 6d 7d BmNPV bacmid BmNPV-CP- bacmid Recombinant bacmid BmNPV BmNPV- BmNPV- CP- CP--Chi- n 5 6 7 5 6 7 5 6 7 P.I.T. (d) 464 (A) (B) MW (kDa) M 75 25 Fig. 2 a Comparison of larval liquefaction. Silkworm larvae were injected with BmNPV (WT) and BmNPV-CP− bacmids. b GFPuv fluorescence of the GFPuv-13CG2 (scFv) fusion protein expressed in the BmNPV/bx-GFPuv-13CG2-, BmNPV-CP-/bx-GFPuv-13CG2-, and BmNPV-CP−-Chi−/bx-GFPuv-13CG2 bacmid-injected silkworm lar- vae. Loaded protein concentrations were 65–95 μg. The arrow indicates the molecular mass of the GFPuv-13CG2 fusion protein glycosylation pathway was reinforced with GlcNAcT II and sialyltransferase. Expression of glycosyltransferase in silkworm larvae and its application to the synthesis of glycomimetics to block infections by avian and human influenza viruses Recently, the glycosyltransferase produced by silkworm larvae was applied to the synthesis of sialoglycopolypeptides for blocking influenza virus infection. Influenza viruses infect host cells through the binding of viral hemagglutinins (HAs) to sialoglycoproteins or sialoglycopeptides of the receptors on the host cell surfaces (Suzuki et al. 1986). Since the molecular recognition process leads to the adhesion of the host cells and virus, molecules with a high affinity for the viral HA act as potent inhibitors of infection by influenza viruses (Ogata et al. 2007; Schengrund 2003). The host cell specificity of the virus is dependent not only on the linkage of SAs to the Appl Microbiol Biotechnol (2010) 85:459–470 penultimate galactose but also on the number of SA residues and the precise nature of the core structure (Gambaryan et al. 2004; Yamada et al. 2006). In order to synthesize an artificial sialoglycoprotein, rat α2,6-sialyltransferase (ST6Gal I) was expressed in fifth-instar silkworm larval hemolymph, using a recombinant bacmid. Approximately 2.2 mg of ST6GalI was purified from the hemolymph of 11 larvae. The substrate specificity of the ST6GalI purified from hemolymph was the same as that of the native rat ST6GalI. By using this ST6GalI, the synthesis of a α2,6-sialoglycopolypeptide as a glycoprotein mimetic was successfully achieved. The syn- thesized α2,6-sialoglycopolypeptide selectively inhibited hemagglutination induced by Sambucus nigra lectin, which specifically binds to the Siaα2-6Gal(GalNAc) structure, and showed approximately 780-fold higher affinity than fetuin as a control (Ogata et al. 2009b). Moreover, Ogata et al. (2009a) designed a series of γ-polyglutamic acid (γ-PGA)-based glycopolypeptides carrying long/short R2,3/6 sialylated glycans to act as inhibitors of the influenza virus. As an alternative design, sialoglycopolypeptides carrying long- spacer-linked glycans were engineered, by the replacement of the N-acetyllactosamine (LN) unit by an alkyl chain. The avian viruses specifically bound to glycopolypeptides carry- ing a short sialoglycan with higher affinity than to a long glycan. In contrast, human viruses preferentially bound not only to a long R2,3/6 sialylated glycan with LN repeats in the receptors but also to more spacer-linked glycans, in which the inner sugar was replaced by a nonsugar structural unit, such as a pentylamido group (Hidari et al. 2007). A spaced tandem/triplet pentylamido repeat is a good mimetic of a tandem/triplet LN repeat and thus provides a facile way to design strong polymeric inhibitors of infection by avian and human influenza viruses (Fig. 4). Surface display system and subunit vaccine production in silkworm Baculovirus particles have become valuable tools in biotechnology and medicine. Even a small virus provides a huge surface, which can be engineered in many ways. Heterologous peptides or proteins can be displayed on the surface of baculovirus particles by fusing the peptide or protein to the baculovirus surface glycoprotein, gp64 (Boublik et al. 1995). Baculoviruses displaying gp64 fusion proteins have proven to be very effective immu- nogens (Kost et al. 2005). Since this approach was first used to raise monoclonal antibodies against the nuclear receptors LXRβ and FXR (Lindley et al. 2000), it has been used for the development of low-cost insect larvae-derived recombinant subunit vaccines. These include H5N1 influ- enza vaccine (Jin et al. 2008), as well as subunit vaccines against rabbit hemorrhagic disease virus (Pérez-Filgueira