Baculogold

The chimeric HPV18L1–45RG1 fusion construct was designed using CLC DNA workbench based on HPV18 L1 (accession number HPV18 AY262282) and HPV45 L2 (X74479), codon optimized for mammalian expression and synthesized by GeneArt (Regensburg, Germany). The gene sequence encoding the 20 aa (residues 16–35) RG1 epitope of HPV45 was inserted into the DE loop of HPV18 L1 between aa positions 134/135. The purified (Qiagen’s Gel extraction kit) L1-RG1 fusion gene was subcloned into the baculovirus transfer vector pSynwtVI^- using BglII and KpnI sites [30 (link)]. Recombinant vectors were verified by bidirectional sequencing (VBC-Biotech, Vienna, Austria).
Transfer vector and linearized baculovirus DNA (BaculoGold; BD Bioscience) were co-transfected into Sf9 cells and recombinant baculoviruses isolated by plaque assay. Following expression in insect cells, VLP were purified as described [30 (link)–32 (link)]. Briefly, Sf9 insect cells were infected with amplified high-titer baculovirus stocks for three days. Cells were harvested and lysed by sonication in a breaking buffer (PBS + 0.8M NaCl + 2mM CaCl₂ + 1mM phenylmethylsulfonyl fluoride (PMSF)) and incubated overnight with addition of 0.5% Brij58. VLP were purified by ultracentrifugation on sucrose-PBS cushions [35% (w/v)] and CsCl-PBS [29% (w/w)] density gradients.

Soluble HLA-DR1*01:01 and DM were produced as described (Kim et al., 2014 (link); Narayan et al., 2007 (link)). Baculovirus DNA (BaculoGold; PharMingen) and transfer vectors carrying DR α- and β-chains were transfected together into Sf9 insect cells to produce recombinant viruses. Recombinant viruses were passaged three times before being used to infect High Five cells in ISFM media (Cleveland et al., 2014 (link); Inlow et al., 1989 (link)). DR1 proteins were purified from culture supernatants using immunoaffinity chromatography with a monoclonal antibody L243 to DR1 (purified from HB-55 hybridoma; American Type Culture Collection). Soluble DM was also expressed by High Five cells transduced with baculovirus containing the extracellular domains of genes encoding the α- and β-chains of human DM. The truncated DM α- and β-chains were modified to contain the FLAG epitope (DYKDDDDK) and c-Myc epitope (EQKLISEEDL), respectively, at their C-termini. DM protein was purified from culture supernatants with a monoclonal antibody to M2 (anti-FLAG) agarose resin (Sigma-Aldrich) and eluted with 5 mg/ml FLAG peptide (Sigma-Aldrich) in tris-buffered saline. DM was further concentrated and buffer exchanged into citric phosphate buffer, pH 6, with 05% wt/vol sodium azide and stored in aliquots at −80°C.

A genotype 1 (G1) HEV strain (GenBank accession no.: DQ079624) was isolated from a patient with acute hepatitis E in Myanmar in 1986. DNA fragments with deletions of the N-terminal 111 amino acids encoded by ORF2 were amplified by PCR with two primers, HEV-D13 (5′-AAGGATCCATGGCGGTCGCTCCAGCCCATGACACCCCGCCAGT-3′) and HEV-U14 (5′-GGTCTAGACTATAACTCCCGAGTTTTACCCACCTTCATCTT-3′). The PCR product contained the BamHI site before the start codon and the XbaI site after the stop codon. Then, the PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA, USA), digested with BamHI and XbaI, and ligated with a baculovirus transfer vector, pVL1393 (Pharmingen, San Diego, CA, USA), to yield plasmid pVL5480/7126. To produce the recombinant baculovirus, the transfer plasmid pVL5480/7126 was mixed with baculoGold (Pharmingen) and lipofectin (GIBCO-BRL, Gaithersburg, MD, USA) and transfected into insect cells Sf9 (Riken Cell Bank, Tsukuba, Japan). The cells were incubated at 26.5 °C in TC-100 medium (GIBCO-BRL) supplemented with 10% FBS and 0.26% tryptose phosphate broth (Difco Laboratories, Sparks, MD, USA). The recombinant viruses were plaque-purified three times in Sf9 cells and designated as Ac5480/7126^{57 (link)}.