Phenotypic characterization of cell culture-derived hepatitis E virus subjected to different chemical treatments: Application in virus removal via nanofiltration.

Safety evaluation for the hepatitis E virus (HEV) is required for plasma fractionation products. Plasma-derived HEV (pHEV) is quite unique in that it is associated with a lipid membrane, which, when stripped during manufacturing processes, induces morphological changes in the virus, making it difficult to select proper HEV phenotypes for clearance studies. We developed a convenient system for the preparation of a high titer cell culture-derived HEV (cHEV). In this system, PLC/PRF/5 cells transfected with the wild-type HEV genome generated lipid membrane-associated cHEV for a long period even after cryopreservation. We also examined how this lipid membrane-associated cHEV can be used to verify the robustness of pHEV removal via 19-nm nanofiltration. Sodium-deoxycholate and trypsin (NaDOC/T) treatment not only dissolved lipid but also digested membrane-associated proteins from pHEV and cHEV, making the resulting cHEV particle smaller in size than any pHEV phenotypes generated by ethanol or solvent-detergent treatment in this study. In both 19-nm and 35-nm nanofiltration, cHEV behaved identically to pHEV. These results indicate that cHEV is a useful resource for viral clearance studies in term of availability, and the use of NaDOC/T-treated cHEV ensured robust pHEV removal capacity via 19-nm nanofiltration.

Quantitative PCR evaluation of parvovirus B19 removal via nanofiltration.

When human parvovirus B19 (B19) is removed from plasma-derived products by nanofiltration, non-infectious fragmented B19 DNA in filtrate prevents quantitative real time PCR (qPCR) from accurately evaluating reduction of the virus particles. To determine optimal target sequence length for detection of full-length B19 genome in the viral particles by qPCR, we analyzed 4 different sequences ranging from 372 to 1,980 bp and found that a 989 bp sequence shows a well-balanced performance for the sensitivity and the run time. Nuclease treatment of filtrates prior to qPCR is also expected to decrease the influence of the residual B19 DNA, but extremely high protein concentration of plasma-derived products in filtrates may result in incomplete digestion of the B19 DNA. In this context, however, our analysis showed that even when B19 genome is incompletely digested, qPCR for the 989 bp sequence successfully eliminates the influence of the B19 DNA. Finally, we verified that when B19-spiked plasma products are subjected to nanofiltration with the resulting filtrates treated with nuclease, qPCR for the 989 bp sequence accurately evaluates B19 removal. These results demonstrate that qPCR for the 989 bp sequence combined with nuclease treatment enables convenient and accurate evaluation of B19 removal by nanofiltration.

Hepatitis E virus derived from different sources exhibits different behaviour in virus inactivation and/or removal studies with plasma derivatives.

Hepatitis E virus (HEV) causes viral hepatitis, and is considered a risk factor for blood products. Although some HEV inactivation/removal studies have been reported, detailed investigations of different manufacturing steps as heat treatment, partitioning during cold ethanol fractionation, low pH treatment, and virus filtration have yet to be reported for plasma-derived medicinal products. In this study, human serum- and swine faeces-derived HEVs, with and without detergent treatment, were used. The kinetic patterns of inactivation, log reduction value, or partitioning during the process were evaluated. In addition, the mouse encephalomyocarditis virus (EMCV) and canine and porcine parvoviruses (CPV/PPV) were also evaluated as model viruses for HEV. Small pore size (19 or 15 nm) virus filtration demonstrated effective removal of HEV. Middle pore size (35 nm) virus filtration and 60 °C liquid heating demonstrated moderate inactivation/removal. Ethanol fractionation steps demonstrated limited removal of HEV. Unpurified HEV exhibited different properties than the detergent-treated HEV, and both forms displayed differences when compared with EMCV, CPV, and PPV. Limited or no inactivation of HEV was observed during low pH treatment. Untreated plasma-derived HEV from humans showed different properties compared to that of HEV treated with detergent or derived from swine faeces. Therefore, HEV spike preparation requires more attention.

Mode of swine hepatitis E virus infection and replication in primary human hepatocytes.

The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcriptase-quantitative PCR. The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes was attributed mainly to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occurred without sequence variation.

Immunization of rabbits with synthetic peptides derived from a highly conserved ß-sheet epitope region underneath the receptor binding site of influenza A virus.

BACKGROUND: There is increasing concern about the speed with which health care providers can administer prophylaxis and treatment in an influenza pandemic. Generally, it takes several months to manufacture an influenza vaccine by propagation of the virus in chicken eggs or cultured cells. Newer, faster protocols for the production of vaccines that induce broad-spectrum immunity are therefore highly desirable. We previously developed human monoclonal antibody B-1 that shows broadly neutralizing activity against influenza A virus H3N2. B-1 recognizes an epitope region that includes an antiparallel ß-sheet structure underneath the receptor binding site of influenza hemagglutinin (HA). In this study, the efficacy of a synthetic peptide vaccine derived from this epitope region against influenza A was evaluated. MATERIALS AND METHODS: Two peptides were synthesized, the upper and lower peptides. These peptides comprise amino acid residues 167-187 and 225-241, respectively, of the B-1 epitope region of HA, which is involved in forming the ß-sheet structure. Both peptides were then coupled to keyhole limpet hemocyanin, and the peptides, alone or in combination, were used to immunize rabbits. The resulting antibody responses were examined by enzyme-linked immunosorbent assay. The upper peptide, but not the lower peptide, elicited antibodies that were reactive to HA. Interestingly, the use of both peptides together could elicit antibodies with a higher reactivity to HA than either peptide alone. The antibodies were found to react to HA at the N-terminus of the upper peptide, which is exposed at the surface of trimeric HA on influenza virions. DISCUSSION: The higher production of HA-reactive antibodies following immunization with both peptides suggests that the upper peptide forms the effective epitope structure in the binding state, and the lower peptide enhances the production of HA antibodies. This study could be the first step towards the development of pandemic viral vaccines that can be produced within short time periods.

Induction of anti-influenza immunity by modified green fluorescent protein (GFP) carrying hemagglutinin-derived epitope structure.

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel ß-sheet structure. Our previous study identified this ß-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this ß-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of ß-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition.

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors.

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.