Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum.

The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein-based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance.

Strain-specific monoclonal antibodies to the E2 protein of classical swine fever virus, Paderborn strain.

Monoclonal antibodies (MAbs) against the E2 protein of classical swine fever virus (CSFV) are useful for diagnosis and strain characterization. A purified, baculovirus-expressed CSFV E2 protein from the Paderborn strain was formulated with a saponin adjuvant and successfully used to induce an antigen-specific immune response in mice. After cell fusion a panel, designated F92G, of 12 mouse hybridomas (5-2, 11-1, 14-1, 25-2, 28-2, 31-1, 34-1, 35-2, 37-3, 38-2, 39-1, 41-1) producing CSFV-E2 specific MAbs were selected based on their Ig subclass and secretion level (µg IgG/mL). Nine IgG 1/k, two IgG 2b/k, and one IgG 2a/k MAbs were further characterized using immunoperoxidase reactivity, ELISA, and Western blot analysis. Immunoglobulin concentration-dependent immunoperoxidase and ELISA reactivity was observed for some of the MAbs with certain antigens. In general there were several reactivity patterns exhibited by the MAbs, with CSFV strains representing different genetic subgroups (by immunoperoxidase staining) and recombinant antigens (by ELISA). It was interesting to note that in some cases the strain-specific reactivity of a MAb was dependent on the test, thereby providing a clue regarding the nature of the binding site.

Effects of N-linked glycosylation of the fusion protein on replication of human metapneumovirus in vitro and in mouse lungs.

The fusion (F) protein is an important membrane glycoprotein necessary for cellular entry and replication of human metapneumovirus (hMPV). Selective prevention of N-linked glycosylation may compromise the catalytic and fusion functions of the F protein. By using site-directed mutagenesis and reverse genetics, recombinant mutant viruses lacking one or two N-linked glycosylation sites in the F protein were constructed. M1, which lacked glycosylation at position 57 of the F protein, had slightly compromised replication, whereas M2 and M4, which lacked glycosylation at position(s) 172 or 57 and 172, respectively, showed profound impairment of replication when compared with wild-type (WT) NL/1/00-GFP virus in both Vero E6 cells and mouse lungs. M2 was less fit than WT virus in vitro and in immunocompromised mouse lungs. The F proteins of WT and mutant viruses were similarly expressed on the infected cell membrane, while the activated fusion protein subunits, F1 of M2 and M4, were produced in lower quantities compared with those of WT and M1 virus. The mutated viruses lacking N-linked glycosylation at position 353, either individually or together with other sites, could not be recovered. Thus, N-linked glycosylation may be involved in the catalysis of the fusion protein from F0 to F1 and F2, which is critical for fusion function. Strategies targeting N-linked glycosylation may be helpful for developing attenuated live vaccines or antiviral drugs for hMPV.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA.

Characterization of the biosynthesis and cell surface expression of avian metapneumovirus attachment glycoprotein.

Biosynthesis, glycosylation and cell surface expression of the AMPV/C G protein were examined in eukaryotic cell lines (LLC-MK2, CHO-K1, CHO-1d1D). Immature G gene products with a molecular mass of 42, 45 and 58-90 kilodaltons (kDa) were identified by SDS-PAGE and represented glycosylated intermediates. Tunicamycin treatment of transfected cells confirmed the presence of N-linked carbohydrate moieties on these intermediate species and identified a 38 kDa unglycosylated precursor. A fully processed, mature form of the protein migrated around 110 kDa. The presence of O-linked sugars on the mature G protein was confirmed by using the O-glycosylation deficient CHO-ldlD cell line supplemented with exogenous Gal and GalNAc. Binding of the lectin Arachis hypogaea (peanut agglutinin) confirmed the presence of O-linked sugars on the mature protein and its intracellular transport to the cell surface was demonstrated by indirect immunofluorescent staining.

Hydrophilicity dependent budding and secretion of chimeric HIV Gag-V3 virus-like particles.

Virus-like particles (VLPs) of numerous viruses have been considered as possible candidates for vaccine development. We have constructed HIV chimeric genes by coupling the gag gene of HIV-2 with the V3 domain of the gp120 gene of either HIV-1 or HIV-2 and expressed the chimeric genes in SF21 cells using the recombinant baculovirus expression system. Although the level of expression of the chimeric HIV-2 gag gene with the V3 domain of either HIV-1 gp120 (gagC-1V3) or HIV-2 gp120 (gagC-2V3) was high, the VLP assembly and extracellular release of GagC-1V3 was very poor. In contrast, GagC-2V3 chimeric proteins formed VLPs and released efficiently. We have constructed substitution mutants to investigate the effects of the hydrophobic region of the V3 domain of HIV-1 Gp120 (1V3) in VLP assembly and release. The substitution mutant analyses revealed that in replacing the hydrophobic region of the 1V3 in GagC-1V3 with the hydrophilic sequence of the V3 domain of HIV-2 Gp120 (2V3) enhanced the extracellular VLP. We demonstrate here that disruption of the hydrophobic character of the C-terminus of the chimeric protein improves assembly and release of the VLPs. Our results suggest that the poor GagC-1V3 VLP release was attributed to the hydrophobic region in the V3 sequence of the chimeric protein, and that not only the N-terminal myristylation and positively charged domain of the Gag protein functioned as a targeting signal to direct membrane binding, but also that the C-terminal hydrophobic region affected release of chimeric VLPs.

Production of a recombinant major inner capsid protein for serological detection of epizootic hemorrhagic disease virus.

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.

Expression of recombinant small hydrophobic protein for serospecific detection of avian pneumovirus subgroup C.

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.

Budding and secretion of HIV Gag-Env virus-like particles from recombinant human adenovirus infected cells.

We have characterized the assembly, budding and extra-cellular release of human immunodeficiency virus (HIV) Gag-Env virus-like particles (VLPs) from human embryonic kidney cells (293 cells expressing the E1a protein of adenovirus) infected with recombinant replication-defective human adenovirus type 5. Recombinant human adenovirus vectors expressing the chimeric Gag-Env protein were constructed by inserting the gag-env fusion gene into the E1a region of the human adenovirus type 5. Biochemical and immunological analyses of VLPs recovered from the culture supernatant revealed that these particles contain the HIV-2 Gag protein and segments of Env protein from the HIV-1 gp120. This chimeric Gag-Env protein interacted with HIV-1 positive patient sera and with HIV-2 Gag monoclonal antibody. Immunoelectron microscopy of the 293 cells infected with the recombinant adenoviruses showed that the HIV Gag-Env antigen is present in the VLPs. Thin-section electron microscopy (EM) revealed that the Gag-Env VLPs bud through the cytoplasmic membrane, as well as through membranes of intracellular vacuoles. The thin-section EM showed that the Gag-Env VLPs have a spherical morphology with an electron-dense ring. The size of VLPs range from 110 to 140 nm in diameter, which is slightly larger than that of the Gag particles without Env protein fusion. Mice immunized with recombinant adenoviruses generated antibodies that specifically reacted with Gag-Env chimeric proteins. Our results support the idea that the replication-defective adenovirus could be used to induce immune responses that might be useful in a vaccine against HIV/AIDS.