Sendai Virus-Based Production of HIV Type 1 Subtype B and Subtype E Envelope Glycoprotein 120 Antigens and Their Use for Highly Sensitive Detection of Subtype-Specific Serum Antibodies

Abstract

We previously described a Sendai virus (SeV)-based expression system for the recombinant gp120 of HIV-1 subtype B (rgp120-B), which has permitted the production of antigenetically and functionally authentic gp120 at a concentration as high as 6 microg/ml of culture supernatant (Yu D et al.: Genes Cells 1997;2:457-466). Here the same procedure was successfully applied to the production of HIV-1 subtype E gp120 (rgp120-E). The remarkable production of the proteins by the SeV expression system enabled us to use crude culture supernatants for serological and functional studies of gp120s. The immunological authenticity of rgp120-E was verified by patient sera and anti-V3 loop monoclonal antibodies specific for HIV-1 subtypes B and E. CD4-binding properties were corroborated by FACS analyses. The rgp120s were then used in an enzyme immunoassay (rgp120-EIA) to detect antibodies in the sera of HIV-1-infected individuals, and the performance was assessed in comparison with a conventional V3 loop peptide EIA (V3-EIA). The initial evaluation of a serum panel (n = 164) consisting of 76 subtype E and 88 subtype B sera revealed that the rgp120-EIA was nearly 1000-fold more sensitive than the V3-EIA and was able to detect subtype-specific antibody with 100% sensitivity and with a complete correlation with the genotypes, whereas the V3-EIA failed to detect 9 and 24% of the same subtype E and B sera, respectively. Furthermore, a study employing a panel of 28 international sera with known genotypes (HIV-1 subtypes A through F) confirmed the remarkable specificity of this method. An EIA reactivity higher than 1.0 was an unambiguous predictor of HIV-1 subtype E and B infections. The data imply the presence of strong subtype-specific epitopes for antibody bindings to these rgp120s.

Proline 78 Is Crucial for Human Immunodeficiency Virus Type 1 Nef To Down-Regulate Class I Human Leukocyte Antigen

“…Unexpectedly, our K3 construct lacked 33 nucleotides spanning from nucleotide 583 to 615 in K3 ORF (26). The rSeV stocks were obtained as previously described (30) and generally had infectious titers of 10 7 to 10 9 PFU/ml. The mutant rSeVs were designated SeV-EEEE/AAAA, SeV-APPP, SeV-PAPP, SeV-PPAP, SeV-PPPA, SeV-PPAA, SeV-PAAA, SeV-PPPP/ AAAA, SeV-K3, and SeV-K5.…”

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration

“…Culture plates (96-well) were coated with purified HEV-VLPs or synthesized oligopeptides (P18) at a concentration of 10 or 100 mg/well, respectively, overnight at 41C followed by 30 min of blocking with PBS containing 0.1% FBS and 0.05% Tween 20. To determine the anti-HIV env gp120 antibody responses, CV-1 cells were seeded in 96-well plates and infected with recombinant Sendai virus expressing HIV env gp120 of NL432 strain (SeV gp120), 32 and then the plates were incubated at 371C. At 3 days after infection, plates were washed and fixed with PBS containing 10% formalin for 10 min.…”

Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus

“…7 Also recombinant virus designed to encode the gp120 glycoprotein resulted in extremely high levels of transgene expression in CV-1 monkey kidney cells. 8 Interestingly, although HVJ liposomes containing Sendai-derived haemaglutinin protein complexed with plasmid DNA carrying the b-galactosidase gene have been applied prenatally by intra-amniotic delivery, which resulted in low and transient expression in rat skin, 9 no previous studies have described the use of recombinant SeV particles in a prenatal context probably due to its intrinsic cytotoxic nature and transient gene expression.…”