Native Viral Antigens Research Articles

Mapping of B-cell determinants in the nucleocapsid protein of Puumala virus: definition of epitopes specific for acute immunoglobulin G recognition in humans

The complete amino acid sequence of the Puumala (PUU) virus nucleocapsid protein (N), deduced from the genome of the prototype strain Sotkamo, was synthesized as decapeptides with 5-amino-acid overlaps. By use of the PEPSCAN method, 86 peptides were examined for reactivity with sera from serologically confirmed nephropathia epidemica (NE) patients and 11 PUU virus N-specific bank vole monoclonal antibodies. The human sera showed reactivity with several different regions, while only one of the monoclonal antibodies reacted with one single peptide. Sequences were selected by this PEPSCAN analysis of human antibody reactivities, and five 15-amino-acid peptides were synthesized and evaluated as antigens by an enzyme-linked immunosorbent assay (ELISA). Peptide-reactive antibodies of the immunoglobulin M (IgM) class were measured in serum samples drawn from patients with acute NE. In comparison with the results of a mu-capture IgM ELISA using native PUU virus antigen, only a few serum samples were found positive (sensitivity, 2 to 10%). Interestingly, when antibodies of the IgG class were measured, the sensitivities of the five peptide ELISAs were found to be 79, 46, 2, 100, and 40%, respectively, as compared with the sensitivity of an IgG ELISA based on native viral antigen. The IgG reactivities of sequentially drawn sera from NE patients with the two peptides giving the highest assay sensitivities were analyzed and compared with their reactivities with native viral antigen. All patients had detectable anti-peptide IgG in the acute-phase sample, which, however, had totally declined in samples drawn after 2 years. The opposite pattern was seen with native viral antigen, in which case all patients showed the highest levels of specific IgG after 2 years. The results suggest the presence of epitopes specific for the acute IgG response.

Comparison of immune responses to a native viral antigen and a synthetic peptide derived from it: implications for vaccine development.

Murine immune responses to the hepatitis B surface antigen (HBsAg) and a synthetic peptide derived from it were compared at the humoral level. Six of nine strains used responded to either peptide or HBsAg, though restriction profiles were not superimposable. Two of three strains non-responsive to HBsAg produced an antibody response on immunization with peptide which was cross-reactive with both peptide and HBsAg. In in vitro lymphocyte stimulation assays, lymphocyte from all six peptide-immunized mouse strains could be induced to proliferate on challenge with HBsAg. However, of the HBsAg-immunized groups, lymphocytes from only three of six responder strains proliferated on in vitro HBsAg challenge. Cumulatively, these results suggest that a vaccine formulation that includes both protein antigens and synthetic peptides derived from these proteins may be more effective at eliciting an immune response in a broader cross-section of target population.

Competitive enzyme-immunoassays using native viral antigens to discriminate between HIV-1 and HIV-2 infections

Human immunodeficiency virus (HIV) strains can be separated into two serotypes: HIV-1 and HIV-2. In the study reported herein, we developed and evaluated competitive enzyme-immunoassays (CEIA-1 for the detection of antibody to HIV-1, CEIA-2 for the detection of antibody to HIV-2) to discriminate serologically between HIV-1 and HIV-2 infections. In most of the cases, the serotyping of known reactive serum samples was done easily with the CEIAs, showing a similar specificity to the Western blot. Such competitive assays could represent alternative procedures for serotyping HIV infection and would limit the need for expensive Western blots, especially in economically poor countries.

The purification of a gs antigen of the murine mammary tumor virus and its quantitation by radioimmunoassay

A method using Concanavalin (ConA) affinity chromatography has been developed for the purification of the mouse mammary tumor virus (MuMTV) gs antigens (gp52) and (p27). The procedure combines ConA chromatography and DEAE cellulose fractionation to yield the gs antigens in both high yield and sufficient purity for use in radioimmunoassay (RIA). The gp52 obtained has been iodinated with the [ 125I]Bolton-Hunter reagent, chromatographed on Sephadex G-100, and utilized in the development of a blocking radio immunoassay (RIA). The sensitivity of this assay (0.5–1.0 ng) is superior to that achieved with whole particle assays. The procedures used for the isolation and iodination of the gp52 avoid the technical problem of incomplete displacement of [ 125I]gp52 by native viral antigen. The resulting RIA provides a sensitive, reliable tool for studying the expression of the gp52 antigen and its relationship to the onset and progression of murine breast cancer.