Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells

Puumala hantavirus (PUUV) glycoproteins G1 and G2 and nucleocapsid protein (N) were expressed in BHK-21 cells by transfection of a plasmid producing a recombinant alphavirus replicon. Coexpression of G1 and G2 from separate constructs seemed to be important for the optimal folding of the glycoproteins, as evaluated by a panel of MAbs detecting conformational epitopes. To evaluate the human antibody response against recombinant G1, G2 and N, several panels of sera were tested by immunofluorescence assay (IFA). Also human sera showed the best reactivity towards G1 and G2 coexpressed from separate transcripts (G1 + G2). Notably, only 2% of the acute sera (total number = 133) contained IgG antibodies against G1 + G2, whereas of old-immunity sera (total number = 100) 87% were G1 + G2 positive. Analysis of a panel of serial patient sera showed that as the immunity matured, IgG antibodies against the recombinant glycoproteins appeared and the titers increased in the course of time, while antibodies against the recombinant N were present already in the acute phase in high titers. The granular fluorescence pattern in PUUV IgG-IFA, associated with the acute phase of immunity, was linked to the presence of antibodies against N, whereas the diffuse fluorescence pattern associated with old-immunity, was linked to the development of antibodies against G1 + G2. The granular fluorescence pattern in PUUV IgG-IFA had a predictive value of 100% for acute PUUV infection. Weak cross-reaction with PUUV glycoproteins was observed in 36% of old-immunity DOBV-specific human sera.