Development of a Luciferase Immunoprecipitation System Assay To Detect IgG Antibodies against Human Respiratory Syncytial Virus

The nucleoprotein of respiratory syncytial virus (RSV-N) is immunogenic and elicits an IgG response following infection. The RSV-N gene was cloned into a mammalian expression vector, pREN2, and the expressed luciferase-tagged protein (Ruc-N) de-tected anti-RSV-N-specific IgG antibodies using a high-throughput immunoprecipitation method (the luciferase immunopre-cipitation system [LIPS]-NRSV assay). The specificity of the assay was evaluated using monoclonal antibodies (MAbs) andmono-specific pre- and postimmunization rabbit antisera. Blood serum samples from chimpanzees and humans with proven/probable RSV infection were also tested. The pre- and postimmunization serum samples from rabbits given humanmetapneumovirus (HMPV) or measles virus were negative when tested by the LIPS-NRSV assay, while antisera obtained after immunization with either the RSV-A or RSV-B strain gave positive signals in a dose-dependent manner. RSV-NMAb 858-3 gave a positive signal in the LIPS-NRSV assay, while MAbs against other paramyxovirus nucleoproteins or RSV-F or RSV-G did not. Serum samples from chimpanzees simultaneously immunized with vaccinia-RSV-F and vaccinia-RSV-G recombinant viruses were negative in the LIPS-NRSV assay; however, anti-RSV-N IgG responses were detected following subsequent RSV challenge. Seven of the 12 infants who were seronegative at 9 months of age had detectable anti-RSV-N antibodies when they were retested at 15 to 18 months of age. The LIPS-NRSV assay detects specific anti-RSV-N IgG responses that may be used as a biomarker of RSV infection. Respiratory syncytial virus (RSV) is an important pathogen forvaccine development since morbidity is high among infants and the elderly (1). Two distinct antigenic subtypes, RSV-A and-B, may either cocirculate or independently dominate durin