Development and Characterization of a Highly Sensitive NanoLuciferase-Based Immunoprecipitation System for the Detection of Anti-Influenza Virus HA Antibodies

Abstract

Antibody detection is crucial for monitoring host immune responses to specific pathogen antigens (Ags) and evaluating vaccine efficacies. The luciferase immunoprecipitation system (LIPS) was developed for sensitive detection of Ag-specific antibodies in sera from various species. In this study, we describe NanoLIPS, an improved LIPS assay based on NanoLuciferase (NLuc), and employ the assay for monitoring antibody responses following influenza virus infection or vaccination. We generated recombinant influenza virus hemagglutinin (HA) proteins tagged with N-terminal (N-NLuc-HA) or C-terminal (C-NLuc-HA) NLuc reporters. NLuc-HA yielded an at least 20-fold higher signal-to-noise ratio than did a LIPS assay employing a recombinant HA-Gaussia princeps luciferase (GLuc) fusion protein. NanoLIPS-based detection of anti-HA antibodies yielded highly reproducible results with a broad dynamic range. The levels of antibodies against C-NLuc-HA generated by mice vaccinated with recombinant vaccinia virus DIs strain expressing an influenza virus HA protein (rDIs-HA) was significantly correlated with the protective effect elicited by the rDIs-HA vaccine. C-NLuc-HA underwent glycosylation with native conformations and assembly to form a trimeric structure and was detected by monoclonal antibodies that detect conformational epitopes present on the globular head or stalk domain of HA. Therefore, NanoLIPS is applicable for evaluating vaccine efficacy. We also showed that C-NLuc-HA is applicable for detection of HA-specific antibodies in sera from various experimental species, including mouse, cynomolgus macaque, and tree shrew. Thus, NanoLIPS-based detection of HA offers a simple and high-sensitivity method that detects native conformational epitopes and can be used in various experimental animal models. IMPORTANCE Influenza virus HA-specific antibodies can be detected via the hemagglutination inhibition (HI) assay, the neutralization (NT) assay, and the enzyme-linked immunosorbent assay (ELISA). However, these assays have some drawbacks, including narrow dynamic range and the requirement for large amounts of sera. As an alternative to an ELISA-based method, luciferase immunoprecipitation system (LIPS) was developed. We focused on NanoLuciferase (NLuc), which has a small size, higher intensity, and longer stability. In this study, we developed a technically feasible and highly sensitive method for detecting influenza virus-specific antibodies using a NLuc-tagged recombinant HA protein produced in mammalian cells. HA with a C-terminal NLuc extension (C-NLuc-HA) was glycosylated and formed trimeric complexes when expressed in mammalian cells. Furthermore, C-NLuc-HA was recognized not only by monoclonal antibodies that bind to the globular head domain but also by those that bind to the stalk domain. We also demonstrated that the data obtained by this assay correlate with the protection of an experimental vaccine in animal models.

Comparative Analysis of Swine Antibody Responses following Vaccination with Live-Attenuated and Killed African Swine Fever Virus Vaccines

“…Because viral proteins might contain a signal sequence at their N terminus to direct the nascent proteins to the appropriate cellular compartments for post-translation modifications [40], we decided to fuse the reporter gene nluc to the carboxy terminus of each ASFV gene to avoid interfering with the signal sequence functions. However, it has been reported in previous studies that antibody binding to an antigen-luciferase fusion protein might be affected by whether the luciferase protein is fused to the C-or N-termini of the antigens [41]. In future studies, we will assess antibody reactivity to two forms of ASFV-luciferase proteins, each carrying luciferases at either the C-or N-terminus.…”

Shedding Light onH. pyloriDetection: A Fusion Protein Approach Unveiled through LIPS Method

“…In a 2023 investigation, HEK 293T cells were utilized for fusion protein production to evaluate Deltacoronavirus porcine (PDCoV), a coronavirus with potential cross-species infectivity (Boley, Lemon, and Kenney 2023). Similarly, in another study, HEK293FT cells were employed for producing recombinant HA proteins tagged with NLuc reporters for influenza virus HA detection (Honda et al 2021). Furthermore, a LIPS assay for detecting IgG antibodies against hRSV’s G-glycoprotein was established using COS-1 cells for fusion protein production (Crim et al 2019).…”

Development of a highly sensitive Gaussia luciferase immunoprecipitation assay for the detection of antibodies against African swine fever virus

“…Various luciferase genes have been used as the reporter gene in LIPS assays, including Renilla luciferase ( Berguido et al., 2021 ; Hachim et al., 2022 ), NanoLuciferase ( Honda et al., 2021 ), and GLuc ( Honda et al., 2021 ). GLuc from the marine copepod Gaussia princeps is naturally secreted from mammalian cells in an active form and can be released efficiently into the conditioned medium of mammalian cells ( Tannous, 2009 ).…”