Detection of anti‐NS1 antibodies after pandemic influenza exposure: Evaluation of a serological method for distinguishing H1N1pdm09 infected from vaccinated cases

Abstract

Background: Reliable exposure information is crucial for assessing health outcomes of influenza infection and vaccination. Current serological methods are unable to distinguish between anti-hemagglutinin (HA) antibodies induced by infection or vaccination. Objectives:We aimed to explore an alternative method for differentiating influenza infection and vaccination. Methods: Sera from animals inoculated with influenza viruses or purified H1N1pdm09HA were obtained. Human samples were selected from a pregnancy cohort established during the 2009 H1N1 pandemic. Unvaccinated, laboratory-confirmed cases (N = 18), vaccinated cases without influenza-like-illness (N = 18) and uninfected, unvaccinated controls (N = 18) were identified based on exposure data from questionnaires, national registries and maternal hemagglutination inhibition (HI) titres at delivery.Animal and human samples were tested for antibodies against the non-structural protein 1 (NS1) and HA from H1N1pdm09, using a Luciferase Immunoprecipitation System (LIPS). Results:Anti-NS1 H1N1pdm09 antibodies were detected in sera from experimentally infected, but not from vaccinated, animals. Anti-HA H1N1pdm09 antibodies were detectable after either of these exposures. In human samples, 28% of individuals with laboratory-confirmed influenza were seropositive for H1N1pdm09 NS1, whereas vaccinated cases and controls were seronegative. There was a trend for H1N1pdm09 NS1 seropositive cases reporting more severe and longer duration of symptomatic illness than seronegative cases. Anti-HA H1N1pdm09 antibodies were detected in all cases and in 61% of controls. Conclusions:The LIPS method could differentiate between sera from experimentally infected and vaccinated animals. However, in human samples obtained more than 6

Beyond the Spike: identification of viral targets of the antibody response to SARS-CoV-2 in COVID-19 patients

“…The Luciferase Immunoprecipitation system (LIPS) assay allows for a comprehensive understanding of the antibody immune response against infectious agents (17) by the expression of any protein or antigen as a recombinant Renilla luciferase (Ruc)-antigen fusion. The LIPS technique has previously been used to distinguish infected versus vaccinated cases for influenza due to the presence of antibodies against non-structural proteins (18), and to characterize human infections by zoonotic spill over from bat viruses (19). In this study, we used LIPS to assess the acute and convalescent antibody responses to a panel of 15 potential SARS-CoV-2 antigens: the 4 structural proteins (S, N, M and E), 3 S subunits (S1, S2, S2’), the 7 available ORFs (ORF3a, 3b, 6, 7a, 7b, 8 and 10) and 1 relevant NSP within ORF1a/b (NSP1) (20).…”

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

“…This study demonstrated that NanoLIPS offers a highly sensitive and simple technique for the detection of Ag-specific Abs present in sera from mouse, macaque, and tree shrew. In recent years, LIPS has been widely used for assessing immune responses in infectious diseases caused by HIV (36), Ebola virus (37), and influenza virus (38). In a previous study, a binding Ab titer against HA-1 of influenza virus, measured via Renilla luciferase-based LIPS, demonstrated a positive correlation with HI-determined titer.…”

Evidence for the extracellular delivery of influenza NS1 protein

“…The expression of the full length NS1 protein of A/WSN/1933 (H1N1) influenza virus on the cell surface was previously described [20]. It is also known that the natural influenza infection in animals [30,31] or people [19] may result in the formation of NS1 specific antibodies indicating the presentation of this antigen to B-lymphocytes in the extracellular compartment. The formation of antibodies to NS1 protein may serve as indirect evidence of its release from the infected cells.…”

“…Since NS1 protein can interact with the RNP complex [18], it is possible to assume that both of them may be delivered to the cell surface in association. Indirect confirmation of this hypothesis is the detection of anti-NS1 antibodies in sera from humans with laboratory-confirmed influenza [19]. In addition, it was demonstrated that nonstructural antigens are detected on the surfaces of the cells infected with influenza A virus [20].…”