Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Serologic Evidence of Occupational Exposure to Avian Influenza Viruses at the Wildfowl/Poultry/Human Interface.

Ecological interactions between wild aquatic birds and outdoor-housed poultry can enhance spillover events of avian influenza viruses (AIVs) from wild reservoirs to domestic birds, thus increasing the related zoonotic risk to occupationally exposed workers. To assess serological evidence of AIV infection in workers operating in Northern Italy at the wildfowl/poultry interface or directly exposed to wildfowl, serum samples were collected between April 2005 and November 2006 from 57 bird-exposed workers (BEWs) and from 7 unexposed controls (Cs), planning three sample collections from each individual. Concurrently, AIV surveillance of 3587 reared birds identified 4 AIVs belonging to H10N7, H4N6 and H2N2 subtypes while serological analysis by hemagglutination inhibition (HI) assay showed recent infections caused by H1, H2, H4, H6, H10, H11, H12, and H13 subtypes. Human sera were analyzed for specific antibodies against AIVs belonging to antigenic subtypes from H1 to H14 by using HI and virus microneutralization (MN) assays as a screening and a confirmatory test, respectively. Overall, antibodies specific to AIV-H3, AIV-H6, AIV-H8, and AIV-H9 were found in three poultry workers (PWs) and seropositivity to AIV-11, AIV-H13-still detectable in October 2017-in one wildlife professional (WP). Furthermore, seropositivity to AIV-H2, accounting for previous exposure to the "extinct" H2N2 human influenza viruses, was found in both BEWs and Cs groups. These data further emphasize the occupational risk posed by zoonotic AIV strains and show the possible occurrence of long-lived antibody-based immunity following AIV infections in humans.

Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens.

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.