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.publishedVersio

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 Lectine Assay (ELLA). IFN?-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 towards 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

Epidemiological characteristics of COVID-19 cases and estimates of the reproductive numbers 1 month into the epidemic, Italy, 28 January to 31 March 2020

BackgroundOn 20 February 2020, a locally acquired coronavirus disease (COVID-19) case was detected in Lombardy, Italy. This was the first signal of ongoing transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the country. The number of cases in Italy increased rapidly and the country became the first in Europe to experience a SARS-CoV-2 outbreak.AimOur aim was to describe the epidemiology and transmission dynamics of the first COVID-19 cases in Italy amid ongoing control measures.MethodsWe analysed all RT-PCR-confirmed COVID-19 cases reported to the national integrated surveillance system until 31 March 2020. We provide a descriptive epidemiological summary and estimate the basic and net reproductive numbers by region.ResultsOf the 98,716 cases of COVID-19 analysed, 9,512 were healthcare workers. Of the 10,943 reported COVID-19-associated deaths (crude case fatality ratio: 11.1%) 49.5% occurred in cases older than 80 years. Male sex and age were independent risk factors for COVID-19 death. Estimates of R0 varied between 2.50 (95% confidence interval (CI): 2.18-2.83) in Tuscany and 3.00 (95% CI: 2.68-3.33) in Lazio. The net reproduction number Rt in northern regions started decreasing immediately after the first detection.ConclusionThe COVID-19 outbreak in Italy showed a clustering onset similar to the one in Wuhan, China. R0 at 2.96 in Lombardy combined with delayed detection explains the high case load and rapid geographical spread. Overall, Rt in Italian regions showed early signs of decrease, with large diversity in incidence, supporting the importance of combined non-pharmacological control measures

International laboratory comparison of influenza microneutralization assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) influenza viruses by CONSISE

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HAMNassay protocols to enable better correlation of these assays in the future

2015/16 seasonal vaccine effectiveness against hospitalisation with influenza a(H1N1)pdm09 and B among elderly people in Europe: Results from the I-MOVE+ project

We conducted a multicentre test-negative caseâ\u80\u93control study in 27 hospitals of 11 European countries to measure 2015/16 influenza vaccine effectiveness (IVE) against hospitalised influenza A(H1N1)pdm09 and B among people aged â\u89¥ 65 years. Patients swabbed within 7 days after onset of symptoms compatible with severe acute respiratory infection were included. Information on demographics, vaccination and underlying conditions was collected. Using logistic regression, we measured IVE adjusted for potential confounders. We included 355 influenza A(H1N1)pdm09 cases, 110 influenza B cases, and 1,274 controls. Adjusted IVE against influenza A(H1N1)pdm09 was 42% (95% confidence interval (CI): 22 to 57). It was 59% (95% CI: 23 to 78), 48% (95% CI: 5 to 71), 43% (95% CI: 8 to 65) and 39% (95% CI: 7 to 60) in patients with diabetes mellitus, cancer, lung and heart disease, respectively. Adjusted IVE against influenza B was 52% (95% CI: 24 to 70). It was 62% (95% CI: 5 to 85), 60% (95% CI: 18 to 80) and 36% (95% CI: -23 to 67) in patients with diabetes mellitus, lung and heart disease, respectively. 2015/16 IVE estimates against hospitalised influenza in elderly people was moderate against influenza A(H1N1)pdm09 and B, including among those with diabetes mellitus, cancer, lung or heart diseases

Ecology of Avian Influenza Viruses in Siberia

Wild aquatic birds represent major reservoirs of the influenza A virus gene pool, from which novel influenza viruses can emerge to infect other avian and mammalian species, including humans. These avian hosts have long been considered the natural reservoir for low pathogenic avian influenza viruses (AIVs) that, in some occasions, can evolve in poultry to become highly pathogenic (HP) AIVs posing a risk for animal and public health. However, changes in viral ecology have been recently observed with the possibility of perpetuation of HPAIVs in the aquatic bird reservoir. Siberia is of great importance in influenza A virus ecology. After a short reproductive season, huge numbers of wild aquatic birds move from different subarctic breeding sites and congregate in pre-migration staging areas of Siberian wetlands. Major intra-and inter-continental bird flyways overlap in these breeding and/or molting grounds where water birds from different wintering regions of the world are brought together, thus providing the influenza A virus gene pool for the possible emergence of novel reassortant viruses. Moreover, in the context of the global ecology of influenza A viruses, Siberian wetlands play a crucial role in the geographical dispersal of the virus, as shown by the HPAI H5 infections, which have spread since the early 2000s via migratory birds from Asia towards Europe, Africa, Middle East and, more recently, North America. Here we consider ecological interactions that take place within the vast region of Siberia among the avian reservoir hosts and viral populations, and the environment they utilize. We also consider ecological drivers which, in the context of the current global change, can modulate in this Asian portion of Russia the influenza A virus circulation and spill-over from wild bird reservoirs to different avian and/or mammalian hosts

Generation of switched memory B cells in response to vaccination in Down syndrome children and their siblings

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Moderate influenza vaccine effectiveness against A(H1N1)pdm09 virus, and low effectiveness against A(H3N2) subtype, 2018/19 season in Italy

Background: Influenza vaccines are updated every year to match the vaccine strains with currently circulating viruses; consequently influenza vaccine effectiveness (IVE) has to be assessed annually.Research design and methods: A test-negative case-control study was conducted within the context of the Italian sentinel influenza surveillance network to estimate IVE by age group, virus subtype, and vaccine brand in medically attended laboratory-confirmed influenza.Results: In Italy, the 2018/19 influenza season was characterized by the co-circulation of influenza A(H1N1)pdm09 and A(H3N2) viruses. The adjusted IVE estimate in preventing influenza was moderate (44.8%, 95% CI: 18.8 to 62.5) against A(H1N1)pdm09, whereas there was no evidence of effectiveness (1.8%, 95% CI: -37.8 to 30.1) in persons affected by A(H3N2). IVE against A(H1N1)pdm09 decreased with age ranging from 65.7% to 13.1% among children/adolescents and elderly, respectively; moreover results suggest that Vaxigrip Tetra® was more effective against A(H1N1)pdm09 compared to Fluarix Tetra® [62.5% (95% CI: 34.3 to 78.6) vs 24.5% (95% CI: -40.6 to 59.6)]. Low effectiveness (35.2%, 95% CI: -50.8 to 72.1) against A(H3N2) was detected only in the elderly immunized with Fluad®.Conclusions: Findings suggest that influenza vaccines were low to moderately effective, probably due to a mismatch between circulating and vaccine strains

Virological Surveillance of Influenza in the eight epidemic seasons after the 2009 pandemic in Emilia-Romagna (Northern Italy)

Background and aim of the work: Influenza virological surveillance is essential for monitoring the evolution of influenza viruses (IVs) as well as for annual updating of the vaccine composition. The aim of this study is to analyse IVs circulation in Emilia-Romagna during the eight epidemic seasons after the 2009 pandemic and to evaluate their match with seasonal vaccine strains. Methods: A total of 7882 respiratory specimens from patients with influenza-like illness (ILI), were collected by regional sentinel practitioners and hospital physicians. Viral investigations were conducted by rRT-PCR assay. Genetic characterization was performed for a spatial-temporal representative number of influenza laboratory-confirmed specimens. Results:Influenza-positive samples per season ranged between 28.9% (2013-2014) and 66.8% (2012-2013). Co-circulation of IVs type A and type B was observed in all seasons, although with a different intensity. In all seasons, the highest number of positive samples was recorded in younger patients aged 5-14 years with relative frequencies ranging from 40% in the 2013-2014 season and 78% in the 2012-2013 season. Since the 2009 pandemic, A/H1N1pdm09 IVs circulating were closely related to the vaccine strain A/California/7/2009. Antigenic mismatch between vaccine strain and A/H3N2 IVs was observed in the 2011-2012 and 2014-2015 seasons. During 2015-2016, 2016-2017 and 2017-2018 seasons a complete or nearly complete mismatch between the predominant influenza B lineage of IVs type B circulating and vaccine B lineage occurred. Conclusions:This analysis confirms the importance of the virological surveillance and highlights the need of a continuous monitoring of IVs circulation, to improve the most appropriate vaccination strategies