Report on eighth WHO meeting on development of influenza vaccines that induce broadly protective and long-lasting immune responses: Chicago, USA, 23-24 August 2016

In August 2016, the World Health Organization (WHO) convened the "Eighth meeting on development of influenza vaccines that induce broadly protective and long-lasting immune responses" to discuss the regulatory requirements and pathway

An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.publishedVersio

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1

Developing Vaccines to Combat Pandemic Influenza

Influenza vaccine manufacturers require antigenically relevant vaccine viruses that have good manufacturing properties and are safe to use. In developing pandemic vaccine viruses, reverse genetics has been employed as a rational approach that can also be used effectively to attenuate the highly virulent H5N1 virus and at the same time place the H5 HA and N1 NA on a background of PR8, a virus that has been used over many decades to provide high yielding vaccine viruses. Reverse genetics has also been used successfully alongside classical reassorting techniques in the development of (swine flu) pandemic A(H1N1)v vaccine viruses

Broad Reactivity Single Domain Antibodies against Influenza Virus and Their Applications to Vaccine Potency Testing and Immunotherapy

The antigenic variability of influenza presents many challenges to the development of vaccines and immunotherapeutics. However, it is apparent that there are epitopes on the virus that have evolved to remain largely constant due to their functional importance. These more conserved regions are often hidden and difficult to access by the human immune system but recent efforts have shown that these may be the Achilles heel of the virus through development and delivery of appropriate biological drugs. Amongst these, single domain antibodies (sdAbs) are equipped to target these vulnerabilities of the influenza virus due to their preference for concave epitopes on protein surfaces, their small size, flexible reformatting and high stability. Single domain antibodies are well placed to provide a new generation of robust analytical reagents and therapeutics to support the constant efforts to keep influenza in check