Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells

We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.     

A sensitive retroviral pseudotype assay for influenza H5N1‐neutralizing antibodies

Background The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. Results Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. Conclusions The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high‐throughput format for human and animal surveillance and for the evaluation of vaccines.     

感染人类的禽流感病毒A（H5N1）研究进展

禽流感病毒A（H5N1）[avian influenza（H5N1） viruses]之前一直存在于鸟类，但却能导致人类疾病，并且具有高致死性和广泛流行的威胁。本文在综合了第二届世界卫生组织（World Health Organization，WHO）感染人类禽流感病毒A（H5N1）临床诊断咨询会议公布的研究信息基础上，对2005年的报告进行了更新。     

Specificity,kinetics and longevity of antibody responses to avian influenza A(H7N9) virus infection in humans

ObjectivesThe long-term dynamics of antibody responses in patients with influenza A(H7N9) virus infection are not well understood.MethodsWe conducted a longitudinal serological follow-up study in patients who were hospitalized with A(H7N9) virus infection, during 2013–2018. A(H7N9) virus-specific antibody responses were assessed by hemagglutination inhibition (HAI) and neutralization (NT) assays. A random intercept model was used to fit a curve to HAI antibody responses over time. HAI antibody responses were compared by clinical severity.ResultsOf 67 patients with A(H7N9) virus infection, HAI antibody titers reached 40 on average 11 days after illness onset and peaked at a titer of 290 after three months, and average titers of ≥80 and ≥40 were present until 11 months and 22 months respectively. HAI antibody responses were significantly higher in patients who experienced severe disease, including respiratory failure and acute respiratory distress syndrome, compared with patients who experienced less severe illness.ConclusionsPatients with A(H7N9) virus infection who survived severe disease mounted higher antibody responses that persisted for longer periods compared with those that experienced moderate disease. Studies of convalescent plasma treatment for A(H7N9) patients should consider collection of donor plasma from survivors of severe disease between 1 and 11 months after illness onset.     

Monitoring of perceptions,anticipated behavioral,and psychological responses related to H5N1 influenza

Background  

The aim of this study was to monitor changes in behavioral and emotional responses to human H5N1 in the community over a 28-month period (from November 2005 to February 2008).     

Review of clinical symptoms and spectrum in humans with influenza A/H5N1 infection

Abstract:   Influenza A/H5N1 infection has become the major emerging infectious disease of global concern again since late 2003. A history of exposure to dead or sick poultry or wild birds occurs in over 60% of cases of human H5N1 infection. The incubation period of avian-to-human transmission is generally between 2 and 5 days and the median duration of symptoms before hospitalization is about 4.5 days. The clinical spectrum has ranged from asymptomatic infection or mild influenza-like illness to severe pneumonia and multi-organ failure. Fever > 38°C, cough and dyspnoea are the major symptoms on presentation, whereas gastrointestinal symptoms such as watery diarrhoea, vomiting and abdominal pain are common early in the course of the disease. In contrast, upper respiratory tract symptoms are less prominent in human H5N1 infection when compared to seasonal influenza. Laboratory features of human H5N1 infection include leucopoenia, especially lymphopenia, elevated amino-transaminases, thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time, increased D-Dimer, increased serum lactate dehydrogenase and creatinine phospho-kinase, and hypoalbuminemia. A low absolute lymphocyte count on admission is associated with more severe disease and death. Radiographic abnormalities include multi-focal airspace consolidation, interstitial infiltrates, patchy or lobar involvement, with rapid progression to bilateral and diffuse ground-glass opacities consistent with ARDS. However, none of the clinical, laboratory and radiographic features are specific to H5N1 infection. A detailed exposure history needs to be elicited, including any close contact with sick or dead poultry, wild birds, other severely ill persons, travel to an area with A/H5N1 activity or work in laboratory handling samples possibly containing A/H5N1 virus.     

Cross-reactive H1N1 antibody responses to a live attenuated influenza vaccine in children: implication for selection of vaccine strains

Cross-reactive antibody responses of 3 trivalent, live attenuated intranasal influenza vaccine (FluMist) formulations containing 3 different H1N1 strains (A/Texas/36/91, A/Shenzhen/227/95, and A/Beijing/262/95) were evaluated in initially seronegative children. FluMist containing A/Shenzhen/227/95 was more likely to induce cross-reactive hemagglutination inhibition (HAI) antibody against A/Texas/36/91 than against A/Beijing/262/95, and FluMist containing A/Beijing/262/95 induced low levels of cross-reactive HAI antibody against A/Shenzhen/227/95 and A/New Caledonia/20/99. The observed differences in HAI cross-reactivity seem to be partly related to the number of amino acid (aa) differences on the hemagglutinin 1 domain (328 aa residues) rather than the hemagglutinin protein (550 aa residues).     

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.