Ritsuko Kubota-Koketsu

Naturally Occurring Antibodies in Humans Can Neutralize a Variety of Influenza Virus Strains, Including H3, H1, H2, and H5

ABSTRACT Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the VH1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use VH1-69. The possible epitope recognized by these clones is discussed.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors.

Abstract Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine–human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

Interleukin-1 Family Cytokines as Mucosal Vaccine Adjuvants for Induction of Protective Immunity against Influenza Virus

ABSTRACT A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1β, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/Wv mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.

A cross-reactive neutralizing monoclonal antibody protects mice from H5N1 and pandemic (H1N1) 2009 virus infection

A novel influenza (H1N1) virus caused an influenza pandemic in 2009, while highly pathogenic H5N1 avian influenza viruses have continued to infect humans since 1997. Influenza, therefore, remains a serious health threat. Currently, neuraminidase (NA) inhibitors are the mainstay for influenza therapy; however, drug-resistant mutants of seasonal H1N1 and H5N1 viruses have emerged highlighting the need for alternative therapeutic approaches. One such approach is antibody immunotherapy. Here, we show that the monoclonal antibody C179, which recognizes a neutralizing epitope common among H1, H2, H5, and H6 hemagglutinins (HAs), protected mice from a lethal challenge with various H5N1 and pandemic (H1N1) 2009 viruses when administered either intraperitoneally or intranasally. The protective efficacy of intranasally inoculated C179 was comparable to that of intraperitoneal administration. Our results suggest that direct administration of this anti-influenza antibody to viral replication sites is an effective strategy for prophylaxis and therapy.

Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

ABSTRACT A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses. IMPORTANCE Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism. Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.

Monoclonal antibodies in man that neutralized H3N2 influenza viruses were classified into three groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

We tried to reveal the strain specificity of neutralizing mAbs against H3N2 influenza viruses in individuals. A large number of B lymphocytes of a pediatrician were collected by apheresis and two Ab libraries were constructed at 2004 and 2007 by using the phage-display technology. The libraries were screened against 12 different H3 strains of flu isolated between 1968 and 2004. Large numbers of clones that bound to the Ags were isolated and mAbs that specifically bound to H3 strain viruses were selected. Their binding activity to the 12 strains and neutralizing activity were studied by ELISA and focus reduction test, respectively. Furthermore, the binding activity to hemagglutinin (HA) was examined by Western blot. The majority of clones showing the neutralizing activity turned out to be anti-HA mAbs and could be divided into three major groups showing distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003.

Highly conserved sequences for human neutralization epitope on hemagglutinin of influenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition

The epitope sequences within the hemagglutinin (HA) of influenza A virus H3N2 at amino acid residues 173-181 and 227-239 that forms anti-parallel beta-sheet structure are similarly recognized by human monoclonal antibodies (HuMAbs), B-1 and D-1 that we recently obtained using the peripheral blood lymphocytes from two influenza-vaccinated volunteers. Both HuMAbs showed strong global neutralization of H3N2 strains. Here we show the significant conservation of the beta-sheet region consisting of the above-mentioned two epitope regions in H3N2. In addition, we also identified the corresponding regions with similar structure in other subtypes such as H1N1 and H5N1. These two regions are similarly located underneath the receptor-binding sites of individual subtypes. Analysis of those regions using sequences available from the Influenza Virus Resource at the National Center for Biotechnology Information revealed that compared with those in the known neutralizing epitopes A-E, those sequences were fairly conserved in human H3N2 (n=7955), swine H1N1 (n=360) and swine H3N2 (n=235); and highly conserved in human H1N1 (n=2722), swine-origin pandemic H1N1 (n=1474), human H5N1 (n=319) and avian H5N1 (n=2349). Phylogenetic tree for these regions formed clearly separable clusters for H1N1, H3N2 and H5N1, irrespective of different host origin. These data may suggest a possible significance of those regions for development of alternative vaccine that could induce neutralizing antibodies reactive against wide-range of influenza virus strains.

Development of Two Types of Rapid Diagnostic Test Kits to Detect the Hemagglutinin or Nucleoprotein of the Swine-Origin Pandemic Influenza A Virus H1N1

ABSTRACT Since its emergence in April 2009, pandemic influenza A virus H1N1 (H1N1 pdm), a new type of influenza A virus with a triple-reassortant genome, has spread throughout the world. Initial attempts to diagnose the infection in patients using immunochromatography (IC) relied on test kits developed for seasonal influenza A and B viruses, many of which proved significantly less sensitive to H1N1 pdm. Here, we prepared monoclonal antibodies that react with H1N1 pdm but not seasonal influenza A (H1N1 and H3N2) or B viruses. Using two of these antibodies, one recognizing viral hemagglutinin (HA) and the other recognizing nucleoprotein (NP), we developed kits for the specific detection of H1N1 pdm and tested them using clinical specimens of nasal wash fluid or nasopharyngeal fluid from patients with influenza-like illnesses. The specificities of both IC test kits were very high (93% for the HA kit, 100% for the NP kit). The test sensitivities for detection of H1N1 pdm were 85.5% with the anti-NP antibody, 49.4% with the anti-HA antibody, and 79.5% with a commercially available influenza A virus detection assay. Use of the anti-NP antibody could allow the rapid and accurate diagnosis of H1N1 pdm infections.