A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.     

H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.     

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.     

H5N1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans

Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.     

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.     

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure—a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.     

Neutralizing Epitopes of Influenza Virus Hemagglutinin: Target for the Development of a Universal Vaccine against H5N1 Lineages

The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection. Particularly, variations within the amino acid sequences of major neutralizing epitopes of influenza virus hemagglutinin (HA) hindered the development of universal vaccines against H5N1 lineages. Based on distribution analyses of the identified major neutralizing epitopes of hemagglutinin, we selected three vaccine strains that cover the entire variants in the neutralizing epitopes among the H5N1 lineages. HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the preclinical efficacy of the vaccine formulations was evaluated in a mouse model. The combination of three selected vaccine strains could effectively neutralize viruses from clades 1, 2.1, 2.2, 4, 7, and 8 of influenza H5N1 viruses. In contrast, a vaccine formulation containing only adjuvanted monovalent BacHA (mono-BacHA) or a single strain of inactivated whole viral vaccine was able to neutralize only clade 1 (homologous), clade 2.1, and clade 8.0 viruses. Also, the trivalent BacHA vaccine was able to protect 100% of the mice against challenge with three different clades (clade 1.0, clade 2.1, and clade 7.0) of H5N1 strains compared to mono-BacHA or inactivated whole viral vaccine. The present findings provide a rationale for the development of a universal vaccine against H5N1 lineages. Furthermore, baculoviruses displaying HA will serve as an ideal choice for a vaccine in prepandemic or pandemic situations and expedite vaccine technology without the requirement of high-level-biocontainment facilities or tedious protein purification processes.The nature of influenza virus to randomly mutate and evolve into new types with diverse antigenic determinants is an important challenge in the control of influenza infection (20). This has been evidently recognized by the recent outbreaks of H5N1 avian influenza virus infection and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV). In fact, it has been well documented in the literature that H5N1 had acquired the ability to infect human tissues due mainly to the occurrence of mutation events (1). Highly pathogenic avian influenza (HPAI) H5N1 viruses are antigenically distinguishable owing to differences in hemagglutinin (HA) sequences, the principal determinant of immunity to influenza virus, resulting in different lineages or clades of H5N1 (13, 33). The control of infection with current H5N1 vaccines does not appear to be effective against heterologous strains or phylogenetically variant clades of H5N1 in part due to variations in the HA sequences, particularly within the neutralizing epitope region. Since present vaccines are based solely on the induction of neutralizing antibodies against these epitopes, differences in these sequences may render current vaccines unqualified for the prevention of influenza globally (15, 28, 31). To overcome such limitations and to completely realize the potential of vaccines worldwide, the concept of universal vaccines based on conserved viral proteins has recently been proposed. The highly conserved ion channel protein (M2) and the nucleoprotein (NP) of influenza virus have been evaluated for the induction of cross-protective cellular immunity and viral clearance (2, 35). Antibodies generated against these conserved proteins may reduce viral spread and accelerate recovery from influenza (14). However, antibodies specific to these proteins are poorly immunogenic and were found previously to be infection permissive (5-7, 13). Thus, the development of a vaccine based on influenza virus hemagglutinin appears to be the only viable option to prevent infections by HPAI viruses such as H5N1 viruses. Nevertheless, amino acid variations within the major antigenic neutralizing epitope regions among H5 subtypes restrict the development of such universal vaccines against different H5N1 lineages.The development of a universal vaccine based entirely on HA of influenza virus is still feasible, if the variation or conservation of neutralizing epitopes among the several HPAI H5N1 virus clades can be identified. An understanding of the distribution pattern of such neutralizing epitopes could help in the design of future vaccines by incorporating two or more ideal H5N1 strains in the vaccine composition. The neutralizing epitopes of the selected viral strains should cover the variations among most H5 subtypes in order to acquire broad-range protective immunity against most H5N1 subtypes. Previous attempts to identify amino acid substitutions within HA sequences of variants that escaped from neutralization by monoclonal antibodies (MAbs) revealed the neutralizing epitope sites of HA (9, 10). Along with previous findings, we report here the identification of other major neutralizing epitopes of H5N1 by mapping their amino acid sequences using neutralizing monoclonal antibodies (n-MAbs). Analysis of the distribution of all identified neutralizing epitopes among H5 subtypes revealed variations within the antigenic determinants of H5N1 subtypes from both human and avian sources. Based on these results, we have selected three vaccine strains comprising the major neutralizing epitopes of HA to cover the entire variants within H5N1 lineages. In order to test our hypothesis in vivo, HA proteins of selected vaccine strains were expressed on the baculovirus surface (BacHA), and the efficacy of the vaccine formulations was evaluated with a mouse model challenged with phylogenetically variant H5N1 strains.     

Highly conserved M2e and hemagglutinin epitope-based recombinant proteins induce protection against influenza virus infection

Highly pathogenic influenza viruses continue to cause serious threat to public health due to their pandemic potential, calling for an urgent need to develop effective, safe, convenient, and universal vaccines against influenza virus infection. In this study, we constructed two recombinant protein vaccines, 2H5M2e-2H7M2e-H5FP-H7FP (hereinafter M2e-FP-1) and 2H5M2e-H5FP-2H7M2e-H7FP (hereinafter M2e-FP-2), by respectively linking highly conserved sequences of two molecules of ectodomain of M2 (M2e) and one molecule of fusion peptide (FP) epitope of hemagglutinin (HA) of H5N1 and H7N9 influenza viruses in different orders. The Escherichia coli-expressed M2e-FP-1 and M2e-FP-2 proteins induced similarly high-titer M2e-FP-specific antibodies in the immunized mice. Importantly, both proteins were able to prevent lethal challenge of heterologous H1N1 influenza virus, with significantly reduced viral titers and alleviated pathological changes in the lungs, as well as increased body weight and complete survivals, in the challenge mice. Taken together, our study demonstrates that highly conserved M2e and FP epitope of HA of H5N1 and H7N9 influenza viruses can be used as important targets for development of safe and economical universal influenza vaccines, and that the position of H7N9 M2e and H5N1 HA epitope sequences in the vaccine components has no significant effects on the immunogenicity and efficacy of M2e-FP-based subunit vaccines.     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.     

A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin

The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. We isolated human monoclonal antibody 5J8, which neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Fine mapping of the interaction unexpectedly revealed a novel epitope between the receptor-binding pocket and the Ca₂ antigenic site on HA. This antibody exposes a new mechanism underlying broad immunity against H1N1 influenza viruses and identifies a conserved epitope that might be incorporated into engineered H1 virus vaccines.     

Generation of High-yield Vaccine Strain Wholly Derived from Avian Influenza Viruses by Reverse Genetics

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the haemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 were generated by cell transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with haemagglutination unit of 1:2 048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce haemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and the maximum geometric mean HI-titers were observed 4–5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1-infected and H5N3-vaccinated animals.     

Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.     

利用反向遗传学技术构建H5亚型禽流感高产疫苗株

采用RT-PCR技术分别扩增了鹅源高产禽流感病毒的6条内部基因片段,近期分离的H5N1亚型禽流感病毒的血凝素基因以及N3亚型参考毒株的神经氨酸酶基因,分别构建了8个基因的转录与表达载体,利用反向遗传学技术拯救出了全部基因都源于禽源的重组流感病毒疫苗株rH5N3。通过对血凝素蛋白HA1和HA2连接肽处的5个碱性氨基酸(R-R-R-K-K)基因缺失与修饰,从而消除了病毒基因的毒力相关序列,拯救的rH5N3疫苗株对鸡和鸡胚均无致病性,病毒在鸡胚尿囊液和细胞培养上清的HA效价得到极大提高,分别为12048和1512。制备的禽流感疫苗免疫动物后4~5周即可诱导产生高效价的HI抗体,鸡免疫后18周依然保持高水平的HI抗体。重组疫苗不论是对于国内早期分离的禽流感病毒A/Goose/Guangdong/1/96还是近期分离的A/Goose/HLJ/QFY/04都能够产生完全的免疫保护作用,免疫鸡攻毒后不发病、不排毒、不死亡。带有N3鉴别诊断标记禽流感疫苗株的研制为H5N1高致病性禽流感的防治提供了新的技术保障。     

Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.     

Identification of a highly conserved H1 subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza A virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ² = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.     

Antibodies to Antigenic Site A of Influenza H7 Hemagglutinin Provide Protection against H7N9 Challenge

Identifying major antigenic and protective epitopes of the H7 hemagglutinin (HA) will be important for understanding the antibody response to vaccines developed against the novel influenza H7N9 viruses that emerged in China in 2013. To facilitate antigenic characterization of the H7N9 HA and to develop reagents for evaluation of H7N9 candidate vaccines, we generated a panel of murine monoclonal antibodies (mAbs) to the HA of A/Shanghai/2/2013 using mammalian cell-derived virus-like particles (VLP) containing the H7 HA. Neutralizing antibodies identified an HA epitope corresponding to antigenic site A on the structurally similar influenza H3 hemagglutinin. Importantly, the neutralizing antibodies protect against A/Shanghai/2/2013 challenge. This antigenic site is conserved among many H7 viruses, including strains of both Eurasian and North American lineage, and the isolated neutralizing antibodies are cross-reactive with older H7 vaccine strains. The results indicate that the identified antigenic site is a potentially important protective epitope and suggest the potential benefit of cross-reactive antibody responses to vaccination with H7 candidate vaccines.     

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A (H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses (compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets

This study focused on identifying the conserved epitopes in a single subtype A (H3N2)-as candidates for vaccine targets. We identified a total of 32 conserved epitopes in four viral proteins [22 HA, 4PB1, 3 NA, 3 NP]. Evaluation of conserved epitopes in coverage during 1968-2010 revealed that (1) 12 HA conserved epitopes were highly present in the circulating viruses; (2) the remaining 10 HA conserved epitopes appeared with lower percentage but a significantly increasing trend after 1989 [p<0.001]; and (3) the conserved epitopes in NA, NP and PB1 are also highly frequent in wild-type viruses. These conserved epitopes also covered an extremely high percentage of the 16 vaccine strains during the 42 year period. The identification of highly conserved epitopes using our approach can also be applied to develop broad-spectrum vaccines.     

Protective cross-reactive cellular immunity to lethal A/Goose/Guangdong/1/96-like H5N1 influenza virus is correlated with the proportion of pulmonary CD8(+) T cells expressing gamma interferon

A/Goose/Guangdong/1/96-like H5N1 influenza viruses now circulating in southeastern China differ genetically from the H5N1 viruses transmitted to humans in 1997 but were their precursors. Here we show that the currently circulating H9N2 influenza viruses provide chickens with cross-reactive protective immunity against the currently circulating H5N1 influenza viruses and that this protective immunity is closely related to the percentage of pulmonary CD8(+) T cells expressing gamma interferon (IFN-gamma). In vivo depletion of T-cell subsets showed that the cross-reactive immunity was mediated by T cells bearing CD8(+) and T-cell receptor (TCR) alpha/beta and that the Vbeta1 subset of TCR alpha/beta T cells had a dominant role in protective immunity. The protective immunity induced by infection with H9N2 virus declined with time, lasting as long as 100 days after immunization. Shedding of A/Goose/Guangdong/1/96-like H5N1 virus by immunized chickens also increased with the passage of time and thus may play a role in the perpetuation and spread of these highly pathogenic H5N1 influenza viruses. Our findings indicate that pulmonary cellular immunity may be very important in protecting na?ve natural hosts against lethal influenza viruses.