Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.     

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.     

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.     

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.     

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.     

Induction of Cross-Reactive Antibodies to Novel H7N9 Influenza Virus by Recombinant Newcastle Disease Virus Expressing a North American Lineage H7 Subtype Hemagglutinin

Severe human disease caused by the emerging H7N9 influenza virus in China warrants a rapid response. Here, we present a recombinant Newcastle disease virus expressing a North American lineage H7 influenza virus hemagglutinin. Sera from immunized mice were cross-reactive to a broad range of H7 subtype viruses and inhibited hemagglutination by the novel H7 hemagglutinin. Immunized mice were protected against a heterologous H7 subtype challenge, and genetic analysis suggested that cross-protective antibodies recognize conserved antigenic sites.     

An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.The highly pathogenic avian influenza A subtype H5N1 virus was first isolated from geese in Guangdong province, China, in 1996 (44). Since 2003, the H5N1 strains have caused major morbidity and mortality in poultry populations across Asia, Europe, and Africa (3, 25). In 1997, the virus was transmitted from chickens to humans in Hong Kong, causing 18 reported cases of illness, including 6 deaths (6, 7, 37). As of September 2009, there were 442 confirmed human infections in 15 countries, with an alarming fatality rate of 59% (42). Although occurrences of human H5N1 infection are sporadic and rare, its rapid dissemination, the ongoing evolution of the avian H5N1 virus, and the absence of anti-H5N1 herd immunity in humans raise concerns regarding a possible H5N1 influenza pandemic (2, 4, 13). Since human infections are associated with severe disease and high mortality, the consequences of a pandemic could be catastrophic.Current strategies against influenza include vaccination and antiviral drug treatment (1). Due to the existence of multiple antigenic clades and subclades of the H5N1 virus, the difficulty of predicting the major strain that may cause the next pandemic is the main obstacle to current vaccine development. Moreover, resistance to M2 ion channel inhibitors (rimantidine and amantidine) has been reported in H5N1 isolates (1, 5), and the neuraminidase inhibitors (oseltamivir and zanamivir) require higher doses and prolonged treatment (45), and resistance has been reported in children (21). Passive immunotherapy is now increasingly used to treat numerous human infectious diseases (28, 33). Convalescent-phase blood and serum products were used to improve clinical outcomes for severely ill influenza patients during the 1918 influenza pandemic (27). Promising results with mouse models using a neutralizing monoclonal antibody (MAb) for H5N1 influenza treatment (17, 26) and a report of the recovery of an H5N1 virus-infected patient after treatment with convalescent-phase plasma (47) indicate that MAbs could be a potential treatment against H5N1 viruses.The hemagglutinin (HA) protein is one of the two major surface glycoproteins on the envelope of influenza A virus, with 16 distinct types identified in the avian species. The HA protein is responsible for receptor binding to host cells and for viral entry and is therefore the primary target of neutralizing antibodies (Abs) (35). It is a homotrimer, with each subunit made up of two disulfide-linked polypeptides, HA1 and HA2. Structurally, each subunit consists of a membrane-proximal helix-rich stem structure and a membrane-distal receptor binding globular domain (35).In this study, we describe a MAb, named MAb 9F4, raised against the recombinant baculovirus-expressed HA protein of A/chicken/Hatay/2004 H5N1 virus. Its neutralizing property was investigated, and epitope mapping was performed. The MAb 9F4 binding site was found to lie outside previously characterized antigenic sites in the HA protein. This epitope is well conserved among the different clades of H5N1 viruses, consistent with the cross-neutralizing activity of MAb 9F4. The mode of inhibition was also investigated, and MAb 9F4 was found to mediate postattachment neutralization in a dose-dependent manner. Finally, the protective ability of MAb 9F4 was also evaluated in a mouse model, and it was shown to protect against lethal H5N1 challenge both prophylactically and therapeutically. Taken together, the data could provide new information for the design of an H5N1 vaccine, and MAb 9F4 may be a possible candidate for use in passive immunotherapy.     

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background

The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.

Methods and Findings

Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM⁺ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.

Conclusions

The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM⁺ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.     

Influenza viruses are T cell-independent B cell mitogens.

UV-inactivated influenza virus A strains of subtypes H1, H2, H3, and H6 were shown to be mitogenic for unprimed splenic lymphocytes from BALB/c mice. Representative viruses of these four subtypes all behaved as T cell-independent B cell mitogens. The magnitude of the proliferative response was determined by the subtype of the hemagglutinin molecule: H2 and H6 viruses were the most potent mitogens, and H3 viruses were moderately mitogenic, whereas H1 viruses induced only low, but significant, levels of proliferation. Mitogenesis was inhibited by antiviral sera and by monoclonal antibodies directed against hemagglutinin.     

A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.     

Production and characterization of monoclonal antibodies against the hemagglutinin of H5N1 and antigenic investigation of avian influenza H5N1 viruses isolated from China

Eight monoclonal antibodies against hemagglutinin of influenza A virus A/Chicken/Henan/01/2004(H5N1) were produced by a DNA prime and inactivated virions-boost immunization strategy. Among the monoclonal antibodies, 3 (H50, H56, and H57) exhibited hemagglutination inhibition activity. Western blot analyses revealed that all the monoclonal antibodies reacted to the prokaryotically expressed HA1 of A/Chicken/Henan/01/2004(H5N1). The monoclonal antibodies were then used to characterize 10 avian influenza H5N1 viruses isolated from China during 2004 to 2007, by using the hemagglutination inhibition test and the antigen-capture enzyme-linked immunosorbent assay. The isolates could be divided into 4 different antigenic groups according to their responses to the monoclonal antibodies. The antigenic grouping of these 10 H5N1 isolates, using these antibodies, did not completely match their phylogenetic classification based on the hemagglutinin sequences. The results showed there were antigenic variations within the subclade 2.3.4 of H5N1, which is predominant in China.     

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.     

Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.     

Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

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.     

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.     

A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

Background

Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.

Methodology/Principal Findings

Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.

Conclusion/Significance

The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.     

Avian influenza h6 viruses productively infect and cause illness in mice and ferrets

Influenza pandemic preparedness has focused on influenza virus H5 and H7 subtypes. However, it is not possible to predict with certainty which subtype of avian influenza virus will cause the next pandemic, and it is prudent to include other avian influenza virus subtypes in pandemic preparedness efforts. An H6 influenza virus was identified as a potential progenitor of the H5N1 viruses that emerged in Hong Kong in 1997. This virus continues to circulate in the bird population in Asia, and other H6 viruses are prevalent in birds in North America and Asia. The high rate of reassortment observed in influenza viruses and the prevalence of H6 viruses in birds suggest that this subtype may pose a pandemic risk. Very little is known about the replicative capacity, immunogenicity, and correlates of protective immunity for low-pathogenicity H6 influenza viruses in mammals. We evaluated the antigenic and genetic relatedness of 14 H6 influenza viruses and their abilities to replicate and induce a cross-reactive immune response in two animal models: mice and ferrets. The different H6 viruses replicated to different levels in the respiratory tracts of mice and ferrets, causing varied degrees of morbidity and mortality in these two models. H6 virus infection induced similar patterns of neutralizing antibody responses in mice and ferrets; however, species-specific differences in the cross-reactivity of the antibody responses were observed. Overall, cross-reactivity of neutralizing antibodies in H6 virus-infected mice did not correlate well with protection against heterologous wild-type H6 viruses. However, we have identified an H6 virus that induces protective immunity against viruses in the North American and Eurasian lineages.     

Naturally occurring antibodies in humans can neutralize a variety of influenza virus strains, including H3, H1, H2, and H5

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 V(H)1-69 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 V(H)1-69. The possible epitope recognized by these clones is discussed.