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.     

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.     

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.     

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.     

Epitope-specific antibody response to murine hepatitis virus-4 (strain JHM)

Monoclonal hybridoma antibodies to the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) were used in a competition binding enzyme immunoassay to analyze at the epitope level the antibody response of mice after infection with MHV-4. Colonized mice often had pre-existing MHV antibodies directed against epitopes on the E2 glycoprotein, the E1 glycoprotein, and the nucleocapsid protein. These mice generated a secondary antibody response after virus inoculation, reaching peak levels 7 days after infection. In contrast, Nude/+ mice raised in a pathogen-free colony had no detectable circulating MHV antibodies and generated a primary antibody response which gradually increased to peak levels 14 to 28 days after infection. Kinetics of antibody responses against specific epitopes usually correlated well with measured total virus-specific antibody responses, but variation was observed. Mice injected with three antigenically distinct strains of MHV made antibody responses to conserved epitopes but not to an antigenic determinant absent in these strains. Measurement of epitope-specific responses in a polyclonal population of viral specific antibodies is feasible and a valuable adjunct in understanding viral immunity.     

Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses

Under selective pressure from the host immune system, antigenic epitopes of influenza virus hemagglutinin (HA) have continually evolved to escape antibody recognition, termed antigenic drift. We analyzed the genomes of influenza A(H3N2) and A(H1N1)pdm09 virus strains circulating in Thailand between 2010 and 2014 and assessed how well the yearly vaccine strains recommended for the southern hemisphere matched them. We amplified and sequenced the HA gene of 120 A(H3N2) and 81 A(H1N1)pdm09 influenza virus samples obtained from respiratory specimens and calculated the perfect-match vaccine efficacy using the p _epitope model, which quantitated the antigenic drift in the dominant epitope of HA. Phylogenetic analysis of the A(H3N2) HA1 genes classified most strains into genetic clades 1, 3A, 3B, and 3C. The A(H3N2) strains from the 2013 and 2014 seasons showed very low to moderate vaccine efficacy and demonstrated antigenic drift from epitopes C and A to epitope B. Meanwhile, most A(H1N1)pdm09 strains from the 2012–2014 seasons belonged to genetic clades 6A, 6B, and 6C and displayed the dominant epitope mutations at epitopes B and E. Finally, the vaccine efficacy for A(H1N1)pdm09 (79.6–93.4%) was generally higher than that of A(H3N2). These findings further confirmed the accelerating antigenic drift of the circulating influenza A(H3N2) in recent years.     

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.     

Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log₁₀ range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.     

Evolution of H3N2 influenza virus in a guinea pig model

Studies of influenza virus evolution under controlled experimental conditions can provide a better understanding of the consequences of evolutionary processes with and without immunological pressure. Characterization of evolved strains assists in the development of predictive algorithms for both the selection of subtypes represented in the seasonal influenza vaccine and the design of novel immune refocused vaccines. To obtain data on the evolution of influenza in a controlled setting, naïve and immunized Guinea pigs were infected with influenza A/Wyoming/2003 (H3N2). Virus progeny from nasal wash samples were assessed for variation in the dominant and other epitopes by sequencing the hemagglutinin (HA) gene to quantify evolutionary changes. Viral RNA from the nasal washes from infection of naïve and immune animals contained 6% and 24.5% HA variant sequences, respectively. Analysis of mutations relative to antigenic epitopes indicated that adaptive immunity played a key role in virus evolution. HA mutations in immunized animals were associated with loss of glycosylation and changes in charge and hydrophobicity in and near residues within known epitopes. Four regions of HA-1 (75–85, 125–135, 165–170, 225–230) contained residues of highest variability. These sites are adjacent to or within known epitopes and appear to play an important role in antigenic variation. Recognition of the role of these sites during evolution will lead to a better understanding of the nature of evolution which help in the prediction of future strains for selection of seasonal vaccines and the design of novel vaccines intended to stimulated broadened cross-reactive protection to conserved sites outside of dominant epitopes.     

The antigenic evolution of influenza: drift or thrift?

It is commonly assumed that antibody responses against the influenza virus are polarized in the following manner: strong antibody responses are directed at highly variable antigenic epitopes, which consequently undergo ‘antigenic drift’, while weak antibody responses develop against conserved epitopes. As the highly variable epitopes are in a constant state of flux, current antibody-based vaccine strategies are focused on the conserved epitopes in the expectation that they will provide some level of clinical protection after appropriate boosting. Here, we use a theoretical model to suggest the existence of epitopes of low variability, which elicit a high degree of both clinical and transmission-blocking immunity. We show that several epidemiological features of influenza and its serological and molecular profiles are consistent with this model of ‘antigenic thrift’, and that identifying the protective epitopes of low variability predicted by this model could offer a more viable alternative to regularly update the influenza vaccine than exploiting responses to weakly immunogenic conserved regions.     

Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

MHC Class I-Presented T Cell Epitopes Identified by Immunoproteomics Analysis Are Targets for a Cross Reactive Influenza-Specific T Cell Response

Influenza virus infection and the resulting complications are a significant global public health problem. Improving humoral immunity to influenza is the target of current conventional influenza vaccines, however, these are generally not cross-protective. On the contrary, cell-mediated immunity generated by primary influenza infection provides substantial protection against serologically distinct viruses due to recognition of cross-reactive T cell epitopes, often from internal viral proteins conserved between viral subtypes. Efforts are underway to develop a universal flu vaccine that would stimulate both the humoral and cellular immune responses leading to long-lived memory. Such a universal vaccine should target conserved influenza virus antibody and T cell epitopes that do not vary from strain to strain. In the last decade, immunoproteomics, or the direct identification of HLA class I presented epitopes, has emerged as an alternative to the motif prediction method for the identification of T cell epitopes. In this study, we used this method to uncover several cross-specific MHC class I specific T cell epitopes naturally presented by influenza A-infected cells. These conserved T cell epitopes, when combined with a cross-reactive antibody epitope from the ectodomain of influenza M2, generate cross-strain specific cell mediated and humoral immunity. Overall, we have demonstrated that conserved epitope-specific CTLs could recognize multiple influenza strain infected target cells and, when combined with a universal antibody epitope, could generate virus specific humoral and T cell responses, a step toward a universal vaccine concept. These epitopes also have potential as new tools to characterize T cell immunity in influenza infection, and may serve as part of a universal vaccine candidate complementary to current vaccines.     

Immunogenic properties of multiple antigen peptide systems containing defined T and B epitopes.

Immunization with chemically defined synthetic polymers, multiple Ag peptide (MAP) systems, containing T and B epitopes of the circumsporozoite protein of P. berghei induce high levels of circulating antibodies that are detectable several months after boosting. The anti-MAP secondary antibody response is characterized by an increase in the levels of circulating IgG and a concomitant decrease in the IgM levels. In vitro and in vivo experiments indicated that Th epitopes included in the MAP are recognized by T cells induced after immunization with the native protein and, also, that MAP-induced T cells can recognize the native protein. In addition to high levels of anti-B epitope antibodies, MAP immunization also induces antibodies against the T epitope. This anti-T epitope immune response does not affect the generation of the anti-B epitope antibodies. Immunization of different strains of mice revealed that the antibody response is consistent with the genetically restricted pattern of recognition of the T epitope. There are, however, significant differences in the levels of antibody responses observed among responder strains. The findings of this study indicate that MAP are potent immunogens capable of inducing immunologic memory and are, thus, good candidates for the development of subunit vaccines designed to induce high levels of circulating antibodies.     

Pre-Existing Immunity with High Neutralizing Activity to 2009 Pandemic H1N1 Influenza Virus in Shanghai Population

Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection.     

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.     

In vitro and in vivo characterization of designed immunogens derived from the CD‐helix of the stem of influenza hemagglutinin

The conserved “stem” domain of influenza virus hemagglutinin (HA) is a target for broadly neutralizing antibodies and a potential vaccine antigen for induction of hetero‐subtypic protection. The epitope of 12D1, a previously reported bnAb neutralizing several H3 subtype influenza strains, was putatively mapped to residues 76–106 of the CD‐helix, also referred to as long alpha helix (LAH) of the HA stem. A peptide derivative consisting of wt‐LAH residues 76–130 conjugated to keyhole limpet hemocyanin was previously shown to confer robust protection in mice against challenge with influenza strains of subtypes H3, H1, and H5 which motivated the present study. We report the design of multiple peptide derivatives of LAH with or without heterologous trimerization sequences and show that several of these are better folded than wt‐LAH. However, in contrast to the previous study immunization of mice with wt‐LAH resulted in negligible protection against a lethal homologous virus challenge, while some of the newly designed immunogens could confer weak protection. Combined with structural analysis of HA, our data suggest that in addition to LAH, other regions of HA are likely to significantly contribute to the epitope for 12D1 and will be required to elicit robust protection. In addition, a dynamic, flexible conformation of isolated LAH peptide may be required for eliciting a functional anti‐viral response. Proteins 2013; 81:1759–1775. © 2013 Wiley Periodicals, Inc.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

Antigenic Characterization of Recombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes

Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA.     

Characterization of Immune Responses Induced by Immunization with the HA DNA Vaccines of Two Antigenically Distinctive H5N1 HPAIV Isolates

The evolution of the H5N1 highly pathogenic avian influenza virus (HPAIV) has resulted in high sequence variations and diverse antigenic properties in circulating viral isolates. We investigated immune responses induced by HA DNA vaccines of two contemporary H5N1 HPAIV isolates, A/bar-headed goose/Qinghai/3/2005 (QH) and A/chicken/Shanxi/2/2006 (SX) respectively, against the homologous as well as the heterologous virus isolate for comparison. Characterization of antibody responses induced by immunization with QH-HA and SX-HA DNA vaccines showed that the two isolates are antigenically distinctive. Interestingly, after immunization with the QH-HA DNA vaccine, subsequent boosting with the SX-HA DNA vaccine significantly augmented antibody responses against the QH isolate but only induced low levels of antibody responses against the SX isolate. Conversely, after immunization with the SX-HA DNA vaccine, subsequent boosting with the QH-HA DNA vaccine significantly augmented antibody responses against the SX isolate but only induced low levels of antibody responses against the QH isolate. In contrast to the antibody responses, cross-reactive T cell responses are readily detected between these two isolates at similar levels. These results indicate the existence of original antigenic sin (OAS) between concurrently circulating H5N1 HPAIV strains, which may need to be taken into consideration in vaccine development against the potential H5N1 HPAIV pandemic.