Characterization Of Epitopes Research Articles

Chemoenzymatic synthesis of HIV‐1 V1V2 glycopeptide antigens for epitope characterization and neutralizing antibody detection (1004.4)

Recent discovery of a new class of glycan‐dependent broadly neutralizing antibodies (bnAbs), including PG9, PG16, and the PGT series antibodies, provides valuable templates for HIV‐1 vaccine design. Epitope mapping suggests that PG9 and PG16 recognize unique glycopeptide antigens located in the V1V2 region of gp120. However, further characterization of the neutralizing epitopes has been difficult due to the glycosylation heterogeneity of gp120. In this paper, we report the design, synthesis, and antigenicity of well‐defined V1V2 glycopeptides aiming to characterize the glycan specificity of the epitopes. Homogeneous V1V2 glycopeptides carrying defined N‐glycans at the glycosylation sites (N160 and N156 or N173) were synthesized by a convergent chemoenzymatic approach. Binding studies confirmed the essence of a Man5GlcNAc2 glycan at Asn160 for recognition by PG9 and PG16 and further revealed a critical role of a sialylated N‐glycan at the secondary site (N156 or N173) in the context of glycopeptides. We have also synthesized novel V1V2 glycopeptides in which the innermost GlcNAc was replaced with a Glc moiety. Binding studies show that the Glc replacement did not impair antibody recognition. The usefulness of the synthetic V1V2 glycopeptides as coating antigens for neutralizing antibody detection was demonstrated in an ELISA format, which enables the detection of PG9 and PG16 at up to 50 pM concentration. The high‐affinity synthetic glycopeptides identified also provides a basis for HIV‐1 vaccine design.Grant Funding Source: Supported by NIH Grant 1R21AI101035 to L‐XW

Synthetic glycopeptide antigens for HIV vaccine design (469.1)

Heavy glycosylation of the HIV‐1 outer envelope glycoprotein gp120 forms a dense glycan shield that constitutes a strong defense mechanism for viral immune evasion. Nevertheless, the recent discovery of a new class of glycan‐dependent broadly neutralizing antibodies (bNAbs) from HIV‐infected individuals, including PG9, PG16, PGT121, PGT128 and PGT135, suggests that the viral defensive carbohydrates can also serve as targets of vaccine. Epitope mapping indicates that these bNAbs target glycopeptide structures at the variable (V1V2 or V3) regions of gp120. However, further characterization of the fine epitopes is complicated by the glycosylation heterogeneity of gp120. We have launched a project aiming to characterize and reconstitute the minimal antigenic glycopeptide structures through synthetic chemistry. A series of homogeneous glycopeptides corresponding to the V1/V2 and V3 domains were synthesized by a chemoenzymatic method, in which defined N‐glycans were installed selectively at the conserved glycosylation sites (N156, N160, N301, and N332). This study permits a detailed assessment of the glycan specificity at the respective glycosylation sites for recognition by antibodies PG9, PG16, PGT121, and PGT128. The ability to reconstitute the minimal neutralizing epitopes using well‐defined synthetic glycopeptides provides a useful template for HIV vaccine design.

Definition of CD4 Immunosignatures Associated with MTB

We have recently described the first true genome-wide screen for CD4+ T-cell reactivity directed against Mycobacterium tuberculosis (MTB) in latent TB-infected individuals. The approach relied on predictions of HLA-binding capacity for a panel of DR, DP, and DQ alleles representative of those most commonly expressed in the general population, coupled with high throughput ELISPOT assays. The results identified hundreds of novel epitopes and antigens, and documented the novel observation that T cells in latent MTB infection are confined to the CXCR3+CCR6+ phenotype and largely directed against three antigenic “islands” within the MTB genome. In parallel, we have made generally available to the scientific community the technical approaches and reagents developed in the process, such as motifs, algorithms, and binding assays for several common HLA class II alleles, and a panel of single allele HLA class II transfected cell lines representative of the most frequent specificities in the general population. Recent efforts have been focused on characterization of epitopes and antigens recognized by patients with active TB and individuals vaccinated with BCG, with the aim of providing the first systematic evaluation of the overlap between latent, active, and BCG cohorts. The definition of a broad range of epitopes restricted by common HLA molecules, will facilitate development of diagnostic reagents, allow a rigorous evaluation of T-cell responses associated with TB infection in humans, and enable the evaluation of the immunogenicity of different vaccine candidates. Furthermore, it might suggest new candidates for vaccine and diagnostic development.

Prediction and characterization of helper T-cell epitopes from pneumococcal surface adhesin A.

Pneumococcal surface adhesin A (PsaA) is a multifunctional lipoprotein known to bind nasopharyngeal epithelial cells, and is significantly involved in bacterial adherence and virulence. Identification of PsaA peptides that optimally bind human leucocyte antigen (HLA) and elicit a potent immune response would be of great importance to vaccine development. However, this is hindered by the multitude of HLA polymorphisms in humans. To identify the conserved immunodominant epitopes, we used an experimental dataset of 28 PsaA synthetic peptides and in silico methods to predict specific peptide-binding to HLA and murine MHC class II molecules. We also characterized spleen and cervical lymph node (CLN) -derived T helper (Th) lymphocyte cytokine responses to these peptides after Streptococcus pneumoniae strain EF3030 challenge in mice. Individual, yet overlapping, peptides 15 amino acids in length revealed residues of PsaA that consistently caused the highest interferon-γ, interleukin-2 (IL-2), IL-5 and IL-17 responses and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo re-stimulated splenic and CLN CD4⁺ T cells isolated from S. pneumoniae strain EF3030-challenged F1 (B6 × BALB/c) mice. In silico analysis revealed that peptides from PsaA may interact with a broad range of HLA-DP, -DQ and -DR alleles, due in part to regions lacking β-turns and asparagine endopeptidase sites. These data suggest that Th cell peptides (7, 19, 20, 22, 23 and 24) screened for secondary structures and MHC class II peptide-binding affinities can elicit T helper cytokine and proliferative responses to PsaA peptides.

A novel approach for characterisation of conformational allergen epitopes combining phage display and high-throughput sequencing

Background Characterization of allergen epitopes is an essential task in unraveling the molecular mechanisms of allergy. At present, the procedures to characterize conformational epitopes are relatively laborious and, consequently, knowledge of conformational allergen epitopes is limited. The aim of this study was to couple an established epitope mapping approach, based on phage-displayed peptide libraries, with high-throughput sequencing in order to make conformational epitope mapping less laborious and increase data output.

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy

BackgroundCharacterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy.ObjectiveWe sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients.MethodsFour distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1–sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA.ResultsrApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions.ConclusionThe Bet v 1–specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific.

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

A Neutralization Epitope in the Hepatitis C Virus E2 Glycoprotein Interacts with Host Entry Factor CD81

The identification of a specific immunogenic candidate that will effectively activate the appropriate pathway for neutralizing antibody production is fundamental for vaccine design. By using a monoclonal antibody (1H8) that neutralizes HCV in vitro, we have demonstrated here that 1H8 recognized an epitope mapped between residues A524 and W529 of the E2 protein. We also found that the epitope residues A524, P525, Y527 and W529 were crucial for antibody binding, while the residues T526, Y527 and W529 within the same epitope engaged in the interaction with the host entry factor CD81. Furthermore, we detected “1H8-like” antibodies, defined as those with amino acid-specificity similar to 1H8, in the plasma of patients with chronic HCV infection. The time course study of plasma samples from Patient H, a well-characterized case of post-transfusion hepatitis C, showed that “1H8-like” antibodies could be detected in a sample collected almost two years after the initial infection, thus confirming the immunogenicity of this epitope in vivo. The characterization of this neutralization epitope with a function in host entry factor CD81 interaction should enhance our understanding of antibody-mediated neutralization of HCV infections.

Structural characterization of viral epitopes recognized by broadly cross-reactive antibodies.

Influenza hemagglutinin (HA) is the major surface glycoprotein on influenza viruses and mediates viral attachment and subsequent fusion with host cells. The HA is the major target of the immune response, but due to its high level of variability, as evidenced by substantial antigenic diversity, it had been historically considered to elicit only a narrow, strain-specific antibody response. However, a recent explosion in the discovery of broadly neutralizing antibodies (bnAbs) to influenza virus has identified two major supersites of vulnerability on the HA through structural characterization of HA-antibody complexes. These commonly targeted epitopes are involved with receptor binding as well as the fusion machinery and, hence, are functionally conserved and less prone to mutation. These bnAbs can neutralize viruses by blocking infection or the spread of infection by preventing progeny release. Structural analyses of these bnAbs show they exhibit striking similarities and trends in recognition of the HA and use recurring recognition motifs, despite substantial differences in their germline genes. This information can be utilized in design of novel therapeutics as well as in immunogens for improved vaccines with greater breadth and efficacy.

Databases for B-cell epitopes.

Identification and characterization of B-cell epitopes in target antigens is one of the key steps in epitope-driven vaccine design, immunodiagnostic tests, and antibody production. For localizing epitopes by experimental methods is time consuming and cost expensive, researchers have been developing in silico or computational models for the prediction of B-cell epitopes, enabling immunologists and clinicians to identify the most promising epitopes for characterization in the laboratory. A sufficient number of available B-cell epitopes are indispensable for establishing the prediction models. To our knowledge, some popular databases associated with the B-cell epitopes are proposed and widely used in the immunoinformatics. In this chapter, we present an overview of the important databases and introduce how to compile datasets for the development of B-cell epitope prediction tools.

Characterization of the Protective HIV-1 CTL Epitopes and the Corresponding HLA Class I Alleles: A Step towards Designing CTL Based HIV-1 Vaccine.

Human immunodeficiency virus (HIV) possesses a major threat to the human life largely due to the unavailability of an efficacious vaccine and poor access to the antiretroviral drugs against this deadly virus. High mutation rate in the viral genome underlying the antigenic variability of the viral proteome is the major hindrance as far as the antibody based vaccine development is concerned. Although the exact mechanism by which CTL epitopes and the restricting HLA alleles mediate their action towards slow disease progression is still not clear, the important CTL restricted epitopes for controlling viral infections can be utilized in future vaccine design. This study was designed for the characterization the HIV-1 optimal CTL epitopes and their corresponding HLA alleles. CTL epitope cluster distribution analysis revealed only two HIV-1 proteins, namely, Nef and Gag, which have significant cluster forming capacity. We have found the role of specific HLA supertypes such as HLA B∗07, HLA B∗58, and HLA A∗03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins, to be presented as epitopes. The analyses revealed that the clusters of optimal epitopes for Nef and p24 proteins of HIV-1 could potentially serve as a source of vaccine.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

In silico prediction and in vitro identification of bluetongue virus 4 VP5 protein B-cell epitopes

VP5, the outer capsid protein of bluetongue virus (BTV), plays an important role in viral penetration and antibody-mediated viral neutralization. Therefore, VP5 represents an important target for development of vaccines and diagnostic tests. In this study, we use bioinformatic tools to predict nine antigenic B cell epitopes in the VP5 protein of a BTV serotype 4 (BTV4) isolate from China. Further, we generate five BTV4 VP5-specific monoclonal antibodies (MAbs) and define their corresponding epitopes using a set of VP5-derived peptides expressed as maltose-binding protein (MBP) fusion proteins. The five identified epitopes map to amino acids 119-134, 257-272, 286-301, 322-337, and 481-496 of the VP5 protein. Importantly, the epitopes identified using VP5-derived peptides do not correlate with our bioinformatic prediction of antibody epitopes. Identification and characterization of BTV4 VP5 protein epitopes may aid the development of diagnostic tools and provide information with which to study the structure of the BTV VP5 protein.

Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

BackgroundThe identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.MethodsTwenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay.ResultsThe peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%.ConclusionsThe epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.

Identification and Characterization of Antibody-Binding Epitopes on the Norovirus GII.3 Capsid

Genotype II.3 (GII.3) noroviruses are a major cause of sporadic gastroenteritis, particularly in children. The greater incidence of GII.3 noroviruses in the pediatric population compared to the adult demographic suggests development of herd immunity to this genotype, possibly as a consequence of limited evolution of immune epitopes. This study aimed to identify and characterize immune epitopes on the GII.3 capsid protein and to determine the level of immune cross-reactivity within the genotype. A panel of seven GII.3 virus-like particles (VLPs), representing norovirus strains isolated during 1975 to 2008, was tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with human sera and a rabbit anti-GII.3 strain-specific polyclonal serum generated against the 2008 GII.3 VLP. Immunoprecipitation of protease-digested GII.3 VLPs and sequencing of bound peptides via mass spectrometry were used to locate epitopes on the capsid. Two epitopes were investigated further using Mimotopes technology. Serum binding studies demonstrated complete intragenotype GII.3 cross-reactivity using both human and rabbit serum. Six immunoreactive regions containing epitopes were located on the GII.3 capsid protein, two within each capsid domain. Epitopes in the S and P1 domains were highly conserved within GII.3 noroviruses. P2 domain epitopes were variable and contained evolutionarily important residues and histo-blood group antigen (HBGA) binding residues. In conclusion, anti-GII.3 antibody-binding epitopes are highly cross-reactive and mostly conserved within GII.3 strains. This may account for the limited GII.3 prevalence in adults and suggests that a GII.3 strain may be a valuable inclusion in a multivalent pediatric targeted VLP vaccine. Exploration of norovirus immune epitopes is vital for effective vaccine design. IMPORTANCE This study represents an important contribution to the understanding of norovirus immunology in a pediatric genotype. The high cross-reactivity and conservation of GII.3 epitopes suggest development of herd immunity against GII.3 and indicate that a GII.3 strain would be a valuable inclusion in a pediatric targeted multivalent vaccine. Immunological understanding of pediatric norovirus strains is important since norovirus vaccines will likely target high-risk groups such as the pediatric population.

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⁺ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

Designing immunogenic peptides

Peptides fulfill many roles in immunology, yet none are more important than their role as immunogenic epitopes driving the adaptive immune response, our ultimate bulwark against infectious disease. Peptide epitopes are mediated primarily by their interaction with major histocompatibility complexes (T-cell epitopes) and antibodies (B-cell epitopes). As pathogen genomes continue to be revealed, both experimental and computational epitope mapping are becoming crucial tools in vaccine discovery. Immunoinformatics offers many tools, techniques and approaches for in silico epitope characterization, which is capable of greatly accelerating epitope design.

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

Meeting abstracts The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated VNTR region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are

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Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses.

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.