Native Hemagglutinin Research Articles

In silico design of a trivalent multi-epitope global-coverage vaccine-candidate protein against influenza viruses: evaluation by molecular dynamics and immune system simulation

Hemagglutinin (HA), a variable viral surface protein, is essential for influenza vaccine development. Annually, traditional trivalent vaccines containing influenza A/H1N1, A/H3N2 and B viruses are administered globally, which are not very effective for the mutations in HA protein. The aim of this study was to design a multi-epitope vaccine containing epitopes of the HA protein of H1N1, H3N2 and B viruses using immunoinformatics methods. The HA protein epitope prediction was performed using Immune Epitope Database. Toxicity, antigenicity and conservancy of the epitopes were evaluated using ToxinPred, VaxiJen and Epitope Conservancy Analysis tools, respectively. Then, nontoxic, antigenic and high conserved epitopes with high prediction scores were selected. Their binding affinity was evaluated against human and mouse MHC class I and II molecules using the HPEPDOCK tool. Physicochemical properties and post-translational modifications were evaluated using ProtParam, SOLpro and MusiteDeep tools, respectively. Top selected epitopes were joined using linkers to produce the best effective recombinant trivalent vaccine candidate to elicit cellular and humoral immune responses in mouse and human host models. These sequences were modeled and verified. By evaluating the results of various analyses of all models and the most similarity to the native HA protein, model 5 was selected as the best model. Finally, in silico cloning of this model as vaccine candidate was performed in pET21. This study was a computer-aided analysis for a multi-epitope trivalent recombinant vaccine candidate against influenza viruses. The efficiency of our best model of vaccine candidates should be validated using in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma

Development of an ELISA-Based Potency Assay for Inactivated Influenza Vaccines Using Cross-Reactive Nanobodies.

Inactivated vaccines are the main influenza vaccines used today; these are usually presented as split (detergent-disrupted) or subunit vaccines, while whole-virus-inactivated influenza vaccines are rare. The single radial immune diffusion (SRD) assay has been used as the gold standard potency assay for inactivated influenza vaccines for decades; however, more recently, various alternative potency assays have been proposed. A new potency test should be able to measure the amount of functional antigen in the vaccine, which in the case of influenza vaccines is the haemagglutinin (HA) protein. Potency tests should also be able to detect the loss of potency caused by changes to the structural and functional integrity of HA. To detect such changes, most alternative potency tests proposed to date use antibodies that react with native HA. Due to the frequent changes in influenza vaccine composition, antibodies may need to be updated in line with changes in vaccine viruses. We have developed two ELISA-based potency assays for group 1 influenza A viruses using cross-reactive nanobodies. The nanobodies detect influenza viruses of subtype H1N1 spanning more than three decades, as well as H5N1 viruses, in ELISA. We found that the new ELISA potency assays are sensitive to the nature of the reference antigen (standard) used to quantify vaccine antigens; using standards matched in their presentation to the vaccine type improved correspondence between the ELISA and SRD assays.

Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA “breathing” on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Generation and Evaluation of Recombinant Thermostable Newcastle Disease Virus Expressing the HA of H9N2 Avian Influenza Virus.

H9N2 avian influenza virus (AIV) has become endemic in many countries, causing great economic losses when co-infected with other pathogens. So far, several live vaccines based on Newcastle disease virus (NDV) vectors expressing influenza hemagglutinin (HA) have been developed. However, the thermostable recombinant NDV is rarely reported. In this study, using a thermostable NDV rAHR09 strain as the vector, three recombinant NDVs expressing native HA, chimeric HA ectodomain with transmembrane domain/C-terminal cytoplasmic tail domain from fusion protein of NDV, and HA ectodomain were generated, designated rAHR09-HA, rAHR09-HAF, and rAHR09-HAE. The MDT value of three recombinant NDVs was above 120 h, their ICPI value was about 0.03, and the recombinant NDVs were still infectious when treated for 100 min under 56 °C, which demonstrated that the recombinant NDVs kept the lentogenic and thermostable nature of rAHR09. The immunization data showed that rAHR09-HA and rAHR09-HAF induced a higher HI antibody titer against H9N2 AIV and NDV. After being challenged with H9N2 AIV, the rAHR09-HA and rAHR09-HAF could significantly reduce the virus shedding in cloacal and tracheal swab samples. Our results suggest that rAHR09-HA and rAHR09-HAF might be vaccine candidates against H9N2 AIV.

Dissecting human, ferret, and murine antibody and B cell responses to generate a broadly protective synthetic influenza virus hemagglutinin

Abstract A major challenge in developing improved influenza vaccines is the identification of broadly reactive and protective epitopes. While it is generally believed that the specificity of the antibody response to hemagglutinin (HA) is relatively narrow, we and others have identified individuals that mount a cross-reactive neutralizing antibody response after natural infection. Using plasmablasts from one such individual we generated broadly cross-reactive HA-specific monoclonal antibodies (mAbs). Six of these mAbs bound to the globular head of the HA and one to the stalk region; all provided broad protection against multiple subtypes of influenza A viruses including zoonotic strains in passively immunized mice. Using a peptide ELISA approach, we identified the binding regions of each mAb and subsequently immunized mice with these peptides. Immunized mice showed less weight loss and significantly improved survival against challenges with multiple subtypes as compared with control mice. We next used a similar mapping approach with broadly reactive B cells from mice and ferrets. Based on all of the collected data we generated three different composite HA antigens (HF1, HF2 and HF3) that contained a different mix of broadly reactive epitopes in one native HA structure. Mice immunized with the HFs were protected from multiple challenges, including lethal doses of H1, H3, H5, H6, H7 and H9 viruses. We also showed the HFs protected ferrets from H1, H3, and H9 viruses. Overall, by combining data generated from an examination of B cell and antibody responses following infection and/or vaccination in humans, mice, and ferrets we were able to construct a composite HA that was able to induce broad protection against multiple subtypes of influenza virus.

Structure and applications of novel influenza HA tri-stalk protein for evaluation of HA stem-specific immunity.

Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally nearly identical to the corresponding region of post-fusion form of native HA. This novel tri-stalk protein was able to differentiate between group 1 and 2 influenza in ELISA with virus-infected mice sera. It was also successfully applied for enzyme-linked immunospot assay to estimate the number of HA stem-reactive antibody (Ab)-secreting cells in mice. An in-house indirect ELISA was developed using a HA tri-stalk protein as a coating antigen for evaluation of HA stem-specific Ab levels in human sera collected in Luxembourg from 211 persons with occupational exposure to swine before the pandemic H1N1/09 virus had spread to Western Europe. Our results show that 70% of these pre-pandemic sera are positive for HA stem-specific Abs. In addition, levels of HA stem-specific Abs have positive correlation with the corresponding IgG titers and neutralizing activities against pandemic H1N1/09 virus.

Hybrid baculovirus-mediated prolonged hemagglutinin expression and secretion in vivo enhances the vaccine efficacy

Abstract Viral vectors expressing the major antigen hemagglutinin (HA) are a promising vaccine platform against avian influenza virus (AIV) which poses serious threat to the poultry industry and human health. However, vector vaccines typically express HA transiently within the cells. Conversely, nanoparticles encapsulating and releasing antigens slowly in vivo potentiate humoral and cellular immune responses, hence underscoring the benefits of long-term antigen release for potent vaccine efficacy. Baculovirus (BV) is a non-pathogenic insect virus capable of transducing mammalian and avian cells and holds promise as a vaccine vector. Here we constructed a hybrid BV (Bac-FCsHW) harboring an Frt sites-flanking gene cassette expressing a fusion HA protein composed of the HA ectodomain and a GCN4 pII peptide to facilitate trimerization. Bac-FCsHW transduction of cells enabled expression and secretion of HA trimers. Co-transduction of mammalian cells with Bac-FCsHW and Bac-FLPo expressing FLPo recombinase led to FLPo recognition of Frt sites, excision and formation of episomal DNA minicircles that encompassed the HA expression cassette. Such FLPo/Frt-mediated minicircle formation remarkably prolonged and enhanced the expression of secreted HA to at least 28 days. Intramuscular co-injection of Bac-FLPo and Bac-FCsHW into BALB/c mice significantly potentiated the humoral and cellular immune responses when compared with the control groups expressing native intracellular HA, either for a short-term or long-term. These data demonstrated that BV-mediated prolonged expression and release of HA substantiated the vaccine efficacy and implicated the potentials of the FLPo/Frt-based hybrid BV vector as a new AIV vaccine platform.

Recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines.

Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs.

Platform technology to generate broadly cross-reactive antibodies to α-helical epitopes in hemagglutinin proteins from influenza A viruses.

ABSTRACTWe have utilized a de novo designed two‐stranded α‐helical coiled‐coil template to display conserved α‐helical epitopes from the stem region of hemagglutinin (HA) glycoproteins of influenza A. The immunogens have all the surface‐exposed residues of the native α‐helix in the native HA protein of interest displayed on the surface of the two‐stranded α‐helical coiled‐coil template. This template when used as an immunogen elicits polyclonal antibodies which bind to the α‐helix in the native protein. We investigated the highly conserved sequence region 421–476 of HA by inserting 21 or 28 residue sequences from this region into our template. The cross‐reactivity of the resulting rabbit polyclonal antibodies prepared to these immunogens was determined using a series of HA proteins from H1N1, H2N2, H3N2, H5N1, H7N7, and H7N9 virus strains which are representative of Group 1 and Group 2 virus subtypes of influenza A. Antibodies from region 449–476 were Group 1 specific. Antibodies to region 421–448 showed the greatest degree of cross‐reactivity to Group 1 and Group 2 and suggested that this region has a great potential as a “universal” synthetic peptide vaccine for influenza A. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 144–159, 2016.

Rapid preparation of mutated influenza hemagglutinins for influenza virus pandemic prevention.

Influenza viruses have periodically caused pandemic due to frequent mutation of viral proteins. Influenza viruses have two major membrane glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin plays a crucial role in viral entry, while NA is involved in the process of a viral escape. In terms of developing antiviral drugs, HA is a more important target than NA in the prevention of pandemic, since HA is likely to change the host specificity of a virus by acquiring mutations, thereby to increase the risk of pandemic. To characterize mutated HA functions, current approaches require immobilization of purified HA on plastic wells and carriers. These troublesome methods make it difficult to respond to emerging mutations. In order to address this problem, a yeast cell surface engineering approach was investigated. Using this technology, human HAs derived from various H1N1 subtypes were successfully and rapidly displayed on the yeast cell surface. The yeast-displayed HAs exhibited similar abilities to native influenza virus HAs. Using this system, human HAs with 190E and 225G mutations were shown to exhibit altered recognition specificities from human to avian erythrocytes. This system furthermore allowed direct measurement of HA binding abilities without protein purification and immobilization. Coupled with the ease of genetic manipulation, this system allows the simple and comprehensive construction of mutant protein libraries on yeast cell surface, thereby contributing to influenza virus pandemic prevention.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0179-y) contains supplementary material, which is available to authorized users.

An avian influenza A (H7N9) virus vaccine candidate based on the fusion protein of hemagglutinin globular head and Salmonella typhimurium flagellin.

BackgroundA novel influenza virus, subtype H7N9, circulated through China in 2013–2014. Its higher rates of human infection in a wide range of locations within China and the associated increased likelihood of human-to-human transmission have caused global concern. Recombinant subunit vaccines provide safe and targeted protection against viral infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than vaccines made from whole viruses. Studies have shown that bacterial flagellin has strong adjuvant activity and induces protective immune responses.ResultsIn this study, we used overlap-PCR to generate an H7N9 influenza recombinant subunit vaccine that fused the globular head domain (HA1-2, aa 62–284) of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, Salmonella typhimurium flagellin (fliC). The resulting fusion protein, HA1-2-fliC, was efficiently expressed in an Escherichia coli prokaryotic expression system, and Western blotting and TLR5-stimulating activity analysis confirmed that the HA1-2-fliC moiety could be faithfully refolded to take on the native HA and fliC conformations. In a C3H/HeJ mouse model of intraperitoneal vaccination, the fusion protein elicited significant and robust HA1-2-specific serum IgG titers, maintaining high levels for at least 3 months in the vaccinated animals, and induced similar levels of HA1-2-specific IgG1 and IgG2a that were detectable 12 days after the third immunization. HA1-2-fliC was also found to be capable of triggering the production of neutralizing antibodies, as assessed by measuring hemagglutination inhibition titers.ConclusionsWe conclude that immunization with HA1-2-fliC induces potent HA1-2-specific responses, offering significant promise for the development of a successful recombinant subunit vaccine for avian influenza A (H7N9).

ISCOMATRIX™ adjuvant promotes epitope spreading and antibody affinity maturation of influenza A H7N9 virus like particle vaccine that correlate with virus neutralization in humans

In a previously reported phase I clinical trial, subjects vaccinated with two doses of an unadjuvanted H7N9 virus like particle (VLP) vaccine responded poorly (15.6% seroconversion rates with 45μg hemagglutinin (HA) dose). In contrast, 80.6% of subjects receiving H7N9 VLP vaccine (5μg HA) with ISCOMATRIX™ adjuvant developed hemagglutination-inhibition (HI) responses. To better understand the role of adjuvant, complete antibody epitope repertoires of post-vaccination sera were investigated using Whole Genome Fragment Phage Display Library (GFPDL). In addition, antibody affinity maturation following vaccination was measured against HA1 and HA2 antigenic domains using real time Surface Plasmon Resonance (SPR) based kinetic assays. Unadjuvanted H7N9-VLP vaccine generated primarily antibodies targeting the C-terminus of the HA1 domain, predicted to be mostly buried on the native HA spikes, while adjuvanted VLP vaccine generated antibodies against large epitopes in the HA1 spanning the receptor binding domain (RBD). SPR analysis using a functional H7-HA1 domain demonstrated that sera from adjuvanted H7N9-VLP vaccine induced higher total binding antibodies and significantly higher antibody affinity maturation to HA1 compared to sera from unadjuvanted vaccine. Total antibody binding and affinity to the HA1 (but not HA2) domain correlated with HI and neutralization titers. This study demonstrates that ISCOMATRIX™ adjuvanted vaccine promotes higher quality antibody immune response against avian influenza in naïve humans.

Generation of monoclonal antibodies reactive against subtype specific conserved B-cell epitopes on haemagglutinin protein of influenza virus H5N1

H5-specific monoclonal antibodies may serve as valuable tools for rapid diagnosis of H5N1 avian influenza virus. Therefore, conserved H5-specific sequences of the haemagglutinin (HA) protein were expressed in Pichia pastoris and used for generation of monoclonal antibodies (mAbs). The two mAbs, FD6 and HE4, were strongly reactive against native HA protein and exhibited specificity for subtype H5. By epitope mapping linear epitopes of mAbs were identified that are highly conserved among influenza A virus of subtype H5. Additionally no sequence similarities to homologous regions on HA proteins of other influenza A virus subtypes (i.e. H1, H3, H7, H9) were detected by sequence alignment analysis. Both mAbs did not cross react with native or denatured HA proteins of other influenza A virus subtypes. Furthermore, using ELISA and immunofluorescence test mAb FD6 reacted only to the native H5 protein of recently circulating highly pathogenic H5N1 influenza viruses but not to low pathogenic H5N1 isolates. In conclusion, the use of the two mAbs in non-molecular tests like antigen-capture-ELISA appears promising for detecting influenza A H5N1 virus.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.

Serum antibodies against native and denaturated hemagglutinin glycoproteins detected by ELISA as correlates of protection after influenza vaccination in healthy vaccinees and in kidney transplant recipients

The microneutralization assay is the standard method to investigate immune responses to influenza vaccination. However there remains some uncertainty as to whether ELISA results are a true measure of immunity in healthy or immuno-compromised vaccines. Furthermore it has been questioned if antibodies against native (“folded”) and against denaturated (“unfolded”) viral glycoproteins can equally be used as a marker of protection. In this study, two different quantitative IgG-ELISA assays detecting (i) antibodies against unfolded recombinant hemagglutinin (HA) (r-ELISA) and (ii) antibodies against the native HA on the influenza virus surface captured by fetuin-linkage (f-ELISA) were compared to microneutralization titers in sera from 29 healthy vaccinees (n=87 sera) and 39 kidney transplant recipients (n=117 sera) collected before, three weeks after and six months after vaccination against influenza A (H1N1) 2009. With both ELISAs a significant increase in antibody levels was detected after vaccination and linear regression analysis demonstrated that r-ELISA and f-ELISA correlated with microneutralization (R=0.622 for r-ELISA vs. R=0.56 for f-ELISA). For the healthy vaccinees both ELISAs were found to be adequate to distinguish protected from non-protected individuals (sensitivity and specificity: 87.5%/85.3% for r-ELISA and 87.5%/88.3% for f-ELISA). Results from the transplant recipients showed a slightly reduced sensitivity of 73.3% for r-ELISA while the f-ELISA demonstrated similar sensitivity and specificity as in the healthy vaccinees. However, in order to obtain these assay performances the cut-off-values for protection had to be adjusted for both assays and both investigation cohorts respectively limiting their application in routine laboratories.

Influenza A Virus Hemagglutinin Trimerization Completes Monomer Folding and Antigenicity

Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAV-infected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.

A Novel Synthetic Receptor-Based Immunoassay for Influenza Vaccine Quantification

Vaccination is the most effective prophylactic method for preventing influenza. Quantification of influenza vaccine antigens is critically important before the vaccine is used for human immunization. Currently the vaccine antigen quantification relies on hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the sialic acid binding property of influenza vaccine antigens. Specifically, we chemically synthesized human and avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two sialyllactose-derivatives was reduced and conjugated to mouse serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived hemagglutinins, respectively, forming the basis of a new, and robust assay for hemagglutinin quantification. Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple influenza vaccines.

Molecular Structures of Native HA Trimers on 2009 H1N1 Pandemic Influenza Virus Complexed with Neutralizing Antibodies

Antigenic variation of influenza virus hemagglutinin (HA) remains the principal challenge in developing more effective vaccines. Here, we report determination of the 3D structures of HA trimers on intact 2009 H1N1 pandemic virus particles in the absence and presence of neutralizing antibodies. We studied HA trimer structures bound to H17-L10, an antibody that recognizes a quaternary epitope near the head region of the trimer, and to C179, a broadly neutralizing antibody that is reactive to the conserved stem region of HA and neutralizes viruses expressing a broad range of HA subtypes (H1, H2, H5, and H9). We deduce the locations of the molecular surfaces of HA involved in interaction with each of these antibodies. Despite the dense packing of HA trimers on the viral surface, and the location of the stem region close to the viral membrane, we show that ∼75% of HA trimers have C179 bound to the stem domain. Thus, the majority of HA trimers on intact virions are available to bind anti-stem antibodies that target conserved HA epitopes, suggesting that universal influenza vaccines that elicit such antibodies could be effective.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Reverse engineering the antigenic architecture of the haemagglutinin from influenza H5N1 clade 1 and 2.2 viruses with fine epitope mapping using monoclonal antibodies

The induction of neutralising antibodies to the viral surface glycoprotein, haemagglutinin (HA) is considered the cornerstone of current seasonal and pandemic influenza vaccines. Mapping of neutralising epitopes using monoclonal antibodies (mAbs) helps define mechanisms of antigenic drift, neutralising escape and facilitates pre-pandemic vaccine design. In the present study we reverse engineered the antigenic structure of the HAs of two highly pathogenic H5N1 vaccine strains representative of currently circulating clade 1 and 2.2 H5N1 viruses. The HA sequence of the A/Vietnam/1194/04 clade 1 virus was progressively mutated into the HA sequence of the clade 2.2 virus, A/Bar-headed Goose/Qinghai/1A/05. Fine mapping of clade-specific neutralising epitopes was performed by examining the cross-reactivity of mAbs raised against the native HA of each parent virus. The reactivity across all clade specific mAbs centred around a constellation of mutations at positions 140, 145, 171 and 172, all of which are proximal to the receptor binding site on the membrane distal globular head of the HA. Overlapping cross-reactivity of these antigenic sites suggests that these amino acid positions relate to the antigenic evolution of the H5 clade 1 and 2.2 viruses. This finding may prove useful for the design of vaccines with broader neutralising cross-reactivity against the different H5 HA sublineages currently in circulation. These findings provide important information about the amino acid changes involved in the cross-clade evolution of H5N1 viruses and their potential for human to human transmission; and facilitates a greater understanding of the pandemic potential of H5N1 isolates.