H3 Stalk-Based Chimeric Hemagglutinin Influenza Virus Constructs Protect Mice from H7N9 Challenge

The recent outbreak of H7N9 influenza virus infections in humans in China has raised concerns about the pandemic potential of this strain. Here, we test the efficacy of H3 stalk-based chimeric hemagglutinin universal influenza virus vaccine constructs to protect against H7N9 challenge in mice. Chimeric hemagglutinin constructs protected from viral challenge in the context of different administration routes as well as with a generic oil-in-water adjuvant similar to formulations licensed for use in humans.     

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

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

Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection

Every year, influenza virus infection causes significant mortality and morbidity in human populations. Although egg-based inactivated viral vaccines are available, their effectiveness depends on the correct prediction of the circulating viral strains and is limited by the time constraint of the manufacturing process. Recombinant subunit vaccines are easier to manufacture with a relatively short lead time but are limited in their efficacy partly because the purified recombinant membrane proteins in the soluble form most likely do not retain their native membrane-bound structure. Nanodisc (ND) particles are soluble, stable, and reproducibly prepared discoid shaped nanoscale structures that contain a discrete lipid bilayer bound by two amphipathic scaffold proteins. Because ND particles permit the functional reconstitution of membrane/envelope proteins, we incorporated recombinant hemagglutinin (HA) from influenza virus strain A/New Caledonia/20/99 (H1N1) into NDs and investigated their potential to elicit an immune response to HA and confer immunity to influenza virus challenge relative to the commercial vaccines Fluzone and FluMist. HA-ND vaccination induced a robust anti-HA antibody response consisting of predominantly the immunoglobulin G1 (IgG1) subclass and a high hemagglutination inhibition titer. Intranasal immunization with HA-ND induced an anti-HA IgA response in nasal passages. HA-ND vaccination conferred protection that was comparable to that of Fluzone and FluMist against challenge with influenza virus strain A/Puerto Rico/8/1934 (H1N1).The influenza A virus-type viral genome encodes 11 proteins including hemagglutinin (HA) and neuraminidase (NA). HA is important in virus transmission and is also a major determinant of host range (16). NA prevents viral aggregation and helps in the release of new viruses from the infected cell (25). These glycoproteins are the principal antigens against which humoral immune responses of the host are directed. Vaccination has been accepted as the most effective method of preventing influenza virus. Current licensed vaccines against influenza virus include conventional inactivated virus vaccine, live-attenuated vaccine, or inactivated “split-virus” vaccines, all grown in embryonated chicken eggs. Influenza virus vaccines may contain residual egg-derived antigens, which is a risk factor for persons with hypersensitivity to eggs. In the case of live-attenuated vaccines that are delivered by the mucosal route, there are several potential safety concerns including the possibility that the vaccine strain could undergo spontaneous genetic change and in a rare case of simultaneous infection with another influenza virus could undergo antigenic shift. These factors are of special concern for children and the elderly, who are the primary populations at risk for influenza virus infection (9). Therefore, there is a continuing need for developing more efficacious and safer vaccines.Apart from licensed vaccines, a number of different vaccine formulations including soluble glycoproteins, virus-like particles, and subunit vaccines (6, 9, 14) with various efficacies have been developed. Recombinant glycoprotein vaccines offer many distinct advantages, including cost, the possibility of adapting them to rapidly changing strains within a short time, and independence from egg-based formulations. In experimental setups, recombinant HA (rHA) and recombinant NA have provided protection against lethal challenge to mice (18, 27). The safety, immunogenicity, and efficacy of trivalent rHA vaccines have been established (26), and a potential trivalent HA vaccine (FluBlok; Protein Sciences Corporation) is currently in phase III clinical trials.Some rHA-based vaccines elicit high titers of anti-HA antibodies. However, these antibodies do not necessarily possess a high capacity for virus neutralization. This apparent discrepancy likely results from the use of soluble HA protein that may not accurately mimic the native structure of the membrane-embedded glycoprotein on the viral envelope for immunization. This could result in a robust antibody response with a limited ability to react with “native epitopes.” This notion is supported by data from previously reported studies that indicated that antigens expressed in their native three-dimensional conformation can elicit a more effective antibody response than proteins in their nonnative forms (19). Therefore, we investigated whether rHA presented in a lipid-bilayer-embedded formulation would elicit a potent neutralizing antibody response.The Nanodisc (ND) system was developed as a novel method for functionally reconstituting membrane proteins into soluble nanoscale lipid bilayers (3, 4, 12, 22). NDs are robust, reproducible, and monodisperse discoidal particles 5.5 nm high and nominally 10 nm in diameter that are formed via a self-assembly process. ND particles contain two copies of an alpha-helical, amphipathic protein, termed membrane scaffold protein (MSP), which encircles a lipid bilayer in a “belt-like” fashion (Fig. ​(Fig.1a).1a). A mixture of phospholipids and MSP are placed in a nonequilibrium solubilized state, for instance, using detergent or high hydrostatic pressure, and the system is then allowed to approach equilibrium by the gentle removal of the perturbant. This initiates a process of self-assembly, wherein the phospholipids and MSP find each other and generate a discoidal phospholipid bilayer encircled by the MSP. The resulting nanostructures represent a highly stable and homogeneous population with an aqueous solubility in the millimolar range (11).Open in a separate windowFIG. 1.Construction of HA-NDs. (a) Schematic showing an ND particle that contains a phospholipid bilayer encircled by membrane scaffold proteins (left) (5) and the same ND particle with an embedded transmembrane protein (right) (17). (b) HA-ND assemblies were first purified by Ni²⁺ affinity chromatography. (Top left) Silver-stained SDS-PAGE showing flowthrough, wash, and elution of HA-ND assembly mix over a Ni-nitrilotriacetic acid column (FT1 and FT2 are flowthrough, and the eluate contains the eluted protein). Arrows show the positions of the 72-kDa HA band and the 25-kDa MSPs. (Top right) Anti-HA Western blotting of the same SDS-PAGE gel. Depending on the quality of purification, a certain fraction of full-length 72-kDa rHA (HA0) can exist as proteolytically cleaved HA1 (∼50-kDa) and HA2 (∼28-kDa) subunits. (Bottom left) Ni²⁺ column eluates were further purified by SEC. Silver-stained SDS-PAGE gel shows size-based fractionation of Ni²⁺ column eluate. The numbers at the bottom correspond to the fractions collected. The MSP amounts are largest at fractions 27 to 30, showing that empty NDs eluted at those fractions. (Bottom right) Anti-HA Western blotting of the same SDS-PAGE gel showing that HA-ND assemblies eluted mainly between fractions 18 and 26. (c) Elution profile of HA-ND following SEC separation. The elution times for protein standards used for calibration are indicated at the top. The control profile for empty NDs is superimposed. HA-ND assemblies have a shorter retention time than empty NDs. inj, injection. (d) HA-ND assemblies from different SEC fractions separate as discrete-sized molecules upon native PAGE separation. Silver staining (left) and anti-HA Western blotting (right) of native PAGE gels from size exclusion fractions show different HA polymers contained in NDs. Earlier fractions are rich in higher-polymeric forms of HA, while later fractions are richer in monomeric HA. Control HA was loaded in the last well to the right in both cases.The value of the ND self-assembly process is that one can simply and reproducibly incorporate membrane proteins into these structures. This is accomplished by including the membrane protein in the initial mixture of MSP, lipid, and detergent prior to the initiation of the self-assembly process. An incorporated membrane protein then finds itself in a native-like environment with stability and activity normally found in vivo. By using phospholipids with different chemical characteristics (charge, degree of unsaturation, and length of acyl chains), the bilayer environment can be optimized to accommodate functional requirements. Furthermore, larger scaffold proteins, which in turn create a larger-diameter particle, can be employed to incorporate multimers or membrane protein complexes. Numerous membrane proteins from the three major classes-integral, tethered, and embedded (including monomers and multimers)-in the lipid bilayer environment created by NDs have been studied (2-5, 8, 10, 13, 20, 23). Since the ND system creates a stable bilayer environment that mimics that encountered by a membrane protein in the cell membrane, membrane proteins display normal folding, native ligand binding kinetics, and intact signaling activity (1, 3, 5, 8, 10, 13, 17, 23).In this study, we successfully incorporated recombinant baculovirus-derived HA into NDs (HA-ND) and compared its efficacy to induce a relevant immune response and confer protection against influenza virus challenge with those of existing licensed vaccines by using a mouse model.     

Adjuvants and Immunization Strategies to Induce Influenza Virus Hemagglutinin Stalk Antibodies

The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C), and an RNA hairpin derived from Sendai virus (SeV) Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C) also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.     

DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.     

A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice

To combat the possibility of a zoonotic H5N1 pandemic in a timely fashion, it is necessary to develop a vaccine that would confer protection against homologous and heterologous human H5N1 influenza viruses. Using a replicating modified vaccinia virus Tian Tan strain (MVTT) as a vaccine vector, we constructed MVTT_HA-QH and MVTT_HA-AH, which expresses the H5 gene of a goose-derived Qinghai strain A/Bar-headed Goose/Qinghai/1/2005 or human-derived Anhui Strain A/Anhui/1/2005. The immunogenicity profiles of both vaccine candidates were evaluated. Vaccination with MVTT_HA-QH induced a significant level of neutralizing antibodies (Nabs) against a homologous strain and a wide range of H5N1 pseudoviruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Neutralization tests (NT) and Haemagglutination inhibition (HI) antibodies inhibit the live autologous virus as well as a homologous A/Xingjiang/1/2006 and a heterologous A/Vietnam/1194/2004, representing two human isolates from clade 2.2 and clade 1, respectively. Importantly, mice vaccinated with intranasal MVTT_HA-QH were completely protected from challenge with lethal dosages of A/Bar-headed Goose/Qinghai/1/2005 and the A/Viet Nam/1194/2004, respectively, but not control mice that received a mock MVTT_S vaccine. However, MVTT_HA-AH induced much lower levels of NT against its autologous strain. Our results suggest that it is feasible to use the H5 gene from A/Bar-headed Goose/Qinghai/1/2005 to construct an effective vaccine, when using MVTT as a vector, to prevent infections against homologous and genetically divergent human H5N1 influenza viruses.     

A型流感病毒血凝素、神经氨酸酶DNA疫苗研究

用BALB/C小鼠为模型,检测A型流感病毒血凝素、神经氨酸酶、基质蛋白DNA疫苗抗流感能力。研究表明:血凝素、神经氨酸酶DNA疫苗能提供有效的抗流感保护;血凝素、神经氨酸酶和基质蛋白联合免疫动物提供最佳免疫保护。     

Structure of a Classical Broadly Neutralizing Stem Antibody in Complex with a Pandemic H2 Influenza Virus Hemagglutinin

We report the structural characterization of the first antibody identified to cross-neutralize multiple subtypes of influenza A viruses. The crystal structure of mouse antibody C179 bound to the pandemic 1957 H2N2 hemagglutinin (HA) reveals that it targets an epitope on the HA stem similar to those targeted by the recently identified human broadly neutralizing antibodies. C179 also inhibits the low-pH conformational change of the HA but uses a different angle of approach and both heavy and light chains.     

Acid Sensitivity of the Influenza Virus Hemagglutinin

Influenza virus hemagglutinin was shown to be acid resistant if precipitates which form during acidification are first removed. Adsorption of virus to precipitates formed during acidification may cause a virus to be described incorrectly as acid sensitive.     

一种含不同流感病毒血凝素抗原表位的核酸疫苗

流感病毒表面抗原血凝素( hemagglutinin,HA)是流感核酸疫苗重要的靶抗原,针对HA的保护性中和抗体主要由HA上的五个抗原表位诱导产生.在本文中,我们构建了一种以新甲型H1N1流感病毒HA1为骨架的含2个A/PR/8( H1N1)流感病毒HA抗原表位和3个新甲型H1N1流感病毒HA抗原表位的核酸疫苗,并在B...     

Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies.     

A Virus-Like Particle That Elicits Cross-Reactive Antibodies to the Conserved Stem of Influenza Virus Hemagglutinin

The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备和鉴定

目的:获得分泌抗H9亚型禽流感病毒(AIV)血凝素单克隆抗体的杂交瘤细胞。方法:以H9N2亚型AIV为免疫原,免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14,在PEG4000的作用下进行细胞融合,通过血凝抑制(HI)试验筛选分泌抗H9亚型AIV血凝素单克隆抗体的杂交瘤细胞。结果:经过连续3～4次克隆化,获得能稳定分泌抗H9亚型AIV血凝素的单克隆抗体细胞系6株,分别命名为1B2、1C10、1G2、2B7、2E3和5E11。6株细胞培养上清HI效价为24～28,腹水HI效价为210～213。除1G2为IgM外,其余5株均为IgG1。Western blotting结果显示,1B2、1C10、2B7和2E3能与AIVH9蛋白在Mr为75000处反应,表明其是针对AIVH9亚型血凝素蛋白的单抗。特异性试验表明该6株单抗均只与H9亚型AIV发生特异性HI反应,而不与其他14个HA亚型的AIV及新城疫病毒、传染性支气管炎病毒发生交叉反应,显示出良好的特异性。结论:制备了针对H9亚型禽流感病毒血凝素的单克隆抗体,为禽流感的快速诊断和病毒的抗原性分析等奠定了基础。     

Mechanisms of Hemagglutinin Targeted Influenza Virus Neutralization

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.