Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

SIVMAC vaccine studies using whole inactivated virus antigen sequentially depleted of viral proteins

Groups of four rhesus monkeys were immunised at 0, 1, 2, and 13 months with whole inactivated SIV_mac32H, SIV_mac depleted of the outer envelope glycoprotein gp130, virus cores depleted of the lipid membrane (and hence transmembrane glycoproteins), or purified gag protein. These macaques plus controls were challenged with either the homologous SIV_mac251–32H. grown in human cells or the same virus passed once through monkey cells. None of those challenged with monkey-grown virus were protected, whereas all in the whole and gp130-depleted virus groups, and one in the core group resisted challenge with human-grown virus. As the only difference between the challenge viruses was a single in vitro passage in monkey cells it can be concluded that protection was solely due to human cell components. Finally, passive transfer of high titer IgG from monkeys infected with the homologous challenge virus failed to protect monkeys from infection despite the presence of circulating neutralising antibodies.     

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.     

Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.     

Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.     

Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus

Through cognate interaction between antigen-specific B-cell and CD4(+) alphabeta T cells, the CD4(+) alphabeta T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4(+) T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4(+) T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8(+) T cells with an anti-CD8 monoclonal antibody. In contrast, alphabeta T-cell-deficient mice (TCRbeta(-/-)) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4(-) CD8(-) double-negative alphabeta T cells are playing a role in regulating Ig class switching in the absence of CD4(+) T cells.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza

Over the past eight years, cases of human infection with highly pathogenic avian influenza viruses have raised international concern that we could be on the brink of a global influenza pandemic. Many of these human infections have proved fatal and if the viruses had been able to transmit efficiently from person to person, the effects would have been devastating. How can we arm ourselves against this pandemic threat when these viruses are too dangerous to use in conventional vaccine production? Recent technological developments (reverse genetics) have allowed us to manipulate the influenza virus genome so that we can construct safe, high-yielding vaccine strains. However, the transition of reverse-genetic technologies from the research laboratory to the manufacturing environment has presented new challenges for vaccine manufacturers as well as veterinary and public health authorities.     

N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.     

Novel dry powder preparations of whole inactivated influenza virus for nasal vaccination

The purpose of these studies was to enhance mucosal and systemic antibody production in response to increased local residence time of a whole inactivated influenza virus administered as a dry powder nasal vaccine formulation. Spray-freeze-drying (SFD) particles suitable for nasal delivery were characterized for physico-chemical properties and stability. Mucoadhesive compounds (MA) were characterized for their effects on nasal residence time of vaccine powders in rats compared with published in vitro data and elicited immune responses. SFD particles (D(50) = 26.9 microm) were spherical with a specific surface area of 1.25 m(2)/g. Thermal analysis indicated SFD powders were amorphous and demonstrated improved stability with respect to liquid formulations under various storage conditions. In vitro physico-chemical studies and in vivo scintigraphic imaging experiments indicated sodium alginate (SA) and carboxymethylcellulose-high molecular weight (CMC-HMW) powder formulations most significantly increased residence time in Brown Norway rats. Intramuscular delivery provided equivalent serum antibody titers to intranasal (IN) powder without MA, in the presence of CMC-HMW, SA, and hydroxypropyl methylcellulose (HPMC-HMW) after initial dosing and all formulations except IN powder with chitosan after boosting. IN liquid provided equivalent serum antibody titers to all IN powders after the initial vaccination and significantly greater serum antibody titers than IN powder with chitosan after boosting. Trends were consistent between residence time studies and immune response; however, no statistically significant differences between powder and liquid formulations were observed. It was concluded that enhanced serum and mucosal antibody responses were elicited by a dry powder nasal vaccine, specifically, administered in the presence of sodium alginate.     

Membrane fusion activity of influenza virus.

A simple assay is described to monitor fusion between fowl plague virus (FPV, an avian influenza A virus) and liposomes which allows the simultaneous quantitation of both lytic and non-lytic fusion events. As in fusion between viruses and the plasma membrane and in FPV-induced cell-cell fusion, the reaction only occurs at pH 5.5 or below, and it is fast, highly efficient, and essentially non-lytic when fresh virus and liposomes are used. The fusion occurs over a broad temperature range, and has no requirement for divalent cations. The fusion factor of influenza virus is a hemagglutinin (HA) spike which protrudes from the virus membrane and which is also responsible for virus binding to the host cell. The finding that fusion occurs as efficiently with liposomes containing or lacking virus receptor structures, further emphasizes the remarkable division of labor in the HA molecule: the receptor-binding sites are located in the globular HA1 domains and the fusion activation peptide is found at the N-terminal of HA2 in the stem region of the protein. The mechanism of fusion is discussed in terms of the three-dimensional structure of the HA and the conformational change which the protein undergoes at the fusion pH optimum.     

鸭病毒性肝炎二价灭活疫苗 (DHAV-SH和DHAV-FS株) 的制备和评价

我国鸭甲型肝炎病毒(DHAV)的快速变异及广泛流行给养鸭业造成了较大的经济损失,为研究鸭病毒性肝炎二价灭活疫苗(DHAV-SH和DHAV-FS株)的制备和评价方法,首先对我国多个鸭场进行血清型流行病学分析,从201株DHAV-1、38株DHAV-3中筛选出6株毒力较强的DHAV,验证了DHAV-1和DHAV-3为我国流行优势株,并对6株DHAV进行ELD50和LD50测定,筛选疫苗候选株后经传代确定疫苗毒种,选取F5代鸭胚胚体研磨液作为疫苗毒种,经甲醛灭活后制成3批水包油包水(W/O/W)型乳剂二价灭活疫苗实验室制品。通过安全性试验、抗体中和试验、攻毒保护及免疫交叉保护试验发现:疫苗安全性良好,雏鸭免疫后第7天可以检测到DHAV中和抗体,第14-21天的攻毒保护率为90%-100%,免疫持续期可达5周以上,DHAV-SH和DHAV-FS之间的交叉保护为20%-30%。研究表明本试验研制的鸭病毒性肝炎二价灭活疫苗实验室制品安全且高效,为我国DHAV预防和控制提供了一种新方法和新产品。     

流行性感冒灭活疫苗人体免疫效果及评价

通过对兰州生物制品研究所试生产的流行性感冒灭活疫苗的体试验研究,兰州所流感灭活疫苗接种后,所有接种对象均未见红肿,硬结等局部反应,仅有1例发生在儿童组的低热全身反应（体温37．4℃）,72小时后恢复正常,整体副反应发生率0．274％,肯定了该疫苗的安全性,疫苗接种前后易人群血清血凝抑制（HI）抗体阳转率100％,非易感人群HI抗体几何平均效价（GMT）增长19－60倍,抗体4倍增长率最低为95％,成人组平均值最高（97．67％）,证实该疫苗具有较好的免疫原性。     

The structure of a membrane fusion mutant of the influenza virus haemagglutinin.

The haemagglutinin glycoprotein (HA) of influenza virus specifically mediates fusion of the viral and host cell endosomal membranes at the acidic pH of endosomes. The HAs from mutant viruses with raised fusion pH optima contain amino acid substitutions in regions of the HA structure thought to be involved in the fusion process [Daniels et al. (1985b) Cell, 40, 431-439]. We have determined the neutral pH crystal structure of one such mutant, HA2 112 Asp----Gly. A water molecule appears to partially replace the aspartate side chain, and no changes are observed in the surrounding structure. It appears that four intra-chain hydrogen bonds that stabilize the location of the N-terminus of HA2 are lost in the mutant, resulting in a local destabilization that facilitates the extrusion of the N-terminus at higher pH.     

Interplay between lipids and viral glycoproteins during hemolysis and fusion by influenza virus

Since mixtures of lipids alone are known to elicit membrane fusion without participation of fusion proteins, the role of viral lipids in the so-called virus-induced hemolysis and cell fusion has been investigated, using as a model the fowl plague virus (influenza A/FPV/Rostock/H7N1). The experiments were planned in a way that allowed quantitative modification of viral lipids without changing envelope glycoproteins. Under the conditions employed, cholesterol oxidase of Nocardia erythropolis and phospholipase C of Bacillus cereus were shown to completely modify their substrates in the virus without altering virus-associated hemagglutinating and neuraminidase activities. It was found with such enzyme treatment that virus-induced hemolysis and cell fusion are greatly influenced by cholesterol and phospholipids of the envelope. It became clear, that hemolysis and fusion are differently dependent on the nature of lipid components even though mediated by the same viral glycoproteins.