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Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses.

ABSTRACT: We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H(3)N(2)) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.

SUBMITTER: Wang BZ

PROVIDER: S-EPMC2583664 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses.

Wang Bao-Zhong BZ Quan Fu-Shi FS Kang Sang-Moo SM Bozja Jadranka J Skountzou Ioanna I Compans Richard W RW

Journal of virology 20080910 23

We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular imm ...[more]

PMID: 18786995

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Project description:Rabies remains an important worldwide public health threat, so safe, effective, and affordable vaccines are still being sought. Virus-like particle-based vaccines targeting various viral pathogens have been successfully produced, licensed, and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs) containing rabies virus (RABV) glycoprotein (G), matrix (M) protein, and membrane-anchored flagellin (EVLP-F) or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L) as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNAs) responses and elicited greater numbers of CD4(+) and CD8(+) T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP) containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin, and heat-labile enterotoxin B subunit possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

Project description:UnlabelledInfluenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates.ImportanceYoung infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population.

Project description:Sudden increases in the number of human A (H7N9) cases reported during December and January have been observed in previous years. Most reported infection cases are due to prior exposure to live poultry or potentially contaminated environments. Low pathogenicity of influenza A (H7N9) virus in avian species complicates timely discovery of infected birds. Therefore, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry to reduce the risk of human infection and prevent the emergence of novel mutated strains. In addition to a good antigen, an effective vaccine also requires an appropriate adjuvant to enhance its immunogenicity. Previously, we generated an H7N9 influenza recombinant subunit vaccine (HA1-2-fliC), in which haemagglutinin globular head domain (HA1-2) was fused with flagellin (fliC), a potent TLR5 ligand, and demonstrated that HA1-2-fliC elicited effective HA1-2-specific immune responses in mice.In this study, we determined flagellin-induced expression profiles of cytokines and chemokines in different types of avian immune cells in vitro and ex vivo. We found that flagellin significantly increased the expression levels of CXCL inflammatory chemokines (CXCLi1 and CXCLi2) and CCL chemokines (MIP-1? and MCP-3) in avian macrophage HD11 cells. In addition, HA1-2-fliC induced significant upregulation of cytokines (IL-1?, IL-6, IL-18 and IFN-?) and chemokines (CXCLi1, CXCLi2 and MIP-1?) in ex vivo splenic lymphocytes and peripheral blood mononuclear cells (PBMCs), suggesting that flagellin promoted immune responses of avian cells in vitro. We also evaluated specific humoural and cellular immune responses induced by HA1-2-fliC and found that chickens immunised intramuscularly with HA1-2-fliC showed significantly higher HA1-2-specific immunoglobulin (Ig)G titers in serum. Furthermore, HA1-2-fliC potentiated cellular immune responses, as reflected by an increase in CD4+ and CD8+ T cells and proliferation of PBMCs. Significantly higher levels of IFN-? and IL-4 in PBMCs from chickens vaccinated with HA1-2-fliC further indicated that HA1-2-fliC promoted a balanced Th1/Th2 immune response.We demonstrated that the use of the flagellin as an adjuvant potentiated immunogenicity of influenza subunit vaccine HA1-2 in vitro and in vivo. These findings provide a basis for the development of H7N9 influenza HA1-2 subunit vaccines for chickens.

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Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses.

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Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses.

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