lipid-a and Orthomyxoviridae-Infections

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Antigens, Viral; CpG Islands; Dogs; Female; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins; Influenza A virus; Influenza Vaccines; Lipid A; Madin Darby Canine Kidney Cells; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Orthomyxoviridae Infections; Squalene; Vaccines, Combined; Vaccines, Synthetic

Influenza A virus (IAV) poses a constant worldwide threat to human health. Although conventional vaccines are available, their protective efficacy is type or strain specific, and their production is time-consuming. For the control of an influenza pandemic in particular, agents that are immediately effective against a wide range of virus variants should be developed. Although pretreatment of various Toll-like receptor (TLR) ligands have already been reported to be effective in the defense against subsequent IAV infection, the efficacy was limited to specific subtypes, and safety concerns were also raised. In this study, we investigated the protective effect of an attenuated bacterial outer membrane vesicle -harboring modified lipid A moiety of lipopolysaccharide (fmOMV) against IAV infection and the underlying mechanisms. Administration of fmOMV conferred significant protection against a lethal dose of pandemic H1N1, PR8, H5N2, and highly pathogenic H5N1 viruses; this broad antiviral activity was dependent on macrophages but independent of neutrophils. fmOMV induced recruitment and activation of macrophages and elicited type I IFNs. Intriguingly, fmOMV showed a more significant protective effect than other TLR ligands tested in previous reports, without exhibiting any adverse effect. These results show the potential of fmOMV as a prophylactic agent for the defense against influenza virus infection.

Topics: Animals; Bacterial Outer Membrane; Escherichia coli; Female; Humans; Influenza A virus; Influenza Vaccines; Influenza, Human; Interferon Type I; Ligands; Lipid A; Macrophages; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Secretory Vesicles; Signal Transduction; Toll-Like Receptors

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential

Topics: Adjuvants, Immunologic; Aged; Animals; Antibodies, Viral; Cells, Cultured; Dendritic Cells; Female; Glucosides; Humans; Immunity; Immunization; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Influenza, Human; Lipid A; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Toll-Like Receptor 4

Microbial biochemicals have been indicated as the primary stimulators of innate immunity, the first line of the body's defence against infections. However, the influence of topological features on a microbe's surface on immune responses remains largely unknown. Here we demonstrate the ability of TiO

Topics: Animals; Dendritic Cells; Influenza A virus; Lipid A; Macrophages; Mice; Mice, Knockout; Nanoparticles; Neoplasms, Experimental; Orthomyxoviridae Infections; Phagocytosis; Titanium

Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5μg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10-20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Body Temperature; Carbohydrates; Drug Compounding; Esters; Fatty Acids; Ferrets; HEK293 Cells; Hemagglutination Inhibition Tests; Humans; Immunity, Humoral; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Influenza, Human; Injections, Intramuscular; Lipid A; Orthomyxoviridae Infections; Polysorbates; Rabbits; Squalene; Toll-Like Receptor 4; Vaccination

Influenza is an acute respiratory disease and a major health problem worldwide. Since mucosal immunity plays a critical role in protection against influenza virus infection, mucosal immunization is considered a promising vaccination route. However, except for live-attenuated vaccines, there are no effective killed or recombinant mucosal influenza vaccines to date. Outer membrane vesicles (OMVs) are nano-sized vesicles produced by gram-negative bacteria, and contain various bacterial components capable of stimulating the immune system of the host. We generated an OMV with low endotoxicity (fmOMV) by modifying the structure of the lipid A moiety of lipopolysaccharide and investigated its effect as an intranasal vaccine adjuvant in an influenza vaccine model. In this model, fmOMV exhibited reduced toll-like receptor 4-stimulating activity and attenuated endotoxicity compared to that of native OMV. Intranasal injection of the vaccine antigen with fmOMV significantly increased systemic antibody and T cell responses, mucosal IgA levels, and the frequency of lung-resident influenza-specific T cells. In addition, the number of antigen-bearing CD103

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Dendritic Cells; Disease Models, Animal; Extracellular Vesicles; Female; Immunoglobulin A; Influenza Vaccines; Lipid A; Lymph Nodes; Mice, Inbred C57BL; Mucous Membrane; Orthomyxoviridae; Orthomyxoviridae Infections; Survival Analysis; T-Lymphocytes; Treatment Outcome

The ongoing threat from Influenza necessitates the development of new vaccine and adjuvant technologies that can maximize vaccine immunogenicity, shorten production cycles, and increase global vaccine supply. Currently, the most successful adjuvants for Influenza vaccines are squalene-based oil-in-water emulsions. These adjuvants enhance seroprotective antibody titers to homologous and heterologous strains of virus, and augment a significant dose sparing activity that could improve vaccine manufacturing capacity. As an alternative to an emulsion, we tested a simple lipid-based aqueous formulation containing a synthetic TLR4 ligand (GLA-AF) for its ability to enhance protection against H5N1 infection. GLA-AF was very effective in adjuvanting recombinant H5 hemagglutinin antigen (rH5) in mice and was as potent as the stable emulsion, SE. Both adjuvants induced similar antibody titers using a sub-microgram dose of rH5, and both conferred complete protection against a highly pathogenic H5N1 challenge. However, GLA-AF was the superior adjuvant in ferrets. GLA-AF stimulated a broader antibody response than SE after both the prime and boost immunization with rH5, and ferrets were better protected against homologous and heterologous strains of H5N1 virus. Thus, GLA-AF is a potent emulsion-free adjuvant that warrants consideration for pandemic influenza vaccine development.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adjuvants, Immunologic; Animals; Antibody Formation; Dogs; Drug Combinations; Emulsions; Female; Ferrets; Humans; Immunity; Influenza A Virus, H5N1 Subtype; Influenza Vaccines; Influenza, Human; Lipid A; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Pandemics; Th1 Cells

The ectodomain of matrix protein 2 (M2e) of influenza virus is suggested to be a rational target for a universal influenza A vaccine. However, there are some concerns that M2e vaccines might not be highly effective in the general population with diverse genetic backgrounds. Here we examined the immunogenicity and protective efficacy of the baculovirus-derived virus-like particles containing multiple M2e (M2eVLP) with AS04 adjuvant in a C57BL/6 mouse strain (H-2(b)). M2eVLP vaccine induced significant levels of M2e-specific IgG in C57BL/6 mice after vaccination. Furthermore, M2eVLP adjuvanted with AS04 was more effective than M2eVLP alone in conferring protection as well as in inducing recall humoral and T cell responses specific for M2e after lethal influenza virus challenge. A mechanistic study provides evidence that activation of dendritic cells by the toll-like receptor 4 agonist MPL in the AS04 adjuvant was associated with interferon-γ producing CD4 T cell responses. Our results suggest that AS04 adjuvanted M2eVLP vaccines have the potential to improve cross-protection.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Antibodies, Viral; CD4-Positive T-Lymphocytes; Disease Models, Animal; Female; Influenza Vaccines; Interferon-gamma; Lipid A; Mice, Inbred C57BL; Orthomyxoviridae Infections; Vaccines, Virus-Like Particle; Viral Matrix Proteins

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on H9N2 swine influenza virus-induced acute lung injury (ALI) were investigated in mice. BALB/c mice were inoculated intranasally with 10(7) 50% tissue culture infective doses (TCID(50)) of A/swine/HeBei/012/2008/(H9N2) viruses with or without NAC treatments to induce ALI model. The result showed that pulmonary inflammation, pulmonary edema, MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6, IL-1β and CXCL-10 in BALF were attenuated by NAC. Moreover, our data showed that NAC significantly inhibited the levels of TLR4 protein and TLR4 mRNA in the lungs. Pharmacological inhibitors of TLR4 (E5564) exerted similar effects like those determined for NAC in H9N2 swine influenza virus-infected mice. These results suggest that antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Topics: Acetylcysteine; Acute Lung Injury; Animals; Antioxidants; Cytokines; Disease Models, Animal; Humans; Inflammation Mediators; Influenza A Virus, H9N2 Subtype; Influenza, Human; Lipid A; Lung; Male; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Peroxidase; Swine; Toll-Like Receptor 4; Virus Replication

Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigens, Surface; Apoptosis Regulatory Proteins; Cattle; Cytokines; Female; Humans; Immunologic Memory; Influenza A virus; Interleukin-6; Lipid A; Mice; Mice, Knockout; Mice, Transgenic; Nucleocapsid Proteins; Orthomyxoviridae Infections; Ovalbumin; Programmed Cell Death 1 Receptor; RNA-Binding Proteins; Serum Albumin, Bovine; T-Lymphocytes, Cytotoxic; Vaccines, Subunit; Viral Core Proteins

While the current influenza vaccine strategy is dependent on eliciting neutralizing antibodies to the hemagglutinin (H or HA) surface glycoprotein, antigenic drifts and occasional antigenic shifts necessitate constant surveillance and annual updates to the vaccine components. The ectodomain of the matrix 2 (M2e) channel protein has been proposed as a universal vaccine candidate, although it has not yet been shown to elicit neutralizing antibodies. Utilizing a liposome-based vaccine technology, an M2e vaccine (L-M2e-HD/MPL) was tested and shown to stimulate the production of anti-M2e antibodies which precipitated with whole virus and inhibited viral cell lysis by multiple type A strains of influenza virus using a novel in vitro assay. The anti-M2e antibodies also conferred complete protection following passive transfer from L-M2e-HD/MPL vaccinated mice to naïve mice challenged with H1N1 virus. Significantly higher levels of IL-4 compared to IFN-γ were secreted by the splenocytes of L-M2e-HD/MPL vaccinated mice incubated with M2e. In addition, depletion of CD4 cells or CD4 cells plus CD8 cells from L-M2e-HD/MPL vaccinated mice using monoclonal antibodies markedly decreased the level of protection of the vaccine when compared to just CD8 depletion of L-M2e-HD/MPL vaccinated mice. These results suggest that the protective immune response elicited by this vaccine is mediated primarily by a Th2 mechanism.

Topics: Amino Acid Sequence; Animals; Antibodies, Viral; Cytokines; Female; Humans; Immunization; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Lipid A; Liposomes; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Orthomyxoviridae Infections; Th2 Cells; Viral Matrix Proteins

Impairments in anti-influenza T helper 1 (Th1) responses are associated with greater risk of influenza-related mortality in the elderly. Addition of adjuvants to existing influenza vaccines could improve immune responses in the elderly. In this study, the activity of three adjuvants, an oil-in-water emulsion and a synthetic lipid A adjuvant formulated with or without the emulsion, is compared. Our results show that Fluzone combined with lipid A plus an emulsion effectively leads to greater vaccine-specific IgG2a and IgG titers, enhances hemagglutination-inhibition titers and induces Type 1 cytokine responses (IFN-gamma and IL-2) to each of the Fluzone components.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Antibody Formation; Cell Line; Emulsions; Female; Hemagglutination Inhibition Tests; Immunity, Cellular; Immunoglobulin G; Influenza Vaccines; Interferon-gamma; Interleukin-2; Lipid A; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Orthomyxoviridae Infections; Th1 Cells; Toll-Like Receptor 4

A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Topics: Animals; Female; Immunity, Innate; Influenza A virus; Lipid A; Listeria monocytogenes; Listeriosis; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Molecular Mimicry; Orthomyxoviridae Infections; Receptors, Cell Surface; Structure-Activity Relationship; Toll-Like Receptor 4; Toll-Like Receptors

After covalent attachment of bacterial lipopolysaccharide to the nucleoprotein of influenza A virus, this water-soluble antigen could be incorporated firmly into ISCOM. This potent "immunostimulating complex" induced the production of high antibody titers in mice and could partially protect the animals from a lethal challenge infection. After immunization with ISCOM preparations NP-specific cytotoxic T cell activity could not be demonstrated.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Cytotoxicity Tests, Immunologic; Immunization; Influenza A virus; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Nucleoproteins; Orthomyxoviridae Infections; Quillaja Saponins; Saponins; Solubility; Viral Core Proteins

The ability of nontoxic monophosphoryl lipid A (MPL) to stimulate nonspecific resistance against viral infection was investigated. Mice pretreated intravenously with squalane-in-water emulsions of MPL, alone or in combination with other immunostimulants, were given an aerosol of influenza virus three weeks after the pretreatment. Complete protection against lethal influenza virus infection was conferred when MPL was combined with trehalose dimycolate (TDM). The protective activity of MPL plus TDM combination was corroborated by a significant reduction of the lung virus titers. Combination of lower doses of MPL with TDM extracted from Mycobacterium bovis, but not with that of M. phlei, induced significant resistance to influenza virus. Preparations containing MPL alone, or combined with mycobacterial cell wall skeleton or muramyl dipeptide, were not effective. The adjuvant activity of MPL on bivalent influenza subunit vaccine was also studied. The primary antibody responses to influenza A and influenza B antigens were enhanced by the addition of MPL and were higher than the vaccine associated with aluminum hydroxide. The adjuvant activity of MPL was confirmed by the elevated secondary response. High levels of circulating antibodies were still present in the MPL group when antibody titers in the controls were waning.

Topics: Adjuvants, Immunologic; Animals; Chick Embryo; Cord Factors; Drug Interactions; Female; Glycolipids; Immunity, Innate; Influenza A virus; Lipid A; Mice; Mice, Inbred Strains; Mycobacterium; Orthomyxoviridae Infections; Salmonella