Influenza virosomes supplemented with GPI-0100 adjuvant:a potent vaccine formulation for antigen dose sparing

Adjuvants can stimulate vaccine-induced immune responses and can contribute decisively to antigen dose sparing when vaccine antigen production is limited, as for example during a pandemic influenza outbreak. We earlier showed that GPI-0100, a semi-synthetic saponin derivative with amphiphilic structure, significantly stimulates the immunogenicity and protective efficacy of influenza subunit vaccine administered via a systemic route. Here, we evaluated the adjuvant effect of GPI-0100 on a virosomal influenza vaccine formulation. In contrast to influenza subunit vaccine adjuvanted with GPI-0100, virosomal vaccine supplemented with the same dose of GPI-0100 provided full protection of mice against infection at the extremely low antigen dose of 2 x 8 ng hemagglutinin. Overall, adjuvanted virosomes elicited higher antibody and T-cell responses than did adjuvanted subunit vaccine. The enhanced immunogenicity of the GPI-0100-adjuvanted virosomes, particularly at low antigen doses, is possibly due to a physical association of the amphiphilic adjuvant with the virosomal membrane. These results show that a combination of GPI-0100 and a virosomal influenza vaccine formulation is highly immunogenic and allows the use of very low antigen doses without compromising the protective potential of the vaccine.</p

The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

Prior associations with the APC/C complex during prometaphase makes cyclin B1 a better substrate for the cell cycle–regulating ubiquitin ligase in metaphase (see also a related paper by Di Fiore et al. in this issue)

Effect of viral membrane fusion activity on antibody induction by influenza H5N1 whole inactivated virus vaccine

Whole inactivated virus (WIV) influenza vaccines are more immunogenic in unprimed individuals than split-virus or subunit vaccines. In mice, this superior immunogenicity has been linked to the recognition of the viral ssRNA by endosomal TLR7 receptors in immune cells, leading to IFN alpha production and Th1-type antibody responses. Recent data suggest that viral membrane fusion in target cell endosomes is necessary for TLR7-mediated IFN alpha induction. If so, virus inactivation procedures that compromise the fusion activity of WIV vaccines, like formaldehyde (FA) treatment, could potentially harm vaccine efficacy. Therefore, we measured the effect of fusion inactivation of H5N1 WIV on TLR7 activation in vitro, and on antibody isotype responses in vivo. Fusion inactivation of WIV reduced, but did not block, TLR7-dependent IFN alpha induction in murine dendritic cells in vitro. In vivo, fusion-inactive WIV was as potent as fusion-active WIV in inducing total H5N1-specific serum IgG and IgG2c subtype antibodies in unprimed mice. Both vaccines induced only small amounts of IgG1. However, FA treatment of WIV did reduce the capacity of the vaccine to induce hemagglutination-inhibiting (HI) antibodies. This possibly relates to modification of epitopes that are targets for HI antibodies rather than to loss of fusion activity. Antibody affinity maturation was not negatively affected by fusion inactivation. In conclusion, fusion activity of H5N1 WIV does not play a major role in Th1-type antibody induction. Yet, to preserve the full immunogenicity of WIV, or possibly also other inactivated influenza vaccines, harsh treatment with formaldehyde should be avoided. (c) 2012 Elsevier Ltd. All rights reserved

Alum boosts TH2-type antibody responses to whole-inactivated virus influenza vaccine in mice but does not confer superior protection

Clinical trials with pandemic influenza vaccine candidates have focused on aluminium hydroxide as an adjuvant to boost humoral immune responses. In this study we investigated the effect of aluminium hydroxide on the magnitude and type of immune response induced by whole-inactivated virus (WIV) vaccine. Balb/c mice were immunized once with a range of antigen doses (0.04-5 mu g) of WIV produced from A/PR/8 virus, either atone or in combination with aluminium hydroxide. The hemagglutination inhibition (HI) titers of mice receiving WIV + aluminium hydroxide were 4-16-fold higher than HI titers in mice receiving the same dose of WIV alone, indicating the boosting effect of aluminium hydroxide. WIV induced a TH1 skewed humoral and cellular immune response, characterized by strong influenza-specific IgG2a responses and a high number of IFN gamma-secreting T cells. In contrast, immunization with WIV adsorbed to aluminium hydroxide resulted in skewing of this response to a TH2 phenotype (high IgG1 levels and a low number of IFN gamma-producing T cells). To assess the effect of the observed immune response skewing on viral clearance from the lungs mice immunized once with 1 mu g WIV without or with aluminium hydroxide were challenged with A/PR/8 virus 4 weeks later. The immunized mice showed a significant decrease in viral lung titers compared to control mice receiving buffer. However, despite higher antibody titers, mice immunized with WIV adsorbed to aluminium hydroxide suffered from more severe weight loss and had significantly higher virus Loads in their lung tissue than mice receiving WIV alone. Major difference between these groups of mice was the type of immune response induced, TH2 instead of TH1, indicating that a TH1 response plays a major role in viral clearance. (C) 2008 Elsevier Ltd. All rights reserved

Preclinical evaluation of the saponin derivative GPI-0100 as an immunostimulating and dose-sparing adjuvant for pandemic influenza vaccines

AbstractWith the current global influenza vaccine production capacity the large demand for vaccines in case of a pandemic can only be fulfilled when antigen dose sparing strategies are employed. Here we used a murine challenge model to evaluate the potential of GPI-0100, a semi-synthetic saponin derivative, to serve as a dose-sparing adjuvant for influenza subunit vaccine. Balb/c mice were immunized with different doses of A/PR8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 significantly stimulated antibody and cellular immune responses, especially of the Th1 phenotype. Furthermore, virus titers detected in the lungs of mice challenged one week after the second immunization were significantly reduced among the animals that received GPI-0100-adjuvanted vaccines. Remarkably, adjuvantation of subunit vaccine with GPI-0100 allowed a 25-fold reduction in hemagglutinin dose without compromising the protective potential of the vaccine

Preservation of the immunogenicity of dry-powder influenza H5N1 whole inactivated virus vaccine at elevated storage temperatures

Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20 degrees C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40 degrees C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40 degrees C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40 degrees C may also point to an extended shelf-life when stored at 4 degrees C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention

Physical and immunogenic stability of spray freeze-dried influenza vaccine powder for pulmonary delivery:Comparison of inulin, dextran, or a mixture of dextran and trehalose as protectants

One of the advantages of dry influenza vaccines over conventional liquid influenza vaccines is that they can be used for alternative routes of administration. Previous studies showed that spray freeze-drying is an excellent technique to prepare vaccine containing powders for pulmonary delivery U.P. Amorij, V. Saluja, A.H. Petersen, W.L.J. Hinrichs, A. Huckriede, H.W. Frijlink, Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice, Vaccine 25 (2007) 8707-8717; S.A. Audouy, G. van der Schaaf, WIT Hinrichs, H.W. Frijlink, J. Wilschut, A. Huckriede. Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization, Vaccine (2011)). The aim of this study was to investigate the physical and immunogenic stability of spray freeze-dried whole inactivated virus influenza vaccine prepared by using inulin, dextran, and a mixture of dextran and trehalose as protectants. Physical and biochemical characteristics of the vaccine powder' were maintained at temperatures up to 30 degrees C for 3 months. In addition, in vivo data indicate that also, the immunogenic properties of the vaccine were maintained under these storage conditions. On the other hand, in vivo results also revealed that subtle changes in powder characteristics were induced during storage at 30 degrees C. However, laser diffraction measurements showed that problems associated with these subtle changes can be overcome by using dry powder inhalers with an efficient powder dispersing capacity. (C) 2013 Elsevier B.V. All rights reserved

Evaluation of monophosphoryl lipid A as adjuvant for pulmonary delivered influenza vaccine

Prophylaxis against influenza could be improved by the development of a stable, easy to deliver, potent mucosal vaccine. In this study, we spray-freeze-dried (SFD) whole inactivated virus influenza vaccine (WIV) alone or supplemented with monophosphoryl lipid A (MPLA) using inulin as a lyoprotectant. Physical characterization revealed that the SFD powder consisted of highly porous particles with a size distribution suitable for pulmonary administration. The receptor-binding properties of WIV and the immunostimulatory properties of MPLA were preserved after spray-freeze-drying as indicated by unchanged hemagglutination titers and a retained ability of the vaccine to activate NFkB after incubation with a reporter cell line, respectively. Pulmonary vaccination of mice with MPLA-adjuvanted liquid or powder WIV resulted in induction of higher mucosal and systemic antibody concentrations than vaccination with non-adjuvanted formulations. When exposed to influenza virus, mice immunized with MPLA-adjuvanted pulmonary vaccine showed similar protection in terms of reduction in lung virus titers and prevention ofweight loss as mice immunized intramuscularly with subunit vaccine. Characterization of the antibody response revealed a balanced IgG2a-to-IgG1 profile along with induction of both memory IgA- and IgG-producing B cells in mice immunized with MPLA-adjuvanted vaccine. These studies suggest that themucosal and systemic immune responses to pulmonary delivered influenza vaccines can be significantly enhanced by using MPLA as adjuvant. MPLA-adjuvanted SFD vaccine was particularly effective implying that delivery of adjuvanted vaccine powder to the lungs can be an attractive way of immunization against influenza. (C) 2013 Elsevier B.V. All rights reserved

Intracytoplasmic Trapping of Influenza Virus by a Lipophilic Derivative of Aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 mu M and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 p,M SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using con focal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance

Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.status: publishe