Anke Huckriede

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.

Development of stable influenza vaccine powder formulations: challenges and possibilities.

Influenza vaccination represents the cornerstone of influenza prevention. However, today all influenza vaccines are formulated as liquids that are unstable at ambient temperatures and have to be stored and distributed under refrigeration. In order to stabilize influenza vaccines, they can be brought into the dry state using suitable excipients, stabilizers and drying processes. The resulting stable influenza vaccine powder is independent of cold-chain facilities. This can be attractive for the integration of the vaccine logistics with general drug distribution in Western as well as developing countries. In addition, a stockpile of stable vaccine formulations of potential vaccines against pandemic viruses can provide an immediate availability and simple distribution of vaccine in a pandemic outbreak. Finally, in the development of new needle-free dosage forms, dry and stable influenza vaccine powder formulations can facilitate new or improved targeting strategies for the vaccine compound. This review represents the current status of dry stable inactivated influenza vaccine development. Attention is given to the different influenza vaccine types (i.e. whole inactivated virus, split, subunit or virosomal vaccine), the rationale and need for stabilized influenza vaccines, drying methods by which influenza vaccines can be stabilized (i.e. lyophilization, spray drying, spray-freeze drying, vacuum drying or supercritical fluid drying), the current status of dry influenza vaccine development and the challenges for ultimate market introduction of a stable and effective dry-powder influenza vaccine.

Safety and efficacy of influenza vaccination in systemic lupus erythematosus patients with quiescent disease

Objective: to assess the safety and efficacy of influenza vaccination in patients with systemic lupus erythematosus (SLE), and to evaluate the influence of immunosuppressive drugs on the immune response. Methods: SLE patients (n = 56) and healthy controls (n = 18) were studied. All patients had quiescent disease (SLE disease activity index ⩽5). Four patient groups were defined on the basis of their drug use: (1) no drug treatment; (2) hydroxychloroquine treatment; (3) azathioprine treatment; (4) prednisone treatment. Participants received trivalent influenza subunit vaccine during October/November 2003. Disease activity scores and side effects were recorded. Antibody titres against influenza virus were measured before and 30 days after vaccination using the haemagglutination inhibition assay. Results: Influenza vaccination did not result in changes in disease activity and was well tolerated. SLE patients had fewer seroconversions or fourfold titre rises for A/H1N1 (p<0.001) and A/H3N2 (p<0.001) than healthy controls, while for B/Hong Kong the difference was of borderline significance (p = 0.051). With regard to immunosuppressive treatment, fewer SLE patients using azathioprine developed fourfold titre rises against A/H3N2 (p = 0.041), and fewer achieved titres of ⩾40 against A/H3N2 (p = 0.030) compared with the other patient groups. Conclusions: Influenza vaccination in SLE patients with quiescent disease is safe but is less effective than in controls. Use of azathioprine was associated with a trend to decreased vaccination efficacy.

Microneedle arrays for the transcutaneous immunization of diphtheria and influenza in BALB/c mice

Transcutaneous immunization (TCI) is limited by poor permeation of macromolecules across the skin. Microneedle arrays form transient conduits and enhance the transport of vaccine molecules across the skin barrier without pain sensation. Here we investigated in mouse the immune responses after TCI using two model antigens, diphtheria toxoid (DT) and influenza subunit vaccine. The electric applicator enabled shorter microneedle (300 microm) to pierce mouse skin effectively, as shown by Trypan blue staining and trans-epidermal water loss measurement. The vaccines were topically applied with and without cholera toxin (CT) on microneedle-treated skin. In DT TCI, microneedle array pretreatment of the skin was essential to achieve substantial IgG and toxin-neutralizing antibody titers. Addition of CT further boosted the immune response to similar levels as observed after subcutaneous injection of AlPO4-adsorbed DT (DT-alum). In contrast, microneedle array pretreatment showed no effect on the immune response to plain influenza vaccine. This response was strongly improved by inclusion of CT, independent of microneedle treatment. These results indicate that TCI of DT and CT with microneedle treatment results in comparable protection as injection of DT-alum, and TCI of influenza vaccine adjuvanted with CT is superior to the injection of plain vaccine.

Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides

Several studies have shown that immunization with heat shock proteins (HSPs) purified from tumors of virus-infected cells induces specific cytotoxic T-cell (CTL) activity. This immune response is directed against peptides bound to the HSPs rather than against the HSPs themselves. The peptides are derived from tumor- or virus-specific proteins which are degraded in the course of normal protein turnover and processing for presentation by MHC class I molecules. The HSPs appear to function as carriers for the antigenic peptides. Upon immunization they ensure their uptake by specialized macrophages and their introduction into the MHC class I presentation route which is otherwise accessible only for intracellular proteins. Using influenza virus nucleoprotein (NP) as a model antigen, we have tested whether an HSP-based vaccine can be preduced by overexpressing an antigen in cultured cells prior to purification of the HSPs. The transfection system based on the Semliki Forest virus (SFV) replicon was employed to achieve high expression of NP. Since SFV-mediated transfection of murine cells was inefficient we used the hamster-derived cell line BHK21, which can be transfected with 100% efficiency, as a source for NP peptide-loaded gp96. The protein was purified from transfected cells and used for first vaccination studies. The hamster gp96 preparation was well tolerated in mice, an antibody response against the foreign protein was not observed. Preliminary results suggest that a cellular immune response against NP was indeed induced. SFV transfection is applicable for any known antigen and is therefore considered to be an elegant means for the production of HSP-based vaccines capable of inducing a cellular immune response.

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 microg) of WIV produced from A/PR/8 virus, either alone 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 IFNgamma-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 IFNgamma-producing T cells). To assess the effect of the observed immune response skewing on viral clearance from the lungs mice immunized once with 1 microg 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.

Needle-free influenza vaccination

Vaccination is the cornerstone of influenza control in epidemic and pandemic situations. Influenza vaccines are typically given by intramuscular injection. However, needle-free vaccinations could offer several distinct advantages over intramuscular injections: they are pain-free, easier to distribute, and easier to give to patients, and their use could reduce vaccination costs. Moreover, vaccine delivery via the respiratory tract, alimentary tract, or skin might elicit mucosal immune responses at the site of virus entry and better cellular immunity, thus improving effectiveness. Although various needle-free vaccination methods for influenza have shown preclinical promise, few have progressed to clinical trials-only live attenuated intranasal vaccines have received approval, and only in some countries. Further clinical investigation is needed to help realise the potential of needle-free vaccination for influenza.

GLA-SE, a Synthetic Toll-like Receptor 4 Agonist, Enhances T-Cell Responses to Influenza Vaccine in Older Adults

BACKGROUND The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza. METHODS The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection. RESULTS GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor α, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon γ to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults. CONCLUSIONS Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults.

Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.

Towards tailored vaccine delivery: Needs, challenges and perspectives

The ideal vaccine is a simple and stable formulation which can be conveniently administered and provides life-long immunity against a given pathogen. The development of such a vaccine, which should trigger broad and strong B-cell and T-cell responses against antigens of the pathogen in question, is highly dependent on tailored vaccine delivery approaches. This review addresses vaccine delivery in its broadest scope. We discuss the needs and challenges in the area of vaccine delivery, including restrictions posed by specific target populations, potentials of dedicated stable formulations and devices, and the use of adjuvants. Moreover, we address the current status and perspectives of vaccine delivery via several routes of administration, including non- or minimally invasive routes. Finally we suggest possible directions for future vaccine delivery research and development.