Most influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of ...dissolving-microneedle-arrays (dMNAs) could serve as minimal invasive alternative to needle injections. However, during the production process of dMNAs antigens are subjected to several stresses, which may reduce their potency. Moreover, the needles need to have sufficient mechanical strength to penetrate the skin and subsequently dissolve effectively to release the incorporated antigen. Here, we investigated whether blends of trehalose and pullulan are suitable for the production of stable dMNA fulfilling these criteria. Our results demonstrate that production of trehalose/pullulan-based dMNAs rendered microneedles that were sharp and stiff enough to pierce into ex vivo human skin and subsequently dissolve within 15 min. The mechanical properties of the dMNAs were maintained well even after four weeks of storage at temperatures up to 37°C. In addition, immunization of mice with influenza antigens via both freshly prepared dMNAs and dMNAs after storage (four weeks at 4°C or 37°C) resulted in antibody titers of similar magnitude as found in intramuscularly injected mice and partially protected mice from influenza virus infection. Altogether, our results demonstrate the potential of trehalose/pullulan-based dMNAs as alternative dosage form for influenza vaccination.
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 µm) 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 AlPO
4-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.
a) Microneedle array, b) Conduits formed in mouse skin stained with Trypan blue and c) Immune responses of topically applied diphtheria toxoid improved by microneedle array pretreatment (MN).
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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.
Abstract In this study pulmonary vaccination with a new influenza subunit vaccine powder was evaluated. Vaccine powder was produced by spray-freeze drying (SFD) using the oligosaccharide inulin as ...stabilizer. Immune responses after pulmonary vaccination of BALB/c mice with vaccine powder were determined and compared to those induced by intramuscular and pulmonary vaccination with a conventional liquid subunit vaccine. All vaccinations were performed without adjuvant. Pulmonary vaccination with liquid subunit vaccine resulted in systemic humoral (IgG) immune responses similar to intramuscular immunization. In contrast, the vaccine powder delivered by the pulmonary route, induced not only systemic humoral (IgG) responses, but also cell-mediated (Il-4, IFN-γ) and mucosal immune responses (IgA, IgG). This study demonstrates that the combination of pulmonary antigen delivery and antigen powder production by SFD improves the immunogenic potential of (influenza subunit) antigen. In conclusion, vaccination with a non-adjuvanted SFD subunit vaccine powder by inhalation might be feasible and could be an alternative to conventional parenteral vaccine administration.
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.
Abstract The development of a stable influenza subunit vaccine in the dry state was investigated. The influence of various carbohydrates, buffer types and freezing rates on the integrity of ...haemagglutinin after freeze-thawing or freeze-drying was investigated with a range of analytical and immunological methods. The use of fast freezing, Hepes buffer and carbohydrates (trehalose, inulin or dextran) as cryo- and lyoprotectants resulted in a significant reduction or even absence of conformational changes of HA as revealed by the used methods. The subunit vaccine in the powder was shown to remain immunogenic in an in vivo study in mice, using reconstituted powder. Moreover, the HA potency of the influenza subunit vaccine powder was stable for at least 26 weeks at room temperature.
Wegener's granulomatosis (WG) is a systemic vasculitis characterised by relapsing and remitting disease activity. Immunosuppressive drugs are used to control disease, but increase susceptibility to ...infection. Therefore, influenza vaccination should be considered in WG patients. This study was performed to assess the immunogenicity of influenza vaccination in WG patients.
A randomised, controlled trial was performed in WG patients with quiescent disease, defined as a Birmingham vasculitis activity score (BVAS) less than 2. Patients were randomly assigned to receive influenza vaccination (n = 49) or to participate as controls (n = 23). In addition, healthy controls (n = 49) were vaccinated. At entry and at 1 and 3-4 months after entry, antibody responses to vaccination were determined. Furthermore, disease activity was measured (BVAS), adverse effects were recorded and antineutrophil cytoplasmic autoantibody (ANCA) titres were determined.
WG patients achieved high seroprotection rates to all three influenza strains, comparable with healthy controls. Only the A/H1N1 strain patients had a lower seroconversion rate (p = 0.002) and geometric mean titre (p = 0.037) than controls. After 1 month, one control and one vaccinated WG patient had developed active disease. At 3-4 months, two additional control patients had developed active disease compared with none of the vaccinated patients (p = 0.099). Vaccination did not influence ANCA titres. Adverse effects did not differ between patients and healthy controls.
Influenza vaccination in WG patients with quiescent disease induced a sufficient antibody response.
NTR1130.
Application of RNA interference for in vivo evaluation of gene function or for therapeutic interventions has been hampered by a lack of suitable delivery methods for small interfering RNA (siRNA). ...Here, we present reconstituted viral envelopes (virosomes) derived from influenza virus as suitable vehicles for in vitro as well as in vivo delivery of siRNAs. Virosomes are vesicles that bear in their membrane the influenza virus spike protein hemagglutinin (HA). This protein mediates binding of native virus to and fusion with cellular target membranes. Accordingly, virosomes with membrane-incorporated HA bind to cells, are taken up by receptor-mediated endocytosis, and fuse with the endosomal membrane to release their contents into the cytoplasm. When complexed to cationic lipids, siRNA was successfully encapsulated in virosomes. Virosomes with encapsulated siRNA fused with target membranes in a pH-dependent manner and delivered the encapsulated siRNA to several cell lines in vitro. Virosome-delivered siRNA markedly downregulated the synthesis of newly induced and constitutively expressed green fluorescent protein. Moreover, intraperitoneal injection of siRNA-loaded virosomes resulted in delivery of the nucleotides to cells in the peritoneal cavity. Our results indicate that virosomes are a promising delivery device for in vivo application, especially where topical administration of siRNA, for example, to the respiratory tract is envisaged.
Abstract Gram-positive enhancer matrix (GEM) particles, produced from non-genetically modified Lactococcus lactis bacteria have an inherent immunostimulatory activity. It was investigated whether ...co-administration of GEM particles can reduce the amount of influenza subunit vaccine (HA) necessary to protect mice from viral infection. Decreasing HA amounts of 5, 1, 0.2 and 0.04 μg admixed with GEM particles were tested in intramuscular immunizations. Combinations of GEM and seasonal HA (A/Wisconsin/67/2005 H3N2) induced significantly higher systemic and better Th1/Th2-type balanced immune responses than HA alone. Addition of GEM to 0.04 μg HA resulted in similar HI titers as 1–5 μg non-adjuvanted HA. To test the protective efficacy of the adjuvanted combination, mice were immunized with influenza subunit vaccine A/PR/8/34 (H1N1) and then challenged with live virus (A/PR/8/34). Mice immunized with 1 μg HA + GEM showed undetectable virus titers in the lungs 5 days after challenge, whereas mice immunized with 1 μg HA alone showed detectable levels of virus in the lungs. Interestingly, mice vaccinated with the 0.04 μg HA + GEM vaccine demonstrated reduced lung virus titers and a reduction in weight that was similar as that in mice vaccinated with 1 μg non-adjuvanted HA. These results indicate that the use of GEM as immunostimulant allows for a strong reduction in the antigen dose as compared to the benchmark vaccine by using GEM particles. Thus, addition of GEM can strongly potentiate immunogenicity of influenza subunit vaccine both quantitatively and qualitatively.
GEM adjuvanted oral influenza vaccine.
In this study, a liquid formulation of influenza subunit vaccine admixed with Gram-positive enhancer matrix (GEM) particles as adjuvant was delivered to upper ...and lower parts of intestinal tract. The aim was to determine the most effective immunization site in the intestines. Mice were vaccinated with a liquid formulation of GEM and influenza subunit vaccine orally and rectally. The oral administration of the vaccine with GEM particles induced a better systemic and mucosal immune response than oral (vaccine only) and rectal (with and without adjuvant) immunizations. Rectal administration elicited high IgG1 responses but little IgG2a, indicating a Th2 dominated immune response. In contrast, the oral immunization with GEM particles elicited a balanced IgG1 and IgG2a response. In conclusion, our results demonstrate that GEM-adjuvanted influenza vaccine should be targeted to the upper part of the intestinal tract.
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