Modern science for better quality control of medicinal products "Towards global harmonization of 3Rs in biologicals": The report of an EPAA workshop.

This article summarizes the outcome of an international workshop organized by the European Partnership for Alternative Approaches to Animal Testing (EPAA) on Modern science for better quality control of medicinal products: Towards global harmonization of 3Rs in biologicals. As regards the safety testing of biologicals, the workshop participants agreed to actively encourage the deletion of abnormal toxicity tests and target animal batch safety tests from all relevant legal requirements and guidance documents (country-specific guidelines, pharmacopoeia monographs, WHO recommendations). To facilitate the global regulatory acceptance of non-animal methods for the potency testing of, e.g., human diphtheria and tetanus vaccines and veterinary swine erysipelas vaccines, international convergence on the scientific principles of the use of appropriately validated in vitro assays for replacing in vivo methods was identified as an overarching goal. The establishment of scientific requirements for new assays was recognized as a further means to unify regulatory approaches in different jurisdictions. It was recommended to include key regulators and manufacturers early in the corresponding discussions. Manufacturers and responsible expert groups, e.g. at the European Directorate for the Quality of Medicines and Health Care of the Council of Europe or the European Medicines Agency, were invited to consider leadership for international collaboration.

A step-by-step approach to study the influence of N-acetylation on the adjuvanticity of N,N,N-trimethyl chitosan (TMC) in an intranasal nanoparticulate influenza virus vaccine.

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper.

Master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortants used in live attenuated influenza vaccine (LAIV) do not display neurovirulent properties in a mouse model.

Demonstration of the absence of neurovirulent properties of reassortant viruses contained in live attenuated influenza vaccine (LAIV) is a regulatory requirement. A mouse model was used to detect neurovirulent properties of the cold-adapted, temperature-sensitive and attenuated influenza master donor viruses (MDVs) A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69 and derived reassortant influenza viruses. A/NWS/33 (H1N1), which is known to be neurovirulent in mice, was used as a positive control. Under conditions where the positive control virus induced symptoms of disease and showed viral replication in the upper respiratory tract as well as in the brain, replication of the influenza master donor viruses and reassortant influenza A and B viruses was limited to the upper respiratory tract where they were administered. None of the mice inoculated with MDVs or reassortant influenza viruses suffered from disease, and no virus or viral replication was observed in the brains of these mice. The results demonstrate the absence of neurovirulent properties of the MDVs and reassortant influenza viruses derived therefrom used in LAIV.

Role of trimethylated chitosan (TMC) in nasal residence time, local distribution and toxicity of an intranasal influenza vaccine.

The nose is a promising immunization site and intranasal (i.n.) vaccination studies with whole inactivated influenza virus (WIV) adjuvanted with N,N,N-trimethylchitosan (TMC-WIV) have shown promising results. In this study, the influence of TMC on the i.n. delivery of WIV was studied in mice by comparing the nasal residence time and the specific location in the nasal cavity of WIV and TMC-WIV. Additionally, the local toxicity profile of the WIV formulations was assessed. In vivo fluorescence imaging was used to study the nasal residence time and the fate of the bulk vaccine in mice that received vaccines fluorescently labeled with IRDye800CW. An immunohistochemical (IHC) staining method for nasal cross-sections was developed to visualize the antigen in the nasal cavity. Therefore, mice were sacrificed at different time points after vaccination with various vaccine formulations and nasal cross-sections were made. The local toxicity was assessed using hematoxylin and eosin staining for the nasal cross-sections. No significant differences in the nasal residence time between WIV and TMC-WIV were observed. However, IHC revealed a striking difference in the location and distribution of WIV in the nasal cavity. When formulated as plain WIV, positive staining was mainly found in the nasal cavity, presumably in mucus blobs. TMC-coated WIV, on the other hand, was mostly present as a thin layer on the epithelial surfaces of the naso- and maxilloturbinates. This difference in staining pattern correlates with the observed differences in immunogenicity of these two vaccines and indicates that TMC-WIV results in a much closer interaction of WIV with the epithelial surfaces than WIV alone, potentially leading to enhanced uptake and induction of immune responses. This study further shows that both WIV and TMC-WIV formulations induce minimal local toxicity. Taken altogether, these results provide more insight in the mode of action and safety of TMC and justify further research to develop TMC-adjuvanted nasal vaccines.

Relationship between structure and adjuvanticity of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine.

The aim of this study was to assess the influence of structural properties of N,N,N-trimethyl chitosan (TMC) on its adjuvanticity. Therefore, TMCs with varying degrees of quaternization (DQ, 22-86%), O-methylation (DOM, 0-76%) and acetylation (DAc 9-54%) were formulated with whole inactivated influenza virus (WIV). The formulations were characterized physicochemically and evaluated for their immunogenicity in an intranasal (i.n.) vaccination/challenge study in mice. Simple mixing of the TMCs with WIV at a 1:1 (w/w) ratio resulted in comparable positively charged nanoparticles, indicating coating of WIV with TMC. The amount of free TMC in solution was comparable for all TMC-WIV formulations. After i.n. immunization of mice with WIV and TMC-WIV on days 0 and 21, all TMC-WIV formulations induced stronger total IgG, IgG1 and IgG2a/c responses than WIV alone, except WIV formulated with reacetylated TMC with a DAc of 54% and a DQ of 44% (TMC-RA44). No significant differences in antibody titers were observed for TMCs that varied in DQ or DOM, indicating that these structural characteristics play a minor role in their adjuvant properties. TMC with a DQ of 56% (TMC56) formulated with WIV at a ratio of 5:1 (w/w) resulted in significantly lower IgG2a/c:IgG1 ratios compared to TMC56 mixed in ratios of 0.2:1 and 1:1, implying a shift towards a Th2 type immune response. Challenge of vaccinated mice with aerosolized virus demonstrated protection for all TMC-WIV formulations with the exception of TMC-RA44-WIV. In conclusion, formulating WIV with TMCs strongly enhances the immunogenicity and induces protection against viral challenge in mice after i.n. vaccination. The adjuvant properties of TMCs as i.n. adjuvant are strongly decreased by reacetylation of TMC, whereas the DQ and DOM hardly affect the adjuvanticity of TMC.

Physicochemical and immunological characterization of N,N,N-trimethyl chitosan-coated whole inactivated influenza virus vaccine for intranasal administration.

PURPOSE: The purpose of this study was the development and physicochemical and immunological characterization of intranasal (i.n.) vaccine formulations of whole inactivated influenza virus (WIV) coated with N,N,N-trimethyl chitosan (TMC). METHODS: Synthesized TMCs with a degree of quarternization of 15% (TMC15) or 37% (TMC37) were tested in vitro for their ability to decrease the transepithelial resistance (TEER) of an epithelial cell monolayer. TMC15- and TMC37-coated WIV (TMC15-WIV and TMC37-WIV) were characterized by zeta potential measurements, dynamic light scattering, electron microscopy and gel permeation chromatography. Mice were vaccinated i.n. with selected vaccine formulations and immunogenicity was determined by measuring serum hemagglutination inhibition (HI) and serum IgG, IgG1 and IgG2a/c titers. Also a pulse-chase study with TMCs in solution administered i.n. 2 h prior to WIV was performed. Protective efficacy of vaccination was determined by an aerosol virus challenge. RESULTS: TMC37 induced a reversible decrease in TEER, suggesting the opening of tight junctions, whereas TMC15 did not affect TEER. Simple mixing of (negatively charged) WIV with TMC15 or TMC37 resulted in positively charged particles with TMCs being partially bound. Intranasal immunization with TMC37-WIV or TMC15-WIV induced stronger HI, IgG, IgG1 and IgG2a/c titers than WIV alone. TMC37-WIV induced the highest immune responses. Both TMC15-WIV and TMC37-WIV provided protection against challenge, whereas WIV alone was not protective. Intranasal administration of TMC prior to WIV did not result in significant immune responses, indicating that the immunostimulatory effect of TMC is primarily based on improved i.n. delivery of WIV. CONCLUSIONS: Coating of WIV with TMC is a simple procedure to improve the delivery and immunogenicity of i.n. administered WIV and may enable effective i.n. vaccination against influenza.

Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model.

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 microg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.

Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate.

Signals delivered by the CD40 ligand, CD154, have crucial roles in immune responses in mammals, being required for development of germinal centres, maturation of T-dependent antibody responses, and generation of B-cell memory. To determine whether these functions were conserved in a non-mammalian species, a putative chicken CD 154 cDNA was used to make an oligomeric fusion protein, and to raise monoclonal antibodies. The antibodies detected surface expression on activated T-cells. The fusion protein detected expression of a receptor on B-cells, thrombocytes and macrophages. Biological effects of the fusion protein included induction of NO synthesis in a macrophage cell line, enhancement of splenic B-cell survival, and induction of apoptosis in a bursal lymphoma cell line. These observations demonstrated substantial functional equivalence with mammalian CD 154 and thus provided evidence for the early evolutionary emergence of the set of functions associated with this molecule, and its central role in the vertebrate immune system.

Large scale protein production of the extracellular domain of the transforming growth factor-beta type II receptor using the Pichia pastoris expression system.

To study the (patho)physiological role of transforming growth factor-beta (TGF-beta), potent and selective inhibitors are necessary. Since TGF-beta signaling is initiated by the high affinity binding to the type II receptor (RII), the extracellular part of RII (solRII) can function as a TGF-beta antagonist. SolRII was cloned and large-scale protein synthesis was performed in the yeast Pichia pastoris expression system. Our results indicate that via this system, high levels of pure concentrated solRII can be obtained. Moreover, purified solRII is an active protein as shown by ELISA and bioassay. In conclusion, our large-scale protein expression procedure results in high quantities of purified solRII, which is a powerful tool to study the natural role of TGF-beta.

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant.

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.

Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair.

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-beta1 and -beta3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-beta on osteophyte formation and articular cartilage, TGF-beta activity was blocked via a scavenging soluble TGF-beta-RII. Our results clearly show that inhibition of endogenous TGF-beta nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-beta-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-beta is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.

Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus.

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime-boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations - on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.