Gideon Kersten

Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice.

In this study, the ability of chitosan microparticles to enhance both the systemic and local immune responses against diphtheria toxoid (DT) after oral and nasal administration in mice was investigated.Firstly, DT was associated to chitosan microparticles to determine antigen loading and release. Then DT loaded chitosan microparticles, DT in phosphate buffered saline (PBS) and empty chitosan microparticles (as controls) were fed intragastrically and administered nasally to mice. Mice were also subcutaneously immunised with DT associated with alum. All mice were vaccinated in week 1 and boosted in week 3. Sera were analysed for anti-DT IgG and nasal washings and faeces for anti-DT IgA titres using an enzyme linked immunosorbent assay. Loading capacities of about 25% and loading efficacies of about 100% were obtained after loading the chitosan microparticles with DT. No DT was released at 37 degrees C in PBS. Compared to intragastrical feeding with DT in PBS, a strong enhancement of the systemic and local immune responses against DT were found in mice fed with DT loaded chitosan microparticles. In addition, a dose-dependent immune reaction was observed for mice vaccinated with different doses of DT associated to chitosan microparticles. Significant systemic humoral immune responses were also found after nasal vaccination with DT associated to chitosan microparticles.DT associated to chitosan microparticles results in protective systemic and local immune response against DT after oral vaccination, and in significant enhancement of IgG production after nasal administration. Hence, these in vivo experiments demonstrate that chitosan microparticles are very promising mucosal vaccine delivery systems.

Liposomes and ISCOMs

Liposomes and ISCOMs have a long history as vehicles for antigen delivery. Liposomes can carry both membrane associated antigens as well as water soluble molecules. Their physical properties are highly variable, depending on composition and manufacturing method. This allows optimised design for specific tasks (targeting, co-incorporation of adjuvants, etc.). ISCOMs already have a build-in adjuvant, Quillaja saponin, which is a structural part of the vehicle. In recent years, considerable progress has been achieved with respect to the use of better defined saponin. Clinical trials with ISCOMs are in progress and registered liposomal vaccines exist. Here, follows a brief overview on recent developments with emphasis on pharmaceutical aspects.

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.

On the structure of immune-stimulating saponin-lipid complexes (iscoms)

Immune-stimulating complexes (iscoms) are stable complexes of cholesterol, phospholipid and Quil A, a triterpene saponin mixture in the size range from 40 to 100 nm. They can be used as antigen carriers in subunit vaccines. In this paper it is demonstrated that iscoms are rigid, negatively charged vesicles in which small water soluble molecules like carboxyfluorescein cannot be retained. The negative zeta-potential prevents iscoms from aggregation. The chemical composition of iscoms in one dispersion varied considerably. A typical example of the composition of iscoms is cholesterol/phospholipid/Quil A = 1.0:1.2:6.2 by weight for the iscom matrix, that is iscoms without antigen, and 1.0:1.3:5.1 for antigen-containing iscoms. A hypothetical model for the structure of the iscom matrix and related structures is presented, based on analytical chemical, physico-chemical and electronmicroscopic data. In this model iscoms are considered to be multi-micellar structures, shaped and stabilized by hydrophobic interactions, electrostatic repulsion, steric factors and possibly hydrogen bonds. The individual micelles are relatively flat, ring-shaped structures, the center offering space for one of the two bulky sugar chains of the saponins.

Efficient induction of immune responses through intradermal vaccination with N-trimethyl chitosan containing antigen formulations.

The function of N-trimethyl chitosan (TMC) in dermal immunisation is unknown. Therefore we investigated the immunogenicity of both antigen-containing TMC nanoparticles and TMC/antigen solutions after intradermal injection. Nanoparticles were prepared with a size around 200 nm and a positive zetapotential. In vitro, TMC nanoparticles increased the uptake of OVA by dendritic cells (DCs) and both nanoparticles and TMC/OVA mixtures were able to induce upregulation of MHC-II, CD83 and CD86. These activated DCs could induce a Th2 biased T cell proliferation. A solution of plain OVA did not induce DC maturation or T cell proliferation. In vivo, mice were injected thrice with TMC based formulations containing either OVA or diphtheria toxoid (DT), a more relevant antigen. All TMC-containing formulations were able to increase the IgG titres compared to unadjuvanted antigen and induced a Th2 biased immune response. When using DT-containing TMC formulations, IgG titres and neutralising antibody titres could match up to those obtained after subcutaneous injection of DT-Alum. In conclusion, both soluble TMC/antigen mixtures and TMC nanoparticles are able to induce DC maturation and enhance immune responses after intradermal injection. This demonstrates that TMC functions as an immune potentiator for antigens delivered via the skin.

Current and next generation influenza vaccines: Formulation and production strategies

Vaccination is the most effective method to prevent influenza infection. However, current influenza vaccines have several limitations. Relatively long production times, limited vaccine capacity, moderate efficacy in certain populations and lack of cross-reactivity are important issues that need to be addressed. We give an overview of the current status and novel developments in the landscape of influenza vaccines from an interdisciplinary point of view. The feasibility of novel vaccine concepts not only depends on immunological or clinical outcomes, but also depends on biotechnological aspects, such as formulation and production methods, which are frequently overlooked. Furthermore, the next generation of influenza vaccines is addressed, which hopefully will bring cross-reactive influenza vaccines. These developments indicate that an exciting future lies ahead in the influenza vaccine field.

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.

Microneedle-Based Transcutaneous Immunisation in Mice with N-Trimethyl Chitosan Adjuvanted Diphtheria Toxoid Formulations

ABSTRACTPurposeThe purpose of this study was to gain insight into the delivery and immunogenicity of N-trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied transcutaneously with microneedles.MethodsMice were vaccinated with DT-loaded TMC nanoparticles, a solution of TMC and DT (TMC/DT) or DT alone. The formulations were applied onto the skin before or after microneedle treatment with two different 300-µm-long microneedle arrays and also injected intradermally (ID). As a positive control, alum-adjuvanted DT (DT-alum) was injected subcutaneously (SC). Ex vivo confocal microscopy studies were performed with rhodamine-labelled TMC.ResultsIndependent of the microneedle array used and the sequence of microneedle treatment and vaccine application, transcutaneous immunisation with the TMC/DT mixture elicited 8-fold higher IgG titres compared to the TMC nanoparticles or DT solution. The toxin-neutralising antibody titres from this group were similar to those elicited by SC DT-alum. After ID immunisation, both TMC-containing formulations induced enhanced titres compared to a DT solution. Confocal microscopy studies revealed that transport of the TMC nanoparticles across the microneedle conduits was limited compared to a TMC solution.ConclusionsIn conclusion, TMC has an adjuvant function in transcutaneous immunisation with microneedles, but only if applied in a solution.

Reduction of animal use in human vaccine quality control: opportunities and problems.

In vivo assays play a crucial role in the assessment of the potency and safety of human vaccines. Robust vaccine production procedures, improved characterisation methods and development of well-characterised vaccines create possibilities to reduce animal use. In this paper the current status in this field is reviewed. Achievements with regard to in vivo and in vitro potency and safety testing are discussed as well as new developments and possibilities in the field of in vitro characterisation of vaccine components. Finally, validation and implementation issues will be dealt with. Although replacement of in vivo tests for batch release of existing vaccines is difficult, emerging technologies allow well-considered reduction of in vivo experiments during product and process development and improvement. Inextricably bound up with this approach is good manufacturing practice (GMP), resulting in robust, validated production processes.

Buccal and sublingual vaccine delivery.

Abstract Because of their large surface area and immunological competence, mucosal tissues are attractive administration and target sites for vaccination. An important characteristic of mucosal vaccination is its ability to elicit local immune responses, which act against infection at the site of pathogen entry. However, mucosal surfaces are endowed with potent and sophisticated tolerance mechanisms to prevent the immune system from overreacting to the many environmental antigens. Hence, mucosal vaccination may suppress the immune system instead of induce a protective immune response. Therefore, mucosal adjuvants and/or special antigen delivery systems as well as appropriate dosage forms are required in order to develop potent mucosal vaccines. Whereas oral, nasal and pulmonary vaccine delivery strategies have been described extensively, the sublingual and buccal routes have received considerably less attention. In this review, the characteristics of and approaches for sublingual and buccal vaccine delivery are described and compared with other mucosal vaccine delivery sites. We discuss recent progress and highlight promising developments in the search for vaccine formulations, including adjuvants and suitable dosage forms, which are likely critical for designing a successful sublingual or buccal vaccine. Finally, we outline the challenges, hurdles to overcome and formulation issues relevant for sublingual or buccal vaccine delivery.