Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP+ memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP+ memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qß-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

Viral particles drive rapid differentiation of memory B cells into secondary plasma cells producing increased levels of antibodies.

Extensive studies have been undertaken to describe naive B cells differentiating into memory B cells at a cellular and molecular level. However, relatively little is known about the fate of memory B cells upon Ag re-encounter. We have previously established a system based on virus-like particles (VLPs), which allows tracking of VLP-specific B cells by flow cytometry as well as histology. Using allotype markers, it is possible to adoptively transfer memory B cells into a naive mouse and track responses of naive and memory B cells in the same mouse under physiological conditions. We have observed that VLP-specific memory B cells quickly differentiated into plasma cells that drove the early onset of a strong humoral IgG response. However, neither IgM(+) nor IgG(+) memory B cells proliferated extensively or entered germinal centers. Remarkably, plasma cells derived from memory B cells preferentially homed to the bone marrow earlier and secreted increased levels of Abs when compared with primary plasma cells derived from naive B cells. Hence, memory B cells have the unique phenotype to differentiate into highly effective secondary plasma cells.

Low-affinity B cells transport viral particles from the lung to the spleen to initiate antibody responses.

The lung is an important entry site for pathogens; its exposure to antigens results in systemic as well as local IgA and IgG antibodies. Here we show that intranasal administration of virus-like particles (VLPs) results in splenic B-cell responses with strong local germinal-center formation. Surprisingly, VLPs were not transported from the lung to the spleen in a free form but by B cells. The interaction between VLPs and B cells was initiated in the lung and occurred independently of complement receptor 2 and Fcγ receptors, but was dependent upon B-cell receptors. Thus, B cells passing through the lungs bind VLPs via their B-cell receptors and deliver them to local B cells within the splenic B-cell follicle. This process is fundamentally different from delivery of blood or lymph borne particulate antigens, which are transported into B cell follicles by binding to complement receptors on B cells.

Innate immunity mediates follicular transport of particulate but not soluble protein antigen.

Ag retention on follicular dendritic cells (FDCs) is essential for B cell activation and clonal selection within germinal centers. Protein Ag is deposited on FDCs after formation of immune complexes with specific Abs. In this study, by comparing the same antigenic determinant either as soluble protein or virus-like particle (VLP), we demonstrate that VLPs are transported efficiently to murine splenic FDCs in vivo in the absence of prior immunity. Natural IgM Abs and complement were required and sufficient to mediate capture and transport of VLPs by noncognate B cells. In contrast, soluble protein was only deposited on FDCs in the presence of specifically induced IgM or IgG Abs. Unexpectedly, IgG Abs had the opposite effect on viral particles and inhibited FDC deposition. These findings identify size and repetitive structure as critical factors for efficient Ag presentation to B cells and highlight important differences between soluble proteins and viral particles.

The antihistamines clemastine and desloratadine inhibit STAT3 and c-Myc activities and induce apoptosis in cutaneous T-cell lymphoma cell lines.

Mycosis fungoides and its leukaemic counterpart Sézary syndrome are the most frequent cutaneous T-cell lymphomas (CTCL), and there is no cure for these diseases. We evaluated the effect of clinically approved antihistamines on the growth of CTCL cell lines. CTCL cell lines as well as blood lymphocytes from patients with Sézary syndrome were cultured with antihistamines, and the cell were analysed for proliferation, apoptosis and expression of programmed death molecules and transcription factors. The two antihistamines clemastine and desloratadine, currently used for symptom alleviation in allergy, induced potent reduction of the activities of the constitutively active transcription factors c-Myc, STAT3, STAT5a and STAT5b in mycosis fungoides and Sézary syndrome cell lines. This inhibition was followed by apoptosis and cell death, especially in the Sézary syndrome-derived cell line Hut78 that also showed increased expression of the programmed death-1 (PD-1) after clemastine treatment. In lymphocytes isolated from Sézary syndrome patients, the CD4-positive fraction underwent apoptosis after clemastine treatment, while CD4-negative lymphocytes were little affected. Because both c-Myc and STAT transcription factors are highly expressed in proliferating tumours, their inhibition by clemastine, desloratadine and other inhibitors could complement established chemotherapies not only for cutaneous T-cell lymphomas but perhaps also other cancers.

Adjusted Particle Size Eliminates the Need of Linkage of Antigen and Adjuvants for Appropriated T Cell Responses in Virus-Like Particle-Based Vaccines.

Since the discovery of the first virus-like particle (VLP) derived from hepatitis B virus in 1980 (1), the field has expanded substantially. Besides successful use of VLPs as safe autologous virus-targeting vaccines, the powerful immunogenicity of VLPs has been also harnessed to generate immune response against heterologous and even self-antigens (2-4). Linking adjuvants to VLPs displaying heterologous antigen ensures simultaneous delivery of all vaccine components to the same antigen-presenting cells. As a consequence, antigen-presenting cells, such as dendritic cells, will process and present the antigen displayed on VLPs while receiving costimulatory signals by the VLP-incorporated adjuvant. Similarly, antigen-specific B cells recognizing the antigen linked to the VLP are simultaneously exposed to the adjuvant. Here, we demonstrate in mice that physical association of antigen, carrier (VLPs), and adjuvant is more critical for B than T cell responses. As a model system, we used the E7 protein from human papilloma virus, which spontaneously forms oligomers with molecular weight ranging from 158 kDa to 10 MDa at an average size of 50 nm. E7 oligomers were either chemically linked or simply mixed with VLPs loaded with DNA rich in non-methylated CG motifs (CpGs), a ligand for toll-like receptor 9. E7-specific IgG responses were strongly enhanced if the antigen was linked to the VLPs. In contrast, both CD4+ and CD8+ T cell responses as well as T cell-mediated protection against tumor growth were comparable for linked and mixed antigen formulations. Therefore, our data show that B cell but not T cell responses require antigen-linkage to the carrier and adjuvant for optimal vaccination outcome.

Incorporation of tetanus-epitope into virus-like particles achieves vaccine responses even in older recipients in models of psoriasis, Alzheimer's and cat allergy.

Monoclonal antibodies are widely used to treat non-infectious conditions but are costly. Vaccines could offer a cost-effective alternative but have been limited by sub-optimal T-cell stimulation and/or weak vaccine responses in recipients, for example, in elderly patients. We have previously shown that the repetitive structure of virus-like-particles (VLPs) can effectively bypass self-tolerance in therapeutic vaccines. Their efficacy could be increased even further by the incorporation of an epitope stimulating T cell help. However, the self-assembly and stability of VLPs from envelope monomer proteins is sensitive to geometry, rendering the incorporation of foreign epitopes difficult. We here show that it is possible to engineer VLPs derived from a non human-pathogenic plant virus to incorporate a powerful T-cell-stimulatory epitope derived from Tetanus toxoid. These VLPs (termed CMVTT) retain self-assembly as well as long-term stability. Since Th cell memory to Tetanus is near universal in humans, CMVTT-based vaccines can deliver robust antibody-responses even under limiting conditions. By way of proof of concept, we tested a range of such vaccines against chronic inflammatory conditions (model: psoriasis, antigen: interleukin-17), neurodegenerative (Alzheimer's, ß-amyloid), and allergic disease (cat allergy, Fel-d1), respectively. Vaccine responses were uniformly strong, selective, efficient in vivo, observed even in old mice, and employing low vaccine doses. In addition, randomly ascertained human blood cells were reactive to CMVTT-VLPs, confirming recognition of the incorporated Tetanus epitope. The CMVTT-VLP platform is adaptable to almost any antigen and its features and performance are ideally suited for the design of vaccines delivering enhanced responsiveness in aging populations.

Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles.

Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.

Vaccination against Alzheimer disease: an update on future strategies.

Alzheimer disease is a devastating chronic disease without adequate therapy. More than 10 years ago, it was demonstrated in transgenic mouse models that vaccination may be a novel, disease-modifying therapy for Alzheimer. Subsequent clinical development has been a roller-coaster with some positive and many negative news. Here, we would like to summarize evidence that next generation vaccines optimized for old people and focusing on patients with mild disease stand a good chance to proof efficacious for the treatment of Alzheimer.

[Transcutaneous applications for vaccination and immunotherapy]. / Transkutane Applikationen bei Impfungen und Immuntherapien.

Although Edward Jenner applied the first vaccines by scratching cow pox material into the skin, the profound immunological properties of the skin have become evident through research and discoveries only in the last 20 years. The immunological cells in the epidermis and the dermis are suitable targets for transcutaneous vaccination and immunotherapy. However, as the skin represents a natural barrier for topically administered large molecules, novel methods to overcome this barrier function have been described. There are chemical, biochemical and physical methods, many of which are pain-free and therefore especially suitable for children. Also for adults non-invasive methods of vaccination and immunotherapy are attractive as self-administration is feasible. Future products are currently undergoing clinical tests which provide promising results.

Alors qu'Edward Jenner a utilisé la peau pour appliquer les premières vaccinations, la peau comme organe immunologique n'a été découverte et étudiée que dans les dernières 20 années. A cause d'une multitude de cellules immunologiques dans l'épiderme et le derme, la peau est appropriée pour la vaccination transcutanée et l'immunothérapie. Cependant, comme la peau est une barrière naturelle pour de grosses molécules, il y a des approches différentes pour surmonter cette barrière. On distingue entre les méthodes chimiques, biochimiques et physiques. Surtout pour les enfants, les traitements sans douleur seraient appropriés. Mais pour les adultes aussi, les méthodes de vaccination et d'immunothérapie non-invasives sont une alternative intéressante, puisqu'elles peuvent être administrées par les patients eux-mêmes. Des produits qui sont à l'essai actuellement dans des études de phase I­III fournissent des résultats prometteurs.

Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo.

In comparison to neutral linear polymers, functional and architecturally complex (that is, non-linear) polymers offer distinct opportunities for enhancing the properties and performance of therapeutic proteins. However, understanding how to harness these parameters is challenging, and studies that capitalize on them in vivo are scarce. Here we present an in vivo demonstration that modification of a protein with a polymer of appropriate architecture can impart low immunogenicity, with a commensurably low loss of therapeutic activity. These combined properties are inaccessible by conventional strategies using linear polymers. For the model protein L-asparaginase, a comb-polymer bio-conjugate significantly outperformed the linear polymer control in terms of lower immune response and more sustained bioactivity. The semi-permeability characteristics of the coatings are consistent with the phase diagram of the polymer, which will facilitate the application of this strategy to other proteins and with other therapeutic models.

Virus-induced humoral immunity: on how B cell responses are initiated.

Most antiviral vaccines are based on viral particles, which are efficient inducers of B cell responses. In addition to their ability to replicate, several features associated with the structure and content of the viral particles are responsible for this high immunogenicity. First, viral particles usually have dimensions between 20 and 200 nm, a size optimal for drainage to lymph nodes and direct interaction with B cells. Second, the surface of most viral particles is highly repetitive, causing efficient cross-linking of B cell receptors, an early and key step of B cell activation. In addition, such repetitive structures bind natural antibodies and fix complement, further enhancing B cell activation as well as transport to and deposition on follicular dendritic cells. Third, viral particles carry ligands for toll-like receptor 7/8 or 9 which activate B cells directly for isotype switching as well as dendritic cells for T cell priming. In this review, we will highlight recent insights in these mechanisms and discuss their impact on antiviral antibody responses.

Bacterially produced recombinant influenza vaccines based on virus-like particles.

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

Carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies.

Pre-existing immunity against vaccine carrier proteins has been reported to inhibit the immune response against antigens conjugated to the same carrier by a process termed carrier induced epitopic suppression (CIES). Hence understanding the phenomenon of CIES is of major importance for the development of conjugate vaccines. Virus-like particles (VLPs) are a novel class of potent immunological carriers which have been successfully used to enhance the antibody response to virtually any conjugated antigen. In the present study we investigated the impact of a pre-existing VLP-specific immune response on the development of antibody responses against a conjugated model peptide after primary, secondary and tertiary immunization. Although VLP-specific immune responses led to reduced peptide-specific antibody titers, we showed that CIES against peptide-VLP conjugates could be overcome by high coupling densities, repeated injections and/or higher doses of conjugate vaccine. Furthermore we dissected VLP-specific immunity by adoptively transferring VLP-specific antibodies, B-cells or T(helper) cells separately into naïve mice and found that the observed CIES against peptide-VLP conjugates was mainly mediated by carrier-specific antibodies.