Publications by authors named "Philippe Saudan"

38 Publications

Induction of Human T-cell and Cytokine Responses Following Vaccination with a Novel Influenza Vaccine.

Sci Rep 2018 12 20;8(1):18007. Epub 2018 Dec 20.

Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos Bldg, Singapore, 138673, Singapore.

Cell mediated immunity plays a vital role in defense against influenza infection in humans. Less is known about the role of vaccine-induced cell mediated immunity and the cytokine responses elicited. We measured CD4 and CD8 T-cell reactivity in human subjects following vaccination with licensed trivalent influenza vaccine and a novel virus-like particle based vaccine. We detected influenza-specific CD4 T-cell responses following vaccination with the licensed trivalent influenza vaccine and found that these correlated with antibody measurements. Administration of the novel virus-like particle based vaccine elicited influenza-specific CD4 and CD8 T-cell responses and the induction of the cytokines IFN-γ, IL-17A, IL17F, IL-5, IL-13, IL-9, IL-10 and IL-21. Pre-existing cytokine responses influenced the profile of the cytokine response elicited by vaccination. In a subset of individuals the VLP vaccine changed pre-vaccination production of type 2 cytokines such as IL-5 and IL-13 to a post-vaccination type 1 cytokine signature characterized by IFN-γ. A transcriptional signature to vaccination was found to correlate with antibody titer, IFN-γ production by T-cells and expression of a putative RNA helicase, DDX17, on the surface of immune cells.
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http://dx.doi.org/10.1038/s41598-018-36703-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301966PMC
December 2018

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

Front Immunol 2017 6;8:226. Epub 2017 Mar 6.

The Jenner Institute, Oxford University, Oxford, UK; Immunology, Inselspital, Bern, Switzerland.

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.
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http://dx.doi.org/10.3389/fimmu.2017.00226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5337491PMC
March 2017

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes.

Malar J 2017 03 13;16(1):115. Epub 2017 Mar 13.

Structural Vaccinology Laboratory, Malaria Vaccine Branch, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD, 20910, USA.

Background: Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic.

Methods: A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP).

Results: Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity.

Conclusions: Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.
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http://dx.doi.org/10.1186/s12936-017-1766-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347822PMC
March 2017

Cystatin F is a biomarker of prion pathogenesis in mice.

PLoS One 2017 8;12(2):e0171923. Epub 2017 Feb 8.

Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171923PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5298286PMC
August 2017

Development of an Interleukin-1β Vaccine in Patients with Type 2 Diabetes.

Mol Ther 2016 05 21;24(5):1003-12. Epub 2015 Dec 21.

Cytos Biotechnology AG, Schlieren, Switzerland.

Interleukin-1β (IL-1β) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 diabetes mellitus, suggesting reduction of IL-1β activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1β. The vaccine hIL1bQb consisting of full-length, recombinant IL-1β coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1β-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1β-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1β and represents a new treatment option for IL-1β-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).
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http://dx.doi.org/10.1038/mt.2015.227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881764PMC
May 2016

Head-to-Head Comparison of Soluble vs. Qβ VLP Circumsporozoite Protein Vaccines Reveals Selective Enhancement of NANP Repeat Responses.

PLoS One 2015 16;10(11):e0142035. Epub 2015 Nov 16.

Structural Vaccinology Laboratory, Malaria Vaccine Branch, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States of America.

Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage Qβ VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to Qβ and the CSP-Qβ vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the Qβ-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by Qβ presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the Qβ-CSP vaccine. The data presented here pave the way for further improvement in the Qβ conjugation chemistry and evaluation of both the Qβ-CSP and soluble CSP vaccines in the non-human primate model.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142035PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646581PMC
June 2016

Safety and immunogenicity of a virus-like particle pandemic influenza A (H1N1) 2009 vaccine: results from a double-blinded, randomized Phase I clinical trial in healthy Asian volunteers.

Vaccine 2014 Sep 18;32(39):5041-8. Epub 2014 Jul 18.

D3 (Drug Discovery and Development), 31 Biopolis Way, #01-02a Nanos, Singapore 138669, Singapore. Electronic address:

Methods: A novel, fully bacterially produced recombinant virus-like particle (VLP) based influenza vaccine (gH1-Qbeta) against A/California/07/2009(H1N1) was tested in a double-blind, randomized phase I clinical trial at two clinical sites in Singapore. The trial evaluated the immunogenicity and safety of gH1-Qbeta in the presence or absence of alhydrogel adjuvant. Healthy adult volunteers with no or low pre-existing immunity against A/California/07/2009 (H1N1) were randomized to receive two intramuscular injections 21 days apart, with 100μg vaccine, containing 42μg hemagglutinin antigen. Antibody responses were measured before and 21 days after each immunization by hemagglutination inhibition (HAI) assays. The primary endpoint was seroconversion on Day 42, defined as percentage of subjects which reach a HAI titer ≥40 or achieve an at least 4-fold rise in HAI titer (with pre-existing immunity). The co-secondary endpoints were safety and seroconversion on Day 21.

Results: A total of 84 Asian volunteers were enrolled in this study and randomized to receive the adjuvanted (n=43) or the non-adjuvanted (n=41) vaccine. Of those, 43 and 37 respectively (95%) completed the study. There were no deaths or serious adverse events reported during this trial. A total of 535 adverse events occurred during treatment with 49.5% local solicited symptoms, of mostly (76.4%) mild severity. The most common treatment-related systemic symptom was fatigue. The non-adjuvanted vaccine met all primary and secondary endpoints and showed seroconversion in 62.2% and 70.3% of participants respectively on Day 21 and Day 42. While the adjuvanted vaccine showed an increased seroconversion from 25.5% (Day 21) to 51.2% (Day 42), it did not meet the immunogenicity endpoint.

Conclusion: In summary, non-adjuvanted gH1-Qbeta showed similar antibody mediated immunogenicity and a comparable safety profile in healthy humans to commercially available vaccines. These results warrant the consideration of this VLP vaccine platform for the vaccination against influenza infection (HSA CTC1300092).
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http://dx.doi.org/10.1016/j.vaccine.2014.07.011DOI Listing
September 2014

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

J Immunol 2014 Jun 12;192(12):5499-508. Epub 2014 May 12.

Department of Dermatology, Zurich University Hospital, 8091 Zurich, Switzerland; Jenner Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom.

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.
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http://dx.doi.org/10.4049/jimmunol.1400065DOI Listing
June 2014

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

PLoS One 2013 18;8(11):e78947. Epub 2013 Nov 18.

Immunodrugs Department, Cytos Biotechnology AG, Schlieren, Zurich, Switzerland.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078947PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832520PMC
July 2014

Enhanced neutralizing antibody titers and Th1 polarization from a novel Escherichia coli derived pandemic influenza vaccine.

PLoS One 2013 18;8(10):e76571. Epub 2013 Oct 18.

ASTAR Program in Translational Research on Infectious Disease, Agency for Science, Technology and Research, Singapore ; Singapore Immunology Network (SIgN), Agency for Science, Technology and Research, Singapore.

Influenza pandemics can spread quickly and cost millions of lives; the 2009 H1N1 pandemic highlighted the shortfall in the current vaccine strategy and the need for an improved global response in terms of shortening the time required to manufacture the vaccine and increasing production capacity. Here we describe the pre-clinical assessment of a novel 2009 H1N1 pandemic influenza vaccine based on the E. coli-produced HA globular head domain covalently linked to virus-like particles derived from the bacteriophage Qβ. When formulated with alum adjuvant and used to immunize mice, dose finding studies found that a 10 µg dose of this vaccine (3.7 µg globular HA content) induced antibody titers comparable to a 1.5 µg dose (0.7 µg globular HA content) of the licensed 2009 H1N1 pandemic vaccine Panvax, and significantly reduced viral titers in the lung following challenge with 2009 H1N1 pandemic influenza A/California/07/2009 virus. While Panvax failed to induce marked T cell responses, the novel vaccine stimulated substantial antigen-specific interferon-γ production in splenocytes from immunized mice, alongside enhanced IgG2a antibody production. In ferrets the vaccine elicited neutralizing antibodies, and following challenge with influenza A/California/07/2009 virus reduced morbidity and lowered viral titers in nasal lavages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076571PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799843PMC
August 2014

Immunotherapy of allergic rhinitis: new therapeutic opportunities with virus-like particles filled with CpG motifs.

Am J Rhinol Allergy 2013 May-Jun;27(3):206-12

Zentrum für Rhinologie and Allergologie, Wiesbaden, Germany.

Background: The incidence of allergic rhinitis (AR) has increased constantly over the last decades. The disease can significantly lower quality of life and subsequently might progress to allergic asthma. Allergen-specific immunotherapy is mostly used to cope with the cause of the disease. However, incidence of systemic reactions or limited compliance hampers the widespread use of this therapeutic approach. Therefore, new candidates are examined to improve immunotherapy of allergies. Recently, a new technology was developed with the aim to positively influence the immune system of allergic patients. Virus-like particles (VLPs) represent a potent vaccine platform that has been proven to be immunogenic and clinically effective. To enhance immune cell activation, addition of Toll-like receptor ligands and/or depot-forming adjuvants seems to be helpful. In this context, CpG motifs represent intensive investigated and potent stimulators of T cells. This article focuses on the function of VLPs and CpG motifs and their clinical experience for treatment of AR.

Methods: A literature review was performed.

Results: Several published studies showed a beneficial impact of the treatment on allergic symptoms. They tested VLPs filled with or without CpG motifs in combination with or without allergen.

Conclusion: Results encourage further investigations of VLPs and CpG motifs as adjuncts to or even alternative candidates for immunotherapy of allergic disorders.
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http://dx.doi.org/10.2500/ajra.2013.27.3875DOI Listing
December 2013

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

Proc Natl Acad Sci U S A 2012 Dec 20;109(50):20566-71. Epub 2012 Nov 20.

Research Department, Cytos Biotechnology, CH-8952 Zurich-Schlieren, Switzerland.

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.
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http://dx.doi.org/10.1073/pnas.1206970109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528531PMC
December 2012

Universal vaccine against influenza virus: linking TLR signaling to anti-viral protection.

Eur J Immunol 2012 Apr;42(4):863-9

Cytos Biotechnology AG, Schlieren-Zürich, Switzerland.

A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.
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http://dx.doi.org/10.1002/eji.201041225DOI Listing
April 2012

Selective utilization of Toll-like receptor and MyD88 signaling in B cells for enhancement of the antiviral germinal center response.

Immunity 2011 Mar 25;34(3):375-84. Epub 2011 Feb 25.

Department of Microbiology & Immunology, University of California, San Francisco, CA 94143, USA.

The contribution of Toll-like receptor (TLR) signaling to T cell-dependent (TD) antibody responses was assessed by using mice lacking the TLR signaling adaptor MyD88 in individual cell types. When a soluble TLR9 ligand was used as adjuvant for a protein antigen, MyD88 was required in dendritic cells but not in B cells to enhance the TD antibody response, regardless of the inherent immunogenicity of the antigen. In contrast, a TLR9 ligand contained within a virus-like particle substantially augmented the TD germinal center IgG antibody response, and this augmentation required B cell MyD88. The ability of B cells to discriminate between antigens based on the physical form of a TLR ligand probably reflects an adaptation to facilitate strong antiviral antibody responses.
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http://dx.doi.org/10.1016/j.immuni.2011.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064721PMC
March 2011

Mechanisms of allergen-specific desensitization.

J Allergy Clin Immunol 2010 Aug 10;126(2):375-83. Epub 2010 Jul 10.

Cytos Biotechnology AG, Zurich-Schlieren, Switzerland.

Background: Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined.

Objective: We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions.

Methods: We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype.

Results: IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed.

Conclusions: These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.
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http://dx.doi.org/10.1016/j.jaci.2010.05.040DOI Listing
August 2010

Versatile virus-like particle carrier for epitope based vaccines.

PLoS One 2010 Mar 23;5(3):e9809. Epub 2010 Mar 23.

Cytos Biotechnology AG, Zurich-Schlieren, Switzerland.

Background: Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.

Methodology/principal Findings: Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.

Conclusions/significance: AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009809PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843720PMC
March 2010

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

Vaccine 2010 Jul 20;28(33):5503-12. Epub 2010 Mar 20.

Cytos Biotechnology AG, Wagistr. 25, 8952 Zürich-Schlieren, Switzerland.

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.
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http://dx.doi.org/10.1016/j.vaccine.2010.02.103DOI Listing
July 2010

Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against influenza A M2 protein.

Virol J 2009 Dec 21;6:224. Epub 2009 Dec 21.

Cytos Biotechnology AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.

Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.
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http://dx.doi.org/10.1186/1743-422X-6-224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2804611PMC
December 2009

Cutting edge: limited specialization of dendritic cell subsets for MHC class II-associated presentation of viral particles.

J Immunol 2010 Jan 30;184(1):26-9. Epub 2009 Nov 30.

Cytos Biotechnology AG, Zürich-Schlieren, Switzerland.

Dendritic cells (DCs) are the most important APC. It was recently reported that there is a dichotomy for Ag presentation by DC subsets; exogenous Ags reach the MHC class I pathway, but not the MHC class II pathway, in CD8(+) DCs, whereas CD8(-) DCs only process Ags for the MHC class II pathway. In this study, we used virus-like particles (VLPs) to show that CD8(+) and CD8(-) DCs efficiently capture and process VLPs for presentation in association with MHC class II in vivo. In contrast, CD8(+) DCs, but not CD8(-) DCs, cross presented VLP-derived peptides. This pattern was changed in an FcgammaR-dependent fashion in the presence of VLP-specific Abs, because under those conditions both DC subsets failed to efficiently cross present. Thus, the presentation of viral particles to CD4(+) T cells is not restricted to distinct DC subsets, whereas the presentation of viral particles to CD8(+) T cells is limited to CD8(+) DCs.
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http://dx.doi.org/10.4049/jimmunol.0901540DOI Listing
January 2010

Innate signaling regulates cross-priming at the level of DC licensing and not antigen presentation.

Eur J Immunol 2010 Jan;40(1):103-12

Cytos Biotechnology AG, Schlieren, Switzerland.

Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of cross-presentation or at the level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-alpha/beta receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.
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http://dx.doi.org/10.1002/eji.200939559DOI Listing
January 2010

Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses.

J Immunol 2009 Sep 26;183(6):3788-99. Epub 2009 Aug 26.

Cytos Biotechnology AG, Zürich-Schlieren, Switzerland.

The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.
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http://dx.doi.org/10.4049/jimmunol.0804004DOI Listing
September 2009

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy.

J Exp Med 2009 Aug 10;206(9):1941-55. Epub 2009 Aug 10.

Department of Immunodrugs, Cytos Biotechnology AG, 8952 Schlieren-Zürich, Switzerland.

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qbeta-derived virus-like particles (Qbeta-Fel d1). Qbeta-Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcgammaRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcgammaRIIb. In addition, treatment with Qbeta-Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qbeta-Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qbeta-Fel d1 may be a safe and effective treatment for cat allergy.
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http://dx.doi.org/10.1084/jem.20090199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737174PMC
August 2009

Follicular and marginal zone B cells fail to cross-present MHC class I-restricted epitopes derived from viral particles.

J Immunol 2009 May;182(10):6261-6

Cytos Biotechnology AG, Schlieren, Switzerland.

Viruses and virus-like particles (VLPs) are known to be potent inducers of B cell as well as Th cell and CTL responses. It is well established that professional APCs such as dendritic cells (DCs) and macrophages efficiently process viral particles for both MHC class I- and MHC class II-associated presentation, which is essential for induction of CTL and Th cell responses, respectively. Less is known, however, about the ability of B cells to present epitopes derived from viral particles to T cells. Using two different VLPs, in this study we show in vitro as well as in vivo that DCs present VLP-derived peptides in association with MHC class I as well as class II. In contrast, although B cells were able to capture VLPs similarly as DCs and although they efficiently processed VLPs for presentation in association with MHC class II, they failed to process exogenous VLPs for presentation in association with MHC class I. Thus, in contrast to DCs, B cells are not involved in the process of cross-priming. This finding is of physiological importance because B cells with the ability to cross-present Ag to specific CD8(+) T cells may be killed by these cells, preventing the generation of neutralizing Ab responses.
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http://dx.doi.org/10.4049/jimmunol.0804035DOI Listing
May 2009

Isolation of human monoclonal antibodies by mammalian cell display.

Proc Natl Acad Sci U S A 2008 Sep;105(38):14336-41

Cytos Biotechnology AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.

Due to their low immunogenicity in patients, humanized or fully human mAbs are becoming increasingly important for the treatment of a growing number of diseases, including cancer, infections, and immune disorders. Here, we describe a technology allowing for the rapid isolation of fully human mAbs. In contrast to previously described methods, B cells specific for an antigen of interest are directly isolated from peripheral blood mononuclear cells (PBMC) of human donors. Recombinant, antigen-specific single-chain Fv (scFv) libraries are generated from this pool of B cells and screened by mammalian cell surface display by using a Sindbis virus expression system. This method allows isolating antigen-specific antibodies by a single round of FACS. The variable regions (VRs) of the heavy chains (HCs) and light chains (LCs) are isolated from positive clones and recombinant fully human antibodies produced as whole IgG or Fab fragments. In this manner, several hypermutated high-affinity antibodies binding the Qbeta virus like particle (VLP), a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high expression levels in cell culture. The human nicotine-specific mAbs were validated preclinically in a mouse model. Thus, the technology presented here allows for rapid isolation of high-affinity, fully human antibodies with therapeutic potential from human volunteers.
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http://dx.doi.org/10.1073/pnas.0805942105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567231PMC
September 2008

Vaccination against GIP for the treatment of obesity.

PLoS One 2008 Sep 9;3(9):e3163. Epub 2008 Sep 9.

Cytos Biotechnology AG, Schlieren, Switzerland.

Background: According to the WHO, more than 1 billion people worldwide are overweight and at risk of developing chronic illnesses, including cardiovascular disease, type 2 diabetes, hypertension and stroke. Current therapies show limited efficacy and are often associated with unpleasant side-effect profiles, hence there is a medical need for new therapeutic interventions in the field of obesity. Gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide) has recently been postulated to link over-nutrition with obesity. In fact GIP receptor-deficient mice (GIPR(-/-)) were shown to be completely protected from diet-induced obesity. Thus, disrupting GIP signaling represents a promising novel therapeutic strategy for the treatment of obesity.

Methodology/principal Findings: In order to block GIP signaling we chose an active vaccination approach using GIP peptides covalently attached to virus-like particles (VLP-GIP). Vaccination of mice with VLP-GIP induced high titers of specific antibodies and efficiently reduced body weight gain in animals fed a high fat diet. The reduction in body weight gain could be attributed to reduced accumulation of fat. Moreover, increased weight loss was observed in obese mice vaccinated with VLP-GIP. Importantly, despite the incretin action of GIP, VLP-GIP-treated mice did not show signs of glucose intolerance.

Conclusions/significance: This study shows that vaccination against GIP was safe and effective. Thus active vaccination may represent a novel, long-lasting treatment for obesity. However further preclinical safety/toxicology studies will be required before the therapeutic concept can be addressed in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003163PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525840PMC
September 2008

Nanoparticles target distinct dendritic cell populations according to their size.

Eur J Immunol 2008 May;38(5):1404-13

Department Immunodrugs, Cytos Biotechnology, Schlieren, Switzerland.

The efficiency of a vaccine largely depends on the appropriate targeting of the innate immune system, mainly through prolonged delivery of antigens and immunomodulatory substances to professional antigen-presenting cells in the lymphoid environment. Particulate antigens, such as virus-like particles (VLP) induce potent immune responses. However, little is known about the relative importance of direct drainage of free antigen to lymph nodes (LN) versus cellular transport and the impact of particle size on the process. Here, we show that nanoparticles traffic to the draining LN in a size-dependent manner. Whereas large particles (500-2000 nm) were mostly associated with dendritic cells (DC) from the injection site, small (20-200 nm) nanoparticles and VLP (30 nm) were also found in LN-resident DC and macrophages, suggesting free drainage of these particles to the LN. In vivo imaging studies in mice conditionally depleted of DC confirmed the capacity of small but not large particles to drain freely to the LN and demonstrated that DC are strictly required for transport of large particles from the injection site to the LN. These data provide evidence that particle size determines the mechanism of trafficking to the LN and show that only small nanoparticles can specifically target LN-resident cells.
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http://dx.doi.org/10.1002/eji.200737984DOI Listing
May 2008

Identification of Ly-6K as a novel marker for mouse plasma cells.

Mol Immunol 2008 May 26;45(10):2727-33. Epub 2008 Mar 26.

Cytos Biotechnology AG, Wagistrasse 25, Schlieren, Switzerland.

Plasma cells are the main producers of antibody and key effector cells of the immune system. Despite their importance, analytics of plasma cells still suffers from the limited availability of specific markers. Currently, plasma cell identification relies on the expression of a single marker, CD138/syndecan-1. However, syndecan-1 is widely expressed on various cell types outside the hematopoietic compartment, and furthermore, not expressed on all subsets of plasma cells. To discover novel surface markers, a differential screening followed by signal sequence trap cloning was developed, leading to the identification of mouse Ly-6K (mLy-6K). Expression profiling confirmed that mLy-6K is expressed by plasma cells but not B cells or tissues not containing plasma cells. Expression at the surface of plasma cells isolated from spleen, lymph node, bone marrow, and lamina propria of the small intestine was demonstrated at the protein level using a polyclonal rabbit antibody. This novel plasma cell marker shows promise to help broaden our understanding of plasma cell differentiation and function.
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http://dx.doi.org/10.1016/j.molimm.2008.02.009DOI Listing
May 2008

Regulation of memory antibody levels: the role of persisting antigen versus plasma cell life span.

J Immunol 2007 Jan;178(1):67-76

Cytos Biotechnology, Zurich-Schlieren, Switzerland.

Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab responses as a function of the life span of plasma cells and tested the role of persisting Ag in maintaining B cell memory. Our analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are long-lived, but decline continuously with a t(1/2) of approximately 80 days, which corresponds to the life span of plasma cells; 2) the germinal center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the maintenance of Ab levels and B cell memory.
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http://dx.doi.org/10.4049/jimmunol.178.1.67DOI Listing
January 2007

VSIG4, a B7 family-related protein, is a negative regulator of T cell activation.

J Clin Invest 2006 Oct;116(10):2817-26

Cytos Biotechnology AG, Zurich-Schlieren, Switzerland.

T cell activation by APCs is positively and negatively regulated by members of the B7 family. We have identified a previously unknown function for B7 family-related protein V-set and Ig domain-containing 4 (VSIG4). In vitro experiments using VSIG4-Ig fusion molecules showed that VSIG4 is a strong negative regulator of murine and human T cell proliferation and IL-2 production. Administration to mice of soluble VSIG4-Ig fusion molecules reduced the induction of T cell responses in vivo and inhibited the production of Th cell-dependent IgG responses. Unlike that of B7 family members, surface expression of VSIG4 was restricted to resting tissue macrophages and absent upon activation by LPS or in autoimmune inflammatory foci. The specific expression of VSIG4 on resting macrophages in tissue suggests that this inhibitory ligand may be important for the maintenance of T cell unresponsiveness in healthy tissues.
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http://dx.doi.org/10.1172/JCI25673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1578631PMC
October 2006

Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.

J Immunol 2006 Feb;176(3):1311-5

Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany.

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and by T cells. In both humans and mice, KLRG1 identifies Ag-experienced T cells that are impaired in their proliferative capacity but are capable of performing effector functions. In this study, we identified E-cadherin as a ligand for murine KLRG1 by using fluorescently labeled, soluble tetrameric complexes of the extracellular domain of the murine KLRG1 molecule as staining reagents in expression cloning. Ectopic expression of E-cadherin in B16.BL6 target cells did not affect cell-mediated lysis by lymphokine-activated NK cells and by CD8 T cells but inhibited Ag-induced proliferation and induction of cytolytic activity of CD8 T cells. E-cadherin is expressed by normal epithelial cells, Langerhans cells, and keratinocytes and is usually down-regulated on metastatic cancer cells. KLRG1 ligation by E-cadherin in healthy tissue may thus exert an inhibitory effect on primed T cells.
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http://dx.doi.org/10.4049/jimmunol.176.3.1311DOI Listing
February 2006