Jörg Willers

A Vaccine against Nicotine for Smoking Cessation: A Randomized Controlled Trial

Background Tobacco dependence is the leading cause of preventable death and disabilities worldwide and nicotine is the main substance responsible for the addiction to tobacco. A vaccine against nicotine was tested in a 6-month randomized, double blind phase II smoking cessation study in 341 smokers with a subsequent 6-month follow-up period. Methodology/Principal Findings 229 subjects were randomized to receive five intramuscular injections of the nicotine vaccine and 112 to receive placebo at monthly intervals. All subjects received individual behavioral smoking cessation counseling. The vaccine was safe, generally well tolerated and highly immunogenic, inducing a 100% antibody responder rate after the first injection. Point prevalence of abstinence at month 2 showed a statistically significant difference between subjects treated with Nicotine-Qβ (47.2%) and placebo (35.1%) (P = 0.036), but continuous abstinence between months 2 and 6 was not significantly different. However, in subgroup analysis of the per-protocol population, the third of subjects with highest antibody levels showed higher continuous abstinence from month 2 until month 6 (56.6%) than placebo treated participants (31.3%) (OR 2.9; P = 0.004) while medium and low antibody levels did not increase abstinence rates. After 12 month, the difference in continuous abstinence rate between subjects on placebo and those with high antibody response was maintained (difference 20.2%, P = 0.012). Conclusions Whereas Nicotine-Qβ did not significantly increase continuous abstinence rates in the intention-to-treat population, subgroup analyses of the per-protocol population suggest that such a vaccination against nicotine can significantly increase continuous abstinence rates in smokers when sufficiently high antibody levels are achieved. Immunotherapy might open a new avenue to the treatment of nicotine addiction. Trial Registration Swiss Medical Registry 2003DR2327; ClinicalTrials.gov NCT00369616

A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity

Nicotine is the principal addictive component in tobacco, and following uptake acts in the central nervous system. The smoking‐cessation efforts of most smokers fail because a single slip often delivers sufficient nicotine to the brain to reinstate the drug‐seeking behaviour. Blocking nicotine from entering the brain by induction of specific antibodies may be an effective means to prevent such relapses. The hapten nicotine was coupled to virus‐like particles (VLP) formed by the coat protein of the bacteriophage Qb. In preclinical experiments, this Nicotine–Qb VLP (NicQb) vaccine induced strong antibody responses. After intravenous nicotine challenge, vaccinated mice exhibited strongly reduced nicotine levels in the brain compared with control mice. In a phase I study, 32 healthy non‐smokers were immunized with NicQb. The vaccine was safe and well‐tolerated. All volunteers who received NicQb showed nicotine‐specific IgM antibodies at day 7 and nicotine‐specific IgG antibodies at day 14. Antibody levels could be boosted by a second injection or the addition of Alum as an adjuvant and the antibodies had a high affinity for nicotine. These data suggest that antibodies induced by NicQb may prevent relapses by sequestering nicotine in the blood of immunized smokers.

Assessment of clinical efficacy of CYT003-QbG10 in patients with allergic rhinoconjunctivitis: a phase IIb study.

Background Allergic symptoms are generally caused by exposure to substances to which people have become sensitized. Associated with this is an ‘unbalanced’ Th1/Th2 immune response with T cell responses skewed towards the production of Th2 cytokines, IL‐4, 5, and 13 and high levels of IgE antibodies. Current immune modulating therapies require the use of allergens, carrying the risk to induce potentially severe allergic reactions.

Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

Pathogenesis of cutaneous lymphomas.

Cutaneous lymphomas are a heterogeneous group of lymphoproliferative disorders derived from T cells, B cells and, in rare cases, natural killer cells. The precise mechanisms of the lymphomagenesis are still obscure. However, there are various factors involved. These factors include environmental, especially infectious factors, translocations, mutations and genetic instability. The special microenvironment in the skin is responsible for the peculiar behavior of these neoplasms by providing various key factors, such as adhesion molecules and cytokines. Newly identified molecular disturbances in cutaneous lymphomas might be targeted by specific molecular or immunologic interventions in the future.

The use of anti-T-cell receptor-Vβ antibodies for the estimation of treatment success and phenotypic characterization of clonal T-cell populations in cutaneous T-cell lymphomas

Summary. Sézary syndrome and Mycosis fungoides are the most common forms of cutaneous T‐cell lymphomas. To assess the response to different therapies especially in Sézary syndrome, it is helpful to monitor the percentage of circulating tumour cells in the blood. The use of T‐cell receptor (TCR)‐Vβ specific monoclonal antibodies provides a suitable tool for detecting Sézary cells. In this study, we analysed the levels of clonal CD4+Vβ+ cells of seven patients with various treatment modalities using flow cytometry and investigated the immunophenotype of the clonal cells by double staining with a panel of antibodies recognizing lymphatic surface markers. Additionally, a polymerase chain reaction‐denaturing gradient gel electrophoresis assay was performed on clonal CD4+Vβ2+ cells, showing that these cells carry a Vγ10/11, JγP1/2 TCR rearrangement. Follow‐up studies revealed close association of the Vβ+ clone developmentwith the clinical response to different therapiesinsixpatients. Intwo cases, the CD4+Vβ+ cells decreased accompanied by partial regression or even complete remission. In four cases, a stable or increasing clonal CD4+Vβ+ population reflected well a stable or progressing course of the disease. Double staining of Vβ+ cells revealed the following pattern, CD3+, CD5+, CD7+, CD28+, CD80–, CD86+ and human leucocyte antigen (HLA) class I+. In contrast, HLA‐DR was heterogeneously expressed. We conclude that identification and monitoring of CD4+Vβ+ clonal T cells by fluorescence‐activated cell sorting with double staining is a suitable method to assess clinical responses to different therapies.

Non-self-discrimination as a driving concept in the identification of an immunodominant HMW-MAA epitopic peptide sequence by autoantibodies from melanoma cancer patients

We analyzed the sera of patients with melanoma to define the human humoral autoantibody profile towards HMW‐MAA. Computational proteome scanning using the non‐self‐discrimination principle as a guide led to the individuation of the low‐similarity HMW‐MAA781–789RATVWMLRL peptide fragment as an immunodominant B‐cell epitope. Linear B‐cell determinant individuation was experimentally validated by dot blot immunoassay and NMR spectroscopy analysis. Regulation of physiologic self‐reactivity by the non‐self‐discrimination principle is discussed.

Proteomic Scan for Tyrosinase Peptide Antigenic Pattern in Vitiligo and Melanoma: Role of Sequence Similarity and HLA-DR1 Affinity

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95–104FMGFNCGNCK; aa175–182 LFVWMHYY; aa176–190FVWMHYYVSMDALLG; aa222–236IQKLTGDENFTIPYW, and aa233–247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95–104FMGFNCGNCK, aa222–236IQKLTGDENFTIPYW, and aa233–247 IPYWDWRDAEKCDIC, but did not react with the aa175–182LFVWMHYY and aa176–190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.

Loss of single HLA Class I allospecificities in melanoma cells due to selective genomic abbreviations

Expression of human leucocyte antigen (HLA) Class I molecules is essential for the recognition of malignant melanoma (MM) cells by CD8+ T lymphocytes. A complete or partial loss of HLA Class I molecules is a potent strategy for MM cells to escape from immunosurveillance. In 2 out of 55 melanoma cell cultures we identified a complete phenotypic loss of HLA allospecificities. Both patients have been treated unsuccessfully with HLA‐A2 peptides. To identify the reasons underlying the loss of single HLA‐A allospecificities, we searched for genomic alterations at the locus for HLA Class I α‐chain on chromosome 6 in melanoma cell cultures established from 2 selected patients with MM in advanced stage. This deficiency was associated with alterations of HLA‐A2 gene sequences as determined by polymerase chain reaction‐sequence specific primers (PCR‐SSP). Karyotyping revealed a chromosomal loss in Patient 1, whereas melanoma cell cultures established from Patient 2 displayed 2 copies of chromosome 6. Loss of heterozygosity (LOH) using markers located around position 6p21 was detected in both cases. By applying group‐specific primer‐mixes spanning the 5′‐flanking region of the HLA‐A2 gene locus the relevant region was amplified by PCR and subsequent sequencing allowed alignment with the known HLA Class I reference sequences. Functional assays using HLA‐A2‐restricted cytotoxic T‐cell clones were performed in HLA‐A2 deficient MM cultures and revealed a drastically reduced susceptibility to CTL lysis in HLA‐A2 negative cells. We could document the occurrence of selective HLA‐A2 deficiencies in cultured advanced‐stage melanoma metastases and identify their molecular causes as genomic alterations within the HLA‐A gene locus.

Definition of anti-tyrosinase MAb T311 linear determinant by proteome-based similarity analysis

Abstract:  Using non‐self discrimination as a driving force in generating peptide immunogenicity, we have developed a computer‐assisted proteomic analysis in order to identify the protein antigenic regions that have evoked humoral response. The purpose of this study was to further validate the computational analysis for melanoma‐associated antigens and, at the same time, to assess the efficacy of the methodology in defining antigenic regions of autoantigens associated to autoimmune diseases. To achieve this two‐fold objective, we have examined the enzyme tyrosinase, a protein that represents an important autoantigen in patients with vitiligo or melanoma. Here, we report that the antigenic linear determinant of the monoclonal antibody (Mab) T311 raised against the melanoma/vitiligo tyrosinase autoantigen is located in the low similarity 15‐mer amino acid sequence tyrosinase 233–247 IPYWDWRDAEKCDIC, within the fragment 237–247. These data confirm non‐similarity to the host proteome as a factor that participates in shaping peptide immune reactivity and may be a first step towards designing tyrosinase antigenic peptides to be used for (i) direct neutralization of harmful melanocytes‐attacking autoantibodies in vitiligo, or (ii) production of antibodies against tyrosinase‐positive melanomas. Moreover tyrosinase peptide antigens might be used as key tools in studying the boundaries between self‐tolerance and autoimmunity phenomena.