Breaking B-cell tolerance and CTL tolerance in three OVA-transgenic mouse strains expressing different levels of OVA.

It is of major importance to overcome the immunological tolerance in attempts to generate efficient tumour vaccines. Here, we describe induction of autoantibodies and self-reactive CTL in three types of OVA-transgenic mouse strains, RIP-OVA(low), RIP-mOVA and RIP-OVA(HI) exhibiting varying levels of OVA expression and tolerance. This was achieved by immunizing with DNA constructs where a foreign T-helper epitope, P30 from tetanus toxin, was inserted into the OVA sequence.

Generation of autoreactive CTL by tumour vaccines containing foreign T helper epitopes.

The aim of this study was to evaluate the effect of including a foreign T helper cell epitope in vaccines designed for generation of CTL against self-antigens and for inhibition of tumour growth. Two different vaccine designs were composed, a minimal epitope vaccine and a modified full length self-antigen, both based on OVA containing either a colinearily synthesized or an inserted Th-epitope, respectively. These vaccines were used for immunization of tolerant OVA transgenic mice (RIP-OVA(low)) and non-tolerant C57BL/6 mice.

Linked foreign T-cell help activates self-reactive CTL and inhibits tumor growth.

Transgenic mice expressing membrane-bound OVA under the rat insulin promoter, RIP-mOVA, has previously been suggested to display deletional tolerance toward the dominant CTL epitope, SIINFEKL, and provide an elegant model system to test the hypothesis that the lack of T cell help contributes to the tolerance. To understand how the CD8 tolerance is maintained in these mice, a set of neo-self-Ags, OVA, modified to contain a foreign Th peptide, were constructed and tested for their ability to induce CTL responses in RIP-mOVA mice. Immunization with these Th peptide-modified OVA molecules and not with the wild-type OVA induced self-reactive CTLs recognizing dominant CTL peptide, SIINFEKL.

Phase I study of TNFalpha AutoVaccIne in patients with metastatic cancer.

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNFalpha in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNFalpha proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNFalpha antibodies.

Insertion of foreign T cell epitopes in human tumor necrosis factor alpha with minimal effect on protein structure and biological activity.

To create a human therapeutic vaccine able to circumvent self-tolerance against tumor necrosis factor (TNF) alpha, foreign T helper epitopes were inserted into human TNFalpha, with minimal effect on the native three-dimensional structure. TNFalpha variants were screened for solubility, structural stability, biological activity, and after immunization, for eliciting inhibitory antibodies. The longest and most flexible loop in TNFalpha, also designated loop 3, is the only region that is not involved in intra- or intermolecular interactions and therefore constitute an attractive insertion site.

Induction of TNF-alpha autoantibody production by AutoVac TNF106: a novel therapeutic approach for the treatment of allergic diseases.

Background: Cytokines play an integral role in the coordination and persistence of allergic inflammatory processes and therefore represent prime targets for novel therapies in diseases such as asthma. Multiple attempts to generate low-molecular-weight cytokine inhibitors have failed, and the main attention has focused on the development of biological agents such as neutralizing antibodies. The present work describes a simple and effective method to induce the production of therapeutic anti-cytokine autoantibodies by active immunization against a modified endogenous cytokine.

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice.

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule.

Active vaccination against IL-5 bypasses immunological tolerance and ameliorates experimental asthma.

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs.

Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and specific immunization against TNFalpha confers no additional benefit.

TNFalpha participates in the pathogenesis of atherosclerosis. The effect of immunization against TNFalpha on development of advanced vascular lesions in atherosclerosis-susceptible apoE-deficient mice was investigated. At 5-7 weeks of age, animals received immunization with either Freunds adjuvant and a recombinant antigenic TNFalpha molecule (TNF106), Freunds adjuvant alone, or no immunization.

Functional screening of a retroviral peptide library for MHC class I presentation.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector.

Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha.

A novel microtiter plate based method for identification of B-cell epitopes.

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B-cell epitopes in two different proteins using a peptide-based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15-mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor-alpha, respectively, was synthesized.

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope.

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I.

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction.

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover.

T-cell recognition of tumor-associated carbohydrates: the nature of the glycan moiety plays a decisive role in determining glycopeptide immunogenicity.

Aberrant glycosylation is one of the most constant traits of the malignant cell phenotype. To study T-cell responses to tumor-associated glycans, the mouse hemoglobin-derived decapeptide Hb(67-76), which binds well to the MHC class II molecule E(k) and is nonimmunogenic in CBA/J mice, was either O- or N-glycosylated at its primary T-cell receptor contact residue, position 72, with different glycans attached to either threonine, serine, or asparagine. The carbohydrate moieties included tumor-associated mucins, i.

Carbohydrate and peptide specificity of MHC class II-restricted T cell hybridomas raised against an O-glycosylated self peptide.

MHC class II E(k)-restricted, IL-2 secreting T cell hybridomas were raised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T72(Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas against the glycan moiety was investigated by testing their response against a panel of Hb(67-76)-derived glycopeptides, all with a glycan attached to serine or threonine at the position 72 in the peptide, but with different glycans. The hybridomas showed a high degree of specificity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of these were structurally very similar to alpha-GalNAc.

Constrained glycopeptide ligands for MPRs. Limitations of unprotected phosphorylated building blocks.

A new methodology for the synthesis of cyclic and phosphorylated glycopeptide templates was developed. First, fully protected building blocks containing mannose and mannose disaccharides with bis-trichloroethyl phosphate on Fmoc-Thr-OPfp were synthesized. These were used in solid-phase assembly through side chain anchoring of glycosylated hexa- and octa-peptides protected at the C-terminal carboxylate as the allyl ester.

Breaking of B cell tolerance toward a highly conserved self protein.

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition.

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice.

Multiple column synthesis of a library of T-cell stimulating Tn-antigenic glycopeptide analogues for the molecular characterization of T-cell-glycan specificity.

A series of peptides and glycopeptides derived by amino acid and glycosyl amino acid scans through the self peptide from CBA/J mouse haemoglobin Hb (67-76). VITAFNEGLK, was synthesized by multiple column peptide synthesis (MCPS). Investigation of glycopeptide binding to the mouse major histocompatibility class II molecule Ek showed that glycans in position 72 did not interfere with the binding to Ek.

T cell recognition of Tn-glycosylated peptide antigens.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice.

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids.

Peptides consisting solely of D-amino acids (D-peptides) as opposed to their L-counterparts (L-peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L- and D-peptides, capable of binding to both avidin and streptavidin, were found. The L-peptides contained the previously described HPQ/M motifs, and among the D-peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position.