Professor Avraham Ben-Nun The Eugene & Marcia Applebaum Professor

 Research Interests:

Our major aims are to identify the potentially pathogenic T-cells involved in demyelinating autoimmune diseases of the central nervous system, multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), and characterize them with regard to epitope specificity and TCR/MHC-peptide interaction. These studies are directed towards devising antigen-specific and non antigen-specific approaches to immunomodulation of the disease.

Demonstration of new targets for T-cell autoreactivity leading to demyelination: Multiplicity of primary target antigens in MS

In addition to autoreactivity against the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), which have long been investigated as potential target antigens in MS, autoreactivity against other myelin proteins, myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocyte basic protein (MOBP) and oligodendrocyte-specific protein (OSP), likely to be involved in structural and/or physiological maintenance of myelin can also induce clinical and pathological EAE in susceptible animals, as indicated by our and other laboratories. Thus, in addition to MBP and PLP, MOG, MOBP and OSP can now be viewed as potential primary target antigens in MS, indicating that the pathogenic autoreactivity in MS may be directed against a multiplicity of antigenic targets, all of which should be taken into account when considering approaches to antigen-directed immune-specific therapy to the disease. Therefore, understanding the pathophysiology of the disease and devising approaches to immune-specific therapy require thorough characterization of the autoreactivity to all these potential primary target antigens.

1. Autoreactivity to myelin oligodendrocyte glycoprotein (MOG)
   The potential importance in the pathogenesis of MS of T-cell autoreactivity to MOG, which we had demonstrated in MS patients, has been further emphasized by our recent work in non-human primates, such as rhesus monkeys closely related to humans at the immunological level. The T-cell autoreactivity to MOG in rhesus monkeys with MOG-induced EAE is directed against three immunodominant epitopes, two of which are also predominantly recognized by MS MOG-reactive T-cells. Most importantly, autoreactivity against one of these epitopes induces demyelinating CNS disease resembling acute MS in these animals, strongly supporting the identification of MOG as a highly relevant potential primary target in MS.

2. Autoreactivity to myelin-associated oligodendrocyte basic protein (MOBP)
   MOBP is situated at the cytoplasmic appositions of the myelin lamellae and seemingly involved in maintaining compactness of the myelin sheath. Immunization of mice with MOBP peptides representing predicted T-cell epitopes on the MOBP molecule identified an encephalitogenic region for H-2b mice, while in H-2s mice encephalitogenic T-cells elicited by immunization with recombinant MOBP recognize a single immunodominant epitope which induces EAE upon immunization. In MS, increased reactivity to these two epitopes has been demonstrated by our and another laboratory.

3. Autoreactivity to oligodendrocyte-specific protein (OSP)
   The recently uncovered highly hydrophobic OSP is a specific component of tight junction strands in CNS myelin, suggesting that it may have a role to play in the integrity of the myelin sheath and/or in physiological control of the permeability to small molecules by the interlamellar tight junctions. We recently demonstrated, using a recombinant preparation of OSP specifically designed for enhanced solubility, that OSP and specific T-cells thereof are highly encephalitogenic for H-2b and H-2s mice. The characterization of the encephalitogenic OSP-reactive T-cells for their epitope specificity revealed an unusual feature, whereby two- and three-way intramolecular cross-reactivity involving OSP epitopes with no primary structural sequence homology, appears to be implicated in pathogenic autoreactivity to OSP. The significance of three-way cross-reactivity in enhancing development and/or perpetuation of autoimmune disease is being further investigated, together with the mode of interaction between a single OSP-specific TCR and three different MHC-OSP peptide complexes.

Therapeutic approaches to Autoimmune Demyelinating CNS Diseases

1. Antigen-specific approaches
   Studies from our and other laboratories have indicated that in EAE induced by PLP, MOG, MOBP or OSP, the encephalitogenic T-cells while displaying a diverse TCR repertoire, are generally directed against a major immunodominant epitope in a given mouse strain, favouring epitope-directed rather than TCR-targeted approaches to immune-specific therapy. In this context, we are investigating the potential protective and/or curative effect of analogs of immunodominant encephalitogenic epitopes where TCR contact residues have been substituted, and which do not stimulate T-cell clonal proliferation, i.e. "altered peptide ligands" (APLs).

   In addition to autoreactivity against the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), which have long been investigated as potential target antigens in MS, autoreactivity against other myelin proteins, myelin oligodendrocyte glycoprotein (MOG), myelin-associated oligodendrocyte basic protein (MOBP) and oligodendrocyte-specific protein (OSP), likely to be involved in structural and/or physiological maintenance of myelin can also induce clinical and pathological EAE in susceptible animals, as indicated by our and other laboratories. Thus, in addition to MBP and PLP, MOG, MOBP and OSP can now be viewed as potential primary target antigens in MS, indicating that the pathogenic autoreactivity in MS may be directed against a multiplicity of antigenic targets, all of which should be taken into account when considering approaches to antigen-directed immune-specific therapy to the disease. Therefore, understanding the pathophysiology of the disease and devising approaches to immune-specific therapy require thorough characterization of the autoreactivity to all these potential primary target antigens.

   1. Epitope-directed APL-mediated immunomodulation
      Most of our studies on the efficacy and mode of action of APL treatment so far have been conducted in H-2b mice on MOG-induced EAE and encephalitogenic MOG-specific T-cells. Following our delineation of the core sequence within the encephalitogenic region of MOG (amino acids 35-55), we have defined residues 41 and 44 as the crucial TCR contact residues for the interaction between the TCR of MOG-specific T-cells and the nonameric core sequence MOG40-48, also found by molecular modelling studies to represent the preferred binding mode for MOG35-55 to I-Ab. On this basis, we have designed APLs with non-conservative substitutions at either or both of these crucial TCR contact residues. In vitro these APLs inhibited the proliferation of MOG35-55-specific T-cell clones to the native epitope, possibly via induction of partial TCR signalling suggesting partial agonist as well as antagonist activities. In vitro inhibition could also be related to an observed shift in cytokine profile of the T-cells from Th1 to Th2-type. Both mechanisms could be at play in the observed effect in vivo whereby i.v. administration of the APLs led to a marked decrease in severity and incidence of the disease induced by the native peptide, together with inhibition of the proliferative response by encephalitogenic T-cells to the native peptide and a marked decrease in secretion of inflammatory cytokines.
      

   2. Multi-targeted immunomodulation of EAE via:
      • Tolerogenic administration of the protein products of synthetic genes encoding multiple disease-relevant epitopes of target autoantigens
        While EAE associated with reactivity to a single epitope can be effectively suppressed by tolerogenic administration of that epitope, attempts to inhibit EAE associated with reactivities to more than one epitope or more than one autoantigen via tolerogenic administration of a single epitope have not been successful. In view of the multiplicity of potential primary target antigens in MS, we have devised a new antigen-specific therapeutic approach which aims at inhibiting concomitantly the potentially pathogenic T-cell autoreactivities to all known primary targets. For such a multi-targeted approach to therapy, we have constructed synthetic genes designed to encode tandemly arranged disease-relevant epitopes clusters of all five encephalitogenic proteins, MBP, PLP, MOG, MOBP and OSP; the purified protein product of such genes were tested for their immunomodulatory effect on EAE associated with a single or with multiple autoreactivities, upon tolerogenic administration. In SJL/J mice, i.v. administration of the purified protein product of a synthetic gene coding for disease-relevant epitopes of MBP, PLP and MOG fully abrogated the severe clinical EAE induced by immunization with PLP139-151. Analysis of T-cell reactivity to the encephalitogenic peptide in the protected animals indicated profound inhibition of the encephalitogenic T-cells. Most importantly, the development of EAE associated with multiple autoreactivities and induced by transfer of T-cells reactive with defined epitopes of MBP, PLP, MOG and MOBP could also be totally suppressed by daily i.v. administration of the purified protein product of a synthetic gene coding for disease-relevant epitopes of MBP, PLP, MOG and MOBP. The relevant PLP or MOG peptides administered singly according to the same regimen had no effect on disease development, while a combination of MOG+PLP only marginally decreased disease severity.

        These data strongly emphasize the necessity to neutralize as many as possible of the relevant multiple autoreactivities for effective immunomodulation of autoimmune diseases associated with a multiplicity of potential primary target antigens. In this context, we are further investigating the concept of multi-targeted immunomodulation to minimize the possible disease-enhancing effect of using synthetic gene products encompassing native epitopes which could still be highly stimulatory for the potentially stimulatory T-cells, even when administered via a tolerogenic route. In view of eventual extrapolation to therapeutic approaches for MS, we are constructing synthetic genes coding for non-stimulatory, non-encephalitogenic APLs of the disease-relevant epitopes, which in vitro inhibit the respective T-cells demonstrated to be potentially pathogenic in MS.

      • DNA vaccination with a synthetic gene encoding multiple disease-relevant epitopes of target autoantigens
        The direct introduction of a plasmid DNA encoding an antigenic protein into the organism's tissue has become an acceptable approach to vaccination. Following gene transfer, the protein expressed in the tissue can trigger the immune system, and the nature of the immune response is much dependent on the imunogenicity of the protein, among other factors. DNA vaccination with a synthetic gene encoding multiple disease-related epitopes may lead to the development of EAE but could also be of value in immunomodulating EAE. We are investigating the conditions whereby DNA vaccination may have an enhancing or downregulating effect on EAE associated with multiple pathogenic autoreactivities. To this end, the synthetic gene encoding disease-relevant epitope clusters of all five encephalitogenic proteins has been cloned into a mammalian vector, pCI. The enhancing or immunoregulatory effect on EAE development is now being investigated in mice injected intramuscularly with pCI/synthetic gene DNA and induced foor EAE associated with single or multiple autoreactivities.

2. Non antigen-specific approaches
   Several years ago, we had demonstrated the influence of environmental factors on the course of autoimmune diseases, in particular the profound immunomodulatory effect on EAE of bacterial components, such as the pertussis toxin of Bordetella pertussis. Our further investigation of the immunomodulatory effect of pertussis toxin (PT) on EAE revealed that the S3-S4 heterodimer of the PT holomer is responsible for protection against the disease by PT, whilst the S1 subunit is essential for disease enhancement. We have purified the S3-S4 dimer in a form whereby native conformation is preserved, and were thus able to demonstrate in vitro that the highly purified S3-S4 dimer is as mitogenic for T cells as PT. Most importantly, investigation of the mitogenic effect of the dimer on T cell subsets showed that in vitro culture of naive T cells with PT or the S3-S4 dimer result in a reversal of the CD4+/CD8+ T cell ratio. These data may suggest a possible mechanism of protection against EAE by the S3-S4 dimer, as a result of enhanced proliferation of suppressor CD8+ T cells in treated mice. As the S3-S4 dimer is totally devoid of toxic activity, it has obvious potential as a non antigen-specific therapeutic agent. Mechanisms underlying the protective mode of action of the S3-S4 dimer are now being tested further.

 Group Members:

Dr. Miriam Rozner-Paz, Post-doctoral Fellow
Nathali Kaushansky , Ph.D. student
Dr. Lydia Cohen, Lab assistant
Dr. Rina Zilkha-Falb, Post-doctoral Fellow

 List of Publications (last 3 years)