Suppression of Immune Response to Adenovirus Serotype 5 Vector by Immunization with Peptides Containing an MHC Class II Epitope and a Thio-Oxidoreductase Motif.
- 1Center for Molecular and Vascular Biology, University of Leuven , Leuven, Belgium.
- 2Imcyse SA, Leuven, Belgium.
The main obstacle to viral vector-mediated gene therapy remains the elicitation of an immune response to the vector, resulting in clearance of transgene and resistance to further transgenesis. Specific antibody production contributes to such immune responses. A single class II-restricted epitope of adenovirus serotype 5 (Ad5) vector hexon-6 capsid protein containing a thiol-oxidoreductase motif was used in an attempt to prevent specific antibody production in response to Ad5 vectors. We demonstrate here that such immunization carried out before intravenous administration of Ad5 vectors prevents antibody production to the ensemble of Ad5 vector proteins in both BALB/c and C57BL/6 mice. The antibody response to Ad5 is dependent on innate immune activation, seemingly involving natural killer T (NKT) cells. We observed that immunization with a class II-restricted Ad5 peptide prevents such NKT cell activation. Increased transgenesis and prolonged transgene expression result from such immunization, providing a simple protocol for improving gene therapy.
- PMID: 26711172
Under many circumstances it would be advantageous to eliminate detrimental immune responses without affecting immune defenses in general. Antigen-specific immunotherapy could cure autoimmune diseases, prevent graft rejection and suppress immune responses elicited by administration of therapeutic proteins such as monoclonal antibodies, or open the possibility to practicing gene therapy with no need for non-specific immunosuppressant.
Adaptive immune responses depend on the recruitment and activation of CD4+ T cells, which, upon formation of a synapse with a cognate epitope presented by class II major histocompatibility complexes (MHC) adopt various effector functions, including activation of B cells and production of antibodies. Suppressing the recruitment and activation of antigen-specific CD4+ T cells therefore represents a logical target to suppress an unwanted immune response without affecting immunity.
Administration of peptides encompassing epitopes recognized by such CD4+ T cells has been carried out over the last 30 years or so, using different formats and modes of administration, yet, unfortunately, with limited success. An exception to this rule is, however, provided by systemic vaccination with high-affinity class II-restricted epitopes in the absence of an adjuvant, which results in induction of CD4+ T cell anergy and infectious tolerance (1).
Although this method of immunomodulation constitutes a very efficient system to switch off polyclonal CD4+ T cell response, it requires regular administration of such epitopes.
Preventing the activation of antigen-specific CD4+ T cells and eliminating them, including polarized cells already recruited into an unwanted immune response, would, however, provide the benefit of preventing as well as suppressing such response. Our group has been working on a method by which antigen-specific CD4+ T cells are converted into potent cytolytic cells. Such a method consists in simple vaccination with an adjuvant, which elicits cells expressing an effector memory phenotype, limiting the number of injections to just a few. Cytolytic CD4+ T cells eliminate by apoptosis both the antigen-presenting cell (APC) with which a synapse is formed, thereby preventing a response, and CD4+ T cells activated at the surface of the same APC, even when such cells are fully differentiated, thereby suppressing ongoing immune responses. Importantly, by inducing cytolytic cells towards a single epitope, the method results in elimination of all CD4+ T cells specific for alternative epitopes of the same protein, as well as epitopes presented by alternative proteins participating to the immune response. In other words, selective apoptosis results in a “purge” of unwanted cells from the immune system, without affecting unrelated responses, as the cytolytic CD4+ T cells obtained by vaccination requires activation by formation of a synapse with its cognate epitope. Such a vaccination might not need to be repeated and could be considered as curative.
The concept emerged after observing that exceptional epitopes, upon presentation by MHC class II molecules, elicited robust CD4+ T cell activation but significant APC cytolysis (2). In an attempt to optimize this phenomenon, we designed a number of epitopes in their natural sequence, but to which an oxido-reductase activity was added within flanking residues. This oxido-reductase activity reduces disulfide bonds such as the one located in the second extracellular domain of the CD4 cofactor molecule, resulting in a net increase in synapse affinity. Signaling consequences within CD4+ T cells push them to acquire stable and seemingly irreversible properties as described above: (1) induction of apoptosis of the APC with which a synapse is formed, and; (2) apoptosis of alternative CD4+ T cells activated at the surface of the same APC (3).
The grounds for a vaccination strategy with the potential to prevent and to suppress unwanted polyclonal CD4+ T cell responses were therefore established, in a setting maintained specific for an antigen. Vaccination based on this concept was then carried out in a number of experimental models, from autoimmune diseases (type 1 diabetes, multiple sclerosis and myasthenia gravis), to graft rejection and allergic asthma. This confirmed the prediction that an antigen-specific immunotherapy was effective in both the prevention and suppression of immune responses (4).
The immune response to viral vectors used for the practice of gene therapy is a typical example of an immune response one would wish to eliminate. However, the rapid activation of innate immunity observed upon administration of such vectors (5) constituted a potential challenge for a therapy orientated towards class II-restricted adaptive responses. Such innate immune response involves many activation pathways, from molecular pattern recognition receptors (e.g. Toll-like receptors) to activation of plasmacytoid dendritic cells and NK cells (6).
However, another T cell lineage soon appeared to participate to innate immunity activation upon IV administration of an adenovirus 5 (Ad5) vector, selected on the basis of its reported strong immunogenicity (7). Early production of specific antibodies of the IgG2a isotype (BALB/c mice, or the equivalent IgG2c in C57BL/6 mice), well before other isotypes, suggested the involvement of cells of the natural killer T cell lineage (NKT cells). This was soon confirmed by two types of experiments: (1) CD1d KO mice, with full deletion of the NKT cell lineage, did not make an immune response to Ad5 viral vector administration, either innate or, more surprisingly, adaptive, and; (2) direct activation of NKT cells upon Ad5 vector administration was confirmed by accumulation of such cells in liver sinusoids (8).
NKT cells are selected in the thymus by homotypic interaction and circulate in peripheral blood with a phenotype of effector memory cells (9). They are activated by presentation of lipids or glycolipids in the context of a MHC-like molecule, CD1d. Such lipids are found from extrinsic sources (e.g. infectious agents such as mycobacterium or Borrelia) or intrinsic, as formed by cells under metabolic stress. CD1d is expressed on a number of cells including plasmacytoid cells, macrophages and Kupffer cells.
If preventing activation of NKT cells was required to prevent the immunogenicity of Ad5 vectors, it remained to be demonstrated that applying a vaccination strategy directed exclusively to the adaptive response would be efficient. This is what is clearly shown in the publication.
The mechanism behind the control of NKT cell activation by cytolytic, class II-restricted CD4+ T cells is under scrutiny. Plasmacytoid dendritic cells, macrophages located in liver sinusoids and spleen marginal zone as well as Kupffer cells are first line target cells for IV administration of viral vectors and express both class II and CD1d restriction elements. Induction of apoptosis of such cells after cognate interaction with cytolytic CD4+ T cells likely participates to the mechanism, yet alternative explanations are under investigation.
A single class II-restricted epitope conditioned as to contain an oxido-reductase activity and administered with an adjuvant (alum) is therefore sufficient as to elicit cytolytic class II-restricted CD4+ T cells, which prevent an immune response towards full viral vectors both at the innate and adaptive levels. Moreover, preliminary evidence suggests that this effect might be all that is required to increase efficacy of transgenesis and prolong transgene expression. These provocative findings have now to be confirmed using alternative vectors, such as the adenovirus associated viral vectors (AAV), most commonly used. AAVs are deemed to be less immunogenic than Ad5 vectors, yet they do elicit class I-restricted CD8+ T cells (10), responsible for hepatocyte lysis. Whether cytolytic CD4+ T cells can keep under control such class I-restricted response is also under investigation.
There is obviously a risk of preventing an immune response to a virus known to be pathogenic in humans and the present report should be taken as a first demonstration of the feasibility of preventing such response. Deriving vectors from viruses deemed to be devoid of pathogenicity in man, such as some AAV species, might still be considered as potentially risky. The strategy adopted here is to design vectors containing an artificial peptidic sequence, which will be used in vaccination, so that cytolytic CD4+ T cells will not be activated unless this added sequence is presented within the vector. It will still be required to evaluate whether preventing or suppressing the immune response towards the limited number of proteins present in vectors would significantly affect the overall response towards the virus.
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