The Immunology of Adeno-Associated Virus Serotype 2
Madsen, Declan Paul (2009) The Immunology of Adeno-Associated Virus Serotype 2. PhD thesis, National University of Ireland Maynooth.
Adeno-associated virus serotype 2 (AAV-2) has been developed as a gene therapy vector. Antibody and cell-mediated immune responses to AAV-2 or AAV-2 transfected cells may confound the therapeutic use of such vectors in the clinic. In this study, cell-mediated and humoral immune responses to AAV-2 were characterised from a panel of healthy blood donors. The extent of AAV-2-specific antibody in humans was determined by examination of circulating AAV-2-specific total IgG levels in plasma from 45 normal donors. 41 donors were seropositive and responses were dominated by IgG1 and IgG2 subclasses. Conversely, AAV-2-specific IgG3 levels were consistently low in all donors. Cell-mediated immune recall responses were detectable in nearly half the population studied. In vitro re-stimulation with AAV-2 of peripheral blood mononuclear cell (PBMC) cultures from 16 donors elicited interferon-gamma (IFN-γ) (10 donors), interleukin-10 (IL-10) (8 donors) and interleukin-13 (IL-13) (4 donors). Using a series of overlapping peptides derived from the sequence of the VP1 viral capsid protein, a total of 59 candidate T-cell epitopes were identified. HLA characterisation of donors revealed that the population studied included diverse haplotypes, but that at least 17 epitopes were recognised by multiple donors and could be regarded as immunodominant. These data indicate that robust immunological memory is established to AAV-2. The diversity of sequences recognised suggests that attempts to modify the AAV-2 capsid, as a strategy to avoid confounding immunity, will not be feasible. It was hoped that murine mesenchymal stem cells (MSC) might be amenable to transformation using an AAV-2-based vector, rendering the MSC as a potential platform to deliver transgene products whilst avoiding the immune memory for AAV-2 characterised herein. However, murine MSC appeared resistant to transduction using an AAV-2 GFP vector. To assess the impact of transfection of MSC with a plasmid vector, a lipofection protocol, utilising a plasmid encoding indoleamine 2,3-dioxygenase (IDO), was instead adopted. Transfection was successful, MSC morphology was not altered and osteogenic and adipogenic functions remained. However, MSC cell numbers in culture declined sharply in the 24 hours following transfection and growth rates were poor for up to 7 days. Cell surface markers were also altered, with increased MHC class I observed in transfected cultures. Perhaps most significantly, transfected MSC displayed a reduced capacity to suppress mitogen-driven lymphocyte proliferation. Further optimisation of the lipofection protocol might reduce MSC death and restore proliferation, but the observed changes in murine MSC immunomodulatory function as a result of IDO transfection coupled with the cell death and loss of proliferation indicates that lipofection is a suboptimal method for the transfection of murine MSC.
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