Hui, et al. (2015). AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes. Molecular Therapy — Methods & Clinical Development. 2, 15029. [PubMedID: 26445723]
Recently published work from Katherine High’s group based in The Children’s Hospital of Philadelphia used ProImmune’s Pro5® MHC Class I Pentamers together with their ProPresent®assay to identify major histocompatability (MHC) class I epitopes in adeno-associated viral (AAV) vectors that are naturally presented by liver cells and trigger cytotoxic T cell responses. Dr. High is a world renowned hematologist who has been focussed for many years on developing genetic therapies for hemophilia.
The inherited blood clotting disorder Hemophilia B, affects approximately 1 in 25000 males. It is caused by deficiency in Factor IX, a protein normally made by hepatocytes. Current treatment involves infusion of patients with recombinant Factor IX protein – in severely affected patients this is carried out several times per week. Because it is a monogenic disorder for which numerous mutations have been characterised, it has for some time been a prime candidate for treatment using gene therapy – adeno-associated viral (AAV) vectors being amongst the most promising gene transfer vectors.
Therapeutic levels of expression of the AAV2-F.IX transgene in severe hemophilia B subjects were reported ten years ago in the first clinical trial for hepatic delivery of the AAV2-Factor IX transgene (Manno et al, 2006). However, Factor IX expression was short-lived (4 weeks at therapeutic levels). Subsequent studies suggested this was due to adaptive immune responses against the AAV vector capsid itself.
As part of their initial work characterising the T cell response to AAV-F.IX transgene Dr High’s group identified AAV capsid T cell epitopes in human splenocytes and confirmed using ProImmune’s MHC class I Pentamers that the AAV vector capsid caused an expansion in capsid-specific CD8+ T cells from splenocytes (Hui et al, 2015).
Having identified a number of immunodominant MHC class I epitopes for common human leukocyte antigen (HLA) types in splenocytes and shown that these did induce cytotoxic T cell responses it was important to determine which if any, of these epitopes were naturally-presented by MHC class I molecules in liver cells. ProImmune were able to carry this out for Dr. High’s group using their ProPresent® assay.
ProPresent® identifies naturally-presented peptides, directly determining which portion of the vector is visible to the immune system. HHL5-B7 hepatocytes were transduced with AAV2, cells harvested and a pan-MHC class I antibody used to pull down the MHC class I alleles. After elution, the associated peptides were identified using high-resolution mass spectrometry and screened against a database which comprises the entire human proteome in addition to the AAV-2 capsid sequence. One particular peptide identified using the ProPresent® assay as being naturally-presented in the MHC class I complex was demonstrated by Katharine High’s group to trigger cytotoxic T cell responses. This epitope is associated with the AAV serotype I vector.
Hui and colleagues stated that the aim of their study into the immunogenicity of the AAV capsid was to
‘perform ex vivo characterisation and validation of HLA-restricted AAV capsid epitopes and use this to improve immunomonitoring through the design of pentamers for specific HLA alleles as well as for future bioengineering efforts to alter the vector to reduce immunogenicity’.
Such understanding of cellular immune responses is pivotal to the development of successful long-term liver-directed gene transfer strategies. Almost 60% of the adult population have neutralizing antibodies against AAV-2 serotype and 50.5% against AAV-1 (Boutin et al, 2010). A number of recent studies (including UniQure’s clinical trials of an AAV5-based gene therapy) are using alternative AAV vectors for which much lower percentages of adults have developed neutralizing antibodies. In all cases immunomonitoring of human T cell responses against the AAV capsid is essential.
ProImmune with their comprehensive range of tools and expert services to characterize and track antigen-specific immune responses right through from preclinical to clinical are well positioned to support gene transfer therapy research.