The HLA class I immunopeptidomes of AAV capsid proteins.

Introduction: Cellular immune responses against AAV vector capsid represent an obstacle for successful gene therapy. Previous studies have used overlapping peptides spanning the entire capsid sequence to identify T cell epitopes recognized by AAV-specific CD8+ T cells. However, the repertoire of peptides naturally displayed by HLA class I molecules for CD8 T cell recognition is unknown. Methods: Using mRNA transfected monocyte-derived dendritic cells (MDDCs) and MHC-associated peptide proteomics (MAPPs), we identified the HLA class I immunopeptidomes of AAV2, AAV6 and AAV9 capsids. MDDCs were isolated from a panel of healthy donors that have diverse alleles across the US population. mRNA-transfected MDDCs were lysed, the peptide:HLA complexes immunoprecipitated, and peptides eluted and analyzed by mass spectrometry. Results: We identified 65 AAV capsid-derived peptides loaded on HLA class I molecules of mRNA transfected monocyte derived dendritic cells. The HLA class I peptides are distributed along the entire capsid and more than 60% are contained within HLA class II clusters. Most of the peptides are organized as single species, however we identified twelve clusters containing at least 2 peptides of different lengths. Only 9% of the identified peptides have been previously identified as T cell epitopes, demonstrating that the immunogenicity potential for the vast majority of the AAV HLA class I immunopeptidome remains uncharacterized. In contrast, 12 immunogenic epitopes identified before were not found to be naturally processed in our study. Remarkably, 11 naturally presented AAV peptides were highly conserved among the three serotypes analyzed suggesting the possibility of cross-reactive AAV-specific CD8 T cells. Discussion: This work is the first comprehensive study identifying the naturally displayed HLA class I peptides derived from the capsid of AAVs. The results from this study can be used to generate strategies to assess immunogenicity risk and cross-reactivity among serotypes during gene therapies.

The HLA class-II immunopeptidomes of AAV capsids proteins.

Introduction: Gene therapies are using Adeno-associated viruses (AAVs) as vectors, but immune responses against the capsids pose challenges to their efficiency and safety. Helper T cell recognition of capsid-derived peptides bound to human leukocyte antigen (HLA) class II molecules is an essential step in the AAV-specific adaptive immunity. Methods: Using MHC-associated peptide proteomics, we identified the HLA-DR and HLA-DQ immunopeptidomes of the capsid proteins of three different AAV serotypes (AAV2, AAV6, and AAV9) from a panel of healthy donors selected to represent a majority of allele usage. Results: The identified sequences span the capsids of all serotypes, with AAV2 having the highest peptide count. For all the serotypes, multiple promiscuous peptides were identified and displayed by both HLA-DR and -DQ. However, despite high sequence homology, there were few identical peptides among AAV2, AAV6, and AAV9 immunopeptidomes, and none were promiscuous. Discussion: Results from this work represent a comprehensive immunopeptidomics research of potential CD4+ T cell epitopes and provide the basis for immunosurveillance efforts for safer and more efficient AAV-based gene therapies.

The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein.

Precise elucidation of the antigen sequences for T cell immunosurveillance greatly enhances our ability to understand and modulate humoral responses to viral infection or active immunization. Mass spectrometry is used to identify 526 unique sequences from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein extracellular domain in a complex with human leukocyte antigen class II molecules on antigen-presenting cells from a panel of healthy donors selected to represent a majority of allele usage from this highly polymorphic molecule. The identified sequences span the entire spike protein, and several sequences are isolated from a majority of the sampled donors, indicating promiscuous binding. Importantly, many peptides derived from the receptor binding domain used for cell entry are identified. This work represents a precise and comprehensive immunopeptidomic investigation with the SARS-CoV-2 spike glycoprotein and allows detailed analysis of features that may aid vaccine development to end the current coronavirus disease 2019 (COVID-19) pandemic.