Background: In last two decades, the world has experienced several outbreaks of potential pandemic viruses including highly pathogenic avian influenza (H5N1), Middle East Respiratory Syndrome Corona Virus (MERS-CoV), influenza A (H1N1)pdm09, Ebola, Zika and, most recently, Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). As emerging pathogens, there is no pre-existing immunity at the population level, generating a need for effective vaccines. In virally infected cells, antigens are processed by proteasome complex and transported to Endoplasmic Reticulum where it further trims to short peptides (8-11 aa) and presented through Major Histocompatibility complex (MHC) on to the cell surface for the T cells scanning and response. Peptide-based T cell vaccines are promising candidates to induce broadly protective immunity against multiple circulating strains of the pathogen (e.g. influenza), by targeting conserved proteins of the pathogen. This study provides an alternative approach to examine the immunopeptidome of avian influenza H5N1 by transfecting antigen-presenting cells (APCs) with targeted influenza proteins and comparing these data with natural influenza infection using mass spectrometry. Given the low avian influenza H5N1 susceptibility incidences in South-Asia region, we are interested in exploring the immunopeptidome presented by Human Leukocyte Antigen (HLA)-A*33:03 allele (highly expressed in South Asian populations) to determine whether there are protective effects in play.
Methods: Class I reduced (C1R) APCs expressing HLA-A*33:03 allele (C1R.A*33:03) were used as an APCs for transfection of targeted genes (hemagglutinin (HA), polymerase basic 2 (PB2), matrix & nucleoprotein (NP)) of avian influenza H5N1 and influenza A/X31. Transfected or infected C1R.A*33:03 was grown to 109 cells to generate cell pellets, underwent immunoaffinity purification, RP-HPLC and LC-MS/MS to identify influenza-specific peptides. Raw data were analysed using PEAKS software version X+.
Results: We have interrogated the HLA-A*33:03-restricted immunopeptidome of selected proteins derived from both avian influenza H5N1 (NP; 18 peptides identified) and influenza A/X31 (total of 40 peptides identified; HA=25, matrix=7, NP=18) transfectants. We have identified 41 peptides (HA=8, matrix=7, NP=22) from the influenza A/X31 naturally infected cells. HLA-binding motifs and length distribution were similar between transfectants and natural infection. Investigation of avian influenza H5N1 for HA, PB2 and matrix proteins is currently underway.
Conclusion: Using this workflow we have successfully identified large numbers of H5N1 derived HLA-A*33:03 restricted peptides. Thus, utilisation of transfectants offers a novel approach for identification of peptide targets derived from highly infectious viruses for development as immunotherapeutics, circumventing the need to handle such pathogens in high containment facilities.