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Immunopeptidomics: Decoding Pathogen Peptides To Deliver More Potent Vaccines

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Human leukocyte antigens (HLAs), also known as major histocompatibility complexes, are the core drivers of adaptive immunity. Class II HLAs (HLA-IIs) are displayed on both dendritic and B cells and instigate immune responses at both the cellular and humoral level through peptide presentation. HLA-IIs are of particular importance as they present extracellular peptides derived from proteins from the external environment and cell surfaces.

It is widely accepted that increasing our knowledge on the peptide-binding motifs displayed by HLA-IIs is critical to understanding the peptide chains they are likely to present and, therefore, the specificity of the immune response to certain proteins displayed by a pathogen.

Until now, algorithms have been used to ascertain the predominant peptide-binding motifs in HLA-II populations when challenged by a particular pathogen. But, these algorithms are error prone and don’t provide an accurate view of peptide antigen presentation. Now, using immunopeptidomics and utilizing the powerful separation and detection capabilities of liquid chromatography with tandem mass spectrometry (LC-MS/MS), HLA-II binding peptides can be accurately mapped. This insight provides the potential to develop vaccines that not only have greater specificity, but also elicit a broader immune response at the population level.

This article explores how immunopeptidomics can be used to accurately determine HLA-II peptide-binding motifs and its recent utilization in exploring peptide presentation in response to SARS-CoV-2, demonstrating the capability of this technique to reveal additional insights to support therapeutic development.

Understanding the pathway to peptide presentation

To understand the importance of HLA-II peptide-binding motifs, we must first explore the role of HLA in adaptive immunity. HLA-II complexes have a dual purpose, both to recognize and raise an immune response to foreign pathogens, such as bacteria, fungi and viruses, and to inhibit immune responses to the body’s own cells through self-identification.

When dendritic cells encounter a pathogen, they become activated, engulfing the pathogen and producing peptide fragments that are displayed on HLA-II structures. The dendritic cells move to the lymph nodes where they activate CD4+ T cells. Receptors present on the CD4+ T cells are highly diverse and where a receptor corresponds with an exact HLA-II complex, together with its displayed peptide, that T cell is activated.

Concurrently, B cells also sample the external environment by using a randomly generated, fixed B-cell antibody. If the specific B-cell receptor has an affinity for a particular protein, it will internalize and process it, displaying select peptides in an HLA-II complex. When an activated CD4+ T cell encounters a B cell with a complementary HLA-II complex to its receptor, it will bind to the B cell and activate it. The B cell then proliferates and begins antibody mutation, at which point high-affinity antibodies are produced in large numbers to drive the humoral response. It is this response that conveys vaccine effectiveness and, by activating both the CD4+ T cells and B cells in this way, researchers hope to develop vaccines that demonstrate broad efficacy across populations.

From predictive algorithms to precise identification

HLA-II receptors are highly diverse and heterogeneous structures; each person has two alleles and although some alleles are very common, many are rare. The peptide-binding motifs corresponding to these alleles are highly diverse and show affinity for certain amino acid sequences, however, heterogenous HLA-II alleles may present the same pathogen peptide fragments. Identifying and isolating pan-binding peptides, those that can attach to heterogeneous HLAs and generate a cluster effect, is of great interest in the production of vaccines that hope to convey broad immunity across populations.

Currently, our knowledge of peptide-binding motifs is based largely on biochemical binding assay data that has been compiled and used to create prediction algorithms, indicating the likely peptide chains that will initiate a cluster effect and convey broader immunity. But these algorithms are not accurate enough to drive the more efficacious vaccine development needed to meet the challenge of novel viruses with pandemic potential, s
uch as SARS-CoV-2. To increase vaccine efficacy and convey more widespread adaptive immunity, scientists can utilize LC-MS/MS technology to identify the precise peptide sequences that will prompt HLA-II cluster effects.

Deciphering the SARS-CoV-2 spike glycoprotein

The field of immunopeptidomics, which investigates peptides presented by HLAs using mass spectrometry, has been used in vaccine design for pathogens such as
Mycobacterium tuberculosis 1 and human herpesvirus 6B,2 but came to the foreground during the COVID-19 pandemic.

As the scientific community developed COVID-19 vaccines, a new immunopeptidomics study was started with the aim to investigate the peptides displayed in HLA-II complexes when challenged by the SARS-CoV-2 spike glycoprotein.3

The spike glycoprotein is of great interest in vaccine development as it is known to drive interactions that enable cellular binding and entry. The receptor-binding protein (RBD), located within the spike structure, interacts with the human angiotensin-converting enzyme (ACE2) to facilitate viral entry and the altered expression of ACE2 is linked to many of the pathologies associated with COVID-19.4 For this reason, many of the approved vaccines being used against SARS-CoV-2 are dependent on humoral responses targeted at the spike glycoprotein.

Using the MHC-Associated Peptide Proteomics (MAPPs) assay, scientists took dendritic cells from blood samples collected before the emergence of COVID-19 to ensure no prior immune knowledge of the virus. Immature dendritic cells were then isolated from the samples and treated with SARS-CoV-2 spike glycoprotein. Following maturation and lysis, HLA-II complexes were isolated from the cells using immunoprecipitation and HLA-II antigen-derived peptides were identified using highly sensitive LC-MS/MS. 876 peptide sequences in total were identified, 526 of which were unique. The distribution of peptide length showed that these peptides were characteristic of an HLA-II display (see figure 1).

Figure 1: Peptide length vs the number of unique peptides.3

Peptides sharing common sequences that highlighted a centralized HLA-II binding core were then grouped in clusters and plotted onto a protein heat map, with each line representing an individual donor and a deeper red color indicating a greater number of peptides (see figure 2).

Figure 2: Heat map indicating peptide clusters throughout the SARS-CoV-2 spike protein. 3

Consensus clusters represent peptides that bind multiple HLA-IIs and occur across donors. Several consensus clusters were observed in the S1-NTD, RBD and S’ regions of the spike glycoprotein. Overall, each donor presented around 17-18 cluster peptides, all donors presented some consensus clusters, and 11 consensus clusters were observed in total.

The results of this experiment were then compared to the predictive algorithms to determine the agreement between the two methods. Of the 11 consensus clusters observed through immunopeptidomics, only two were predicted by the software.

Cracking the code for more effective vaccines

During the initial stages of COVID-19 vaccine development, some early models targeted the RBD region of the SARS-CoV-2 spike glycoprotein.5 With the early observations of RBD-specific antibodies in COVID-19 patients, this would have seemed a logical pathway to follow. However, the heat map generated through immunopeptidomics3 demonstrates that consensus clusters are found throughout the S1-NTD, RBD and S’ regions of the spike glycoprotein, indicating that an RBD-specific vaccine may not confer adequate immunity across populations. By using the full spike glycoprotein, as is the case in many COVID-19 vaccines, the risk of point mutations rendering certain HLA-II clusters inactive would be lessened due to the presence of other active clusters.

Immunopeptidomics has shown that predictive algorithms do not provide the full picture on consensus clusters
and could inhibit scientists’ ability to perform rapid and accurate vaccine development, which could be particularly detrimental when working against novel viruses where swift, efficacious development may be needed to prevent a pandemic from taking hold. With the latest developments in high-sensitivity LC-MS/MS, it is now possible to accurately detect and isolate HLA-II peptide-binding motifs and relate these to the pathogen peptides that could drive adaptive immunity through vaccination.

When novel pathogens emerge, determining the peptides that illicit an HLA-II response in as large a population as possible is a critical stage in creating effective vaccines that deliver broad and long-lasting immunity. This deep insight will also help scientists understand the potential effects on adaptive immunity when viruses undergo rapid mutations. As scientists battle to control COVID-19 on a global level, taking this detailed approach to understand the fundamental drivers of adaptive immunity will ensure the rapid development of broader, more effective and longer-lasting vaccines in the future.


Bettencourt P, Müller J, Nicastri A, et al. Identification of antigens presented by MHC for vaccines against tuberculosis. NPJ Vaccines. 2020;5(1):2. doi: 10.1038/s41541-019-0148-y
2. BecerraArtiles A, Cruz J, Leszyk JD, et al. Naturally processed HLADR3restricted HHV6B peptides are recognized broadly with polyfunctional and cytotoxic CD4 Tcell responses. Eur J Immunol. 2019;49(8):1167-1185. doi: 10.1002/eji.201948126
3. Knierman MD, Lannan MB, Spindler LJ, McMillian CL, Konrad RJ, Siegel RW. The human leukocyte antigen class II immunopeptidome of the SARS-CoV-2 spike glycoprotein. Cell Rep. 2020;33(9):108454. doi: 10.1016/j.celrep.2020.108454
4. Bourgonje AR, Abdulle AE, Timens W, et al. Angiotensin‐converting enzyme 2 (ACE2), SARS‐CoV ‐2 and the pathophysiology of coronavirus disease 2019 (COVID ‐19). J Pathol. 2020;251(3):228-248. doi: 10.1002/path.5471
5. Yang J, Wang W, Chen Z, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature. 2020;586(7830):572-577. doi: 10.1038/s41586-020-2599-8