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A Mutagenesis Study for Potential T1D Vaccine Design

Posted on: 2023-04-11 12:22

In the immune system, the two main lymphocytes, B cells and T cells, work together to fight off foreign pathogens. Type 1 diabetes (T1D) is an autoimmune disease in which the immune system attacks pancreatic beta cells, resulting in insulin deficiency and high blood glucose levels. The prevalence of T1D is currently on the rise worldwide. Approximately 5 in every 100 people with diabetes have T1D, especially in children and adolescents with diabetes, with rates of T1D at 80-90%. In patients with T1D, insulin, glutamate deacidase (GAD65), heat shock protein (HSP), and human zinc transporter 8 (ZnT8) can be identified as antigens. Currently, the treatment of T1D relies on exogenous insulin injections, but this therapy does not accurately regulate the body's blood glucose levels and suffers from side effects such as hypoglycemia. Therefore, the search and development of new treatments for people with T1D is urgent.
In 2019, Professor Abdel Rahim A. Hamad and Professor Zhou Ruhong and their colleagues discovered a new autoantigen, X-autoantigen (also known as X-idiotype), that binds about 1,000 times more strongly to the antigen-presenting protein HLA-DQ8 (the major risk allele for T1D) than insulin (Cell 2019). On 11 April 2023, the two team co-published a paper titled A mutagenesis study of autoantigen optimization for potential T1D vaccine design was published in Proceedings of the National Academy of Science, provides deeper insight into the mechanism of interaction between X autoantigen and HLA-DQ8, and designs a series of autoantigens that can be used as HLA blockers or modulators of TCR function, providing new ideas for developing more effective, safer, and personalized immunotherapies.

First, the authors simulated the dynamic interaction of the X autoantigen with HLA-DQ8 with all-atom molecular dynamics. By analyzing the HLA-antigen contact, they generated point mutations at relevant epitopes. Using free energy perturbation (FEP) calculations with point mutations, they found that a methionine Met residue (Met4) at position p4 of the core antigen epitope can increase the binding affinity of the antigen to HLA when mutated to isoleucine-Ile (M4I) or leucine-Leu (M4L) (Figure 1). As can be seen from the detailed structural diagram, the Met4 residue is deeply buried in HLA and is in contact with the disulfide bond of HLA as well as hydrophobic residues. Mutation of Met4 to hydrophobic residues in M4I and M4L reduces the conflict with the disulfide bonded atoms and thus increases the affinity.

The authors further employed exchange mutation and double mutation to investigate mutants that enhance the binding affinity of the antigen to HLA. Among the initially designed residue exchange mutations, the two that increased the binding affinity of the X autoantigen to HLA most were Val5->Tyr + Tyr6->Val (V5Y_Y6V) and Tyr6->Val + Tyr7->Val (Y6V_Y7V) (Figure 1C). They also investigated favorable double mutations in the Val5-Tyr6-Tyr7 triplet. From the breakdown of the energy contribution of the favorable double mutation, it was found that the Tyr (Tyr6) mutation at position p6 of the epitope played a key role in increasing the binding affinity of the antigen to HLA (Figure 2). The calculations showed a moderate correlation between the affinity of the mutant and the volume of residues (R2 = 0.37), but not with Eisenberg hydrophobicity (R2 = 0.02).

Based on the above HLA antigen (pHLA) analysis, the authors further investigated the effects of antigen mutations on pHLA-TCR binding (Figure 3). The computational results showed that the Tyr6 mutants Y6C and Y6I increase the binding affinity of pHLA to TCR and may became candidates for modulating TCR function. On the other hand, unfavorable TCR binding may reduce the risk of autoimmune diseases, and in this case, mutants V5Y_Y6V and Y6Q_Y7Q are more effective. Mutants such as Y6V and Y6V_Y7V, which exhibit enhanced binding to pHLA dimers but have little effect on binding affinity to TCR trimers, can also be used as HLA blockers. In summary, the above analysis shows that mutation of epitope p6 to smaller hydrophobic/hydrophilic residues improves the binding affinity of X autoantigen to HLA-DQ8. The p6 mutant (Y6V), the p5-p6 exchange mutant (V5Y_Y6V), and the p6-p7 double mutant (Y6Q_Y7Q) could be used as potential therapeutics to modulate pHLA-TCR binding.

To verify the computational predictions above, the authors also performed CFSE-based T-cell proliferation experiments (Figure 4). Of the experimental results, the M2 (Y6V_Y7V), M3 (Y6Q_Y7Q), M4 (Y6C), and M5 (Y6V) mutants predicted pHLA-TCR affinity in a manner consistent with CD4 T-cell proliferation (compared with the X idiotype), whereas the M1 (V5Y_Y6V) exchange mutant and the M6 (Y6I) point mutant differed most from the experimental results. The TCR conformation at the contact interface predicted by the M1 and M6 mutants differed from the native Tyr6-X idiotype (Figure 3), which may be explained by the structural changes of pHLA-TCR were not detected over the long period of these systems.

In summary, the exact autoimmune mechanism responsible for causing Type 1 diabetes (T1D) remains unknown, but is thought to be a result of autoimmune activation by some potent self-antigen. A recent work isolated a unique cell set, termed the X-cell, that displays both T cell receptors (TCR) and B cell receptors (BCR) and encodes an autoantigen that produces a strong immune response in cells from T1D patients. Here, we explored the presentation of the autoantigen mutants to the HLA-TCR immune complex with a combined theoretical and experimental approach and identified several mutated sequences that modify HLA-antigen-TCR binding and therefore change immune responses. Specifically, we identify mutants that bind more strongly to the HLA and hold relevance to unique T1D immunotherapies.
This work was partially supported by the National Key R&D Program of China, the National Natural Science Foundation of China, the National Independent Innovation Demonstration Zone Shanghai Zhangjiang Major Projects, the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study, and the W. M. Keck Foundation etc.
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