AntigenBoost: enhanced mRNA-based antigen expression through rational amino acid substitution

Abstract

Messenger RNA (mRNA) vaccines represent a groundbreaking advancement in immunology and public health, particularly highlighted by their role in combating the COVID-19 pandemic. Optimizing mRNA-based antigen expression is a crucial focus in this emerging industry. We have developed a bioinformatics tool named AntigenBoost to address the challenge posed by destabilizing dipeptides that hinder ribosomal translation. AntigenBoost identifies these dipeptides within specific antigens and provides a range of potential amino acid substitution strategies using a two-dimensional scoring system. Through a combination of bioinformatics analysis and experimental validation, we significantly enhanced the in vitro expression of mRNA-derived Respiratory Syncytial Virus fusion glycoprotein and Influenza A Hemagglutinin antigen. Notably, a single amino acid substitution improved the immune response in mice, underscoring the effectiveness of AntigenBoost in mRNA vaccine design.

Keywords: amino acid substitution; mRNA optimization; mRNA vaccine and therapeutics; nascent peptides.

Figure 1

Overview of the AntigenBoost workflow. The input is the target antigen sequence for optimization, and a reference protein structure helps to identify the β-strand segments of the target sequence. The reference should either be a homology structure from the PDB database or a predicted structure of the target sequence. Step 1. Use the reference structure to predict the β-strand segments of the target sequence through sequence alignment. Step 2. Search for destabilizing dipeptides in the target sequence. Step 3. Identify the LEDipep sites by combining the results from Step 1 and Step 2. Step 4. Provide mutational strategies for each LEDipep site by calculating the 〖ΔSeq〗_score for the potential improvement in mRNA expression and conservative score (BLOSUM62) for evolutionary acceptance. Step 5. Experimental validation for promising mutants.

Figure 2

AntigenBoost identified LEDipeps on RSV-F. (A) Linear diagram of the antigen design for RSV-F protein variants NR091 and NR135. NR091 is derived from DS-Cav1 and includes furin cleavage sites (residues 109 and 137), resulting in the cleavage of F0 into F1 and F2 in vivo. NR135 replaces these furin cleavage sites and links F1 and F2 with a GS linker. The cytoplasmic tail is also removed in NR135. Both NR091 and NR135 preserve the transmembrane domain. LEDipep sites identified by AntigenBoost are highlighted in the inset. SP, signal peptide; p27, the p27 peptide removed by furin cleavage in NR091; TM, transmembrane domain; CT, cytoplasmic tail. (B) Identification of LEDipep sites on the structure of RSV-F. The reference RSV-F structure (PDB ID, 8WSQ) is shown in surface representation, sharing a sequence identity of 98% with NR135. One monomer of internal protein structure is shown in cartoon. LEDipep sites, I252I253 and K320V321, are shown in sticks.

Figure 3

Amino acid substitution increased RSV-F expression in vitro. (A) RSV-F amino acid substitution strategies from AntigenBoost with a 2D scoring system. Each amino acid substitution strategy is represented in a scatter plot according to its 〖ΔSeq〗_score against conservative score. Left: I252I253. Right: K320V321. The shaded area includes promising strategies given by AntigenBoost with 〖ΔSeq〗 _Score ≥ 0.4 and Conservative Score ≥ −1. Selected mutants for experimental validation are depicted by triangles, while other strategies are shown as dots. (B) Conservative substitutions of I252 and K320 on NR135 largely improved RSV-F expression in HEK293T cells. (C) Conservative substitutions of I290 and K368 on NR091 improved RSV-F expression in HEK293T cells. Cells were transfected with an equal amount of control (ctrl, unmuted) or mutated mRNA. Mock cells were not transfected. Cells were lysed 24 h after transfection, and total protein was analyzed by western blot. RSV-F and reference protein α-tubulin were detected by specific antibodies.

Figure 4

Amino acid substitution induced stronger humoral immune responses in vivo. (A) Immunization plan. BALB/c mice were primed by 1 μg LNP-encapsulated NR091 (ctrl) or I290L mutant RSV-F mRNA on Day 0 and boosted with the same vaccine on Day 21. PBS was injected as a negative control. Serum was collected on Day 35. (B) I290L mutated RSV-F vaccine induced higher levels of specific IgG. Serum IgG titers from individual mice are represented by dots, with the geometric mean and 95% confidence intervals for each group. The limit of detection was shown by a dashed line. Statistical difference compared to the control group was assessed using GraphPad Prism 8 t-test. ^*P < .05. (C) The tested RSV-F vaccines did not affect mice growth. Weekly body weights were recorded, and the mean growth values for each group were shown with their standard errors (SEM).

Figure 5

AntigenBoost improved influenza HA mRNA expression in vitro. (A) Identification of LEDipep sites on the predicted structure of HA. The predicted monomeric structure of HA by AlphaFold2 is shown in cartoon, overlaid with trimeric surface representation (PDB ID: 6PDX). Functional regions comprise the head domain, stem domain, and TM domain. LEDipep sites on the stem domain, I252I253 and K320V321, are shown in sticks. (B) HA amino acid substitution strategies from AntigenBoost with a 2D scoring system. Each amino acid substitution strategy is represented in a scatter plot according to its 〖ΔSeq〗_Score against Conservative Score. Left: K275I276. Right: K484I485. The shaded area includes promising strategies given by AntigenBoost with 〖ΔSeq〗_Score ≥ 0.4 and Conservative Score ≥ −1. Selected mutants for experimental validation are depicted by triangles, while other strategies are shown as dots. (C, D) The effects of single/double amino acid substitutions on HA expression in vitro. HEK293T cells were transfected with an equal amount of WT or mutated mRNA. Mock cells were not transfected. Cells were lysed 24 h after transfection, and total protein was analyzed by western blot. Influenza A HA and reference protein α-tubulin were detected by specific antibodies.