Impact of genotypic variability of measles virus T-cell epitopes on vaccine-induced T-cell immunity

Abstract

After the COVID-19 pandemic, significant increases in measles cases were observed globally. Community-wide vaccination remains the most effective strategy for preventing measles. However, it is crucial to understand whether prevalent genotypes, when circulating in populations with suboptimal vaccination coverage, may undergo adaptive mutations that allow them to escape vaccine-induced immunity. In this study, a bioinformatics-guided approach was used to predict universal helper T-cell epitopes specific to the measles vaccine virus (vaccine-MeV) presented by multiple HLA-DR, -DP, and -DQ alleles to achieve population-wide coverage. By using MeV-specific T-cell lines, we identified 37 functional epitopes out of 83 predicted candidates, including 25 novel ones. Strikingly, 73% of these epitope regions were associated with sequence variations in wild-type viruses. More importantly, we demonstrated that mutations disrupted the ability of vaccine-induced CD4⁺ T cells to respond to circulating viruses. Consequently, mutations in epitope regions of circulating viruses may affect the effectiveness of vaccine-induced T-cell immunity.

Fig. 1. Heatmap of immunogenicity scores for predicted helper T-cell epitope candidates of vaccine-MeV.

Heatmap depicting the 83 selected helper epitope candidates of MeV (Edmonston) with the best T-cell immunogenicity scores for 20 different common HLA-DR, DP, and DQ alleles indicated below. The green color scale indicates differences in predicted HLA-II binding affinity scores (left panel) or elution scores (right panel) to the various HLA-II alleles indicated below. Peptides with lower rank scores of 0–0.5% (dark green) represent the best-predicted T-cell epitopes. On the left side, identified universal CD4⁺ T-cell epitope candidates (all 15-mers) are indicated as the location of the first and last amino acid position of the corresponding MeV proteins (nucleoprotein (N), phosphoprotein (P), C protein (C), matrix protein (M), fusion protein (F), hemagglutinin (H), and large protein (L)). Epitopes that were later confirmed as functional T-cell epitopes are shown in red font. The novel-confirmed epitopes that were not previously described as CD4⁺ T-cell epitopes are indicated in (red and) bold.

Fig. 2. Identification of functional helper T-cell epitopes in the vaccine-MeV sequence.

Polyclonal MeV-specific T-cell lines obtained from two convalescent measles cases and three vaccinated subjects were stimulated with 19 various peptide pools (1 µM/peptide) in a two-dimensional peptide matrix. T-cell responses were measured by IFN-ɣ ELISPOT assay. MeV-specific T-cell frequencies are depicted in box plots and shown as means ± SD of triplicates. a T-cell frequencies of two convalescent donors (#A, #B) are presented as a spot-forming unit (SFU) per 1 × 10⁴ cells in the left panel. b T-cell frequencies of three recently measles, mumps, rubella (MMR) vaccinated donors (#1, #2, #3) are presented as SFU/ 5 × 10⁴ cells in the lower panel. c Peptide pools that show a “positive” T-cell response from a representative vaccinated donor (#1) are indicated in yellow boxes. Peptides that are present in two “positive-tested peptide pools” are indicated in green boxes. Responsiveness of the T-cell lines to individual epitope candidates, selected as peptides present in two “positive-tested peptide pools”, were tested by flow cytometry for expression of intracytoplasmic IFN-ɣ and CD154 activation marker (“positive-confirmed” individual epitopes are indicated in blue boxes). d Representative flow cytometry plots of expression of IFN-ɣ (Y-axis) and CD154 (X-axis) of gated CD4⁺ T cells of T-cell line (donor #1) after 6 h stimulation with medium/0.3% DMSO (negative control), PMA/ionomycin (positive control), peptide mix (containing all 83 candidate epitopes), and six representative individual peptides from positive-tested pools. Individual peptides (all 15-mers) are indicated as the location of the first amino acid position of the corresponding MeV proteins (nucleoprotein (N), phosphoprotein (P), C protein (C), matrix protein (M), fusion protein (F), hemagglutinin (H), and large protein (L)).

Fig. 3. Screening for nonsynonymous mutations in regions of confirmed CD4 + T-cell epitopes in WT-MeV sequences.

a Number of the 628 screened wild-type measles viruses (WT-MeVs) having nonsynonymous mutation(s) (Y-axis) are presented per confirmed T-cell epitope (predicted HLA-II-binding core sequence) (X-axis). Peptides (15-mers) are indicated as the location of the first and last amino acid position of the corresponding MeV protein (nucleoprotein (N), phosphoprotein (P), C protein (C), matrix protein (M), fusion protein (F), hemagglutinin (H), and large protein (L)). b Two T-cell epitopes (i.e., phosphoprotein (P_108-122) and fusion protein (F_409-423) (a, b indicated in red box) were selected for further study as they showed to have nonsynonymous mutations (Y111H, V120I, K121E and H419R, H419Y) in many WT-MeVs of the B3 (dark blue), H1 (light gray), D4 (dark gray), and/or D8 genotype (turquoise bars). The conserved T-cell epitope (F_314-328) was selected as a control for further study on the impact of the mutations on T-cell responsiveness (a, green box).

Fig. 4. Mutations in vaccine-MeV epitopes disrupt the function of specific T-cell clones.

a Two vaccine-MeV epitope-specific T-cell clones (E9/E11) against P_108-122 were tested for their reactivity upon stimulation with antigen-presenting cells (APC) pulsed with the original vaccine epitope (orange) and to peptide variants with wild-type mutations, Y111H (turquoise) or V120I (gray) by measuring the percentage of cells expressing both the activation marker CD154 and intracellular IFN-ɣ. b The percentage of cells expressing both the activation marker CD154 and intracellular IFN-ɣ of four F_409-423-epitope-specific T-cell clones (B5/C4/D9/E11) was measured upon stimulation with APC pulsed with original vaccine epitope (orange) and peptide variant with wild-type mutation, H419R (dark blue). All data (in triplicate) are shown as means ± SD. On the Y-axis, the percentage of CD154⁺ /IFN-γ⁺ T cells of total T cells is presented. T-cell response was measured at Effector (T-cell): Target (APC) (E:T) ratio of 1:1. All measured T-cell responses (CD154/IFN-ɣ) against the various peptide variants are statistically significantly different from the responses against vaccine-MeV peptides (unpaired T-test (b) or one-way ANOVA for multiple comparisons (a), p ≤ 0.01).

Fig. 5. Vaccine-specific T-cell clones do not upregulate CD154 in response to mutated wild-type MeV isolates.

a MeV epitope-specific T-cell clones against phosphoprotein (P_108-122; representative clone E9), fusion protein (F_409-423; representative clone D9), or against the conserved F_314-328 epitope were tested for their capacity to upregulate CD154 expression upon stimulation with antigen-presenting cells (APC) infected with vaccine-measles (MeV) strain (orange line) versus infected with wild-type (WT-)MeV of B3 (dark blue line) or D8 (turquoise line) genotype. b MeV epitope-specific T-cell clones were tested for their capacity to upregulate CD154 expression upon stimulation with APC pulsed with peptides of P_108-122, F_409-423, F_314-328 derived from vaccine-MeV (orange dotted line) versus the corresponding variant peptides with relevant WT mutation (P: Y111H (turquoise dotted line) or F: H419R (dark blue dotted lines). All data (in triplicate) were shown as means ± SD. On the Y-axis, the percentage of CD154⁺ T cells of total T cells is presented. T-cell response was measured at various Effector (T-cell) : Target (APC) (E:T) ratios as presented on the X-axis.

Fig. 6. Impaired production of cytokines by vaccine-specific T-cell clones due to wild-type MeV mutations.

a Cytokine secretion (IL-2, IFN- γ, TNF-α, IL-4, IL-5, IL-13, IL-6, IL-9, IL-10, IL-17(A or F), IL-22) by representative P_108-122-epitope-specific T-cell clone (E9) was measured upon stimulation with antigen-presenting cells (APC) infected with vaccine-MeV strain (orange) versus infected with wild-type (WT-)MeV of B3 (dark blue) or D8 (turquoise) genotype. b Cytokines produced by F_409-423-epitope-specific T-cell clone (D9) was also measured upon stimulation with APC infected with vaccine-MeV strain (orange) versus infected with WT-MeV of B3 (dark blue) or D8 (turquoise) genotype. c Cytokines produced by control T-cell clone against the conserved vaccine-MeV F_314-328 epitope upon stimulation with APC infected with vaccine-MeV strain (orange) versus infected with WT-MeV of B3 (dark blue) or D8 (turquoise) genotype. d Cytokine secretion by P_108-122-epitope-specific T-cell clone (E9) was also measured upon stimulation with APC pulsed with vaccine-measles virus (MeV) peptide of phosphoprotein (P_108-122) (orange), the corresponding peptide variants with the V120I (dark blue), or Y111H (turquoise) mutation. e Cytokines produced by a representative F_409-423-epitope-specific T-cell clone (D9) was measured upon stimulation with APC pulsed with vaccine-measles virus (MeV) peptide of fusion protein (F_409-423) (orange) or the corresponding peptide variant with H419R mutation (dark blue). T-cell response was measured at Effector (T-cell) : Target (APC) (E:T) ratio of 1:1.