Specificities of human CD4+ T cell responses to an inactivated flavivirus vaccine and infection: correlation with structure and epitope prediction

Abstract

Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4(+) T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4(+) T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4(+) T cell epitopes. Importance: Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection.

FIG 1

CD4⁺ T cell response to the TBE virus structural proteins C, prM/M, and E. (A) Schematic representation of a flavivirus particle, showing an immature and mature virion. The virion has three structural proteins: C (capsid), prM/M (membrane), and E (envelope). The capsid contains the positive-stranded RNA and several copies of the capsid protein C. Immature virions are covered with prM-E heterodimers. The proteolytic cleavage of prM leads to the reorganization of the E proteins and the formation of particles covered with E dimers. sE, soluble form of E lacking the membrane anchor and stem; M, membrane-anchored cleavage product of prM. (Adapted from PLoS Pathogens [84].) (B) Magnitude of individual CD4⁺ T cell responses to TBE virus C, prM/M, and E from 40 booster-vaccinated, 45 infected, and 5 TBE-naive individuals determined by IL-2 ELISpot assay. Statistical comparisons between the data from vaccinated and infected individuals were performed using a Kruskal-Wallis test (P < 0.0001) and Dunn's multiple comparison tests. Significant differences were observed for the responses to C and E (indicated by stars). Medians are indicated by red lines. (C and D) Spearman correlation of individual C- and E-protein-specific CD4⁺ T cell responses of booster-vaccinated (Vacc) and infected (Inf) individuals. (E) E/C ratios of individual CD4⁺ T cell responses. Values below the cutoff of 21 spots/10⁶ cells were set at 10 for this analysis. (F) Percentage of spots contributed by C, prM/M, and E peptides in vaccinated (n = 40) and infected (n = 45) individuals.

FIG 2

Correlation of CD4⁺ T cell and antibody responses. The magnitude of individual CD4⁺ T cell responses to TBE virus C (A and B) and E (C and D) peptide maxipools was plotted against the corresponding ELISA units (solid dots) and NT titers (empty circles) in vaccinated (A and C) and infected (B and D) individuals. Correlations were calculated using a Spearman correlation coefficient. Linear regressions are indicated by solid (ELISA) or dashed (NT) lines.

FIG 3

SDS-PAGE of TBE virus as well as its envelope proteins and capsid fractions after solubilization with DDM. The soluble fraction (containing the membrane-associated proteins E and prM/M) and aggregated fraction (containing protein C) were separated by low-speed centrifugation. Identical aliquots of the supernatant (SN) and the pellet (P) were analyzed by SDS-PAGE and stained with Coomassie blue. V, untreated virus control. The positions of E, prM, C, and M are indicated.

FIG 4

Individual variation of CD4⁺ T cell responses. CD4⁺ T cell responses against TBE C, prM/M, and E single peptides were measured using an IL-2 ELISpot assay. Examples of two vaccinated (A and B) and two infected (C and D) individuals are shown.

FIG 5

Mapping of immunodominant experimental and predicted CD4⁺ T cell responses. (A and B) Percentage of positively tested vaccinated (A) and infected (B) individuals recognizing a specific single peptide within the C (n = 31 vaccinated; n = 13 infected) and E (n = 34 vaccinated; n = 26 infected) proteins. prM/M-specific single-peptide responses were too low for evaluation. Peptides recognized significantly more often than the average were identified (Fisher's exact or chi-square test; significance level of P < 0.05, separately for each protein and each group) and are indicated by asterisks. Clusters of these peptides are numbered 1 to 9. (C) Crystallographic structure of the flavivirus Kunjin C protein (24) consisting of four helices (H1 to H4; left panel). For the N-terminal region (gray line), no crystallographic data exist. Crystallographic structure of the TBE virus soluble E (23) consisting of three domains (DI, DII, and DIII; right panel). Recent data suggest that the stem-anchor region consists of three alpha-helices in the stem and two alpha-helices in the transmembrane (TM) anchor (34) (boxes). Dominant clusters labeled in panels A and B are colored as follows: C, green; E DI, red; E DII, yellow; E DIII, blue; E stem, magenta. N- and C-terminal residues are indicated. (D and E) Cumulative computer prediction of CD4⁺ T cell epitopes for HLA class II alleles from vaccinated (n = 39 subjects; number of predicted alleles, 233) (D) and infected (n = 44 subjects; number of predicted alleles, = 264) (E) individuals. The percentage of alleles predicting a specific single peptide is shown for peptides of all three structural proteins (C, prM/M, and E). In panels A, B, D, and E, lines below the x axes indicate the TM anchor domain of prM/M (black) and the domains of the E protein (DI, red; DII, yellow; DIII, blue; stem, magenta; TM, black).

FIG 6

Computer prediction of potential CD4⁺ T cell epitopes from envelope proteins of different viruses. CD4⁺ T cell epitopes for surface proteins of hepatitis B virus (HBV), influenza virus, vaccinia virus (VACV) (extracellular envelope virion [EV]), SARS-coronavirus (SARS-CoV), herpes simplex virus 1 (HSV-1), HIV, and yellow fever (YF) virus were predicted using common human HLA class II alleles. Positions of transmembrane domains within the respective amino acid sequences are indicated by open rectangles. The prediction patterns were very similar for YF virus and dengue virus, and the latter was therefore not included in the figure.