The identification of HLA class II-restricted T cell epitopes to vaccinia virus membrane proteins

Abstract

Three decades after the eradication of smallpox, the threat of bioterrorism and outbreaks of emerging diseases such as monkeypox have renewed interest in the development of safe and effective next-generation poxvirus vaccines and biodefense research. Current smallpox vaccines contain live virus and are contraindicated for a large percentage of the population. Safer, yet still effective inactivated and subunit vaccines are needed, and epitope identification is an essential step in the development of these subunit vaccines. In this study we focused on 4 vaccinia membrane proteins known to be targeted by humoral responses in vaccinees. In spite of the narrow focus of the study we identified 36T cell epitopes, and provide additional support for the physical linkage between T and B epitopes. This information may prove useful in peptide and protein-based subunit vaccine development as well as in the study of CD4 responses to poxviruses.

Fig. 1

Cellular and humoral responses to vaccinia. A) The histogram on the left shows the range of IFNγ ELISPOT results as stimulation index (S.I.: average spot forming units in vaccinia stimulated wells divided by the average spot forming units in background wells). X-axis scale indicates upper bound of each bin, i.e. two subjects had an S.I. less than 2, and six subjects had S.I. values between 2.1 and 6. B) The histogram on the right shows the range of 50% inhibitory dose (ID₅₀: reciprocal of the serum dilution which inhibits 50% of viral activity). X-axis scale indicates upper bound of each bin, i.e. no subjects had ID₅₀ values less than 20, while 3 subjects had an ID₅₀ between 21 and 60.

Fig. 2

Immune responses to pooled peptides from a single individual are illustrated. Bars indicate the average number of IFNγ producing spots for wells (3–5 replicates) stimulated with the antigen indicated on the X-axis. Error bars represent the standard deviation. Dark gray bars indicate responses significantly above background (p < 0.05). Data illustrated in this figure to Fig.–4 come from the same individual.

Fig. 3

Immune responses to individual peptides from a single individual. The positive pools shown in Fig. 2 were used to select peptides for individual screening. Graph layout is as described in Fig. 2.

Fig. 4

CD4⁺ T cell responses to selected peptides from a single individual. Prior to stimulation with the indicated peptides (X-axis) CD8⁺ T cells were removed by magnetic bead depletion. Resulting cell populations contained < 2% CD8⁺ T cells (data not shown). The graph layout is as described in Fig. 2.

Fig. 5

Correlation between immune response and number of epitopes identified. A) Scatter plot shows vaccinia-specific IFNγ ELISPOT S.I. on the X-axis and the number of identified epitopes on the Y-axis. The best fit line is depicted in gray with the r² value indicated on the graph. Each diamond represents a single subject's response. B) Scatter plot shows the vaccinia-specific neutralizing antibody ID₅₀ on the X-axis and the number of identified epitopes on the Y-axis. The best fit line is depicted in gray with the r² value indicated on the graph. Each diamond represents a single subject's response.

Fig. 6

Comparison of immune responses in subjects with and without identified epitopes. Average immune responses to vaccinia virus (left panel = humoral response represented by ID₅₀ values, right panel = cellular immunity denoted by IFNγ ELISPOT S.I.) for subjects with identified T cell epitopes are compared to the anti-viral immune responses of subjects for which no epitopes were found. Student's t test comparison p-values are indicated on each graph.