B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines

Abstract

Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD) vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T) or four (B4T) copies of the B-cell epitope from type O FMDV (a widespread circulating serotype) were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFN γ . Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

Figure 1

Time course study of antibody responses to FMDV in sera analyzed by ELISA. Specific antibody titers against FMDV (O UKG 11/2001) measured by ELISA in serum samples collected at days 0, 14, 20, and 39 postimmunization. Antibody titers were expressed as the reciprocal log⁡₁₀ of the last dilution calculated by interpolation to give an absorbance of 1 above background. Each point corresponds to the geometric mean of a least two determinations. In all cases the standard deviation was <1.0. Arrows indicate immunization (first dose and boost) dates.

Figure 2

Antibody responses to FMDV in sera analyzed by neutralization assay at day 39 postimmunization. The VNT titers determined in each group of mice were expressed as the reciprocal (log⁡₁₀) of the last dilution able to neutralize 100 TICD₅₀ of homologous virus. Animals with VNT ≥ 1 are considered seropositive. Each symbol represents the value for an individual animal. Horizontal lines indicate the mean value for each group of animals. Statistical differences between B₂T group and the others are indicated by P values.

Figure 3

Isotype-specific antibody (IgG1 and IgG2a/G2b) responses to FMDV in sera analyzed by ELISA at day 39 postimmunization. Endpoint titers were expressed as the reciprocal log⁡₁₀ of serum dilutions giving the absorbance recorded in the control wells (serum collected day 0) plus twice standard deviation. Each symbol represents the value of individual mouse. Horizontal lines indicate the mean value for each group of animals.

Figure 4

Systemic (serum) and local (nasal) IgA antibody responses to FMDV determined by ELISA at day 39 postimmunization. (a) IgA antibody titers against FMDV. Endpoint titers were expressed as described in Figure 3. Each symbol represents the value for an individual mouse. Horizontal lines indicate the mean value for each group of animals. (b) IgA antibody titers against FMDV determined in nasal fluids. Titers are expressed as the OD at 450 nm value obtained using a 1 : 10 dilution.

Figure 5

Frequency of peptide- and virus-specific IFNγ-producing cells in the spleen of immunized mice. IFNγ-producing cells were measured by ELISPOT assay in four animals from each group. Cells were stimulated with peptide (white bars) or FMDV (grey bars). Data are presented as the mean for four mice; error bars represent standard deviation.