Immunogenicity of a Promiscuous T Cell Epitope Peptide Based Conjugate Vaccine against Benzo[a]pyrene: Redirecting Antibodies to the Hapten

The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142–51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15–56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted

A coculture system composed of THP-1 cells and 3D reconstructed human epidermis to assess activation of dendritic cells by sensitizing chemicals after topical exposure

A key event of the adverse outcome pathway for skin sensitization is the activation of dendritic cells (DC). To be most close to the in vivo situation, we combined for the first time reconstructed human epidermis (RHE) in coculture with THP-1 cells, as surrogate for DC. THP-1 cells were placed underneath RHE (SkinEthic™, OS-REp). Cell activation was measured by analyzing cell surface expression of co-stimulatory molecules CD86 and CD40, adhesion molecule CD54 and of human leukocyte antigen class II-related subtypes (HLA-DR) on THP-1 cells by flow cytometry. Both models were suitable to measure DC activation but basal CD54 levels are significantly increased compared to THP-1 cells in monoculture for OS-REp. Chemical-induced activation of THP-1 cells was investigated after topical exposure on SkinEthic™. As proof of concept we analyzed three sensitizers and lactic acid (non-sensitizer). We observed differential, dose dependent levels of CD86 and/or CD54 on THP-1 cells in response to the skin sensitizers. We conclude that the RHE/THP-1 coculture using topical exposure complements our HaCaT/THP-1 coculture (COCAT) based on a submersed exposure and may allow the analysis of DC activation by various kind of test items including chemicals with pronounced lipophilicity, mixtures and possibly finished products

Metabolic activation of B[a]P.

<p>(A) During detoxification a small fraction of B[a]P is activated to 7-8-diol-B[a]P which is further converted to the highly reactive 7,8-dihydroxy-9,10-epoxy-B[a]P (BPDE) the ultimate DNA carcinogen. (B) Chemical structure of the Benzo[a]pyrene butyric acid isomeric mixture (B[a]P-BA), the derivative used for the conjugation to T cell epitope peptides.</p

Anti-B[a]P antibody concentration determined by indirect ELISA and correlation with antibody levels.

<p>To estimate absolute antibody concentrations a standard curve (insert in panel B) was used, that was based on dilution curves (1/200–1/437,400) of different known concentrations (0.01–1 mg/ml, represented by a line for each concentration in panel A) of purified monoclonal antibody against B[a]P (P9E1R4). 1/16,200 dilution (dashed line) was chosen to plot the standard curve (Insert in panel B, R² = 0.9709) and to calculate the antibody concentration. (B) Example of a titration curve of serum antibodies of mice immunised with B[a]P conjugated TT 2 weeks after the fourth injection. Values are presented as mean ± S.E.M of 6 mice per group. (C, D) Correlation between antibody level and [³H]-B[a]P recovery after a single i.p. injection of 2 μg/kg [³H]-B[a]P in individual mice mock immunised and immunised against B[a]P using B[a]P-peptide or B[a]P-TT conjugates. [³H]-B[a]P recovery in the blood (C) and liver (D). The statistical significant relationship between the two variables as shown in C and D was estimated by linear regression.</p

B[a]P recovery in mice vaccinated against B[a]P and tetanus toxoid.

<p>[³H]-B[a]P recovered in blood 24 h after a single i.p. injection of [³H]-B[a]P (2 µg/kg) in mice immunised with B[a]P-peptide or B[a]P-TT conjugates, with (open bars) or without (closed bars) tetanus toxoid (TT) pre-vaccination. Results are expressed as mean ± S.E.M of 5 mice per group. **p<0.01, ***p<0.001 significant difference from controls (Two Way ANOVA followed by Bonferoni). Control groups are No pre-vaccination (closed bars) or animals without B[a]P vaccination (No vaccination).</p

B[a]P recovery in mice immunised against B[a]P.

<p>[³H]-B[a]P (in Bq/g tissue weight or Bq) recovered in blood (A), tissues (B–liver, C–brain, D–spleen, E–kidney, F–lung), urine (G) and faeces (H) 24 h after a single i.p. injection of [³H]-B[a]P (2 μg/kg) in B[a]P-peptide, B[a]P-TT or mock immunised mice. Results are expressed as mean ± S.E.M of 5 mice per group. *p<0.05; statistical significant difference from control (Mock) (Student-Newman-Keuls-t test for multiple comparisons).</p

Antibody selectivity determined by competitive ELISA in sera immunised with B[a]P peptide or TT conjugates.

<p>(A) B[a]P-BA, (B) B[a]P, and (C) 7,8-diol-B[a]P were used as competitors to compete for binding of specific antibodies to B[a]P-ovalbumin as the coated antigen. The IC₅₀ (concentration of competitor for 50% inhibition) was calculated as a measure of antibody selectivity for each tested competitor. The IC₅₀ is inversely correlated to the antibody selectivity. Results are represented for each mouse (○) and median value (–).*p<0.05, statistically significant difference from TT immunised mice (One Way ANOVA on Ranks followed by Dunn's method).</p

Summery of peptides and their expected and measured masses (mutations in the peptide sequence are in bold).

<p>Tetanus toxoid (TT), wild type (wt).</p

Immunogenicity of B[a]P-peptide conjugates after tetanus toxoid pre-vaccination.

<p>(A) Endpoint titers (serum dilution reaching 5 times the background) for TT specific IgG antibodies determined by indirect ELISA for sera pre-immunised with tetanus toxoid (TT) followed by B[a]P-peptide or B[a]P-TT conjugate vaccination. (B) B[a]P specific IgG antibodies with and without pre-vaccination. Results are presented for each mouse (○) and median value (–). There was no statistical significant difference between animal with and without pre-vaccination (One way ANOVA procedure followed by Student-Newman-Keuls-t test). (C, D) Antibody selectivity determined by competitive ELISA in sera immunised with B[a]P peptide or B[a]P-TT conjugates with (○) and without (•) tetanus toxoid (TT) pre-vaccination. B[a]P (C) and 7,8-diol-B[a]P (D) were used as competitors to compete for binding of specific antibodies to B[a]P-ovalbumin as the coated antigen. The IC₅₀ (concentration of competitor for 50% inhibition) was calculated to determine the antibody specificity for each tested competitor. The IC₅₀ is inverse correlated to the antibody affinity. Results are presented for each mouse (circle) and median value (–).</p

Antibody titration curves of sera from mice immunised with B[a]P-peptide conjugates.

<p>(A) VNNESSE-variants, (B) FIGITEL-variants, (C) SYFPSV-variants and (D) PNRDIL-variants. Control mice were immunised with B[a]P conjugated to TT-protein (dashed line). Values are mean of 6 mice per group determined by indirect ELISA using heterologous conjugates (B[a]P-ovalbumin) as coated antigen. (E) Serum endpoint titers (serum dilution reaching 5 times the background) of B[a]P specific IgG antibodies of individual mice (○) and median value (–). Groups correspond to a selection of panel A to D. Dashed line represents the endpoint titer for immunisation with tetanus toxoid. (F) Endpoint titers against homologous carrier peptide. For sera with no detectable antibodies endpoint titers were set to 1/200. Results are presented for each mouse (○) and median value (–). ***p<0.001, statistically significant difference from TT immunised mice (One Way ANOVA test followed by Student-Newman-Keuls t-test for multiple comparison).</p