Abstract
[Immunoprophylaktische Anwendung gegen chemische Karzinogene]
The main objective of the present thesis was to investigate whether antibody effects observed in earlier in vitro studies can translate into the protection against chemical carcinogenesis in vivo as the basis of an immunoprophylactic approach against carcinogens. As model for chemical carcinogenesis, we selected B[a]P the prototype polycyclic aromatic hydrocarbon (PAH), an environmental pollutant emanating from both natural and anthropogenic sources. Many in vivo models conveniently use high doses of carcinogens mostly given as single bolus, which provides simple surrogate readouts, but poorly reflects chronic exposure to the low concentrations found in the environment. In addition, these concentrations cannot be matched with equimolar antibody concentrations obtained by immunisation. However, low B[a]P concentrations do not permit to directly measure chemical carcinogenesis. Therefore, in the present thesis, the pharmacokinetic, metabolism and B[a]P mediated immunotoxicity were chosen as experimental read-outs. B[a]P conjugate vaccines based on ovalbumin, tetanus toxoid and diphtheria toxoid (DT) as carrier proteins were developed to actively immunise mice against B[a]P. B[a]P-DT conjugate induced the most robust immune response. The antibodies reacted not only with B[a]P but also with the proximate carcinogen 7,8-diol-B[a]P. Antibodies modulated the bioavailability of B[a]P and its metabolic activation in a dose-dependent manner by sequestration in the blood. In order to further improve the vaccination, we replaced the protein carrier by promiscuous T-helper cell epitopes to induce higher antibody titer with increased specificity for the B[a]P hapten. We hypothesised that a reduction of B cell binding sites on the carrier, compared to whole protein carrier, should favour the activation of B cells recognising the hapten instead of the carrier protein. An internal processing of the carrier and cleavage of the B[a]P-BA and subsequent presentation of the carrier peptide by MHC II molecules to T cell receptor should induce a B cell dependent immune response by activating B cells capable to recognise B[a]P. We demonstrated that a vaccination against B[a]P using promiscuous T-helper cell epitopes as a carrier is feasible and some tested peptide conjugates were more immunogenic as whole protein conjugates with increased specificity. We showed that vaccination against B[a]P reduces immunotoxicity. B[a]P suppressed the proliferative response of both T and B cells after a sub-acute administration, an effect that was completely reversed by vaccination. In immunized mice the immunotoxic effect of B[a]P on IFN-γ, Il-12, TNF-� production and B cell activation was restored. In addition, specific antibodies inhibited the induction of Cyp1a1 by B[a]P in lymphocytes and Cyp1b1 in the liver, enzymes that are known to convert the procarcinogen B[a]P to the ultimate DNA-adduct forming metabolite, a major risk factor of chemical carcinogenesis. In order to replace Freund adjuvant and to improve the immunisation strategy in terms of antibody quantity and quality, several adjuvants that are potentially compatible with their use in humans were tested. In combination with Freund adjuvant, the conjugate-vaccine induced high levels of B[a]P-specific antibodies. We showed that all adjuvants tested induced specific antibodies against B[a]P and 7,8-diol-B[a]P, its carcinogenic metabolite. The highest antibody levels were obtained with Quil A, MF-59 and Alum. Biological activity in terms of enhanced retention of B[a]P was confirmed in mice immunised with Quil A, Montanide, Alum and MF-59. Our findings demonstrate that a vaccination against B[a]P is feasible in combination with adjuvants licensed in humans. Based on these results and with the current understanding of the mechanisms of chemical carcinogenesis of the ubiquitous carcinogen B[a]P and of the effects of specific antibodies, an immunoprophylactic approach against chemical carcinogenesis is absolutely warranted. Nevertheless, the direct effects of B[a]P-specific antibodies on the different stages of carcinogenesis (e.g. adduct formation) and whether these effects may translate into long-term protective effect against tumourigenesis needs to be proven in further experiments.
The main objective of the present thesis was to investigate whether antibody effects observed in earlier in vitro studies can translate into the protection against chemical carcinogenesis in vivo as the basis of an immunoprophylactic approach against carcinogens. As model for chemical carcinogenesis, we selected B[a]P the prototype polycyclic aromatic hydrocarbon (PAH), an environmental pollutant emanating from both natural and anthropogenic sources. Many in vivo models conveniently use high doses of carcinogens mostly given as single bolus, which provides simple surrogate readouts, but poorly reflects chronic exposure to the low concentrations found in the environment. In addition, these concentrations cannot be matched with equimolar antibody concentrations obtained by immunisation. However, low B[a]P concentrations do not permit to directly measure chemical carcinogenesis. Therefore, in the present thesis, the pharmacokinetic, metabolism and B[a]P mediated immunotoxicity were chosen as experimental read-outs. B[a]P conjugate vaccines based on ovalbumin, tetanus toxoid and diphtheria toxoid (DT) as carrier proteins were developed to actively immunise mice against B[a]P. B[a]P-DT conjugate induced the most robust immune response. The antibodies reacted not only with B[a]P but also with the proximate carcinogen 7,8-diol-B[a]P. Antibodies modulated the bioavailability of B[a]P and its metabolic activation in a dose-dependent manner by sequestration in the blood. In order to further improve the vaccination, we replaced the protein carrier by promiscuous T-helper cell epitopes to induce higher antibody titer with increased specificity for the B[a]P hapten. We hypothesised that a reduction of B cell binding sites on the carrier, compared to whole protein carrier, should favour the activation of B cells recognising the hapten instead of the carrier protein. An internal processing of the carrier and cleavage of the B[a]P-BA and subsequent presentation of the carrier peptide by MHC II molecules to T cell receptor should induce a B cell dependent immune response by activating B cells capable to recognise B[a]P. We demonstrated that a vaccination against B[a]P using promiscuous T-helper cell epitopes as a carrier is feasible and some tested peptide conjugates were more immunogenic as whole protein conjugates with increased specificity. We showed that vaccination against B[a]P reduces immunotoxicity. B[a]P suppressed the proliferative response of both T and B cells after a sub-acute administration, an effect that was completely reversed by vaccination. In immunized mice the immunotoxic effect of B[a]P on IFN-γ, Il-12, TNF-� production and B cell activation was restored. In addition, specific antibodies inhibited the induction of Cyp1a1 by B[a]P in lymphocytes and Cyp1b1 in the liver, enzymes that are known to convert the procarcinogen B[a]P to the ultimate DNA-adduct forming metabolite, a major risk factor of chemical carcinogenesis. In order to replace Freund adjuvant and to improve the immunisation strategy in terms of antibody quantity and quality, several adjuvants that are potentially compatible with their use in humans were tested. In combination with Freund adjuvant, the conjugate-vaccine induced high levels of B[a]P-specific antibodies. We showed that all adjuvants tested induced specific antibodies against B[a]P and 7,8-diol-B[a]P, its carcinogenic metabolite. The highest antibody levels were obtained with Quil A, MF-59 and Alum. Biological activity in terms of enhanced retention of B[a]P was confirmed in mice immunised with Quil A, Montanide, Alum and MF-59. Our findings demonstrate that a vaccination against B[a]P is feasible in combination with adjuvants licensed in humans. Based on these results and with the current understanding of the mechanisms of chemical carcinogenesis of the ubiquitous carcinogen B[a]P and of the effects of specific antibodies, an immunoprophylactic approach against chemical carcinogenesis is absolutely warranted. Nevertheless, the direct effects of B[a]P-specific antibodies on the different stages of carcinogenesis (e.g. adduct formation) and whether these effects may translate into long-term protective effect against tumourigenesis needs to be proven in further experiments.
| Original language | English |
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| Award date | 1 Jun 2011 |
| Publication status | Published - 1 Jun 2011 |