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Establishing the pig as a large animal model for vaccine development against human cancer.

Overgaard NH, Frøsig TM, Welner S, Rasmussen M, Ilsøe M, Sørensen MR, Andersen MH, Buus S, Jungersen G - Front Genet (2015)

Bottom Line: Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01.This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes.By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark Copenhagen, Denmark.

ABSTRACT
Immunotherapy has increased overall survival of metastatic cancer patients, and cancer antigens are promising vaccine targets. To fulfill the promise, appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice, and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart, we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC, both important in human cancer development and progression, were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09, CASAC, Montanide ISA 51 VG, or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer.

No MeSH data available.


Related in: MedlinePlus

IFN-γ responses to the stable binders in the 9-11mer peptide library. PBMCs purified from immunized pigs at day −2, 12, 33, 40, and 54 were stimulated with 80 peptides found to be stably binding to one or more of SLA-1*04:01, SLA-1*07:02, SLA-2*04:01, SLA-2*05:02, and SLA-3*04:01. A biological relevant vaccine-induced response was defined as a 2-fold increase as compared to day −2 (dashed line, x-axis) and a concentration of IFN-γ equaling at least 25 pg/ml (dashed line, y-axis). For each animal, peptides were divided into two groups: stable (filled circles) and non-stable (open circles) referring to the binding stability of a given peptide correlated with the SLA-profile of each pig. Responses occurring repeatedly in individual animals are highlighted by peptide name with stably binding peptides being underlined. Animals with no responses at any time point, and time points for included animals with data not fulfilling the quality parameters were left out of the analyses.
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Figure 3: IFN-γ responses to the stable binders in the 9-11mer peptide library. PBMCs purified from immunized pigs at day −2, 12, 33, 40, and 54 were stimulated with 80 peptides found to be stably binding to one or more of SLA-1*04:01, SLA-1*07:02, SLA-2*04:01, SLA-2*05:02, and SLA-3*04:01. A biological relevant vaccine-induced response was defined as a 2-fold increase as compared to day −2 (dashed line, x-axis) and a concentration of IFN-γ equaling at least 25 pg/ml (dashed line, y-axis). For each animal, peptides were divided into two groups: stable (filled circles) and non-stable (open circles) referring to the binding stability of a given peptide correlated with the SLA-profile of each pig. Responses occurring repeatedly in individual animals are highlighted by peptide name with stably binding peptides being underlined. Animals with no responses at any time point, and time points for included animals with data not fulfilling the quality parameters were left out of the analyses.

Mentions: To monitor the induction of specific T cell populations, co-cultures with peptide and PBMCs obtained at different time points prior to and after the immunizations were analyzed for IFN-γ release after ~70 h. From the vaccine-induced responses, a biologically relevant IFN-γ response following peptide co-culture was defined as a 2-fold increase (stimulation index = 2) as compared to pre-immunization (day -2) and with a concentration of 25 pg/ml or more as depicted by the threshold lines (Figure 2). Three pigs were excluded from the analysis due to high non-specific background. For each animal, the peptide responses were divided into two groups based on the measured ability to form stable complexes with the SLA molecules found in the SPA analysis (Table 1, Figure 2). In general, we found responses in all pigs to both stable and non-stable binders (Figure 3, Table 2, Supplementary Table 2). Despite similar SLA profiles the pigs did not respond to the same peptides (Table 2). When comparing the total number of responses of all animals per adjuvant group, CASAC was shown to be slightly superior especially at day 12 after the first immunization; however in general the adjuvant systems performed similarly (Figure 4A). Comparison of the average IFN-γ production (total amount of IFN-γ divided by total number of responses) also revealed that the adjuvants induced a similar level of this cytokine (Figure 4B). Since the stability obtained in the SPA analysis has been used as a determinant for selecting the ligands most likely to be CD8+ T cell epitopes, comparisons of the peptide-MHC class I half-lives and IFN-γ production (Figure 4C) as well as stimulation index (Figure 4D) were carried out. Surprisingly, no significant correlations could be drawn from this analysis. More data points are needed, especially for complexes with long half-lives, to fully determine this, but our findings apparently do not support the idea of stably binding peptides being more immunogenic as shown by Harndahl et al. (2012). It should be noted, though, that this study was performed with viral T cell epitopes. As shown in Supplementary Table 2 we find responses against the vast majority of stably binding peptides, however, for a significant part of these there was a mismatch between the predicted SLA restriction and the observed responses.


Establishing the pig as a large animal model for vaccine development against human cancer.

Overgaard NH, Frøsig TM, Welner S, Rasmussen M, Ilsøe M, Sørensen MR, Andersen MH, Buus S, Jungersen G - Front Genet (2015)

IFN-γ responses to the stable binders in the 9-11mer peptide library. PBMCs purified from immunized pigs at day −2, 12, 33, 40, and 54 were stimulated with 80 peptides found to be stably binding to one or more of SLA-1*04:01, SLA-1*07:02, SLA-2*04:01, SLA-2*05:02, and SLA-3*04:01. A biological relevant vaccine-induced response was defined as a 2-fold increase as compared to day −2 (dashed line, x-axis) and a concentration of IFN-γ equaling at least 25 pg/ml (dashed line, y-axis). For each animal, peptides were divided into two groups: stable (filled circles) and non-stable (open circles) referring to the binding stability of a given peptide correlated with the SLA-profile of each pig. Responses occurring repeatedly in individual animals are highlighted by peptide name with stably binding peptides being underlined. Animals with no responses at any time point, and time points for included animals with data not fulfilling the quality parameters were left out of the analyses.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4584933&req=5

Figure 3: IFN-γ responses to the stable binders in the 9-11mer peptide library. PBMCs purified from immunized pigs at day −2, 12, 33, 40, and 54 were stimulated with 80 peptides found to be stably binding to one or more of SLA-1*04:01, SLA-1*07:02, SLA-2*04:01, SLA-2*05:02, and SLA-3*04:01. A biological relevant vaccine-induced response was defined as a 2-fold increase as compared to day −2 (dashed line, x-axis) and a concentration of IFN-γ equaling at least 25 pg/ml (dashed line, y-axis). For each animal, peptides were divided into two groups: stable (filled circles) and non-stable (open circles) referring to the binding stability of a given peptide correlated with the SLA-profile of each pig. Responses occurring repeatedly in individual animals are highlighted by peptide name with stably binding peptides being underlined. Animals with no responses at any time point, and time points for included animals with data not fulfilling the quality parameters were left out of the analyses.
Mentions: To monitor the induction of specific T cell populations, co-cultures with peptide and PBMCs obtained at different time points prior to and after the immunizations were analyzed for IFN-γ release after ~70 h. From the vaccine-induced responses, a biologically relevant IFN-γ response following peptide co-culture was defined as a 2-fold increase (stimulation index = 2) as compared to pre-immunization (day -2) and with a concentration of 25 pg/ml or more as depicted by the threshold lines (Figure 2). Three pigs were excluded from the analysis due to high non-specific background. For each animal, the peptide responses were divided into two groups based on the measured ability to form stable complexes with the SLA molecules found in the SPA analysis (Table 1, Figure 2). In general, we found responses in all pigs to both stable and non-stable binders (Figure 3, Table 2, Supplementary Table 2). Despite similar SLA profiles the pigs did not respond to the same peptides (Table 2). When comparing the total number of responses of all animals per adjuvant group, CASAC was shown to be slightly superior especially at day 12 after the first immunization; however in general the adjuvant systems performed similarly (Figure 4A). Comparison of the average IFN-γ production (total amount of IFN-γ divided by total number of responses) also revealed that the adjuvants induced a similar level of this cytokine (Figure 4B). Since the stability obtained in the SPA analysis has been used as a determinant for selecting the ligands most likely to be CD8+ T cell epitopes, comparisons of the peptide-MHC class I half-lives and IFN-γ production (Figure 4C) as well as stimulation index (Figure 4D) were carried out. Surprisingly, no significant correlations could be drawn from this analysis. More data points are needed, especially for complexes with long half-lives, to fully determine this, but our findings apparently do not support the idea of stably binding peptides being more immunogenic as shown by Harndahl et al. (2012). It should be noted, though, that this study was performed with viral T cell epitopes. As shown in Supplementary Table 2 we find responses against the vast majority of stably binding peptides, however, for a significant part of these there was a mismatch between the predicted SLA restriction and the observed responses.

Bottom Line: Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01.This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes.By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Vaccinology, National Veterinary Institute, Technical University of Denmark Copenhagen, Denmark.

ABSTRACT
Immunotherapy has increased overall survival of metastatic cancer patients, and cancer antigens are promising vaccine targets. To fulfill the promise, appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice, and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart, we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC, both important in human cancer development and progression, were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09, CASAC, Montanide ISA 51 VG, or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs), the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01, -1(*)07:02, -2(*)04:01, -2(*)05:02, and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-γ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses, and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer.

No MeSH data available.


Related in: MedlinePlus