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Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response

View Article: PubMed Central - PubMed

ABSTRACT

Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses.

No MeSH data available.


Related in: MedlinePlus

Splenocyte culture 3H-Thymidine incorporation following ex vivo OVA challenge. Splenocytes from naïve* (A) and monomer (B) or aggregate (B) immunized mice (as described in Figure 6 legend; n = 3 per group) were co-cultured with BMDC and challenged with 100 µg/ml monomeric OVA (Mono), stir aggregated OVA (Agg) or media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 and 144 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Proliferation measured as DPM is shown ± SEM (2 bars represent 72 and 144 h time points). Statistical significance of differences in proliferation between all groups were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, ****p P < .0001). * Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
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kfw121-F7: Splenocyte culture 3H-Thymidine incorporation following ex vivo OVA challenge. Splenocytes from naïve* (A) and monomer (B) or aggregate (B) immunized mice (as described in Figure 6 legend; n = 3 per group) were co-cultured with BMDC and challenged with 100 µg/ml monomeric OVA (Mono), stir aggregated OVA (Agg) or media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 and 144 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Proliferation measured as DPM is shown ± SEM (2 bars represent 72 and 144 h time points). Statistical significance of differences in proliferation between all groups were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, ****p P < .0001). * Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

Mentions: Cellular assays with splenocyte cultures were conducted to measure antigen driven lymphocytes proliferative responses and cytokine expression. Naïve splenocyte cultures did not proliferate in response to OVA (Fig. 7A). Splenocytes from monomer and aggregate immunized mice each responded similarly in the proliferation assay (Figs. 7B and C); splenocyte-DC co-cultures proliferated significantly more in response to monomer and aggregate compared with medium controls (****P < .0001). Additionally, cultures proliferated more vigorously in response to aggregated protein than to monomer (**P < .01).FIG. 7


Editor ’ s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response
Splenocyte culture 3H-Thymidine incorporation following ex vivo OVA challenge. Splenocytes from naïve* (A) and monomer (B) or aggregate (B) immunized mice (as described in Figure 6 legend; n = 3 per group) were co-cultured with BMDC and challenged with 100 µg/ml monomeric OVA (Mono), stir aggregated OVA (Agg) or media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 and 144 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Proliferation measured as DPM is shown ± SEM (2 bars represent 72 and 144 h time points). Statistical significance of differences in proliferation between all groups were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, ****p P < .0001). * Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036615&req=5

kfw121-F7: Splenocyte culture 3H-Thymidine incorporation following ex vivo OVA challenge. Splenocytes from naïve* (A) and monomer (B) or aggregate (B) immunized mice (as described in Figure 6 legend; n = 3 per group) were co-cultured with BMDC and challenged with 100 µg/ml monomeric OVA (Mono), stir aggregated OVA (Agg) or media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 and 144 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Proliferation measured as DPM is shown ± SEM (2 bars represent 72 and 144 h time points). Statistical significance of differences in proliferation between all groups were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001, ****p P < .0001). * Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
Mentions: Cellular assays with splenocyte cultures were conducted to measure antigen driven lymphocytes proliferative responses and cytokine expression. Naïve splenocyte cultures did not proliferate in response to OVA (Fig. 7A). Splenocytes from monomer and aggregate immunized mice each responded similarly in the proliferation assay (Figs. 7B and C); splenocyte-DC co-cultures proliferated significantly more in response to monomer and aggregate compared with medium controls (****P < .0001). Additionally, cultures proliferated more vigorously in response to aggregated protein than to monomer (**P < .01).FIG. 7

View Article: PubMed Central - PubMed

ABSTRACT

Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 &micro;m) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1&thinsp;mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses.

No MeSH data available.


Related in: MedlinePlus