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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 scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend), or naïve* mice (n = 3 per group) were cultured alone and in co-culture with BMDC and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv or with media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Group mean proliferation measured as DPM is shown ±SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
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kfw121-F4: Splenocyte culture 3H-thymidine incorporation following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend), or naïve* mice (n = 3 per group) were cultured alone and in co-culture with BMDC and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv or with media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Group mean proliferation measured as DPM is shown ±SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.

Mentions: As antibody production profiles suggested that aggregated protein was inducing Th1 type skewing (associated with selective IgG2a antibody responses) we sought to examine this at the cellular level. To this end, antigen-driven proliferation and cytokine production by cultured lymphocytes prepared from the spleens of immunized mice were measured. Splenocytes from immunized and naïve mice were cultured either alone or in the presence of BMDC, the latter included to enhance assay sensitivity. Cultures were primed with monomeric or aggregated scFv and proliferation measured using 3H-thymidine incorporation at a 72 h time point (Fig. 4).FIG. 4


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

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getmorefigures.php?uid=PMC5036615&req=5

kfw121-F4: Splenocyte culture 3H-thymidine incorporation following ex vivo scFv challenge. Splenocytes from monomer (Mono), heat (Heat Agg) or stir aggregated (Stir Agg) scFv immunized (as described in Figure 3 legend), or naïve* mice (n = 3 per group) were cultured alone and in co-culture with BMDC and challenged with 100 µg/ml monomeric scFv, heat or stir aggregated scFv or with media alone. Cells were pulsed with 3H-thymidine 24 h before harvesting at 72 h and β scintillation counting. Each culture condition was performed in triplicate for splenocytes derived from each individual animal and a mean calculated. Group mean proliferation measured as DPM is shown ±SEM. Statistical significance of differences were calculated using a 2-way ANOVA (*P < .05, ** P < .01, *** P < .001). *Experiments with naïve mice were conducted independently but utilized the same batches of mice and protein.
Mentions: As antibody production profiles suggested that aggregated protein was inducing Th1 type skewing (associated with selective IgG2a antibody responses) we sought to examine this at the cellular level. To this end, antigen-driven proliferation and cytokine production by cultured lymphocytes prepared from the spleens of immunized mice were measured. Splenocytes from immunized and naïve mice were cultured either alone or in the presence of BMDC, the latter included to enhance assay sensitivity. Cultures were primed with monomeric or aggregated scFv and proliferation measured using 3H-thymidine incorporation at a 72 h time point (Fig. 4).FIG. 4

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