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Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

Becker PD, Nörder M, Weissmann S, Ljapoci R, Erfle V, Drexler I, Guzmán CA - Vaccines (Basel) (2014)

Bottom Line: T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses.Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced.The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags.

View Article: PubMed Central - PubMed

Affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. pablo.becker@kcl.ac.uk.

ABSTRACT
Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

No MeSH data available.


Related in: MedlinePlus

Expression under control of mPH5 results in degradation of gene products at early time points. Human A375 cells were infected with either MVA-OVA P7.5 or MVA-OVA mPH5 at MOI 10. Cells were harvested at the indicated time points (h p.i., hours post infection) and cell lysates were prepared. (A) Cytosine β-d-arabinofuranoside (AraC), inhibited intermediate and late gene expression; (B) Epoxomicin (Epox), inhibited intermediate and late gene expression and protein degradation by blocking the proteasome.
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vaccines-02-00581-f001: Expression under control of mPH5 results in degradation of gene products at early time points. Human A375 cells were infected with either MVA-OVA P7.5 or MVA-OVA mPH5 at MOI 10. Cells were harvested at the indicated time points (h p.i., hours post infection) and cell lysates were prepared. (A) Cytosine β-d-arabinofuranoside (AraC), inhibited intermediate and late gene expression; (B) Epoxomicin (Epox), inhibited intermediate and late gene expression and protein degradation by blocking the proteasome.

Mentions: We investigated Ag expression by two recombinant viruses in which the gene coding for the model Ag ovalbumin (OVA) was under the control of promoters with differential activity at the early and late phases of infection. More specifically, the P7.5 exhibits moderate early and strong late activity, whereas the mPH5 shows a stronger early activity than the P7.5 and similar activity during the late phase of infection [29]. As shown by Western blot analysis, cells infected in vitro with both MVA-OVA P7.5 and MVA-OVA mPH5 led to efficient expression of OVA. In contrast to our expectations MVA-OVA mPH5 seemed to express a lower amount of protein than MVA-OVA P7.5 at 2 h post infection (p.i.) under normal conditions (Figure 1A, -AraC). Infected cells were then incubated with proteasomal or viral inhibitors to discriminate if a lower expression or a higher protein turnover was the cause of this finding. Cytosine β-d-arabinofuranoside (AraC) inhibits DNA replication and consequently vaccinia virus intermediate and late gene expression, whereas expression of early viral genes remains unaffected [47]. The results obtained in the presence of AraC clearly show that Ag expression from the mPH5 is stronger than that from P7.5 when only early gene expression is licenced (Figure 1A). The proteasome inhibitor epoxomicin not only abrogates MVA intermediate and late gene expression, while early gene expression remains unaffected, but also prevents proteolytic degradation via the proteasomal pathway, thereby allowing proteins to accumulate [48]. As shown in Figure 1B, the addition of epoxomicin allowed protein accumulation at 2 h p.i. and increased over time, demonstrating that mPH5 indeed has a stronger early activity and OVA expressed under the control of mPH5 is rapidly degraded at an early time point. To rule out any possible artefact due to increased Ag secretion, we analyzed the OVA secreted to the medium. Under normal conditions OVA was not detected in the medium before 4 h p.i. and treatment with brefeldin A, an inhibitor of the secretory pathway, confirmed that OVA started to accumulate in the cells at a late time point (data not shown). Thus, we conclude that there is indeed a rapid turnover of OVA by MVA-OVA mPH5 infected cells at 2 h. These data collectively suggest that the OVA protein expressed under the control of the mPH5 promoter could serve as substrate for Ag processing by proteasomes, therefore increasing the formation of defective ribosomal products (DRiPs), which in turn leads to the apparent faster turnover of the Ag [49,50].


Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

Becker PD, Nörder M, Weissmann S, Ljapoci R, Erfle V, Drexler I, Guzmán CA - Vaccines (Basel) (2014)

Expression under control of mPH5 results in degradation of gene products at early time points. Human A375 cells were infected with either MVA-OVA P7.5 or MVA-OVA mPH5 at MOI 10. Cells were harvested at the indicated time points (h p.i., hours post infection) and cell lysates were prepared. (A) Cytosine β-d-arabinofuranoside (AraC), inhibited intermediate and late gene expression; (B) Epoxomicin (Epox), inhibited intermediate and late gene expression and protein degradation by blocking the proteasome.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494220&req=5

vaccines-02-00581-f001: Expression under control of mPH5 results in degradation of gene products at early time points. Human A375 cells were infected with either MVA-OVA P7.5 or MVA-OVA mPH5 at MOI 10. Cells were harvested at the indicated time points (h p.i., hours post infection) and cell lysates were prepared. (A) Cytosine β-d-arabinofuranoside (AraC), inhibited intermediate and late gene expression; (B) Epoxomicin (Epox), inhibited intermediate and late gene expression and protein degradation by blocking the proteasome.
Mentions: We investigated Ag expression by two recombinant viruses in which the gene coding for the model Ag ovalbumin (OVA) was under the control of promoters with differential activity at the early and late phases of infection. More specifically, the P7.5 exhibits moderate early and strong late activity, whereas the mPH5 shows a stronger early activity than the P7.5 and similar activity during the late phase of infection [29]. As shown by Western blot analysis, cells infected in vitro with both MVA-OVA P7.5 and MVA-OVA mPH5 led to efficient expression of OVA. In contrast to our expectations MVA-OVA mPH5 seemed to express a lower amount of protein than MVA-OVA P7.5 at 2 h post infection (p.i.) under normal conditions (Figure 1A, -AraC). Infected cells were then incubated with proteasomal or viral inhibitors to discriminate if a lower expression or a higher protein turnover was the cause of this finding. Cytosine β-d-arabinofuranoside (AraC) inhibits DNA replication and consequently vaccinia virus intermediate and late gene expression, whereas expression of early viral genes remains unaffected [47]. The results obtained in the presence of AraC clearly show that Ag expression from the mPH5 is stronger than that from P7.5 when only early gene expression is licenced (Figure 1A). The proteasome inhibitor epoxomicin not only abrogates MVA intermediate and late gene expression, while early gene expression remains unaffected, but also prevents proteolytic degradation via the proteasomal pathway, thereby allowing proteins to accumulate [48]. As shown in Figure 1B, the addition of epoxomicin allowed protein accumulation at 2 h p.i. and increased over time, demonstrating that mPH5 indeed has a stronger early activity and OVA expressed under the control of mPH5 is rapidly degraded at an early time point. To rule out any possible artefact due to increased Ag secretion, we analyzed the OVA secreted to the medium. Under normal conditions OVA was not detected in the medium before 4 h p.i. and treatment with brefeldin A, an inhibitor of the secretory pathway, confirmed that OVA started to accumulate in the cells at a late time point (data not shown). Thus, we conclude that there is indeed a rapid turnover of OVA by MVA-OVA mPH5 infected cells at 2 h. These data collectively suggest that the OVA protein expressed under the control of the mPH5 promoter could serve as substrate for Ag processing by proteasomes, therefore increasing the formation of defective ribosomal products (DRiPs), which in turn leads to the apparent faster turnover of the Ag [49,50].

Bottom Line: T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses.Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced.The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags.

View Article: PubMed Central - PubMed

Affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. pablo.becker@kcl.ac.uk.

ABSTRACT
Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

No MeSH data available.


Related in: MedlinePlus