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Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

Choi JH, Schafer SC, Freiberg AN, Croyle MA - Mol. Pharm. (2015)

Bottom Line: An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50).Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations.These effects were not compromised by PEI.

View Article: PubMed Central - PubMed

Affiliation: †Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACT
The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

No MeSH data available.


Related in: MedlinePlus

Formulated preparations maintain antigenspecific polyfunctionalT cell responses in naive mice and those with prior exposure to adenovirus.Characterization of the immune response to Ebola glycoprotein wasperformed in B10.Br mice as described previously.12−14,24 (A) Magnitude of the systemic CD8+ T cellresponse against Ebola glycoprotein. The number of IFN-γ secretingmononuclear cells was quantitated in isolates taken 10 days afterimmunization from the spleen of naive B10.Br mice and those with prior-exposureto adenovirus by ELISpot. (B) Magnitude of the mucosal CD8+ T cell response against Ebola glycoprotein. The number of IFN-γsecreting mononuclear cells was quantitated 10 days after immunizationin bronchoalveolar lavage (BAL) fluid of naive mice and those withprior-exposure to adenovirus by ELISpot. (C) Polyfunctionality ofthe Ebola glycoprotein-specific T cell response in naive mice. Tendays after immunization, splenocytes from 5 mice per treatment groupwere pooled and stimulated with an Ebola glycoprotein-specific peptide.Bar graphs illustrate the percentage of CD8+ tumor necrosisfactor α (TNF-α)-, interleukin 2 (IL-2)-, and interferonγ (IFN-γ)-producing cells detected after 5 h of antigenstimulation. Distribution of single-, double-, and triple-cytokine-producingCD8+ T cells is shown as various colors in pie chart diagrams.The relative frequency of cells that produce all three cytokines definesthe quality of the vaccine-induced CD8+ T cell response.The proportion of these cells (IFN-γ+IL-2+TNF-α+) generated in response to each treatmentis written in the red section of each pie chart while the proportionof cells producing a single cytokine are represented by the lightblue, purple, and yellow sections of each pie chart. (D) Polyfunctionalityof the Ebola glycoprotein-specific T cell response in mice with priorexposure to adenovirus. Pre-existing immunity to adenovirus 5 wasinduced by instilling 5 × 1010 virus particles ofAdNull, an E1/E3 deleted virus that does not contain a transgene cassette,in the nasal cavity of mice 28 days prior to immunization. Ten daysafter immunization, splenocytes were harvested and pooled as describedin panel C. An increase in the number of polyfunctional cells, asindicated by an increase in the size of the red section of each piegraph, was fostered by several of the test formulations with respectto that produced by unformulated vaccine. (E) Quantitative analysisof the effector memory T cell response. Splenocytes were harvested42 days after immunization, stained with CFSE, and stimulated withthe TELRTFSI peptide for 5 days. Cells positive for CD8+, CD44HI, and CD62LLOW markers were then evaluatedfor CFSE by four-color flow cytometry. Data represent the averagevalues obtained from three separate experiments each containing 5mice per treatment. Error bars reflect the standard error of the data.*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni/Dunn post hoc analysis.
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fig3: Formulated preparations maintain antigenspecific polyfunctionalT cell responses in naive mice and those with prior exposure to adenovirus.Characterization of the immune response to Ebola glycoprotein wasperformed in B10.Br mice as described previously.12−14,24 (A) Magnitude of the systemic CD8+ T cellresponse against Ebola glycoprotein. The number of IFN-γ secretingmononuclear cells was quantitated in isolates taken 10 days afterimmunization from the spleen of naive B10.Br mice and those with prior-exposureto adenovirus by ELISpot. (B) Magnitude of the mucosal CD8+ T cell response against Ebola glycoprotein. The number of IFN-γsecreting mononuclear cells was quantitated 10 days after immunizationin bronchoalveolar lavage (BAL) fluid of naive mice and those withprior-exposure to adenovirus by ELISpot. (C) Polyfunctionality ofthe Ebola glycoprotein-specific T cell response in naive mice. Tendays after immunization, splenocytes from 5 mice per treatment groupwere pooled and stimulated with an Ebola glycoprotein-specific peptide.Bar graphs illustrate the percentage of CD8+ tumor necrosisfactor α (TNF-α)-, interleukin 2 (IL-2)-, and interferonγ (IFN-γ)-producing cells detected after 5 h of antigenstimulation. Distribution of single-, double-, and triple-cytokine-producingCD8+ T cells is shown as various colors in pie chart diagrams.The relative frequency of cells that produce all three cytokines definesthe quality of the vaccine-induced CD8+ T cell response.The proportion of these cells (IFN-γ+IL-2+TNF-α+) generated in response to each treatmentis written in the red section of each pie chart while the proportionof cells producing a single cytokine are represented by the lightblue, purple, and yellow sections of each pie chart. (D) Polyfunctionalityof the Ebola glycoprotein-specific T cell response in mice with priorexposure to adenovirus. Pre-existing immunity to adenovirus 5 wasinduced by instilling 5 × 1010 virus particles ofAdNull, an E1/E3 deleted virus that does not contain a transgene cassette,in the nasal cavity of mice 28 days prior to immunization. Ten daysafter immunization, splenocytes were harvested and pooled as describedin panel C. An increase in the number of polyfunctional cells, asindicated by an increase in the size of the red section of each piegraph, was fostered by several of the test formulations with respectto that produced by unformulated vaccine. (E) Quantitative analysisof the effector memory T cell response. Splenocytes were harvested42 days after immunization, stained with CFSE, and stimulated withthe TELRTFSI peptide for 5 days. Cells positive for CD8+, CD44HI, and CD62LLOW markers were then evaluatedfor CFSE by four-color flow cytometry. Data represent the averagevalues obtained from three separate experiments each containing 5mice per treatment. Error bars reflect the standard error of the data.*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni/Dunn post hoc analysis.

Mentions: Since the transductionefficiency data in mice with pre-existing immunity to adenovirus lookedpromising for each formulation, the immune response elicited by eachformulation was evaluated in B10.Br mice with the Ad-CAGoptZGP vectoras described previously.13,14,24 The systemic antigen-specific T cell response generated by eachformulation candidate was evaluated by quantitation of IFN-γsecreting mononuclear cells (MNCs) in the spleen by ELISpot. Therewas no significant difference in the amount of antigen-specific cellspresent in samples obtained from naive animals immunized with PEGylated,PLGA encapsulated, or unformulated virus (p >0.05,Figure 3A). Samples from mice immunized withthe F3 formulation contained slightly more antigen-specific cellsthan those from mice given unformulated vaccine (486.7 ± 4.8spot-forming cells (SFCs)/million MNCs, F3, vs 414.7 ± 27.6 SFCs/millionMNCs, unformulated). In contrast to what was observed in naive animals,PEI significantly decreased the number of activated IFN-γ secretingMNCs in the spleens of animals given each preparation except in thosegiven the PEGylated vaccine (317.3 ± 58.2 spot-forming cells(SFCs)/million mononuclear cells (MNCs), naive, vs 234.7 ± 54.3SFCs/million MNCs, PEI, Figure 3A). The mostsignificant reduction in IFN-γ secreting MNCs was observed inanimals given the microsphere preparation (426.7 ± 33.8 SFCs/millionMNCs, naive, vs 57.3 ± 7.1 SFCs/million MNCs, PEI). Pre-existingimmunity also significantly reduced the number of IFN-γ secretingcells recovered from bronchoalveolar lavage (BAL) fluid in mice givenunformulated vaccine (1,513.3 ± 63.6 SFCs/million MNCs, naive,vs 526.7 ± 98.2 SFCs/million MNCs, PEI, p <0.01, Figure 3B). This response was not compromisedin animals given the PEGylated and PLGA encapsulated vaccines (PEG,2,666.7 ± 54.6 SFCs/million MNCs, naive, vs 580 ± 61.1 SFCs/millionMNCs, PEI; PLGA, 1,280 ± 90.2 SFCs/million MNCs, naive, vs 1,360± 231.8 SFCs/million MNCs, PEI).


Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

Choi JH, Schafer SC, Freiberg AN, Croyle MA - Mol. Pharm. (2015)

Formulated preparations maintain antigenspecific polyfunctionalT cell responses in naive mice and those with prior exposure to adenovirus.Characterization of the immune response to Ebola glycoprotein wasperformed in B10.Br mice as described previously.12−14,24 (A) Magnitude of the systemic CD8+ T cellresponse against Ebola glycoprotein. The number of IFN-γ secretingmononuclear cells was quantitated in isolates taken 10 days afterimmunization from the spleen of naive B10.Br mice and those with prior-exposureto adenovirus by ELISpot. (B) Magnitude of the mucosal CD8+ T cell response against Ebola glycoprotein. The number of IFN-γsecreting mononuclear cells was quantitated 10 days after immunizationin bronchoalveolar lavage (BAL) fluid of naive mice and those withprior-exposure to adenovirus by ELISpot. (C) Polyfunctionality ofthe Ebola glycoprotein-specific T cell response in naive mice. Tendays after immunization, splenocytes from 5 mice per treatment groupwere pooled and stimulated with an Ebola glycoprotein-specific peptide.Bar graphs illustrate the percentage of CD8+ tumor necrosisfactor α (TNF-α)-, interleukin 2 (IL-2)-, and interferonγ (IFN-γ)-producing cells detected after 5 h of antigenstimulation. Distribution of single-, double-, and triple-cytokine-producingCD8+ T cells is shown as various colors in pie chart diagrams.The relative frequency of cells that produce all three cytokines definesthe quality of the vaccine-induced CD8+ T cell response.The proportion of these cells (IFN-γ+IL-2+TNF-α+) generated in response to each treatmentis written in the red section of each pie chart while the proportionof cells producing a single cytokine are represented by the lightblue, purple, and yellow sections of each pie chart. (D) Polyfunctionalityof the Ebola glycoprotein-specific T cell response in mice with priorexposure to adenovirus. Pre-existing immunity to adenovirus 5 wasinduced by instilling 5 × 1010 virus particles ofAdNull, an E1/E3 deleted virus that does not contain a transgene cassette,in the nasal cavity of mice 28 days prior to immunization. Ten daysafter immunization, splenocytes were harvested and pooled as describedin panel C. An increase in the number of polyfunctional cells, asindicated by an increase in the size of the red section of each piegraph, was fostered by several of the test formulations with respectto that produced by unformulated vaccine. (E) Quantitative analysisof the effector memory T cell response. Splenocytes were harvested42 days after immunization, stained with CFSE, and stimulated withthe TELRTFSI peptide for 5 days. Cells positive for CD8+, CD44HI, and CD62LLOW markers were then evaluatedfor CFSE by four-color flow cytometry. Data represent the averagevalues obtained from three separate experiments each containing 5mice per treatment. Error bars reflect the standard error of the data.*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni/Dunn post hoc analysis.
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fig3: Formulated preparations maintain antigenspecific polyfunctionalT cell responses in naive mice and those with prior exposure to adenovirus.Characterization of the immune response to Ebola glycoprotein wasperformed in B10.Br mice as described previously.12−14,24 (A) Magnitude of the systemic CD8+ T cellresponse against Ebola glycoprotein. The number of IFN-γ secretingmononuclear cells was quantitated in isolates taken 10 days afterimmunization from the spleen of naive B10.Br mice and those with prior-exposureto adenovirus by ELISpot. (B) Magnitude of the mucosal CD8+ T cell response against Ebola glycoprotein. The number of IFN-γsecreting mononuclear cells was quantitated 10 days after immunizationin bronchoalveolar lavage (BAL) fluid of naive mice and those withprior-exposure to adenovirus by ELISpot. (C) Polyfunctionality ofthe Ebola glycoprotein-specific T cell response in naive mice. Tendays after immunization, splenocytes from 5 mice per treatment groupwere pooled and stimulated with an Ebola glycoprotein-specific peptide.Bar graphs illustrate the percentage of CD8+ tumor necrosisfactor α (TNF-α)-, interleukin 2 (IL-2)-, and interferonγ (IFN-γ)-producing cells detected after 5 h of antigenstimulation. Distribution of single-, double-, and triple-cytokine-producingCD8+ T cells is shown as various colors in pie chart diagrams.The relative frequency of cells that produce all three cytokines definesthe quality of the vaccine-induced CD8+ T cell response.The proportion of these cells (IFN-γ+IL-2+TNF-α+) generated in response to each treatmentis written in the red section of each pie chart while the proportionof cells producing a single cytokine are represented by the lightblue, purple, and yellow sections of each pie chart. (D) Polyfunctionalityof the Ebola glycoprotein-specific T cell response in mice with priorexposure to adenovirus. Pre-existing immunity to adenovirus 5 wasinduced by instilling 5 × 1010 virus particles ofAdNull, an E1/E3 deleted virus that does not contain a transgene cassette,in the nasal cavity of mice 28 days prior to immunization. Ten daysafter immunization, splenocytes were harvested and pooled as describedin panel C. An increase in the number of polyfunctional cells, asindicated by an increase in the size of the red section of each piegraph, was fostered by several of the test formulations with respectto that produced by unformulated vaccine. (E) Quantitative analysisof the effector memory T cell response. Splenocytes were harvested42 days after immunization, stained with CFSE, and stimulated withthe TELRTFSI peptide for 5 days. Cells positive for CD8+, CD44HI, and CD62LLOW markers were then evaluatedfor CFSE by four-color flow cytometry. Data represent the averagevalues obtained from three separate experiments each containing 5mice per treatment. Error bars reflect the standard error of the data.*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, Bonferroni/Dunn post hoc analysis.
Mentions: Since the transductionefficiency data in mice with pre-existing immunity to adenovirus lookedpromising for each formulation, the immune response elicited by eachformulation was evaluated in B10.Br mice with the Ad-CAGoptZGP vectoras described previously.13,14,24 The systemic antigen-specific T cell response generated by eachformulation candidate was evaluated by quantitation of IFN-γsecreting mononuclear cells (MNCs) in the spleen by ELISpot. Therewas no significant difference in the amount of antigen-specific cellspresent in samples obtained from naive animals immunized with PEGylated,PLGA encapsulated, or unformulated virus (p >0.05,Figure 3A). Samples from mice immunized withthe F3 formulation contained slightly more antigen-specific cellsthan those from mice given unformulated vaccine (486.7 ± 4.8spot-forming cells (SFCs)/million MNCs, F3, vs 414.7 ± 27.6 SFCs/millionMNCs, unformulated). In contrast to what was observed in naive animals,PEI significantly decreased the number of activated IFN-γ secretingMNCs in the spleens of animals given each preparation except in thosegiven the PEGylated vaccine (317.3 ± 58.2 spot-forming cells(SFCs)/million mononuclear cells (MNCs), naive, vs 234.7 ± 54.3SFCs/million MNCs, PEI, Figure 3A). The mostsignificant reduction in IFN-γ secreting MNCs was observed inanimals given the microsphere preparation (426.7 ± 33.8 SFCs/millionMNCs, naive, vs 57.3 ± 7.1 SFCs/million MNCs, PEI). Pre-existingimmunity also significantly reduced the number of IFN-γ secretingcells recovered from bronchoalveolar lavage (BAL) fluid in mice givenunformulated vaccine (1,513.3 ± 63.6 SFCs/million MNCs, naive,vs 526.7 ± 98.2 SFCs/million MNCs, PEI, p <0.01, Figure 3B). This response was not compromisedin animals given the PEGylated and PLGA encapsulated vaccines (PEG,2,666.7 ± 54.6 SFCs/million MNCs, naive, vs 580 ± 61.1 SFCs/millionMNCs, PEI; PLGA, 1,280 ± 90.2 SFCs/million MNCs, naive, vs 1,360± 231.8 SFCs/million MNCs, PEI).

Bottom Line: An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50).Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations.These effects were not compromised by PEI.

View Article: PubMed Central - PubMed

Affiliation: †Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, United States.

ABSTRACT
The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

No MeSH data available.


Related in: MedlinePlus