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. 2010 Mar 8;5(3):e9579.
doi: 10.1371/journal.pone.0009579.

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target

Daniel M Appledorn  1 , Yasser A Aldhamen, William Depas, Sergey S Seregin, Chyong-Jy J Liu, Nathan Schuldt, Darin Quach, Dionisia Quiroga, Sarah Godbehere, Igor Zlatkin, Sungjin Kim, J Justin McCormick, Andrea Amalfitano
Affiliations

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target

Daniel M Appledorn et al. PLoS One. .

Abstract

Background: Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.

Methodology/principal findings: In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+) and CD8(-) T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.

Conclusion/significance: The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Heightened Ad-GFP/rEA induced cytokine responses.
(A) C57BL/6 mice were mock injected (n = 4), or IV injected with 7.5×1010 vps of Ad-GFP, 1 ng purified rEA protein, a mix of Ad-GFP and rEA protein, or Ad-GFP/rEA. Plasma was harvested and cytokines and chemokines were measured at 6 hpi (n = 5-10). Bars represent mean ± SE. #, ## represent significantly different from mock injected animals (p<0.05, p<0.01 respectively). *,**, *** represent statistical differences (p<0.05, p<0.01, p<0.001, respectively). ns, not significant.
Figure 2
Figure 2. Ad-GFP and Ad-GFP/rEA induced NK and NKT cell activation in liver and spleen in vivo.
C57BL/6 mice (N = 3) were either mock injected or injected with 7.5×1010 vps of either Ad-GFP or Ad-GFP/rEA. Lymphocytes from liver and spleen tissue were harvested at 6 hpi, stained for expression of surface markers for (A) NK cells or (B) NKT cells and FACS sorted. Bars represent mean ± SE. #, ##, ### represent significantly different from mock injected mice (p<0.05, p<0.01 respectively). *,**,*** represent statistical differences (p<0.05, p<0.01, p<0.001, respectively). Representative contour plots are illustrated at right. ns, not significant; MFI, mean fluorescence intensity.
Figure 3
Figure 3. Ad-GFP and Ad-GFP/rEA induced DC activation in spleen in vivo.
C57BL/6 mice (N = 3) were either mock injected or injected with 7.5×1010 vps of either Ad-GFP or Ad-GFP/rEA. Lymphocytes from spleen tissue were harvested at 6 hpi, stained for expression of surface markers Cd11c, Cd11b, CD80, CD86 and MHC-II. CD-19, NK1.1, and CD3 expressing cells were eliminated using PerCP-Cy5.5 labeled antibodies. Cd11cHigh, Cd11b- cells are represented. Bars represent mean ± SE. #, ##, ### represent significantly different from mock injected mice (p<0.05, p<0.01, p<0.001 respectively). *,**,*** represent statistical differences (p<0.05, p<0.01, p<0.001, respectively). Representative histograms are found at right. ns, not significant; MFI, mean fluorescence intensity.
Figure 4
Figure 4. Heightened transgene specific cellular responses as a result of Ad-GFP/rEA injection.
(A) C57BL/6 mice were mock injected (n = 3) or IV injected with 1×1010 vp of either Ad-GFP or Ad-GFP/rEA (n = 8). Splenocytes were harvested 7 dpi and used to complete IFNγ ELISpot analyses. Spot forming cells (SFCs) are reported. (B) C57BL/6 mice (n = 6) were injected IM as above. IFNγ ELISpot analyses were completed at 14 dpi. Bars represent mean ± SE. ## represent significantly different from mock injected mice (p<0.01). *,** represent statistical differences (p<0.01).
Figure 5
Figure 5. Increased HIV-Gag specific CMI responses in mice co-injected with Ad-GFP/rEA.
(A) Wild-type (N = 3) or MyD88/TRIF double knockout (M88-TRIF DKO; N = 3) mice were co-immunized IM with equivalent viral particles of Ad-Gag mixed with either Ad-GFP or Ad-GFP/rEA (total of 1×107 vps mixed prior to injection). At 14 dpi IFNγ ELISpot analyses were completed using QBI# 304796 to restimulate cells ex vivo. Data are presented as mean ± SE. *** denotes statistically significant difference (p<0.001). (B, C) Balb/c mice (n = 3) were co-immunized IM as above (total of 1×106 vps). At 8 dpi, Gag specific CD3+/CD8+ T-cells in PBMC preparations were analyzed by FACS. (C) At 14 dpi, splenocytes were stimulated ex vivo with the indicated peptide to complete IFNγ ELISpot analyses. Data represent the mean ± SE. *, *** represent p<0.05, p<0.001 respectively. This experiment was completed in triplicate at various doses yielding identical results (two doses are illustrated in this figure).
Figure 6
Figure 6. Ad-GFP/rEA improves antigen specific CD8+ and CD8- cellular responses.
At 14 dpi, splenocytes from vaccinated C57BL/6 (1×107 total vps) (A) or Balb/c (1×108 total vps) (B) mice were equivalently pooled (N = 3 mice per treatment) and CD8+ cells were depleted using magnetic beads and IFNγ ELISpot analyses were completed. %SFC that are CD8- = (#SFCs CD8 dep/#SFCs CD8+)*100. This experiment was repeated two independent times with the same result.
Figure 7
Figure 7. Increased quantity and quality of Gag-specific T-cells in Ad-GFP/rEA co-injected mice.
At 14 dpi, splenocytes from vaccinated C57BL/6 (1×107 total vps) (A) or Balb/c (1×108 total vps) (B) mice were equivalently pooled (N = 3 mice per treatment) and individual wells were stimulated ex vivo with a pool of 2-4 15-mer peptides spanning the complete Gag protein sequence, not including peptides included in Figure 5 (AMQ, or 304796). Inset graphs indicate the number of wells with >10 SFCs. This experiment was carried out two independent times with the same result. (C) Balb/c mice were vaccinated as above (total of 1×107 vp). At 14 dpi, an in vivo CTL assay was completed and Gag specific CTL activity was quantified (N = 2 for all treatments). This experiment was carried out two independent times, each with the same result.
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