In vivo targeting of protein antigens to dendritic cells using anti-DEC-205 single chain antibody improves HIV Gag specific CD4+ T cell responses protecting from airway challenge with recombinant vaccinia-gag virus

Abstract

Introduction: Targeting antigens to dendritic cells (DCs) in vivo via a DC-restricted endocytic receptor, DEC205, has been validated to enhance immunity in several vaccine platforms. Particularly atttractive is selected delivery of proteins to DCs in vivo because it enables proteins to be more immunogenic and provides a cheaper and effective way for repeated immunizations.

Methods: In this study, we tested the efficacy of a single chain antibody to DEC205 (scDEC) to deliver protein antigens selectively to DCs in vivo and to induce protective immunity.

Results: In comparison to soluble Ovalbumin (OVA) antigen, when recombinant scDEC:OVA protein was injected subcutaneously (s.c.) into mice, the OVA protein was selectively presented by DCs to both TCR transgenic CD8⁺ and CD4⁺ T cells approximately 500 and 100 times more efficient than soluble OVA, respectively, and could persist for seven days following s.c. injection of the scDEC205:OVA. Similarly selective targeting of HIV Gag P24 to DCs in vivo using scDEC-Gag protein plus polyICLC vaccine resulted in strong, long lasting, polyfuntional CD4⁺ T cells in mice which were protective against airway challenge by a recombinant vaccinia-gag virus.

Conclusion: Thus targeting protein antigens to DCs using scDEC can be used either alone or in combination with other strategies for effective immunization.

Keywords: Antigens; dendritic cells; immunization.

Figure 1

Antigen presentation to OT‐I T cells following scDEC targeting in vivo. scDEC‐OVA induces stronger in vivo proliferation of OT‐I T cells than OVA alone. C57BL/B6 mice were injected intravenously with 2 × 10⁶ CFSE‐labelled OT‐I T‐cells then graded doses of scDEC‐OVA (A) or OVA (B) subcutaneously 24 h later. Three days after scDEC injection, draining lymph node cells were harvested and the expansion of CD8⁺ Vα₂ß_5.1/5.2 evaluated by flow cytometry for CFSE dilution. scDEC‐OVA elicited better presentation of OVA derived peptide than the scCon‐OVA and soluble OVA. In contrast to C57BL/B6 (B6) presentation of peptide from scDEC‐OVA could not be seen in DEC205 K (DEC^−/−) and TAP knock out (TAP^−/−) mice. On the contrary OVA peptide from B6 and DEC^−/− could be presented (C). In (D) to (F) similar data is shown for the spleen. Here as shown in the KO controls presentation was less efficient in the spleen (F). The experiment was repeated several times with similar results.

Figure 2

Antigen presentation to OT‐II T cells following scDEC targeting in vivo.scDEC‐OVA induces stronger in vivo proliferation of OT‐I I T cells than OVA alone. C57BL/B6 mice were injected intravenously with 3 × 10⁶ CFSE‐labelled OT‐II T‐cells then graded doses of scDEC‐OVA (A) or OVA (B) subcutaneously 24 h later. Three days after scDEC injection, draining lymph node cells were harvested and the expansion of CD4⁺ Vα₂/20.1 evaluated by flow cytometry for CFSE dilution. scDEC‐OVA elicited better presentation of OVA derived peptide than the scCont‐OVA and soluble OVA in both the lymph node (B) and spleen (E). On the other hand presentaion was abrogated in DEC^−/− mice in both the lymph nodes and spleen (compare C and F). In contrast to Figure 2 OT‐II presentation of peptide from scDEC‐OVA could be seen in both TAP knock out (TAP^−/−) and B6 mice but not in DEC205 (DEC^−/−). On the contrary OVA peptide form B6 and DEC^−/− mice was well presented. The experiment was repeated several times with similar results.

Figure 3

Comparison of Antigen presentation to T cells following scDEC‐OVA and αDECmAb‐OVA targeting in situ. B6 mice were injected intravenously with 2 × 10⁶ or 3 × 10⁶ CFSE‐labeled OT‐I and OT‐II T‐cells respectively 24 h prior to the s.c. injection of graded doses of scDEC‐OVA and αDECmAb‐OVA as depicted. Three days after protein injection draining lymph nodes cells were harvested and the expansion of CD8⁺ Vα₂ß_5.1/5.2 (A) or CD4⁺ Vα₂/20.1 (B) T cells evaluated by flow cytometry. B6 mice were injected s.c with 5 ug of scDEC‐OVA or αDECmAb‐OVA or the isotype control and then evaluated for peptide presentation 1, 7, or 15 days later. Treated mice were injected intravenously with 2 × 10⁶ or 3 × 10⁶ CFSE‐labeled OT‐I and OT‐II T‐cells respectively. Three days later draining lymph nodes cells were harvested and the expansion of CD8⁺ Vα₂ß_5.1/5.2 (C) or CD4⁺ Vα₂/20.1 (D) T cells evaluated by flow cytometry. For mice targeted with scDEC‐OVA presentation to OT‐I T cells persisted for up 7 days as compared to 15 days for αDECmAb‐OVA targeted mice (A). Similar data for the expansion of OT‐1 T cells in the spleen is shown in (E) for scDEC‐OVA and (F) for aDECmAb‐OVA.

Figure 4

scDEC‐Gag plus polyICLC vaccination results to robust polyfunctional CD4⁺ T cell responses. C57BL/6 mice were immunized twice s.c. at 4 weeks interval as indicated on the Y‐axis 14 after the last vaccination bulk splenocytes were assessed for HIV Gag specific T cell immunity. Splenocytes were restimulated either with unreactive peptides or with an HIV Gag p24 peptide mix then IFN‐γ, IL‐2, and TNF‐α production in response to peptide was evaluated by intracellular cytokine staining 6 h later in T cell (B). In Figure 4A data is shown for the gating schema of the cytokine forming cells while in (B) is dot plot is shown for IFN‐γ production by CD4⁺ T cells in both wild type B6 and DEC205KO mice. Polyfunctional Gag specific CD4⁺ T cell responses 14 days after the last boost in DKO mice (C) or wildtype C57BL/6 mice (D). All data are mean ± SD of three repeat experiments involving five female C57BL/6 mice per group.

Figure 5

Comparing the immunogenicity of parental αDECmAb‐Gag plus polyICLC with scDEC‐Gag plus polyICLC. Groups of five female 6–10 weeks C57BL/6 mice vaccinated twice 4 weeks apart either with graded doses of scDEC‐Gag or αDECmAb‐Gag in combination with 50 μg polyIC as indicated on the X‐axis. T cells responses were monitored 1 week after the boost. (A) mean percentage IFNγ producing CD4⁺ T cells (*P = 0.32; t‐test). (B) as in (A) but shows IL‐2 production (*P = 0.09; t‐test). (C) as in (A) but showing TNF‐α producing CD4 T cells 7 days post boost (*P = 0.14; t‐test). Data represents mean ± SD of 3 three repeat experiments with five mice per group per experiment.

Figure 6

Dendritic cell targeted scDEC‐Gag protein vaccine results to protection in the airway. Groups of five female 6–10 weeks C57BL/6 mice vaccinated twice with 5 μg scDEC‐Gag with 50 μg polyIC as adjuvant 4 weeks apart. Twelve weeks after the boost, the mice received a lethal dose (10⁵ PFU) of recombinant vaccinia‐gag intranasally. Weight loss was monitored daily for 6 days after challenge for (A) DEC205KO or (B) wild type B6 mice, and vaccinia virus titres in the lung (PFU/lung) was measured after euthanizing at 6 days for (C) DEC205KO or (D)WTB6 mice. (mean ± SD of three experiments).