Potent HIV-1-Specific CD8 T Cell Responses Induced in Mice after Priming with a Multiepitopic DNA-TMEP and Boosting with the HIV Vaccine MVA-B

Abstract

An effective vaccine against Human Immunodeficiency Virus (HIV) still remains the best solution to provide a sustainable control and/or eradication of the virus. We have previously generated the HIV-1 vaccine modified vaccinia virus Ankara (MVA)-B, which exhibited good immunogenicity profile in phase I prophylactic and therapeutic clinical trials, but was unable to prevent viral rebound after antiretroviral (ART) removal. To potentiate the immunogenicity of MVA-B, here we described the design and immune responses elicited in mice by a new T cell multi-epitopic B (TMEP-B) immunogen, vectored by DNA, when administered in homologous or heterologous prime/boost regimens in combination with MVA-B. The TMEP-B protein contained conserved regions from Gag, Pol, and Nef proteins including multiple CD4 and CD8 T cell epitopes functionally associated with HIV control. Heterologous DNA-TMEP/MVA-B regimen induced higher HIV-1-specific CD8 T cell responses with broader epitope recognition and higher polyfunctional profile than the homologous DNA-TMEP/DNA-TMEP or the heterologous DNA-GPN/MVA-B combinations. Moreover, higher HIV-1-specific CD4 and Tfh immune responses were also detected using this regimen. After MVA-B boost, the magnitude of the anti-VACV CD8 T cell response was significantly compromised in DNA-TMEP-primed animals. Our results revealed the immunological potential of DNA-TMEP prime/MVA-B boost regimen and supported the application of these combined vectors in HIV-1 prevention and/or therapy.

Keywords: HIV-1; MVA; immunogenicity; multiepitopic vaccine.

Figure 1

T cell multiepitopic B (TMEP-B) design, construction of pcDNA-TMEP-B plasmid, and TMEP-B expression analysis by Western blot. (A) Scheme of TMEP-B protein. (B) Map of the plasmid pcDNA-TMEP-B. (C) Expression of TMEP-B construct by Western blot. The 293T cells were mock-infected or infected with 5 pfu/cell of Western Reserve (WR) or vaccinia virus (VACV) that expresses the T7 RNA polymerase (VT7) viruses, and transfected 1 h later with 5 μg of pcDNA-TMEP-B or pMax-GFP. At 6 h post-infection, cells were harvested and lysed in Laemmli buffer with mercapoethanol and cell extracts were fractionated by 8% SDS-PAGE and analyzed by Western blot using mouse monoclonal anti-FLAG M2 antibody to evaluate TMEP-B expression.

Figure 2

Polyfunctional Gag–Pol–Nef (GPN)-specific CD4 and CD8 T cell immune responses elicited in spleen after prime/boost immunization of mice with TMEP-B construct and modified vaccinia virus Ankara (MVA)-B recombinant virus. (A) Immunization schedule. Groups of 6–8-week-old female mice (n = 4) received 50 μg of pcDNA-_IIIBGPN, pcDNA-TMEP-B, or pcDNA-φ by bilateral intramuscular route (i.m.); three weeks later, they received a bilateral i.m. inoculation of 50 μg of DNA (pcDNA-_IIIBGPN, pcDNA-TMEP-B, or pcDNA-φ) or 1 × 10⁷ pfu of MVA-wild type (WT) or MVA-B viruses. At 10 days after the last immunization, mice were sacrificed and spleens were processed for intracellular cytokine staining (ICS) assay to analyze Gag–Pol–Nef-specific T cell immune responses. Two independent experiments have been performed for the different groups. Magnitude of the Gag- and GPN-specific CD4 (B) or CD8 (C) T cell immune responses were measured at 10 days post-boost by ICS assay after stimulation of splenocytes from immunized animals with the different HIV-1 clade B peptide pools. The total value in each group indicates the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting CD107a and/or IFN-γ and/or IL-2 and/or TNF-α against Gag/GPN peptide pools. Data are background subtracted. *** p < 0.001. (D) Flow cytometry profiles of vaccine-induced CD8 T cell responses against Gag-1 or GPN-3 peptide pools in the groups immunized with the heterologous combinations DNA/MVA. (E) Functional profile of the Gag/GPN-specific CD4 (left panel) or CD8 (right panel) T cell responses in the different immunization groups. Positive combinations of the responses are represented on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD4 or CD8 T cells are shown on the y axis. Responses are grouped and colour-coded based on the number of functions. Non-specific responses obtained in the control groups were subtracted in all populations. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α.

Figure 3

Gag–Pol–Nef-specific Tfh T cell immune responses elicited in spleen and draining lymph nodes (DLNs) after prime/boost immunization of mice with TMEP-B construct and MVA-B virus. Magnitude of the total CD4 T cells with Tfh phenotype (CXCR5⁺PD1⁺) in spleen (A) and DLNs (B) measured 10 days after the last immunization by ICS assay in the non-stimulated samples (RPMI). *** p < 0.001. (C) Magnitude of the Gag/GPN-specific Tfh cells in spleen measured 10 days after the last immunization by ICS assay following stimulation of splenocytes derived from immunized animals with the different HIV-1 clade B Gag and GPN peptide pools. The total value in each group indicates the sum of the percentages of Tfh⁺ T cells secreting IL-4 and/or IFN-γ against Gag/GPN peptide pools. Data are background subtracted. *** p < 0.001. (D) Flow cytometry profiles of vaccine-induced Tfh cell responses in spleen against Gag and GPN peptide pools in the groups immunized with the heterologous combinations DNA/MVA.

Figure 4

Env-specific T cell immune responses (CD4, CD8, and Tfh) elicited in spleen and DLNs after prime/boost immunization of mice with TMEP-B construct and MVA-B virus. (A) Magnitude of the Env-specific CD4 or CD8 T cell immune responses measured at 10 days post-boost by ICS assay after stimulation of splenocytes from immunized animals with HIV-1 clade B Env peptide pool. The total value in each group indicates the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting CD107a and/or IFN-γ and/or IL-2 and/or TNF-α against Env peptide pool. Data are background subtracted. (B) Functional profile of the Env-specific CD4 (left panel) or CD8 (right panel) T cell responses in the different immunization groups. Positive combinations of the responses are represented on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD4 or CD8 T cells are shown on the y axis. Responses are grouped and colour-coded based on the number of functions. Non-specific responses obtained in the control groups were subtracted in all populations. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α. (C, left panel) Magnitude of the Env-specific Tfh cells in spleen and DLNs measured as in (A). The total value in each group indicates the sum of the percentages of Tfh⁺ T cells secreting IL-4 and/or IFN-γ against Env pool. Data are background subtracted. (C, right panel) Flow cytometry profiles of vaccine-induced Tfh cell responses in spleen against Env pool in the groups immunized with the heterologous combinations DNA/MVA.

Figure 4

Env-specific T cell immune responses (CD4, CD8, and Tfh) elicited in spleen and DLNs after prime/boost immunization of mice with TMEP-B construct and MVA-B virus. (A) Magnitude of the Env-specific CD4 or CD8 T cell immune responses measured at 10 days post-boost by ICS assay after stimulation of splenocytes from immunized animals with HIV-1 clade B Env peptide pool. The total value in each group indicates the sum of the percentages of CD4⁺ or CD8⁺ T cells secreting CD107a and/or IFN-γ and/or IL-2 and/or TNF-α against Env peptide pool. Data are background subtracted. (B) Functional profile of the Env-specific CD4 (left panel) or CD8 (right panel) T cell responses in the different immunization groups. Positive combinations of the responses are represented on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD4 or CD8 T cells are shown on the y axis. Responses are grouped and colour-coded based on the number of functions. Non-specific responses obtained in the control groups were subtracted in all populations. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α. (C, left panel) Magnitude of the Env-specific Tfh cells in spleen and DLNs measured as in (A). The total value in each group indicates the sum of the percentages of Tfh⁺ T cells secreting IL-4 and/or IFN-γ against Env pool. Data are background subtracted. (C, right panel) Flow cytometry profiles of vaccine-induced Tfh cell responses in spleen against Env pool in the groups immunized with the heterologous combinations DNA/MVA.

Figure 5

VACV vector E3-specific CD8 T cell immune responses elicited in spleen after prime/boost immunization of mice with TMEP-B construct and MVA-B virus. (A) Magnitude of the E3-specific CD8 T cell immune responses measured at 10 days post-boost by ICS assay after stimulation of splenocytes from immunized animals with VACV E3 peptide. The magnitude in each group indicates the sum of the percentages of CD8⁺ T cells secreting CD107a and/or IFN-γ and/or IL-2 and/or TNF-α against E3 peptide. Data are background subtracted. *** p < 0.001. (B) Functional profile of the E3-specific CD8 T cell responses in the different immunization groups. Positive combinations of the responses are represented on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cells are shown on the y axis. Responses are grouped and colour-coded based on the number of functions. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α. (C) Flow cytometry profiles of vaccine-induced CD8 T cell responses in spleen against E3 peptide in the groups immunized with the heterologous combinations DNA/MVA.

Figure 5

VACV vector E3-specific CD8 T cell immune responses elicited in spleen after prime/boost immunization of mice with TMEP-B construct and MVA-B virus. (A) Magnitude of the E3-specific CD8 T cell immune responses measured at 10 days post-boost by ICS assay after stimulation of splenocytes from immunized animals with VACV E3 peptide. The magnitude in each group indicates the sum of the percentages of CD8⁺ T cells secreting CD107a and/or IFN-γ and/or IL-2 and/or TNF-α against E3 peptide. Data are background subtracted. *** p < 0.001. (B) Functional profile of the E3-specific CD8 T cell responses in the different immunization groups. Positive combinations of the responses are represented on the x axis, whereas the percentages of the functionally distinct cell populations within the total CD8 T cells are shown on the y axis. Responses are grouped and colour-coded based on the number of functions. C: CD107a; I: IFN-γ; 2: IL-2; T: TNF-α. (C) Flow cytometry profiles of vaccine-induced CD8 T cell responses in spleen against E3 peptide in the groups immunized with the heterologous combinations DNA/MVA.

Figure 6

Anti-p24 humoral responses elicited in serum from immunized mice after prime/boost immunization with TMEP-B construct and MVA-B virus. The graph represents the values of optical density at 450 nm (OD₄₅₀) for each animal at a serum dilution of 1:50 (dots) and the mean value (solid line) and standard deviation of each group. ** p < 0.005; *** p < 0.001.