Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein

Abstract

Background: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load.

Methods: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4⁺ and T CD8⁺) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen.

Results: T CD4⁺ and T CD8⁺ lymphocytes cells stimulated by HBZ-peptides (HBZ_42-50 and HBZ_157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8⁺ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44^+High/CD62L^-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8⁺ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA.

Conclusions: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

Keywords: HBZ; Human T-lymphotropic virus 1; Plasmid DNA vaccine (pcDNA3.1(+)-HBZ); Recombinant Modified Vaccinia virus Ankara (MVA-HBZ); Vaccine.

Fig. 1

Schematic diagram of HTLV-1 proviral genome and the HBZ multiepitope cassette construction. A HTLV-1 proviral genome pointing out the HTLV-1 basic leucine zipper factor (HBZ) in the antisense strand. The sHBZ box demonstrate in the Activator Domain (AD) and bZIP region the position of peptides 1 to 3 (in silico analysis), peptide 4 (MacNamara et al. [23]), and peptide 5 (Sugata et al. [17]) that compound the multiepitope recombinant protein. B HBZ multiepitope cassette presenting the selected peptides (pep1 to pep5), spacer regions (GSGSG), signal peptide (MDAMKRGLCCVLLLCGAVFVDSVTG), and a flag tag (DYKDDDDK). C Construction of recombinant MVA-HBZ by homologous recombination expressing the HBZ multiepitope protein. A pLW44 plasmid that contains the gene of interest is used to transfect an MVA-infected cell. The pcDNA3.1(+)-HBZ express the same HBZ multiepitope protein

Fig. 2

Mice immunization protocol. Group 1 received: Prime—pcDNA3.1(+)-HBZ (100 µg); Boost—MVA-HBZ 10⁷ PFU and 2nd Boost—MVA-HBZ 10⁷ PFU. Group 2 received: Prime—Saline; Boost—MVA (wt) 10⁷ PFU and 2nd Boost—MVA (wt) 10⁷ PFU. Group 3 received: Prime—Addavax 1:1; Boost—Saline and 2nd Boost—Saline. N = 7. Immunizations were performed at 14 days-intervals

Fig. 3

Microscopy of MVA-HBZ infected foci in BHK-21 cells monolayer. Fluorescence microscopy shows green fluorescence, due to GFP-tag 24 h post-infection in BHK-21 cells. Fluorescence microscope EVOS® FL Cell Imaging System (Thermo Fisher), at 400× magnification

Fig. 4

Analysis of HBZ multiepitope protein expression by the pcDNA and MVA vector. A Representative graphs of the gating strategy used in the flow cytometry expression assays. Total percentage of HBZ expression in BHK-21 transfected / infected cells. Subset low (subset low), subset (subset medium) and subset high (subset high) according to cell size (FSC) and granularity (SSC). A1 for pcDNA3.1(+)-HBZ and A2 for MVA-HBZ. B–B1 In black: pcDNA3.1(+)-HBZ; In gray: Mock transfected cell control. B2 In black: MVA-HBZ; In gray: MVA-GFP control. Both vectors showed efficient recombinant protein expression in vitro mainly in subsets medium and high

Fig. 5

Recruitment of T CD8⁺ lymphocytes and cytokine production in mice immunized with the recombinant DNA-MVA. A Representative graphs of the gating strategy used in the flow cytometry assays. The A3 peptide and PMA / Ionomycin were used as positive controls. B–B1 Graph of the total percentage of CD8⁺ T lymphocytes, double-positives for INF-γ and TNF-α recruited after restimulation. Statistical differences were observed between the groups DNA-MVA vs MVA (wt). B2 Graph of the total percentage of T CD8⁺ lymphocytes producers of TNF-α and IL-2 recruited after restimulation with HBZ peptides. Legend (symbols): Circle: Cells from mice immunized with the recombinant DNA-MVA regimen; Square: Cells from mice inoculated only with the MVA (wt) vector; Triangle: Cells from mice inoculated only with the Addavax adjuvant. N = 7

Fig. 6

Recruitment of T CD8⁺ lymphocytes, TNF-α and CD107a production in mice immunized with the recombinant DNA-MVA. A Representative graphs of the gating strategy used in the flow cytometry assays. The A3 peptide and PMA/Ionomycin were used as positive controls. B Graph of the total percentage of T CD8⁺ lymphocytes producers of TNF-α, expressing CD107a, recruited after restimulation with HBZ peptides. Statistical differences were observed between the group’s DNA-MVA vs Adjuvant. Legend (symbols): Circle: Cells from mice immunized with the recombinant DNA-MVA regimen; Square: Cells from mice inoculated only with the MVA (wt) vector; Triangle: Cells from mice inoculated only with the Addavax adjuvant. N = 7

Fig. 7

Recruitment of T CD8^+EM, CD44^+High/CD62L^−Low, granzyme B⁺ in mice immunized with the recombinant DNA-MVA. A Representative graphs of the gating strategy used in the flow cytometry assays. The A3 peptide and PMA/Ionomycin were used as positive controls. B Graph of the total percentage of T CD8^+EM lymphocytes CD44^+High / CD62L^−Low producers of granzyme B recruited after restimulation with HBZ peptides. Legend (symbols): Circle: Cells from mice immunized with the recombinant DNA-MVA regimen; Square: Cells from mice inoculated only with the MVA (wt) vector; Triangle: Cells from mice inoculated only with the Addavax adjuvant. N = 7

Fig. 8

Recruitment of T CD4⁺ lymphocytes and cytokine production in mice immunized with the recombinant DNA-MVA. A Representative graphs of the gating strategy used in the flow cytometry assays. The A3 peptide and PMA/Ionomycin were used as positive controls. B–B1 Graph of the total percentage of CD4⁺ T lymphocytes, double-positives for INF-γ and TNF-α recruited after restimulation. B2 Graph of the total percentage of T CD4⁺ lymphocytes producers of TNF-α and IL-2 recruited after restimulation with HBZ peptides. Legend (symbols): Circle: Cells from mice immunized with the recombinant DNA-MVA regimen; Square: Cells from mice inoculated only with the MVA (wt) vector; Triangle: Cells from mice inoculated only with the Addavax adjuvant. N = 7

Fig. 9

Evaluation of humoral immune response in mice immunized with the recombinant DNA-MVA through ELISA assays. Legend (symbols): Circle: Sera from mice immunized with the recombinant DNA-MVA regimen; Square: Sera from mice inoculated only with the MVA (wild type) vector; Triangle: Sera from mice inoculated only with the Addavax adjuvant. N = 7. Data represented graphically as mean and standard error of the media (SEM)