PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Abstract

Viral vector-based vaccines that induce protective CD8+ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non-DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8+ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1-based vaccination potentiated CD8+ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12-producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8+ T cell immunity induced by soluble PD1-based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.

Figures

Figure 1. Expression and binding characteristics of DNA vaccine constructs.

(A) Schematic representation of constructs encompassing the soluble form of PD1 (sPD1) or with 2 amino acid deletions essential for binding with PD-L1/L2 (sΔPD1), p24, and rabbit Fc under the CMV promoter, denoted as sPD1-p24fc, sΔPD1-p24fc, and p24fc, respectively. All constructs contain a tissue plasminogen activator (tPA) signal sequence. Rabbit Fc was used as a tag for purification and detection purposes. (B) Expression of fusion constructs as purified recombinant proteins examined by Western blot. Upper and lower blots show proteins detected by anti-HIV GAG or anti-rabbit Fc antibody, respectively. Smaller-sized bands (lower panel arrows) represent p24fc, while the larger-sized bands (upper panel arrows) represent sPD1-p24fc or sΔPD1-p24fc. Lanes are identified by the legend and numbers in kDa indicate marker sizes. (C) 293T cells were transiently transfected with PD-L1 or PD-L2 expression vectors, and the binding profiles of recombinant proteins were examined by flow cytometry using anti-rabbit Fc-FITC detection antibody (gray line). Controls included transfected 293T cells stained with anti-rabbit Fc-FITC antibody (negative, shaded line) or anti-mouse PD-L1 or L2 antibodies (positive, solid line).

Figure 2. Induction of enhanced and long-lasting p24-specific immune responses by sPD1-p24fc vaccination in optimized conditions.

(A) DNA vaccine immunization schedule for BALB/c mice. Mice were immunized at weeks 0, 3, and 6. For assessing overall immune response, mice were sacrificed 2 weeks after the last immunization to collect spleen and blood for analysis of cellular and humoral immune responses. (B) BALB/c mice were immunized with sPD1-p24fc, sΔPD1-p24fc, and p24fc at a dose of 20 μg DNA delivered via i.m./EP. Mice that received PBS only served as negative controls. Two weeks after each vaccination, ELISPOT assays for CD8+ T cells and CD4+ T cells were performed to test the ability of T cells to produce IFN-γ in specific response to HIV-1 GAG p24 epitopes GAG A-I (CD8) and GAG 26 (CD4), respectively, as well as to (C) IgG1 and IgG2a antibodies specific to HIV-1 p24 in sera detected by ELISA. To determine long-lived immunity, mice were rested for 7.5 months following the immunization regimen before being sacrificed. ELISPOT assays were performed for (D) CD8+ T cells and (E) CD4+ T cells to test their ability to produce IFN-γ in specific response to HIV-1 GAG p24 epitopes GAG A-I and GAG 26, respectively. (F) Specific IgG1 and IgG2a antibodies against HIV-1 GAG p24 detected by ELISA. Data show the means with standard error from 3 independent immunization experiments with 3 mice per group. *P < 0.05; **P < 0.01.

Figure 3. Comparison of sPD1-p24fc elicited antigen-specific immunity in 2 mice models.

BALB/c and C57BL/6 mice were vaccinated with sPD1-p24fc, sΔPD1-p24fc, and p24fc at a dose of 100 μg DNA i.m/EP according to the immunization schedule. (A) IFN-γ–producing CD8+ and (B) CD4+ cells were measured by ELISPOT assay in BALB/c splenocytes stimulated using the specific peptides GAG A-I and GAG 26, respectively. (C) Specific antibodies against HIV-1 GAG p24 detected by ELISA. IgG1 (Th2) was tested in both strains of mice, while IgG2a was detected in BALB/c mice and IgG2c was detected in C57BL/6 mice. (D) IFN-γ–secreting cells from splenocytes isolated from BALB/c or C57BL/6 mice in response to stimulation using 3 different nonoverlapping peptide pools derived from 59 peptides spanning the entire HIV-1 GAG p24 region as detected by ELISPOT. Data show the means with standard error from 3 independent immunization experiments with 3 mice per group. *P < 0.05; ***P < 0.001.

Figure 4. Increased dose of DNA vaccine further enhanced p24-specific tetramer-positive and long-term memory CD8+ T cell responses.

sPD1-p24fc, sΔPD1-p24fc, and p24fc were administered to BALB/c mice at a dose of 100 μg DNA delivered by i.m./EP. (A) HIV-1 p24-specific H2-Kd-AMQMLKDTI-PE tetramer staining of CD8+ T cell populations 2 weeks after the final immunization is shown in flow cytometric plots of 1 representative experiment, or (B) in data represented as a column graph of immunization with 2 doses of 20 μg and 100 μg DNA. PBS represents the negative control. (C) Thirty weeks after the final i.m./EP vaccination at a dose of 100 μg DNA, IFN-γ–producing CD8+ T cells were measured by ELISPOT assay in splenocytes stimulated using specific GAG A-I epitope. (D) Splenocytes labeled with CFSE and stimulated with purified CD11c+ BM-DCs plus p24 peptide pools and anti-CD28 for 5 days. Addition of anti-CD3 antibody served as a positive control. CFSE signals on CD8+ T cell populations were detected by flow cytometry. Data show the SEM from 3 independent immunization experiments with 3 mice per group. *P < 0.05; **P < 0.01.

Figure 5. Polyfunctionality and cytotoxicity effects of sPD1-p24fc–induced T cells.

BALB/c mice were immunized by i.m./EP with 3 injections of 100 μg sPD1-p24fc and sΔPD1-p24fc. Splenocytes were collected and analyzed by flow cytometry following intracellular staining using antibodies against IFN-γ, TNF-α, and IL-2. (A) Gating strategy for flow cytometric scatter plots to analyze the frequency of CD8+ or CD4+ T cells positive for IFN-γ, TNF-α, and/or IL-2. (B) Column graphs depicting subpopulations of single-, double-, or triple-positive CD8+ or (C) CD4+ T cells releasing the cytokines IFN-γ, TNF-α, and IL-2 induced by DNA vaccination of sPD1-p24fc and sΔPD1-p24fc (and PBS control). (D) Pie chart analysis representing subpopulations of total cytokine-secreting CD8+ or CD4+ T cells positive for combinations of IFN-γ, TNF-α, and IL-2. Columns and pie regions represent the mean values of 2 independent experiments with 3 mice per group, with error bars representing the SEM. (E) Purified CD8+ or (F) CD4+ T cells from sPD1-p24fc– and p24fc-immunized mice were stimulated by p24 peptide pools plus anti-CD28 overnight before cytotoxicity assay with an AB1-HIV-1-GAG cell line as the target. Line graphs show the percentages of dead cells as a result of cell-mediated killing, with the baseline of natural target cell death percentage subtracted. *P < 0.05; **P < 0.01.

Figure 6. sPD1-based DNA vaccination–induced antigen-specific immunity confers protection against virulent viral challenge.

(A) Mice previously immunized with 20-μg DNA vaccines were challenged with 4 × 107 PFUs VTTgagpol three weeks after immunization to examine immune protection. Virus titers in the lungs of immunized mice were evaluated by examining plaque formation on Vero cell monolayers 3 days after challenge. Mice immunized with 100-μg DNA vaccines were challenged with 2 × 106 PFUs WRgagpol three weeks after immunization and weighed daily for 8 days. (B) Body weight percentage loss from day 0. (C) Mice were sacrificed on day 8 and virus titers in the lungs were evaluated by examining plaque formation on Vero cell monolayers. Data represent the SEM of 2 independent experiments with 5 mice per group. **P < 0.01; ***P < 0.001.

Figure 7. DNA immunization of mice with sPD1-based vaccines elicits stronger antigen-specific CD8+ T cell responses compared with DEC205-based vaccines.

BALB/c mice were administered 2 different DC-targeting DNA vaccines of sPD1-p24fc and psc-DEC205-p41 at a dose of 100 μg delivered via i.m./EP. p24fc, (psc-)Cont-p41, and PBS were used as controls. (A) IFN-γ–producing CD8+ and (B) CD4+ cells were measured by ELISPOT assay in splenocytes stimulated with the specific epitopes GAG A-I and GAG 26, respectively. (C) HIV-1 p24–specific H2-Kd-AMQMLKDTI-PE tetramer staining of CD8+ T cell populations is represented as a column graph of immunization. (D) Specific IgG1 and IgG2a antibodies against HIV-1 GAG p24 were detected in sera by ELISA. Intracellular staining performed on both CD4+ and CD8+ T cell populations in splenocytes is shown in column graphs depicting subpopulations of single-, double-, and triple-positive (E) CD8+ or (F) CD4+ T cells releasing the cytokines IFN-γ, TNF-α, and IL-2 induced by DNA vaccination. Data show the SEM with 5 mice per group. *P < 0.05.

Figure 8. sPD1-based vaccine functions by activating DCs and engaging in cross-presentation.

Mice were immunized with sPD1-p24fc, anti–DEC205-p41, and p24fc DNA vaccines, or PBS placebo, at a 100-μg dose administered by i.m./EP. Column graphs show (A) surface expression of CD40 and (B) MHC II, as well as intracellular staining of (C) IL-12 and (D) IFN-γ in draining lymph node CD11c+ DCs 16 hours after immunization. Data show the SEM with 5 mice per group. *P < 0.05. (E) Purified splenic CD11c+ DCs were treated with the proteins sPD1-p24, anti–DEC205-p24, or p24 for 15 minutes and 1 hour. Formaldehyde-fixed DCs were immunostained for HIV-1 GAG, Rab 14, and Lamp1. Nuclei were counterstained with DAPI. Images are overlays of the 3 signals (green: GAGp24; red: endosomal marker; blue: DAPI). (F) Schematic representation of the underlying mechanisms of sPD1-based vaccine. Following DC targeting of sPD1-p24fc (red arrows), antigen uptake into Rab5 endosomes was diverted to: (a) Rab14 endosomal compartment for subsequent MHC I cross-presentation; or (b) Rab7, Rab9, and Lamp1 endosomes, leading to MHC II presentation. Antigen presentation and activation of respective T cells are likely accompanied by increased costimulatory CD40 expression and production of Th1 cytokines IFN-γ and IL-12. By comparison, DC targeting of anti–DEC205-p24 (green arrows) was only routed to Lamp1 endosomes with induced IFN-γ production. Nontargeting p24 was only found in Rab7 endosomes (dotted blue arrow). The red cross symbol represents the HIV-1 antigen p24.