The modified HSV-ΔICP34.5-based constructs reactivated HIV latency more efficiently than wild-type HSV counterparts.

(A) J-Lat 10.6 cells (1×106) were infected with varying MOIs of wild-type HSV-1 17 strain for 30 h. The increasing expression level of GFP+ cells with increasing MOI of HSV-1 was displayed with the pseudocolor plot (left) and the corresponding bar chart (right). (B) The mRNA levels of HIV-1 LTR, Tat, Gag, Vpr, and Vif in J-Lat 10.6 cells following infection with the wild-type HSV-1 17 strain were shown with the histogram. (C) J-Lat 10.6 cells (1×106) were infected with HSV-wt or HSV-ΔICP34.5 at an MOI of 0.1 for 30 h, and then the mRNA levels of HIV-1 LTR, Tat, Gag, Vpr, Vif, and (D) HSV-1 UL27 were shown with the histogram. (E) ACH-2 cells (1×106) were infected with HSV-wt or HSV-ΔICP34.5 at an MOI of 0.1 for 30 h, and then the p24 protein level was detected using an HIV-1 p24 ELISA kit. (F) ACH-2 cells (1×106) were infected with HSV-wt or HSV-ΔICP34.5 at an MOI of 0.1 for 30 h, and then the mRNA levels of HIV-1 LTR, Tat, Gag, Vpr and Vif were shown with the histogram. (G) J-Lat 10.6 and J-Lat 10.6-ICP34.5 cells were infected with HSV-wt or HSV-ΔICP34.5, and then the mRNA levels of HIV-1 Tat were shown with the histogram (left). The blotting showed that the J-Lat 10.6 cells stably expressing HSV ICP34.5 (J-Lat 10.6-ICP34.5) can appropriately express ICP34.5 protein using Flag-tag antibodies (right). (H) J-Lat 10.6 and J-Lat 10.6-ICP34.5 cells were respectively stimulated with PMA (10 ng/mL) and TNF-α (10 ng/mL), and the expression level of GFP+ cells was displayed with the corresponding bar chart. Data shown are mean ± SD. **P<0.01, ***P<0.001, ****P<0.0001. ns: no significance.

The modified HSV-based constructs effectively reactivated HIV latency by modulating the NF-κB pathway and HSF1 pathway.

(A) J-Lat 10.6 cells were infected with HSV-wt and HSV-ΔICP34.5 at an MOI of 0.1. The cytoplasmic and nuclear proteins were separated to detect the expression level of p65, p-IKKα/β and IkBα. GAPDH and Lamin B1 served as loading controls for cytoplasmic and nuclear proteins, respectively. (B) 293T cells were transfected with Flag-ICP34.5, IKKα (left), or IKKβ (right), and the cell lysates and IP complexes were analyzed through Co-IP assays. (C) 293T cells were transfected with Flag-ICP34.5 or empty vector (Vec) for 24h, and then treated with LPS (1μg/mL) for 8h. The cytoplasmic and nuclear proteins were analyzed by Western blotting (WB) assay. (D-E) J-Lat 10.6 cells were infected with HSV-ΔICP34.5 and then treated with different concentrations of KRIBB11. The relative fold change in LTR and Tat mRNA was analyzed by qPCR. (F) J-Lat 10.6 cells were infected with HSV-wt or HSV-ΔICP34.5 at MOI of 0.1 for 36h. ChIP-qPCR was conducted to evaluate the ability of HSF1 to bind to the LTR. IgG and Histone antibody (His) were used as negative and positive controls, respectively. (G) 293T cells were transfected with Flag-ICP34.5 and Myc-HSF1, and the cell lysates and IP complexes were analyzed through Co-IP assays. (H) 293T cells were transfected with 0, 1, 2, 4 μg of Flag-ICP34.5 and analyzed by WB analysis. (I) 293T cells were transfected with Flag-ICP34.5 and HA-PP1α, and the cell lysates and IP complexes were analyzed through Co-IP assays. (J) 293T cells were transfected with 0, 0.5, 1, 2 μg of HA-PP1α and analyzed by WB analysis. 293T cells were transfected with Myc-HSF1 with or without Flag-ICP34.5 (K), or were transfected with Myc-HSF1 and HA-PP1α (L), and then the cell lysates and IP complexes were analyzed through Co-IP assays. Data shown are mean ± SD. **P<0.01, ****P<0.0001. ns: no significance.

Recombinant HSV-1 vector-based SIV vaccines induce specific T cell immune responses in mice.

(A) The schematic diagram illustrated the process of constructing recombinant HSV through the BAC/galK selection system. Firstly, ICP34.5 gene was replaced with galK gene through homologous recombination. Subsequently, galK was substituted with the target gene expression cassette containing the hCMV promoter and BGH terminator. Finally, the ICP47 gene was deleted. (B) The image of one clone of the rescued recombinant HSV with the brightfield (top) and the fluorescence channel (bottom). (C) Vero cells were infected with recombinant HSV constructs, and the protein expression of targeted genes was detected by using SIV-infected monkey serum. (D) HeLa cells were transfected with Myc-PDL1, followed by infection with HSV-empty, HSV-SIVgag, or HSV-sPD1-SIVgag at an MOI of 0.1 for 24 h. The cell lysates were subjected to Co-IP analysis. (E) Schematic schedule of vaccinated mice. Twenty-five mice were randomly allocated to five groups, HSV-empty, HSV-sPD1, HSV-SIVgag, HSV-sPD1-SIVgag, and HSV-SIVenv. At week 0 and week 2, mice were injected with the corresponding vaccines. At week 4, mice were sacrificed and spleen lymphocytes were collected to evaluate immune response. (F-G) Column graphs depicted the Gag or Env1, Env2-specific spot-forming cells (SFCs) per 106 spleen lymphocytes as measured by IFN-γ ELISpot assay. (H) The pseudocolor plot of flow cytometry illustrated the gating strategy. Column graphs depicted the frequencies of IFN-γ, IL-2, and TNF-α production from gag-specific CD3+ T (I), CD4+ T (J), and CD8+ T cells (K). (L) The bar chart illustrated the proportion of Tem of CD4+ T and CD8+ T cells under stimulation with the SIV Gag peptide pools. (M) The bar chart illustrated the frequencies of Env2-specific IFN-γ+ CD4+ T cells. These data were expressed as the mean±SD from five mice samples. Three independent experiments for the animal immunization were repeated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns: no significance.

The modified HSV-based constructs efficiently elicited SIV-specific immune responses in chronically SIV-infected macaques.

(A) Schematic schedule of the macaque experiment. Nine chronically SIV-infected macaques were assigned into three groups: ART+saline group (n=3), ART+HSV-empty group (n=3), and ART+HSV-sPD1-SIVgag/SIVenv group (n=3). All SIV-infected macaques received ART treatment (FTC/6.7 mg/kg/once daily, PMPA/10 mg/kg/once daily) for 33 days. On day 33 and day 52, macaques were immunized with saline, HSV-empty, and HSV-sPD1-SIVgag/SIVenv respectively. On day 70 after the second vaccination, ART treatment was interrupted in all macaques. Samples were collected at different time points to monitor virological and immunological parameters. (B) The representative images for the Gag or Env-specific spots (2.5×105 cells per well) of each macaque pre-vaccination (before, day 33) and post-vaccination (after, day 70) by ELISpot assay. (C-D) The difference in SIV-specific IFN-γ-secreting cells (ΔSFCs) between pre-immunization and post-immunization for the assessment of the immune response induced by HSV-vectored SIV vaccines. (E) The difference in SIV-specific TNF-α/IFN-γ/IL-2-secreting CD4+ T and CD8+ T subsets between pre-immunization and post-immunization was detected by ICS assay.

The modified HSV-based constructs effectively reactivated SIV latency in vivo in chronically SIV-infected, ART-treated macaques.

(A-D) The viral load (VL) change in plasma for each animal was monitored during the whole experiment by y real-time PCR. The detection limit is 100 copies per mL plasma. The shadow represented the duration of ART administration. (E) The VL change in plasma between pre-ART and the peak value in the rebound stage after ART discontinuation. (F) The change of total SIV DNA copies between pre-ART and viral rebound after ART discontinuation. (G) The change of the SIV Pol-specific IFN-γ-secreting cells between pre-immunization (day 33) and post-immunization (day 70) was detected by ELISpot assay. (H) The change of the CD4+ T/ CD8+ T ratio.

Pattern to illustrate the proof-of-concept strategy based on a bifunctional HSV-vectored therapeutic vaccine for HIV functional cure.

In the present study, the modified HSV-ΔICP34.5-based constructs effectively reactivated HIV/SIV latency by modulating the IKKα/β-NF-κB pathway and PP1-HSF1 pathway (Shock) and simultaneously elicited antigen-specific polyfunctional CD8+ T cells to eliminate cells infected with the reactivated virion (Kill). BAC: bacterial artificial chromosome; rHSV: recombinant HSV; TCR: T-cell receptor; PD1: Programmed Cell Death Protein 1; CD40L: CD40 Ligand.