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 Mckrae strain for 30 h. The proportion of GFP+ cells, indicating activated latent cells, is shown in the pseudocolor plot (left) and the corresponding bar chart (right). (B) J-Lat 10.6 cells (1×106) were infected with varying MOIs of HSV-1 17 strain containing GFP (HSV-GFP) for 30 h, and then the mRNA levels of HIV-1 LTR, Tat, Gag, Vpr, Vif are shown with the histogram. (C)J-Lat 10.6 cells (1×106) were infected with HSV-GFP or HSV-ΔICP34.5 at an MOI of 0.1 for 30 h. The mRNA levels of HIV-1 LTR, Tat, Gag, Vpr, Vif, and (D) HSV-1 UL27 are shown with the histogram. (E) ACH-2 cells (1×106) were infected with HSV-GFP or HSV-ΔICP34.5 at an MOI of 0.1 for 30 h. The p24 protein level was detected using an HIV-1 p24 ELISA kit, and the mRNA levels of HIV-1 LTR, Tat, Gag, Vpr, and Vif are shown in the histogram (F). (G) J-Lat 10.6 and J-Lat 10.6-ICP34.5 cells were infected with HSV-GFP or HSV-ΔICP34.5, and the mRNA levels of HIV-1 Tat were shown with the histogram (left). Blotting showed that 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 is displayed with the corresponding bar chart. (I-K) Primary CD4+ T cells from people living with HIV (PLWH) were infected with HSV-GFP or HSV-ΔICP34.5. The inflammatory response was assessed by evaluating mRNA levels of IL-6, IL-1β and TNF-α using qPCR. 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 infected with HSV-GFP and HSV-ΔICP34.5 at MOI 0.1. Cytoplasmic and nuclear proteins were analyzed for p65, p-IKKα/β and IkBα levels. GAPDH and Lamin B1 served as loading controls for cytoplasmic and nuclear proteins, respectively. (B) 293T cells transfected with Flag-ICP34.5, IKKα (left), or IKKβ (right) analyzed through Co-IP assays. (C) 293T cells were transfected with Flag-ICP34.5 or empty vector (Vec) for 24h, treated with LPS (1μg/mL) for 8h. Cytoplasmic and nuclear proteins analyzed by Western blot (WB). (D-E) J-Lat 10.6 cells infected with HSV-ΔICP34.5 treated with various KRIBB11 concentrations. LTR and Tat mRNA levels analyzed by qPCR. (F) J-Lat 10.6 cells infected with HSV-wt or HSV-ΔICP34.5 at MOI 0.1 for 36h. ChIP-qPCR assessed HSF1 binding to LTR. IgG and Histone antibody (His) served as negative and positive controls. (G) 293T cellstransfected with Flag-ICP34.5 and Myc-HSF1, analyzed by Co-IP assays. (H) The immunoblot depicts the alterations in protein levels in 293T cells transfected with either empty vector or ICP34.5 for 6 h, followed by 24 h of treatment with 10 μM MG132. (I) 293T cells transfected with Flag-ICP34.5 and HA-PP1α, analyzed by Co-IP assays. (J) The immunoblot depicts the alterations in protein levels in 293T cells transfected with either empty vector or HA-PP1α for 6 h, followed by 24 h of treatment with 10 μM MG132. (K) 293T cells transfected with Myc-HSF1 with or without Flag-ICP34.5, analyzed by Co-IP assays. (L) 293T cells transfected with Myc-HSF1 and HA-PP1α, 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) Schematic diagram illustrating the construction of recombinant HSV through the BAC/galK selection system. The ICP34.5 gene was replaced with the galK gene via homologous recombination, followed by substituting galK with a target gene expression cassette containing the hCMV promoter and BGH terminator. Finally, the ICP47 gene was deleted. (B) Brightfield (top) and fluorescence (bottom) images of a clone of the rescued recombinant HSV. (C) Vero cells infected with recombinant HSV constructs, with protein expression of targeted genes detected using SIV-infected monkey serum. (D) HeLa cells transfected with Myc-PDL1 and then infected with HSV-empty, HSV-SIVgag, or HSV-sPD1-SIVgag at an MOI of 0.1 for 24 h. Cell lysates were subjected to Co-IP analysis. (E) Schematic schedule of mouse vaccination. Twenty-five mice were randomly allocated to five groups: HSV-empty, HSV-sPD1, HSV-SIVgag, HSV-sPD1-SIVgag, and HSV-SIVenv. Mice were injected with the corresponding vaccines at weeks 0 and 2. At week 4, mice were sacrificed, and spleen lymphocytes were collected to evaluate immune response.(F-G) Column graphs showing the number of Gag or Env1, Env2-specific spot-forming cells (SFCs) per 106 spleen lymphocytes, as measured by IFN-γ ELISpot assay. (H) Pseudocolor plot of flow cytometry illustrating the gating strategy. Column graphs showing 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) Bar chart showing the proportion of Tem (effector memory T cells) among CD4+ T and CD8+ T cells upon stimulation with the SIV Gag peptide pools.(M) Bar chart showing the frequencies of Env2-specific IFN-γ+ CD4+ T cells. Data were expressed as 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 days 33 and 52, macaques were immunized with saline, HSV-empty, and HSV-sPD1-SIVgag/SIVenv respectively. ART treatment, was interrupted in all macaques on day 70 after the second vaccination. Samples were collected at various time points to monitor virological and immunological parameters. (B) Representative images of Gag or Env-specific spots (2.5×105 cells per well) from each macaque pre-vaccination (before, day 33) and post-vaccination (after, day 70) by ELISpot assay. (C-D) Difference in SIV-specific IFN-γ-secreting cells (ΔSFCs) between pre- and post-immunization, assessing the immune response induced by HSV-vectored SIV vaccines. (E) Difference in SIV-specific TNF-α/IFN-γ/IL-2-secreting CD4+ T and CD8+ T subsets between pre- and post-immunization, detected by ICS assay.

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

(A-D) Viral load (VL) changes in plasma for each animal were monitored throughout the experiment using real-time PCR. The detection limit is 100 copies per mL plasma. The shaded area representsthe 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) Change in total SIV DNA copies between pre-ART and viral rebound after ART discontinuation. (G) SIV integrated DNA (iDNA) copy numbers detected by Alu-PCR at different time points. (H) Change in the number of SIV Pol-specific IFN-γ-secreting cells between pre-immunization (day 33) and post-immunization (day 70), asdetected by ELISpot assay. (I) Change in 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.