Introduction

The epidemic of acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus type I (HIV-1), is still a huge challenge for global public health, with approximately 39 million people living with HIV-1 as of 2022. To date, there is neither a curable drug nor a prophylactic vaccine for clinical use against HIV-1 infections (Sun et al., 2023b, Sun et al., 2023a). Antiretroviral therapy (ART) can effectively control HIV-1 replication to an undetectable level, but the termination of ART usually results in prompt viral rebound from latent viral reservoirs (Archin et al., 2014, Looker et al., 2017, Calistri et al., 2003, Heng et al., 1994). Thus, it is of great priority to explore novel strategies for curing HIV latency. The “Shock and Kill” strategy is considered a promising approach for purging HIV-1 reservoirs, involving the activation of latently infected cells to express viral products (Shock), followed by viral cytopathic effects or specific cytolytic T lymphocytes (CTLs) to eliminate the activated cells (Kill) (Wu et al., 2022, Yang et al., 2019, Kim et al., 2018). Numerous latency-reversing agents (LRAs) (Yang et al., 2019, Wu et al., 2022), including methylation inhibitors, histone deacetylase (HADC) inhibitors (Archin et al., 2017, Lehrman et al., 2005), and bromodomain and extra terminal domain (BET) protein inhibitors (Li et al., 2013, Bisgrove et al., 2007), have been identified to reactivate latent HIV-1 in preclinical studies, but there is no ideal LRA available for clinical patients yet.

Herpes simplex virus (HSV), a human herpesvirus, features a 152-kb double-stranded DNA genome encoding over 80 proteins (Poh, 2016). Owing to its distinctive genetic background, high capacity, broad tropism, thermostability, and excellent safety profile, the modified HSV constructs have extensive applications in gene therapy and oncolytic virotherapy. For example, talimogene laherparepvec (T-VEC), an HSV-1 variant with ICP34.5 deletion and GM-CSF insertion, received FDA approval in 2015 for treating malignant melanoma, showcasing notable safety and efficacy in clinical practice. Recombinant HSV-based constructs have also emerged as efficacious gene delivery vectors against infectious diseases. Early studies indicated that prophylactic vaccines based on HSV, expressing simian immunodeficiency virus (SIV) antigens, could elicit robust antigen-specific CTL responses in mice and monkeys, providing enduring and partial protection against pathogenic SIVmac239 challenges (Kaur et al., 2007, Murphy et al., 2000). Moreover, increasing data suggest the crucial role of HIV-specific CTL in controlling viral replication and eliminating potential HIV reservoirs (Collins et al., 2020, Leitman et al., 2017). Significantly, epidemiological research suggests a synergistic effect between HSV and HIV infections, with HSV infection in HIV patients being associated with increased HIV-1 viral load and disease progression (Looker et al., 2017, Calistri et al., 2003, Heng et al., 1994). Some studies have further unveiled the ability of HSV to activate HIV latent reservoirs (Amici et al., 2004, Amici et al., 2001, Pierce et al., 2023). Given the potential of HSV to simultaneously induce antigen-specific immune responses and reactivate latent viral reservoirs, we propose a proof-of-concept strategy to achieve an HIV functional cure using a modified bifunctional HSV-vectored therapeutic vaccine.

Results

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

The J-Lat 10.6 cell line, derived from Jurkat T cells containing latent HIV-1 provirus, was infected with wild-type HSV-1 Mckrae strain at different multiplicities of infection (MOI) to assess its capability to reactivate HIV latency. Flow cytometry analysis showed a dose-dependent increase in green fluorescent protein (GFP) expression (Figure 1A), indicating that HSV-1 can reactivate latent HIV. Subsequently, we infected J-Lat 10.6 cells with an HSV-1 17 strain containing GFP (HSV-GFP) and found significant upregulation of mRNA levels of HIV-1 LTR, Tat, Gag, Vif, and Vpr, demonstrating that the attenuated HSV-1 strain can reactivate latent HIV at different MOIs (Figure 1B).Using the bacterial artificial chromosome (BAC)/galactokinase (galK) system, we then constructed the recombinant HSV deleted with ICP34.5 (HSV-ΔICP34.5) based on HSV-GFP. Notably, we discovered for the first time that the HSV-ΔICP34.5 reactivated HIV latency more efficiently than its parental strain (HSV-GFP). Specifically, mRNA levels of HIV genes (LTR, Tat, Gag, Vpr, Vif) were significantly higher in J-Lat 10.6 cells infected with HSV-ΔICP34.5 compared to those treated with HSV-GFP (Figure 1C), despite the weaker replication ability of HSV-ΔICP34.5 in these cells,as indicated by the mRNA level of HSV UL27 (Figure 1D, Figure S1).

Figure 1.

Furthermore, this finding was validated in ACH-2 cells, which are derived from T cells latently infected with replication-competent HIV-1. A significantly higher level of p24 protein, a key indicator of HIV replication, was found in the HSV-ΔICP34.5-infected ACH-2 cells compared to HSV-GFP-treated cells (Figure 1E), and the mRNA levels of HIV-related genes (LTR, Tat, Gag, Vpr, Vif) were also significantly increased (Figure 1F). Subsequently, we generated J-Lat 10.6 cells stably expressing ICP34.5-Flag-Tag (J-Lat 10.6-ICP34.5) using a recombinant lentivirus system and confirmed the expression of the ICP34.5 protein. Our research revealed that HSV-ΔICP34.5 displayed a reduced capacity to reverse HIV latency in J-Lat 10.6-ICP34.5 cells compared to J-Lat 10.6 cells (Figure 1G). Moreover, in J-Lat 10.6-ICP34.5 cells, the potency of latent reversal agents such as phorbol 12-myristate 13-acetate (PMA) and TNF-α was notably reduced when contrasted with J-Lat 10.6 cells (Figure 1H). We also confirmed the enhanced reactivation of HIV latency by HSV-ΔICP34.5 in primary CD4⁺ T cells from people living with HIV (PLWH) (Figure S2). In addition, HSV-ΔICP34.5 induced a lower level of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in primary CD4⁺ T cells from PLWH compared to HSV-GFP stimulation, likely due to its lower virulence and replication ability (Figure 1I-K). Thus, these data suggested that the safety of HSV-ΔICP34.5 in PLWH might be tolerable. In addition, both adenovirus and vaccinia virus cannot reactivate HIV latency in this study (Figure S3), and the deletion of ICP0 gene from HSV-1 diminished the reactivation effect of HIV latency by HSV-1 (Figure S4), implying this reactivation might be virus-specific. Overall, these findings indicate that the recombinant HSV-ΔICP34.5 construct specifically reactivate HIV latency with high efficiency.

The modified HSV-based constructs effectively reactivated HIV latency by modulating the IKKα/β-NF-κB pathway and PP1-HSF1 pathway

Next, based on RNA-seq analysis for HSV-ΔICP34.5-induced signaling pathways (Figure S5) and previous literature, we explored the mechanism of reactivating viral latency by HSV-ΔICP34.5-based constructs. J-Lat10.6 cells were infected with HSV-GFP or HSV-ΔICP34.5, and results showed that HSV-ΔICP34.5 significantly enhanced the phosphorylation of IKKα/β, promoted the degradation of IKBα, and thus led to the accumulation of p65 in the nucleus (Figure 2A). NF-κB is a well-known host transcription factor that exists in the form of the NF-κB-IκB complex in resting cells, but IκB can degrade and release the NF-κB dimer to enter the nucleus and promote gene transcription in response to external stimulation. Using the coimmunoprecipitation (Co-IP) assay, we verified that the ICP34.5 protein had a specific interaction with IKKα/β, and then ICP34.5 could dephosphorylate IKKα/β. Moreover, the overexpression of ICP34.5 effectively inhibited lipopolysaccharide (LPS) -induced NF-κB pathway activation by inhibiting p65 entry into the nucleus (Figure 2B-C).

Figure 2.

To further clarify the underlying mechanism, we performed the immunoprecipitation-mass spectrometry (IP-MS) analysis in ICP34.5 overexpressing cells to identify other potential molecules contributing to this reactivation, and we found that ICP34.5 can also interact with heat shock 1 protein (HSF1) (Table S1). HSF1 has been reported as a transcription factor correlated with the reactivation of HIV latency (Xu et al., 2022, Lin et al., 2018, Zeng et al., 2017). To test whether HSF1 contributes to the reactivation of HIV latency by HSV-ΔICP34.5-based constructs, KRIBB11, an inhibitor of HSF1, was administered to HSV-ΔICP34.5-infected J-Lat 10.6 cells. The results indicated that the reactivation ability of HSV-ΔICP34.5 was significantly inhibited by KRIBB11 treatment in a dose-dependent manner (Figure 2D-E). Furthermore, a significant enhancement of the binding of HSF1 to the HIV LTR was observed upon HSV-ΔICP34.5 infection, leading to an increase in the reactivation of HIV latency (Figure 2F). The direct interaction between ICP34.5 and HSF1 was also identified by Co-IP assay. Importantly, HSF1 was effectively dephosphorylated at Ser320 as a result of the overexpression of ICP34.5, while no influence on the level of HSF1 expression was observed (Figure 2G-H, Figure S6). Considering that protein phosphatase 1 (PP1) can interact with ICP34.5 and dephosphorylate eIF2α (Li et al., 2011), we then investigated the interaction between PP1α and ICP34.5 (Figure 2I). Additionally, a direct interaction between PP1α and HSF1 was found, allowing for the dephosphorylation of HSF1 and then affecting its ability to reactivate HIV latency (Figure 2J-L). Collectively, these findings demonstrated that our modified HSV-ΔICP34.5-based constructs effectively reactivated HIV latency by modulating the IKKα/β-NF-κB pathway and PP1-HSF1 pathway.

Recombinant HSV-ΔICP34.5 constructs expressing SIV antigens elicited robust immune responses in mice

Subsequently, we explored the potential of HSV-ΔICP34.5 as a bifunctional therapeutic vector to not only reactivate latent viral reservoirs but also induce antigen-specific immune responses against viral replication. To achieve this, SIV antigen genes were introduced into HSV-ΔICP34.5 vector based onBAC/ galK system. Additionally, the ICP47 gene was ablated to augment the immunogenicity of the HSV vector (Figure 3A, Figure S7). A series of recombinant HSV-ΔICP34.5ΔICP47-based vectors expressing SIV gag and env antigen were constructed, and the antigen expression of these constructs was confirmed by Western blotting assay (Figure 3B and C). Furthermore, to improve the immunogenicity of the targeted antigen, we fused the SIV gag with soluble PD1 (sPD1), enabling it to competitively bind with PD-L1 and thereby block the PD1/PD-L1 immune inhibitory pathway (Figure 3D). Consistent with the above findings, these HSV-ΔICP34.5ΔICP47-based SIV vaccines also efficiently reactivated HIV latency (Figure S8). Then, the immunogenicity of the above modified HSV-ΔICP34.5ΔICP47-based SIV vaccine was assessed in mice (Figure 3E). Our results showed that these constructs effectively elicited SIV antigen-specific T cell immune responses using the interferon γ (IFN-γ) ELISpot assay and the intracellular cytokine staining (ICS) assay. Of note, the frequency of SIV Gag-specific IFN-γ -secreting spot-forming cells (SFCs) in the HSV-sPD1-SIVgag group (1350 SFCs per 10⁶ splenocytes) was significantly higher than that in the HSV-SIVgag group (498 SFCs per 10⁶ splenocytes) (Figure 3F). The frequency of SIV Env2-specific IFN-γ -secreting SFCs in the HSV-SIVenv group was significantly higher than the HSV-empty group (Figure 3G). Furthermore, the polyfunctionality of antigen-specific T cell subsets in response to SIV antigen stimulation was confirmed using the ICS assay (Figure 3H-M). Consistently, the HSV-sPD1-SIVgag group showed a significantly higher frequency of SIV-specific CD3⁺ T, CD4⁺ T, and CD8⁺ T cell subsets secreting IFN-γ, IL-2, and TNF-α cytokines compared to the HSV-SIVgag group (Figure 3I-K). Notably, a heightened proportion of Gag-specific effector memory T cells (Tem) of CD8⁺ T cell subset in HSV-sPD1-SIVgag group was observed in comparison to the HSV-Gag group (Figure 3L). In addition, a higher frequency of SIV-Env2-specific CD4⁺ T cells secreting IFN-γ was observed in the HSV-SIVenv group than in the HSV-empty group (Figure 3M). These data indicated that the vaccines constructed in this study elicited a robust T cell immune response in mice. Moreover, the blockade of PD1/PDL1 signaling pathway effectively enhanced vaccine-induced T cell immune responses, which was consistent with our and other previous studies (Zhou et al., 2013, Wu et al., 2022, Pan et al., 2018, Xiao et al., 2014).

Figure 3.

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

Next, the immunogenicity of these HSV-vectored SIV vaccines was further investigated in chronically SIV_mac239-infected rhesus macaques (RMs). To mimic chronically infected HIV patients in clinic practice, all RMs used in this study were chronically infected with SIV and received ART treatment for several years, as reported in our previous studies (Pan et al., 2018, Yang et al., 2019, Wu et al., 2021, Wu et al., 2022, He et al., 2023). Based on sex, age, viral load, and CD4 count, nine RMs 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) (Table S2). All RMs received ART (FTC/6.7 mg/kg/once daily, PMPA/10 mg/kg/once daily) treatment to avoid the interference of free SIV particles. On day 33 and day 52, these RMs were immunized with saline, HSV-empty, or HSV-sPD1-SIVgag/SIVenv respectively. On day 70, ART treatment in all RMs was discontinued to evaluate the time interval of viral rebound. Samples were collected at different time points to monitor virological and immunological parameters (Figure 4A). To reduce the impact of individual RM variations, the difference in SIV-specific IFN-γ-secreting SFCs between post-immunization and pre-immunization (ΔSFCs) was used to evaluate the immune response induced by HSV-vectored SIV vaccines. The results showed that SIV Gag-specific ΔSFCs in the ART+HSV-sPD1-SIVgag/SIVenv group were greatly increased when compared with those in the ART+HSV-empty group and ART+saline group (Figure 4B-D). A similar enhancement of SIV Gag-specific TNF-α -secreting CD4⁺ T and CD8⁺ T subsets was also verified by ICS assay (Figure 4E). Collectively, these data demonstrated that the HSV-sPD1-SIVgag/SIVenv construct elicited robust SIV-specific T cell immune responses in ART-treated, SIV-infected RMs.

Figure 4.

The modified HSV-based constructs effectively reactivated SIV latency in vivo and delayed viral rebound in chronically SIV-infected, ART-treated macaques

Finally, we investigated the therapeutic efficacy of HSV-sPD1-SIVgag/SIVenv in chronically SIV-infected, ART-treated RMs. Consistent with our previous studies, the plasma viral load (VL) in these RMs was effectively suppressed during ART treatment, but rebounded after ART discontinuation. The VL in the ART+saline group promptly rebounded after ART discontinuation, with an average 8.63-fold increase in the rebounded peak VL compared with the pre-ART VL (Figure 5A, D and E). However, plasma VL in the ART+HSV-empty group and the ART+HSV-sPD1-SIVgag/SIVenv group exhibited a delayed rebound interval (Figure 5B-D). Remarkably, there was a lower rebounded peak VL than pre-ART VL in the ART+HSV-sPD1-SIVgag/SIVenv group (average 12.20-fold decrease), while a higher rebounded peak VL than pre-ART VL in the ART+HSV-empty group (average 2.74-fold increase) (Figure 5E). Then, we assessed the potential effect on the latent SIV reservoirs in vivo by administering our modified HSV-based SIV therapeutic constructs in these RMs. Although there were no obvious viral blips observed in these RMs, we found significant suppression of total SIV DNA and integrated SIV DNA provirus in the ART+HSV-sPD1-SIVgag/SIVenv group. However, the copies of the SIV DNA provirus were significantly improved in the ART+HSV-empty group and ART+saline group (Figure 5F-G). More interestingly, we assessed the magnitude of SIV Pol antigen-specific immune responses, which could represent to some extent the level of newly generated virions from the reactivated SIV reservoirs, because SIV Pol antigen was not included in our designed vaccine constructs. Specifically, the Pol-specific SFCs amount on Day 70 (511 SFCs/10⁶ PBMCs, post-vaccination) was higher than that on Day 33 (315 SFCs/10⁶ PBMCs, pre-vaccination) in the ART+HSV-sPD1-SIVgag/SIVenv group. In addition, there was a similar observation in the ART+HSV-empty group. In contrast, the Pol-specific SFCs gradually decreased with the duration of ART treatment in the ART+saline group (Figure 5H). In addition, the CD4⁺ /CD8⁺ T cell ratio (Figure 5I) and body weight (Figure S9) after treatment were effectively ameliorated in the RMs of the ART+HSV-sPD1-SIVgag/SIVenv group, but not in the ART+HSV-empty group and ART+saline group. Our data also demonstrated that there was no significant effect on the cell composition of peripheral blood in the macaques of ART+HSV-sPD1-SIVgag/SIVenv group (Figure S10). Taken together, these findings suggested that the latent SIV reservoirs might be effectively purged because of the effect of simultaneously reactivating viral latency and eliciting SIV-specific immune responses by our modified HSV-based SIV therapeutic constructs, thus resulting in a delayed viral rebound in chronically SIV-infected, ART-treated macaques.

Figure 5.

Discussion

To conquer the continuous epidemic of AIDS, exploring novel strategies to render and eliminate HIV latency should stand as a pivotal pursuit. Currently, numerous strategies, including shock and kill, block and lock, chimeric antigen receptor T-cell therapy, therapeutic vaccination, and gene editing, have been extensively investigated to target the latent HIV reservoirs for an HIV functional cure (Deeks, 2012, Yeh and Ho, 2021, Maldini et al., 2020, Herzig et al., 2019, Dash et al., 2023, Dashti et al., 2023, Walker-Sperling et al., 2022). However, there is no safe and effective approach for clinical use in HIV patients yet. In the present study, we occasionally found that the modified HSV-ΔICP34.5-based constructs could reactivate HIV latency more efficiently than wild-type HSV counterpart, which inspired us to develop a proof-of-concept strategy based on a bifunctional HSV-vectored therapeutic vaccine, aiming to simultaneously reactivate viral latency and elicit HIV/SIV-specific immune responses for HIV functional cure. Our results indicated that these modified HSV-based constructs efficiently elicited antigen-specific immune responses in mice and chronically SIV-infected macaques, and further therapeutic efficacy experiments showed that this strategy effectively reactivated SIV latency in vivo and delayed viral rebound in chronically SIV-infected, ART-treated macaques (Figure 6).

Figure 6.

The latent HIV proviruses can harbor it into the host genome with a quiescent transcription state, and thus cannot be recognized by immune surveillance or drug killing (Churchill et al., 2016, Pierson et al., 2000). Therefore, it is critical to disrupt viral latency for developing an HIV cure strategy. Based on our experimental data, the mechanism for efficiently reactivating viral latency by the modified HSV-ΔICP34.5-based constructs may involve regulating the IKKα/β-NF-κB pathway and PP1-HSF1 pathway. Indeed, during its replication, HSV can activate the double-stranded RNA-dependent protein kinase (PKR) pathway, and thus phosphorylate the protein translation initiation regulator eIF2α, resulting in the initiation of protein translation (Farassati et al., 2001). Previous studies have shown that the reactivation potential of HSV might be intertwined with NF-κB, Sp1, and other unknown transcription factors by ICP0, ICP4, and ICP27 (Chapman et al., 1991, Amici et al., 2004, Vlach and Pitha, 1992, Mosca et al., 1987b, Golden et al., 1992, Vlach and Pitha, 1993, Mosca et al., 1987a). However, the underlying mechanism by which the ICP34.5-deleted HSV construct can greatly improve the reactivation efficacy of HIV latency remains elusive. ICP34.5 is a neurotoxicity factor that can antagonize innate immune responses, including PKR, TANK binding kinase (TBK1) signaling, and Beclin1-mediated apoptosis (He et al., 1997, Manivanh et al., 2017, Orvedahl et al., 2007). ICP34.5 binds to host PP1 and mediates the dephosphorylation of eIF2α, thus allowing protein synthesis and reversing the effects of PKR and host antiviral functions (He et al., 1997, He et al., 1998). In this study, our findings further unveiled an interaction between ICP34.5 and HSF1, resulting in reduced HSF1 phosphorylation via recruitment of PP1α. Interestingly, previous reports indicated that HSF1 could positively regulate HIV gene transcription (Rawat and Mitra, 2011), which is facilitated by its nuclear entry post-phosphorylation and subsequent recruitment of p300 for self-acetylation, along with binding to the HIV-1 LTR. Studies have also shown that HSF1 could further orchestrate p-TEFb recruitment to promote HIV-1 transcriptional elongation (Peng et al., 2020, Lin et al., 2018, Pan et al., 2016b, Pan et al., 2016a). Under stress-induced conditions, phosphorylation triggers the formation of the HSF1 trimer, thus facilitating its nuclear entry to bind to heat shock elements (HSEs) for gene transcription regulation (Bonner et al., 2000, Timmons et al., 2020). Additionally, we also demonstrated that ICP34.5 interacted with IKKα and IKKβ, thereby impeding NF-κB nuclear entry and further curbing HIV latency. Consistently, previous studies have also suggested that ICP34.5 could disrupt the NF-κB pathway and possibly affect the maturation of dendritic cells (Jin et al., 2011). Intriguingly, these findings collectively indicated that ICP34.5 might play an antagonistic role in the reactivation of HIV by HSV-1, and thus our modified HSV-ΔICP34.5 constructs can effectively reactivate HIV/SIV latency through the release of imprisonment from ICP34.5. However, ICP34.5 overexpression had only a partial effect on the reduction of the HIV latency reactivation, indicating that HSV-ΔICP34.5-based constructs can also reactivate HIV latency through other yet-to-be-determined mechanisms.

Another observation in this study is that the HSV-sPD1-SIVgag/SIVenv construct elicited robust and persistent SIV-specific T cell immune responses in ART-treated, chronically SIV-infected macaques. Increasing evidence has indicated that HIV-specific cytolytic T lymphocytes (CTLs) can facilitate the suppression of latent viral reservoirs, and thus, the induction of robust and persistent HIV-specific CTL responses is essential for achieving long-term disease-free and transmission-free HIV control (Collins et al., 2020). Featured polyfunctional CD8⁺ T cells may contribute to HIV elite controllers or long-term non-progressors, a rare proportion of HIV-infected individuals who can spontaneously control viral replication even without ART treatment (Owen et al., 2010, Ferre et al., 2009, O’Connell et al., 2009). In addition, both our previous study and others have demonstrated that strong antigen-specific CD8⁺ T cell immune responses, especially effector memory CD8⁺ T cells, were associated with a lower viral load, and in vivo CD8⁺ lymphocyte depletion with intravenous infusion of anti-CD8 monoclonal antibody could lead to dramatical viral rebound in these vaccinated elite macaques (Perdomo-Celis et al., 2022, Pandrea et al., 2011, Sun et al., 2013, Sun et al., 2010, Pan et al., 2018, Sun et al., 2012). Notably, previous studies have shown that HSV-based vaccines expressing HIV/SIV antigen did elicit specific CD8⁺ T cell immune responses in mice and macaques, but the magnitude was not as strong as other viral vectors (Parker et al., 2007, Murphy et al., 2000, Kaur et al., 2007). Therefore, to further improve its immunogenicity in vivo, some modifications were adopted to optimize the HSV-vectored vaccine. 1) The ICP47 protein, encoded by the US12 gene, can bind to the transporter associated with antigen presentation (TAP) 1/2, inhibiting the transport of viral peptides into the endoplasmic reticulum and the loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules to activate CD8⁺ T cells (Orr et al., 2005). Therefore, the ICP47 gene was deleted from our developed HSV vector. 2) Negatively immunoregulatory molecules, including PD1, TIM-3, and LAG-3, are usually involved in the pathogenesis of HIV infection, as well as in chronically SIV-infected macaques. Among them, PD1 upregulation can result in the exhaustion and dysfunction of CD8⁺ T cells (Trautmann et al., 2006). In addition, PD1 expression on memory CD4⁺ T cells might be linked with HIV latency. In this study, we modified the SIV antigen by fusing to soluble PD1 (sPD1), which can block the PD1/PDL1 pathway by competitively binding with PDL1 and thus improve HIV/SIV vaccine-induced CD8⁺ T cell immune responses (Zhou et al., 2013, Wu et al., 2022, Pan et al., 2018, Xiao et al., 2014).

Although promising, there are still some limitations to our study. Firstly, this is a pilot study with a relatively small numbers of RMs, and future studies with a larger number of animals can be conducted to better verify our strategy. Secondly, the HSV-sPD1-SIVgag/SIVenv vaccine resulted in delayed viral rebound and a lower peak viral load post-rebound than pre-ART treatment but did not completely suppress SIV virus rebound in this study, which may be attributed to suboptimal doses and treatment, implying that we should further optimize this regimen for eventually achieving an HIV functional cure in future studies. The current consensus on HIV/AIDS vaccines emphasizes the importance of simultaneously inducing broadly neutralizing antibodies and cellular immune responses. Therefore, we believe that incorporating the induction of broadly neutralizing antibodies into our future optimizing approaches may lead to better therapeutic outcomes. Taken together, these findings demonstrated that our modified HSV-ΔICP34.5-based constructs potentially reactivated HIV/SIV latency by modulating the IKKα/β-NF-κB pathway and PP1-HSF1 pathway, and thereby we developed a proof-of-concept strategy based on a bifunctional HSV-vectored therapeutic vaccine, which can provide insights into the rational design of novel strategies for pursuing an HIV functional cure.

Methods

Mouse ethics statement and vaccination

Female BALB/c mice, aged six to eight weeks, were procured from the Experimental Animal Center of Sun Yat-sen University. A total of twenty-five mice were randomly divided into five groups to evaluate the immunogenicity of the recombinant HSV-vector-based SIV vaccine. During the initial week, each mouse was subcutaneously administered a vaccination of 1×10⁶ PFU of the respective recombinant HSV-vector vaccines (HSV-empty, HSV-sPD1, HSV-SIVgag, HSV-sPD1-SIVgag, HSV-SIVenv). Following a two-week interval, a booster vaccination was administered via the subcutaneous route using 2×10⁶ PFU of recombinant HSV-vector vaccines. The subsequent assessment of the immune response involved ELISpot and intracellular cytokine staining (ICS) assays in accordance with the vaccination schedule.

Ethics statement and vaccination of macaques

A total of 9 chronically SIV_mac239-infected rhesus macaques (RMs) were included in this study. All RMs received ART treatment (FTC/6.7 mg/kg/once daily, PMPA/10 mg/kg/once daily) for a duration of 33 days. The RMs were allocated into three groups based on their baseline plasma SIV viral loads (VL): ART + saline (n=3) as the control group, ART+ HSV-empty (n=3) as the sham group, and ART+ HSV-sPD1-SIVgag/SIVenv (n=3) as the vaccinated group. On day 0, all RMs received ART treatment. Once the VL dropped below the limit of detection (1×10² copies/mL), the vaccinated group and sham group were primed with a subcutaneous vaccination of 1×10⁷ PFU HSV-sPD1-SIVgag/SIVenv or HSV-empty, respectively, while the control group received an injection of 0.9% saline. After three weeks of the prime vaccination, the vaccinated group and sham group RMs received a subcutaneous booster vaccination of 5×10⁷ PFU HSV-sPD1-SIVgag/SIVenv or HSV-empty, respectively. After two weeks of booster vaccination, the ART treatment for all RMs was interrupted. The immune response was evaluated by IFN-γ ELISpot and ICS assays. Plasma viral load was regularly monitored throughout the studied period.

Peptide pools

The peptide pools, encompassing the complete sequences of SIV Gag, Env, and Pol proteins, comprising 15 amino acids with 11 overlaps, were generously provided by the HIV Reagent Program, National Institutes of Health (NIH), USA. Gag pools comprise 125 peptides, while Env and Pol pools are subdivided into Env1 (109 peptides) and Env2 (109 peptides) pools, as well as Pol1 (131 peptides) and Pol2 (132 peptides) pools. These peptide pools were dissolved in dimethyl sulfoxide (DMSO, Sigma) at a concentration of 0.4 mg/ peptide/mL for subsequent immunological assays.

Construction of recombinant HSV

For the generation of recombinant HSV-vector-based vaccines, we implemented modifications at the ICP34.5 loci through homologous recombination within the BAC-HSV-1 system. Specifically, double copies of the ICP34.5 and ICP47 genes were either deleted or inserted into the respective counterparts: sPD1, SIVgag, sPD1-SIVgag, and SIVenv genes. The sPD1-SIVgag gene was created by fusing the N-terminal region of mouse soluble PD1 (sPD1) with the C-terminal segment of SIVgag, connected by a GGGSGGG linker, which was engineered through overlapping PCR.

Plaque Assay

Vero cells were seeded at a density of 2 × 10⁵ cells per well in a 12-well plate and cultured for 24 h. Virus samples were serially diluted and then added to the wells. After 2 h of incubation at 37°C with 5% CO₂, the supernatant was aspirated, and the cells were washed with PBS. Then, a medium containing 1% FBS, 1% low-melting-point agarose, and 1% penicillin-streptomycin was added to each well. After 3 days of incubation, cells were fixed with 4% paraformaldehyde at room temperature for 2 h and stained with 1% crystal violet for 10 min. Plaques were visualized and counted, and virus titers were calculated as plaque-forming units per milliliter (PFU/mL).

IFN-γ ELISpot assay

The IFN-γ-ELISpot assay was conducted in accordance with our previous study (Sun et al., 2010). In the mouse experiment, 2.5×10⁵ freshly isolated mouse spleen lymphocytes were simulated with SIV Gag, Env1, and Env2 peptide pools. In the monkey experiment, 2×10⁵ peripheral blood mononuclear cells (PBMCs) were seeded and simulated with the SIV Gag, Env, and Pol peptide pools. Mock stimulation utilized DMSO (Sigma), while concanavalin A (ConA, 10 μg/mL) was employed as a positive control. Spot quantification was performed using an ELISpot reader (Mabtech), and peptide-specific spot counts were determined by subtracting the spots from mock stimulation.

Intracellular cytokine staining (ICS)

The ICS assay was performed as described previously (Wu et al., 2022). In the mouse experiment, 2×10⁶ freshly isolated mouse spleen lymphocytes were stimulated with SIV Gag, Env1, and Env2 peptide pools. For the monkey experiment, 2×10⁶ PBMCs were seeded and simulated with the SIV Gag, Env, and Pol peptide pools. DMSO and PMA (Thermo Scientific) plus ionomycin were used as the mock and positive controls, respectively. Data analysis was performed using FlowJo software (version 10.8.1), and the antibodies used are listed in Table S3. The frequencies of cytokines produced from peptide-specific cells were analyzed by subtracting mock stimulation.

SIV viral RNA and DNA copy assays and absolute T cell count

Absolute T cell count and the levels of plasma mRNA and SIV total DNA were quantified as described previously (Wu et al., 2022). Plasma viral RNA copy numbers were determined via SYBR green-based real-time quantitative PCR (Takara), using SIV gag-specific primers (Table S4). Viral RNA copy numbers were calculated based on the standard curve established using SIV_mac239 gag standards. The lower limit of detection for this assay was 100 copies/mL of plasma. Total cellular DNA was extracted from approximately 0.5 to 5 million cells using a QIAamp DNA Blood Minikit (Qiagen). PCR assays were performed with 200 ng samples of DNA, and SIV viral DNA was quantified using a pair of primers targeting a conserved region of the SIV gag gene, as previously described. Quantitation was performed by comparing the results to the standard curve of SIV gag copies. Integrated SIVmac239 DNA was measured using nested PCR with two sets of primers. The first round used SIVgag-reverse and Alu-forward primers, while the second round used primers specific to a conserved SIVgag region. Quantification was compared to a standard of CEMss/pWPXLD-rc cell genome as our previously described (Yang et al., 2019).

Plasmids, cells, and viruses

pVAX-Myc-PDL1, pVAX-Flag-ICP34.5, pVAX-Myc-HSF1, pVAX-HA-PP1α: full-length mouse PDL1, full-length HSV-1 ICP34.5, and full-length human HSF1 and human PP1α with N or C-terminal Tag were cloned into the pVAX vector. pVAX-empty, pcDNA3.1-IKKα, and pcDNA3.1-IKKβ plasmids were stored in our laboratory. pVAX-empty was used as a mock transfection in our study.

293T cells (from the embryonic kidney of a female human fetus), Vero cells (from the kidney of a female normal adult African green monkey), and Hela cells (from the cervical cancer cells of an African American woman) were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in an atmosphere of 5% CO₂. The J-Lat 10.6 cells (Jurkat cells contain the HIV-1 full-length genome whose Env was frameshifted and inserted with GFP in place of Nef) and the HIV-1 latently infected CD4⁺ CEM cells ACH-2 (A3.01 cell integrated HIV-1 proviral DNA) were cultured in complete RPIM640 (Gibco) containing 10% FBS and 1% penicillin/streptomycin at 37°C in an atmosphere of 5% CO₂. J-Lat 10.6-ICP34.5 cells were constructed in our laboratory.

The HSV-1 (McKrae) strains were stored in our laboratory. HSV-GFP (17 strain) was rescued from pBAC-GFP-HSV in Vero cells. HSV-ΔICP34.5 (17 strain) was rescued from pBAC-GFP-HSV-ΔICP34.5 (with deletion of double copies of ICP34.5 gene) in Vero cells.

RT-qPCR

RT-qPCR was performed as described in our previous study (Zhao et al., 2022). Data were normalized to β-actin. The primer sequences are listed in Supplemental Table 4. Fold changes in the threshold cycle (Ct) values were calculated using the 2^-ΔΔCt method.

Chromatin immunoprecipitation (ChIP) assay

ChIP analysis was conducted using the SimpleChIP Enzymatic Chromatin IP kit (Agarose Beads) (CST), following the manufacturer’s instructions. Quantitative real-time PCR was employed for detecting the LTR sequence. The sequences of primers used in the LTR ChIP are listed in Supplemental Table 4. % Input= 2% × 2^{[(ct)2% input sample (ct) IP sample]}.

Co-immunoprecipitation (Co-IP)

Cells were harvested and subjected to Co-IP assay, following the protocol outlined in our previous study (Zhao et al., 2022).

Protein extraction and western blot

Nuclear and cytoplasmic proteins were extracted by kits following the manufacturer’s instructions (Beyotime). The Western blotting assay was performed as previously described (Zhao et al., 2022).

Statistical analysis

GraphPad Prism 8.0 (GraphPad Software, San Diego, California) was used for statistical analysis. For intragroup direct comparisons, Student’s unpaired two-tailed t test was performed to analyze significant differences. For comparisons of multiple groups, one-way ANOVAs were performed. Significance levels are indicated as *P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001.

Study approval

Mice experiment was approved by the Laboratory Animal Ethics Committee guidelines at Sun Yat-sen University (approval number: SYSU-IACUC-2021-000185). Chinese rhesus macaques (Macaca mulatta) were housed at the Landau Animal Experimental Center, Guangdong Landau Biotechnology Co., Ltd. (approval number: N2021101). The primary PBMC samples were isolated from the chronically HIV-1-infected participants who were recruited from Guangzhou Eighth People’s Hospital (Guangzhou, Guangdong, China), which was approved by the Medical Ethics Review Board of the School of Public Health (Shenzhen), Sun Yat-sen University (2022-037). All participants provided written informed consent, agreeing to participate in this study. The enrollment criteria for HIV-1-infected individuals included substained suppression of plasma HIV-1 viremia under ART, with undetectable plasma HIV-1 RNA levels (less than 50 copies/mL) for at least 12 months and a CD4⁺ T cell count more than 350 cells/μL).

Data Availability Statement

Source data contain the numerical data used to generate the figures.

Acknowledgements

We appreciate the staff at the Animal Center of GIBH for their excellent technical assistance. We thank all other members of our group for their helpful advice and discussion to improve this project. We also appreciate the NIH AIDS Research and Reference Reagent Program for providing SIV peptide pools.

Additional information

Competing interests

The authors have declared that no conflict of interest exists.

Funding

This project was supported by National Natural Science Foundation of China (82271786, 81971927, 32370171), Science and Technology Planning Project of Guangdong Province, China (2021B1212040017), Shenzhen Key Laboratory of Pathogenic Microbes and Biosafety (ZDSYS20230626091203007), the Sanming Project of Medicine in Shenzhen Nanshan (SZSM202103008), and the Key Subject of Nanshan district of Shenzhen for AIDS surveillance and prevention.

Author Contributions

All authors were involved in drafting or critically revising the manuscript for important intellectual content, and all authors approved the final version for publication. CS conceived the project and provided the main funding. CS and LC designed the experiments and reviewed the manuscript. ZW, PL, YY, and CW performed most of the experiments and analyzed the data. ZW, PL, YY, CW, ML, HW, MS, YH, and MC established the animal models and performed animal experiments. ZW and CS interpreted the data and wrote the manuscript.