
==== Front
Signal Transduct Target Ther
Signal Transduct Target Ther
Signal Transduction and Targeted Therapy
2095-9907
2059-3635
Nature Publishing Group UK London

39245675
1943
10.1038/s41392-024-01943-9
Article
Anti-PD-L1 antibody ASC22 in combination with a histone deacetylase inhibitor chidamide as a “shock and kill” strategy for ART-free virological control: a phase II single-arm study
http://orcid.org/0000-0002-9918-9047
Wu Luling 12
Zheng Zhihang 13
Xun Jingna 14
Liu Li 1
Wang Jiangrong 1
Zhang Xinyu 1
Shao Yueming 1
Shen Yinzhong 1
Zhang Renfang 1
Zhang Min 5
Sun Meiyan 1
Qi Tangkai 1
Wang Zhenyan 1
Xu Shuibao 1
Song Wei 1
Tang Yang 1
Zhao Bihe 1
Song Zichen 1
Routy Jean-Pierre 6
Lu Hongzhou luhongzhou@fudan.edu.cn

37
Chen Jun qtchenjun@163.com

1
1 grid.8547.e 0000 0001 0125 2443 Department of Infectious Diseases and Immunology, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
2 grid.8547.e 0000 0001 0125 2443 Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China
3 grid.8547.e 0000 0001 0125 2443 Shanghai Institute of Infectious Disease and Biosecurity, Fudan University, Shanghai, China
4 grid.8547.e 0000 0001 0125 2443 State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology, Ministry of Education, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China
5 grid.8547.e 0000 0001 0125 2443 Department of Clinical Laboratory, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
6 https://ror.org/04cpxjv19 grid.63984.30 0000 0000 9064 4811 Infectious Disease and Immunity in Global Health Program, Research Institute of McGill University Health Centre, Montreal, QC Canada
7 https://ror.org/04xfsbk97 grid.410741.7 Department of Infectious Diseases and Nursing Research Institution, National Clinical Research Center for Infectious Diseases, The Third People’s Hospital of Shenzhen, Guangdong, China
9 9 2024
9 9 2024
2024
9 2318 12 2023
7 6 2024
4 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The combination of ASC22, an anti-PD-L1 antibody potentially enhancing HIV-specific immunity and chidamide, a HIV latency reversal agent, may serve as a strategy for antiretroviral therapy-free virological control for HIV. People living with HIV, having achieved virological suppression, were enrolled to receive ASC22 and chidamide treatment in addition to their antiretroviral therapy. Participants were monitored over 24 weeks to measure changes in viral dynamics and the function of HIV-specific CD8+ T cells (NCT05129189). 15 participants completed the study. At week 8, CA HIV RNA levels showed a significant increase from baseline, and the values returned to baseline after discontinuing ASC22 and chidamide. The total HIV DNA was only transiently increased at week 4 (P = 0.014). In contrast, integrated HIV DNA did not significantly differ from baseline. Increases in the proportions of effector memory CD4+ and CD8+ T cells (TEM) were observed from baseline to week 24 (P = 0.034 and P = 0.002, respectively). The combination treatment did not succeed in enhancing the function of HIV Gag/Pol- specific CD8+ T cells. Nevertheless, at week 8, a negative correlation was identified between the proportions of HIV Gag-specific TEM cells and alterations in integrated DNA in the T cell function improved group (P = 0.042 and P = 0.034, respectively). Nine adverse events were solicited, all of which were graded 1 and resolved spontaneously. The combined treatment of ASC22 and chidamide was demonstrated to be well-tolerated and effective in activating latent HIV reservoirs. Further investigations are warranted in the context of analytic treatment interruption.

Subject terms

Clinical trials
Infectious diseases
issue-copyright-statement© West China Hospital, Sichuan University 2024
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pmcIntroduction

Combination antiretroviral therapy (ART) significantly improves the prognosis for people living with HIV (PLWH) by effectively reducing the plasma human immunodeficiency virus (HIV) viral load to undetectable levels.1,2 Despite of effective ART, HIV may persist in both latent and transcriptionally active states, within quiescent or proliferating cells, across various T cell sub-populations encompassing defective and intact viruses.3 These T cells constitute a potential HIV latent reservoir that has evaded immune detection and remained unresponsive to ART.4 HIV reactivation from latency after ART treatment interruption occurs, on average, every 5–8 days, which has implications for HIV remission. Therefore, innovative therapeutic approaches targeting persistent HIV reservoirs are necessary to achieve an ART-free virological control.5,6 To date, latency-reversing agents evaluated in HIV-infected individuals receiving ART, such as histone deacetylase (HDAC) inhibitors and toll-like receptor agonists alone, have failed to consistently demonstrate latency reversal.7 No intervention other than allogeneic stem cell transplantation has shown sustained reduction or elimination of cells containing replication-competent viruses.8 The most in-depth investigated HIV eradication is the “shock and kill” strategy, which seeks to activate the latent reservoir with latency reversing agents (LRAs) and promote elimination through viral cytopathic effects or immune-mediated clearance.9

PD-1, a transmembrane immune receptor belonging to the CD28 family, has been identified as a crucial negative regulator of T cell function.10 It interacts with its ligand, programmed death ligand 1 (PD-L1), which is widely expressed on T cells, B cells, dendritic cells, and macrophages.11 Envafolimab (ASC22), a humanized anti-PD-L1 antibody derived from camel single domains and used with a human immunoglobulin Fc fragment to extend its half-life, is approved for treating advanced solid tumors.12–14Recently, anti-programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) checkpoint inhibitors have drawn considerable attention for their potential for cancer and persistent viral infections therapeutic applications.10,11,15 Numerous experiments both in vivo and in vitro have shown that elevated levels of antigenemia following HIV infection can lead to impaired T cell function, mediated by PD-1 and its ligands via immunomodulatory signaling pathways, as evidenced by studies in HIV-infected humanized mice.16–18 T cell exhaustion features multiple deficiencies in effector functions, such as impaired proliferation, cytotoxicity, and cytokines production. These deficiencies contribute to weakening the immune system’s capacity to control HIV viral infections. Recent researches indicate that inhibiting the interaction between PD-1 and its ligand, PD-L1 can partially restore T cell exhaustion.19,20 Furthermore, there is evidence suggesting that using anti-PD-1/PD-L1 antibody compounds may effectively activate HIV viral expression in latently infected CD4+ T cells.21 Immune checkpoint inhibitors (ICIs) have the capability to enhance the clearance of cells harboring latent infections, offering a promising approach towards achieving ART-free virological control for HIV infection.5,22,23 Nonetheless, the lack of PLWH in a substantial number of clinical trials has restricted our understanding of the safety and durability of HIV-targeted effects associated with immune checkpoint inhibitor therapies in this population. A few reports of ART-treated PLWH with tumors receiving anti-PD-1 therapy have documented transient increases in cell-associated (CA) HIV RNA or plasma HIV RNA, suggesting that immune checkpoint inhibition may impact the reservoir of latent HIV.24,25 Additionally, small-scale studies on HIV-associated tumors have demonstrated favorable safety profiles for combined treatment with ICIs.7,26 However, there is still lacking of clinical trials assessing the efficacy of anti-PD-1/PD-L1 inhibitors for achieving ART-free virological control.

Various latency-reversing agents (LRAs) have been developed to target different HIV transcriptional regulatory pathways. These mainly include PKC agonists, atypical NF-κB agonists, and HDAC inhibitors (HDACis).27 The regulation of HDAC activity on the HIV long terminal repeat (LTR) promoter has been extensively documented as critical for enhancing HIV expression.28,29 Chidamide, a HDAC inhibitor in the benzamide category, is specifically designed for oral administration to inhibit HDAC1, 2, 3, and 10 selectively and was approved by the China Food and Drug Administration (CFDA) in December 2014 for treating relapsed or refractory PTCL. Chidamide has also proven effective in enhancing the cytotoxic response of immune cells.30,31 Recent studies have revealed a property of chidamide as a HIV latency reversing agent, capable of reactivating latent HIV in primary CD4+ T cells.28,32 In vivo researches have shown the safe and effective disruption of HIV-1 latency by chidamide, indicating its potential as a promising candidate for clinical investigation in activating the HIV viral reservoir.33 Several studies indicate that HDAC inhibitors can rapidly reduce PD-L1 expression at the surface of cells across various cancers, enhancing the anti-tumor efficacy of ICIs and facilitating activated T cell infiltration into tumor microenvironments.34,35 Therefore, ASC22 may synergize with chidamide in activating the HIV reservoir.

Collectively, we hypothesize that treatment with the combination of ASC22 and chidamide may potentially enhance the removal of infected cells and bolster the immunity of HIV specific CD8+ T cell, resulting in ART-free viral control. During this phase II clinical trial, our objective was to assess the effectiveness and safety of using the combination of ASC22 and chidamide as a “shock and kill” strategy for ART-free virological control in PLWH.

Results

Baseline characteristics of the participants

From July 1, 2022 to March 6, 2023, 18 participants were screened, and 3 individuals were considered ineligible for various reasons (Supplementary Fig. 1). These included having a plasma HIV viral load exceeding 50 copies/mL, experiencing renal insufficiency, or refusing to adhere to the prescribed medication. All 15 enrolled participants completed the clinical trial (Fig. 1 and Table 1). The study comprised solely male participants of Asian ethnicity. Their median age was 36 years, with a median duration of ART was 61.27 months (Interquartile range (IQR), 25–174.17 months). At baseline, the median CD4+ T cell counts and the ratios of CD4/CD8 were 433 cells/μL (IQR, 348–730 cells/µl) and 0.6 (IQR, 0.52–0.74), respectively.Fig. 1 Flowchart of specimens collection and research protocol in relation to ASC22 combined with chidamide administration. The duration of ART treatment is indicated by blue shading, while the duration of chidamide treatment is indicated by red shading. Figure 1 was created with biorender.com. ART antiviral therapy

Table 1 Participant characteristics

No.	Age (years)	Gender	Ethnicity	BMI (kg /m2)	Months since HIV diagnosis	Days from HIV diagnosis to ART initiation	Months on ARTs	Current ART regimens	Months with HIV RNA < 50 copies/mL	Baseline CD4 counts (cells/μL)	Baseline CD8 counts (cells/μL)	Baseline CD4/CD8 ratios	
01	29	Male	Asian	20.6	52.1	0	52.1	ABC, 3TC, DTG	45.3	304	571	0.5	
02	28	Male	Asian	19.2	158.1	2570	72.5	TAF, 3TC, DTG	32.8	539	814	0.7	
03	53	Male	Asian	28.1	39	19	38.4	ABC, 3TC, DTG	33.4	370	879	0.4	
04	25	Male	Asian	21.9	35.9	251	27.6	TDF, 3TC, EFV	21.8	405	670	0.6	
05	30	Male	Asian	22.7	57.1	54	55.3	TDF, 3TC, EFV	49.6	433	578	0.8	
06	38	Male	Asian	30.0	111	1291	68	TDF, 3TC, EFV	49.2	633	1355	0.5	
08	51	Male	Asian	25.1	103.6	197	97	ABC, 3TC, DTG	49.8	902	1462	0.6	
09	34	Male	Asian	28.1	57.6	18	57	TAF, FTC, DTG	50.9	785	998	0.8	
10	31	Male	Asian	25.0	62.1	26	61.3	TDF, 3TC, LPV/r	30.5	730	1319	0.6	
11	33	Male	Asian	22.5	85	1311	41.3	TDF, 3TC, EFV	35.7	844	989	0.9	
12	53	Male	Asian	18.4	144.5	23	143.7	TDF, 3TC, EFV	98.4	284	547	0.5	
13	42	Male	Asian	26.9	25	0	25	TDF, 3TC, EFV	24.8	670	991	0.7	
14	36	Male	Asian	21.7	75.6	41	74.2	TDF, 3TC, EFV	69	398	712	0.6	
15	37	Male	Asian	20.7	198.7	735	174.2	AZT, 3TC, EFV	102.9	349	1074	0.3	
16	38	Male	Asian	20.8	87.5	163	82.1	TDF, 3TC, EFV	76.3	327	444	0.7	
Median (range)	36 (25–53)			22.5 (20.7–26.9)	75.6 (25–198.7)	54 (0–2570)	61.3 (25–174.1)		49.2 (21.8–102.9)	433 (284–902)	879 (444–1462)	0.6 (0.3–0.9)	
Continuous variables are presented as median [interquartile range, (IQR)]

ABC Abacavir, ART antiviral therapy, AZT azidothymidine, BMI body mass index, DTG Dolutegravir, TAF tenofovir alafenamide fumarate, TDF tenofovir disoproxil fumarat, EFV Efavirenz, LPV/r lopinavir/ritonavir, 3TC lamivudine

ASC22 and chidamide potentiates HIV latency reversal

After administering ASC22 and chidamide, a gradual elevation in the levels of CA RNA: total HIV DNA ratios were observed from baseline to week 4 (Fig. 2a–c, Supplementary Figs. 2a, 3). At week 8, CA HIV RNA levels showed a significant increase from baseline, with an average rise of 4.27-fold (P = 0.004). The CA HIV RNA exhibited a gradual downward trend at week 8, returning to baseline levels by week 24. Interestingly, The CA HIV RNA: total DNA ratios were significantly higher at weeks 8 and 12 compared to baseline, showing an increase of 1.87-fold and 2.14-fold, respectively (P < 0.001, P = 0.007). At week 24, the ratios of CA-RNA to total HIV DNA returned to baseline. No significant difference in plasma HIV viral loads were detected at both weeks 4 and 8 compared to the baseline. However, at week 12, the plasma HIV viral loads increased in three participants. Plasma HIV RNA levels of 105 copies/ml and 205 copies/ml were observed in two participants at week 24, respectively. Only one of these four participants had sustained HIV viral loads elevation at both weeks 12 and 24 (Fig. 2d). The proportion of participants exhibiting HIV reservoir activation increased gradually, observed in 53% (8/15), 73% (11/15), and 73% (11/15) of participants at weeks 4, 8, and 12, respectively (Fig. 2e). By week 24, this had dropped to 33% (5/15).Fig. 2 ASC22 combine with chidamide treatment and latency reversal. a Changes in cell-associated (CA) HIV RNA levels at weeks 0 (baseline), 4, 8, 12, and 24 for each individual. b Mean fold increase in CA HIV RNA levels from baseline. Changes in the ratios of CA HIV RNA to total HIV DNA (c) and individual dynamics of plasma HIV viral loads (d) at weeks 0 (baseline), 4, 8, 12, and 24. The black dotted line represents the plasma HIV RNA less than 50 copies/ml, and the gray shade represents the plasma HIV RNA less than 20 copies/ml. e Proportion of participants with activated latent HIV-1 reservoirs across different treatment cycles and observation periods. Activation of the latent HIV reservoir is indicated by either a greater than twofold increase in CA HIV RNA or plasma HIV viral loads exceeding the threshold of 50 copies/ml. ns means not significant

Combination of ASC22 and chidamide did not reduce the HIV reservoir size

In this cohort, we did not observe a significant decrease in total or integrated HIV DNA. Compared with the baseline, the total HIV DNA exhibited an increasing trend at week 4 (P = 0.014), but subsequently demonstrated a declining trajectory at week 8, ultimately returning to baseline levels by week 12 and week 24 (Fig. 3a, b, Supplementary Figs. 2b, 3). As the duration of therapy prolonged, the proportion of participants experiencing a decrease in total HIV DNA levels gradually increased. This proportion reached 13% (2/15) at week 4, further climbed to 33% (5/15) at week 8, and peaked at 73%(11/15) by week 12. However, by week 24, the proportion regressed to 33% (5/15) (Fig. 3c). The levels of integrated HIV DNA did not significantly change from baseline, despite the activation of the HIV reservoir (P = 0.114) (Fig. 3d, e, Supplementary Figs. 2c,3). However, at week 4, 33% (5/15) of participants exhibited a composite decline in integrated HIV DNA, which increased to 67% (10/15) at week 8. At weeks 12 and 24, 53% (8/15) and 40% (6/15) of participants showed a decrease in integrated HIV DNA (Fig. 3f).Fig. 3 ASC22 combined with chidamide treatment and HIV DNA. a Changes in total HIV DNA levels at weeks 0 (baseline), 4, 8, 12, and 24. b Mean fold change in total HIV DNA from baseline. c The proportion of participants exhibiting a reduction in total HIV DNA levels was measured during both treatment and observation periods. d Alterations in integrated HIV DNA at weeks 0 (baseline), 4, 8, 12, and 24 in diverse participants. e Mean fold increase in integrated HIV DNA from baseline. f The histograms represent the proportion of participants with decreasing integrated HIV DNA levels assessed across various treatment and observation periods. ns means not significant

Combination of ASC22 and chidamide raised the proportion of effector memory CD4+ and CD8+ T cells (TEM)

The CD4+ T cells primarily included central memory (TCM), TEM, and naïve (TN) subsets, whereas the CD8+ T cells predominantly consisted of TEM, terminally differentiated memory (TEMRA), and TN (Supplementary Figs. 4, 5a). The percentage of CD4+ and CD8+ TEM cell subsets tended to increase with prolonged treatment, showing significant elevation compared to their respective baseline levels at week 24 (P = 0.034; P = 0.002) (Supplementary Fig. 4a). In contrast, CD4+ TN displayed an overall declining trend, especially at week 24 when compared with the baseline levels (P = 0.04) (Supplementary Fig. 4b). The ratios of CD8+ TN, CD4+ and CD8+ TCM and TEMRA cells subsets did not show significant changes during the treatment and observation periods (Supplementary Fig. 4b, c, d). CD4+ and CD8+ T cell counts, as well as CD4/CD8 ratios in blood remained stable throughout the entire period (Supplementary Fig. 5b–d).

Combination of ASC22 and chidamide did not improve HIV Gag- and Pol-specific CD8+ T cells responses significantly

Throughout both treatment and observation phases, the capacity of HIV Gag-specific CD8+ T cells was not enhanced by the combined administration of multiple treatment cycles. However, the functional cytokine secretion by HIV Gag-specific CD8+ T cells and CD8+ TEM cells gradually increased as the treatment progressed (Fig. 4a, b). At week 8, the ratios of participants exhibiting functional improvement reached their highest level, with rates of 27% (4/15) and 53% (8/15) observed for HIV Gag-specific CD8+ T cells and CD8+ TEM cells, respectively (Fig. 4c, d). The percentages gradually declined, and at week 24, 20% (3/15) and 13% (2/15) of participants sustained elevated levels of TNF-α secretion by HIV Gag-specific CD8+ T cells and CD8+ TEM cells, respectively. Out of the participants, only 7% (1/15) demonstrated a sustained elevation above baseline in HIV Gag-specific CD8+ T cell functionality. All individuals in the study were observed to have their HIV Gag-specific CD8+ TEM cell functionality return to baseline. Similarly, no enhancement in the functionality of HIV Pol-specific CD8+ T cells and CD8+ TEM cells was observed (Supplementary Fig. 6). Compared to baseline levels, there were no significant alterations observed in PD-1 fluorescence expression on CD4+ and CD8+ T cells or TEM subset.(Supplementary Fig. 7).Fig. 4 Impact of combination therapy involving ASC22 and chidamide on the responses of HIV Gag-specific CD8+ T cells. a Individual dynamics of HIV Gag-specific CD8+ T cells responses for each participant at weeks 0 (baseline), 4, 8, 12, and 24, indicated for IFN-γ (left) and TNF-α (right). b Individual dynamics of HIV Gag-specific CD8+ TEM cells responses at weeks 0 (baseline), 4, 8, 12, and 24 for each participant shown for IFN-γ (left) and TNF-α (right). c, d Changes in the ratios of participants with improvement in HIV Gag-specific CD8+ T cells (left) and CD8+ TEM cells (right) secreting functional cytokines from baseline to week 24. Improvement in T cell immune function was defined as a more than 2-fold increase in the expression of IFN-γ or TNF-α in HIV Gag- specific CD8+ T cells

Subgroup with boosted T cell functions tend to have more pronounced reduction in HIV reservoir size compared to those with unboosted T cell function

At week 8, we observed that the proportion of participants exhibiting T cell functional improvement and a decline in integrated HIV DNA reached a peak. Subsequently, we performed a correlation analysis to investigate further. In the subgroup with T cell function improvement, a negative correlation was detected between the proportions of IFN-γ/TNF-α secreting HIV Gag-specific CD8+ TEM cells and alterations in integrated HIV DNA at week 8 (P = 0.04, R2 = −0.89 and P = 0.03, R2 = −0.79, respectively) (Fig. 5a, b). No correlation was observed between the expression of IFN-γ/TNF-α in the group with unimproved HIV Gag-specific CD8+ TEM cell responses and alterations in integrated HIV DNA (P = 0.14, R2 = −0.53 and P = 0.65, R2 = 0.19, respectively) (Fig. 5c, d). Additionally, integrated HIV DNA showed no significant correlation with weak HIV Gag-specific CD8+ T cell responses, such as in weeks 4, 12, and 24 (data not shown). At weeks 8 and 24, integrated HIV DNA and total HIV DNA were lower in the T cell function improved group compared to the T cell function unimproved group, although these differences did not observed statistical significance (Fig. 5e, f).Fig. 5 Correlation between fold change of integrated HIV DNA and expression of IFN-γ (left) and TNF-α (right) in the improved (a, b) and unimproved (c, d) HIV Gag-specific CD8+ TEM cells response group at week 8. Comparison of integrated HIV DNA (e) and total HIV DNA (f) in T cell function improved and unimproved groups at weeks 4, 8, 12 and 24

Combination of ASC22 and chidamide represented safety in HIV virologically suppressed participants

A total of 18 AEs were reported, 9 were classified as solicited systemic AEs, and the remaining 9 were unsolicited (Table 2). Among these, 7 were immune-related adverse events (irAEs), including drug eruption, blood thyroid stimulating hormone decreased, hypertriglyceridemia and liver function test abnormal, which may typically associated with ASC22. Overall, 46.67% (7/15) participants reported at least one AE and 6.67% (1/15) of participants reported more than one solicited AE. All drug-related AEs were grade 1 and resolved spontaneously. Two participants developed blood thyroid stimulating hormone decreased, which recovered without adjusting the dose of ASC22 and chidamide. After receiving the initial dose, one participant experienced drug eruption on the trunk and extremities, which resolved within 24 h. Another individual experienced generalized rash 16 days after dosing, which spontaneously resolved by day 13. Additionally, a different participant developed generalized rash 22 days after receiving the initial dose, which persisted for 5 days and then resolved spontaneously. After the completion of ASC22, one participant developed bilateral lower extremity edema while receiving chidamide alone, which self-recovered without adjusting the dose.Table 2 Adverse events (AEs)

Type of AEs	Grade of adverse events (N = 15)	No. of participants	
1	2	3	4	5	
Drug eruption	3 (20)	0 (0)	0 (0)	0 (0)	0 (0)	Participants 2, 12, 15	
Gravitational edema	1 (6.67)	0 (0)	0 (0)	0 (0)	0 (0)	Participant 2	
Blood thyroid stimulating hormone decreased	2 (13.33)	0 (0)	0 (0)	0 (0)	0 (0)	Participants 1,2	
Coronavirus disease 2019 (COVID-19)	4 (26.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participants 1,2	
Nephrolithiasis	1 (6.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participants 1,4	
Weight increased	1 (6.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participant 2	
Hepatic steatosis	1 (6.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participant 2	
Injury	1 (6.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participant 11	
Chest discomfort	1 (6.67)	0 (0)	0 (0)	0 (0)	0 (0)	Participant 9	
Blood creatine phosphokinase abnormal	1 (6.67)a	0 (0)	0 (0)	0 (0)	0 (0)	Participant 15	
Hypertriglyceridemia	1 (6.67)	0 (0)	0 (0)	0 (0)	0 (0)	Participant 4	
Liver function test abnormal	1 (6.67)	0 (0)	0 (0)	0 (0)	0 (0)	Participant 11	
1 a represented unsolicited systemic AEs

Discussion

To our knowledge, the study represents the first prospective cohort investigation involving PLWH who have received a combination of ICI and HDAC inhibitors, with the aim of comprehensively assessing the reversal of HIV latency and immune function in individuals under ART suppression. The simultaneous administration of ASC22 and chidamide over 12 weeks was safe and well-tolerated. Importantly, it can effectively activate latent HIV reservoirs and promote the differentiation of CD4+ and CD8+ TN cells into functional TEM cells. In the subgroup displaying stronger T cell function, the combination treatment strategy results in a more pronounced reduction in the HIV reservoir. These findings also tend to have a small size of HIV reservoir in populations with boosted T cell function compared with those without T cell functional improvement.

The “shock and kill” strategy, a promising approach for achieving ART-free virological control, harnesses the immune response to target and clear latent HIV infections by selectively inducing the reactivation of latent HIV transcription while avoiding full activation of T cells.36,37 During our clinical trial, we noted a substantial rise in CA HIV RNA relative to baseline upon treatment with ASC22 and chidamide. Furthermore, we observed a notable increase in the ratios of CA HIV RNA to total HIV DNA, and some participants displayed an elevation in their peripheral plasma HIV viral loads, indicating a marked activation of the HIV reservoir. Furthermore, levels of the CA HIV RNA returned to the baseline in 67% (10/15) of the participants after discontinuation of ASC22 and chidamide, providing further evidence that the observed changes in HIV transcription were a result of the synergistic effect of the combination therapy strategy. However, due to combined treatment, we cannot distinguish whether the therapeutic effect is solely attributed to this combination or if similar effects would have been observed with either drug alone. Four participants remained that did not respond to during treatment. Since the level of reversal of the latent reservoir is also correlated with its size, in these participants who are difficult to reverse the latent reservoir may have been smaller. At the same time, with long-term ART therapy, the reservoir may become more difficult to reverse, and more research is needed to confirm this. During the treatment periods, we identified a disconnect between CA HIV RNA and plasma HIV viral loads, like in another study.33 We speculate that fluctuations in HIV viral loads were not detected due to relatively long monitoring intervals. In our study, during the treatment periods, we identified a disconnect between CA HIV RNA and plasma HIV viral loads, like in another study.33 We speculate that fluctuations in HIV viral loads were not detected due to relatively long monitoring intervals. In our study, plasma HIV viral load rebounded in four participants after discontinuation of drug treatment. However, the underlying mechanism behind this phenomenon remains unclear. To explore the potential impact of ASC22 and chidamide on clonal expansion, we endeavored to perform HIV deep sequencing at both baseline and during the rebound of HIV plasma load. Regrettably, we were unable to obtain sufficient HIV sequence information in the plasma samples, likely attributable to the low viral load present in the plasma.

The HIV latent reservoir is commonly believed to evade immune surveillance. Should there be a robust immune response upon HIV reactivation, it could potentially shift the balance in favor of the host in controlling the virus.38 However, a sustained decrease in the latent HIV reservoir size was not shown following treatment with ASC22 and chidamide. The failure to adequately eliminate the HIV reservoir may be attributed to immunological recognition impairments in CD8+ T cells function.39,40 Despite the observed increase in CD4+ and CD8+ TEM cells, the present study reveals a notable absence of substantial enhancement in the function of HIV-specific CD8+ T cells. Firstly, the infection with HIV reduces phosphatase and tensin homolog (PTEN) levels.41 Prior oncology investigations revealed that constitutive PD-L1 expression due to PTEN loss might lead to resistance to other cancer immunotherapy approaches by actively suppressing antitumor T cells.42,43 Hence, we hypothesized that the lack of PD-L1 response may be due to the decrease in PTEN levels after HIV infection. Secondly, other research has shown the developmental biology of exhausted CD8+ T cells (Tex) may contribute to the restoration of T cell function.44 T cells converted the Tex subset in the third stage of exhaustion, and T cells demonstrated a partial restoration of their functionalities after PD-L1 blockade.45 However, once the T cells progress into the terminal exhaustion stage, they exhibit unresponsiveness to PD-L1 blockade.46 Thirdly, promoting immune-mediated mechanisms like boosting cytotoxic T cell responses can achieve prolonged viral control, contingent upon continual viral replication and the expression of viral antigens.47 Previous studies in non-human primates indicate that achieving viral control may require a phase of rebound viremia after ART interruption (ATI); PD-1 blockade administered post-ATI has been shown to significantly enhance the functionality of CD8+ T cells and contribute to better viral control.47,48 Therefore, the lack of significant improvement in T cell function in our study may be partially attributed to the absence of ATI. Fourthly, due to the lack of T Cell Receptor stimulation in suppressed individuals, these cells may not be adequately activated by even a slight rise in CA HIV RNA to effectively assess the impact of ICIs.49 Thus, optimizing the reversal of the HIV-1 latency may prove advantageous in enhancing specific T cell responses. The absence of significant differences observed in our study may also stem from our utilization of total and integrated HIV DNA to represent HIV viral reservoir, rather than intact HIV DNA. Additionally, T cell responses are not assessed immediately after each injection but rather 4 weeks later, which may be too delayed to detect any effect. Future studies with ICIs may incorporate higher doses, multiple dose administration, combination therapy with various ICIs, e.g., PD-1/PD-L1 inhibitors and CTLA-4 inhibitors or enhancers, recombinant human interleukin-15 (rhIL-15)50,51 and closer follow-up of T cell function to validate the potential to improve T cell function.

The incidence of AEs attributed to ASC22 and chidamide was low, with all being of low grade (CTCAE grade 1). These events primarily encompassed rash and subclinical hyperthyroidism. Importantly, none of the participants required pharmacological intervention, and no AEs necessitated discontinuation of the study treatment, which supports further clinical trials.

This study has several limitations. Firstly, baseline ART was consistently maintained throughout the study duration, with no assessments conducted for ATI. Secondly, the quantification of surrogate markers of the HIV reservoir relies on PCR-based techniques, which may lead to an overestimation of their magnitude as a significant proportion of the detected viral genomes are non-replicable. Due to limitations in sample size, we were unable to simultaneously measure replication-competent HIV and conduct Quantitative Viral Outgrowth Assays (QVOA), which would have contributed to a more comprehensive study.52 We also do not have enough samples to sort CD4+ T cells for HIV DNA determination, so we chose PBMCs for quantification. Thirdly, the enrollment for clinical trials exclusively included male participants. Therefore, the results may not be generalized to females. The study had limitations in terms of its single-arm design. Fourthly, Plasma drug concentrations were not monitored. Thus, the increased plasma viremia, elevated levels cell-associated unspliced HIV RNA, and decline in HIV DNA observed could potentially be attributed to naturally occurring longitudinal variation. However, this explanation seems unlikely, considering all participants had maintained stable suppression viremia for minimum of 2 years prior to enrollment.

The co-administration of ASC22 and chidamide demonstrates efficacy in potentiating HIV latency reversal; however, it falls short in effectively eradicating the HIV reservoir. Additionally, this combined approach significantly augments the ratios of CD4+ and CD8+ TEM cells, it does not effectively enhance the function of HIV-specific CD8+ T cells. This strategy tends to have a more pronounced decrease in the viral reservoir of HIV in populations with stronger T cell functionality. The combination strategy shows the potential of effectively activate and reduce latent HIV reservoirs and highlights the role of enhanced HIV-specific T cell functionality in reducing viral reservoirs.

Materials and methods

Study design and participants

We performed an open-label, non-randomized, controlled study phase II clinical trial at the Shanghai Public Health Clinical Center, Shanghai, China. Our study enrolled adult PLWH who had received at least 24 months of ART and had two consecutive episodes of plasma HIV viral load less than 50 copies/mL, separated by a minimum of 12 months. All enrolled participants were receiving plasma HIV RNA testing at least once every six months or one year. Participants had baseline CD4+ T cell counts exceeding 250 cells/μL and CD4/CD8 ratios <0.9. The main exclusion criteria included prior administration or exposure to ICIs, the presence of active autoimmune disease, and the occurrence of any severe acute illness within an eight-week timeframe. The clinical trial was approved by the Research Ethics Committee of the Shanghai Public Health Clinical Center (Ethics committee number: 2021-E040-01), and all participants signed written informed consent before being included in the study. The study has been registered on the ClinicalTrials.gov (NCT05129189).

Procedures

Participants were administered ASC22 (1 mg/kg) via subcutaneous injection every 4 weeks for a total of 3 times. Chidamide (10 mg) was administered orally twice weekly for 12 weeks, while continuing ART (Fig. 1). ASC22 was supplied by Ascletis Pharma Inc., China, while chidamide was supported by Shenzhen Chipscreen Biosciences, China. The treatment duration of ASC22 combined with chidamide was 12 weeks, and the observation periods were 12 weeks after discontinuation of ASC22 and chidamide. Participants had a total of 11 visits during the study: once every 2 weeks during the treatment period, and once every 4 weeks during the observation period (Supplementary Table. 1). During the treatment period, samples were collected at weeks 0 (baseline), 4, 8, 12, and 24. ASC22 was administered at least 1 h after chidamide. Blood samples were collected each time before ASC22 administration. We evaluate the treatment effect of the previous cycle through each sampling period. Peripheral blood mononuclear cells (PBMCs) and plasma were immediately processed and cryopreserved for subsequent testing.

Outcomes

The primary outcomes were the changes in total and integrated HIV DNA of PBMCs from baseline to week 24. The secondary endpoints included assessed by quantifying CA HIV RNA in PBMCs, and plasma HIV viral loads, as well as analyzing variations in the functionality of HIV Gag/Pol specific CD8+ T cells from baseline to post-treatments.53 Additionally, changes in CD4+ and CD8+ T cell counts, as well as CD4/CD8 ratios were monitored at weeks 0 (baseline), 4, 8, 12, and 24. Other outcome was determined by assessing the incidence and severity of adverse events (AEs), which were evaluated following the Adverse Event Common Terminology Criteria, version 6.0 (CTCAE V6.0). AEs were recorded from the initial dose of ASC22 through the safety follow-up period, extending to 24 weeks after the final dose. Solicited and unsolicited adverse reactions were registered from all volunteers. The causality of suspected adverse drug reactions was assessed using the WHO-Uppsala Monitoring Center (WHO-UMC) system.54 AEs were documented using Medical Dictionary for Regulatory Activities (MedDRA®; version 26.1) terms.

A greater than 2-fold increase in CA HIV RNA or plasma HIV viral loads exceeding the threshold of 50 copies/ml were considered indicative of HIV reservoir activation. If the fold change of total and integrated HIV DNA was less than 1, this was defined as a reduction in the HIV reservoir size. Improvement in T cell immune function was defined as a more than 2-fold rise in the proportions of IFN-γ or TNF-α expression by HIV Gag- or Pol-specific CD8+ T cells after HIV peptide pool stimulation.

Quantification of CD4+ and CD8+ T cell counts and plasma HIV viral loads

Flow cytometry (BD FACS CantoTM, New Jersey) assessed CD4+ and CD8+ T cell counts, while plasma HIV viral loads were quantified via polymerase chain reaction (PCR) (Cobas X 480 and Cobas Z 480, Roche, Basel, Switzerland). All assays were performed at the clinical laboratory of the Shanghai Public Health Clinical Center.

Quantitative detection of CA HIV RNA and total HIV DNA

Total cellular RNA and DNA were amplified and quantified from PBMCs using HIV DNA quantitative detection kit (SUPBIO, China) and CA HIV RNA quantitative detection kit (SUPBIO, China).55 The quantitative values for total HIV DNA (copy/μL) and the quantitative numbers of cells (unit/μL) were obtained, which were then used to calculate the copy number of total HIV DNA and CA HIV RNA per million PBMCs. The total HIV DNA assay displayed a lower detection limit of 20 copies/million PBMCs and a quantification range of 50-1 × 106 copies/million PBMCs. For the CA HIV RNA, the lower detection limit was found to be 2 copies/reaction, while the linear range spanned from 2–2 × 105 copies/reaction.

A quantitative assay was conducted to measure HIV DNA integration

Integrated HIV DNA Quantitative was conducted using an integrated HIV DNA quantitative detection kit (SUPBIO, China), and the principle of nested fluorescent quantitative PCR was adopted. In the first round of PCR, upstream and downstream primers were designed on the HIV gene and the Alu fragment of the human genome, respectively. In the second round of fluorescent quantitative PCR, the first round of PCR products (2 μL) were quantified with HIV-specific primers and probe detection. After obtaining the quantitative value of integrated HIV DNA (copies/μL) and the quantitative numbers of cells (cells/μL), the integrated HIV DNA copy number per million PBMCs was calculated. The integrated HIV DNA assay detected a lower limit of 10 copies/106 PBMCs, and the quantitative range was 10–106 copies/106 PBMCs.

Peptides

The CD8+ T cell epitope of the HIV-1 Gag and Pol protein were sourced from the HIV database (https://www.hiv.lanl.gov/mojo/immunology/search/ctl/form.html), which consists of 8 to 11 amino acids. A total of 32 CD8+ T cell epitopes were selected and submitted to GenScript Biotech Corp company for chemical synthesis. The peptides sequences were Gag (18–27) KIRLRPGGKK, Gag (45–53) AVNPGLLET, Gag (63–72) QLQPSLQTGS, Gag (77–86) SLYNTVATLY, Gag (87–95) CVHQRIEVK, Gag (148–158) SPRTLNAWVKV, Gag (193–203) GHQAAMQMLKE, Gag (214–224) RLHPVHAGPIA, Gag (240–251) TSTLQEQIGWMT, Gag (249–258) WMTNNPPIPV, Gag (267–274) ILGLNKIV, Gag (275–283) RMYSPTSIL, Gag (291–301) EPFRDYVDRFY, Gag (349–359) ACQGVGGPGHK, Gag (362–370) VLAEAMSQV, Gag (368–376) (SQVTNSATI) and Pol (101–110) KMIGGIGGFI, Pol (132–140) LVGPTPANI, Pol (158–167) SPIETVPVKL, Pol (188–196) ALVEICTEM, Pol (248–257) GIPHPAGLKK, Pol (263–273) VLDVGDAYFSV, Pol (282–290) YTAFTIPSI, Pol (313–321) AIFQSSMTK, Pol (336–344) YQYMDDLYV, Pol (464–473) ILKEPVHGVY, Pol (495–505) QIYQEPFKNLK, Pol (571–579) FVNTPPLVK, Pol (640–648) ALQDSGLEV, Pol (886–895) HLKTAVQMAV, Pol (894–903) AVFIHNFKRK, Pol (918–926) (IIATDIQTK), respectively. Each peptide, synthesized at a quantity of 4 mg, was dissolved in DMSO first, then diluted to 1 mg/ml in PBS, and stored at −80 °C in the end for future use.

Flow cytometry to measure HIV-specific CD8+ T cells responses

PBMCs were resuspended into 96 U-well plates at 6 × 105 cells/well, then stimulated with Gag and Pol peptide pool (5 μg/ml of each selected single peptide), and PMA/Ionomycin cocktail (eBioscience, USA) as a positive control. After a 2-h incubation at 37 °C, adding protein transport inhibitor cocktail (eBioscience, USA), and incubated for an additional 10 h. The stimulated cells were stained with various cell surface markers, including anti-human CD3- APC-H7 (Clone SK7, BD Pharmingen, USA), anti-Human CD4-FITC (Clone RPA-T4, BD Pharmingen, USA), anti-human CD8a-BV650 (Clone RPA-T8, Biolegend, USA), anti-Human CCR7-PE (Clone REA108, MACS, GER), anti-Human CD45RA-APC (Clone HI100, BD Pharmingen, USA), anti-human CD279 (PD-1)-BV785 (Clone EH12.2H7, Biolegend, USA), and LIVE/DEAD Aqua reagent (Invitrogen, USA). Subsequently, the stimulated cells were incubation in fixation and permeabilization solution (BD Biosciences, USA) half hours, then stained with intracellularly markers including anti-human TNF-α-PE-Cy7 (Clone MAb11, Invitrogen, USA) and anti-human IFN-γ-BV421 (Clone 4 S.B3, BD Horizon, USA). Stimulated PBMCs were analyzed using BD LSRFortessa flow cytometer, with 100,000 events recorded for each sample (Supplementary Fig. 8).

Statistical analysis

We employed repeated measures ANOVA to evaluate whether there were significant longitudinal changes in numerical outcome measures compared to baseline. Paired t-tests, Mann-Whitney U or Wilcoxon signed-rank tests were employed to evaluate changes from baseline to specific time points, with the choice of test depending on the data distribution. For post hoc analyses exploring correlations, we employed Spearman’s or Pearson’s rank correlation, depending on the nature of the data. Statistical analyses were conducted using SPSS 15.0 statistical software (Chicago, IL, USA) and GraphPad Prism 8.0 (San Diego, CA, USA). P value < 0.05 was deemed statistically significant.

Supplementary information

Supplementary_Materials

Study protocol

Supplementary information

The online version contains supplementary material available at 10.1038/s41392-024-01943-9.

Acknowledgements

We extend our gratitude to all participants who enrolled in this study. We also thank Dan Yin, Jiadan Gu, Lin Gu and Huiyan Yu for their invaluable assistance with this study. This study was funded by a grant from Ascletis Pharma Inc. and the Shanghai Commission of Science and Technology (grant no. 20MC1920100; grant no. 20Y31900400 and grant no. 21Y11901200). Ascletis Pharma Inc. did not participate in the study design or execution.

Author contributions

Each author has significantly contributed to this study, and has approved this article. H.Z.L. and J.C. designed the clinical protocol and provided oversight for the conduct of the trial. L.LW., Z.H.Z. and J.N.X. analyzed and interpreted the data. L.L., J.R.W., Y.Z.S., R.F.Z., T.K.Q., Z.Y.W., S.B.X., W.S., Y.T., B.H.Z. and M.Y.S. contributed to identifying participants and following them through the clinical protocol. Z.H.Z., L.L.W., J.N.X. and X.Y.Z. and M.Z. performed laboratory specimens testing work. Y.M.S. and Z.C.S. management the specimens. L.L.W. and Z.H.Z. prepared the manuscript. L.L.W. drew the figures. H.Z.L., J.C., Routy J.P. and J.N.X. critically revised and finally approved the manuscript.

Data availability

The dataset produced during this study is available upon request from the corresponding author (qtchenjun@163.com).

Competing interests

The authors declare no competing interests.

These authors contributed equally: Luling Wu, Zhihang Zheng
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References

1. Abdel-Mohsen M Recommendations for measuring HIV reservoir size in cure-directed clinical trials Nat. Med. 2020 26 1339 1350 10.1038/s41591-020-1022-1 32895573
Abdel-Mohsen, M. et al. Recommendations for measuring HIV reservoir size in cure-directed clinical trials. Nat. Med. 26, 1339–1350 (2020).32895573 10.1038/s41591-020-1022-1
2. Wandeler G Johnson LF Egger M Trends in life expectancy of HIV-positive adults on antiretroviral therapy across the globe: Comparisons with general population Curr. Opin. HIV AIDS 2016 11 492 500 10.1097/COH.0000000000000298 27254748
Wandeler, G., Johnson, L. F. & Egger, M. Trends in life expectancy of HIV-positive adults on antiretroviral therapy across the globe: Comparisons with general population. Curr. Opin. HIV AIDS 11, 492–500 (2016).27254748 10.1097/COH.0000000000000298
3. Dufour C Gantner P Fromentin R Chomont N The multifaceted nature of HIV latency J. Clin. Invest 2020 130 3381 3390 10.1172/JCI136227 32609095
Dufour, C., Gantner, P., Fromentin, R. & Chomont, N. The multifaceted nature of HIV latency. J. Clin. Invest 130, 3381–3390 (2020).32609095 10.1172/JCI136227
4. Vigon L Provirus reactivation is impaired in HIV-1 infected individuals on treatment with dasatinib and antiretroviral therapy Biochem Pharm. 2021 192 114666 10.1016/j.bcp.2021.114666 34186065
Vigon, L. et al. Provirus reactivation is impaired in HIV-1 infected individuals on treatment with dasatinib and antiretroviral therapy. Biochem Pharm. 192, 114666 (2021).34186065 10.1016/j.bcp.2021.114666
5. Pitman MC Lau JSY McMahon JH Lewin SR Barriers and strategies to achieve a cure for HIV Lancet HIV 2018 5 e317 e328 10.1016/S2352-3018(18)30039-0 29893245
Pitman, M. C., Lau, J. S. Y., McMahon, J. H. & Lewin, S. R. Barriers and strategies to achieve a cure for HIV. Lancet HIV 5, e317–e328 (2018).29893245 10.1016/S2352-3018(18)30039-0
6. Chargin A Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood J. Clin. Microbiol 2015 53 60 66 10.1128/JCM.02539-14 25339403
Chargin, A. et al. Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood. J. Clin. Microbiol 53, 60–66 (2015).25339403 10.1128/JCM.02539-14
7. Uldrick TS Pembrolizumab induces HIV latency reversal in people living with HIV and cancer on antiretroviral therapy Sci. Transl. Med 2022 14 eabl3836 10.1126/scitranslmed.abl3836 35080914
Uldrick, T. S. et al. Pembrolizumab induces HIV latency reversal in people living with HIV and cancer on antiretroviral therapy. Sci. Transl. Med 14, eabl3836 (2022).35080914 10.1126/scitranslmed.abl3836
8. Gupta RK HIV-1 remission following CCR5Delta32/Delta32 haematopoietic stem-cell transplantation Nature 2019 568 244 248 10.1038/s41586-019-1027-4 30836379
Gupta, R. K. et al. HIV-1 remission following CCR5Delta32/Delta32 haematopoietic stem-cell transplantation. Nature 568, 244–248 (2019).30836379 10.1038/s41586-019-1027-4
9. Deeks SG HIV: Shock and kill Nature 2012 487 439 440 10.1038/487439a 22836995
Deeks, S. G. HIV: Shock and kill. Nature 487, 439–440 (2012).22836995 10.1038/487439a
10. Dai B Xiao L Bryson PD Fang J Wang P PD-1/PD-L1 blockade can enhance HIV-1 Gag-specific T cell immunity elicited by dendritic cell-directed lentiviral vaccines Mol. Ther. 2012 20 1800 1809 10.1038/mt.2012.98 22588271
Dai, B., Xiao, L., Bryson, P. D., Fang, J. & Wang, P. PD-1/PD-L1 blockade can enhance HIV-1 Gag-specific T cell immunity elicited by dendritic cell-directed lentiviral vaccines. Mol. Ther. 20, 1800–1809 (2012).22588271 10.1038/mt.2012.98
11. Chen L Co-inhibitory molecules of the B7–CD28 family in the control of T-cell immunity Nat. Rev. Immunol. 2004 4 336 347 10.1038/nri1349 15122199
Chen, L. Co-inhibitory molecules of the B7–CD28 family in the control of T-cell immunity. Nat. Rev. Immunol. 4, 336–347 (2004).15122199 10.1038/nri1349
12. Zhang F Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade Cell Discov. 2017 3 17004 10.1038/celldisc.2017.4 28280600
Zhang, F. et al. Structural basis of a novel PD-L1 nanobody for immune checkpoint blockade. Cell Discov. 3, 17004 (2017).28280600 10.1038/celldisc.2017.4
13. Li J Subcutaneous envafolimab monotherapy in patients with advanced defective mismatch repair/microsatellite instability high solid tumors J. Hematol. Oncol. 2021 14 95 10.1186/s13045-021-01095-1 34154614
Li, J. et al. Subcutaneous envafolimab monotherapy in patients with advanced defective mismatch repair/microsatellite instability high solid tumors. J. Hematol. Oncol. 14, 95 (2021).34154614 10.1186/s13045-021-01095-1
14. Papadopoulos KP First-in-Human Phase I Study of Envafolimab, a Novel Subcutaneous Single-Domain Anti-PD-L1 Antibody, in Patients with Advanced Solid Tumors Oncologist 2021 26 e1514 e1525 10.1002/onco.13817 33973293
Papadopoulos, K. P. et al. First-in-Human Phase I Study of Envafolimab, a Novel Subcutaneous Single-Domain Anti-PD-L1 Antibody, in Patients with Advanced Solid Tumors. Oncologist 26, e1514–e1525 (2021).33973293 10.1002/onco.13817
15. Tio M Anti-PD-1/PD-L1 immunotherapy in patients with solid organ transplant, HIV or hepatitis B/C infection Eur. J. Cancer 2018 104 137 144 10.1016/j.ejca.2018.09.017 30347289
Tio, M. et al. Anti-PD-1/PD-L1 immunotherapy in patients with solid organ transplant, HIV or hepatitis B/C infection. Eur. J. Cancer 104, 137–144 (2018).30347289 10.1016/j.ejca.2018.09.017
16. Day CL PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression Nature 2006 443 350 354 10.1038/nature05115 16921384
Day, C. L. et al. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443, 350–354 (2006).16921384 10.1038/nature05115
17. Porichis F Responsiveness of HIV-specific CD4 T cells to PD-1 blockade Blood 2011 118 965 974 10.1182/blood-2010-12-328070 21652684
Porichis, F. et al. Responsiveness of HIV-specific CD4 T cells to PD-1 blockade. Blood 118, 965–974 (2011).21652684 10.1182/blood-2010-12-328070
18. Palmer BE In vivo blockade of the PD-1 receptor suppresses HIV-1 viral loads and improves CD4+ T cell levels in humanized mice J. Immunol. 2013 190 211 219 10.4049/jimmunol.1201108 23209326
Palmer, B. E. et al. In vivo blockade of the PD-1 receptor suppresses HIV-1 viral loads and improves CD4+ T cell levels in humanized mice. J. Immunol. 190, 211–219 (2013).23209326 10.4049/jimmunol.1201108
19. Trautmann L Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction Nat. Med 2006 12 1198 1202 10.1038/nm1482 16917489
Trautmann, L. et al. Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat. Med 12, 1198–1202 (2006).16917489 10.1038/nm1482
20. Munoz O Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells PLoS Pathog. 2022 18 e1010673 10.1371/journal.ppat.1010673 35788752
Munoz, O. et al. Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells. PLoS Pathog. 18, e1010673 (2022).35788752 10.1371/journal.ppat.1010673
21. Gubser C Chiu C Lewin SR Rasmussen TA Immune checkpoint blockade in HIV EBioMedicine 2022 76 103840 10.1016/j.ebiom.2022.103840 35123267
Gubser, C., Chiu, C., Lewin, S. R. & Rasmussen, T. A. Immune checkpoint blockade in HIV. EBioMedicine 76, 103840 (2022).35123267 10.1016/j.ebiom.2022.103840
22. McGary CS Silvestri G Paiardini M Animal models for viral infection and cell exhaustion Curr. Opin. HIV AIDS 2014 9 492 499 10.1097/COH.0000000000000093 25023622
McGary, C. S., Silvestri, G. & Paiardini, M. Animal models for viral infection and cell exhaustion. Curr. Opin. HIV AIDS 9, 492–499 (2014).25023622 10.1097/COH.0000000000000093
23. Gay CL Clinical Trial of the Anti-PD-L1 Antibody BMS-936559 in HIV-1 Infected Participants on Suppressive Antiretroviral Therapy J. Infect. Dis. 2017 215 1725 1733 10.1093/infdis/jix191 28431010
Gay, C. L. et al. Clinical Trial of the Anti-PD-L1 Antibody BMS-936559 in HIV-1 Infected Participants on Suppressive Antiretroviral Therapy. J. Infect. Dis. 215, 1725–1733 (2017).28431010 10.1093/infdis/jix191
24. Evans VA Programmed cell death-1 contributes to the establishment and maintenance of HIV-1 latency AIDS 2018 32 1491 1497 10.1097/QAD.0000000000001849 29746296
Evans, V. A. et al. Programmed cell death-1 contributes to the establishment and maintenance of HIV-1 latency. AIDS 32, 1491–1497 (2018).29746296 10.1097/QAD.0000000000001849
25. Guihot A Drastic decrease of the HIV reservoir in a patient treated with nivolumab for lung cancer Ann. Oncol. 2018 29 517 518 10.1093/annonc/mdx696 29206889
Guihot, A. et al. Drastic decrease of the HIV reservoir in a patient treated with nivolumab for lung cancer. Ann. Oncol. 29, 517–518 (2018).29206889 10.1093/annonc/mdx696
26. Gonzalez-Cao M Assessment of the Feasibility and Safety of Durvalumab for Treatment of Solid Tumors in Patients With HIV-1 Infection: The Phase 2 DURVAST Study JAMA Oncol. 2020 6 1063 1067 10.1001/jamaoncol.2020.0465 32271353
Gonzalez-Cao, M. et al. Assessment of the Feasibility and Safety of Durvalumab for Treatment of Solid Tumors in Patients With HIV-1 Infection: The Phase 2 DURVAST Study. JAMA Oncol. 6, 1063–1067 (2020).32271353 10.1001/jamaoncol.2020.0465
27. Rodari A Darcis G Van Lint CM The Current Status of Latency Reversing Agents for HIV-1 Remission Annu Rev. Virol. 2021 8 491 514 10.1146/annurev-virology-091919-103029 34586875
Rodari, A., Darcis, G. & Van Lint, C. M. The Current Status of Latency Reversing Agents for HIV-1 Remission. Annu Rev. Virol. 8, 491–514 (2021).34586875 10.1146/annurev-virology-091919-103029
28. Kuai Q Histone deacetylase inhibitor chidamide promotes reactivation of latent human immunodeficiency virus by introducing histone acetylation J. Med Virol. 2018 90 1478 1485 10.1002/jmv.25207 29704439
Kuai, Q. et al. Histone deacetylase inhibitor chidamide promotes reactivation of latent human immunodeficiency virus by introducing histone acetylation. J. Med Virol. 90, 1478–1485 (2018).29704439 10.1002/jmv.25207
29. Abbas W Herbein G Molecular Understanding of HIV-1 Latency Adv. Virol. 2012 2012 574967 10.1155/2012/574967 22548060
Abbas, W. & Herbein, G. Molecular Understanding of HIV-1 Latency. Adv. Virol. 2012, 574967 (2012).22548060 10.1155/2012/574967
30. Ning ZQ Chidamide (CS055/HBI-8000): a new histone deacetylase inhibitor of the benzamide class with antitumor activity and the ability to enhance immune cell-mediated tumor cell cytotoxicity Cancer Chemother. Pharm. 2012 69 901 909 10.1007/s00280-011-1766-x
Ning, Z. Q. et al. Chidamide (CS055/HBI-8000): a new histone deacetylase inhibitor of the benzamide class with antitumor activity and the ability to enhance immune cell-mediated tumor cell cytotoxicity. Cancer Chemother. Pharm. 69, 901–909 (2012).10.1007/s00280-011-1766-x
31. Shi Y Chidamide in relapsed or refractory peripheral T cell lymphoma: a multicenter real-world study in China J. Hematol. Oncol. 2017 10 69 10.1186/s13045-017-0439-6 28298231
Shi, Y. et al. Chidamide in relapsed or refractory peripheral T cell lymphoma: a multicenter real-world study in China. J. Hematol. Oncol. 10, 69 (2017).28298231 10.1186/s13045-017-0439-6
32. Yang W Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus Microbes Infect. 2018 20 626 634 10.1016/j.micinf.2017.10.003 29126877
Yang, W. et al. Chidamide, a histone deacetylase inhibitor-based anticancer drug, effectively reactivates latent HIV-1 provirus. Microbes Infect. 20, 626–634 (2018).29126877 10.1016/j.micinf.2017.10.003
33. Li JH The histone deacetylase inhibitor chidamide induces intermittent viraemia in HIV-infected patients on suppressive antiretroviral therapy HIV Med 2020 21 747 757 10.1111/hiv.13027 33369029
Li, J. H. et al. The histone deacetylase inhibitor chidamide induces intermittent viraemia in HIV-infected patients on suppressive antiretroviral therapy. HIV Med 21, 747–757 (2020).33369029 10.1111/hiv.13027
34. Booth L Roberts JL Poklepovic A Dent P [pemetrexed + sildenafil], via autophagy-dependent HDAC downregulation, enhances the immunotherapy response of NSCLC cells Cancer Biol. Ther. 2017 18 705 714 10.1080/15384047.2017.1362511 28812434
Booth, L., Roberts, J. L., Poklepovic, A. & Dent, P. [pemetrexed + sildenafil], via autophagy-dependent HDAC downregulation, enhances the immunotherapy response of NSCLC cells. Cancer Biol. Ther. 18, 705–714 (2017).28812434 10.1080/15384047.2017.1362511
35. Booth L Roberts JL Poklepovic A Kirkwood J Dent P HDAC inhibitors enhance the immunotherapy response of melanoma cells Oncotarget 2017 8 83155 83170 10.18632/oncotarget.17950 29137331
Booth, L., Roberts, J. L., Poklepovic, A., Kirkwood, J. & Dent, P. HDAC inhibitors enhance the immunotherapy response of melanoma cells. Oncotarget 8, 83155–83170 (2017).29137331 10.18632/oncotarget.17950
36. Siliciano JD Siliciano RF HIV-1 eradication strategies: design and assessment Curr. Opin. HIV AIDS 2013 8 318 325 23698561
Siliciano, J. D. & Siliciano, R. F. HIV-1 eradication strategies: design and assessment. Curr. Opin. HIV AIDS 8, 318–325 (2013).23698561
37. Archin NM Margolis DM Emerging strategies to deplete the HIV reservoir Curr. Opin. Infect. Dis. 2014 27 29 35 10.1097/QCO.0000000000000026 24296585
Archin, N. M. & Margolis, D. M. Emerging strategies to deplete the HIV reservoir. Curr. Opin. Infect. Dis. 27, 29–35 (2014).24296585 10.1097/QCO.0000000000000026
38. Davenport MP Functional cure of HIV: the scale of the challenge Nat. Rev. Immunol. 2018 19 45 54 10.1038/s41577-018-0085-4
Davenport, M. P. et al. Functional cure of HIV: the scale of the challenge. Nat. Rev. Immunol. 19, 45–54 (2018).10.1038/s41577-018-0085-4
39. McBrien JB Kumar NA Silvestri G Mechanisms of CD8(+) T cell-mediated suppression of HIV/SIV replication Eur. J. Immunol. 2018 48 898 914 10.1002/eji.201747172 29427516
McBrien, J. B., Kumar, N. A. & Silvestri, G. Mechanisms of CD8(+) T cell-mediated suppression of HIV/SIV replication. Eur. J. Immunol. 48, 898–914 (2018).29427516 10.1002/eji.201747172
40. Fenwick C T-cell exhaustion in HIV infection Immunol. Rev. 2019 292 149 163 10.1111/imr.12823 31883174
Fenwick, C. et al. T-cell exhaustion in HIV infection. Immunol. Rev. 292, 149–163 (2019).31883174 10.1111/imr.12823
41. Chugh P Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy Retrovirology 2008 5 11 10.1186/1742-4690-5-11 18237430
Chugh, P. et al. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy. Retrovirology 5, 11 (2008).18237430 10.1186/1742-4690-5-11
42. Parsa AT Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma Nat. Med 2007 13 84 88 10.1038/nm1517 17159987
Parsa, A. T. et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat. Med 13, 84–88 (2007).17159987 10.1038/nm1517
43. Sharma P Hu-Lieskovan S Wargo JA Ribas A Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy Cell 2017 168 707 723 10.1016/j.cell.2017.01.017 28187290
Sharma, P., Hu-Lieskovan, S., Wargo, J. A. & Ribas, A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell 168, 707–723 (2017).28187290 10.1016/j.cell.2017.01.017
44. Zhang J Lei F Tan H The development of CD8 T-cell exhaustion heterogeneity and the therapeutic potentials in cancer Front Immunol. 2023 14 1166128 10.3389/fimmu.2023.1166128 37275913
Zhang, J., Lei, F. & Tan, H. The development of CD8 T-cell exhaustion heterogeneity and the therapeutic potentials in cancer. Front Immunol. 14, 1166128 (2023).37275913 10.3389/fimmu.2023.1166128
45. Sun C Mezzadra R Schumacher TN Regulation and Function of the PD-L1 Checkpoint Immunity 2018 48 434 452 10.1016/j.immuni.2018.03.014 29562194
Sun, C., Mezzadra, R. & Schumacher, T. N. Regulation and Function of the PD-L1 Checkpoint. Immunity 48, 434–452 (2018).29562194 10.1016/j.immuni.2018.03.014
46. Beltra JC Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms Immunity 2020 52 825 841.e828 10.1016/j.immuni.2020.04.014 32396847
Beltra, J. C. et al. Developmental Relationships of Four Exhausted CD8(+) T Cell Subsets Reveals Underlying Transcriptional and Epigenetic Landscape Control Mechanisms. Immunity 52, 825–841.e828 (2020).32396847 10.1016/j.immuni.2020.04.014
47. Zheng L Tierney C Bosch RJ Analytical Treatment Interruption in HIV Trials: Statistical and Study Design Considerations Curr. HIV/AIDS Rep. 2021 18 475 482 10.1007/s11904-021-00569-8 34213731
Zheng, L., Tierney, C. & Bosch, R. J. Analytical Treatment Interruption in HIV Trials: Statistical and Study Design Considerations. Curr. HIV/AIDS Rep. 18, 475–482 (2021).34213731 10.1007/s11904-021-00569-8
48. Borducchi EN Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys Nature 2018 563 360 364 10.1038/s41586-018-0600-6 30283138
Borducchi, E. N. et al. Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys. Nature 563, 360–364 (2018).30283138 10.1038/s41586-018-0600-6
49. Clutton GT CD3 downregulation identifies high-avidity human CD8 T cells Clin. Exp. Immunol. 2024 215 279 290 10.1093/cei/uxad124 37950348
Clutton, G. T. et al. CD3 downregulation identifies high-avidity human CD8 T cells. Clin. Exp. Immunol. 215, 279–290 (2024).37950348 10.1093/cei/uxad124
50. Rotte A Combination of CTLA-4 and PD-1 blockers for treatment of cancer J. Exp. Clin. Cancer Res. 2019 38 255 10.1186/s13046-019-1259-z 31196207
Rotte, A. Combination of CTLA-4 and PD-1 blockers for treatment of cancer. J. Exp. Clin. Cancer Res. 38, 255 (2019).31196207 10.1186/s13046-019-1259-z
51. Knudson KM Hicks KC Alter S Schlom J Gameiro SR Mechanisms involved in IL-15 superagonist enhancement of anti-PD-L1 therapy J. Immunother. Cancer 2019 7 82 10.1186/s40425-019-0551-y 30898149
Knudson, K. M., Hicks, K. C., Alter, S., Schlom, J. & Gameiro, S. R. Mechanisms involved in IL-15 superagonist enhancement of anti-PD-L1 therapy. J. Immunother. Cancer 7, 82 (2019).30898149 10.1186/s40425-019-0551-y
52. Bruner KM A novel quantitative approach for measuring the reservoir of latent HIV-1 proviruses Nature 2019 566 120 125 10.1038/s41586-019-0898-8 30700913
Bruner, K. M. et al. A novel quantitative approach for measuring the reservoir of latent HIV-1 proviruses. Nature 566, 120–125 (2019).30700913 10.1038/s41586-019-0898-8
53. Hong F Novel assays for measurement of total cell-associated HIV-1 DNA and RNA J. Clin. Microbiol 2016 54 902 911 10.1128/JCM.02904-15 26763968
Hong, F. et al. Novel assays for measurement of total cell-associated HIV-1 DNA and RNA. J. Clin. Microbiol 54, 902–911 (2016).26763968 10.1128/JCM.02904-15
54. Behera SK Das S Xavier AS Velupula S Sandhiya S Comparison of different methods for causality assessment of adverse drug reactions Int J. Clin. Pharm. 2018 40 903 910 10.1007/s11096-018-0694-9 30051231
Behera, S. K., Das, S., Xavier, A. S., Velupula, S. & Sandhiya, S. Comparison of different methods for causality assessment of adverse drug reactions. Int J. Clin. Pharm. 40, 903–910 (2018).30051231 10.1007/s11096-018-0694-9
55. Wan LY Distinct inflammation-related proteins associated with T cell immune recovery during chronic HIV-1 infection Emerg. Microbes Infect. 2023 12 2150566 10.1080/22221751.2022.2150566 36408648
Wan, L. Y. et al. Distinct inflammation-related proteins associated with T cell immune recovery during chronic HIV-1 infection. Emerg. Microbes Infect. 12, 2150566 (2023).36408648 10.1080/22221751.2022.2150566
