Immune restoration by TIGIT blockade is insufficient to control chronic SIV infection

ABSTRACT

TIGIT is a negative immune checkpoint receptor associated with T cell exhaustion in cancer and HIV. TIGIT upregulation in virus-specific CD8⁺ T cells and NK cells during HIV/SIV infection results in dysfunctional effector capabilities. In vitro studies targeting TIGIT on CD8⁺ T cells suggest TIGIT blockade as a viable strategy to restore SIV-specific T cell responses. Here, we extend these studies in vivo using TIGIT blockage in nonhuman primates in an effort to reverse T cell and NK cell exhaustion in the setting of SIV infection. We demonstrate that in vivo administration of a humanized anti-TIGIT monoclonal antibody (mAb) is well tolerated in both cynomolgus macaques and rhesus macaques. Despite sustained plasma concentrations of anti-TIGIT mAb, we observed no consistent improvement in NK or T cell cytolytic capacity. TIGIT blockade minimally enhanced T cell proliferation and virus-specific T cell responses in both magnitude and breadth though plasma viral loads in treated animals remained stable indicating that anti-TIGIT mAb treatment alone was insufficient to increase anti-SIV CD8⁺ T cell function. The enhancement of virus-specific T cell proliferative responses observed in vitro with single or dual blockade of TIGIT and/or PD-1 highlights TIGIT as a potential target to reverse T cell dysfunction. Our studies, however, reveal that targeting the TIGIT pathway alone may be insufficient in the setting of viremia and that combining immune checkpoint blockade with other immunotherapeutics may be a future path forward for improved viral control or elimination of HIV.

IMPORTANCE

Upregulation of the immune checkpoint receptor TIGIT is associated with HIV-mediated T cell dysfunction and correlates with HIV disease progression. Compelling evidence exists for targeting immune checkpoint receptor pathways that would potentially enhance immunity and refocus effector cell efforts toward viral clearance. In this report, we investigate TIGIT blockade as an immunotherapeutic approach to reverse immune exhaustion during chronic SIV/SHIV infection in a nonhuman primate model of HIV infection. We show that interfering with the TIGIT signaling axis alone is insufficient to improve viral control despite modest improvement in T cell immunity. Our data substantiate the use of targeting multiple immune checkpoint receptors to promote synergy and ultimately eliminate HIV-infected cells.

INTRODUCTION

Immune checkpoint receptors, or inhibitory receptors, are upregulated upon T cell activation and are necessary for balancing the quality of T cell responses to effectively combat infection without driving autoimmunity. Conditions of persistent antigen exposure and T cell activation seen in chronic viral infections and cancer lead to a state of immune dysfunction deemed “exhaustion,” where immune checkpoint receptor expression fails to decline and T cell effector functions remain attenuated and incapable of clearing the offending antigen (1). This defect in critical T cell function is a hallmark of human and simian immunodeficiency virus (HIV/SIV) infection in humans and nonhuman primates (NHP) and contributes to virus persistence (2, 3). We and others have shown several immune checkpoint receptors including programmed death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin-domain containing-3 (TIM-3), and T cell immunoreceptor with Ig and ITIM domain (TIGIT) are highly expressed by exhausted T cells in the context of HIV and SIV infection (4–12). Immune checkpoint blockade (ICB), namely targeting PD-1 and CTLA-4 for blockade by monoclonal antibodies, has demonstrated clinical efficacy in cancer models, reversing T cell exhaustion and preventing further attrition of T cells (13, 14). Due to these promising results observed in cancer clinical trials, ICB has been a focus as a viable route to reinvigorate immune function in people living with HIV (PLWH).

For over a decade, substantial efforts to reverse immune dysfunction in the context of HIV/SIV infection have been limited to PD-1 and CTLA-4 blockade in preclinical and clinical studies yielding variable results. During chronic SIV infection, PD-1 blockade decreased set-point viremia and improved virus-specific T cell responses but failed to improve antiviral immunity in the context of antiretroviral therapy (ART) (15–17). Studies investigating the blockade of CTLA-4 in SIV-infected rhesus macaques (RM) have shown inconsistent effects on both viral replication and CD8⁺ T cell immunity (18, 19). More recently, simultaneous targeting of PD-1 and CTLA-4 pathways in SIV-infected, ART-suppressed macaques showed that dual blockade was more successful than PD-1 alone in improving T cell responses and inducing viral latency (20). Given that signaling pathways activated downstream of immune checkpoint receptors are non-redundant, targeting multiple pathways may have a synergistic effect and lead to more robust immunity. Current ICB interventions have failed to consistently improve anti-HIV immunity and decrease the latent reservoir; therefore, there is a need for fundamentally different approaches and/or targets to enhance existing HIV cure strategies. We previously identified TIGIT as a correlate of HIV-associated immune dysfunction and a novel ICB target to reverse T cell exhaustion (10).

TIGIT belongs to the immunoglobulin superfamily, and its ligands include poliovirus receptor (PVR), also known as CD155, as well as CD112 (21). TIGIT binds to PVR by outcompeting other receptors, CD226 (DNAM-1), a costimulatory molecule, and CD96 (TACTILE), a coinhibitory molecule. Due to its high affinity binding to PVR, TIGIT can effectively block the binding of PVR to CD226 and CD96, thus favoring cellular inhibition and ultimately dampening the immune response (22–24). TIGIT can also form a heterodimer with CD226, preventing effective CD226/ligand interactions and further reducing cellular costimulation (25). We and others have shown that TIGIT expression is upregulated on dysfunctional HIV and SIV-specific CD8⁺ T cells and NK cells and correlates with HIV disease progression (10, 26, 27). Additionally, CD4⁺ T cells harboring the HIV reservoir express immune checkpoint receptors PD-1, LAG-3, and TIGIT either alone or in combination (28). Given that TIGIT impacts various signaling cascades in a variety of effector cell types, targeting the TIGIT signaling axis via ICB is a compelling strategy to mediate HIV cure. Indeed, in vitro studies using single and dual blockade of TIGIT/PD-1 enhanced proliferation of HIV- and SIV-specific CD8⁺ T cells and reinvigorated NK cell HIV-1-specific antibody-dependent cytotoxicity underscoring the importance of this pathway in modulating immune function during HIV/SIV infection (10, 28). Here, we use a nonhuman primate model of HIV infection to evaluate the safety and efficacy of in vivo TIGIT blockade on restoration of T cell immunity and ultimately control of virus infection.

RESULTS

Characterization of human anti-TIGIT mAb, WBP2117

We and others have shown in vitro blockade of the TIGIT axis can lead to enhanced NK cell and T cell effector function in HIV (10, 29) as well as in various cancer models (30). Thus, here, we evaluated whether the human TIGIT-specific humanized IgG1 monoclonal antibody, WBP2117, could contain SIV replication in a pre-clinical NHP model of HIV infection (10). We first measured the ability of WBP2117 to bind to TIGIT on the surface of human, Mauritian cynomolgus macaque (MCM), and rhesus macaque (RM) peripheral blood mononuclear cells (PBMC). We directly conjugated WBP2117 to fluorescein isothiocyanate (FITC) and compared its binding to a phycoerythrin (PE) labeled, commercially available anti-TIGIT mAb, clone MSBA43 via sequential costaining, and flow cytometry (Fig. 1). As expected, WBP2117 effectively cross-reacted with human TIGIT expressed on NK, CD4⁺, and CD8⁺ T cells, binding to about 70% of TIGIT expressed on the surface of human PBMC. WBP2117 displayed improved binding to RM TIGIT compared to MCM TIGIT, recognizing about 30% of total RM TIGIT on T cells and about 40% on NK cells in vitro and, in some cases, recognizing upward of 50% of RM TIGIT on CD8⁺ T cells and NK cells. For MCM TIGIT, WBP2117 was minimally reactive, binding an average of 10%–20% of TIGIT on T cells and NK cells. These results indicate that WBP2117 binds to RM and MCM TIGIT to varying degrees. The variation in reactivity between RM and MCM must be accounted for, but overall provides us with two potential nonhuman primate models to evaluate the in vivo safety and efficacy of WBP2117 as an ICB in the context of SIV/SHIV infection.

Fig 1.

WBP2117 binding to human, RM, and MCM white blood cells in vitro. Isolated peripheral blood mononuclear cells (PBMC) from human, Mauritian cynomolgus macaques (MCM), and rhesus macaques (RM) were stained with a commercially available anti-TIGIT antibody (eBioscience, clone MBSA43) and anti-TIGIT monoclonal antibody, WBP2117. Costaining was assessed on CD8⁺ T cells, CD4⁺ T cells, and NK cells. (A) Representative flow cytometry plots showing TIGIT costaining on CD8⁺ T cells, CD4⁺ T cells, and NK cells. (B) Graph shows compiled data of TIGIT costaining with human PBMC (n = 10), MCM PBMC (n = 10), and RM PBMC (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

TIGIT blockade is well-tolerated in both rhesus macaques and Mauritian cynomolgus macaques

To determine the efficacy of TIGIT blockade in an NHP model of HIV, we treated both chronically SIV-infected MCM (n = 3) and SHIV-infected RM (n = 2) with WBP2117. The anti-TIGIT mAb was administered as biweekly, intravenous infusions at a dose of 10 mg/kg for a total of five infusions over the course of nine weeks (Fig. 2A). Three SIV-infected MCM were left untreated and served as experimental controls. WBP2117 was well-tolerated with no evidence of treatment-related clinical pathology with no notable changes in hematology parameters measured by complete blood count and no evidence of hepatoxicity or nephrotoxicity measured by serum concentrations of alkaline phosphatase (ALP), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (Fig. S1). We monitored the concentration of WBP2117 in plasma throughout the study and multiple infusions of WBP2117 resulted in sustained plasma concentrations with peak plasma concentrations ranging between 10 and 80 μg/mL (Fig. 2B). Animals elicited anti-drug antibody (ADA) responses after the first infusion, but the development of ADA did not lead to increased clearance of WBP2117 in plasma. One animal (MCM4) developed ADA after the first infusion of WBP2117, concomitant with rapid clearance of the mAb in plasma (Fig. 2C). Because WBP2117 is a human antibody, MCM4 experienced symptoms of allergic reaction including tachycardia, hypotension, and hypoxemia at week 4 after the third infusion and was subsequently removed from further study at week 6.

Fig 2.

In vivo administration of anti-TIGIT mAb, WBP2117. (A) SIV-infected Mauritian cynomolgus macaques (MCM, n = 3) and SHIV-infected rhesus macaques (RM, n = 2) were treated with five biweekly intravenous infusions of anti-TIGIT mAb, WBP2117 at a dose of 10 mg/kg. Three additional SIV-infected Mauritian cynomolgus macaques were left untreated. Blood samples were collected weekly, as well as additional blood samples at 1, 8, 24, 48 h post-mAb infusion. (B) Plasma was assessed for WBP2117 levels and (C) antidrug antibodies (ADA) against WBP2117 over the course of the study via ELISA. Blood was drawn prior to WBP2117 infusions. Vertical dashed lines indicate WBP2117 administration.

Transient changes in T cell proliferative and effector responses after TIGIT blockade

In the context of HIV and SIV infection, TIGIT is upregulated on virus-specific CD8⁺ T cells and NK cells, even in the presence of ART, contributing to the state of immune dysfunction characteristic of chronic HIV/SIV infection. Additionally, TIGIT has been shown to be enriched, alone or in combination with other immune checkpoint receptors, in a subset of CD4⁺ T cells harboring latent HIV (28). Given the significance of TIGIT expression in the setting of HIV and SIV infection, we investigated the impact of sustained TIGIT blockade on T cells and NK cells in blood. In MCM, the frequency and absolute numbers of CD4⁺ and CD8⁺ T cells as well as NK cells in blood were not significantly influenced by TIGIT blockade. In RM, CD4⁺ T cell frequencies transiently increased concordant with WBP2117 administration, which was reflected in the absolute counts as compared to control animals. The frequency and absolute counts of CD8⁺ T cells, however, were largely unimpacted. NK cells in RMs, notably in RM2, exhibited transient increases in frequency and absolute counts over the course of the study (Fig. 3). Additionally, we noted no consistent change in T cell memory populations nor SIV/SHIV-specific binding antibodies during TIGIT blockade (Fig. S2 and S3).

Fig 3.

Longitudinal T cell and NK cell frequencies and absolute counts in blood. (A) CD4⁺ T cell and (B) CD8⁺ T cell frequency as a percent of CD3⁺ T cells and as a percent of CD45⁺ cells, as well as absolute counts were monitored over the course of the study. (C) CD3-CD8⁺ NK cell frequency and absolute counts were also monitored. Absolute counts were calculated based on the percentage of the cell subset and the WBC count. Vertical dashed lines indicate WBP2117 administration. Weekly timepoints shown here are predose samples.

Targeting the TIGIT axis is predicted to ameliorate the proliferative and cytotoxic capabilities of CD8⁺ T cells and NK cells. Both MCM and RM experienced transient increases in Ki67 expression in CD4⁺ and CD8⁺ T cells in peripheral blood shortly after WBP2117 administration, but not after every dose (Fig. 4A). We also evaluated the impact of TIGIT blockade on the intracellular granular content and cellular proliferation of blood CD4⁺ and CD8⁺ T cells and NK cells. Our immunophenotypic analysis revealed little change in perforin and granzyme B in T cells or NK cells in animals that received WBP2117 (Fig. 4B). We also assessed TIGIT blockade and its ability to enhance NK cell or CD8⁺ T cell-mediated viral suppression in vitro (Fig. S4). In accordance with our in vivo observations, WBP2117 failed to improve cytotoxic capacity of NK cells or CD8⁺ T cells. Interestingly, we compared TIGIT blockade to PDL1 blockade and found that PDL1 blockade significantly improved NK cell-mediated killing of virus-infected cells. This effect of improved NK cell activity with PDL1 blockade has been demonstrated in cancer models, but this is evidence of the potential benefits of PDL1 inhibition on NK cell cytolytic capacity in the context of SIV infection, which warrants further exploration (31). Taken together, these results show that WBP2117 mAb treatment moderately increased T cell and NK cell proliferation but did not enhance the cytotoxic effects of T cells or NK cells.

Fig 4.

TIGIT blockade induces transient fluctuations in T cell and NK cell activation and proliferation. (A) Proliferation of CD4⁺ T cells, CD8⁺ T cells, and NK cells measured by nuclear antigen Ki67 was longitudinally evaluated in peripheral blood. (B) Perforin and granzyme B were also monitored longitudinally as a measure of cytotoxicity in CD4+ and CD8+ T cells as well as NK cells. Vertical dashed lines indicate WBP2117 administration. Weekly timepoints shown here are predose samples.

In vivo TIGIT blockade in RM increases SIV Gag-specific T cell responses in both magnitude and breadth but is insufficient to impact viral load

To further explore the impact of TIGIT blockade on the virus-specific T cell responses in MCM and RM, longitudinal PBMC from all study animals were stimulated ex vivo with SIV Gag peptide pool covering the entire open reading frame (ORF) and assayed for secretion of IFN-γ, TNF-α, and IL-2. TIGIT blockade in both MCM and RM failed to consistently enhance production and secretion of these critical effector cytokines (Fig. 5A). Additionally, we used an IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay to more closely evaluate changes in the magnitude and breadth of Gag-specific T cells responses in ex vivo PBMC over the course of the study. These analyses revealed an increase in the magnitude of SIV Gag-specific T cell responses to Gag peptide pools and whole Gag ORF in the RM group and in MCM5 compared to baseline levels but not in the control animals (Fig. 5B and C). These animals also exhibited increased cellular immune breadth measured by positive responses against subpools of Gag. The increased Gag-specific T cell responses observed were short-lived however, peaking between weeks 4 and 6 after 2–3 repeated mAb infusions and then declining over time. No differences in the magnitude and breadth of SIV Gag responses were observed in MCM treated with TIGIT blockade. Collectively, these results indicate that TIGIT blockade transiently improves SIV-specific T cell responses in NHP.

Fig 5.

In vivo TIGIT blockade increases the magnitude and breadth of Gag-specific CD8+ T cells. (A) Longitudinal monitoring of CD4+ (left) and CD8+ (right) T cell responses against SIV Gag from control and WBP2117-treated MCM and RMs as measured by cytokine secretion of TNFα (top), IFNγ (middle) and IL-2 (bottom). Positive responses shown are background subtracted from unstimulated cells at each time point. Vertical dashed lines indicate WBP2117 administration. (B) IFNγ spot-forming cells (SFC) per 10⁵ mononuclear cells were quantified by ELISPOT upon SIV-Gag stimulation using 13 pools of overlapping Gag peptides (Gag A-M) spanning the entire Gag protein sequence or (C) the entire Gag open reading frame (Gag ORF). Note, there is no ELIspot data from MCM6 as there were insufficient cells to carry out the assay. Weekly timepoints shown here are predose samples.

TIGIT contributes to HIV persistence by facilitating CD8⁺ T cell dysfunction and promoting virus latency (10, 27, 28, 32). Thus, inhibition of the TIGIT signaling axis may not only improve T cell responses but may also function as a latency-reversing agent (LRA) by increasing viral transcription. To evaluate the efficacy of TIGIT blockade on viral replication, we longitudinally measured SIV and SHIV RNA in plasma. All animals were chronically infected with either SIVmac239 or SHIVAD8EOM and had varying plasma viremia (10²–10⁶ copies/mL plasma) prior to TIGIT blockade. We observed stochastic fluctuations in plasma viral loads in the control animals as well as in WBP2117-treated MCM and RM (Fig. 6). Animals that received TIGIT blockade showed no sustained effect on plasma viral loads revealing that TIGIT blockade alone does not have a measurable impact on viral replication. RM1, however, did experience a 4-fold decrease in plasma RNA over the course of the study, dropping from 1.8 × 10³ to 4.3 × 10² SHIV RNA copies/mL, concomitant with an increased Gag-specific T cell response observed via ELISPOT (Fig. 5B). This result shows that although TIGIT blockade with WBP2117 may improve T cell responses, it is insufficient to significantly reduce plasma viral loads in SIV/SHIV-infected macaques.

Fig 6.

In vivo TIGIT blockade during chronic SIV and SHIV infection has no sustained effect on plasma viral loads. (A) Longitudinal plasma viral loads. Vertical dashed lines indicate WBP2117 administration at weeks 0, 2, 4, 6, 8. Horizontal dashed line indicates the limit of quantification (LOQ), which is 15 copies/mL plasma. (B) Fold change in plasma viral load between week 0 and week 9. Weekly timepoints shown here are predose samples.

Our results demonstrate that in vivo blockade of TIGIT is overall safe and well-tolerated in NHP and results in a transient restoration of virus-specific T cell responses. Despite improved T cell function, WBP2117 mAb treatment failed to significantly impact plasma viral loads, demonstrating that TIGIT blockade is not sufficient for sustained reversal of T cell dysfunction to ultimately eliminate HIV-infected cells.

DISCUSSION

The challenge of achieving functional cure of HIV is multifactorial including, but not limited to long-term persistence of HIV in latently infected cells, lack of reliable latency-reversing agents to induce potent viral reactivation, and T cell dysfunction driven by chronic immune activation. Immune checkpoint blockade has long been implicated in the reinvigoration of exhausted T cells in the context of cancer and may have a dual role as an HIV cure strategy by both restoring HIV-specific immunity and targeting latently infected cells. TIGIT is one of several immune checkpoint receptors that is both upregulated on exhausted CD8⁺ T cells and NK cells in PLWH and expressed on CD4⁺ T cells harboring latent HIV making it an attractive target to mediate HIV cure (10, 26, 28, 32, 33).

In this study, we evaluated the safety and efficacy of TIGIT-blocking monoclonal antibody, WBP2117 in SIV-infected MCM and SHIV-infected RM. We administered WBP2117 to five chronically SIV/SHIV-infected NHP in a biweekly dosing schedule where animals received five total doses of the mAb. Repeated monoclonal antibody infusion was well-tolerated in NHP indicated by stable serum chemistries and resulted in sustained mAb concentrations in plasma. One animal, MCM4, experienced a mild allergic reaction after the third dose of WBP2117 and out of abundance of caution the animal was removed from the study. Given that our study did not evaluate receptor occupancy on T cells or NK cells after WBP2117 administration, we cannot rule out the possibility that the absence of immune-related adverse events that we observed is due to low receptor occupancy as opposed to inherent tolerance of antibody-mediated TIGIT blockade. While WBP2117 mAb treatment elicited transient T cell activation and restored Gag-specific T cell responses, TIGIT blockade failed to improve NK cell or CD8⁺ T cell-mediated killing of infected cells in vitro and had no impact on plasma viral loads in chronically-infected NHP. Our results indicate that although in vivo TIGIT blockade may temporarily improve virus-specific immunity, it is not sufficient for prolonged reversal of T cell exhaustion needed to eliminate virus-infected cells, further validating the use of combination immune blockade or immunotherapy as a tactic for HIV cure.

To date, there have been a number of NHP studies evaluating the effects of immune checkpoint blockade on the reversal of T cell exhaustion in the context of SIV infection, specifically focusing on PD-1 and CTLA-4. CTLA-4 blockade has had varying outcomes in the setting of SIV infection, with or without ART, showing variable effects on virus-specific CD8⁺ T cell immunity and viral control (18, 19). In vivo treatment targeting PD-1 has similarly produced unremarkable results with short-lived enhancement of T cell immunity and transient decreases in plasma viremia during chronic SIV infection or post analytical treatment interruption (15, 34). This study is the first to soley target the TIGIT signaling axis in the setting of HIV/SIV infection. Similar to previous studies evaluating single blockade of immune checkpoint receptors, our study using a humanized IgG1 mAb against TIGIT resulted in transient immunological benefits that were ultimately insufficient to reduce plasma viremia. Given that the antibody is specific to human TIGIT and RM/MCM TIGIT shares about 90% sequence homology with human TIGIT, the incomplete binding to RM/MCM TIGIT we observed in vitro likely impacts its lack of efficacy in vivo (2). An important caveat of the study is that the data collected are exclusively generated from the blood, whereas T cell responses could differ in tissues particularly in lymph nodes. Indeed, it was recently shown that TIGIT expression is highly expressed in lymphatic tissue and reduced tissue penetrance of the mAb could result in suboptimal restoration of antiviral T cell responses (10, 20, 35). Nonetheless, TIGIT blockade did not reduce plasma viremia indicating it alone lacks the therapeutic potential necessary for HIV cure. Our data mirror previous studies in the LCMV model (36). Moving forward, immune checkpoint blockade as a therapeutic strategy for HIV could be enhanced by combining multiple immune checkpoint targets including combinations aimed at TIGIT, CTLA-4, LAG-3, and the PD-1/PD-L1 axis, which are currently being tested in clinical trials for multiple cancers (NCT01714739, NCT04150965, NCT04995523, NCT05130177, NCT05695898). It is important to note, however, that efficacy in oncology does not guarantee success in achieving HIV remission. For instance, combination immune checkpoint blockade with anti-CTLA-4 and anti-PD-1 showed superior therapeutic efficacy in clinical trials for melanoma patients but failed to impact reservoir clearance in SIV-infected macaques (37, 38), highlighting the need for additional and more potent interventions to achieve HIV remission while maintaining acceptable toxicity profiles. People living with HIV have been frequently excluded from clinical trials exploring immune checkpoint blockade for cancer malignancies; thus, there is limited data on the safety and efficacy of these therapies in the setting of HIV. Therefore, emerging clinical data using novel targets such as TIGIT and LAG-3 will be highly informative for future HIV studies. Likewise, preclinical studies in non-human primate models of HIV are indispensable for understanding how to balance therapeutic efficacy and immune-related toxicities while achieving HIV remission.

This study highlights the utility of NHP models for the pre-clinical development of interventions for HIV cure. We have demonstrated that although WBP2117 displayed poor crossreactivity to NHP TIGIT, TIGIT blockade in chronically-infected NHP without ART was still able to transiently trigger T cell proliferation and improve virus-specific T cell immunity but did not affect viral control of SIV/SHIV. Thus, it will be critical to improve the therapeutic potential of TIGIT blockade in NHP by increasing its binding efficacy and potentially testing it in combination with other ICB therapies or with other strategies such as therapeutic vaccination or broadly neutralizing antibodies. In conclusion, TIGIT blockade is insufficient to achieve HIV remission and may yield more promising results when given in concert with additional therapeutics.

MATERIALS AND METHODS

Reagents, animals, and veterinary procedures

A total of two female rhesus macaques (Macaca mulatta) and four female and one male Mauritian-origin cynomolgus macaques (Macaca fascicularis) were used in this study and were housed at the Oregon National Primate Research Center (ONPRC) and utilized for studies under the approval of the Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee (IACUC). All macaques in this study were managed according to the ONPRC animal care program, which is fully accredited by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and is based on the laws, regulations, and guidelines set forth by the United States Department of Agriculture (e.g., the Animal Welfare Act and its regulations, and the Animal Care Policy Manual), Institute for Laboratory Animal Research (e.g., Guide for the Care and Use of Laboratory Animals, 8th edition), and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The nutritional plan utilized by the ONPRC is based on National Research Council recommendations and supplemented with a variety of fruits, vegetables, and other edible objects as part of the environmental enrichment program established by the Behavioral Services Unit. Animals were socially housed when possible. All efforts were made to minimize suffering through the use of minimally invasive procedures, anesthetics, analgesics, and environmental enrichment. Study animal demographics are detailed in Table S1.

WBP2117 formulation and preparation

Anti-TIGIT mAb (WBP2117 lot no. 2117S170508Y) provided by Arcus Biosciences was prepared to meet dosing level requirements by dilution of the test article in test article diluent (a histidine-based buffer pH 5.8 provided by ARCUS) (e.g., 10 mg/kg/dose requires a 1 mg/mL dose concentration at a dose volume of 10 mL/kg). The test article was stored in refrigeration at 2–8°C prior to final dilution to final concentration. The dose formulations were prepared the day of each dosing day and allowed to warm to room temperature (RT) for at least 30 min before dosing. All WBP2117 injections were given as intravenous doses of 10 mg/kg over 30 min on weeks 0, 2, 4, 6, and 8 for a total of five doses. Weekly blood samples were collected prior to dosing with WBP2117, with the exception of weeks 0 and 8, where predose blood was taken as well as blood at 1-, 8-, 24-, and 48 h post-dosing. 36,960 initially served as a control and was subsequently repurposed to receive WBP2117 infusions.

Viral stocks and viral detection assay

MCM were infected with a SIVmac239 stock (stock 20082, 9 × 104 TCID50/mL, 2.15 × 10^8 copies/mL, 318 ng/mL p27, harvested 05 February 2011) generated by transfection of 293T cells and subsequent infection of rhesus macaque PBMC with 293T supernatant. RM were infected with a SHIV-AD8-EOM stock (SP2105A_2019, 2 × 105 TCID50/mL in TZMbl cells) provided by Dr. Brandon Keele. Plasma and cell-associated SIV/SHIV viral loads were performed as previously described (39–41). Briefly, nucleic acid was extracted from plasma and viral RNA copies were measured by RT-qPCR, targeting a conserved sequence of Gag. The limit of quantification was 15 copies viral RNA per mL for plasma viral loads.

Blood processing

Whole blood was collected into EDTA-treated tubes (BD Biosciences, San Jose, Ca, USA). Blood was assessed for complete blood counts using an ABX Pentra 60 C⁺ Hematology Analyzer (Horiba, Irvine, CA, USA). Blood in non-anticoagulant tubes was spun down at 1,860 × g for 10 min to separate serum from clotted blood and then assessed for serum chemistry values using an ABX Pentra 400 Chemistry Analyzer. PBMC and plasma were isolated from whole blood in EDTA-treated tubes by density gradient centrifugation using Ficoll-Hypaque.

In vitro sequential TIGIT costaining of PBMC

Cryopreserved PBMC were incubated with anti-TIGIT mAb WBP2117 conjugated to fluorescein isothiocyanate (FITC) for 30 min at RT and then washed with 1× PBS. Cells were then incubated with commercially available anti-TIGIT-phycoerythrin (eBioscience, clone MBSA43) for 30 min at RT and then washed with 1× PBS. Cells were then stained with antibodies against CD3, CD4, and CD8 and an amine-reactive viability dye for 30 min at RT in the dark. Cells were washed twice with 1× PBS and fixed with 2% PFA before acquiring data via flow cytometry on a FACSymphony A5 Cell Analyzer (Becton Dickinson, Franklin Lakes, NJ, USA). Flow cytometric data were analyzed using FlowJo, version 10 (TreeStar Ashland, OR, USA). Total TIGIT was determined by the percent of anti-TIGIT-PE (clone MBSA43) bound to that particular cell population.

Immune cell phenotyping and flow cytometry

Blood lymphocyte frequencies were monitored by whole blood staining as previously described (42). Briefly, EDTA-treated whole blood (50–100 µL) was washed twice with 1× PBS and then stained for surface markers and viability (Live/dead Yellow Fixable, Invitrogen) for 30 min at RT. After staining, whole blood was resuspended in 1 mL 1× FACS Lysing Solution (BD) to lyse red blood cells and fix remaining cells, incubated for 8 min, and washed three times with 1× PBS supplemented with 10% FCS (FACS buffer). After the last wash, 500 μL FACS perm buffer (1× BD FACS Lysing Solution + 0.05% Tween-20) was added and incubated for 10 min at RT. Cells were then washed three times with FACS buffer. For Ki67, perforin, and granzyme B assessment, fixed and permeabilized cells were stained for 45 min at RT. Following intracellular staining, samples were washed twice with FACS buffer and data were acquired on an LSR II (Becton Dickinson, Franklin Lakes, NJ, USA). Flow cytometric data were analyzed using FlowJo, version 10 (TreeStar Ashland, OR, USA). Antibodies against the following antigens were used: CD4 BUV395 (clone: L200), CD8 BUV737 (clone: SK1), CD3 Pacific Blue (clone: SP34-2), CD28 PE-Cy7 (clone: CD28.2), CD95 APC (clone: DX2), CD45 BV786 (clone: D058-1283), Ki67 PerCP-Cy5.5 (clone: B56), Granzyme B PE (clone: GB12), Perforin FITC (clone: Pf-344), Live/Dead Qdot585. Absolute lymphocyte counts were calculated by multiplying whole blood staining frequencies within the live CD45⁺ gate by the white blood cell count (WBC), as determined by complete blood count (CBC) performed on an additional aliquot of EDTA-treated whole blood. Gating strategy for immune cell populations is shown in Fig. S5.

IFN-γ enzyme-linked immunosorbent spot assay

Responses against SIV Gag peptide pools and full open reading frame (ORF) were tested in an IFN-γ ELISPOT using cryopreserved peripheral blood mononuclear cells. PBMC were incubated with peptides overnight (16 h). Peptides were tested in duplicate wells at a concentration of 10 μg/mL or 10 μM (either individually or in pools) with 100,000 PBMC per well. The total numbers of spots for duplicate wells were averaged, and all the numbers of spots were normalized to the numbers of IFN-γ spot-forming cells (SFC) per 1 × 10⁵ PBMC. Responses comprising <50 SFC per 10⁵ cells were considered negative and were not tested statistically. Positive responses were determined by using a one-tailed t test and an alpha level of 0.05, where the null hypothesis was that the background level would be greater than or equal to the treatment level. If determined to be positive statistically, the values were reported as the average of the test wells minus the average of all negative control wells (i.e., wells with no peptide added).

T cell stimulation and intracellular cytokine staining assay

Gag-specific CD8⁺ and CD4⁺ T cells responses were measured in PBMC preparations from blood by flow cytometric intracellular cytokine staining (ICS) assay as previously described (43). PBMC were incubated with Gag ORF peptide pool in addition to costimulatory molecules CD28 and CD49d (BD Biosciences, San Jose, Ca, USA) for 1 h, followed by the addition of Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) for an additional 8 h. Stimulated cells were stained for surface markers, CD4 and CD8, for 20 min at RT. For the intracellular stains (CD3, IL-2, TNF-α, and IFN-γ), cells were washed twice with 1× FACS buffer, fixed in 2% paraformaldehyde, and subsequently washed in saponin buffer. Cells were then stained for 1 h at RT. Following staining, cells were washed in saponin buffer and analyzed via flow cytometry as described above.

WBP2117 quantification in plasma

Enzyme-linked immunosorbent assay (ELISA) was used to detect free WBP2117 in plasma. Half-area 96-well Costar Assay Plates (Corning) were coated with the recombinant human TIGIT His-tag protein (R&D Systems) at a concentration of 0.5 μg/mL in carbonate-bicarbonate buffer (Thermo Fisher) and incubated overnight at 4°C. Plates were washed three times with wash buffer (1× PBS + 0.1% Triton X-100) and blocked with blocking buffer (1× PBS  +  1% normal goat serum + 5% dry milk) for 1 h at RT. After incubation, blocking buffer was discarded and diluted plasma and standard were added to the plates. WBP2117 concentration was calculated with a standard curve created with a serial titration of WBP2117 diluted in wash buffer with a range of 0.013–10 μg/mL. Heat-inactivated plasma samples were serially diluted in wash buffer. After incubation for 1 h at RT, plates were washed three times with wash buffer and incubated with 1:5,000 dilution of Peroxidase-conjugated AffiniPure goat anti-human IgG, Fcγ fragment specific (Jackson ImmunoResearch) in wash buffer for 1 h at RT. Plates were washed five times with wash buffer and left to incubate in the final wash for 5 min before being discarded. Plates were then developed for 2–10 min using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Southern Biotech). Reaction was stopped with 1 N H₂SO₄. Plates were read on the Synergy HTX Milti-Mode Microplate Reader (BioTek), and data were collected using software Gen5 v3.09 at two absorbance wavelengths: 650 nm for the developing reaction and 450 nm for the developed reaction after the reaction was stopped with 1 N H₂SO₄. Final OD was determined by OD_{450 nm} minus OD_{650 nm}.

Measurement of anti-WBP2117 antibodies in plasma

Half-area 96-well Costar Assay Plates (Corning) were coated with 2  µg/mL WBP2117. Plates were washed with wash buffer (1× PBS + 0.1% Triton X-100) once and blocked with blocking buffer (1× PBS + 1% normal goat serum + 5% dry milk) for 1 h at RT. After incubation, blocking buffer was discarded and heat-inactivated plasma samples were serially diluted in wash buffer, added to the plates in duplicate, and incubated for 1 h at RT. Plates were then washed three times with wash buffer and then incubated with a 1:3,000 dilution of detection antibody, mouse anti-macaque IgG-HRP (1B3-HRP, NHP Reagent Resource) for 1 h at RT. Plates were then washed five times with wash buffer and left to incubate in the final wash for 5 min before being discarded. Plates were developed for 2–10 min using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Southern Biotech). Reaction was stopped with 1 N H₂SO₄. Plates were read on the Synergy HTX Milti-Mode Microplate Reader (BioTek) and data were collected using software Gen5 v3.09 at two absorbance wavelengths: 650 nm for the developing reaction and 450 nm for the developed reaction after the reaction was stopped with 1 N H₂SO₄. Final OD was determined by OD_{450 nm} minus OD_{650 nm}. Anti-drug antibody (ADA) titers are defined as the reciprocal of the highest dilution of the sample that yields a positive result (e.g., dilution of 1/2,460  =  titer of 2,460). A positive result was defined as twice that of background values.

Detection of Env-binding antibodies in plasma

Antibody responses were determined by ELISA, measuring binding of plasma IgG to recombinant HIV-1 SF162 or SIVmac239 gp140 trimer. The gp140 trimer was produced as previously described (44). Half-well ELISA plates (Costar) were coated with SF162 or SIVmac239 gp140 trimer at 0.5 µg/mL or 2 µg/mL, respectively, in carbonate/bicarbonate buffer and incubated overnight at 4°C. Plates were washed one time with wash buffer (0.1% Triton X-100 in 1× PBS) and blocked with 1% normal goat serum/5% nonfat dried milk in 1× PBS for 1 h at RT. Serially diluted plasma samples were then incubated for 1 h at RT followed by three washes with wash buffer. Plates were probed with a horseradish peroxidase-conjugated goat anti-human IgG Fc fragment-specific polyclonal antibody (1:5,000, Jackson ImmunoResearch) for 1 h at RT. After five washes, TMB substrate (SouthernBiotech) was added and incubated for 2–10 min before quenching with H₂SO₄. Final OD was determined by OD_{450 nm} minus OD_{650 nm}. Plates were read on the Synergy HTX Milti-Mode Microplate Reader (BioTek), and data were collected using software Gen5 v3.09 at two absorbance wavelengths: 650 nm for the developing reaction and 450 nm for the developed reaction after the reaction was stopped with 1 N H₂SO₄. Each sample was assayed in duplicate.

Viral suppression assay

We performed in vitro viral suppression assays (VSAs) as previously described (45). Briefly, we enriched rhesus macaque splenocytes for CD4⁺ T cells using NHP-specific CD4 microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturers protocol. CD4⁺ T cells were then activated by overnight stimulation with staphylococcus enterotoxin B (SEB), CD3, CD28, and CD49d antibodies. After activation, CD4⁺ target cells were infected with sucrose-purified SIV_mac239 via spinoculation for 2 h. Infected targets remained in culture for 24 h prior to coculture with effector cells. To generate NK effector cells, we used CD8β-depleted and NKG2a-enriched autologous splenocytes, and for CD8β⁺ T cells, we used CD8β-enriched autologous splenocytes. On the day of coculture, we stained target cells with PKH26 and added 2.5e5 PKH26-stained target cells to each well of a 48-well plate. Effector cells were isolated and added to the appropriate wells. NK cells were added at an effector-to-target (E:T) ratio of 1:1, and CD8β⁺ T cells were added at an E:T of 1:8. Either anti-PDL1 mAb (BMS-936559, Bristol-Myers Squibb) or WBP2117 were added to the cocultures at a concentration of 10 µg/mL. Cocultures were maintained in R15-100 media containing RPMI 1,640 + 15% fetal bovine serum supplemented with 100 units of interleukin-2/mL (NIH AIDS Reagent Program). NK cell cocultures were maintained for 24 h, whereas CD8β⁺ T cell cocultures were maintained for 5 days. At the end of the assay (24 h for NK cell VSA and 5 days for CD8β T cell VSA), intracellular Gag p27 staining was performed to measure SIV_mac239 infection. We first stained for cell surface markers CD3, CD4, CD8, followed by intracellular Gag p27 staining (FITC-conjugated 55-2F12 Gag p27 anitbody, NIH Reagent Resource Program) using Fixation/Permeabilization Buffer set (eBioscience, Carlsbad, CA). Following staining, cells were analyzed via flow cytometry as described above.

Statistics

Statistical significance was assessed by two-way ANOVA test with Tukey’s multiple comparisons test (Fig. 1) and repeated-measures one-way ANOVA test with Holm-Sidak’s multiple comparison test (Fig. S4). One-way ANOVA with Tukey’s multiple comparison test was used to compare fold change in plasma viral loads. Statistical analyses were conducted using GraphPad Prism version 9.0 (GraphPad Software, La Jolla, California, USA). Statistical significance of the findings was set at a P-value of less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

DATA AVAILABILITY

All data supporting the findings of the study are available from the corresponding author upon request.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.00273-24.

Figure S1. jvi.00273-24-s0001.eps.

Complete blood counts and serum chemistry.

Figure S2. jvi.00273-24-s0002.eps.

T cell memory subpopulations remain stable throughout WBP2117 treatment.

Figure S3. jvi.00273-24-s0003.eps.

WBP2117 treatment does not impact longitudinal gp140-binding antibodies.

Figure S4. jvi.00273-24-s0004.eps.

TIGIT blockade does not improve NK cell or CD8+ T cell-mediated killing of SIV-infected cells.

Figure S5. jvi.00273-24-s0005.eps.

Representative gating strategy to evaluate T cells and NK cells.

Supplemental legends. jvi.00273-24-s0006.docx.

Legends for Fig. S1 to S5 and Table S1.

Table S1. jvi.00273-24-s0007.docx.

Study animal demographics.

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