Article Text

Original research
Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment
  1. Josipa Raguz1,
  2. Catarina Pinto1,
  3. Theresa Pölzlbauer1,
  4. Mohamed Habbeddine1,
  5. Sandra Rosskopf1,
  6. Judith Strauß1,
  7. Valentin Just1,
  8. Sarah Schmidt1,
  9. Katell Bidet Huang1,
  10. Felix Stemeseder1,
  11. Timo Schippers1,
  12. Ethan Stewart2,
  13. Jakub Jez2,
  14. Pedro Berraondo3,4,
  15. Klaus K. Orlinger1 and
  16. Henning Lauterbach1
  1. 1Hookipa Pharma Inc, New York, NY, USA
  2. 2Vienna BioCenter Core Facilities GmbH (VBCF), Vienna, Austria
  3. 3Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Pamplona, Spain
  4. 4Navarra Institute for Health Research (IDISNA), Pamplona, Spain
  1. Correspondence to Henning Lauterbach; Henning.Lauterbach{at}


Background Engineered arenavirus vectors have recently been developed to leverage the body’s immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic choriomeningitis virus (artLCMV) encoding a non-oncogenic fusion protein of human papillomavirus (HPV)16 E6 and E7 are currently being tested in patients with HPV16+ cancer, showing a favorable safety and tolerability profile and unprecedented expansion of tumor-specific CD8+ T cells. Although the strong antigen-specific immune response elicited by artLCMV vectors has been demonstrated in several preclinical models, PICV-based vectors are much less characterized.

Methods To advance our understanding of the immunobiology of these two vectors, we analyzed and compared their individual properties in preclinical in vivo and in vitro systems. Immunogenicity and antitumor effect of intratumoral or intravenous administration of both vectors, as well as combination with NKG2A blockade, were evaluated in naïve or TC-1 mouse tumor models. Flow cytometry, Nanostring, and histology analysis were performed to characterize the tumor microenvironment (TME) and T-cell infiltrate following treatment.

Results Despite being phylogenetically distant, both vectors shared many properties, including preferential infection and activation of professional antigen-presenting cells, and induction of potent tumor-specific CD8+ T-cell responses. Systemic as well as localized treatment induced a proinflammatory shift in the TME, promoting the infiltration of inducible T cell costimulator (ICOS)+CD8+ T cells capable of mediating tumor regression and prolonging survival in a TC-1 mouse tumor model. Still, there was evidence of immunosuppression built-up over time, and increased expression of H2-T23 (ligand for NKG2A T cell inhibitory receptor) following treatment was identified as a potential contributing factor. NKG2A blockade improved the antitumor efficacy of artARENA vectors, suggesting a promising new combination approach. This demonstrates how detailed characterization of arenavirus vector-induced immune responses and TME modulation can inform novel combination therapies.

Conclusions The artARENA platform represents a strong therapeutic vaccine approach for the treatment of cancer. The induced antitumor immune response builds the backbone for novel combination therapies, which warrant further investigation.

  • Immunotherapy
  • Immunogenicity, Vaccine
  • Tumor Microenvironment
  • Drug Therapy, Combination
  • Head and Neck Cancer

Data availability statement

Data are available on reasonable request.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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  • Engineered arenavirus vectors based on Pichinde virus (artPICV) and lymphocytic choriomeningitis virus (artLCMV) encoding a non-oncogenic fusion protein of human papillomavirus 16 E6 and E7 are characterized by a favorable safety and tolerability profile and robust induction of tumor-specific CD8+ T-cell responses. Clinical responses on monotherapy are hampered by immunosuppressive mechanisms. Identification of those might help to maximize the potential benefit of arenavirus vectors through rational combination with other therapeutic modalities.


  • Both artLCMV and artPICV vectors showed preferential infection of professional antigen-presenting cells. Additionally, a single administration of both vectors led to tumor-specific CD8+ T-cell responses which infiltrated the tumor mass, inflamed the tumor microenvironment (TME), and mediated prolonged survival in a TC-1 mouse tumor model. NKG2A blockade was identified as a potential combination to further improve the antitumor efficacy.


  • This study highlights the need for characterization of TME modulation on arenavirus vector-mediated treatment, which can inform novel combination therapies. These results might affect clinical testing and biomarker strategies.


High-risk human papillomavirus (HPV) is one of the main causes of infection-related cancers and a major risk factor for developing a subset of head and neck squamous cell carcinomas (HNSCC) and cervical and anogenital cancers.1 While increased awareness decreased carcinogen-related HNSCC incidence, HPV infections led to a rise in HPV-driven HNSCC over recent decades. These mostly develop in the oropharynx and around 90% can be attributed to the HPV16 variant.2 Prophylactic vaccines are available; however, the incidence and mortality rates do not yet reflect the success of the vaccination process.3 Most patients present with advanced-stage HNSCC, requiring a multimodal therapeutic approach. Standard of care (SoC) includes surgery, radiation, and platinum-based chemotherapy, typically cisplatin.2 4 More recently, anti-programmed cell death (PD)-1 antibodies were approved for metastatic, platinum-refractory HNSCCs.5–7 Yet, the lack of predictive biomarkers beyond PD-L1 expression hinders patient stratification,7 and most responders develop mechanisms of acquired resistance, which are not fully understood.8 Still, the clinical benefit from immunotherapy for HNSCC was established, encouraging the development of more effective approaches to improve outcomes.

Therapeutic vaccines emerged as a promising modality to trigger cellular immunity against HPV+ tumors.3 HPV16 oncogenic proteins E6 and E7 are required for malignant transformation and remain constitutively expressed throughout tumor progression, making them ideal target antigens.9 The detection of E6-specific/E7-specific T cells was associated with better prognosis and improved tumor control,10 11 and several therapeutic vaccine approaches targeting E6 and E7 are under clinical development. However, efficacy is limited to precancerous lesions and patients with low tumor burden, and none were approved so far.12 Treatment of advanced tumors will likely require combination approaches to overcome the immunosuppressive mechanisms within the tumor microenvironment (TME).13

We recently described the development of a novel, live attenuated, replicating, non-lytic, arenavirus-based vector platform (artARENA).14 15 Vectorization of the two arenaviruses lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV) allows for incorporation of selected tumor antigens and de novo vector generation by reverse genetics. These serve as antigen-delivery platforms that infect and activate multiple cell types, including professional antigen-presenting cells (APCs), leading to efficient T-cell priming.14 Vaccination with LCMV-based vectors encoding non-oncogenic versions of E7 and E6 viral proteins (artLCMV-E7E6) was shown to induce a robust polyfunctional T-cell response and lead to tumor regression in a mouse model of HPV16+ tumors.16 Sequential alternating administration of PICV-based vectors encoding the same HPV16-derived antigens (artPICV-E7E6) followed by artLCMV-E7E6 further improved the antitumor efficacy by focusing CD8+ T-cell responses to the encoded cargo, while limiting vector-targeted immunity.17 Importantly, a clinical trial in HPV16+ HNSCC patients showed that this alternating two-vector therapy led to the generation of antigen-specific T-cell responses constituting up to 48% of the circulating CD8+ T-cell pool.18 19 Both vectors (HB-201: artLCMV-E7E6; HB-202: artPICV-E7E6) are currently evaluated in a phase I/II trial, alone or in combination with pembrolizumab in metastatic or recurrent HPV16+ HNSCC patients (NCT04180215), and in a window of opportunity trial in patients with newly diagnosed HPV16+ cancers (NCT04630353) with a single dose of HB-201.

Given the potential of the artARENA platform for therapeutic vaccination, in-depth characterization of each vector’s immune response will be key to informing clinical strategies. Thus, here, we investigated the innate and adaptive immune responses elicited by either artLCMV-E7E6 or artPICV-E7E6, and explored combination approaches that enhance their efficacy in the treatment of HPV-driven tumors. Using human in vitro systems and in vivo mouse models we found that, despite being phylogenetically distant, the two vectors exhibited similar infection and activation patterns of human and mouse APCs. In a TC-1 mouse tumor model, both vectors triggered CD8+ T-cell mediated control of tumor growth and improved median survival following a single intravenous or intratumoral administration. Both vectors induced extensive remodeling of the TME, increased T-cell infiltration, and activated critical immune pathways. Finally, we identified NKG2A blockade as a possible combination strategy to enhance the efficacy of the artARENA platform.


Cells and cell lines

TC-1 cells expressing HPV16 E6 and E7 (Johns Hopkins University20) were cultured in RPMI1640, 10% heat-inactivated fetal bovine serum (FBS), non-essential amino acids (NEAA), 2 mM GlutaMAX, 1 mM sodium pyruvate, 0.4 mg/mL geneticin, 50 U/mL penicillin/streptomycin (Gibco) and split twice weekly using trypsin-EDTA (ThermoFisher). BHK21 LCMV-GP-expressing cells and HEK293 suspension cells (Institute of Experimental Immunology, University of Zurich) were cultured in DMEM, 10% FBS, 4 mM GlutaMAX, 10 mM HEPES (ThermoFisher), and Puromycin (Sigma Aldrich) or in CDM4HEK293 (GE Healthcare), 4 mM GlutaMAX, respectively. HEK293 adherent cells (ATCC) were cultured in MEM (ThermoFisher), 10% FBS, 2 mM GlutaMAX, NEAA, 1 mM Sodium Pyruvate. HEK293-GP adherent cells (Department of Experimental Virology, University of Basel) were cultured in DMEM, 10% FBS, 2 mM GlutaMAX. ARPE-19 cells (ATCCs) were cultured in IMDM (ThermoFisher), 20% FBS and DMEM-F12 (ThermoFisher), 10% FBS. J-E6 and J-E7 reporter cell lines (Promega) were cultured in RPMI1640, 10% FBS, 400 µg/mL hygromycin B, 10 µg/mL blasticidin-S-HCl, NEAA, 1 mM sodium pyruvate (ThermoFisher), 100 µg/mL Zeocin (Invitrogen). Growth conditions were 5% of CO2 and 37°C in a water-saturated atmosphere.

Vector generation

artLCMV vectors and artPICV vectors encoding either E7E6, GFP or NanoLuc were generated as described previously.14 16 17 21 The vector-encoded antigen (fusion protein of E7 and E6: GenBank accession #K02718 with five mutations to abrogate oncogenic potential22 coding sequence for artLCMV-E7E6 was synthesized by GenScript and inserted into two plasmids encoding the S-Segment of LCMV (clone 13). S-Segment #1 encoded LCMV NP, and S-Segment #2 encoded LCMV GP (LCMV strain WE). For the generation of artPICV-E7E6, the identical HPV16-derived antigen was inserted into plasmids encoding the S-Segment of PICV (strain p18), with S-Segment #1 encoding PICV-NP and S-Segment #2 encoding PICV-GP. The plasmids encoding S-segments of artARENA vectors encoding GFP (Department of Experimental Virology, University of Basel) or NanoLuc (Promega) were generated similarly. Vectors were generated by transient transfection of BHK21 cells stably expressing LCMV-GP.23

Infection of human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) isolated from buffy coats (Red Cross) using standard gradient density centrifugation were spinoculated with vectors at 2671 g for 2 hours, RT, MOI5. After 20 hours, PBMCs were harvested using 0.05% Trypsin-EDTA, seeded in 96-well plates, washed using FACS buffer (PBS (Phosphate Buffered Saline), 2.5% FBS, 10 mM EDTA) with 0.01% sodium azide, stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher) and incubated for 1 hour in CD56-BV650, CD19-BV650, HLA-DR-BV421, CD14-AF700, CD11c-PE-Dazzle594, CD303-PerCP-Cy5.5, CD1c-APC-Cy7, CD141-PE-Cy7, CD80-BV711 and CD40-BV605 (BioLegend), CD86-BUV737, CD16-BV786 (BD Biosciences). After washing, cells were fixed with Cytofix (BD Biosciences), resuspended in FACS buffer, and analyzed using a BD Fortessa flow cytometer and FlowJo v10.8 Software (BD Life Sciences).

HPV J-E6 and J-E7 reporter assay

ARPE-19 human leucocyte antigen (HLA)-A2 expression status was verified using an anti-HLA-A2 antibody (BioLegend). Their infectibility with artARENA vectors was tested with spinoculation at 2671 g for 2 hours, RT, MOI1. GFP-expression levels were analyzed by flow cytometry after 24 hours. For the reporter assay, ARPE-19 cells were seeded in white 96-well plates (Costar) for 4 hours, incubated with vectors at MOI2 for 20 hours, washed and cocultured for 6 hours with Jurkat IL-2/NanoLuc reporter cells transgenic for HLA-A2-restricted TCRs against HPV E6 (TIHDIILECV)-derived and HPV E7 (YMLDLQPET)-derived epitopes. ARPE-19 cells loaded with 6.25 ng/mL TIH and YML or ELAGIGILTV (MART-1) peptides were used as a positive and a negative control, respectively. Reporter induction was measured with the Bio-Glo-NL Luciferase Assay Detection Kit (Promega) and relative light units (RLUs) were measured using GloMax Discover microplate reader (Promega).

Syngeneic TC-1 tumor model

Age-matched (4–7 weeks) C57BL/6J or C57BL/6N strain female mice (Janvier Labs or Charles River Laboratories) were used.

For the generation of a stringent TC-1 tumor model, mice were subcutaneously inoculated with 1×105 TC-1 cells. Clinical signs and body weight were evaluated. Tumor size was measured at least twice weekly (starting from day 4 post-inoculation) in two dimensions using a caliper, and the tumor volume was expressed in mm3 using the formula: V=0.5×a×b2 (a and b are the long and short diameters of the tumor in mm). At tumor volume of ~100 mm3, mice were assigned to groups by randomized block design based on the tumor volume. Mice were treated intravenously or intratumorally with indicated doses of vectors, formulated in PSB buffer (10 mM HEPES, 150 mM NaCl, 20 mM Glycine, pH 7.4, GE Healthcare), 10% D-sorbitol (Sigma) or with PSB buffer only as control. For cell depletion, mice were intraperitoneally injected with InVivoMAb anti-mouse CD4 (clone GK1.5), InVivoMAb anti-mouse-CD8α (clone 2.43), InVivoMAb anti-mouse NK1.1 (clone PK136), or InVivoMAb rat IgG2b isotype control antibodies (BioXCell) diluted in InVivoMAb dilution buffer. For combination treatments, mice were intraperitoneally injected with 4 mg/kg of Cisplatin (Sigma) diluted in PBS, or with 200 µg/dose of InVivoMAb anti-mouse NKG2A/C/E (BioXCell). Animals were euthanized by cervical dislocation when predefined endpoints were reached.

Blood and tissue preparation

Blood was collected from the facial vein into EDTA-coated tubes (Sarstedt), inverted 5x, and left at RT for at least 30 min. Tubes were centrifuged at 12 000 g for 1.5 min. The supernatant was stored at ≤80°C until further analysis. Fresh spleens or lymph nodes were forced twice through a 70 µm cell strainer (Falcon), washed using FACS buffer, and treated with Pharm Lyse Lysing Buffer (BD Biosciences) for 3 min, 37°C. Tumors were excised, weighed, and cut into pieces (2–4 mm). These were transferred into gentleMACS C tubes and dissociated in Tumor Dissociation Kit on a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec). Cells were washed, strained through 70 µm and 30 µm MACS SmartStrainers (Miltenyi Biotec), resuspended in MACS BSA Stock Solution and CD45+ cells were separated using CD45 (TIL) MicroBeads, on an autoMACS Pro Separator (Miltenyi Biotec). The viability of single-cell suspensions was evaluated using Cell Viability Acridine Orange & Propidium Iodide Cell Viability Kit (Biozym), and cells were counted in a Luna FL Dual Fluorescence Cell Counter. Cells were stored in FACS buffer at 4°C until further analysis.

Flow cytometry

Cells were blocked (Mouse BD Fc Block, BD Biosciences) and viability evaluated using LIVE/DEAD Fixable Aqua Dead Cell Stain kit or Fixable Viability Stain 700 (BD Biosciences). Cells were incubated in antibody cocktails for 15–30 min, RT, dark, fixed using Cytofix (BD Biosciences), and resuspended in FACS buffer for analysis. For intracellular staining cells were fixed/permeabilized using Mouse FoxP3 Buffer Set (BD Biosciences). For major histocompatibility complex (MHC) multimer staining, cells were incubated in MHC dextramer mix for 15 min, RT, dark, prior to antibody staining. MHC dextramers (Immudex): HPV16 E7-PE (H-2 Db/RAHYNIVTF), LCMV NP396-APC (H-2 Db/FQPQNGQFI), PICV NP38-APC (H-2 Kb/SALDFHKV). Antibodies: CD11b-BUV395, CD11b-PE-Cy5, CD26-BUV737, CD86-FITC, CD3e-BUV395, CD45-BUV737, CD45-APC-Cy7, CD4-BV421, CD44-APC-Cy7, NKp46-BV510, CD8a-BB515, CD25-BV605, FoxP3-PE-CF594, TCF-7/TCF-1-PE (BD Biosciences); Ly-6G-BV650, CD64-BV711, Ly-6C-BV785, XCR1-PE, CD3e-PE-Cy5, CD19-PE-Cy5, NK1.1-PE-Cy5, CD11c-PE-Cy7, F4/80-APC, CD45-AF700, Siglec-H-BV421, CD169-BV605, MHCII-APC-Cy7, CD8-PerCPCy5.5, CD69-BV785, NK1.1-PE, GranzymeB-FITC, IFNγ-APC, CD45R/B220-PE-Cy5, ICOS-BV7855 (BioLegend).

Cells were defined by pregating on singlets and live CD45+ cells and determined as follows: pDC (LinMHCII+ Siglec-H+), cDC1 (LinMHCII+ Siglec-H CD11c+ CD26+ XCR1+ CD172a), cDC2 (Lin-MHCII+ Siglec-H CD11c+ CD26+ XCR1 CD172a+), iMO (Lin MHCII CD11b+ Ly6G- Ly6Chi). B cells (CD19+), CD8+ T cells (CD19 CD3+ NK1.1 CD8+), CD4+ T cells (CD19 CD3+ NK1.1 CD4+), NK cells (CD19 CD3 NK1.1+), CD8+ NKT cells (CD19 CD3+ NK1.1+ CD8+). For the flow cytometry analysis of TILs, spleen, and lymph node, populations were defined by pregating on singlets and live CD45+ cells and determined as follows: CD8+ T cells (B220 CD11b NKp46 CD3+ CD8+), CD4+ T cells (B220 CD11b NKp46 CD3+ CD4+), Tregs (B220 CD11b NKp46 CD3+ CD4+ FoxP3+ CD25+).

For the intracellular cytokine staining, splenocytes were restimulated in vitro for 1 hour using overlapping peptide sets for HPV16 E6 and E7 (JPT) and incubated in Brefeldin A (Sigma Aldrich) for 4 hours. During incubation, anti-mouse CD107a-BV786 (BD Biosciences) was added to respective wells. Cells were washed with FACS buffer, live cells were stained using Aqua Live/Dead Stain (ThermoFisher Scientific) and incubated for 30 min, at 4°C, in the dark with the following antibodies: CD3e-BUV395, CD8a-BB515, CD44-APC-Cy7 (BD Biosciences), CD4-BV650 and CD45R/B220-PE-Cy5 (BioLegend). Subsequently, cells were fixed using 4% paraformaldehyde and permeabilized with 0.1% Saponin (Sigma Aldrich). Intracellular staining was performed with TNFα-BV421 and IFNγ-APC (BD Biosciences) antibodies for 30 min, 4°C, dark. Background values (medium only) were subtracted, and only peptide-specific responses were analyzed.

Samples were acquired on a BD LSR Fortessa (Beckton Dickinson) and analyzed using FlowJo V.10.8 Software (BD Life Sciences).

Cytokine analysis in serum/multiplex immunoassay

Analysis of serum samples was performed using the U-PLEX Biomarker Group 1 (MSD). Data were acquired in MESO QuickPlex SQ 120 MM instrument and analyzed using DISCOVERY WORKBENCH V.4.0 Analysis Software.

Bioluminescence imaging

Experiments were conducted at CIMA, University of Navarra. Murine TC-1/A9 cell line was used,24 25 and cells were maintained in RPMI1640 GlutaMAX, 10% FBS, 1% penicillin/streptomycin, 0.4 mg/mL Geneticin, 0.05 mM 2-mercaptoethanol (Gibco) and split twice weekly. Six-week-old female immunocompetent C57BL/6J mice (Harlan, Barcelona, Spain) were housed at the animal facility of the University of Navarra. Tumor cells (1×105) were inoculated subcutaneously in 100 µL PBS on the right flank. At a tumor diameter of ~5 mm, mice were randomized and administered the treatments. Mice were anesthetized using ketamine/xylazine, and the Nano-Glo In Vivo Substrate, fluorofurimazine (Promega), was administered at 7.5 µmol/kg intraperitoneally in 50 µL PBS based on recent body weights. After 5 min, animals were imaged using the PhotonIMAGER Optima system (Biospace labs). A color-scale photograph of the animals was acquired, followed by bioluminescent acquisition. Ventral and dorsal images were acquired and analyzed. Regions of interest were drawn over the animal image. Regions of no signal were used as background. Light intensity was quantified using photons/s (RLU). The color-scale photograph and data images were superimposed using M3 Vision software (Biospace labs). For the ex vivo biodistribution analysis, mice were sacrificed, and organs were isolated and imaged in a 12-well plate. Mice were sacrificed using CO2 at predefined end-point criteria.

Immunohistochemistry staining and quantification

Immunohistochemistry (IHC) stainings were performed at the VBCF Histology facility. Formalin-fixed paraffin-embedded tumors from treated mice were cut into 2 µm sections, mounted onto Superfrost Ultra Plus slides (Thermofisher), deparaffinized in xylene and hydrated in ethanol gradients (100%, 95%, 70%–50%). Stainings were performed using Leica Bond III automated stainer, BOND Epitope Retrieval Solution 1 (AR9961 Leica) and Bond Polymer Refine Detection kit (DS9800 LEICA). Tissue sections were incubated for 60 min with primary antibodies against CD4, CD8 (Thermofisher), Ki-67, CD8, Foxp3 (Abcam). Negative control studies were performed using an isotype-matched antibody. As positive control, a multitissue block containing samples of spleen, liver, lymph node, lung, long bone, tongue, heart, pancreas, and thymus from a healthy control mouse were stained. Whole slide digitization and digital analysis were performed using PANNORAMIC 250 Flash III scanner (3DHISTECH) with a 20 x objective. Analysis was performed by a veterinary pathologist using Qpath software.

For Ki-67 quantification, 206 images of individual tumor pieces were exported from whole slide scans in .tif format at half resolution corresponding to a pixel scale of 0.243 µm per pixel. Tumor images were split into 512×512 pixel patches. Nuclei in 32 randomly selected patches were manually annotated as positive or negative for Ki-67 staining by a trained pathologist and further divided into 128×128 pixel patches to facilitate model training. Annotated patches were split 70:20:10 into training, validation, and test sets, respectively. The manually annotated training and validation sets were used to train a Mask RCNN Deep learning model.26 The model was built with a resnet101 backbone and initiated with pretrained weights from the MS-COCO dataset.27 The training set was augmented by flipping, rotating, stretching, and zooming the images. The network heads were trained for four epochs and the entire network was trained for a further two epochs with a learning rate of 0.001. The accuracy of the trained model was assessed on the remaining test set. Mean average precision was calculated with an intersection over union (IOU) threshold of 0.50. The IOU was calculated for each image by flattening the predicted and ground truth masks to 512×512×1 dimension binary masks.

NanoString analysis

20–170 mg pieces of snap-frozen tumors were subjected to RNA isolation and NanoString Analysis at Targos Molecular Pathology Gmbh (Kassel, Germany). Gene expression signatures were evaluated using the nCounter PanCancer IO 360 Panel (NanoString Technologies). This panel included 770 genes (including internal reference genes) which allowed the calculation of 48 gene signatures measuring biological variables crucial to the tumor-immune interaction. Data QC and cleaning were performed using the nSolver v.4.0 software (NanoString Technologies). Differential expression analysis, pathway analysis, and cell type profiling were performed using the nCounter Advanced Analysis Software (NanoString Technologies).


artARENA immunization induces a robust innate immune response and strong antigen-specific effector T-cell responses

Both artLCMV-E7E6 alone and artPICV-E7E6/artLCMV-E7E6 alternating prime-boost vaccination regimens have previously shown immunogenicity and therapeutic efficacy in preclinical mouse models.16 17 Interestingly, the order of administration influenced the outcome, with artPICV prime followed by artLCMV boost outperforming the inverse sequence.17 Therefore, we sought to analyze the immune response elicited by artPICV in comparison to artLCMV and uncover specific characteristics of each vector, that could ultimately inform about optimal regimens and promising combinational therapies. For this, naïve C57BL/6 mice were intravenously administered a single dose of either artLCMV-E7E6 or artPICV-E7E6, and the immune response was analyzed 24 hours and 7 days later (figure 1A).

Figure 1

artARENA immunization induces a robust innate immune response, as well as strong antigen-specific effector T cell responses. (A) Experimental design (created with C57BL/6 mice were immunized intravenously with 1×105 (B, C) or with 1×106 (D–H) RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors. Control animals received formulation buffer. Blood and/or spleen were collected 24 hours and/or 7 days postimmunization. (B) Serum cytokines and chemokines measured by MSD multiplex assay. Log2 fold change calculated relative to control animals (n=5–7/group, t-test). (C) Immune cell activation measured by flow cytometry of splenocytes (mean±SD, n=5–7/group, one-way ANOVA and Tukey’s multiple comparisons test). (D–G) Characterization of E7-specific CD8+ T cell response, measured by flow cytometry of splenocytes, 7 days postimmunization (n=5/group). (D) Percentage of E7-specific CD8+ T cells. Boxplots show median±IQR, min/max, and individual mice (unpaired t-test). (E) Flow cytometry histograms showing PD-1 expression. (F) Expression of CD127 and KLRG1 on E7-specific CD8+ T cells (mean±SD; two-way ANOVA and Tukey’s multiple comparisons test). For control samples, CD8+ T cells were used. (G) Geometric mean fluorescence intensity (gMFI) of CD127 on E7-specific CD8+ T cells (mean±SD, unpaired t-test). (H) Polyfunctional CD8+ T cell response in immunized mice. Splenocytes harvested 7 days postimmunization were ex vivo restimulated with E6 or E7 peptides. Total percentage of IFN-y-expressing CD8+ T cells (left). Percentage of CD8+ T cells expressing different combinations of IFN-γ, TNF-α, and CD107a (right). Boxplots show median±IQR, min/max, and individual mice (n=5/group, one-way (left) or two-way (right) ANOVA and Dunnett’s multiple comparisons test). For each test: ns=non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Single immunization with either vector led to an increase in key systemic proinflammatory cytokines and chemokines 24 hours after treatment (figure 1B). Substantial levels of type I IFN (IFN-α, IFN-β) and robust induction of IFN-γ were detected in the serum (figure 1B). Increased levels of myeloid and lymphoid cell chemoattractant chemokines CCL2 and CXCL10 and other proinflammatory mediators further marked the increased inflammation shortly after treatment (figure 1B). Flow cytometry analysis of splenocytes revealed a concomitant increase in the activation of different subsets of myeloid cells as well as plasmacytoid dendritic cells (pDCs), as measured by CD86 expression (figure 1C, left). Of note, conventional DC1 (cDC1), key players in antitumor immunity,28 29 showed the highest increase in numbers and activation, with around 90% of these cells expressing CD86 following treatment with both vectors (figure 1C, online supplemental figure 1A). Lymphocyte populations also showed increased activation at this time point (figure 1C, right). Expression of early activation marker CD6930 was seen in all analyzed populations, including CD4+, CD8+ T, NK, NKT, and B cells (figure 1C, right). On the other hand, expression of granzyme B (GzmB), a potent cytotoxicity marker,31 was robustly upregulated in NK cells, and to a lower extent also in NKT and CD8+ T cells (figure 1C, right). Although a strong systemic immune activation was observed following immunization with either vector, significantly lower percentages of activated cells were detected following artPICV administration (figure 1B,C), possibly indicating a slower or overall lower induction of inflammation by this vector.

Supplemental material

To further dissect the immunogenicity of each vector, de novo priming of antigen-specific T cells was analyzed 7 days post-treatment. Flow cytometry analysis of splenocytes revealed a significant increase in E7-specific T cells following administration of either vector, reaching in average 2%–3% of CD8+ T cells present (figure 1D, online supplemental figure 1B). These cells had increased expression of PD-1 (figure 1E), denoting their activated phenotype.32 While in control animals CD8+ T cells have a naïve KLRG1 CD127+ phenotype, a large proportion (up to 90%) of treatment-induced E7-specific CD8+ T cells displayed KLGR1+ CD127 and KLGR1 CD127 effector phenotypes (figure 1F, online supplemental figure 1C). A small but distinct population (3%–16%) expressed CD127 (figure 1F), a marker linked to the development of memory T cells.33 Interestingly, the CD127+ KLGR1 T cell population was significantly larger following artPICV administration (figure 1F), and their CD127 expression levels per cell were also significantly higher when compared with artLCMV treatment-induced cells (figure 1G).

Finally, effector function was analyzed by measuring CD8+ T cell cytokines and CD107a expression after ex vivo restimulation with E6 and E7 peptides of splenocytes from treated animals (figure 1H). At this time point, E6-specific and E7-specific CD8+ T cells from animals treated with both vectors mainly coexpressed IFN-γ and CD107a, with or without TNF-α, and only a minor population expressed IFN-γ alone (figure 1H). E7-specific effector T cells were more abundant overall (figure 1H).

These data indicate that artARENA immunization quickly and efficiently induces systemic activation of innate and adaptive immune responses, which translate into robust antigen-specific T-cell responses. Seven days post-treatment, the majority of E6-specific and E7-specific CD8+ T cells were differentiated into an effector phenotype with polyfunctional cytokine expression, while artPICV immunization may have increased potential to develop long-lived antigen-specific memory T cells.

Human APCs are the preferential target for artARENA vectors, leading to T-cell activation through direct presentation of the encoded antigens on MHC class I molecules

While direct infection of APCs is known to mediate the strong immunogenicity triggered by artLCMV vectors,14 the infection pattern of arenavirus-based vectors, especially artPICV, toward different human myeloid cell subsets has not been fully investigated. Therefore, we sought to analyze the infection profile of both artLCMV and artPICV by infecting human PBMCs with vectors encoding GFP as a reporter gene. PBMCs were harvested 20 hours postinfection, and different myeloid populations were identified and analyzed by flow cytometry (figure 2A). All populations showed GFP expression after infection with either vector, indicating a similar infection profile (figure 2B). Infection rates were comparable and ranged from 50% to 80% for each cell type, with cDC1 infected at the highest rate (figure 2B). APC activation was analyzed by flow cytometry analysis of CD86, with all populations showing increased CD86 expression postinfection (figure 2C), providing strong costimulatory signals for T cells. Of note, artARENA vectors’ infection is specific to myeloid cells and pDCs, as little to no infection was observed in lymphocytes (data not shown).

Figure 2

Human APCs are the preferential target for artARENA vectors and present encoded antigens on MHC class I, leading to T cell activation. (A–C) PBMCs from healthy donors were infected with artLCMV-GFP or artPICV-GFP vectors (MOI5) and analyzed 20 hours postinfection. (A) Flow cytometry gating strategy for obtaining the different human APC subsets. (B) GFP and (C) CD86 expression (mean fluorescence intensity) in human APC subsets analyzed by flow cytometry. Values indicate the percentage of GFP+ cells. Plots are representative of two replicates. Error bars, mean±SD of duplicate wells, one-way ANOVA. (D) Flow cytometry analysis of HLA-A2 expression on the human cell line ARPE-19 (top) stained with HLA-A2 antibody (gray) or isotype control (open histogram). Flow cytometry analysis of GFP expression in ARPE-19 cells (bottom) 24 hours postinfection with artLCMV-GFP or artPICV-GFP (MOI1). (E) artLCMV-E7E6 or artLCMV-GFP and artPICV-E6E7 or artPICV-GFP infected ARPE-19 cells (MOI2) were cocultured for 6 hours with Jurkat IL-2/NanoLuc reporter cells transgenic for HLA-A2-restricted TCRs against HPV E6 (TIHDIILECV)- and HPV E7 (YMLDLQPET)-derived epitopes. ELAGIGILTV (MART-1) was used as an irrelevant peptide control and TIH and YML peptide-loaded ARPE-19 cells were used as positive controls. Error bars, mean±SD of duplicate wells, one-way ANOVA and Dunnett’s multiple comparisons test. For each test: ns=non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; APC, antigen-presenting cells; artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus; PBMCs, peripheral blood mononuclear cells.

The capacity of infected cells to prime and activate T cells is crucial for the efficacy of the artARENA platform and for the development of antitumor immunity. Therefore, to investigate antigen expression and presentation in human cells following infection, we used ARPE-19 cells, a human HLA-A*02:01+ cell line amenable to artARENA infection (figure 2D). artLCMV-E7E6 or artPICV-E7E6 infected ARPE-19 cells were cocultured with E6-specific or E7-specific Jurkat reporter cells. These express an HLA-A*02:01-restricted E6-specific or E7-specific TCR and were engineered to express Nanoluc luciferase under the control of the interleukin-2 (IL-2) promoter. Six hours after coculture, we measured high levels of luciferase, indicating robust activation of the reporter cells (figure 2E).

Our results show that both artLCMV and artPICV vectors infect and activate human APC subsets. Moreover, encoded antigens are expressed, processed, and presented by human cells, inducing T-cell activation.

Therapeutic immunization with artARENA vectors mediates potent antitumor activity and control of bilateral tumors

To analyze the antitumor efficacy of artARENA vectors, C57BL/6 mice subcutaneously grafted with 1×105 TC-1 cells (HPV16 E7/E6-expressing syngeneic tumor model) were intravenously or intratumorally administered 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 when tumors reached ~100 mm3 (figure 3A). Monitoring of tumor size showed that artARENA immunization suppressed tumor growth in all four treatment groups (figure 3B, online supplemental figure 2A). Further, survival was significantly prolonged, increasing 1.8-fold on average in this model (figure 3C). The observed antitumor effect was dependent on HPV-specific immunity induced by the artLCMV-E7E6 or artPICV-E7E6 vectors, as only a minor and transient delay in tumor growth and no survival benefit was observed in animals treated with vectors encoding an irrelevant protein (data not shown).16 34

Figure 3

Therapeutic immunization with artARENA vectors mediates potent antitumor activity and control of bilateral tumors. (A) Experimental design (created with TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm3. Control animals received formulation buffer. Blood was collected 6 and 15 days postimmunization and cells were analyzed by flow cytometry. (B) Tumor volume monitored over time (mean±SEM, n=5/group). (C) Kaplan-Meier survival curves, with indicated median survival and long-term survivors (**p<0.01, Log-rank test). (D) Percentage of E7-specific CD8+ T cells. Boxplots show median±IQR, min/max, and individual mice. (E) Expression of CD127 and KLRG1 on E7-specific CD8+ T cells (mean±SD). For control animals, total CD8+ T cells were used. (F) Experimental design. TC-1 cells were grafted subcutaneously in both flanks of C57BL/6 mice. When tumors reached a volume of ~100 mm3, mice were immunized with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors. Immunization was done intravenously or intratumorally (on the right flank tumor only). Control animals received formulation buffer. (G) Tumor volume monitored over time (mean±SEM, n=10/group, data are shown until the time point at which more than 50% of the animals in the group were euthanized). Tumor growth data were analyzed by fitting a linear mixed model (fixed effect terms: treatment, day, and treatment:day interaction; subject was specified as a random factor nested within treatment). Data were log-transformed prior to analysis and a separate model was fitted for the pretreatment and post-treatment periods). From the model, the difference in mean effects between treatments was calculated and assessed by Tukey’s test. (H) Kaplan-Meier survival curves, with median survival indicated (ns=non-significant, log-rank test). artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Expansion of E7-specific CD8+ T cells was observed in the blood of immunized mice, 6 days post-treatment (figure 3D). While all treatment groups elicited comparable frequencies of E7-specific CD8+ T cells (ranging from 1% to 13%), two animals treated with artLCMV-E7E6 intravenously and intratumorally showed frequencies of 25% and 43%, respectively, which were maintained at d15 (figure 3D). Most treatment-induced E7-specific CD8+ T cells exhibited a CD127 effector phenotype (figure 3E), likely contributing to the tumor control observed until d25 (figure 3B). At day 15, we observed a contraction of the early effector cell population, and a relative increase of both CD127+ KLRG1+ and CD127+ KLRG1 cells (figure 3E), potentially indicating the development of long-term antitumor immunity.33 35 The increased frequency of memory CD8+ T cells was more pronounced following artPICV treatment via either administration route, whereas for artLCMV this increase was more pronounced for the intratumoral route (figure 3E).

Since combination therapies will likely be required for effective long-term tumor control, and improvement in response rates for current SoC treatments would have a greater and possibly more immediate impact on HPV-driven HNSCC patients, we sought to understand if cisplatin combination could be leveraged to increase the efficacy of artARENA vectors. As high doses of cisplatin were shown to have toxic side effects in the clinic,36 we investigated a lower dose for beneficial therapeutic effects. Although cisplatin treatment alone at this dose had a modest effect on tumor growth and median survival, we observed an improvement in the therapeutic responses when cisplatin was administered in combination with each vector (online supplemental figure 2B,C).

To explore the potential antitumor efficacy of each vector towards metastatic tumors, we compared local (intratumoral) and systemic (intravenous) vector administration in mice bearing TC-1 tumors in both the left and right flanks (figure 3F). Monitoring of tumor volume showed that both intratumoral administration into the right tumor and intravenous administration controlled the tumor growth in both flanks (figure 3G, online supplemental figure 2D). The rate of tumor control between flanks was comparable even in the intratumorally treated animals (figure 3G), and there were no significant differences in survival (figure 3H), suggesting that both regimens promote a systemic immune response. To analyze the biodistribution and transgene expression following either administration route, artARENA vectors encoding the Nanoluc luciferase were intravenously or intratumorally injected into tumor-bearing mice, and the bioluminescence was measured in vivo over time (online supplemental figure 2E,F), and ex vivo in different organs at 24 hours postimmunization (online supplemental figure 2G,H). We observed a similar infection pattern in multiple organs (online supplemental figure 2E–H). The spread of the vectors was similar for both administration routes and possibly explained the induction of systemic immunity in the intratumoral setting. Nanoluc expression was stable or even increased until the end of the observation period (online supplemental figure 2E,F). The in vivo bioluminescence was higher following artLCMV treatment.

Overall, the strong immunogenicity elicited by artARENA vectors translates into potent antitumor activity, with the potential for controlling tumor growth and prolonging survival in the presence of metastatic lesions, as monotherapy or in combination with chemotherapy.

artARENA immunization induces T-cell infiltration in tumors

CD8+ T-cell infiltration is prognostic for good clinical outcome and increased survival in several human cancers.37 38 Therefore, we analyzed the magnitude and phenotype of T-cell infiltration following treatment. For this, TC-1 tumor-bearing C57BL/6 mice were intravenously or intratumorally treated with either vector, tumors were harvested on days 7, 14, and 21 post-treatment, and tumor-infiltrating lymphocytes were analyzed by flow cytometry and IHC (figure 4A).

Figure 4

artARENA immunization induces T cell infiltration in tumors. (A) Experimental design (created with TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm3. Control animals received formulation buffer. Tumors were harvested on days 7, 14, and 21 postimmunization. (B–E) Tumors were dissociated and cells were analyzed by flow cytometry (n=3–5/group, mean±SD). (B) Absolute numbers of CD8+ T cells, calculated per gram of tissue. (C) CD8+/Treg ratio, calculated by dividing the total number of CD8+ by the total number of CD4+CD25+FoxP3+ T cells. (D) Percentage of ICOS+ T cells (within CD8+T cell population) in TILs, spleen, and lymph node. (E) Percentage of PD-1+ subsets (low, intermediate, and high expression) in ICOS+CD8+ T cells. (F) Tumor tissue was subjected to immunohistochemical analysis. Representative whole staining scans (top, scale bar=1000 µm), and inserts (bottom, scale bar=50 µm) of respective conditions are shown (scans representative of 1–5 animals/group). Statistical analysis: two-way ANOVA and Dunnett’s multiple comparisons test, ns=non-significant, *p<0.05, **p<0.01, ***p<0.001,. ANOVA, analysis of variance.

Flow cytometry analysis revealed robust tumor infiltration of CD8+ T cells (figure 4B). Although some variability was observed, all artARENA-treated groups showed a comparable increase in infiltrating CD8+ T cells (around 300×104 cells/g of tumor) 7 days postimmunization and promoted proinflammatory ratios of CD8+/Tregs within the TME (figure 4B,C). Around 60% of tumor-infiltrating CD8+ T cells expressed inducible T cell costimulator (ICOS), highlighting their activated effector phenotype (figure 4D).39 40 These cells were primarily accumulating at the tumor site, as low to no ICOS+ CD8+ T cells were detected in the spleen or lymph nodes of treated animals (figure 4D). However, T cell numbers tended to decrease over time, as fewer infiltrating CD8+ T cells were measured at days 14 and 21, along with a concomitant decrease in CD8+/Treg ratio, and increase in PD-1 expression (figure 4B,C and E). Cells expressing higher levels of PD-1 constituted most of the infiltrating CD8+ T cells by day 21 (figure 4E). Finally, IHC staining of tumor sections also showed robust infiltration of CD8+ T cells, penetrating the tumor mass (figure 4F). FoxP3+ T-cells were visible within the tumor mass at later time points (figure 4F). Total CD3+ and CD4+ T cell-infiltration can be seen in online supplemental figure 3A. Quantification of cells within the stained histological sections corroborated the flow cytometry results, with higher infiltration of CD8+ T cells at earlier time points, and gradually higher accumulation of Tregs at later time points (online supplemental figure 3B). In addition, quantification of Ki-67 expressing cells showed that these were reduced in vector-treated groups, 7 days post-treatment (online supplemental figure 3C).

These data show that both vectors triggered strong intratumoral infiltration of cytotoxic T cells; however, immunosuppression built up over time suggested that combination treatments might be required for adequate long-term tumor control.

Antitumor efficacy of artARENA is mediated by CD8+ T cells

To understand the relative contribution of the T cell and NK cell response in mediating tumor control, animals grafted with TC-1 cells were depleted of either CD4+, CD8+ T or NK cells, and tumor size was monitored following intravenous or intratumoral administration of either vector (figure 5A, online supplemental figure 4A). Depletion of CD8+ T cells abrogated the therapeutic effect from both vectors, suggesting a critical role for cytotoxic T cells in mediating tumor growth control (figure 5B,C). Depletion of CD4+ T cells had the opposite effect, leading to increased tumor growth control and to a moderate increase in survival for most treatment groups (figure 5B,C).

Figure 5

Antitumor efficacy of artARENA is mediated by CD8+ T cells. (A) Experimental design (created with TC-1 tumor-bearing C57BL/6 mice were immunized intravenously (B) or intratumorally (C) with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm3. Control animals received formulation buffer. Mice were depleted of CD8+, CD4+ T cells or NK cells by the addition of neutralizing antibodies three times over the course of the study. (B, C) Tumor volume monitored over time (left, mean±SEM, n=7/group, data are shown until the time point at which more than 50% of the animals in the group were euthanized). Kaplan-Meier survival curves, with indicated median survival and long-term survivors (right, *p<0.05, **p<0.01, ***p<0.001 Log-rank test). artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

Unexpectedly, upon depletion of NK cells, the antitumor immune response was either not affected (intravenous groups) or further improved (intratumoral groups). This suggests that the NK cell response may be unfavorable in this model.

Overall, the antitumor effect of both artLCMV and artPICV vectors is mediated by CD8+ T cells, and CD4+ T cell-mediated immunosuppression may be partially impairing antitumor immunity following artARENA treatment.

artARENA treatment induces proinflammatory changes in the TME and reveals NKG2A blockade as a potential new target for combination

Having shown that the artARENA platform triggers T-cell infiltration, we sought to obtain a more comprehensive view of cancer and immune-related changes within the TME resulting from the two vectors and administration routes. For this, we performed NanoString nCounter PanCancer IO 360 analysis on tumor samples harvested 7 days post-treatment. This time point was chosen to analyze gene expression changes within the TME mediated by both, innate immune responses induced by the viral vectors as well as tumor antigen-specific T-cell responses.

Differential gene expression analysis identified a high number of genes upregulated in all treatment groups. Each sample’s gene expression profile was condensed into a set of pathway scores, whereas increasing scores correspond to mostly increasing expression (specifically, each pathway score has positive weights for at least half its genes). Pathway analysis revealed a deep rearrangement of the TME on administration of the vectors, but no distinct clustering of the different treatment groups (figure 6A,B). Pathways potentially promoting tumor growth including cell proliferation, autophagy, and DNA damage repair41 42 as well as TGF-ß signaling, which is known to potentiate an immunosuppressive TME,43 had a lower score in vector-treated groups. Conversely, pathways associated with immune activation scored higher (figure 6A). Enhanced activity of the myeloid and lymphoid compartments further underscored the increased immune infiltration observed previously (figure 4). Higher scores of costimulatory signaling, cytotoxicity, and antigen presentation indicated increased activation of the infiltrating immune cells. Additional pathways (figure 6A—other) with multiple described roles in the function of both tumor and immune cells were enhanced in treated samples; however, further studies are needed to better dissect their contribution to the artARENA-mediated antitumor response.

Figure 6

artARENA treatment induces proinflammatory changes in the TME and identifies NKG2A blockade as a potential new target for combination. (A, B) Similar to previous experiments, TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm3. Control animals received formulation buffer. 7 days postimmunization tumors were harvested, and gene expression was analyzed using the nCounter PanCancer IO 360 Panel (Nanostring Technologies). (A) Heatmap of pathway scores of the differentially expressed genes in each sample is shown. Each column represents one sample (one mouse). Scores were normalized via z-transformation. (B) Volcano plots displaying each gene’s -log10 adjusted p value and log2 fold change between buffer and indicated treatment groups. The top 16 genes upregulated genes across all groups are displayed in red and indicated in the table. (C) TC-1 tumor-bearing C57BL/6 mice were immunized intravenously or intratumorally with 1×105 RCV FFU of artLCMV-E7E6 or artPICV-E7E6 vectors when tumors reached a volume of ~100 mm3. αNKG2A antibody was administered intraperitoneally 3 times over the course of the study: on day 0 (same day of vector administration), on days 3–5, and on days 7–8 (data from three independent experiments). Control animals received formulation buffer. Tumor volume was monitored over time (left, mean±SEM, n=10/group, shown is one experiment representative of three independent experiments, data are shown until the time point at which more than 50% of the animals in the group were euthanized or maximum d23). Kaplan-Meier survival curves, with indicated median and long-term survivors (right, *p<0.05, Log-rank test, data pooled from three independent experiments). (D) Animals showing complete response were rechallenged with TC-1 cells in the opposite flank. Control animals correspond to age-matched naïve mice grafted with TC-1. Kaplan-Meier survival curves are shown. artLCMV, arenavirus vectors based on lymphocytic choriomeningitis virus; artPICV, arenavirus vectors based on Pichinde virus.

A closer look at the top 20 upregulated genes identified 16 to be consistently upregulated across all treated groups (figure 6B—red dots). In line with the pathways identified above, several of these were indicative of increased accumulation of T cells within the TME (Cd3g, Cxcl9), while others denoted their potentially increased cytotoxic capacity (Gzmb, Nkg7, Irf1). The upregulation of IFN-induced genes indicated a strong IFN response triggered post-treatment. Gbp2 and Gbp3 encode guanylate binding proteins which contribute to protective immunity during infection, inflammation, and cancer.44 IL18 binding protein (IL18bp), on the other hand, is a high-affinity decoy receptor that limits the proinflammatory antitumor activity of IL18.45 Its upregulation may, therefore, represent a potential therapeutic target for enhancing antitumor efficacy.46 In addition, multiple genes involved in antigen processing and presentation were identified (figure 6B). Upregulation of Psmb8 and Psmb9, immunoproteasome subunits involved in MHC class I antigen processing, and upregulation of Tap1, Tap2, and Tapbpl, involved in peptide loading onto MHC class I molecules, suggested a strong presence of functional APCs within the infiltrating myeloid cells.47 48

Among the top differentially expressed genes was the non-classical MHC class I molecule H2-T23, alternatively named Qa-1b (figure 6B—bold). This is a homolog of the HLA-E, the main ligand of inhibitory receptor NKG2A49 50 expressed in NK, NKT, and a subset of CD8+ T cells.51 The increased expression of H2-T23 following treatment prompted us to investigate whether a combination with an anti-NKG2A blocking antibody could enhance the therapeutic efficacy of artARENA vectors. Using the same TC-1 tumor model described in previous experiments, we observed that NKG2A blockade indeed improved the antitumor response in combination with intravenous administration of artLCMV-E7E6 (figure 6C, online supplemental figure 5A). We observed prolonged tumor growth control in this combination group and a significant increase in median survival, with 19% of animals achieving a complete response. Combination with artPICV-E7E6 had a less pronounced effect on tumor control, but we could still observe an increase in median survival (figure 6C, online supplemental figure 5A). Animals that remained tumor-free until the end of the study were rechallenged with TC-1 tumor cells in the opposite flank. All previously treated groups were protected from rechallenge (figure 6D), indicating the generation of long-term antitumor immunity.

Overall, transcriptomic characterization of tumors showed a profound proinflammatory shift within the TME, with active antigen presentation, influx of activated lymphocytes, and strong IFN response. Furthermore, it revealed that upregulation of an NKG2A ligand could be negatively impacting the immune response following treatment and identified the blockade of NKG2A as a potential new combination approach to further enhance the therapeutic efficacy of artARENA.


To overcome current hurdles and maximize clinical benefit, novel therapeutic vaccines need to generate high numbers of tumor antigen-specific T cells with potent effector functions and memory T cells for long-term tumor control. In addition, rational drug combinations based on the underlying immunosuppression mechanisms associated with therapy resistance will be key in ensuring sustained tumor regression. As such, this study provides an overview of the strong immunogenicity elicited by artARENA vectors, inducing a proinflammatory shift in the TME, with increased infiltration of ICOS+CD8+ T cells that mediate tumor control. In-depth characterization of the TME provided the rationale for a potential new combination approach to increase its therapeutic efficacy. NKG2A blockade could potentially counteract an adaptive resistance mechanism to therapeutic vaccination (increased expression of its inhibitory ligand), thus synergizing with artARENA therapy.

We found that a single administration of either artLCMV or artPICV vectors triggered a systemic proinflammatory response within 24 hours and induced profound changes in the TME up to 2 weeks post-treatment, indicative of an active antitumor immune response. In line with previous reports on artLCMV-mediated immune response,14 16 we showed that artPICV vectors also led to priming and expansion of polyfunctional E6-specific and E7-specific CD8+ T cells coexpressing effector molecules (CD107a) and cytokines (IFN-γ and TNF-α) by days 6–7 post-treatment. The peak T-cell response was previously observed to occur at this time point16; however, by day 15, we could still detect antigen-specific T cells within the spleens of treated mice, indicating durable responses to both vectors. Non-lytic infection and activation of human and mouse professional APCs, together with a proinflammatory milieu and the release of danger-associated molecular patterns (current study and Kallert et al14) generate ideal conditions for antigen presentation and T-cell priming. These hallmarks of artARENA vectors, along with prolonged transgene expression observed using bioluminescence studies, mark a clear advantage over other antigen-delivering platforms and oncolytic viruses. In addition, we evaluated immunogenicity after immunization of naïve as well as after treatment of tumor-bearing mice and observed higher frequencies of E7-specific CD8+ T cells in the latter. Thus, apart from inducing de novo T-cell responses, this platform could also amplify preexisting tumor-specific T-cell responses, which were detected at very low levels in control animals. This is confirmed by early data from the ongoing Phase I trial in heavily pretreated patients with HPV16+ cancers, where up to 48% E6/E7 specific CD8+ T cells were measured in blood after alternating HB-202 (artPICV-E7E6) and HB-201 (artLCMV-E7E6) therapy.18 19 Further studies are needed to understand this in preclinical models and in the clinical setting, where broadening and amplification of the T-cell repertoire was linked to a better outcome.10 11 38

Antigen-specific CD8+ T cells triggered by both vectors presented in various stages of differentiation, with most exhibiting a KLGR1+CD127 effector phenotype at the time points measured. Potent effector and cytotoxic functions were evidenced by ICOS expression in a large proportion of T cells only at the tumor site.39 40 ICOS agonists are under clinical development and demonstrated a favorable toxicity profile but modest clinical activity.52 Still, it would be interesting to investigate a combination with artARENA vectors in preclinical models once commercial ICOS agonists become available. Nevertheless, the generation of long-lasting memory T cells capable of mediating durable clinical responses is also required for successful therapeutic vaccination.53 Both artLCMV and artPICV vector-induced E7-specific T cells were composed of distinct populations of KLGR1CD127+ and KLGR1+CD127+ T cells, although these were more abundant following artPICV treatment. While a previous report54 suggested that KLGR1-expressing T cells may have poor proliferative capacity, other reports55 indicate that these cells also exhibit properties of long-lived effector and memory cells. The latter further shows that these cells undergo homeostatic proliferation and that downregulation of KLGR1 is not necessary for T cells to develop long-lived protective memory function. Although we do not phenotype antigen-specific T cells at later time points, we do see a persistence of KLGR1+CD127+ T cells on day 15 post-treatment, further suggesting the development of long-term antitumor immunity following treatment.33 35 The increased potential for these cells to develop memory phenotypes suggested that artPICV is better suited to generate long-term antitumor immunity. In fact, lower exposure to inflammatory signals during priming was suggested to promote the development of effector CD8+ T cells with increased potential for memory formation.33 The lower levels of inflammatory cytokines detected in the serum of artPICV-treated animals, along with lower frequencies of activated myeloid and lymphoid cells, would suggest lower levels of inflammation elicited by this vector. This, in turn, provides a possible explanation for the better outcome observed in the alternating prime-boost protocol tested preclinically, in which artPICV priming followed by artLCMV boosting yields stronger CD8+ T-cell responses than immunization in the reverse order.17

Both artLCMV and artPICV single administration inhibited tumor growth and prolonged survival in a stringent TC-1 mouse tumor model, which was largely driven by CD8+ T cells. Unlike many previous reports, where treatment was initiated when tumors became palpable, here we focused on larger, harder-to-treat tumors, and still were able to demonstrate the efficacy of this platform. Furthermore, our results provide evidence of the generation of a robust systemic immune response following both systemic (intravenous) and local (intratumoral) administration of either vector. While the broad tropism of artLCMV vectors and their ability to modulate stromal cells within the TME of B16.F10 tumors on intratumoral administration was previously linked to their antitumor efficacy,34 our results indicate that artARENA vectors also reach secondary lymphoid organs after intratumoral injection. On the other hand, intravenously injected vectors also reach tumor tissue. Thus, the widespread distribution allows antigen presentation and modification of stromal cells in lymphoid and non-lymphoid tissue following either administration route. Differences within the TME of each tumor indication could be driving the disparate outcomes, highlighting the need for in-depth characterization of the TME on treatment.56

Treatment with artLCMV and artPICV led to a remodeling of the TME, with increased T-cell infiltration. Transcriptomic analysis of tumor samples evidenced the switch into a more proinflammatory environment, with T cells presenting with high cytolytic activity and activation of effector signaling pathways. A strong myeloid compartment, with active antigen presentation, was also clear from transcriptomic studies. Still, there was evidence of increased immunosuppression over time. Tumor outgrowth was seen at later stages, along with decreasing T-cell infiltration and lower CD8+ T/Treg ratio. Differential gene expression analysis identified the increased expression of NKG2A inhibitory receptor ligand H2-T23 as a possible contributing factor. Although its human ortholog HLA-E expression is seen in several tumors, recent reports suggest it is further upregulated within an immune reactive environment.57 Indeed, NKG2A blockade alone had no effect on tumor growth in this model system, suggesting that the inflammatory conversion of the TME on artARENA treatment is driving its overexpression. NKG2A blockade in combination with artLCMV improved antitumor efficacy, validating it as a potential combination partner. While previous reports found no indication that NKG2A expression would inhibit the cytotoxic function of LCMV-induced T cells,58 more recent studies show that its blockade potentiates CD8+ T-cell immunity induced by cancer vaccines.59 The latter also shows that NKG2A blockade effects were exerted through CD8+ T cells and independent of NK cells. This is in line with our results showing that depletion of NK cells either improves or has no effect on the antitumor efficacy of the artARENA platform and suggests that the positive impact of blocking NKG2A is mediated by CD8+ T cells in this model. NKG2A-blocking antibody monalizumab showed a good safety profile and promising clinical responses in refractory HNSCC.60 The INTERLINK-1 phase III trial investigating monalizumab in combination with cetuximab, however, was stopped after a failed futility analysis (press release Innate Pharma on August 1, 2022). To evaluate a potential combination, it would be interesting to analyze NKG2A and HLA-E expression in tumors of HB-202/HB-201 treated patients.

In conclusion, this work highlights the artARENA platform as a promising contender in the therapeutic vaccine space, providing large numbers of tumor-specific cytotoxic T cells that infiltrate the tumor mass and mediate tumor control. The greater understanding of the artPICV-mediated immune response supports its use in an alternating two-vector therapy together with artLCMV. Finally, a potential combination partner here explored (NKG2A inhibition) is under clinical development. This accelerates the further work needed to evaluate its safety and efficacy in clinical settings and to fully realize its potential.

Data availability statement

Data are available on reasonable request.

Ethics statements

Patient consent for publication

Ethics approval

In vivo mouse experiments performed at Hookipa Pharma were approved by the Austrian authorities and were carried out in accordance with the approved guidelines for animal experiments at Hookipa Pharma. In vivo mouse experiments performed at the University of Navarra were approved by the Ethics Committee of the University of Navarra (055-21).


The Vienna BioCenter Core Facilities (VBCF) Plant Sciences Facility acknowledges funding from the Austrian Federal Ministry of Education, Science & Research; and the City of Vienna. The computational results presented were obtained using the CLIP cluster ( We are grateful to Johns Hopkins University for providing HPV16 E6 and E7-positive TC-1 tumor cells. The authors acknowledge the Histology Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria, and Anoop Kavirayani for the histopathology analysis. The authors acknowledge Barbara Haigl for her contribution to the revision of figure 2.


Supplementary materials

  • Supplementary Data

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  • Contributors Conceptualization and design: JR, TP, MH, KKO and HL. Design and generation of vectors: SS, FS and TS. Data acquisition and analysis: JR, CP, TP, MH, SR, JS, VJ, KBH, ES, JJ and PB. Writing: JR, CP and HL. All authors performed a critical revision of the manuscript and approved the submitted version. Guarantor: HL.

  • Funding This study was supported by Hookipa Pharma, and by an Innovation Voucher from the Austrian Research Promotion Agency, voucher 882888.

  • Competing interests JR, CP, TP, SR, JS, VJ, SS, KBH, FS, TS, KKO and HL are employees of Hookipa Pharma and its subsidiary Hookipa Biotech. MH was an employee of Hookipa Pharma and its subsidiary Hookipa Biotech GmbH when this study was conducted. PB, ES and JJ received research funding from Hookipa Pharma.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.