Engineering antiviral immune-like systems for autonomous virus detection and inhibition in mice

Viral diseases pose significant public health and economic threats, disproportionately affecting immunocompromised individuals who are at higher risk of severe outcomes. Herpes simplex virus type 1 (HSV-1), for example, can cause herpetic simplex keratitis (HSK), a leading cause of corneal blindness and viral encephalitis if untreated. Innate immune sensing, particularly via the STING pathway, detects cytosolic viral DNA/RNA and triggers IRF3-mediated transcription of antiviral responses. Motivated by the need for broad-spectrum and autonomous antivirals for pandemic preparedness and to support individuals with weak innate immunity, the authors sought to engineer mammalian cells that can detect viral infection and conditionally express antiviral effectors. The central hypothesis is that coupling virus detection to inducible expression of antiviral cytokines, CRISPR-Cas9 nucleases targeting viral genomes, and neutralizing antibodies can create an autonomous sense-and-destroy system to prevent and treat viral infections. The study introduces autonomous, intelligent, virus-inducible immune-like (ALICE) systems that rewire endogenous signaling to link viral detection to antiviral outputs and evaluates their efficacy in vitro and in mouse models.

Emerging synthetic biology diagnostics leverage programmable components for pathogen detection, including toehold RNA switches and freeze-dried, paper-based cell-free systems enabling point-of-care viral detection. CRISPR-based diagnostics (SHERLOCK, DETECTR, HOLMES) exploit collateral nuclease activity of Cas13/Cas12a for sensitive, specific nucleic acid detection. Beyond detection, CRISPR-Cas9 has been explored to degrade viral genomes (e.g., SARS-CoV-2, influenza, HIV, HBV, HPV, HSV-1), but constitutive Cas9 expression from lentiviral/AAV vectors raises safety concerns: sustained nuclease presence, anti-Cas9 immunity, off-target editing, and emergence of CRISPR-resistant viral escape mutants. Advances in immune-like designer cells suggest engineered cellular therapies can autonomously respond to infection, as shown in self-regulated systems against MRSA, inspiring development of programmable antiviral cells. This work builds on these foundations by integrating STING-based sensing with inducible effector expression to overcome limitations of constitutive antiviral expression systems.

Design of virus sensor (ALICEsen): The authors engineered a destabilized STING-based sensor in mammalian cells. A proteolytic PEST tag was fused to STING (STING-PEST) to minimize basal activity and increase inducibility. Viral sensing activates endogenous TBK1, leading to IRF3 phosphorylation/dimerization and nuclear translocation. A synthetic IRF3-responsive promoter (PALICE, optimized as PALICE6) drives genes of interest (GOI) such as reporters, cytokines, or effectors. Sensor optimization used HEK-293T cells (endogenous cGAS/STING absent) and HSV-1 as a model. Baseline stable STING yielded ~3.3-fold induction; STING-PEST increased induction to ~20-fold. The combination of PhCMV-driven STING-PEST and PALICE6 achieved ~23-fold SEAP induction; using an EGFP reporter achieved ~412-fold maximal induction. Sensor breadth was assessed across viruses: activation by STING-dependent viruses (DENV-2, SARS-CoV-2, hCoV-229E, HCV, HBV, adenovirus 5, HSV-1) and no activation by STING-suppressing/irrelevant viruses (H1N1, EV-A71, PRV2P, VSV, lentivirus, AAV). Kinetics and dose responses were profiled; reversibility was shown using acyclovir.

ALICEim (inducible cytokines): PALICE6 drove human IFN-α or IFN-β in response to infection (HCV, HBV, ADV, HSV-1). Cytokine levels were quantified by ELISA at 2 and 4 dpi; antiviral efficacy was assessed by qPCR of viral genes relative to ALICEsen-SEAP controls.

ALICECas9 (inducible Cas9): SEAP was replaced with SpCas9 under PALICE6 control. HSV-1 multiplicity of infection correlated with Cas9 expression (immunoblot). Initial validation targeted host loci (CCR5, d2EYFP) with constitutive sgRNAs to confirm HSV-1-inducible editing. For antiviral testing, sgRNAs targeted HSV-1 essential genes: US8 (glycoprotein E), UL29 (ssDNA-binding), UL52 (helicase-primase). HSV-1 life cycle markers UL23 (immediate early), UL30 (early), and US2 (late) were monitored by RT-qPCR. EGFP-HSV-1 infection assessed infected cell fluorescence and plaque-forming units. Comparisons were made to constitutive Cas9 expression. Multiplexing was evaluated by tandem sgRNAs targeting ADV (E1A) and HSV-1 (UL52), followed by qPCR for ADV E1B/E2 and HSV-1 UL23/UL30.

ALICEAb (inducible neutralizing antibodies): For HSV-1, PALICE6 drove secretion of His-tagged human mAb E317 (targets HSV-1 gD). Function was validated by neutralization assays against EGFP-HSV-1 at MOIs 1–5. For SARS-CoV-2, PALICE6 drove REGN10989+REGN10987 antibody cocktail; infection reduction was quantified (~70% reduction in vitro).

ALICECas9+Ab (dual-output): Stable HEK-293T-derived lines expressing PALICE6-Cas9 and PALICE6-E317Ab with PhCMV-STING-PEST were generated (Sleeping Beauty transposition; puromycin/zeocin/blasticidin selections). Inducible expression upon HSV-1 infection was confirmed (qPCR, immunoblot). Antiviral performance was quantified versus single-output systems (ALICECas9, ALICEAb) by RT-qPCR of HSV-1 transcripts, fluorescence readouts, and live virus titers. Longitudinal expression over 7 days was assessed. Comparison with acyclovir (10 µM, 50 µM) benchmarked potency. Transwell assays evaluated: (i) protection of bystander HEK-293T cells from spread (designer cells in inner chamber, HEK-293T in outer); and (ii) self-protection (infected HEK-293T in outer chamber; ALICE cells in inner chamber), read by EGFP intensity.

In vivo cell therapy in mice (BALB/c): Hydrogels (hyaluronic acid-based) encapsulated engineered cells (Control HEK-293T; ALICECas9; ALICEAb; ALICECas9+Ab). For prophylaxis, hydrogels were implanted intraperitoneally; 20 h later mice were challenged IP with HSV-1 (2×10^7 PFU). At 2, 4, 6 dpi, liver/spleen/kidney were assessed for HSV-1 UL23/US2 mRNA (ΔΔCt to WT) and viral titers; serum E317Ab monitored by His-tag ELISA; scaffold protein extracts blotted for Cas9. Long-term study: stable ALICECas9+sgRNAs+Ab hydrogels implanted; mice challenged at 28 days, analyzed at day 30; cytokines (IL-6, CCL5, CXCL10, TNF-α, IFN-α) by flow assay; IgG by ELISA. Transmission model: hydrogels pre-loaded with HSV-1-infected control or ALICECas9+Ab cells implanted to mimic infected graft; organ viral RNAs/titers and serum IL-6/E317Ab measured.

In vivo treatment post-infection: Mice first infected IP with HSV-1 (2×10^7 PFU), then implanted with ALICECas9+Ab or control hydrogels 20 h later; assessed at day 6 (organ viral RNAs/titers, scaffold Cas9/E317Ab, serum E317Ab/IgG).

AAV-mediated gene therapy in HSK model: Two AAVs were co-administered via retro-orbital injection 6 days before ocular HSV-1 infection: AAVrh10-ALICEsaCas9 (PALICE6-driven SaCas9; U6-driven HSV-1 ICP4 sgRNA) and AAV1-ALICEAb (PALICE6-driven E317Ab-P2A-NanoLuc; PhCMV-STING-PEST). HSK was induced by corneal scarification and EGFP-HSV-1 infection (9×10^5 PFU/eye) at days 0 and 20. Outcomes at 14 and 25 days post-initial infection included body weight, HSV-1 UL23/US2 mRNA in eye/trigeminal ganglia/brain, brain titers, serum NanoLuc and E317Ab induction, cytokines (IL-6, CCL5, CXCL10, TNF-α, IFN-α), and IgG. Statistical analyses used t-tests or ANOVA with Bonferroni/Dunnett post hoc as appropriate.

• Sensor performance: STING-PEST with PALICE6 minimized basal expression and maximized inducibility. Initial stable STING yielded ~3.3-fold induction; adding PEST achieved ~20-fold; optimized ALICEsen achieved ~23-fold SEAP induction and up to ~412-fold with an EGFP reporter. ALICEsen responded to multiple STING-dependent viruses (DENV-2, SARS-CoV-2, hCoV-229E, HCV, HBV, ADV, HSV-1) but not to H1N1, EV-A71, PRV2P, VSV, lentivirus, or AAV. Responses were dose- and time-dependent and reversible with acyclovir.
• ALICEim cytokine module: Infection with HCV, HBV, ADV, or HSV-1 induced IFN-α and IFN-β production (ELISA), and reduced viral gene expression compared to ALICEsen-SEAP controls (qPCR), demonstrating broad-spectrum, nonspecific antiviral activity.
• ALICECas9 antiviral editing: Upon HSV-1 infection, inducible Cas9 plus sgRNAs targeting US8, UL29, or UL52 significantly reduced HSV-1 transcripts UL23 (immediate early), UL30 (early), and US2 (late), decreased infected-cell fluorescence, and reduced live virus titers, with efficacy comparable to constitutive Cas9 but avoiding constitutive nuclease exposure. Multiplexed sgRNAs targeting ADV (E1A) and HSV-1 (UL52) simultaneously lowered ADV E1B/E2 and HSV-1 UL23/UL30 RNA levels, indicating multi-virus targeting capability.
• ALICEAb neutralization: ALICEAb secreting HSV-1 mAb E317 reduced HSV-1 infection at MOIs 1–5. For SARS-CoV-2, a REGN10989/REGN10987 ALICEAb reduced infection by ~70.3 ± 4.3% in vitro.
• Dual-output synergy (ALICECas9+Ab): The combined system outperformed single-output ALICECas9 or ALICEAb in reducing HSV-1 mRNAs and replication, sustained Cas9 and E317Ab expression over one week during infection, matched the efficacy of high-dose ACV (50 µM) and exceeded low-dose ACV (10 µM). Transwell assays showed strong protection against virus spread to bystander cells and self-protection of ALICE cells.
• In vivo prophylaxis (hydrogel implants): In mice challenged with HSV-1 after implantation, ALICECas9+Ab showed the strongest reduction in UL23/US2 mRNAs and viral titers in liver, spleen, kidney at 2, 4, and 6 dpi compared with controls; single-output systems had intermediate effects (antibody slightly better than Cas9). Infection induced robust Cas9 and E317Ab expression in scaffolds/serum. No significant differences in host IgG were observed between control and treated groups.
• Long-term functionality: At 30 days post-implantation, ALICECas9+sgRNAs+Ab mice challenged with HSV-1 showed higher inducible Cas9 and E317Ab, significantly reduced organ viral mRNAs and titers, and markedly lower HSV-1 mRNA within control hydrogels (1000–3000-fold increase upon challenge versus unchallenged hydrogels). Elevated inflammatory cytokines induced by HSV-1 (IL-6, CCL5, CXCL10, TNF-α, IFN-α) were reduced after ALICE treatment; IgG levels remained unchanged.
• Transmission model: Mice receiving HSV-1-infected ALICECas9+Ab hydrogels had lower viral RNAs/titers in nearby organs and lower serum IL-6 than mice receiving infected control hydrogels, evidencing reduced transmission.
• Post-infection therapy: Implantation of ALICECas9+Ab into already infected mice reduced organ viral RNAs/titers; infection induced Cas9 and E317Ab in scaffolds/serum.
• AAV-ALICEsaCas9+Ab in HSK: Retro-orbital delivery of AAVrh10-ALICEsaCas9 + AAV1-ALICEAb before ocular infection reduced weight loss, and significantly decreased HSV-1 mRNAs in cornea and trigeminal ganglia and brain viral titers at 14 and 25 days. HSV-1 challenge induced systemic NanoLuc and E317Ab in treated mice. Pro-inflammatory cytokine elevations (IL-6, CCL5, CXCL10, TNF-α, IFN-α) were blocked by AAV-ALICE treatment; total IgG was unchanged.

The study demonstrates that coupling a destabilized STING-based sensor to an IRF3-responsive synthetic promoter can endow mammalian cells with autonomous detection of multiple DNA and some RNA virus infections and trigger targeted antiviral responses. By restricting effector expression (IFNs, Cas9, antibodies) to infection contexts, ALICE systems address key limitations of constitutive antiviral strategies, potentially reducing off-target effects, immune responses against effectors, and selection for resistant viral escape mutants. The dual-output ALICECas9+Ab exhibited synergistic inhibition of HSV-1 via orthogonal mechanisms (genome cleavage and entry neutralization), rivaling high-dose acyclovir and offering a path to mitigate drug resistance. In vivo, hydrogel-embedded designer cells provided both prophylactic and therapeutic benefits, reducing viral burdens and inflammatory responses in multiple organs, with sustained functionality up to at least 30 days. AAV delivery of ALICE components extended efficacy to a clinically relevant HSK model, lowering corneal/TG/brain viral loads and systemic inflammatory markers while enabling noninvasive monitoring via NanoLuc. The modularity of ALICE allows retargeting by swapping sgRNAs or antibodies and adapting the sensing module (e.g., RIG-I) to address RNA viruses, broadening potential applications. These results support the relevance of engineered immune-like cells and gene therapy vectors as programmable, pathogen-responsive antivirals capable of early detection and intervention, particularly beneficial for immunocompromised populations.

This work establishes ALICE, a modular class of autonomous, virus-inducible immune-like systems that sense infection via a destabilized STING circuit and conditionally deploy antiviral cytokines, CRISPR-Cas9 genome editing, and neutralizing antibodies. In vitro, ALICE detected multiple STING-activating viruses and robustly inhibited HSV-1; in vivo, both cell-based hydrogel implants and AAV-mediated delivery reduced viral loads, transmission, and inflammatory responses, including in a herpetic keratitis model. The dual-output ALICECas9+Ab achieved synergistic, sustained antiviral activity comparable to high-dose acyclovir while being virus-inducible. The platform’s modularity enables retargeting to different pathogens by changing sgRNAs, antibodies, or the sensing module, suggesting broad applicability. Future work should extend ALICE to RNA viruses using alternative sensors (e.g., RIG-I), optimize delivery and implantation materials for long-term performance, evaluate safety and immunogenicity comprehensively, and explore patient-derived cell chassis for personalized, durable antiviral therapies.

• Sensing scope: The STING-based ALICEsen did not activate in response to several viruses (e.g., H1N1, EV-A71, PRV2P, VSV, lentivirus, AAV), limiting applicability to STING-activating infections. For RNA viruses, alternative sensors (e.g., RIG-I) would be required.
• Delivery constraints: AAV packaging limits necessitated use of SaCas9 and split-vector designs, potentially complicating vector manufacturing and co-transduction efficiency.
• Leaky expression and safety: Although low, some basal (leaky) Cas9/antibody expression was observed in the absence of virus; comprehensive off-target editing, anti-Cas9 immune responses, and long-term biosafety were not fully assessed.
• Model specificity: Most in vivo data focus on HSV-1 in BALB/c mice; generalizability to other pathogens and to human physiology remains to be established.
• Implantation and durability: Hydrogel-embedded cell therapies showed functionality up to 30 days; longer-term stability, host responses, and retrievability require further study.