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

The escalating threat of viral diseases, exemplified by the COVID-19 pandemic, underscores the critical need for effective, broad-spectrum antiviral strategies. Individuals with compromised immune systems are particularly vulnerable to severe viral complications. The human innate immune system, with STING (stimulator of interferon genes) as a key player, represents the first line of defense. STING recognizes viral nucleic acids, triggering interferon production and immune cell recruitment. However, individual variations in innate immune function, especially in immunocompromised individuals, necessitate the development of robust, artificial antiviral systems. Synthetic biology offers a powerful approach to engineer such systems. Prior work has explored synthetic sensors (toehold switches) and CRISPR-Cas systems for virus detection and elimination. CRISPR-Cas9, while demonstrating in vitro efficacy against various viruses, presents challenges related to constitutive expression, off-target effects, and the potential for viral escape mutants. This research aims to address these limitations by engineering "immune-like designer cells" that autonomously detect and respond to viral infection with multiple antiviral mechanisms.

Existing antiviral strategies rely heavily on traditional small-molecule antivirals, which often exhibit limited broad-spectrum activity and are prone to the development of drug resistance. Synthetic biology provides a potential solution through the engineering of artificial immune systems capable of broad-spectrum antiviral activity. The use of programmable RNA sensors, such as toehold switches, has shown promise in detecting a wide range of RNA viruses. CRISPR-Cas-based systems have also emerged as powerful tools for virus detection and gene editing. CRISPR-Cas systems, such as SHERLOCK, DETECTR, and HOLMES, offer highly sensitive and specific detection of target-specific viruses. Furthermore, CRISPR-associated protein 9 (Cas9)-nuclease-based technologies show promise in degrading viral genomic material. However, constitutive expression of Cas9 can lead to undesired off-target effects, immune responses, and the emergence of CRISPR-resistant virus strains. The concept of "immune-like designer cells" offers a more sophisticated approach, leveraging the natural capabilities of cells to detect and respond to pathogens in a controlled manner. Such cells can provide self-regulated, targeted antiviral actions, potentially overcoming limitations of traditional therapies.

The researchers engineered autonomous, intelligent, virus-inducible immune-like (ALICE) cells. These cells incorporate a destabilized STING protein as a sensor for viral nucleic acids. STING activation triggers a synthetic signaling pathway, leading to the expression of multiple antiviral effectors. Several iterations of ALICE cells were created, including: ALICEim (inducible expression of antiviral cytokines IFN-α and IFN-β), ALICECas9 (Cas9-based viral degradation), and ALICEAb (secretion of a neutralizing antibody). In vitro experiments used HEK-293T cells and other mammalian cell lines to assess the system's functionality using various viruses, including HSV-1, DENV-2, SARS-CoV-2, and others. Viral detection was measured using a secreted alkaline phosphatase (SEAP) reporter, while antiviral efficacy was assessed via qPCR and fluorescence assays. In vivo experiments employed mouse models of HSV-1 infection, using hyaluronic acid-based hydrogels for cell delivery. Mice were either prophylactically treated with ALICE cells or treated after infection. Viral loads in different organs (liver, spleen, kidney) were measured at various time points post-infection. A herpetic simplex keratitis (HSK) mouse model was used to evaluate the efficacy of an AAV-delivered dual-output ALICEsaCas9+Ab system. Various assays, including qPCR, ELISA, western blotting, flow cytometry, and plaque assays, were used to assess viral loads, cytokine levels, and expression levels of antiviral effectors (Cas9, E317Ab).

The engineered ALICE sensor (ALICEsen) effectively detected various viruses belonging to seven different genera. The system exhibited minimal basal expression in the absence of virus and high induction ratios upon viral infection. The ALICEim system successfully induced production of IFN-α and IFN-β, resulting in broad-spectrum antiviral activity. ALICECas9 cells demonstrated targeted deletion of viral genes, leading to significant reduction in viral replication. ALICEAb cells efficiently reduced viral load through antibody-mediated neutralization. The dual-output ALICEsaCas9+Ab system showed significantly enhanced antiviral activity compared to single-output systems, both in vitro and in vivo. In mouse models, ALICEsaCas9+Ab cells, delivered via hyaluronic acid hydrogels, effectively reduced viral titers in various organs even when treatment was initiated after infection. The long-term antiviral activity of ALICEsaCas9+Ab cells in mice was also confirmed. In an HSK mouse model, AAV-delivered ALICEsaCas9+Ab effectively eliminated viruses from corneas to trigeminal ganglia (TG), reducing disease severity. Importantly, the dual-output system showed synergy, outperforming single-output systems and low-dose acyclovir (ACV). The ALICE system demonstrated self-protection against viral infection. In vivo studies demonstrated that dual-output ALICE systems significantly reduced viral loads in mouse organs at various times after infection.

This study successfully demonstrated the development and application of engineered ALICE cells as a novel antiviral platform. The modular design of the system allows for flexibility in tailoring the antiviral response, including incorporating different sensors, effectors, and delivery methods. The ALICE system's ability to detect and mitigate infections before symptom onset is particularly valuable, especially in immunocompromised individuals. The combination of multiple antiviral mechanisms (cytokines, Cas9, neutralizing antibodies) within a single cellular system enhances antiviral efficacy and reduces the likelihood of drug resistance. The successful use of AAV vectors for delivering the system to the trigeminal ganglia in the HSK mouse model opens the way for long-term therapy. The results highlight the potential of ALICE systems to address challenges associated with conventional antiviral therapies. The demonstrated antiviral efficacy in mice suggests that these immune-like designer cells could have significant clinical applications in combating refractory viral infections.

This research successfully engineered and validated autonomous, intelligent, virus-inducible immune-like (ALICE) cells as a novel antiviral platform. The modular design and demonstrable in vivo efficacy in multiple mouse models suggest broad potential for clinical application against a wide range of viral diseases. Future research should focus on optimizing the system for different viral targets, exploring alternative delivery methods, and conducting comprehensive preclinical studies to advance towards clinical translation.

The study primarily focused on HSV-1 infection in mice. Further research is needed to assess the effectiveness of ALICE systems against other viruses and in different animal models, particularly those reflecting human immune responses. The long-term safety and biocompatibility of the ALICE system in humans remain to be evaluated. The hydrogel-based delivery method, while effective in this study, may not be optimal for all clinical applications. While the study showed promising results, further investigation is needed to explore the system's utility in humans.