Background/Objectives: Although influenza A viruses with partially truncated NS1 proteins are substantially attenuated and immunogenic due to enhanced innate immune activation; residual NS1-mediated antagonism of antiviral innate responses may support viral replication in the lower respiratory tract and constrain optimal immune responses. Strategies to further improve their immunogenicity and protective efficacy by incorporating immunomodulatory cytokines, such as IL-2, have been successfully explored. Methods: Here, we extended this approach to chemokine expression by engineering an NS1-truncated PR8-based virus (PR8/NS124) to express the immunomodulatory chemokine CXCL10 from the NS segment and compared it with the parental vector. Results: The recombinant NS124_SS_CXCL10 virus replicated to high titers in embryonated chicken eggs and MDCK cells. In vivo, however, CXCL10 expression reduced viral replication in mouse lungs by ~10⁴-fold, resulting in a near-non-replicating phenotype. In contrast to the parental virus, the vector did not induce weight loss and exhibited a strongly attenuated phenotype. This effect was associated with altered innate immune signaling, including increased IRF7 expression and early induction of IFN-α responses in the lungs, together with modulation of TLR-dependent sensing pathways in the upper respiratory tract. Despite severely impaired replication, intranasal immunization induced antigen-specific T-cell responses comparable to those elicited by the parental vector. Following intraperitoneal immunization, when replication of both vectors was minimal, the CXCL10-expressing vector induced significantly higher frequencies of antigen-specific CD8⁺ and CD4⁺ effector-memory T cells. This was accompanied by enhanced antigen-specific T-cell recall responses in the lungs following intranasal challenge. Importantly, the CXCL10-expressing vector demonstrated protective efficacy comparable to that of the parental NS124 vector against heterologous H3N2 challenge while exhibiting an improved safety profile. Conclusions: These findings support the incorporation of CXCL10 as a strategy to improve the safety and T-cell immunogenicity of NS1-truncated influenza vectors.

АННОТАЦИЯ: Глиобластома остается опухолью с одним из самых неблагоприятных прогнозов в клинической онкологии. На фоне недостаточной эффективности стандартных методов лечения активно развивается онколитическая виротерапия. Однако вирус гриппа все еще остается на периферии внимания. В данный обзор мы включили ключевые характеристики и особенности глиом, определяющие резистентность к терапии; рассмотрели иммуносупрессивное микроокружение и стратегии его коррекции; обобщили текущий статус клинических исследований и доклинических разработок вирусных препаратов для терапии глиом и выделили перспективные точки приложения для комбинированных подходов. Особое внимание мы уделили потенциалу гриппозных векторов как онколитических агентов, рассматривая преимущества и возможные ограничения этой платформы.

Glioblastoma remains a tumor with one of the most unfavorable prognoses in clinical oncology. Given the insufficient effectiveness of standard treatment modalities, oncolytic virotherapy is being actively developed. However, the influenza virus has so far remained on the periphery of scientific attention. In this review, we outline the key characteristics and features of gliomas that determine their resistance to therapy; examine the immunosuppressive microenvironment and strategies for its modulation; summarize the status of clinical trials and preclinical development of viral agents for glioma therapy; and identify promising targets for combination approaches. Attention is paid to the potential of influenza-based vectors as oncolytic agents, with consideration of both the advantages and possible limitations of this platform.

Influenza A virus (IAV) vectors with truncated NS1 proteins combine strong innate adjuvanticity with genetic flexibility and are attractive platforms for immune modulation. We engineered an NS1-truncated A/Puerto Rico/8/34 (H1N1) virus, PR8/NS124_SS_CXCL10, to express human CXCL10 from the NS segment and compared its biological and immunological properties with the parental NS124 vector in mice. The CXCL10-expressing virus replicated efficiently in embryonated chicken eggs and MDCK cells and secreted high levels of CXCL10, but showed profoundly reduced replication in mouse lungs and peritoneal cavity, indicating a strongly attenuated in vivo phenotype. After intraperitoneal immunization, both vectors induced rapid local cytokine and innate-cell recruitment, although early inflammatory responses and viral RNA loads were lower with PR8/NS124_SS_CXCL10 than with NS124. Despite this attenuation, PR8/NS124_SS_CXCL10 elicited significantly higher frequencies of systemic antigen-specific CD8⁺ and CD4⁺ effector-memory T cells producing IFN-γ, TNF-α, and IL-2, and promoted robust recall CD8⁺ and CD4⁺ T-cell responses in the lungs following low-dose homologous challenge. In a stringent heterologous challenge model with A/Aichi/2/68 (H3N2), however, mice primed intranasally with the CXCL10 vector experienced greater weight loss than NS124-primed animals, consistent with enhanced T-cell–driven immunopathology in the context of insufficient early viral control. These data show that CXCL10 expression in an NS1-attenuated IAV backbone simultaneously enforces replication restriction and amplifies T-cell immunogenicity, supporting its potential as a chemokine-armed platform for immune modulation and oncolytic virotherapy while underscoring the need to carefully balance mucosal priming and recall in chemokine-expressing influenza vaccines.

INTRODUCTION: Oncolytic viruses are a promising approach for treating malignant brain tumors as part a of combination therapy. OBJECTIVE: To develop reporter influenza A viruses expressing NanoLuc luciferase and evaluate their safety following intracranial administration in rats. METHODS: Chemiluminescent reporter influenza A virus strains were obtained by reverse genetics. The NS genetic segment of the T_NS124-Luc and E_NS124-Luc strains encoded a fusion protein that combined NS1₁₂₄ and NanoLuc. In the T_NS124-2A-Luc and E_NS124-2A-Luc strains, the NS1₁₂₄ and NanoLuc sequences were separated by a 2A co-translational cleavage site. To enhance the tumor specificity of the viruses, the trypsin cleavage site (T) in the hemagglutinin (HA) protein was replaced with an elastase cleavage site (E) by introducing S342→P and R343→I substitutions in the HA region of the E_NS124-Luc and E_NS124-2A-Luc constructs. RESULTS: The obtained constructs demonstrated comparable reproductive and luminescent activity in MDCK cells. However, vectors containing the 2A site upstream of the transgene infected the glioma cell lines C6, A172, and T98G more effectively. Intracranial administration of a high dose of the virus was safe, causing no neurological or other pathological symptoms in rats. In addition, the luminescent reporter NanoLuc was expressed at the injection site without the formation of active viral progeny. CONCLUSION: This study demonstrated that a chemiluminescent influenza A virus strain can induce transgene expression at the site of intracranial injection without active viral replication.

ВВЕДЕНИЕ: Онколитические вирусы представляют собой перспективный инструмент для лечения злокачественных опухолей головного мозга в составе комплексной терапии. ЦЕЛЬ ИССЛЕДОВАНИЯ: Разработка репортерных рекомбинантных вирусов гриппа А, экспрессирующих люциферазу NanoLuc, и оценка их безопасности при интракраниальном введении у крыс. МЕТОДЫ: Репортерные штаммы вируса гриппа А, обладающие хемилюминесцентной активностью, были получены методом обратной генетики. Генетический сегмент NS штаммов Т_NS124-Luc и Е_NS124-Luc кодировал слитую белковую последовательность NS1₁₂₄ вируса гриппа и NanoLuc. В штаммах Т_NS124-2А-Luc и Е_NS124-2А-Luc белок-кодирующие последовательности были разделены 2А сайтом, обеспечивающим ко-трансляционное разделение белков. Для повышения специфичности вирусов к опухолевым клеткам в двух конструкциях (Е_NS124-Luc и Е_NS124-2А-Luc) в сайт протеолитического расщепления гемагглютинина были внесены точечные мутации S342→P и R343→I, приводящие к замене сайта протеолитического расщепления с трипсинового (T) на эластазный (E). РЕЗУЛЬТАТЫ: Все исследованные конструкции имели сопоставимую репродуктивную и люминесцентную активность при культивировании в клетках MDCK. Однако при инфицировании клеточных линий глиом (С6, A172 и T98G) векторы с 2А сайтом перед трансгеном продемонстрировали более высокий потенциал роста. Интракраниальное введение высокой дозы вируса крысам было безопасным и не вызывало неврологических и других патологических симптомов. При этом люминесцентный репортер NanoLuc экспрессировался в месте введения, без формирования инфекционного вирусного потомства. ЗАКЛЮЧЕНИЕ: Показана возможность экспрессии трансгена в зоне интракраниального введения репортерного штамма вируса гриппа А, обладающего хемилюминесцентной активностью, в отсутствии активной вирусной репликации.