Seasonal influenza causes 290,000-650,000 deaths annually, with vaccination efficacy ranging from 10 to 60%. The emergence of drug-resistant and highly pathogenic avian influenza viruses underscores the urgent need for novel protective strategies. Epidemiological observations have long suggested that certain vaccines, such as Bacillus Calmette-Guérin (BCG), can provide protection against diverse pathogens (S. Biering-Sørensen, P. Aaby, N. Lund, et al., Clin Infect Dis 65:1183-1190, 2017, https://doi.org/10.1093/cid/cix525; M.-L. Garly, C. L. Martins, C. Balé, et al., Vaccine 21:2782-2790, 2003, https://doi.org/10.1016/s0264-410x(03)00181-6; C. A. G. Timmermann, S. Biering-Sørensen, P. Aaby, et al., Trop Med Int Health 20:1733-1744, 2015, https://doi.org/10.1111/tmi.12614). While the cellular and molecular mechanisms underlying such protection remain incompletely understood, emerging research offers critical insights into innate immune system modulation (B. Cirovic, L. C. J. de Bree, L. Groh, et al., Cell Host Microbe 28:322-334, 2020, https://doi.org/10.1016/j.chom.2020.05.014; L. Kong, S. J. C. F. M. Moorlag, A. Lefkovith, et al., Cell Rep 37:110028, 2021, https://doi.org/10.1016/j.celrep.2021.110028; H. Mohammadi, N. Sharafkandi, M. Hemmatzadeh, et al., J Cell Physiol 233:4512-4529, 2018, https://doi.org/10.1002/jcp.26250; S. J. C. F. M. Moorlag, Y. A. Rodriguez-Rosales, J. Gillard, et al., Cell Rep 33:108387, 2021, https://doi.org/10.1016/j.celrep.2020.108387). We investigated whether a trained innate immune system with non-replicating adenoviruses could provide protection against diverse influenza virus strains. We demonstrated that replication-defective human adenoviruses can effectively train the innate immune system, conferring protective immunity in mice against multiple influenza virus strains, including H1N1, H3N2, H5N2, H7N9, and H9N2. In addition, bovine and chimpanzee adenoviruses can also activate human innate lymphoid cells (ILCs) and confer protection against challenge with influenza H3N2 virus in mice. Remarkably, this protection occurs in the complete absence of influenza-specific adaptive immune responses (influenza virus-specific hemagglutination-inhibiting antibodies, neutralizing antibodies, and influenza nucleoprotein-specific CD8 T cells). Key protective mechanisms include increased activation of ILC1, ILC2, and ILC3 populations, enhanced expression of interferon-stimulated genes (ISGs), upregulation of antiviral signaling pathways, and metabolic reprogramming of ILC subsets. Adoptive transfer experiments demonstrated that trained ILCs were sufficient to protect against influenza H1N1 infection in ILC-deficient mice. This research establishes a novel strategy for enhancing innate antiviral immunity, offering broad-spectrum protection against diverse influenza strains, a promising approach for not only pandemic preparedness but also against emerging infectious diseases. Training innate lymphoid cells through non-replicating adenoviral vectors represents a promising approach to enhancing broad-spectrum antiviral immunity, complementing traditional vaccination strategies.IMPORTANCEThe findings represent a potential game-changer for fighting influenza, which kills hundreds of thousands of people worldwide each year despite our best vaccination efforts. Current flu vaccines often provide limited protection because they must be reformulated annually to match circulating strains, and their effectiveness varies dramatically from year to year. The scientists discovered something remarkable: common adenoviruses (which typically cause mild cold-like symptoms) can essentially "train" our immune system's first line of defense to recognize and fight off multiple types of flu viruses simultaneously. This protection works through a completely different mechanism than traditional vaccines-it does not rely on creating specific antibodies against flu proteins. Instead, the treatment activates special immune cells called innate lymphoid cells (ILCs), which act like the body's rapid response team. These trained cells provide broad protection against various flu strains, including dangerous bird flu variants that could cause future pandemics. The significance lies in potentially creating a universal flu protection strategy that could work against unknown future flu strains, offering hope for better pandemic preparedness and reducing seasonal flu's devastating global impact.

Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8(+) T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8(+) T cells. Our results indicate that both CD8(+) T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8(+) T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8(+) T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8(+) T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8(+) T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.

Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to approximately 300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-beta (IFN-beta) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-beta by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.