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.

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.

Influenza infections cause several million cases of severe respiratory illness, hospitalizations, and hundreds of thousands of deaths globally. Secondary infections are a leading cause of influenza's high morbidity and mortality, and significantly factored into the severity of the 1918, 1968, and 2009 pandemics. Furthermore, there is an increased incidence of other respiratory infections even in vaccinated individuals during influenza season. Putative mechanisms responsible for vaccine failures against influenza as well as other respiratory infections during influenza season are investigated. Peripheral blood mononuclear cells (PBMCs) are used from influenza vaccinated individuals to assess antigen-specific responses to influenza, measles, and varicella. The observations made in humans to a mouse model to unravel the mechanism is confirmed and extended. Infection with influenza virus suppresses an ongoing adaptive response to vaccination against influenza as well as other respiratory pathogens, i.e., Adenovirus and Streptococcus pneumoniae by preferentially infecting and killing activated lymphocytes which express elevated levels of sialic acid receptors. These findings propose a new mechanism for the high incidence of secondary respiratory infections due to bacteria and other viruses as well as vaccine failures to influenza and other respiratory pathogens even in immune individuals due to influenza viral infections.

Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet or in vivo-jetPEI((R)), and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.