Mucosal Immunology and Virology

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

The overarching goal of our research program is to understand the immune mechanisms that protect against respiratory viruses, with a particular focus on mucosal immunity in the upper and lower respiratory tract. By studying how different vaccine platforms, delivery routes, and viruses influence immune responses, we aim to inform the rational design of next-generation vaccines that are not only effective systemically but also at the site of viral entry. Over the past six months, our group has made substantial progress across multiple complementary areas: 1. Intranasal Vaccination and Mucosal Immunity to SARS-CoV-2 We published a study demonstrating that intranasal booster vaccination enhances mucosal immune responses in a mouse model of SARS-CoV-2 (Koolaparambil Mukesh et al., Scientific Reports). Mice primed with an intramuscular vaccine and boosted intranasally with a recombinant adenovirus vector developed durable mucosal immune responses, including elevated antigen-specific IgA and tissue-resident memory T cells in the respiratory tract. Importantly, these mice were protected in the upper respiratory tract (URT) longer than their intramuscularly vaccinated counterparts. These findings support a heterologous prime-boost strategy as a means to improve local immunity and long-term protection against SARS-CoV-2, and reinforce the importance of targeting mucosal sites for respiratory virus vaccines. 2. Targeting the Upper vs. Lower Respiratory Tract for Vaccination We performed a series of studies in mice using ChAdOx1-GFP, a replication-deficient adenoviral vector expressing green fluorescent protein, to compare delivery to the URT alone versus both the URT and lower respiratory tract (LRT). By varying the volume of intranasal instillation, we achieved targeted deposition and tracked antigen expression and cellular tropism over time. Flow cytometry was used to identify specific epithelial, endothelial, and immune cell populations expressing GFP, as well as the persistence of expression in each region. This work establishes a foundation for mechanistically dissecting how the anatomical site of vaccination influences immune priming and protective outcomes. 3. Development of a Self-Amplifying RNA Vaccine Platform To expand our vaccine development toolbox, we initiated the design and production of a self-amplifying RNA (saRNA) vaccine platform. saRNA offers the advantage of prolonged antigen expression from a smaller initial dose compared to conventional mRNA, and may prove particularly beneficial for mucosal immunization where antigen persistence can enhance immune imprinting. While this platform is still under development, it is intended for direct comparisons with existing modalities (e.g., protein, adenoviral, conventional mRNA) to evaluate immunogenicity and durability in respiratory virus models. 4. Refinement of Animal Models for Respiratory Virus Pathogenesis We have continued to refine our animal models to study infection, immunity, and pathogenesis of respiratory viruses. We contributed to a study demonstrating that Omicron lineage variants have evolved toward increased replication in the URT, accompanied by reduced lung pathology in mice (Wickenhagen et al, Nature Communications). Additionally, in two separate studies, we examined the pathogenicity of a bovine-origin H5N1 clade 2.3.4.4b isolate. In macaques, this virus caused systemic infection and severe disease (Rosenke et al, Nature), while in mice, we observed enhanced neurotropism and broader dissemination compared to a Vietnam-origin strain (Goldin et al, npj Viruses). To better understand the cellular architecture and immune composition of nasal-associated lymphoid tissue (NALT) following vaccination, we optimized tissue preparation and staining protocols for use with the MACSima™ platform. This high-plex spatial imaging approach allowed us to simultaneously visualize dozens of markers in intact tissue sections, yielding detailed maps of immune cell organization. We intend to use this technology to investigate critical insights into local immune activation and help identify correlates of protection at the site of pathogen entry. 5. Establishment of Human Tonsil Organoids as a Mucosal Model System We have successfully established a 3D human tonsil organoid culture system, derived from primary tonsillar tissue. Upon stimulation with antigen, these organoids exhibit robust activation of both T and B cells, including germinal center-like structures. This ex vivo system provides a promising platform for studying human immune responses. Together, these studies reflect our integrated approach to dissecting mucosal immune responses to respiratory viruses, spanning in vivo mouse models, human organoids, spatial imaging, and vaccine platform development. Our ongoing work aims to define the tissue-specific correlates of protection that can guide more effective and targeted vaccine strategies against current and emerging respiratory threats.