DOI: 10.25442/hku.30517886.v1
title: <b>Supporting data for "Human Respiratory Organoids as A Platform for Antibody Neutralization Evaluation And Mucosal Vaccine Development"</b>

description: The advancement of therapeutics for respiratory viruses—such as those enabling high-throughput screening of neutralizing antibodies and assessment of mucosal vaccines—is critically dependent on the development of robust and physiologically meaningful in vitro models. Conventional cell lines are limited by their lack of cellular heterogeneity and physiological receptor expression, often resulting in poor clinical translation. To overcome these limitations, we developed human respiratory organoids derived from two clinically accessible sources: adult stem cells (ASCs) isolated from primary lung tissue and nasal epithelial cells collected noninvasively via brushing from individual donors. The resulting 3D nasal and lung organoids support long-term expansion for over six months. Through directed differentiation, we generated mature nasal, airway, and alveolar organoids that closely replicate the genetic and functional properties of native respiratory epithelia, including tissue-specific cellular composition, key epithelial functions, and authentic virus-host interactions. These organoids effectively recapitulate the structural and functional complexity of the human respiratory tract, offering a highly representative ex vivo platform for studying viral infection and host immune responses.<br>This study highlights the value of human nasal organoids as a dual-purpose platform for critical applications in respiratory medicine: first, the precise evaluation of neutralizing antibody efficacy, and second, the comprehensive assessment and mechanistic investigation of mucosal vaccines. Traditional neutralization assays failed to predict the clinical performance of VIR-7831, a monoclonal antibody that showed efficacy in trials but was withdrawn due to insufficient activity against Omicron variants in standard cell line-based tests. In contrast, nasal organoids—which faithfully mimic the native tissue environment with low ACE2 expression—accurately restored VIR-7831’s neutralizing profile and served as a reliable correlate of <i>in vivo</i> protection. Moreover, this system exclusively reproduced the <i>in vivo</i> protection of S2 mAbs due to the high TMPRSS2 expression, reminiscent of native human respiratory epithelial cells.<br>We further applied this platform to assess pemivibart, a recently authorized monoclonal antibody, against emerging SARS-CoV-2 variants such as KP.3.1.1. Our data revealed a significant reduction in pemivibart’s neutralization capacity against this TMPRSS2-dependent variant. However, this deficit was effectively rescued through synergistic combination with broad-spectrum S2 antibodies. Additionally, the organoid model confirmed the potent and broad neutralizing activity of SA55, which has since successfully completed Phase I clinical trials.<br>Beyond antibody testing, nasal organoids integrated with single-cell RNA sequencing offered unprecedented insights into mucosal immunity induced by live attenuated influenza vaccine. We found that vaccine delivery triggers a robust and sustained antiviral response across distinct epithelial cell types, mediated through ZBP1-mediated necroptosis pathways. Co-culture models further demonstrated the essential role of epithelial cells in activating antigen-presenting cells, highlighting the central role of the epithelium in coordinating innate immunity.<br>In conclusion, human nasal organoids bridge a critical gap between reductionist in vitro models and clinical reality. They enhance the accuracy of neutralization profiling for therapeutic antibodies against evolving viruses and provide a powerful tool for elucidating the mechanisms underpinning mucosal vaccine efficacy. This platform offers a promising path toward the development of more effective countermeasures against respiratory pathogens.