Immunopathogenesis of lethal H5N1 avian influenza virus clade 2.3.4.4b infection in macaques

The H5N1 avian influenza virus clade 2.3.4.4b outbreak represents a major pandemic threat for humans, with some reported cases of severe and fatal respiratory illness. A key unanswered question is the pathogenesis of severe H5N1 disease following respiratory infection. In this study, we explored mechanisms of pathogenesis of severe H5N1 disease in cynomolgus and rhesus macaques following infection with the H5N1 isolate A/Texas/37/2024 (huTX37-H5N1). Cynomolgus macaques developed severe pneumonia that was lethal in 100% of macaques by 7 days post-infection. By contrast, rhesus macaques demonstrated dose-dependent mortality, and surviving animals showed protective immunity against high-dose re-challenge. A multi-omics analysis demonstrated that H5N1 infection was characterized by robust induction of proinflammatory cytokines, innate immune cells, complement, coagulation, apoptosis, and immune exhaustion pathways. Taken together, our data indicate inflammation and immune dysregulation as key mechanisms of H5N1 pathogenesis in nonhuman primates. Overall design: This study was performed in accordance with an approved Animal Care and Use Committee protocol and following recommendations from the Guide for the Care and Use of Laboratory Animals of the Office of Animal Welfare, National Institutes of Health and the Animal Welfare Act of the US Department of Agriculture, in an AAALAC-accredited facility. The macaques were placed in high biocontainment (BSL-3) inside a climate-controlled room with a fixed 12-hour light-dark cycle. Animals were singly housed during the H5N1 challenge period in HEPA filtered microisolator caging (Rockville Steel, MD). They were provided a commercial monkey chow, treats, and fruit twice daily with water ad libitum. Animals that were noted to have reduced appetite were provided Gatorade. Environmental enrichment was provided with manipulanda and commercial toys. The animals were monitored at least twice daily (AM/PM) throughout the study. All sample processing and sample removal from high biocontainment followed approved biosafety protocols. 9 outbred Mauritian-origin cynomolgus monkeys (Macaca fascicularis) and 9 outbred Indian-origin rhesus macaques (Macaca mulatta), adult male and female ages 3-13 were randomly allocated to groups. All monkeys were housed at Bioqual, Rockville, MD. In the first study, 9 cynomolgus macaques were inoculated with 109 TCID50 H5N1 by the combination IN+IT route (N=6) or by the IN only route (N=3). In the IN+IT group, 3 animals were utilized for PET-CT imaging and were not sampled for BAL to avoid interference with the imaging. The virus was administered as 1 ml by the intranasal (IN) route and/or 1 ml by the intratracheal (IT) route. In the second study, 9 rhesus macaques were inoculated with 109, 107, or 105 TCID50 (N=3/group) H5N1 by the IN+IT routes. On day 28 following initial infection, the surviving rhesus macaques (N=6) were re-challenged with 109 TCID50 H5N1.