Lethal Disease Following 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. Although most human cases to date have been mild, several severe cases of respiratory illness have been reported. A key unanswered question is the pathogenesis of H5N1 infection in primates following respiratory infection. In this study, we observed severe clinical disease in cynomolgus and rhesus macaques following respiratory infection with the H5N1 isolate A/Texas/37/2024 (hu-TX37-H5N1). Cynomolgus macaques inoculated with a high dose of hu-TX37-H5N1 developed severe consolidative necrotizing pneumonia with extrapulmonary spread. Clinical disease was rapidly progressive and lethal in 100% (9 of 9) of macaques by day 5-9 following challenge. Rhesus macaques inoculated with varying doses of hu-TX37-H5N1 demonstrated dose-dependent mortality, and surviving animals showed robust natural protective immunity against high dose re-challenge. H5N1 infection in both cynomolgus and rhesus macaques was characterized by upregulation of proinflammatory cytokine, innate immune cell, complement and coagulation, apoptosis, and immune exhaustion pathways and downregulation of NK, T, and B cell activation and differentiation pathways. Taken together, our data demonstrate severe respiratory disease with H5N1 clade 2.3.4.4b in nonhuman primates and suggest that acute inflammation and immune dysregulation are key contributors to disease pathogenesis. 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.