Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 1 Mucosal neutrophil activation predisposes to respiratory viral infection 2 Running title: Roles of neutrophils & IL-17 in RSV infection 3 One sentence summary: Mucosal neutrophil activation at the time of respiratory virus exposure 4 enhances infection and opposes early prodromal mucosal inflammatory responses that prevent disease. 5 Authors: Maximillian S Habibi1*, Ryan S Thwaites1*, Meiping Chang2, Agnieszka Jozwik1, Allan 6 Paras1, Freja Kirsebom1, Augusto Varese1, Amber Owen1, Leah Cuthbertson1, Phillip James1, 7 Tanushree Tunstall1, David Nickle3, Trevor T Hansel1, Miriam F Moffatt1, Cecilia Johansson1, 8 Christopher Chiu4ǂ and Peter J. M. Openshaw1ǂ 9 Affiliations: 10 1National Heart and Lung Institute, Imperial College London, London, United Kingdom 11 2Merck &Co., Inc., Kenilworth, NJ, USA 12 3Genetics & Pharmacogenomics, Department of Translational Medicine, Merck &Co., Inc., Boston, 13 Massachusetts, USA 14 4Department of Infectious Diseases, Imperial College London, London, United Kingdom 15 *, ǂ these authors contributed equally 16 17 Correspondence should be addressed to: Peter J. M. Openshaw FRCP PhD, National Heart and Lung 18 Institute, Imperial College London, London W2 1PG, UK. E-mail: [email protected]; 19 or Christopher Chiu MRCP FRCPath Ph.D, Department of Infectious Disease, Imperial College 20 London, London W12 0NN, UK. E-mail: [email protected]; 21 or Cecilia Johansson PhD, National Heart and Lung Institute, Imperial College London, London W2 22 1PG, UK. E-mail: [email protected] 1 Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 23 Abstract: 24 The variable outcome of viral exposure is only partially explained by known factors. We administered 25 respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil 26 activation prior to exposure was highly predictive of symptomatic RSV disease. This was associated 27 with rapid pre-symptomatic decline in mucosal IL-17A and other mediators. Conversely, those who 28 resisted infection showed pre-symptomatic activation of IL-17 and TNF-related pathways. 29 Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by 30 pre-infection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease 31 mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at respiratory 32 virus exposure enhances susceptibility, revealing dynamic time-dependent local immune responses 33 before symptom onset and explaining hitherto unpredictable outcomes of pathogen exposure. 2 Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 34 Main Text: 35 The respiratory tract is by necessity an open portal, highly vulnerable to invasion by many pathogens. 36 A complex system of defenses counters this vulnerability, but current knowledge of what governs 37 susceptibility fails to fully explain the erratic transmission of infectious agents (1, 2). For some 38 infections, antibodies in the respiratory mucosa are clearly protective (3). Furthermore, infectious 39 agents can also be blocked by antimicrobial peptides (4), entrapment in mucus (5), the downstream 40 effects of pattern recognition receptor activation (1, 2, 6), as well as direct inactivation by immune cells 41 (6). The SARS-CoV-2 pandemic has brought into sharp focus the need for greater understanding of the 42 role of mucosal innate immunity in protection against respiratory infection. Better understanding of 43 such defenses may offer ways to prevent or modulate viral disease, even against novel and emerging 44 pathogens. Among innate immune cells, neutrophils are classically ascribed roles in defense against 45 bacteria and fungi, but their role in antiviral responses is less clear. Although they are sometimes found 46 in the lungs of patients with severe viral infections and have a demonstrable antiviral role in animal 47 models, information concerning how they contribute to defense and disease in human respiratory viral 48 infections is largely lacking (7). 49 Respiratory syncytial virus (RSV) is the leading cause of infant hospitalization worldwide, infecting 50 around 34 million children each year (8) and contributing prominently to morbidity and mortality in 51 elderly and immunosuppressed adults (9, 10). Though RSV infection is essentially confined to the 52 respiratory epithelium, resultant neutrophilic lung inflammation can be life-threatening, particularly in 53 at-risk children (11, 12). Although prophylaxis with palivizumab (a monoclonal anti-RSV antibody) 54 can prevent hospitalization of premature infants (13), no specific therapeutic intervention is currently 55 available and no vaccine has yet been licensed that protects against RSV (14). This is despite the 56 existence of several promising candidates (15), including enhancement of placental anti-RSV IgG 57 transfer by maternal immunization. There is therefore a pressing need to further identify interventions 58 that prevent RSV and other respiratory viral diseases. 3 Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 59 Animal models of respiratory viral infection provide useful mechanistic insights but do not fully 60 recapitulate human disease (6). Conversely, observational studies of hospitalized children and high-risk 61 adults are limited by heterogeneity of populations and disease severity, an inability to dynamically 62 assess the status of host immunity and, specifically, the mucosa before and immediately after viral 63 exposure. Experimental infection of human volunteers therefore provides a unique opportunity to 64 identify pre-existing immune factors and pre-symptomatic responses in a controlled setting that 65 correlate with protection and disease (16, 17). 66 All adults have RSV-specific antibodies, reflecting multiple rounds of infection throughout the course 67 of their lives. Nevertheless, symptomatic re-infection occurs repeatedly even in healthy people and is 68 associated with unusually short-lived humoral and cellular memory responses (3). Such defective 69 defense against reinfection is also observed following some other respiratory viral infections, including 70 coronaviruses (18). Furthermore, variations in antibody levels do not accurately predict susceptibility 71 to RSV infection in experimental studies of volunteers, those with the lowest antibody levels being only 72 modestly more susceptible to viral challenge (3, 19). Although mucosal IgA, circulating IgG (3), and 73 resident memory CD8+ T cells (20) show some association with resistance to infection and reduced 74 disease severity, these factors do not completely explain who resists or succumbs. Additionally, 75 although RSV viral burden approximately correlates with symptoms in experimentally infected adults 76 (16) and moderately ill infants (21, 22), viral load may be paradoxically reduced in the most severely 77 affected children (23). 78 How the very early events following viral exposure influence clinical outcomes has not been studied in 79 either adults or children. Studying the respiratory mucosa before, during, and after human RSV 80 challenge, we found that prior neutrophil activation in the upper respiratory tract predisposes to 81 symptomatic viral infection. The neutrophilic mucosal environment was strongly associated with a 82 reduction in antiviral mucosal inflammatory responses immediately after viral exposure that was 83 followed by the onset of disease. By contrast, a protective response characterized by rapid activation of 84 type-17 inflammation was observed in those resisting infection. Moreover, studies in mice showed that 4 Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 85 recruitment of airway neutrophils prior to viral administration enhanced the cytotoxic CD8+ T cell 86 response and disease severity. 5 Habibi, Thwaites, et al. Roles of neutrophils & IL-17 in RSV infection Science 87 Results 88 Characterization of time-course of responses to human RSV challenge 89 Fifty-eight healthy adult volunteers were inoculated with RSV Memphis 37. Nasal sampling was 90 performed at baseline (7-14 days pre-inoculation) and then repeatedly up to 14 days after inoculation 91 (Fig. 1A). Symptoms were quantified using a previously validated self-reported symptom scale (24). 92 Twenty-three participants developed PCR-positive RSV infection with symptoms of upper respiratory 93 tract disease (“Cold”), whereas 25 had no evidence of RSV infection and did not develop symptoms 94 (“No Cold”). Ten participants had PCR evidence of RSV infection, but had self-reported symptom 95 scores that did not reach the threshold required for the “Cold” group. This indeterminate group was 96 excluded from subsequent analyses, which focused on identifying factors associated with the most 97 distinct outcomes. No significant demographic differences were observed between the Cold and No 98 Cold groups (table S1). 99 In infected volunteers, viral load (determined by qPCR from daily nasal washes) broadly indicated three 100 phases: (1) a pre-symptomatic incubation phase during the first 3 days post-inoculation when virus was 101 undetectable; (2) a viral replication phase from days 3-7; and (3) a viral clearance phase from day 8 102 (previously shown to be associated with the rise in virus-specific CD8+ T cells in both blood and lower 103 airway (20))