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Microbiome research has transformed our understanding of microbes and human health. Resident bacteria can protect the host from pathogens by shaping immunological responses. These new insights suggest the microbiome could be a target for preventing influenza virus infection, a major cause of illness and death worldwide. In this study, we explore the relationship between the nose/throat microbiome and influenza virus among Nicaraguan households. Household members were enrolled immediately after one member was diagnosed with influenza virus infection. This study design allowed us to identify an association between the microbiome structure and influenza susceptibility. Further, this association may be due differences in the abundance of Veillonella. We also explored whether influenza virus infection altered the microbiome structure and found short-term changes were common among both secondary cases and household members who remained uninfected. Age played a major role in influenza susceptibility and in short-terms changes in the microbiome. Although much work is needed, our findings suggest strategies that appropriately modify the microbiome might be useful in preventing influenza virus infections.

Influenza is a major cause of morbidity and mortality worldwide. However, vaccine effectiveness has been low to moderate in recent years and vaccine coverage remains low, especially in low- and middle-income countries. Supplementary strategies for prevention should be explored. The epithelial cells of the respiratory tract are a primary target for influenza virus infection and replication. They also are enveloped by complex bacterial communities, a respiratory microbiome, which plays a critical role in shaping host immunity. Using a household transmission study, we examined whether influenza susceptibility was associated with the nose/throat microbiome structure among participants exposed to influenza in the household. We also described changes in the nose/throat microbiome following influenza virus infection. We identified a single bacterial community type associated with decreased susceptibility to influenza using a mixed model accounting for household clustering of secondary cases, community types, and other risk factors. The community type – characterized by a low abundance of Veillonella – was rare and transitory among young children but a prevalent and stable community type among adults. We found high rates of change in the microbiome structure following influenza virus infection as well as among household contacts who were never infected with influenza during follow up. Further, age strongly influenced susceptibility to influenza and short-term dynamics of the nose/throat microbiome. These results suggest that the nose/throat microbiome might mediate influenza risk and may be a target for future interventions.

3.3 Introduction

Influenza is a major contributor of human illness and death worldwide, estimated to cause 3-5 million cases of severe illness [1] and 400,000 deaths during interpandemic years [2]. Vaccination is the best available means of influenza prevention. However, low vaccine

effectiveness has been low to moderate in recent year [3,4]. Further, vaccine coverage remains low, especially in low- and middle-income countries [5]. With increasing support for the microbiome shaping host immunity [6–8], exploring whether these effects extend to influenza risk could contribute to supplementary methods of prevention.

We hypothesized that the nose/throat microbiome is an unrecognized factor associated with susceptibility to influenza virus. Murine and human studies support this assertion. Compared to controls, mice treated with oral antibiotics exhibited enhanced degeneration of the bronchiole epithelial layer and increased risk of death following intranasal infection with influenza virus [7]. In two separate randomized controlled trials, newborns fed prebiotics and probiotics had significantly lower incidence of respiratory tract infections compared to placebo [9,10]. These studies suggest the manipulation of the microbiome, either through disruption or supplementation, can alter risk of respiratory tract infections.

The epithelial cells of the upper and lower respiratory tracts are the primary targets for influenza virus infection and replication [11]. However, these cells are enveloped by complex bacterial communities that may directly or indirectly interact with influenza virus to mediate risk of infection. Commensal bacteria may prevent infection by regulating innate and adaptive host immune responses [6,7], but immune response to infection also might stimulate changes to the microbiome [12–14]. Alternatively, or in addition, infection may directly act on the microbiome [15,16]. A human experimental trial has shown intranasal administration of live attenuated influenza vaccine can alter the nasal microbiota of young adults by increasing taxa richness [17]. Further, influenza-related changes in the bacterial community structure might explain the enhanced risk of bacterial pneumonia and otitis media following influenza virus infection [18–21]. The most commonly detected causative organisms of bacterial pneumonia and otitis media

increase in abundance in the upper respiratory tract following respiratory virus infection [22,23]. We previously showed that adults in the US with influenza virus infection expressed increased nose/throat carriage of Streptococcus pneumoniae and Staphylococcus aureus [22]. Similarly, other studies have observed an increase in pneumococcal density following rhinovirus infection [23] and changes in the microbiota during rhinovirus and respiratory syncytial virus infection [24]. Increased carriage elevates risk of invasive disease [25,26], potentially through more frequent microaspiration into the lung or migration to the middle ear [27]. However, an association between the nose/throat microbiome and influenza risk has not been demonstrated in human populations.

In this study, we used data from a longitudinal household transmission study of influenza to assess the relationship between the nose/throat microbiota and susceptibility to influenza virus infection and to determine whether influenza virus infection alters the bacterial community structure using an untargeted 16S rRNA (V4) taxonomic screen. We classified the nose/throat microbiota into five community types using the Dirichlet multinomial mixture method [28] (Graphical abstract available in Figure 3.1).