**1. Introduction**

Human respiratory syncytial virus (hRSV) is the leading cause of acute lower respiratory tract infection (ALRTI) in children under five-year-old, displaying high rates of hospitalization and over 90,000 death-related cases [1,2]. Children under two years of age, people with comorbidities, such as cardiac and pulmonary affections, and the elderly, are the most susceptible to developing hRSV bronchiolitis and pneumonia [3]. HRSV infections cause airway inflammatory hyperresponsiveness characterized by the secretion of pro-inflammatory cytokines, including interleukin (IL)-6, IL-4, IL-13, and tumor necrosis factor (TNF)-α, leading to the recruitment of innate leukocytes, such as neutrophils, as well as adaptive immune cells including T and B cells [4].

The consequences associated with hRSV infection during the early stages of infection include poor adaptive immunity that allows reinfections [5,6] and increased susceptibility to the development of allergies [7], post-bronchiolitis wheeze (PBW), and asthma [8]. Another important consequence of hRSV infections is the frequent establishment of secondary bacterial infections, such as *Streptococcus pneumoniae* (*S. pneumoniae*), which can lead to more severe clinical symptoms of pulmonary pathology, and are considered an essential factor that contributes to mortality rate [9–12]. Different studies have suggested that primary respiratory viral infections can induce host susceptibility to secondary infections, either acute or concomitant bacterial infection (*S. pneumoniae*) or established bacterial infection after viral clearance, such as *Mycobacterium tuberculosis* (*M. tuberculosis*) [13]. However, the susceptibility that an initial infection with hRSV promotes for secondary infections with *M. tuberculosis* has not been evaluated. Here we sought to evaluate whether hRSV infection shapes the lung immune response and promotes pulmonary inflammatory hyperresponsiveness in a chronic mycobacterial infection model.

The *Mycobacterium bovis* (*M. Bovis*) strain, referred to as Bacillus Calmette-Guerin (BCG), is an attenuated version of a known species belonging to the *M. tuberculosis* complex [14]. Although infections with BCG do not recapitulate all pathological features observed with *M. tuberculosis*, BCG is a valuable model for studying anti-mycobacterial immune responses mimicking important aspects of bacilli-host interaction [15,16]. Moreover, BCG provides a functional model for chronic infections as viable bacilli can persist for up to 10 months in the lungs [17].

Heme Oxygenase (HO)-1 is an enzyme that catalyzes a reaction in which the heme group turns into carbon monoxide (CO), biliverdin, and free iron [18]. HO-1 promotes heme group homeostasis and has a cryoprotective role against tissue damage. Additionally, it has been reported that the activity of HO-1 has an antiviral effect on pathogens such as hRSV, among others [19–21]. In the case of mycobacteria, the function of HO-1 in the host is induced by the dormancy mechanism activated by the mycobacteria, promoting an anti-inflammatory response that avoids the immune system and ensures their survival [22,23]. The expression of HO-1 is controlled by a transcription factor named nuclear factor erythroid 2-related factor (Nrf2), which can be activated as a consequence of stimuli such as hypoxia [24]. The OX-2 glycoprotein membrane (CD200) is an important molecule expressed on the surface of alveolar epithelial cell type II, which can bind to the receptor CD200R on the surface of alveolar macrophages [25]. The union of CD200 with its receptor on the alveolar macrophages leads to their inhibition, downregulating the inflammatory response in the pulmonary epithelium [25]. By doing this, CD200 can regulate the airway immunological response against infections.

As mentioned above, hRSV-infection can cause airway inflammation and hyperresponsiveness [4]. However, the link between hyperresponsiveness of the airways and long-term pulmonary sequels regarding *Mycobacterium* infections is unclear. Therefore, we evaluated whether the lungs of hRSV-infected mice are immunologically susceptible to secondary bacterial colonization along with pulmonary pathology. With this aim, we performed long-term bacterial colonization using BCG as the *Mycobacterium* infectious model after

a primary infection with hRSV in C57BL/6 mice [26,27]. Additionally, HO-1 induction during hRSV-infection produces an anti-inflammatory environment and reduction of viral loads [20]. Since the HO-1 effect in BCG immunization has not been evaluated, we decided to test whether the effect previously described was increased using a BCG-based vaccine prototype against hRSV. This study aimed to determine the contribution of HO-1 during a mycobacterial infection that followed an infection by hRSV and after an immunization scheme followed by an hRSV challenge.

#### **2. Materials and Methods**

#### *2.1. Ethics Statements*

All experimental protocols followed guidelines from the Sanitary Code of Terrestrial Animals of the World Organization for Animal Health (OIE, 24. Edition, 2015) and were reviewed and approved by the Scientific Ethical Committee for Animal and Environment Care of the Pontificia Universidad Católica de Chile (Protocol number 160915010 and 160405005). All mouse experiments were conducted in agreement with international ethical standards and according to the local animal protection law number 20,800.
