Mucosal immunization with a low-energy electron inactivated respiratory syncytial virus vaccine protects mice without Th2 immune bias.

The respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections associated with numerous hospitalizations. Recently, intramuscular (i.m.) vaccines against RSV have been approved for elderly and pregnant women. Noninvasive mucosal vaccination, e.g., by inhalation, offers an alternative against respiratory pathogens like RSV. Effective mucosal vaccines induce local immune responses, potentially resulting in the efficient and fast elimination of respiratory viruses after natural infection. To investigate this immune response to an RSV challenge, low-energy electron inactivated RSV (LEEI-RSV) was formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) or 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DD-LEEI-RSV) for vaccination of mice intranasally. As controls, LEEI-RSV and formalin-inactivated-RSV (FI-RSV) were used via i.m. vaccination. The RSV-specific immunogenicity of the different vaccines and their protective efficacy were analyzed. RSV-specific IgA antibodies and a statistically significant reduction in viral load upon challenge were detected in mucosal DD-LEEI-RSV-vaccinated animals. Alhydrogel-adjuvanted LEEI-RSV i.m. showed a Th2-bias with enhanced IgE, eosinophils, and lung histopathology comparable to FI-RSV. These effects were absent when applying the mucosal vaccines highlighting the potential of DD-LEEI-RSV as an RSV vaccine candidate and the improved performance of this mucosal vaccine candidate.

Maternal awareness, acceptability and willingness towards respiratory syncytial virus (RSV) vaccination during pregnancy in Ireland.

BACKGROUND: Respiratory syncytial virus (RSV) is the world's leading cause of viral acute lower respiratory infections (ALRI) in infants. WHO has identified maternal RSV vaccination a priority and candidate vaccines are in development; however, vaccine hesitancy remains an impediment to successful implementation of maternal immunization. This study, the largest antenatal survey conducted to-date, aimed to examine maternal RSV awareness, likely acceptance of RSV vaccination in pregnancy, and attitudes to maternal vaccination. METHODS: Pregnant women of all gestations attending antenatal clinic of a university maternity hospital in Ireland were invited to participate. An information leaflet provided, consent obtained, and survey administered examining RSV awareness, willingness to avail of antenatal RSV vaccination, factors influencing acceptability and preferred sources of assistance. Research Ethics Committee (REC) approval obtained, and general data protection regulation (GDPR) guidelines followed. RESULTS: 528 women completed the survey. A large proportion (75.6%) had never heard of RSV, yet 48.5% would still avail of a vaccine, 45.8% were undecided and only 5.3% would not. The main factor making vaccination acceptable to women (76.4%) was that it protects their infant from illness (p < .001, CV 0.336 for association with acceptance) and general practitioner (GP) was the preferred guidance source in decision-making (57.7%). CONCLUSIONS: Despite low levels of maternal awareness of RSV, pregnant women in Ireland are open to availing of antenatal vaccination. Maternal immunization strategies need to focus on infant's protection from RSV-associated ALRI along with vaccine safety, and build on an interdisciplinary collaboration of maternal, neonatal, primary care and public health services.

RSV awareness, risk perception, causes, and terms: Comment.

This correspondence discusses on res awpiratory syncytial virus areness, risk perception and causes. Important limitations and possible furture direction for researching are mentioned.

Prenatal exposure to RSV season influences first-year risk of RSV lower respiratory illness and RSV-specific immune responses assessed at birth.

Maternal-to-fetal transmission of respiratory syncytial virus (RSV) has been shown to occur but whether late prenatal exposure to RSV season influences offspring postnatal RSV-lower respiratory illness (LRI) risk in early life or RSV immune status at birth is unclear. In this study, the duration of third trimester RSV season exposure was determined for 1,094 newborns of the Tucson Children's Respiratory Study (TCRS) and found to show an inverse relation to risk for first RSV-LRI in the first year. Cord blood anti-RSV antibody is related to third trimester RSV season exposure but not to first year RSV-LRI risk. In a separate birth cohort (the Infant Immune Study), supernatants from cord blood mononuclear cells stimulated with the recall antigen, UV-inactivated RSV, were assayed for IFN-γ and IL-4. The frequency of detectable IFN-γ (but not IL-4) was increased for those with at least 2 mo of third trimester RSV season exposure, suggestive of a fetal immune response to RSV. IMPORTANCE Our study found that duration of third trimester exposure to RSV season related inversely to subsequent risk of postnatal RSV-LRI in the first year, thus implicating this exposure as an important factor in reducing risk of postnatal RSV-LRIs, a risk reduction that appears to be independent of maternally transferred anti-RSV antibody level. The increase in frequency of detectable IFN-γ and not IL-4 in response to UV-inactivated RSV in cord blood immune cells for infants with greater third trimester exposure to RSV season is suggestive of a Type-1 immune response to RSV occurring in utero.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

DTX3L Enhances Type I Interferon Antiviral Response by Promoting the Ubiquitination and Phosphorylation of TBK1.

Studies already revealed that some E3 ubiquitin ligases participated in the immune response after viral infection by regulating the type I interferon (IFN) pathway. Here, we demonstrated that type I interferon signaling enhanced the translocation of ETS1 to the nucleus and the promoter activity of E3 ubiquitin ligase DTX3L (deltex E3 ubiquitin ligase 3L) after virus infection and thus increased the expression of DTX3L. Further experiments suggested that DTX3L ubiquitinated TBK1 at K30 and K401 sites on K63-linked ubiquitination pathway. DTX3L was also necessary for mediating the phosphorylation of TBK1 through binding with the tyrosine kinase SRC: both together enhanced the activation of TBK1. Therefore, DTX3L, being an important positive-feedback regulator of type I interferon, exerted a key role in antiviral response. IMPORTANCE Our present study evaluated DTX3L as an antiviral molecule by promoting IFN production and establishing an IFN-ß-ETS1-DTX3L-TBK1 positive-feedback loop as a novel immunomodulatory step to enhance interferon signaling and inhibit respiratory syncytial virus (RSV) infection. Our finding enriches and complements the biological function of DTX3L and provides a new strategy to protect against lung diseases such as bronchiolitis and pneumonia that develop with RSV.

IL-17A plays a critical role in RSV infection in children and mice.

BACKGROUND: IL-17A is a pleiotropic cytokine and intimately associated with asthma, but its role in respiratory syncytial virus (RSV) infection is conflicting in the literature. METHODS: Children hospitalized in the respiratory department with RSV infection during RSV pandemic season of 2018-2020 were included. Nasopharyngeal aspirates were collected for pathogen and cytokines determination. In the murine model, RSV intranasal administrations were performed in wild-type and IL-17A-/- mice. Leukocytes and cytokines in bronchoalveolar lavage fluid (BALF), lung histopathology, and airway hyperresponsiveness (AHR) were measured. RORγt mRNA and IL-23R mRNA were semi-quantified by qPCR. RESULTS: IL-17A increased significantly in RSV-infected children and was positively associated with pneumonia severity. In the murine model, IL-17A significantly increased in BALF of mice with RSV infection. Airway inflammation, lung tissue damage and AHR were significantly alleviated in wild-type mice following IL-17A neutralization and in the IL-17A-/- mice. IL-17A decreased by removing CD4+ T cells but increased by depleting CD8+ T cells. IL-6, IL-21, RORγt mRNA and IL-23R mRNA dramatically increased in parallel with the rise of IL-17A. CONCLUSIONS: IL-17A contributes to the airway dysfunctions induced by RSV in children and murine. CD3+CD4+T cells are its major cellular sources and the IL-6/IL-21-IL-23R-RORγt signaling pathway might participate in its regulation.

Efficacy and Safety of an Ad26.RSV.preF-RSV preF Protein Vaccine in Older Adults.

BACKGROUND: Respiratory syncytial virus (RSV) can cause serious lower respiratory tract disease in older adults, but no licensed RSV vaccine currently exists. An adenovirus serotype 26 RSV vector encoding a prefusion F (preF) protein (Ad26.RSV.preF) in combination with RSV preF protein was previously shown to elicit humoral and cellular immunogenicity. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 2b, proof-of-concept trial to evaluate the efficacy, immunogenicity, and safety of an Ad26.RSV.preF-RSV preF protein vaccine. Adults who were 65 years of age or older were randomly assigned in a 1:1 ratio to receive vaccine or placebo. The primary end point was the first occurrence of RSV-mediated lower respiratory tract disease that met one of three case definitions: three or more symptoms of lower respiratory tract infection (definition 1), two or more symptoms of lower respiratory tract infection (definition 2), and either two or more symptoms of lower respiratory tract infection or one or more symptoms of lower respiratory tract infection plus at least one systemic symptom (definition 3). RESULTS: Overall, 5782 participants were enrolled and received an injection. RSV-mediated lower respiratory tract disease meeting case definitions 1, 2, and 3 occurred in 6, 10, and 13 vaccine recipients and in 30, 40, and 43 placebo recipients, respectively. Vaccine efficacy was 80.0% (94.2% confidence interval [CI], 52.2 to 92.9), 75.0% (94.2% CI, 50.1 to 88.5), and 69.8% (94.2% CI, 43.7 to 84.7) for case definitions 1, 2, and 3, respectively. After vaccination, RSV A2 neutralizing antibody titers increased by a factor of 12.1 from baseline to day 15, a finding consistent with other immunogenicity measures. Percentages of participants with solicited local and systemic adverse events were higher in the vaccine group than in the placebo group (local, 37.9% vs. 8.4%; systemic, 41.4% vs. 16.4%); most adverse events were mild to moderate in severity. The frequency of serious adverse events was similar in the vaccine group and the placebo group (4.6% and 4.7%, respectively). CONCLUSIONS: In adults 65 years of age or older, Ad26.RSV.preF-RSV preF protein vaccine was immunogenic and prevented RSV-mediated lower respiratory tract disease. (Funded by Janssen Vaccines and Prevention; CYPRESS ClinicalTrials.gov number, NCT03982199.).

The Respiratory Syncytial Virus (RSV) G Protein Enhances the Immune Responses to the RSV F Protein in an Enveloped Virus-Like Particle Vaccine Candidate.

Respiratory syncytial virus (RSV) is a serious human respiratory pathogen, but no RSV vaccine has been licensed. Many vaccine candidates are focused on the viral F protein since the F protein is more conserved than the viral G protein across RSV strains and serotypes; thus, the F protein is thought more likely to induce a broader range of protection from infection. However, it is the G protein that binds the likely receptor, CX3CR1, in lung ciliated epithelial cells, raising the question of the importance of the G protein in vaccine candidates. Using virus-like particle (VLP) vaccine candidates, we have directly compared VLPs containing only the prefusion F protein (pre-F), only the G protein, or both glycoproteins. We report that VLPs containing both glycoproteins bind to anti-F-protein-specific monoclonal antibodies differently than do VLPs containing only the prefusion F protein. In RSV-naive cotton rats, VLPs assembled with only the pre-F protein stimulated extremely weak neutralizing antibody (NAb) titers, as did VLPs assembled with G protein. However, VLPs assembled with both glycoproteins stimulated quite robust neutralizing antibody titers, induced improved protection of the animals from RSV challenge compared to pre-F VLPs, and induced significantly higher levels of antibodies specific for F protein antigenic site 0, site III, and the AM14 binding site than did VLPs containing only the pre-F protein. These results indicate that assembly of pre-F protein with G protein in VLPs further stabilized the prefusion conformation or otherwise altered the conformation of the F protein, increasing the induction of protective antibodies. IMPORTANCE Respiratory syncytial virus (RSV) results in significant disease in infants, young children, and the elderly. Thus, development of an effective vaccine for these populations is a priority. Most ongoing efforts in RSV vaccine development have focused on the viral fusion (F) protein; however, the importance of the inclusion of G in vaccine candidates is unclear. Here, using virus-like particles (VLPs) assembled with only the F protein, only the G protein, or both glycoproteins, we show that VLPs assembled with both glycoproteins are a far superior vaccine in a cotton rat model compared with VLPs containing only F protein or only G protein. The results show that the presence of G protein in the VLPs influences the conformation of the F protein and the immune responses to F protein, resulting in significantly higher neutralizing antibody titers and better protection from RSV challenge. These results suggest that inclusion of G protein in a vaccine candidate may improve its effectiveness.

PLZF promotes the development of asthma tolerance via affecting memory phenotypes of immune cells.

Clarifying the pathogenesis of asthma and/or identifying the specific pathway underlying oral asthma tolerance (OT) would be of great significance. In our previous study, promyelocytic leukemia zinc finger (PLZF), which reportedly regulates memory phenotypes, was found to promote ovalbumin (OVA)-induced OT. Therefore, this study aimed to explore the regulatory effects of PLZF on memory phenotypes in asthma and OT mouse models. We found that Zbtb16 (encoding PLZF) and PLZF+ cells were highly increased in OT lungs compared with asthmatic lungs. PLZF was co-expressed with GATA3, and IL-4+PLZF+ cells were significantly lower in OT lungs than in asthmatic lungs. Notably, memory cells were decreased in OT mice, and these mice had PLZF+ cells that expressed lower levels of CD44 than those of asthmatic mice. When Zbtb16 was overexpressed in splenic lymphocytes, the number of CD44+ cells decreased. There were increased memory cells in splenic lymphocytes after treatment with the supernatant of OVA-treated airway epithelial cells; however, this was reversed by Zbtb16 overexpression. Early respiratory syncytial virus infection increased memory cells and reduced PLZF+ cells in the OT mice. Collectively, these results indicate that PLZF may reduce the proportion of memory cells, thereby, promoting the establishment of OT.

Prefusion F Protein-Based Respiratory Syncytial Virus Immunization in Pregnancy.

BACKGROUND: Respiratory syncytial virus (RSV), a major cause of illness and death in infants worldwide, could be prevented by vaccination during pregnancy. The efficacy, immunogenicity, and safety of a bivalent RSV prefusion F protein-based (RSVpreF) vaccine in pregnant women and their infants are uncertain. METHODS: In a phase 2b trial, we randomly assigned pregnant women, at 24 through 36 weeks' gestation, to receive either 120 or 240 µg of RSVpreF vaccine (with or without aluminum hydroxide) or placebo. The trial included safety end points and immunogenicity end points that, in this interim analysis, included 50% titers of RSV A, B, and combined A/B neutralizing antibodies in maternal serum at delivery and in umbilical-cord blood, as well as maternal-to-infant transplacental transfer ratios. RESULTS: This planned interim analysis included 406 women and 403 infants; 327 women (80.5%) received RSVpreF vaccine. Most postvaccination reactions were mild to moderate; the incidence of local reactions was higher among women who received RSVpreF vaccine containing aluminum hydroxide than among those who received RSVpreF vaccine without aluminum hydroxide. The incidences of adverse events in the women and infants were similar in the vaccine and placebo groups; the type and frequency of these events were consistent with the background incidences among pregnant women and infants. The geometric mean ratios of 50% neutralizing titers between the infants of vaccine recipients and those of placebo recipients ranged from 9.7 to 11.7 among those with RSV A neutralizing antibodies and from 13.6 to 16.8 among those with RSV B neutralizing antibodies. Transplacental neutralizing antibody transfer ratios ranged from 1.41 to 2.10 and were higher with nonaluminum formulations than with aluminum formulations. Across the range of assessed gestational ages, infants of women who were immunized had similar titers in umbilical-cord blood and similar transplacental transfer ratios. CONCLUSIONS: RSVpreF vaccine elicited neutralizing antibody responses with efficient transplacental transfer and without evident safety concerns. (Funded by Pfizer; ClinicalTrials.gov number, NCT04032093.).

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G (H. C. Bergeron, J. Murray, A. M. Nuñez Castrejon, et al., Viruses 13:352, 2021, https://doi.org/10.3390/v13020352). However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. IMPORTANCE Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

Multiple Respiratory Syncytial Virus (RSV) Strains Infecting HEp-2 and A549 Cells Reveal Cell Line-Dependent Differences in Resistance to RSV Infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors. IMPORTANCE Infection with the respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. Most RSV studies have involved either of two historic RSV/A strains infecting one of two cell lines, HEp-2 or A549 cells. However, RSV contains ample variation within two evolving subgroups (A and B), and HEp-2 and A549 cell lines are genetically distinct. Here, we measured viral action and host response in both HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporary strains. We discovered a subgroup-dependent difference in viral gene expression and found A549 cells were more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings revealed important differences between RSV subgroups and two widely used cell lines and provided baseline data for experiments with model systems better representative of natural RSV infection.

RSV Infection in Neonatal Mice Induces Pulmonary Eosinophilia Responsible for Asthmatic Reaction.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. Severe respiratory viral infection in early life is intimately associated with childhood recurrent wheezing and is a risk factor for asthma later in life. Although eosinophilic airway inflammation is an important trait in asthma of children, the roles of pulmonary eosinophils in the disease have been inadequately understood. Here, we show that RSV infection in neonatal mice causes eosinophilia after allergen stimulation. We showed that RSV infection in neonatal mice exacerbated allergic asthma to allergen stimulation that was accompanied with increased detection of eosinophils in the lungs. In addition, we also detected accumulation of ILC2, CD4+ T cells, and macrophages. Importantly, adoptive transfer of eosinophils from asthmatic mice with early-life RSV infection exacerbated pulmonary pathologies associated with allergic respiratory inflammation in naive mice in response to foreign antigen. The induction of asthmatic symptoms including AHR, tracheal wall thickening, and mucus production became more severe after further stimulation in those mice. The expression of antigen presentation-related molecules like CD80, CD86, and especially MHC II was markedly induced in eosinophils from OVA-stimulated asthmatic mice. The accumulation of CD4+ T cells in the lungs was also significantly increased as a result of adoptive transfer of eosinophils. Importantly, the deterioration of lung pathology caused by adoptive transfer could be effectively attenuated by treatment with indomethacin, a nonsteroidal anti-inflammatory drug. Our findings highlight the significance of eosinophil-mediated proinflammatory response in allergic disease associated with early-life infection of the respiratory tract.

How RSV Proteins Join Forces to Overcome the Host Innate Immune Response.

Respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Although several pattern recognition receptors (PRRs) can sense RSV-derived pathogen-associated molecular patterns (PAMPs), infection with RSV is typically associated with low to undetectable levels of type I interferons (IFNs). Multiple RSV proteins can hinder the host's innate immune response. The main players are NS1 and NS2 which suppress type I IFN production and signalling in multiple ways. The recruitment of innate immune cells and the production of several cytokines are reduced by RSV G. Next, RSV N can sequester immunostimulatory proteins to inclusion bodies (IBs). N might also facilitate the assembly of a multiprotein complex that is responsible for the negative regulation of innate immune pathways. Furthermore, RSV M modulates the host's innate immune response. The nuclear accumulation of RSV M has been linked to an impaired host gene transcription, in particular for nuclear-encoded mitochondrial proteins. In addition, RSV M might also directly target mitochondrial proteins which results in a reduced mitochondrion-mediated innate immune recognition of RSV. Lastly, RSV SH might prolong the viral replication in infected cells and influence cytokine production.

Type I interferons and MAVS signaling are necessary for tissue resident memory CD8+ T cell responses to RSV infection.

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (TRM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8+ TRM cells correlates with a better outcome. However, the requirements for generating and maintaining lung TRM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the TRM cell pool during RSV infection. We show that CD8+ TRM cells expand independently from systemic CD8+ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/ß), display impaired expansion of CD8+ TRM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored TRM cell expansion upon re-challenge but failed to recover TRM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8+ TRM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.

Respiratory Syncytial Virus Provides Protection against a Subsequent Influenza A Virus Infection.

Respiratory infections are a leading cause of morbidity and mortality. The presence of multiple heterologous virus infections is routinely observed in a subset of individuals screened for the presence of respiratory viruses. However, the impact overlapping infections has on disease severity and the host immune response is not well understood. Respiratory syncytial virus (RSV) and influenza A virus (IAV) are two of the most common respiratory infections observed in hospitalized patients, particularly in the very young and aged populations. In this study, we examined how the order in which BALB/c mice were infected with both RSV and IAV impacts disease severity. RSV infection prior to an IAV infection was associated with decreased weight loss and increased survival as compared with IAV infection alone. In contrast, IAV infection prior to an RSV infection was associated with similar morbidity and mortality as compared with an IAV infection alone. Our results suggest that the order in which viral infections are acquired plays a critical role in the outcome of disease severity and the host immune response.