Development of an efficacious universal influenza vaccines remains a long-sought goal. Current vaccines have shortfalls such as mid/low efficacy and needing yearly strain revisions to account for viral drift/shift. Horses undergo bi-annual vaccines for the H3N8 equine influenza virus, and surveillance of sera from vaccinees demonstrated very broad reactivity and neutralization to many influenza strains. Subsequently, vaccinating mice using the equine A/Kentucky/1/1991 strain or recombinant hemagglutinin (HA) induced similar broadly reactive and neutralizing antibodies to seasonal and high pathogenicity avian influenza strains. Challenge of vaccinated mice protected from lethal virus challenges across H1N1 and H3N2 strains. This protection correlated with neutralizing antibodies to the HA head, esterase, and stem regions. Vaccinated ferrets were also protected after challenge with H1N1 influenza A/07/2009 virus using whole viral or HA. These data suggest that equine H3N8 induces broad protection against multiple influenzas using a unique antigen that diverges from other universal vaccine approaches.

With circulation of SARS-CoV-2, fears about coinfection with other respiratory viruses such as influenza and RSV were significant, but the opposite was observed. Distancing/barriers played a major role in reducing other viral co-infections, however, some infrequent co-infections still occurred. We investigated the relationship between SARS-CoV-2 and RSV during coinfection to understand how they might compete or synergize. We found only RSV's replication was significantly reduced when coinfected with SARS-CoV-2. Investigation of the mechanism revealed that the SARS-CoV-2 protein Nsp1 disrupts the RSV M2-2 protein but not the upstream M2-1 protein on the same biscistronic mRNA transcript. The impact of Nsp1 on M2-2 was not dependent on M2-2 being the second ORF in a bicistronic mRNA transcript, but likely from prevention of ribosomal termination-reinitiation necessary for M2-2 production. Additional viral ORFs from influenza A, influenza B, or Sendai virus dependent on the same or other ribosomal initiation mechanisms were tested and we found only influenza B M/M2 which likely uses a similar method as M2-2 was disrupted. Various M2-2 constructs, with/without the proposed site of ribosomal termination-reinitiating, co-transfected with Nsp1 and were in agreement that disruption to M2-2 expression occurs if the site of re-initiation was present upstream. These data not only suggest Sars-CoV-2 can outcompete RSV through suppression of M2, but may also point to potential ways to interfere with RSV by targeted therapies. ### Competing Interest Statement The authors have declared no competing interest.

Respiratory Syncytial Virus (RSV) is a negative stranded RNA virus with a high incidence of secondary bacterial infections. RSV contains two broad immune inhibitory proteins Ns1 and Ns2 which are not present in any other viruses of the Mononegavirales Order. Here we report that expression of Ns2 is attenuated during RSV infection of neutrophils and that RSV is indeed infecting neutrophils rather than simply being phagocytosed by them. Infection was determined by intracellular staining and coinfection studies of uninfected Hep2 cells. The significant attenuation of Ns2 in vivo along with the low abundance of coinfected cells indicates that RSV infection is likely functionally non permissive in in vivo infection. The implications of RSV infection of neutrophils may explain the previously observed phenomenon of decreased phagocytosis in neutrophils exposed to RSV and the lack of Ns2 expression within neutrophils may provide avenues of study to attenuate viral infection through therapy development. ### Competing Interest Statement The authors have declared no competing interest.

An analysis that combined bioinformatics, comparative sequence/structural analysis, and experimental assays has been completed on respiratory syncytial virus (RSV). Both the genomic RNA and its reverse complement were studied using the novel bioinformatics pipeline ScanFold, which predicted 49 regions on RSV RNAs that appear to encode functional secondary structures (based on their unusually stable sequence order). Multiple motifs appear to be conserved between RSV and related virus strains, including one region within the F gene, which had a highly favorable overall prediction metric of a paired secondary structure. This motif was subjected to additional experimental analyses using SHAPE analysis to confirm ScanFold predicted secondary structure. In subsequent analysis, RSV F mRNA transcripts were made by in vitro transcription using T7 polymerase and transcripts which relaxed the predicted secondary structure yielded slightly higher mRNA transcripts and protein expression levels as wildtype F. However, using reverse genetics for comparison of viruses containing wildtype or relaxed F suggested that the predicted secondary structures may be critical for RSV replication in cells. To our knowledge, this is the first study to examine conserved RNA structures across multiple RSV strains and may help identify potential therapeutic targets to inhibit. ### Competing Interest Statement The authors have declared no competing interest.

AbstractTooth brushing and flossing are usually both hallmarks of a good oral hygiene routine to prevent decay, gingivitis, and periodontitis. While brushing removes much of the oral bacteria from the front and backs of the teeth, flossing is believed to be necessary to remove bacteria between the teeth. However, the effectiveness of self-flossing has not been established very well. Flossing effectiveness was evaluated two ways in this study: adults and 12 year old children were instructed on how to floss and bacterial colonies were determined before or after 7 days or pediatric and family dentists were blinded to patient surveys that asked about flossing frequency and evaluated the patient for gingivitis. We found a significant number of children did not floss at all despite brushing daily. However, flossing had no effect on the number of bacteria in their mouths nor did flossing have any correlation with reducing gingivitis development. Taken together, self-flossing did not appear to be an effective strategy for reduction of gingivitis in children or adults but could stem from improper technique or simply a lack of doing it.

Young children, especially those aged 4 months to 2 years of age, frequently exhibit severe morbidity during respiratory viral infections. For influenza infections, macrophages/monocytes serve as front line defenses against early viral replication in the lungs until the adaptive immune system arrives to clear virus. However, infiltrating inflammatory monocytes are a significant cause of influenza induced lung pathology/morbidity. We utilized a young murine model of respiratory viral infections using 21-day-old mice to investigate the mechanisms driving the heightened influenza induced morbidity observed in human young children. We hypothesized that macrophages/monocytes responses to influenza would diverge between young and older mice despite our evidence that macrophages from both groups appear to control viral replication at similar rates. While inflammatory monocyte infiltration contributed to influenza induced morbidity/lung inflammation in adults, they did not appear to contribute to morbidity in young mice. Instead, young mice appeared to develop lung inflammation through a lack of interferon gamma (IFNg) and infection of macrophage populations. In contrast, adult mice controlled early viral replication through macrophage populations (alveolar, interstitial, and inflammatory) and inflammatory monocytes. While intrinsic limitations in anti-viral cytokine responses, especially IFNg, characterized the macrophage response to viral infection in young mice, the innate immune response to infection appears diminished compared to adults. This study highlights the intrinsic limitations in macrophage effector functions that may arise in young children but that also contribute to disease pathology. ### Competing Interest Statement The authors have declared no competing interest.

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH33) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH33 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH33 and polymeric nanoadjuvant platforms.

Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5' untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 μM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.

Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, especially the co-pathologies between RSV and Spn , is known. Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups were mock nebulized. At day three post-RSV infection, one RSV group (RSV /Spn) and one mock-nebulized group ( Spn only ) were inoculated with Spn intratracheally. At day six post-RSV infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/ Spn trended with higher RSV titers, but lower Spn . Additionally, lung lesions were observed to be more frequent in the RSV/ Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration and higher myeloperoxidase. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts.

Exercise has substantial health benefits, but the effects of exercise on immune status and susceptibility to respiratory infections are less clear. Furthermore, there is limited research examining the effects of prolonged exercise on local respiratory immunity and antiviral activity. To assess the upper respiratory tract in response to exercise, we collected nasal lavage fluid (NALF) from human subjects (1) at rest, (2) after 45 min of moderate-intensity exercise, and (3) after 180 min of moderate-intensity exercise. To assess immune responses of the lower respiratory tract, we utilized a murine model to examine the effect of exercise duration on bronchoalveolar lavage (BAL) fluid immune cell content and lung gene expression. NALF cell counts did not change after 45 min of exercise, whereas 180 min significantly increased total cells and leukocytes in NALF. Importantly, fold change in NALF leukocytes correlated with the post-exercise fatigue rating in the 180-min exercise condition. The acellular portion of NALF contained strong antiviral activity against Influenza A in both resting and exercise paradigms. In mice undergoing moderate-intensity exercise, BAL total cells and neutrophils decreased in response to 45 or 90 min of exercise. In lung lobes, increased expression of heat shock proteins suggested that cellular stress occurred in response to exercise. However, a broad upregulation of inflammatory genes was not observed, even at 180 min of exercise. This work demonstrates that exercise duration differentially alters the cellularity of respiratory tract fluids, antiviral activity, and gene expression. These changes in local mucosal immunity may influence resistance to respiratory viruses, including influenza or possibly other pathogens in which nasal mucosa plays a protective role, such as rhinovirus or SARS-CoV-2.

Peptide nucleic acids (PNAs) are charge-neutral oligonucleotides with emerging potential for treatment of genetic, acquired, and viral diseases, including COVID-19. Their challenging synthesis, however, limits their use for rapid therapeutic intervention and widespread application. Here, we report a highly efficient technology that utilizes a fully automated fast-flow instrument to manufacture cell-penetrating peptide-conjugated PNAs (PPNAs) in a single shot. The machine is rapid: each amide bond is formed in 10 seconds and the synthesis of an 18-mer bioactive PNA is complete in one hour. Anti-IVS2-654 PPNA synthesized in a single shot with this instrument presented over 16-fold activity compared to unmodified PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing an eight-member anti-SARS-CoV-2 PPNA library within one day. A designer PPNA targeting the 5’ untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in live virus infection assays (IC 50 : 0.8 mM). Our technology can rapidly yield on-demand PPNA candidates to tackle newly-emerging viral pathogens.

The antisense phosphorodiamidate morpholino oligomer (PMO) drugs Eteplirsen and Golodirsen are improving the lives of some Duchenne muscular dystrophy (DMD) patients, but treating all DMD subtypes would require the development of over 50 novel antisense therapies. To rapidly prototype personalized PMO for diseases such as DMD, we designed a fully automated flow-based oligonucleotide synthesizer. Our optimized high temperature synthesis platform reduces coupling times by up to 22-fold compared to previously reported batch methods. We demonstrate the power of our new automated technology with the synthesis of milligram quantities of an 18-mer reporter PMO sequence in 3.5 hours, three new potential therapeutic PMO sequences targeted to exon 46 of the dystrophin gene in a single day, and a candidate antiviral PMO sequence targeted to the SARS-CoV-2 genomic mRNA in 3.5 hours. This flexible flow synthesis platform can be used for on-demand production of a broad range of personalized therapeutic polymers.

Respiratory Syncytial Virus (RSV) is a highly prevalent virus that affects the majority of the population. The virus can cause severe disease in vulnerable populations leading to high hospitalization rates from bronchiolitis or secondary bacterial infections leading to pneumonia. Two early and non-structural proteins (Ns1 and Ns2), strongly over-ride the antiviral innate system but also diminish the adaptive response as well. This review will cover interactions of Ns1 and Ns2 with the host antiviral response with a focus on alterations to signaling pathways, cytokine gene expression, and effects of the Ns proteins on mitochondria.

Respiratory Syncytial Virus is a yearly respiratory virus that causes significant frequencies of morbidities, particularly in the young and elderly populations. However, preventive vaccines and/or treatment therapies are generally lacking, although much attention is now being placed on this virus. Moreover, there are now multiple strategies currently being explored in a race to the first licensed vaccine. While vaccines are being developed, multiple treatment strategies are being explored to attenuate the severity of infection and thus reduce hospitalization rates in vulnerable populations. This review outlines current strategies to prevent or treat this virus in the hopes of reducing significant human morbidity and mortality that occurs yearly with this seasonal virus.

Immunosenescence poses a formidable challenge in designing effective influenza vaccines for aging populations. While approved vaccines against influenza viruses exist, their efficacy in older adults is significantly decreased due to the diminished capabilities of innate and adaptive immune responses. In this work, the ability of a combination nanovaccine containing both recombinant hemagglutinin and nucleoprotein to provide protection against seasonal influenza virus infection was examined in young and aged mice. Vaccine formulations combining two nanoadjuvants, polyanhydride nanoparticles and pentablock copolymer micelles, were shown to enhance protection against challenge compared to each adjuvant alone in young mice. Nanoparticles were shown to enhance in vitro activation of dendritic cells isolated from aged mice, while both nanoadjuvants did not induce proinflammatory cytokine secretion which may be detrimental in aged individuals. In addition, the combination nanovaccine platform was shown to induce demonstrable antibody titers in both young and aged mice that correlated with the maintenance of body weight post-challenge. Collectively, these data demonstrate that the combination nanovaccine platform is a promising technology for influenza vaccines for older adults.

Although children growing from birth into young adulthood undergo rapid physiological maturation, their immune systems are also undergoing significant changes that may affect how they respond to microbes and especially respiratory pathogens. A key component of control over microbes is the innate immune system that sustains pathogen suppression/elimination until the adaptive immune system can instigate clearance. Here, this review will summarize key characteristics of the developing innate immune system of neonates, infants, and toddlers. In addition, a brief summary of how immunometabolism affects the innate immune system, and its ramifications on the developing innate immune cells will also be covered. Given the key differences between innate immunity of young children and older children/adults and the generally higher levels of morbidity associated with respiratory viral infections of the former, not many studies have examined how metabolic or mitochondrial differences may be influencing their generally limited responses. Further studies in immunometabolism in the young could elucidate keys mechanisms causing the typical diminished responses observed in this population.

Respiratory syncytial virus (RSV) is a very frequent viral respiratory pathogen of the young (<5 years old) with a significant portion of young toddlers having been infected before 2 years of age. Although we understand that some of the morbidity associated with RSV in neonates is due to immunological maturation that favors immunosuppression over antiviral innate and/or adaptive immune responses, the rapid development of the immune system right after birth suggests that each age group (newborn, early infant, older infant, toddler, and older) may respond to the virus in different ways. In this study, we summarize the morbidity associated with infection in young children in the context of immunological maturation of monocytes/macrophages and the ramifications for poor innate control of viral pathogenesis. We also summarize key mechanisms that contribute to the diminished antiviral innate immune responses of these young children.

Respiratory syncytial virus (RSV) is the main cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection especially by Streptococcus pneumoniae (Sp) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co‐infection, human meta‐studies have been mixed. Moreover, less is known about pathogenesis of Sp serotype 22 and especially the co‐pathologies between RSV and Sp dual infections.Here, we sought to examine mechanisms contributing to co‐pathogen‐induced morbidity using a large neonatal lamb animal model naturally permissive to infection by both pathogens.Colostrum deprived lambs (aged 3–5 days) were randomly divided into four groups. Two of the groups were nebulized with RSV M37 (1.27 × 107 IFFU/mL), and the other two group nebulized with cell–conditioned mock media. At day 3 post‐infection, one RSV group (RSV/Sp) and one mock‐nebulized group (Sp only) were infected with (2×106 cfu of Sp) intratracheally. At day 6 post‐infection all lambs were humanely euthanized and bacterial/viral pathogeneses were assessed by culture, focus forming unit, qPCR, IHC, and histopathology.Lambs dually infected with RSV and Sp had higher RSV titers by qPCR but lower Sp than the other comparable groups. Additionally, lung lesions were more intense in the RSV/Sp group as characterized by increased interalveolar wall thickness with neutrophils and lymphocyte infiltration.Despite lower Sp in lungs, lambs co‐infected with RSV exhibited greater morbidity and tissue histopathology. Thus, enhanced disease severity may be due more to elevated immunopathogenesis than elevated bacterial pathogenesisSupport or Funding Informationfunded by Merck & Company Inc.55211 Verhoeven, 11/01/16–10/31/18This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Young children (<5 years of age but especially those <2-year old) exhibit high rates of morbidity and frequently require hospitalizations due to complications from respiratory viral infections. This is also a population for which we understand less about how their unique level of immunological maturation affects their antiviral immune responses. However, we do know from prior studies that their T cells appear to apoptose in the lungs owing to limited interferon (IFN)γ autocrine signaling during infection. To begin to further understand additional limits, we utilized an infant/toddler murine model infected with influenza virus with an adult comparator. In our model, young mice exhibited lower interleukin (IL)-10 + IFNγ + co-producing CD4 T cells infiltrating the lungs that paralleled with a failed switch from an innate to adaptive immune response at the mid infection stage. Specifically, limited co-IL-10 production correlated with a lack of influenza-specific antibodies and subsequent complement receptor signaling (complement receptor type-1 related gene Y (CCRY)/p65) to the lung infiltrating CD4 T cells therefore limiting their IKAROs upregulation. Thus, limited IL-10 production appeared to diminish signaling to lung macrophages to stop accumulating late into infection. Taken together, our results suggest a novel role for complement mediated signaling in CD4 T cells with respect to IL-10 co-production. Furthermore, a subsequent failure to shift from the unfocused innate immune response to the specific adaptive responses may be a principle cause in the enhanced morbidity common in respiratory viral infection of young children.