Differential mucosal IL-10-induced immunoregulation of innate immune responses occurs in influenza infected infants/toddlers and adults.

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.

Enhanced innate antiviral gene expression, IFN-α, and cytolytic responses are predictive of mucosal immune recovery during simian immunodeficiency virus infection.

The mucosa that lines the respiratory and gastrointestinal (GI) tracts is an important portal of entry for pathogens and provides the first line of innate immune defense against infections. Although an abundance of memory CD4(+) T cells at mucosal sites render them highly susceptible to HIV infection, the gut and not the lung experiences severe and sustained CD4(+) T cell depletion and tissue disruption. We hypothesized that distinct immune responses in the lung and gut during the primary and chronic stages of viral infection contribute to these differences. Using the SIV model of AIDS, we performed a comparative analysis of the molecular and cellular characteristics of host responses in the gut and lung. Our findings showed that both mucosal compartments harbor similar percentages of memory CD4(+) T cells and displayed comparable cytokine (IL-2, IFN-γ, and TNF-α) responses to mitogenic stimulations prior to infection. However, despite similar viral replication and CD4(+) T cell depletion during primary SIV infection, CD4(+) T cell restoration kinetics in the lung and gut diverged during acute viral infection. The CD4(+) T cells rebounded or were preserved in the lung mucosa during chronic viral infection, which correlated with heightened induction of type I IFN signaling molecules and innate viral restriction factors. In contrast, the lack of CD4(+) T cell restoration in the gut was associated with dampened immune responses and diminished expression of viral restriction factors. Thus, unique immune mechanisms contribute to the differential response and protection of pulmonary versus GI mucosa and can be leveraged to enhance mucosal recovery.

Differential impact of respiratory syncytial virus and parainfluenza virus on the frequency of acute otitis media is explained by lower adaptive and innate immune responses in otitis-prone children.

BACKGROUND: Acute otitis media (AOM) is a leading cause of bacterial pediatric infections associated with viral upper respiratory infections (URIs). We examined the differential impact of respiratory syncytial virus (RSV) and parainfluenza virus URIs on the frequency of AOM caused by Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) in stringently defined otitis-prone (sOP) and non-otitis-prone (NOP) children as a potential mechanism to explain increased susceptibility to AOM. METHODS: Peripheral blood and nasal washes were obtained from sOP and NOP children (n = 309). Colonization events and antiviral responses consisting of total specific immunoglobulin G (IgG) responses, neutralizing antibody responses, and T-cell responses were determined. Isolated neutrophils were infected with varying multiplicities of infection of both viruses, and opsonophagocytosis potential was measured. RESULTS: A significant increase was found in frequency of AOM events caused by Spn and NTHi, with a concurrent RSV infection in sOP children. These results correlated with diminished total RSV-specific IgG, higher viral nasal burdens, and lower IgG neutralizing capacity. The sOP children had diminished T-cell responses to RSV that correlated with lower Toll-like receptor 3/7 transcript and decreased expression of HLA-DR on antigen-presenting cells. RSV interfered with the Spn phagocytic capacity of neutrophils in a dose-dependent manner. Parainfluenza virus infections did not differentially affect AOM events in sOP and NOP children. CONCLUSIONS: Lower innate and adaptive immune responses to RSV in sOP children may slow the kinetics of viral clearance from the nasopharynx and allow for viral interference with antibacterial immune responses, thus contributing to increased frequency of AOMs.

Lower nasopharyngeal epithelial cell repair and diminished innate inflammation responses contribute to the onset of acute otitis media in otitis-prone children.

About 30 % of young children experience excessive, frequent episodes of middle ear infection and are classified as acute otitis media prone (OP). Streptococcus pneumoniae (Spn) is a predominant otopathogen in OP and non-OP (NOP) children. The pathogenesis of middle ear infection involves otopathogen nasopharyngeal (NP) colonization followed by an upper respiratory viral infection that modifies the NP environment to allow a sufficient inoculum of bacteria to reflux via the Eustachian tube into the middle ear space. Here, we analyzed the NP mucosal repair response between age-matched stringently defined OP (sOP) and NOP children who progressed to middle ear infection caused by Spn. We found lower epidermal growth factor, epidermal growth factor receptor, and angiogenin cytokine concentrations in nasal washes of sOP compared with NOP children. Despite higher expression of TLR2/4 transcript expression in nasal epithelium and in polymorphonuclear cells present in nasal secretions in sOP children, sOP children had lower expression of proinflammatory cytokines such as IL-6 and IL-8 in the NP. Chemotaxis-associated cytokine expression at onset of AOM in sOP children was also lower compared with NOP children, possibly indicating a lower capacity to signal the innate immune system. We conclude that lower epithelial cell repair responses during viral infection in the NP combined with diminished innate inflammatory responses potentiate Spn pathogenesis in the sOP child.

Structural Stability and Antigenicity of Universal Equine H3N8 Hemagglutinin Trimer upon Release from Polyanhydride Nanoparticles and Pentablock Copolymer Hydrogels.

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.

Automated Flow Synthesis of Peptide-PNA Conjugates.

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.

Memory CD4 T cells direct protective responses to influenza virus in the lungs through helper-independent mechanisms.

Memory CD4 T cells specific for influenza virus are generated from natural infection and vaccination, persist long-term, and recognize determinants in seasonal and pandemic influenza virus strains. However, the protective potential of these long-lived influenza virus-specific memory CD4 T cells is not clear, including whether CD4 T-cell helper or effector functions are important in secondary antiviral responses. Here we demonstrate that memory CD4 T cells specific for H1N1 influenza virus directed protective responses to influenza virus challenge through intrinsic effector mechanisms, resulting in enhanced viral clearance, recovery from sublethal infection, and full protection from lethal challenge. Mice with influenza virus hemagglutinin (HA)-specific memory CD4 T cells or polyclonal influenza virus-specific memory CD4 T cells exhibited protection from influenza virus challenge that occurred in the presence of CD8-depleting antibodies in B-cell-deficient mice and when CD4 T cells were transferred into lymphocyte-deficient RAG2(-/-) mice. Moreover, the presence of memory CD4 T cells mobilized enhanced T-cell recruitment and immune responses in the lung. Neutralization of gamma interferon (IFN-gamma) production in vivo abrogated memory CD4 T-cell-mediated protection from influenza virus challenge by HA-specific memory T cells and heterosubtypic protection by polyclonal memory CD4 T cells. Our results indicate that memory CD4 T cells can direct enhanced protection from influenza virus infection through mobilization of immune effectors in the lung, independent of their helper functions. These findings have important implications for the generation of universal influenza vaccines by promoting long-lived protective CD4 T-cell responses.

TLR2 engagement on dendritic cells promotes high frequency effector and memory CD4 T cell responses.

Ligation of TLR by distinct pathogen components provides essential signals for T cell priming, although how individual TLR engagement affects primary and memory T cell responses is not well defined. In this study, we demonstrate distinct effects of TLR2 vs TLR4 engagement on primary and memory CD4 T cell responses due to differential effects on APC. Priming of influenza hemagglutinin (HA)-specific naive CD4 T cells with HA peptide and the TLR2 agonist Pam3CysK in vivo resulted in a high frequency of activated HA-specific CD4 T cells that predominantly produced IL-2 and IL-17, whereas priming with HA peptide and the TLR4 agonist LPS yielded a lower frequency of HA-specific CD4 T cells and predominant IFN-gamma producers. TLR2 agonist priming depended on TLR2 expression by APC, as wild-type CD4 T cells did not expand in response to peptide and Pam3CysK in TLR2-deficient hosts. TLR2-mediated priming also led to an increased frequency of Ag-specific memory CD4 T cells compared with TLR4 priming and mediated enhanced secondary responses to influenza challenge. Our results show that TLR engagement on APC influences both primary and secondary CD4 T cell responses, and suggest that long-term functional capacities of T cells are set by innate signals during early phases of an infection.

Costimulation modulation uncouples protection from immunopathology in memory T cell responses to influenza virus.

The rapid effector functions and tissue heterogeneity of memory T cells facilitate protective immunity, but they can also promote immunopathology in antiviral immunity, autoimmunity, and transplantation. Modulation of memory T cells is a promising but not yet achieved strategy for inhibiting these deleterious effects. Using an influenza infection model, we demonstrate that memory CD4 T cell-driven secondary responses to influenza challenge result in improved viral clearance yet do not prevent the morbidity associated with viral infection, and they exacerbate cellular recruitment into the lung, compared with primary responses. Inhibiting CD28 costimulation with the approved immunomodulator CTLA4Ig suppressed primary responses in naive mice infected with influenza, but was remarkably curative for memory CD4 T cell-mediated secondary responses to influenza, with reduced immunopathology and enhanced recovery. We demonstrate that CTLA4Ig differentially affects lymphoid and nonlymphoid responses to influenza challenge, inhibiting proliferation and egress of lymphoid naive and memory T cells, while leaving lung-resident memory CD4 T cell responses intact. Our findings reveal the dual nature of memory T cell-mediated secondary responses and suggest costimulation modulation as a novel strategy to optimize antiviral immunity by limiting the memory T cell response to its protective capacities.

Streptococcus pneumoniae serotype 22F infection in respiratory syncytial virus infected neonatal lambs enhances morbidity.

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 duration modulates upper and lower respiratory fluid cellularity, antiviral activity, and lung gene expression.

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.

Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

Respiratory Syncytial Virus's Non-structural Proteins: Masters of Interference.

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 syntycial virus: Current treatment strategies and vaccine approaches.

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.

Antiviral therapy during primary simian immunodeficiency virus infection fails to prevent acute loss of CD4+ T cells in gut mucosa but enhances their rapid restoration through central memory T cells.

Gut-associated lymphoid tissue (GALT) is an early target of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) and a site for severe CD4+ T-cell depletion. Although antiretroviral therapy (ART) is effective in suppressing HIV replication and restoring CD4+ T cells in peripheral blood, restoration in GALT is delayed. The role of restored CD4+ T-cell help in GALT during ART and its impact on antiviral CD8+ T-cell responses have not been investigated. Using the SIV model, we investigated gut CD4+ T-cell restoration in infected macaques, initiating ART during either the primary stage (1 week postinfection), prior to acute CD4+ cell loss (PSI), or during the chronic stage at 10 weeks postinfection (CSI). ART led to viral suppression in GALT and peripheral blood mononuclear cells of PSI and CSI animals at comparable levels. CSI animals had incomplete CD4+ T-cell restoration in GALT. In PSI animals, ART did not prevent acute CD4+ T-cell loss by 2 weeks postinfection in GALT but supported rapid and complete CD4+ T-cell restoration thereafter. This correlated with an accumulation of central memory CD4+ T cells and better suppression of inflammation. Restoration of CD4+ T cells in GALT correlated with qualitative changes in SIV gag-specific CD8+ T-cell responses, with a dominance of interleukin-2-producing responses in PSI animals, while both CSI macaques and untreated SIV-infected controls were dominated by gamma interferon responses. Thus, central memory CD4+ T-cell levels and qualitative antiviral CD8+ T-cell responses, independent of viral suppression, were the immune correlates of gut mucosal immune restoration during ART.

Influence of Immunological Maturity on Respiratory Syncytial Virus-Induced Morbidity in Young Children.

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.

Immunometabolism and innate immunity in the context of immunological maturation and respiratory pathogens in young children.

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.

Single dose combination nanovaccine provides protection against influenza A virus in young and aged mice.

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.

Heterogeneous memory T cells in antiviral immunity and immunopathology.

Memory T cells are generated following an initial viral infection, and have the potential for mediating robust protective immunity to viral re-challenge due to their rapid and enhanced functional responses. In recent years, it has become clear that the memory T cell response to most viruses is remarkably diverse in phenotype, function, and tissue distribution, and can undergo dynamic changes during its long-term maintenance in vivo. However, the role of this variegation and compartmentalizationof memory T cells in protective immunity to viruses remains unclear. In this review,we discuss the diverse features of memory T cells that can delineate different subsets, the characteristics of memory T cells thus far identified to promote protective immune responses, and how the heterogeneous nature of memory T cells may also promote immunopathology during antiviral responses. We propose that given the profound heterogeneity of memory T cells, regulation of memory T cells during secondary responses could focus the response to participation of specific subsets,and/or inhibit memory T-cell subsets and functions that can lead to immunopathology.

Control of influenza infection is impaired by diminished interferon-γ secretion by CD4 T cells in the lungs of toddler mice.

Respiratory viral infections, such as influenza, can lead to delayed viral clearance in toddlers, possibly exacerbating disease morbidity. We hypothesized that defective CD4 T cells in toddlers may contribute to a failure to clear virus at a similar rate to adults. Thus, we developed a young mouse model to examine potential divergent responses between toddlers and adults. We determined that young mice (toddler mice, 21 d old) were actively generating and recruiting effector/memory T cells, whereas memory populations were firmly established in older, adult mice (8-10 wk old). We infected toddler and adult mice with influenza A/PR8/34 (H1N1) and found young mice had elevated morbidity, as measured by enhanced weight loss and lower partial pressure of oxygen levels, throughout the infection, thus, modeling the higher morbidity observed in children (<2 y old) during infection. Early viral loads were comparable to adult mice, but toddler mice failed to clear virus by 10 d postinfection. This delayed clearance corresponded to poor lung recruitment of CD4 T cells, lower antiviral T cell responses, and lower B cell/antibodies in the lungs. Mechanistically, diminished interferon-γ was detected in the lungs of toddler mice throughout the infection and corresponded to intrinsic, rather than extrinsic, CD4 T cell limitations in interferon-γ transcription. Moreover, defects in interferon-γ production appeared downstream from signal transducer and activator of transcription 4 in the interleukin-12 signaling pathway, suggesting maturational delays different from neonates. Importantly, recombinant interferon-γ supplementation rescued CD4 T cell numbers in the lungs and influenza-specific antibody formation. This study highlights the intrinsic limitations in CD4 T cell effector functions that may arise in toddlers and contribute to disease pathology.