An RSV Live-Attenuated Vaccine Candidate Lacking G Protein Mucin Domains Is Attenuated, Immunogenic, and Effective in Preventing RSV in BALB/c Mice.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. METHODS: Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. RESULTS: Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. CONCLUSIONS: Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

Enhancing the Thermostability and Immunogenicity of a Respiratory Syncytial Virus (RSV) Live-Attenuated Vaccine by Incorporating Unique RSV Line19F Protein Residues.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, and an effective vaccine is not yet available. We previously generated an RSV live-attenuated vaccine (LAV) candidate, DB1, which was attenuated by a low-fusion subgroup B F protein (BAF) and codon-deoptimized nonstructural protein genes. DB1 was immunogenic and protective in cotton rats but lacked thermostability and stability of the prefusion conformation of F compared to strains with the line19F gene. We hypothesized that substitution of unique residues from the thermostable A2-line19F strain could thermostabilize DB1 and boost its immunogenicity. We therefore substituted 4 unique line19F residues into the BAF protein of DB1 by site-directed mutagenesis and rescued the recombinant virus, DB1-QUAD. Compared to DB1, DB1-QUAD had improved thermostability at 4°C and higher levels of prefusion F as measured by enzyme-linked immunosorbent assays (ELISAs). DB1-QUAD was attenuated in normal human bronchial epithelial cells, in BALB/c mice, and in cotton rats but grew to wild-type titers in Vero cells. In mice, DB1-QUAD was highly immunogenic and generated significantly higher neutralizing antibody titers to a panel of RSV A and B strains than did DB1. DB1-QUAD was also efficacious against wild-type RSV challenge in mice and cotton rats. Thus, substitution of unique line19F residues into RSV LAV DB1 enhanced vaccine thermostability, incorporation of prefusion F, and immunogenicity and generated a promising vaccine candidate that merits further investigation.IMPORTANCE We boosted the thermostability and immunogenicity of an RSV live-attenuated vaccine candidate by substituting 4 unique residues from the RSV line19F protein into the F protein of the heterologous vaccine strain DB1. The resultant vaccine candidate, DB1-QUAD, was thermostable, attenuated in vivo, highly immunogenic, and protective against RSV challenge in mice and cotton rats.

STAT1 Represses Cytokine-Producing Group 2 and Group 3 Innate Lymphoid Cells during Viral Infection.

The appropriate orchestration of different arms of the immune response is critical during viral infection to promote efficient viral clearance while limiting immunopathology. However, the signals and mechanisms that guide this coordination are not fully understood. IFNs are produced at high levels during viral infection and have convergent signaling through STAT1. We hypothesized that STAT1 signaling during viral infection regulates the balance of innate lymphoid cells (ILC), a diverse class of lymphocytes that are poised to respond to environmental insults including viral infections with the potential for both antiviral or immunopathologic functions. During infection with respiratory syncytial virus (RSV), STAT1-deficient mice had reduced numbers of antiviral IFN-γ+ ILC1 and increased numbers of immunopathologic IL-5+ and IL-13+ ILC2 and IL-17A+ ILC3 compared with RSV-infected wild-type mice. Using bone marrow chimeric mice, we found that both ILC-intrinsic and ILC-extrinsic factors were responsible for this ILC dysregulation during viral infection in STAT1-deficient mice. Regarding ILC-extrinsic mechanisms, we found that STAT1-deficient mice had significantly increased expression of IL-33 and IL-23, cytokines that promote ILC2 and ILC3, respectively, compared with wild-type mice during RSV infection. Moreover, disruption of IL-33 or IL-23 signaling attenuated cytokine-producing ILC2 and ILC3 responses in STAT1-deficient mice during RSV infection. Collectively, these data demonstrate that STAT1 is a key orchestrator of cytokine-producing ILC responses during viral infection via ILC-extrinsic regulation of IL-33 and IL-23.

Infant Viral Respiratory Infection Nasal Immune-Response Patterns and Their Association with Subsequent Childhood Recurrent Wheeze.

RATIONALE: Recurrent wheeze and asthma are thought to result from alterations in early life immune development following respiratory syncytial virus (RSV) infection. However, prior studies of the nasal immune response to infection have assessed only individual cytokines, which does not capture the whole spectrum of response to infection. OBJECTIVES: To identify nasal immune phenotypes in response to RSV infection and their association with recurrent wheeze. METHODS: A birth cohort of term healthy infants born June to December were recruited and followed to capture the first infant RSV infection. Nasal wash samples were collected during acute respiratory infection, viruses were identified by RT-PCR, and immune-response analytes were assayed using a multianalyte bead-based panel. Immune-response clusters were identified using machine learning, and association with recurrent wheeze at age 1 and 2 years was assessed using logistic regression. MEASUREMENTS AND MAIN RESULTS: We identified two novel and distinct immune-response clusters to RSV and human rhinovirus. In RSV-infected infants, a nasal immune-response cluster characterized by lower non-IFN antiviral immune-response mediators, and higher type-2 and type-17 cytokines was significantly associated with first and second year recurrent wheeze. In comparison, we did not observe this in infants with human rhinovirus acute respiratory infection. Based on network analysis, type-2 and type-17 cytokines were central to the immune response to RSV, whereas growth factors and chemokines were central to the immune response to human rhinovirus. CONCLUSIONS: Distinct immune-response clusters during infant RSV infection and their association with risk of recurrent wheeze provide insights into the risk factors for and mechanisms of asthma development.

Nasopharyngeal Lactobacillus is associated with a reduced risk of childhood wheezing illnesses following acute respiratory syncytial virus infection in infancy.

BACKGROUND: Early life acute respiratory infection (ARI) with respiratory syncytial virus (RSV) has been strongly associated with the development of childhood wheezing illnesses, but the pathways underlying this association are poorly understood. OBJECTIVE: To examine the role of the nasopharyngeal microbiome in the development of childhood wheezing illnesses following RSV ARI in infancy. METHODS: We conducted a nested cohort study of 118 previously healthy, term infants with confirmed RSV ARI by RT-PCR. We used next-generation sequencing of the V4 region of the 16S ribosomal RNA gene to characterize the nasopharyngeal microbiome during RSV ARI. Our main outcome of interest was 2-year subsequent wheeze. RESULTS: Of the 118 infants, 113 (95.8%) had 2-year outcome data. Of these, 46 (40.7%) had parental report of subsequent wheeze. There was no association between the overall taxonomic composition, diversity, and richness of the nasopharyngeal microbiome during RSV ARI with the development of subsequent wheeze. However, the nasopharyngeal detection and abundance of Lactobacillus was consistently higher in infants who did not develop this outcome. Lactobacillus also ranked first among the different genera in a model distinguishing infants with and without subsequent wheeze. CONCLUSIONS: The nasopharyngeal detection and increased abundance of Lactobacillus during RSV ARI in infancy are associated with a reduced risk of childhood wheezing illnesses at age 2 years.

Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses.

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N4-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from "mucogenic" strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Using urine metabolomics to understand the pathogenesis of infant respiratory syncytial virus (RSV) infection and its role in childhood wheezing.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants causes significant morbidity and is the strongest risk factor associated with asthma. Metabolites, which reflect the interactions between host cell and virus, provide an opportunity to identify the pathways that underlie severe infections and asthma development. OBJECTIVE: To study metabolic profile differences between infants with RSV infection, and human rhinovirus (HRV) infection, and healthy infants. To compare infant metabolic differences between children who do and do not wheeze. METHODS: In a term birth cohort, urine was collected while healthy and during acute viral respiratory infection with RSV and HRV. We used 1H-NMR to identify urinary metabolites. Multivariate and univariate statistics were used to discriminate metabolic profiles of infants with either RSV ARI, or HRV ARI, and healthy infants. Multivariable logistic regression was used to assess the association of urine metabolites with 1st-, 2nd-, and 3rd-year recurrent wheezing. RESULTS: Several metabolites in nicotinate and nicotinamide metabolism pathways were down-regulated in infants with RSV infection compared to healthy controls. There were no significant differences in metabolite profiles between infants with RSV infection and infants with HRV Infection. Alanine was strongly associated with reduced risk of 1st-year wheezing (OR 0.18[0.0, 0.46]) and 2nd-year wheezing (OR 0.31[0.13, 0.73]), while 2-hydroxyisobutyric acid was associated with increased 3rd-year wheezing (OR 5.02[1.49, 16.93]) only among the RSV infected subset. CONCLUSION: The metabolites associated with infant RSV infection and recurrent-wheezing are indicative of viral takeover of the cellular machinery and resources to enhance virulence, replication, and subversion of the host immune-response, highlighting metabolic pathways important in the pathogenesis of RSV infection and wheeze development.

Interference Between Respiratory Syncytial Virus and Human Rhinovirus Infection in Infancy.

BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are the most common viruses associated with acute respiratory tract infections in infancy. Viral interference is important in understanding respiratory viral circulation and the impact of vaccines. METHODS: To study viral interference, we evaluated cases of RSV and HRV codetection by polymerase chain reaction in 2 prospective birth cohort studies (the Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure [INSPIRE] study and the Tennessee Children's Respiratory Initiative [TCRI]) and a double-blinded, randomized, controlled trial (MAKI), using adjusted multivariable regression analyses. RESULTS: Among 3263 respiratory tract samples, 24.5% (798) and 37.3% (1216) were RSV and HRV positive, respectively. The odds of HRV infection were significantly lower in RSV-infected infants in all cohorts, with adjusted odds ratios of 0.30 (95% confidence interval [CI], .22-.40 in the INSPIRE study, 0.18 (95% CI, .11-.28) in the TCRI (adjusted for disease severity), and 0.34 (95% CI, .16-.72) in the MAKI trial. HRV infection was significantly more common among infants administered RSV immunoprophylaxis, compared with infants who did not receive immunoprophylaxis (OR, 1.65; 95% CI, 1.65-2.39). CONCLUSIONS: A negative association of RSV on HRV codetection was consistently observed across populations, seasons, disease severity, and geographical regions. Suppressing RSV infection by RSV immunoprophylaxis might increase the risk of having HRV infection.

Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein.

UNLABELLED: Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCE: RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

Priming of the Respiratory Tract with Immunobiotic Lactobacillus plantarum Limits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM).

UNLABELLED: Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces many clinical and pathological features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, bacterial artificial chromosome-based recombinant PVM strain J3666 that incorporates the fluorescent tag monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. Using recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c(+) major histocompatibility complex class II(+)) and alveolar macrophages (AMs; CD11c(+) sialic acid-binding immunoglobulin-like lectin F(+)) in vivo and likewise detect mKATE2(+) DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum, a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM and a reduction from ∼ 40% to <10% mKATE2(+) AMs in association with a 2-log drop in the release of infectious virions. In contrast, AMs from L. plantarum-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response. IMPORTANCE: Pneumonia virus of mice (PVM) is a natural mouse pathogen that serves as a model for severe human respiratory syncytial virus disease. We have developed a fully functional recombinant PVM strain with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably, dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum.

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats.

UNLABELLED: Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCE: RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo.

Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-ß as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5bfl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5bfl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5bfl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Cross-resistance mechanism of respiratory syncytial virus against structurally diverse entry inhibitors.

Respiratory syncytial virus (RSV) is a leading pediatric pathogen that is responsible for a majority of infant hospitalizations due to viral disease. Despite its clinical importance, no vaccine prophylaxis against RSV disease or effective antiviral therapeutic is available. In this study, we established a robust high-throughput drug screening protocol by using a recombinant RSV reporter virus to expand the pool of RSV inhibitor candidates. Mechanistic characterization revealed that a potent newly identified inhibitor class blocks viral entry through specific targeting of the RSV fusion (F) protein. Resistance against this class was induced and revealed overlapping hotspots with diverse, previously identified RSV entry blockers at different stages of preclinical and clinical development. A structural and biochemical assessment of the mechanism of unique, broad RSV cross-resistance against structurally distinct entry inhibitors demonstrated that individual escape hotspots are located in immediate physical proximity in the metastable conformation of RSV F and that the resistance mutations lower the barrier for prefusion F triggering, resulting in an accelerated RSV entry kinetics. One resistant RSV recombinant remained fully pathogenic in a mouse model of RSV infection. By identifying molecular determinants governing the RSV entry machinery, this study spotlights a molecular mechanism of broad RSV resistance against entry inhibition that may affect the impact of diverse viral entry inhibitors presently considered for clinical use and outlines a proactive design for future RSV drug discovery campaigns.

Respiratory syncytial virus infection activates IL-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin.

BACKGROUND: Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response. OBJECTIVE: We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. METHODS: Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. RESULTS: RSV induced a 3-fold increase in the number of IL-13-producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13-producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13-producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13-producing ILC2s through a TSLP-dependent mechanism. CONCLUSION: These data demonstrate that multiple pathogenic strains of RSV induce IL-13-producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection.

Differences in the Nasopharyngeal Microbiome During Acute Respiratory Tract Infection With Human Rhinovirus and Respiratory Syncytial Virus in Infancy.

Respiratory viruses alter the nasopharyngeal microbiome and may be associated with a distinct microbial signature. To test this hypothesis, we compared the nasopharyngeal microbiome of 135 previously healthy infants with acute respiratory infection due to human rhinovirus (HRV; n = 52) or respiratory syncytial virus (RSV; n = 83). The nasopharyngeal microbiome was assessed by sequencing the V4 region of the 16S ribosomal RNA. Respiratory viruses were identified by quantitative reverse-transcription polymerase chain reaction. We found significant differences in the overall taxonomic composition and abundance of certain bacterial genera between infants infected with HRV and those infected with RSV. Our results suggest that respiratory tract viral infections are associated with different nasopharyngeal microbial profiles.