IFN-λ drives distinct lung immune landscape changes and antiviral responses in human metapneumovirus infection.

Human metapneumovirus (HMPV) is a primary cause of acute respiratory infection, yet there are no approved vaccines or antiviral therapies for HMPV. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways. Type III interferon (IFN-λ) displays potent antiviral activity against respiratory viruses and is being investigated for therapeutic use. However, its role in HMPV infection remains largely unknown. Here, we show that IFN-λ is highly upregulated during HMPV infection in vitro in human and mouse airway epithelial cells and in vivo in mice. We found through several immunological and molecular assays that type II alveolar cells are the primary producers of IFN-λ. Using mouse models, we show that IFN-λ limits lung HMPV replication and restricts virus spread from upper to lower airways but does not contribute to clinical disease. Moreover, we show that IFN-λ signaling is predominantly mediated by CD45- non-immune cells. Mice lacking IFN-λ signaling showed diminished loss of ciliated epithelial cells and decreased recruitment of lung macrophages in early HMPV infection along with higher inflammatory cytokine and interferon-stimulated gene expression, suggesting that IFN-λ may maintain immunomodulatory responses. Administration of IFN-λ for prophylaxis or post-infection treatment in mice reduced viral load without inflammation-driven weight loss or clinical disease. These data offer clinical promise for IFN-λ in HMPV treatment. IMPORTANCE: Human metapneumovirus (HMPV) is a common respiratory pathogen and often contributes to severe disease, particularly in children, immunocompromised people, and the elderly. There are currently no licensed HMPV antiviral treatments or vaccines. Here, we report novel roles of host factor IFN-λ in HMPV disease that highlight therapeutic potential. We show that IFN-λ promotes lung antiviral responses by restricting lung HMPV replication and spread from upper to lower airways but does so without inducing lung immunopathology. Our data uncover recruitment of lung macrophages, regulation of ciliated epithelial cells, and modulation of inflammatory cytokines and interferon-stimulated genes as likely contributors. Moreover, we found these roles to be distinct and non-redundant, as they are not observed with knockout of, or treatment with, type I IFN. These data elucidate unique antiviral functions of IFN-λ and suggest IFN-λ augmentation as a promising therapeutic for treating HMPV disease and promoting effective vaccine responses.

Structural basis for ultrapotent antibody-mediated neutralization of human metapneumovirus.

Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.

Inhibition of PI3Kδ Differentially Regulates Poly I:C- and Human Metapneumovirus-Induced PD-L1 and PD-L2 Expression in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are front sentinels eliciting innate and adaptive immunity to respiratory viral pathogens. Recognition of viral double-stranded RNA induces antiviral interferon (IFN) responses in bronchial epithelial cells. Co-inhibitory molecules programmed cell death 1 ligand 1 (PD-L1) and ligand 2 (PD-L2) were also induced on bronchial epithelial cells, which bind programmed cell death 1 on T cell and inhibit the function of virus-specific cytotoxic T lymphocyte. A previous study showed that antiviral type I IFN increased PD-L1 and PD-L2 expression in cultured melanoma cells. However, it remains unknown whether antiviral IFNs affect PD-L1 and PD-L2 expression in bronchial epithelial cells. In addition, we previously reported that inhibition of PI3Kδ signaling enhanced antiviral IFN responses in human primary bronchial epithelial cells (PBECs). Here we assessed the effect of exogenous IFNs or a selective PI3Kδ inhibitor IC87114 on PD-L1 and PD-L2 in PBECs stimulated with a synthetic double-stranded RNA poly I:C or human metapneumovirus. Treatment with IFNß or IFNλ increased PD-L1 and PD-L2, and IFNß or IFNλ treatment plus poly I:C further increased both expressions. Treatment with IC87114 or transfection with siRNA targeting PI3K p110δ enhanced poly I:C-induced gene and protein expression of PD-L2, whereas IC87114 suppressed poly I:C-induced PD-L1. IC87114 enhanced poly I:C-induced gene expression of IFNß, IFNλ, and IFN-regulated genes via increased TBK1 and IRF3 phosphorylation. Transfection with siIRF3 counteracted the enhancement of poly I:C-induced PD-L2 by IC87114, whereas IC87114 suppressed poly I:C-induced PD-L1 regardless of transfection with siNC or siIRF3. Similar effects of IC87114 on PD-L1 and PD-L2 expression were observed in human metapneumovirus-infected PBECs. We showed for the first time that type I and type III IFNs induced the expression of PD-L1 and PD-L2 in PBECs. Our findings suggest that during viral infections, inhibition of PI3Kδ differentially regulates PD-L1 and PD-L2 expression in bronchial epithelial cells.

Novel HLA-B7-restricted human metapneumovirus epitopes enhance viral clearance in mice and are recognized by human CD8+ T cells.

Human metapneumovirus (HMPV) is a leading cause of acute lower respiratory tract illness in children and adults. Repeated infections are common and can be severe in young, elderly, and immunocompromised persons due to short-lived protective humoral immunity. In turn, few protective T cell epitopes have been identified in humans. Thus, we infected transgenic mice expressing the common human HLA MHC-I allele B*07:02 (HLA-B7) with HMPV and screened a robust library of overlapping and computationally predicted HLA-B7 binding peptides. Six HLA-B7-restricted CD8+ T cell epitopes were identified using ELISPOT screening in the F, M, and N proteins, with M195-203 (M195) eliciting the strongest responses. MHC-tetramer flow cytometric staining confirmed HLA-B7 epitope-specific CD8+ T cells migrated to lungs and spleen of HMPV-immune mice. Immunization with pooled HLA-B7-restricted peptides reduced viral titer and protected mice from virulent infection. Finally, we confirmed that CD8+ T cells from HLA-B7 positive humans also recognize the identified epitopes. These results enable identification of HMPV-specific CD8+ T cells in humans and help to inform future HMPV vaccine design.

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.

Human Metapneumovirus Induces IRF1 via TANK-Binding Kinase 1 and Type I IFN.

The innate immune and host-protective responses to viruses, such as the airway pathogen human metapneumovirus (HMPV), depend on interferons (IFNs) that is induced through TANK-binding kinase 1 (TBK1) and IFN regulatory factors (IRFs). The transcription factor IRF1 is important for host resistance against several viruses and has a key role in induction of IFN-λ at mucosal surfaces. In most cell types IRF1 is expressed at very low levels, but its mRNA is rapidly induced when the demand for IRF1 activity arises. Despite general recognition of the importance of IRF1 to antiviral responses, the molecular mechanisms by which IRF1 is regulated during viral infections are not well understood. Here we identify the serine/threonine kinase TBK1 and IFN-ß as critical regulators of IRF1 mRNA and protein levels in human monocyte-derived macrophages. We find that inhibition of TBK1 activity either by the semi-selective TBK1/IKKε inhibitor BX795 or by siRNA-mediated knockdown abrogates HMPV-induced expression of IRF1. Moreover, we show that canonical NF-κB signaling is involved in IRF1 induction and that the TBK1/IKKε inhibitor BX795, but not siTBK1 treatment, impairs HMPV-induced phosphorylation of the NF-κB subunit p65. At later time-points of the infection, IRF1 expression depended heavily on IFN-ß-mediated signaling via the IFNAR-STAT1 pathway. Hence, our results suggest that TBK1 activation and TBK1/IKKε-mediated phosphorylation of the NF-κB subunit p65 control transcription of IRF1. Our study identifies a novel mechanism for IRF1 induction in response to viral infection of human macrophages that could be relevant not only to defense against HMPV, but also to other viral, bacterial and fungal pathogens.

Structure, Immunogenicity, and Conformation-Dependent Receptor Binding of the Postfusion Human Metapneumovirus F Protein.

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.

Host Components That Modulate the Disease Caused by hMPV.

Human metapneumovirus (hMPV) is one of the main pathogens responsible for acute respiratory infections in children up to 5 years of age, contributing substantially to health burden. The worldwide economic and social impact of this virus is significant and must be addressed. The structural components of hMPV (either proteins or genetic material) can be detected by several receptors expressed by host cells through the engagement of pattern recognition receptors. The recognition of the structural components of hMPV can promote the signaling of the immune response to clear the infection, leading to the activation of several pathways, such as those related to the interferon response. Even so, several intrinsic factors are capable of modulating the immune response or directly inhibiting the replication of hMPV. This article will discuss the current knowledge regarding the innate and adaptive immune response during hMPV infections. Accordingly, the host intrinsic components capable of modulating the immune response and the elements capable of restricting viral replication during hMPV infections will be examined.

Efficacy of a novel avian metapneumovirus live vaccine candidate based on vaccination route and age.

This article describes a series of animal studies for the development of an avian metapneumovirus (aMPV) live vaccine. Although aMPV causes continual economic loss in the poultry industry, there are no live aMPV vaccines available in Korea. Furthermore, information is limited with respect to standard field practices for vaccinations at an early age. Here, the development of an aMPV live vaccine was attempted, and its efficacy was investigated with respect to the vaccination route and age to develop a method for controlling aMPV. Before vaccine development, an animal challenge model was established using the aMPV field isolate to identify the most effective time and site for collecting samples for evaluation. After attenuation of the virulent aMPV in Vero cells, a safety and efficacy test was conducted for the vaccine candidate. As a novel aMPV live vaccine candidate, aMPV K655/07HP displayed sufficient safety in day-old chicks with 10 vaccine doses. The efficacy test using 1-week-old chicks showed weaker humoral immune response than that in 4-week-old chicks. However, the candidate vaccine provided complete protection against infection caused by the challenge virus for all ages of vaccinated chicks. In conclusion, an effective aMPV challenge model was established for studying aMPV in chickens, which offered important, insightful information. The safety and efficacy study suggested that the new aMPV candidate vaccine could be used to effectively reduce the economic losses incurred because of aMPV infection.

Development of A recombinant nucleocapsid based indirect ELISA for the detection of antibodies to avian metapneumovirus subtypes, A, B, and C.

Nucleocapsid (N) protein is the most highly expressed of all avian metapneumovirus (aMPV) viral proteins and stimulates a substantial immune response in infected animals. Codon optimized recombinant N (rec-N) protein from aMPV subtypes A, B, and C were expressed using the baculoviral expression system in Trichoplusia ni (Tni) insect cells. A mixture of purified rec-N antigens from each subtype was used as a coating antigen and was evaluated in indirect ELISA (iELISA) to assess antibody response in serum samples collected from experimentally infected chickens and turkeys with different aMPV subtypes. Also, archived field serum samples that were collected from different poultry submissions were used. Receiver operating characteristic (ROC) analysis was performed using chicken and turkey serum samples that were confirmed by indirect fluorescent antibody (IFA) test for serostatus (positive n = 270, negative n = 610). The ROC analysis showed sensitivity and specificity of 97 % at a cut-off value of 0.25. The rec-N iELISA was compared with a commercial whole virus-based APV kit. The rec-N iELISA showed comparable results in detecting antibody response in aMPV infected chicken sera but was more sensitive in detecting early antibody response in aMPV infected turkey serum samples. Our results further confirm the presence of aMPV antibodies in Canadian domestic poultry populations. The developed aMPV-rec N iELISA offers a safe and valuable alternative to whole virus-based iELISA for serodiagnosis and seroepidemiological surveillance of the disease in domestic poultry.

Antibody recognition of the Pneumovirus fusion protein trimer interface.

Human metapneumovirus (hMPV) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. However, there remain no licensed vaccines or specific treatments for hMPV infection. Although the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological properties of hMPV F remain poorly understood. To further define the humoral immune response to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and MPV465. Both mAbs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. We determined both MPV458 and MPV465 have higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV F protein, suggesting significant breathing of the hMPV F protein must occur for host immune recognition of the novel epitope. In addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. The data presented identifies a novel epitope on the hMPV F protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins.

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

Cell-Mediated Responses to Human Metapneumovirus Infection.

Viruses are the most common cause of acute respiratory tract infections (ARTI). Human metapneumovirus (hMPV) frequently causes viral pneumonia which can become life-threatening if the virus spreads to the lungs. Even though hMPV was only isolated in 2001, this negative-stranded RNA virus has probably been circulating in the human population for many decades. Interestingly, almost all adults have serologic evidence of hMPV infection. A well-established host immune response is evoked when hMPV infection occurs. However, the virus has evolved to circumvent and even exploit the host immune response. Further, infection with hMPV induces a weak memory response, and re-infections during life are common. In this review, we provide a comprehensive overview of the different cell types involved in the immune response in order to better understand the immunopathology induced by hMPV. Such knowledge may contribute to the development of vaccines and therapeutics directed against hMPV.

N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I.

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.

Evaluation of pre- and post-fusion Human metapneumovirus F proteins as subunit vaccine candidates in mice.

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.

Induction of Trained Immunity by Recombinant Vaccines.

Vaccines represent an important strategy to protect humans against a wide variety of pathogens and have even led to eradicating some diseases. Although every vaccine is developed to induce specific protection for a particular pathogen, some vaccine formulations can also promote trained immunity, which is a non-specific memory-like feature developed by the innate immune system. It is thought that trained immunity can protect against a wide variety of pathogens other than those contained in the vaccine formulation. The non-specific memory of the trained immunity-based vaccines (TIbV) seems beneficial for the immunized individual, as it may represent a powerful strategy that contributes to the control of pathogen outbreaks, reducing morbidity and mortality. A wide variety of respiratory viruses, including respiratory syncytial virus (hRSV) and metapneumovirus (hMPV), cause serious illness in children under 5 years old and the elderly. To address this public health problem, we have developed recombinant BCG vaccines that have shown to be safe and immunogenic against hRSV or hMPV. Besides the induction of specific adaptive immunity against the viral antigens, these vaccines could generate trained immunity against other respiratory pathogens. Here, we discuss some of the features of trained immunity induced by BCG and put forward the notion that recombinant BCGs expressing hRSV or hMPV antigens have the capacity to simultaneously induce specific adaptive immunity and non-specific trained immunity. These recombinant BCG vaccines could be considered as TIbV capable of inducing simultaneously the development of specific protection against hRSV or hMPV, as well as non-specific trained-immunity-based protection against other pathogenic viruses.

Cell-Type-Specific Transcription of Innate Immune Regulators in response to HMPV Infection.

Human metapneumovirus (HMPV) may cause severe respiratory disease. The early innate immune response to viruses like HMPV is characterized by induction of antiviral interferons (IFNs) and proinflammatory immune mediators that are essential in shaping adaptive immune responses. Although innate immune responses to HMPV have been comprehensively studied in mice and murine immune cells, there is less information on these responses in human cells, comparing different cell types infected with the same HMPV strain. The aim of this study was to characterize the HMPV-induced mRNA expression of critical innate immune mediators in human primary cells relevant for airway disease. In particular, we determined type I versus type III IFN expression in human epithelial cells and monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). In epithelial cells, HMPV induced only low levels of IFN-ß mRNA, while a robust mRNA expression of IFN-λs was found in epithelial cells, MDMs, and MDDCs. In addition, we determined induction of the interferon regulatory factors (IRFs) IRF1, IRF3, and IRF7 and critical inflammatory cytokines (IL-6, IP-10, and IL-1ß). Interestingly, IRF1 mRNA was predominantly induced in MDMs and MDDCs. Overall, our results suggest that for HMPV infection of MDDCs, MDMs, NECs, and A549 cells (the cell types examined), cell type is a strong determinator of the ability of HMPV to induce different innate immune mediators. HMPV induces the transcription of IFN-ß and IRF1 to higher extents in MDMs and MDDCs than in A549s and NECs, whereas the induction of type III IFN-λ and IRF7 is considerable in MDMs, MDDCs, and A549 epithelial cells.

Characterization of cellular transcriptomic signatures induced by different respiratory viruses in human reconstituted airway epithelia.

Acute respiratory infections, a large part being of viral origin, constitute a major public health issue. To propose alternative and/or new therapeutic approaches, it is necessary to increase our knowledge about the interactions between respiratory viruses and their primary cellular targets using the most biologically relevant experimental models. In this study, we used RNAseq to characterize and compare the transcriptomic signature of infection induced by different major respiratory viruses (Influenza viruses, hRSV and hMPV) in a model of reconstituted human airway epithelia. Our results confirm the importance of several cellular pathways commonly or specifically induced by these respiratory viruses, such as the innate immune response or antiviral defense. A very interesting common feature revealed by the global virogenomic signature shared between hRSV, hMPV and influenza viruses is the global downregulation of cilium-related gene expression, in good agreement with experimental evaluation of mucociliary clearance. Beyond providing new information about respiratory virus/host interactions, our study also underlines the interest of using biologically relevant experimental models to study human respiratory viruses.

A Potent Neutralizing Site III-Specific Human Antibody Neutralizes Human Metapneumovirus In Vivo.

Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV; however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs, termed MPV196, MPV201, MPV314, and MPV364, are described. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro Binding studies with monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity for monomeric hMPV F compared to the other three mAbs, yet binding experiments with trimeric hMPV F showed limited differences in binding affinity, suggesting that MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, both of which cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests that it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope yet is unique from previously discovered site III mAbs, as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo and found that it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development and provide insights into hMPV vaccine development.

Human metapneumovirus activates NOD-like receptor protein 3 inflammasome via its small hydrophobic protein which plays a detrimental role during infection in mice.

NOD-like receptor protein 3 (NLRP3) inflammasome activation triggers caspase-1 activation-induced maturation of interleukin (IL)-1ß and IL-18 and therefore is important for the development of the host defense against various RNA viral diseases. However, the implication of this protein complex in human metapneumovirus (HMPV) disease has not been fully studied. Herein, we report that NLRP3 inflammasome plays a detrimental role during HMPV infection because NLRP3 inflammasome inhibition protected mice from mortality and reduced weight loss and inflammation without impacting viral replication. We also demonstrate that NLRP3 inflammasome exerts its deleterious effect via IL-1ß production since we observed reduced mortality, weight loss and inflammation in IL-1ß-deficient (IL-1ß-/-) mice, as compared to wild-type animals during HMPV infection. Moreover, the effect on these evaluated parameters was not different in IL-1ß-/- and wild-type mice treated with an NLRP3 inflammasome inhibitor. The production of IL-1ß was also abrogated in bone marrow derived macrophages deficient for NLRP3. Finally, we show that small hydrophobic protein-deleted recombinant HMPV (HMPV ΔSH) failed to activate caspase-1, which is responsible for IL-1ß cleavage and maturation. Furthermore, HMPV ΔSH-infected mice had less weight loss, showed no mortality and reduced inflammation, as compared to wild-type HMPV-infected mice. Thus, NLRP3 inflammasome activation seems to be triggered by HMPV SH protein in HMPV disease. In summary, once activated by the HMPV SH protein, NLRP3 inflammasome promotes the maturation of IL-1ß, which exacerbates HMPV-induced inflammation. Therefore, the blockade of IL-1ß production by using NLRP3 inflammasome inhibitors might be a novel potential strategy for the therapy and prevention of HMPV infection.