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

ABSTRACT 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.

Download Full-text

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609449113 ◽

2016 ◽

Vol 113 (44) ◽

pp. E6849-E6858 ◽

Cited By ~ 25

Author(s):

Jarrod J. Mousa ◽

Marion F. Sauer ◽

Alexander M. Sevy ◽

Jessica A. Finn ◽

John T. Bates ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Severe Disease ◽

Antigenic Site ◽

Structural Basis ◽

Human Antibody ◽

Human Mabs ◽

F Protein ◽

Syncytial Virus

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix–loop–helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

Download Full-text

Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus

Journal of General Virology ◽

10.1099/vir.0.80219-0 ◽

2004 ◽

Vol 85 (11) ◽

pp. 3229-3238 ◽

Cited By ~ 28

Author(s):

Carolina Johnstone ◽

Patricia de León ◽

Francisco Medina ◽

José A. Melero ◽

Blanca García-Barreno ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Vaccinia Virus ◽

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Recombinant Vaccinia Virus ◽

Recombinant Vaccinia ◽

F Protein ◽

Syncytial Virus ◽

Lymphocyte Responses

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85–93 and F92–106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85–93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249–258, which is presented by Kd as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous Kd ligand. The CD8+ T-lymphocyte responses to epitopes F85–93 and F249–258 present in the F protein of RSV Long were found to be strongly skewed to F85–93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8+ T-lymphocyte responses to F85–93 and F249–258 epitopes was observed in vivo during a primary response.

Download Full-text

Differential Mucin Expression by Respiratory Syncytial Virus and Human Metapneumovirus Infection in Human Epithelial Cells

Mediators of Inflammation ◽

10.1155/2015/347292 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Ma. Del Rocío Baños-Lara ◽

Boyang Piao ◽

Antonieta Guerrero-Plata

Keyword(s):

Immune Response ◽

Respiratory Syncytial Virus ◽

Epithelial Cells ◽

Viral Infections ◽

Human Metapneumovirus ◽

Human Epithelial Cells ◽

Mucin Expression ◽

Rsv Infection ◽

Syncytial Virus ◽

Tract Disease

Mucins (MUC) constitute an important component of the inflammatory and innate immune response. However, the expression of these molecules by respiratory viral infections is still largely unknown. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two close-related paramyxoviruses that can cause severe low respiratory tract disease in infants and young children worldwide. Currently, there is not vaccine available for neither virus. In this work, we explored the differential expression of MUC by RSV and hMPV in human epithelial cells. Our data indicate that the MUC expression by RSV and hMPV differs significantly, as we observed a stronger induction of MUC8, MUC15, MUC20, MUC21, and MUC22 by RSV infection while the expression of MUC1, MUC2, and MUC5B was dominated by the infection with hMPV. These results may contribute to the different immune response induced by these two respiratory viruses.

Download Full-text

RESPIRATORY SYNCYTIAL VIRUS LUNG INFECTION IN INFANTS: IMMUNOREGULATORY ROLE OF INFECTED ALVEOLAR MACROPHAGES

PEDIATRICS ◽

10.1542/peds.96.2.391 ◽

1995 ◽

Vol 96 (2) ◽

pp. 391-391

Author(s):

Leon S. Greos

Keyword(s):

Immune Response ◽

Respiratory Syncytial Virus ◽

Lung Injury ◽

Alveolar Macrophages ◽

Lung Infection ◽

Local Immune Response ◽

Rsv Infection ◽

Syncytial Virus

Alveolar macrophages are infected by RSV in vivo and coexpress potent immunomodulatory molecules that potentially regulate local immune response or lung injury caused by RSV infection.

Download Full-text

Human metapneumovirus induces more severe disease and stronger innate immune response in BALB/c mice as compared with respiratory syncytial virus

Respiratory Research ◽

10.1186/1465-9921-8-6 ◽

2007 ◽

Vol 8 (1) ◽

Cited By ~ 29

Author(s):

Barbara Huck ◽

Dieter Neumann-Haefelin ◽

Annette Schmitt-Graeff ◽

Markus Weckmann ◽

Jörg Mattes ◽

...

Keyword(s):

Immune Response ◽

Respiratory Syncytial Virus ◽

Innate Immune Response ◽

Severe Disease ◽

Human Metapneumovirus ◽

Innate Immune ◽

Syncytial Virus

Download Full-text

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches

Journal of General Virology ◽

10.1099/vir.0.82753-0 ◽

2007 ◽

Vol 88 (10) ◽

pp. 2719-2723 ◽

Cited By ~ 33

Author(s):

Sheng-Jiun Wu ◽

Albert Schmidt ◽

Eric J. Beil ◽

Nicole D. Day ◽

Patrick J. Branigan ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Antigenic Site ◽

Neutralizing Antibody ◽

Human Respiratory Syncytial Virus ◽

Peptide Sequence ◽

F Protein ◽

Syncytial Virus ◽

A Minor ◽

Key Residues

Chimeric 101F (ch101F) is a mouse–human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422–438 spanning antigenic site IV, V, VI was analysed. Residues 423–436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

Download Full-text

Global Molecular Epidemiology of Respiratory Syncytial Virus from the 2017−2018 INFORM-RSV Study

Journal of Clinical Microbiology ◽

10.1128/jcm.01828-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e01828-20

Author(s):

David E. Tabor ◽

Fiona Fernandes ◽

Annefleur C. Langedijk ◽

Deidre Wilkins ◽

Robert Jan Lebbink ◽

...

Keyword(s):

South Africa ◽

Respiratory Syncytial Virus ◽

Molecular Epidemiology ◽

Antigenic Site ◽

Warning System ◽

F Protein ◽

Viral Spread ◽

Antigenic Sites ◽

Geographic Clustering ◽

Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017–2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of <1 year of age (81.2%), males (56.3%), and patients hospitalized for >24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017–2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.

Download Full-text

Role of human metapneumovirus and respiratory syncytial virus in asthma exacerbations: where are we now?

Clinical Science ◽

10.1042/cs20160011 ◽

2017 ◽

Vol 131 (14) ◽

pp. 1713-1721 ◽

Cited By ~ 5

Author(s):

Penny A. Rudd ◽

Belinda J. Thomas ◽

Ali Zaid ◽

Martin MacDonald ◽

Keiko Kan-o ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Animal Models ◽

Immune Responses ◽

Human Metapneumovirus ◽

Innate Immune Responses ◽

Adaptive Responses ◽

Hmpv Infection ◽

Acute Exacerbations ◽

Syncytial Virus

Since its discovery in 2001, human metapneumovirus (hMPV) has been identified as an important cause of respiratory tract infection in young children, second only to the closely related respiratory syncytial virus (RSV). Clinical evidence suggests that hMPV is associated with acute exacerbations of asthma in both children and adults, and may play a role in initiating asthma development in children. Animal models have demonstrated that airway hyperresponsiveness (AHR) and inflammation are triggered following hMPV infection, and hMPV is able to persist in vivo by inhibiting innate immune responses and causing aberrant adaptive responses. In this review, we discuss the prevalence of hMPV infection in pediatric and adult populations and its potential role in asthma exacerbation. We also review recent advances made in animal models to determine immune responses following hMPV infection, and compare to what is known about RSV.

Download Full-text