Potently Neutralizing and Protective Anti-Human Metapneumovirus Antibodies Target Diverse Sites on the Fusion Glycoprotein

Garrett Rappazzo, Ching-Lin Hsieh, Scott A. Rush, Emma S. Esterman, Teresa Delgado, James C. Geoghegan, Anna Z. Wec, Mrunal Sakharkar, Vicente Más, Jason S. McLellan, Laura M. Walker

Immunity 55, 1710–1724.e8. DOI: 10.1016/j.immuni.2022.07.003 

Overview  

Human metapneumovirus (hMPV) is a leading cause of acute lower respiratory infections, responsible for significant hospitalizations in high-risk populations such as young children, the elderly, and immunocompromised individuals. Despite having a clinical burden comparable to respiratory syncytial virus (RSV) and influenza in adults, there are no approved vaccines or antiviral therapies for hMPV to date. hMPV encodes three surface glycoproteins: the attachment (G), small hydrophobic (SH), and the fusion (F) glycoproteins. The hMPV F glycoprotein is required for infection and is the only known target of neutralizing antibodies. Mature hMPV F is a trimer of disulfide-linked heterodimers that exists in two conformations: a metastable pre-fusion (preF) form and a stable post-fusion (postF) form. The preF form is targeted by neutralizing antibodies that inhibit its transition to the postF form, thereby preventing viral fusion with host cells. Understanding which epitopes on the F glycoprotein elicit the most protective antibody responses is critical for the rational design of vaccines and therapeutic antibodies. This study comprehensively interrogated the memory B cell responses to hMPV fusion (F) glycoprotein in young adult and elderly donors using a high-throughput single B-cell sorting technology to identify and characterize potent neutralizing antibody candidates and inform vaccine immunogen design. 

Key hypotheses and objectives 

By analogous comparison with RSV, where the most potently neutralizing antibodies target preF-specific sites at the trimer apex, potently neutralizing preF-specific antibodies may exist within the human memory B cell (MBC) response to hMPV. Such antibodies would be expected to be rare given the glycan shielding of analogous apical sites on the hMPV preF glycoprotein, but their existence and structural basis had not previously been defined.  

The objectives of this study were to: 

• Comprehensively profile the human MBC antibody repertoire for the hMPV F glycoprotein across young adult and elderly donor cohorts. 

• Identify and structurally characterize broadly neutralizing antibodies, particularly for those targeting preF-specific epitopes (as binding to preF is required for neutralization). 

• Evaluate the prophylactic efficacy of potently neutralizing antibodies in a rodent model of hMPV infection. 

Approach and techniques 

Serum and peripheral blood samples were collected from 16 hMPV convalescent donors (eight young and eight elderly adults). Serum IgG binding and neutralizing activity against hMPV F glycoprotein were confirmed in all donors prior to B cell isolation. MBCs reactive with preF were isolated from the peripheral blood of six donors by flow cytometry and individually sorted for antibody cloning. A total of 435 antibodies were cloned and expressed as full-length IgG1 proteins, of which 375 were confirmed to bind hMPV preF glycoprotein and carried forward for characterization of binding affinity, conformational selectivity (preF versus postF), cross-subtype neutralization breadth, and cross-reactivity with the RSV F glycoprotein. A subset of antibodies with sufficient binding affinity were assigned to epitope competition groups corresponding to seven distinct sites on hMPV F using a panel of well-characterized competitor Fab fragments. The binding site of a rare, potentially neutralizing preF-specific antibody was structurally defined by cryo-electron microscopy. Three potently neutralizing antibodies targeting non-overlapping epitopes were evaluated for prophylactic efficacy in a cotton rat model of hMPV infection. 

Major findings and impact 

• Pre/PostF cross-reactive antibodies dominate the neutralizing immune response: Across all donors and hMPV subtypes, the majority of neutralizing antibodies recognized epitopes present on both preF and postF conformations primarily targeting antigenic sites analogous to sites II, III, and IV on RSV F glycoprotein. PreF-specific antibodies were abundant but largely non-neutralizing, likely due to recognition of cryptic epitopes within the trimer interior that are poorly accessible in the context of the native F trimer. 

• Potently neutralizing preF-specific antibodies are rare: A small subset of preF-specific antibodies displayed potent and broad cross-subtype neutralization. Structural characterization of one of these, ADI-61026, revealed that it recognizes a rarely targeted site of vulnerability at the apex of the preF trimer (antigenic site Ø). The epitope is situated directly between two N-linked glycans, and the key interaction involves the insertion of a large hydrophobic sidechain from the heavy chain CDR3 into a deep pocket formed by both F subunits.  

• MBC responses are similar across age groups: The sequence features, binding affinities, epitope specificities, and neutralizing potencies of hMPV F-specific antibodies were highly similar between young adult and elderly donors, suggesting that immunosenescence may not substantially alter the hMPV F-specific MBC repertoire. 

• Monotherapy provides robust protection: Three potently neutralizing antibodies targeting non-overlapping epitopes each provided robust protection against lower respiratory tract infection in cotton rats when administered as monotherapies. Two of the three antibodies also markedly reduced upper respiratory tract viral titers, while the third did not. 

This study provides a comprehensive characterization of the human MBC antibody response to hMPV F, revealing that neutralization is highly epitope-dependent and dominated by antibodies targeting postF-accessible sites. The structural definition of a rare but potently neutralizing antibody at the preF trimer apex illuminates a previously uncharacterized site of vulnerability and provides a molecular template for the rational design of hMPV vaccines and preF-based immunogens that preferentially induce neutralizing antibody responses. 

Implications for therapeutic antibody development 

This study identified three potently neutralizing antibodies that are promising candidates for passive immunoprophylaxis against hMPV, given the clinical successes of analogous mAbs palivizumab and nirsevimab in the prevention of severe RSV disease. Their recognition of non-overlapping epitopes raises the possibility of combining them to mitigate the emergence of resistant variants, as previously described in the context of other viral infections. The study notes that increased dosages of potently neutralizing hMPV antibodies may provide increased protection against upper respiratory tract infection since neutralizing antibody titers are the primary correlate of protection from nasal viral replication in the context of the related respiratory virus RSV.