Development of a potent monoclonal antibody for treatment of human metapneumovirus infections

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: major_revision

Abstract
Human metapneumovirus (HMPV) is a major cause of respiratory infections, particularly among vulnerable populations, yet effective therapeutics remain unavailable. Monoclonal antibodies (mAbs) offer a promising approach for treatment and prevention. We describe the discovery and characterization of 4F11, a highly potent neutralizing mAb with in vitro and in vivo efficacy against HMPV. Using cryo-electron microscopy, we define a unique mechanism of binding employed by 4F11. 4F11 targets an epitope located at the apex of the prefusion F protein (site Ø) with a 1:1 stoichiometry of Fab to trimer, distinct from the 3:1 stoichiometry observed with other HMPV site Ø antibodies. Unlike other site Ø antibodies which penetrate the glycan shield between Asn57 and Asn172, 4F11 binds vertically and directly interacts with the Asn172 glycan. In vitro, 4F11 displays high neutralization potency across diverse HMPV strains. It also shows low propensity for resistance development, with only a single escape mutation (K179E) identified, a mutation not found in any published HMPV sequence to date. Viruses rescued with the K179E escape mutation have significantly decreased fitness in vitro. In male hamsters, 4F11 significantly reduces viral loads in the lungs and nasal turbinates. These findings highlight 4F11 as a promising candidate for therapeutic development.

Introduction
Human metapneumovirus (HMPV) is a ubiquitous respiratory virus. The global burden of infection is large, with an estimated 14.2 million acute lower respiratory infections annually associated with HMPV in children under 5 years of age1. Disease burden is also high for adults aged 60 years or older2,3. While several vaccines for older adults to prevent infection caused by respiratory syncytial virus (RSV), a virus related to HMPV, were approved by the Food and Drug Administration in 20234–7, protective vaccines for HMPV are not yet clinically available. Even if protective vaccines existed for HMPV, vaccination of highly immunocompromised individuals rarely achieves protective immunity. Additionally, vaccination prior to immune-ablative therapies is often ineffective or wanes quickly, failing to maintain durable protection8–10. HMPV represents a serious threat to immunocompromised patients, particularly hematopoietic stem cell transplant recipients who have mortality rates as high as 43%11–13. In adults with other risk factors, the burden of disease from HMPV is comparable to RSV14,15. The administration of neutralizing monoclonal antibodies (mAbs) provides an effective alternative to vaccination for prophylaxis and is a relatively safe therapeutic modality. Despite advances in mAb development for the prevention of RSV16,17 and SARS-CoV-218,19, no mAbs are currently available in the clinic that can prevent or treat HMPV infection. The efficacy of mAbs for the treatment of respiratory viral infections is generally lower compared to prophylaxis, because mAbs in the therapeutic setting are administered after the virus has already established a foothold in the host20–26. As a result, most mAbs targeting respiratory viruses in development and in clinical use are intended for prophylaxis rather than therapy. To achieve efficacy as a therapeutic modality for HMPV, we sought to identify mAbs with the following three characteristics: (1) high potency; (2) the ability to neutralize multiple strains of HMPV; and (3) low susceptibility to resistance development. Ideally, a single mAb would neutralize all four major subtypes of HMPV (A1, A2, B1, and B2), which cause disease and circulate globally27,28.

HMPV expresses a class I fusion (F) protein, an essential surface glycoprotein, specialized in mediating fusion between viral and host cell membranes during viral entry. The F protein transitions between a metastable prefusion (preF) conformation and a stable postfusion (postF) conformation29,30. Since preF is the major conformation on infectious virions, antibodies to preF tend to be the most potent at neutralizing virus31–34. The rapid demise of several mAb candidates authorized for use during the COVID-19 pandemic has also underscored the formidable force of viral evolution and the need to determine how and if a virus can develop mAb resistance. Unlike SARS-CoV-2, which has a high mutation rate, particularly in the spike protein targeted by therapeutic mAbs35,36, HMPV evolves more slowly, and the F protein has shown minimal antigenic drift over time37,38. This slower rate of antigenic drift in HMPV offers a strategic advantage when designing neutralizing mAbs, as the risk of rapid resistance development is comparatively lower. In the present study, we leveraged recombinant F proteins stabilized in the preF conformation to identify and clone a highly potent neutralizing mAb, 4F11, from human memory B cells. 4F11 targets a novel, apical epitope that is present only in the preF conformation. 4F11 neutralizes diverse strains of HMPV, has low susceptibility to the development of viral escape, and has therapeutic efficacy in a hamster challenge model. These features together indicate that 4F11 has potential as a therapeutic modality in humans, which could be beneficial to at-risk populations who are at a significant immunological disadvantage in the setting of an infection with HMPV.

Results
Identification of a highly potent neutralizing antibody to HMPV
HMPV is divided into two major subtypes, A and B. Although the majority of HMPV-neutralizing B cells (approximately 75%) produce antibodies that neutralize both subtypes A and B39, we leveraged a “bait and switch” strategy to further enrich for B cells that could neutralize both strains. This strategy is based on the rationale that B cells capable of binding to one strain, while neutralizing another strain, are more likely to cross-neutralize both strains (Fig. 1a). We used fluorescent tetrameric probes to identify individual B cells able to bind the recombinant F protein of HMPV subtype B2, which was stabilized in the preF conformation by introduction of a GCN4 trimerization domain at the C-terminus40. A total of 600 million human splenocytes from two donors and 320 million peripheral blood mononuclear cells from another two donors were mixed with tetramers of HMPV B2 preF conjugated to allophycocyanin (APC) and control tetramers of His tag peptide conjugated to APC/Dylight 755. This was followed by magnetic enrichment with anti-APC microbeads. Since virtually all adults have been exposed to HMPV41, it was not necessary to pre-screen donors for seropositivity. A total of 1140 isotype-switched (IgD-) B cells that bound HMPV B2 preF tetramers, but not control tetramers of RSV preF, were individually sorted into wells containing CD40L/IL2/IL21-producing 3T3 feeder cells and then incubated for 13 days to stimulate antibody secretion into culture supernatants (Fig. 1b). To identify wells containing candidate B cells expressing cross-neutralizing antibodies, culture supernatants from the individually sorted HMPV B2 preF-binding B cells were mixed with live HMPV subtype A2 virus and screened for their ability to reduce plaque formation. Six out of the 1140 HMPV-binding B cells produced antibodies that could neutralize HMPV A2. From these 6 cells, the expressed heavy and light chain genes of 3 B cell clones were sequenced successfully, and their antibodies were named 4F11, 4E11, and 3B5. Based on sequencing analysis, 4E11 and 4F11 were derived from B cells expressing the IgG isotype. Our sequencing analysis was unable to determine the isotype for 3B5. Since palivizumab, nirsevimab, and other mAbs currently approved or authorized for respiratory viral infections utilize an IgG1 constant region, the B cell receptors from the 3 B cell clones were cloned and produced for further study as IgG1. All 3 mAbs had neutralizing activity against HMPV A2 strain CAN97-83, with 4F11 being the most potent in a 50% plaque reduction neutralization test (PRNT50) at 5.1 ng/mL (Fig. 1c). For comparison, the RSV and HMPV cross-neutralizing mAb, MxR, was less potent with a PRNT50 of 229.4 ng/mL. To assess the neutralization capacity of the 4F11 antibody against different HMPV strains, we used a panel of clinical isolates representing all four subtypes of HMPV (A1, A2, B1, and B2). The 4F11 antibody demonstrated neutralizing activity against all tested subtypes (PRNT50 range 0.97 ng/mL for subtype A1 to 10.7 ng/mL for A2 strain IA27-2004) (Fig. 1d). These results highlight 4F11’s neutralizing activity across diverse HMPV strains.

Molecular structure of 4F11 in complex with HMPV F
To map the antigenic binding site of our most potent mAb, 4F11, we performed cross-competition binding experiments with other previously mapped mAbs using the HMPV preF that was partially stabilized by a GCN4 trimerization domain. The 4F11 epitope did not appear to overlap with the epitope of the MxR mAb42, which we previously isolated and mapped to the equator (site III) of HMPV preF (Figs. 2a and S1a). However, 4F11 did compete, albeit only partially, with the ADI-61026 and SAN32-2 mAbs39,43 (Figs. 2a and S1b), both of which represent a rare class of antibodies that can penetrate the dense glycan shield to bind at the apex (site Ø) of HMPV preF.

To determine the molecular interactions that mediate how 4F11 binds to HMPV preF, cryoEM analysis was used. We first attempted to complex 4F11 with the HMPV preF trimer that was partially stabilized by a GCN4 trimerization domain, but the trimer dissociated into monomers that were bound to 4F11 Fab. In an attempt to obtain a structure with an intact HMPV preF trimer, we turned to alternative versions of prefusion-stabilized HMPV F trimers. We first turned to DS-CavEs244, which includes a Pro185 mutation at site Ø, introduced to confer local rigidity and disfavor helix formation required during the transition to the postfusion conformation. However, 4F11 was unable to bind to this stabilized trimer, whereas the site III mAb MxR retained binding to DS-CavEs2 (Fig. S1c, d). Since our competition experiments indicated that 4F11 bound at or near the apex of GCN4-stabilized HMPV preF (Figs. 2a and S1b), we hypothesized that the Pro185 mutation is part of the 4F11 epitope and could disrupt binding of 4F11. Indeed, reverting proline 185 back to an alanine rescued binding of 4F11 (Fig. S1c). We then attempted to complex 4F11 Fab with the reverted DS-CavEs2 P185A trimer at a 1.2:1 molar ratio of Fab to trimer, but we still only observed monomers bound to 4F11 Fab, indicating that 4F11 does not stabilize the trimer. In contrast to 4F11, MxR binds across protomers and could help stabilize the trimer. The addition of MxR at a 3:1 molar ratio of Fab:DS-CavEs2 P185A trimer led to a peak in the SEC representing intact trimer bound to three Fabs (Fig. S2). To help stabilize the trimer, MxR Fab was precomplexed with DS-CavEs2 P185A trimer before the addition of 4F11 Fab. After addition of 4F11 to this complex, the peak representing the trimer prebound to three MxR Fabs disappeared, and a new peak appeared that was shifted to the right and exited the SEC at a fraction corresponding to the size of the F monomer bound to two Fabs (one Fab each of MxR and 4F11) (Fig. S2). We obtained a 4.03 Å resolution cryoEM structure of 4F11 and Fabs bound to the DS-CavEs2 P185A monomer (Figs. 2b and S3a–c, and Table S1). As predicted from the earlier binding competition experiments, the structure showed that 4F11 binds site Ø almost vertically at the apex of HMPV F in the prefusion conformation (Fig. 2b).

Since a structure of 4F11 bound to an intact trimer could not be obtained using DS-CavEs2, newer stabilized versions of HMPV preF were explored. Recombinant v3B is a prefusion-stabilized variant of HMPV F that includes amino acid substitutions V84C-A249C to generate interprotomer disulfide bonds, similar to another variant DS-CavEs2-IPDS45,46. Attempts to complex 4F11 Fab to the v3B trimer45 with Pro185 reverted to alanine similarly resulted in trimer dissociation upon the addition of 4F11 Fab, as observed with the GCN4-stabilized MxR and DS-CavEs2 trimers. We next turned to the most recently published, stabilized version of the HMPV preF trimer, MPV-2c47. The MPV-2c trimer does not rely on stabilization with Pro185 and was co-transfected with 4F11 Fab due to low expression of the trimer. Fortunately, the MPV-2c trimer remained intact when co-transfected with 4F11 Fab. We obtained a 4.13 Å resolution cryoEM structure of this complex (Figs. 2c, S3d–f and S4, and Table S1). As expected, 4F11 binds site Ø at the apex of preF, confirming what was determined with the monomer structure (Fig. 2b), and only one Fab is bound per trimer (1:1 stoichiometry of 4F11 Fab:MPV-2c trimer). Indeed, when we model the trimer with two or three Fabs of 4F11 bound, the Fabs clash with each other. This suggests that there is structurally only enough room for binding by one 4F11 Fab at a time to the trimer (Fig. S5). M8C10 is another mAb that causes trimer dissociation, but it binds an epitope that is buried deep within the trimerization interface48. M8C10 interferes with trimer formation likely by clashing with the non-binding protomers of the trimer (Fig. S6). In contrast, 4F11 likely facilitates trimer dissociation when more than one Fab tries to bind the trimer, because the Fabs clash with each other (Fig. S6).

4F11 binds site Ø with a total buried surface area (BSA) of ~895 Å2, ~600 Å2 of which are contributed by the heavy chain interaction with protomer A, ~220 Å2 from the light chain interaction with protomer A, and ~75 Å2 from the light chain interaction with protomer B (Figs. 2d and S7a, b). As expected, since 4F11 interacts with residues on two HMPV F protomers, 4F11 has higher affinity for the MPV-2c trimer than for the monomer (4-fold) (Figs. 2e and S8). When the F trimer transitions from the prefusion conformation to the postfusion conformation, site Ø undergoes large rearrangements that would cause 4F11 to clash with portions of the postfusion trimer (Fig. S9). Therefore, 4F11 preferentially binds a quaternary prefusion-specific epitope.

Of the 4F11 residues that contact the trimer, only 3 out of 9 in the VH and 4 out of 17 in the VL are mutated from their germline genes, VH5-51*01 and VK2-28*01, respectively (Fig. S7b). Site Ø is highly conserved across the four HMPV subtypes (Fig. S7a), which explains why 4F11 is able to neutralize all four subtypes (Fig. 1d). Since Asp185 is in the epitope of 4F11, the stabilizing Pro185 mutation in v3B and DS-CavEs2 likely disrupts the interactions between Asp185 and 4F11. Notably, 4F11 accommodates the glycan shield at site Ø that consists of the Asn57 and Asn172 glycans. While 4F11 avoids the Asn57 glycan by binding vertically toward the top of the trimer, its light chain interacts with the Asn172 glycan (glycan total BSA of ~379 Å2) (Fig. 2f and S7b), which is necessary for binding since removal of the Asn172 glycan by mutagenesis or EndoH treatment abrogates 4F11 binding (Figs. 2g and S10a). Since contact by 4F11 with the glycan at Asn172 contributes only a fraction of the overall buried surface area, we would not expect 4F11 to bind non-specifically to other viral glycoproteins. Indeed, 4F11 does not bind to RSV preF (Fig. S10b), as expected, since our B cell sorting strategy already selected for B cells that could bind HMPV but not RSV preF (Fig. 1a, b). In contrast to 4F11, two other site Ø antibodies, SAN32-237 and ADI-6102636 bind equatorially between the Asn57 and Asn172 glycans and penetrate the glycan shield without interacting with the glycans (Fig. S11). Thus, 4F11 is a quaternary-preferring, prefusion-specific, and glycan-dependent antibody with a unique binding mode, distinct from other site Ø antibodies.

HMPV escape mutants
To select for viable escape mutations, we passaged HMPV A2 expressing a GFP reporter in the presence of serial dilutions of either 4F11 or no antibody as a negative control for lab-adapted mutations. We included MxR as a positive control, since substitutions at D280 in HMPV F have been previously reported as a mechanism of escape to site III mAbs49. By passage 3 in the presence of MxR, most cells were infected and expressing GFP even at 15 µg/mL of MxR, which is a concentration approximately 100-fold higher than its PRNT50 (148 ng/mL)42 (Fig. 3a). Deep sequencing revealed a mixed population consisting of viruses with mutations at S237P, D280N, D280G, and S483N (Fig. 3b). These mutations were introduced into recombinant HMPV F protein, and MxR lost binding to HMPV F with S237P, D280N, and D280G, but not S483N (Fig. 3c). Notably, the yield of recombinant F with the S237P mutation was substantially lower compared to wild-type F (36%) and F with the other escape mutations (Fig. 3b). We attempted to isolate viruses containing the individual escape mutations by plaque purification. Out of 22 plaques picked, 6 were cultured and sequenced successfully. We isolated a pure population of virus with D280G from 4 plaques. The D280N and S483N mutations co-occurred in the other 2 plaque-purified viruses, which is consistent with their identical allele frequencies during passaging (Fig. 3b). We were unable to plaque purify virus with S237P, which had the lowest allele frequency during passaging, and which had a F protein that was difficult to produce recombinantly.

In contrast to MxR, infection and GFP expression was negligible at concentrations of 4F11 at or above 470 ng/mL, even after 5 passages (Fig. 3a). At the next dilution of 235 ng/mL, less than 25% of cells were infected at passages 1 through 4, and the level of infection did not appreciably increase with each successive passage. Instead, GFP expression was lost by passage 5. Despite the low level of infection, viruses from the third passage were successfully expanded in the presence of 235 ng/mL of 4F11, and deep sequencing identified a K179E mutation that was present in 100% of the virus population (Fig. 3b). 4F11, but not MxR, lost binding to recombinant HMPV F protein containing the K179E mutation (Fig. 3c). In contrast, the mutations derived during HMPV passaging in the presence of MxR (S237P, D280N, D280G, and S483N) had no effect on 4F11 binding to recombinant HMPV F protein (Fig. 3c).

Following plaque purification, we successfully cultured and sequenced 5 out of 11 plaques. All 5 contained a pure population of virus with the K179E mutation, except for one, which also carried a new N466K mutation in the F protein, a mutation not detected in our initial metagenomic sequencing. The N466K mutation in isolation had no effect on 4F11 binding to recombinant HMPV F protein (Fig. 3d). The K179E and K179E+N466K viruses were then used to evaluate the in vitro neutralization potency of the 4F11 antibody and to assess replication kinetics relative to wild-type (WT) HMPV. The neutralizing potency of 4F11 was determined by calculating the PRNT50 in Vero cells infected with WT, K179E, or K179E+N466K mutant viruses. 4F11 still neutralized HMPV carrying the K179E mutation but had a PRNT50 of 192.3 ng/mL, compared to a PRNT50 of 2.2 ng/mL to WT virus (Fig. 3e). The combined K179E+N466K mutant virus had an approximately 2-fold further increase in PRNT50 to 430.5 ng/mL. To assess whether these mutations impact viral fitness, we compared viral growth kinetics in Vero cells. The K179E virus showed reduced growth kinetics compared to WT HMPV, with titers approximately 2 logs lower by day 7 post-infection (Fig. 3f). The virus containing both K179E and N466K together showed slightly improved growth kinetics relative to K179E alone, suggesting that while N466K does not mediate antibody escape, it may partially compensate for the fitness cost associated with K179E.

To investigate whether the K179E mutation is observed in circulating HMPV strains, we analyzed global sequence data using Nextstrain50. Among 782 publicly sequenced strains, 0 contained an E at position 179 (Fig. S12). At position 179, only 2 strains had a conservative replacement with a Q, 179 strains had an R, and the remaining 601 had a K. The structure of 4F11 bound to the HMPV F trimer revealed that Lys179 interacts with a negatively charged patch of 4F11 that includes residues in the heavy chain (Figs. 2d and 3g) and contributes to ~12% total BSA of HMPV F (Fig. 2d). The K179E mutation would introduce a negatively charged residue that would repel the negatively charged patch on 4F11 (Fig. 3g), providing a mechanism for this resistance mutation. Unlike K179E, which was not found in any publicly sequenced isolates of HMPV, replacement at position 179 with Q, R, or K residues, which are either neutral or positively charged, would not lead to repulsion.

Since 4F11 binding to HMPV F is glycan-dependent, mutation of the Asn172 glycan motif could represent another mechanism of escape. Although we failed to isolate any live virus with a mutation at glycosylation sites, we observed that 4F11 failed to bind recombinant MPV-2c trimer with an N172T mutation that removes the Asn172 glycan (Fig. 2g). The N172T substitution was chosen because we found a single HMPV F sequence with a threonine at position 172 in Nextstrain (Fig. S13). All other 998 sequences (99.9%) in Nextstrain had an asparagine.

In vivo protection
We next investigated whether the in vitro binding and neutralization data would translate into in vivo protection in an animal challenge model. The hamster model has been used extensively to evaluate vaccine and antibody candidates for HMPV42,51–55. Although some studies have observed higher titers of HMPV in the lungs of cotton rats compared to hamsters56,57, this could be related to differences in the viral strain used, the size of the inoculum, and the timing of lung sampling. Since our primary objective was the development of a mAb potent enough to be used as therapy, we performed preclinical testing in the Golden Syrian hamster model in which 4F11 was administered post-infection. In treating respiratory viral infections with mAbs, the greatest clinical benefit is generally observed with early administration to prevent progression to severe disease58,59. Therefore, 4F11 was administered intramuscularly to hamsters 24 h after intranasal inoculation with HMPV. Lungs and nasal turbinates were harvested on day 4 post-infection to determine the titer of HMPV in the lower and upper respiratory tracts, respectively (Fig. 4a). This dosing and route of administration are similar to those used in cotton rat models of HMPV prophylaxis, except that we administered mAb after infection instead of before infection42,43. At a dose of 2.5 mg/kg, 4F11 fully suppressed viral replication of HMPV in the lungs of 7 out of 10 hamsters and significantly reduced viral replication in nasal turbinates (Fig. 4b, c). The remaining 3 hamsters had viral titers at or near the limit of detection. At a reduced dose of 1.25 mg/kg, 4F11 fully suppressed viral replication of HMPV in 2 of 5 animals and still significantly reduced viral replication in the nasal turbinates. In contrast to 4F11, the cross-neutralizing mAb MxR failed to block viral replication in the lungs of any animals at a dose of 2.5 mg/kg (Fig. 4c). For comparison, RSV antibodies such as palivizumab and nirsevimab are clinically administered for prophylaxis in infants at doses higher than that used in the present study (15 mg/kg and approximately 10–20 mg/kg, respectively)60–62. Therefore, the dosage used in our in vivo experiments is lower than the dosages of other mAbs in clinical use, which is an important consideration from the standpoint of potential toxicity in humans.

Methods
Study design
This study complies with all relevant ethical regulations and was reviewed and approved by the Fred Hutchinson Cancer Center Institutional Review Board (IRB). Peripheral blood was obtained by venipuncture from healthy, HIV-seronegative adult volunteers enrolled in the Seattle Area Control study after informed consent (Protocol #5567). PBMCs were isolated from whole blood using Accuspin System Histopaque-1077 (Sigma-Aldrich, cat#10771). Studies involving human spleens were deemed non-human subjects research since tissue was de-identified. Spleen samples received an exempt determination by the Fred Hutchinson Institutional Review Board, as defined by the Common Rule, from the Office for Human Research Protections. Tissue was de-identified and originated from deceased donors in which the spleen would have otherwise been discarded during procurement of other organs (e.g., liver) for donation. Tissue fragments were passed through a basket screen, centrifuged at 300 × g for 7 min, incubated with ACK lysis buffer (Thermo Fisher, cat#A1049201) for 3.5 min, resuspended in RPMI (Gibco, cat#11875093), and passed through a stacked 500 µm and 70 µm cell strainer. Cells were resuspended in 10% dimethyl sulfoxide in heat-inactivated fetal calf serum (Gibco, cat#16000044) and cryopreserved in liquid nitrogen before use.

Cell lines
293F cells (Thermo Fisher, cat#R79007) were cultured in Freestyle 293 media (Thermo Fisher, cat#12338026). Vero cells (ATCC CCL-81) and LLC-MK2 cells (ATCC CCL-7.1) were cultured in DMEM (Gibco, cat#12430054) supplemented with 10% fetal bovine serum and 100 U/mL penicillin plus 100 µg/mL streptomycin (Gibco, cat#15140122).

Viruses
The recombinant virus HMPV-GFP has been previously described81 and was modified from HMPV CAN97-83 (GenBank accession number AY297749) to express enhanced GFP. HMPV was cultured on LLC-MK2 cells with DMEM (Gibco, cat#12430054) supplemented with 100 U/mL penicillin plus 100 µg/mL streptomycin (Gibco, cat#15140122) and 1.2% of 0.05% Trypsin (Gibco, cat#25300054). Virus was purified by centrifugation in a discontinuous 30%/60% sucrose gradient with 0.05 M HEPES and 0.1 M MgSO4 (Sigma-Aldrich, cat#H4034 and 230391, respectively) at 120,000 × g for 90 min at 4°C. Virus titers were determined by infecting Vero cell monolayers in 24-well plates with serial 10-fold dilutions of virus, overlaying with DMEM containing 0.8% methylcellulose (Sigma-Aldrich, cat#M0387) and 2.4% of 0.05% Trypsin (Gibco, cat#25300054). Fluorescent plaques were counted using a Typhoon scanner (GE Life Sciences) at 5 days post-infection.

Clinical strains of HMPV were obtained from ZeptoMetrix, including A1 (strain IA10-2003, lot#331204, cat#0810161CF), A2 (strain IA27-2004, lot#331149, cat#0810164CF), B1 (strain Peru3-2003, lot#328937, cat#0810158CF), and B2 (strain IA18-2003, lot#329066, cat#0810162CF). Plaques were counted by fixing wells with 10% formalin for 1 h, washing three times with 1× PBS with 0.02% Tween 20 (PBST), and then incubating with 1× DPBS containing 10% goat serum (Southern Biotech, cat#0060-01) for 1 h at room temperature. Wells were washed three times with 1× PBST and then incubated with the 4F11 antibody for 1 h. After 3 additional washes with 1× PBST, wells were incubated for 1 h with Alexa Fluor 647-conjugated goat anti-human IgG at a dilution of 1:2000 (Invitrogen, cat#A-21445) and scanned using a Typhoon scanner (GE Life Sciences).

Expression and purification of antigens
Expression plasmids for His-tagged RSV preF and HMPV preF are previously described40,45,47,82. 293F cells were transfected at a density of 106 cells/mL in Freestyle 293 media using 1 mg/mL PEI Max (Polysciences, cat#24765). Transfected cells were cultured for 7 days with gentle shaking at 37°C. Supernatant was collected by centrifuging cultures at 2500 × g for 30 min followed by filtration through a 0.2 µm filter. The clarified supernatant was incubated with Ni Sepharose beads overnight at 4°C, followed by washing with wash buffer containing 50 mM Tris, 300 mM NaCl, and 8 mM imidazole. His-tagged protein was eluted with an elution buffer containing 25 mM Tris, 150 mM NaCl, and 500 mM imidazole. The purified protein was run over a 10/300 Superose 6 size exclusion column (GE Life Sciences, cat#17–5172–01). Fractions containing the trimeric F proteins were pooled and concentrated by centrifugation in a 50 kDa Amicon ultrafiltration unit (Millipore, cat#UFC805024). The concentrated sample was stored in 50% glycerol at −20°C.

For structural characterization, MPV-2c trimer was co-transfected with 4F11 His-Fab and Furin in a 3:1:1:0.6 (MPV-2c:4F11 His-Fab HC:4F11 His-Fab LC:Furin) ratio in HEK293E cells at a density of 106 cells/mL using 2 mL of 1 mg/mL PEI Max per liter of cells transfected. Transfected cells were cultured for 6 days at 37°C with shaking. Supernatant was collected by centrifuging and then filtering the cell cultures using a 0.2 µm filter. The supernatant was incubated with Ni-NTA resin (Takara Bio) for 1 h at room temperature with shaking, followed by washing with wash buffer containing 50 mM Tris, 15 mM imidazole, and 0.3 M NaCl at pH 8.0 and eluted with an elution buffer containing 50 mM Tris, 150 mM imidazole, and 0.3 M NaCl at pH 8.0. The complex was then further purified with size exclusion chromatography (SEC) over a Superdex 200 16/600 on an AKTA pure system (GE Biosciences) equilibrated in 1× Tris-buffered saline (TBS). The final complex was concentrated to 2 mg/mL with a 10 kDa cutoff Amicon ultrafiltration unit (Millipore, cat#UFC8010).

Tetramerization of antigens
Purified F antigens were biotinylated using an EZ-link Sulfo-NHS-LC-Biotinylation kit (Thermo Fisher, cat#A39257) with a 1:1.3 molar ratio of biotin to F. Unconjugated biotin was removed by centrifugation using a 50 kDa Amicon Ultra size exclusion column (Millipore, cat#UFC805024). To determine the average number of biotin molecules bound to each molecule of F, streptavidin-PE (ProZyme, cat#PJRS25) was titrated into a fixed amount of biotinylated F at increasing concentrations and incubated at room temperature for 30 min. Samples were run on an SDS-PAGE gel (Invitrogen, cat#NW04127-BOX), transferred to nitrocellulose, and incubated with streptavidin–Alexa Fluor 680 (Thermo Fisher, cat#S32358) at a dilution of 1:10,000 to determine the point at which there was excess biotin available for the streptavidin–Alexa Fluor 680 reagent to bind. Biotinylated F was mixed with streptavidin-allophycocyanin (APC) or streptavidin-PE at the ratio determined above to fully saturate streptavidin and incubated for 30 min at room temperature. Unconjugated F was removed by centrifugation using a 300 K Nanosep centrifugal device (Pall Corporation, cat#OD300C33). APC/DyLight 755 and PE/DyLight 650 tetramers were created by mixing F with streptavidin-APC pre-conjugated with DyLight 755 (Thermo Fisher, cat#62279) or streptavidin-PE pre-conjugated with DyLight 650 (Thermo Fisher, cat#62266), respectively, following the manufacturer’s instructions. On average, APC/DyLight 755 and PE/DyLight 650 contained 4–8 DyLight molecules per APC and PE. The concentration of each tetramer was calculated by measuring the absorbance of APC (650 nm, extinction coefficient=0.6 µM−1 cm−1) or PE (566 nm, extinction coefficient=2.0 µM−1 cm−1).

Tetramer enrichment
1–2 × 108 frozen PBMCs or spleen cells were thawed into DMEM with 10% fetal calf serum and 100 U/mL penicillin plus 100 µg/mL streptomycin. Cells were centrifuged and resuspended in 50 µL of ice-cold fluorescence-activated cell sorting (FACS) buffer composed of phosphate-buffered saline (PBS) and 1% fetal calf serum (Fisher Scientific, cat#SH3007103). His tag APC/DyLight 755 and PE/Dylight 650-conjugated tetramers were added at a final concentration of 25 nM in the presence of 2% rat and mouse serum (Thermo Fisher) and incubated at room temperature for 10 min. PreF APC and PE tetramers were then added at a final concentration of 5 nM and incubated on ice for 25 min, followed by a 10 mL wash with ice-cold FACS buffer. Next, 50 µL each of anti-APC-conjugated (Miltenyi Biotec, cat#130-090-855) and anti-PE-conjugated (Miltenyi Biotec, cat#130-048-801) microbeads were added and incubated on ice for 30 min, after which 3 mL of FACS buffer was added and the mixture was passed over a magnetized LS column (Miltenyi Biotec, cat#130-042-401). The column was washed once with 5 mL ice-cold FACS buffer and then removed from the magnetic field, and 5 mL ice-cold FACS buffer was pushed through the unmagnetized column twice using a plunger to elute the bound cell fraction.

Flow cytometry
Cells were incubated in 50 µL of FACS buffer containing a cocktail of antibodies for 30 min on ice prior to washing and analysis on a FACS Aria (BD). Antibodies included anti-CD19 BUV395 (SJ25C1, BD, cat#563551, 1:20 dilution), anti-CD3 BV711 (UCHT1, BD, cat#563725, 1:50 dilution), anti-CD14 BV711 (M0P-9, BD, cat#563372, 1:50 dilution), anti-CD16 BV711 (3G8, BD, cat#563127, 1:50 dilution), anti-IgD BV605 (IA6-2, BD, cat#563313, 1:50 dilution), and a fixable viability dye (Tonbo Biosciences, cat#13–0870–T500, 1:250 dilution). B cells were individually sorted into flat-bottom 96-well plates containing feeder cells that had been seeded at a density of 28,600 cells/well 1 day prior in 100 µL of IMDM media (Gibco, cat#31980030) containing 10% fetal calf serum, 100 U/mL penicillin plus 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin. B cells sorted onto feeder cells were cultured at 37°C for 13 days.

B cell receptor sequencing
For individual B cells sorted onto feeder cells, supernatant was removed after 13 days of culture, plates were immediately frozen on dry ice, stored at −80°C, thawed, and RNA was extracted using the RNeasy Micro Kit (Qiagen, cat#74034)42,83,84. The entire eluate from the RNA extraction was used instead of water in the RT reaction. Following RT, 2 µL of cDNA was added to 19 µL PCR reaction mix so that the final reaction contained 0.2 µL (0.5 U) HotStarTaq Polymerase (Qiagen, cat#203607), 0.075 µL of 50 µM 3′ reverse primers, 0.115 µL of 50 µM 5′ forward primers, 0.24 µL of 25 mM dNTPs, 1.9 µL of 10× buffer (Qiagen), and 16.5 µL of water. The PCR program was 50 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 55 s, followed by 72°C for 10 min for heavy and kappa light chains. The PCR program was 50 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 55 s, followed by 72°C for 10 min for lambda light chains. After the first round of PCR, 2 µL of the PCR product was added to 19 µL of the second-round PCR reaction so that the final reaction contained 0.2 µL (0.5 U) HotStarTaq Polymerase, 0.075 µL of 50 µM 3′ reverse primers, 0.075 µL of 50 µM 5′ forward primers, 0.24 µL of 25 mM dNTPs, 1.9 µL 10× buffer, and 16.5 µL of water. PCR programs were the same as the first round of PCR. 5 µL from the PCR reactions was mixed with 2 µL of ExoSAP-IT (Thermo Fisher, cat#78201) and incubated at 37°C for 15 min followed by 80°C for 15 min to hydrolyze excess primers and nucleotides. Hydrolyzed second-round PCR products were sequenced by Genewiz with the respective reverse primer used in the second-round PCR, and sequences were analyzed using IMGT/V-Quest to identify V, D, and J gene segments. Paired heavy chain VDJ and light chain VJ sequences were cloned into pTT3-derived expression vectors containing the human IgG1, IgK, or IgL constant regions using In-Fusion cloning (Clontech, cat#638911)85.

Monoclonal antibody and Fab production
Secretory IgG and His-Fabs were produced by co-transfecting HEK293E cells at a density of 106 cells/mL with the paired heavy and light chain expression plasmids at a ratio of 1:1 in Freestyle 293 media using 1 mg/mL PEI Max. Transfected cells were cultured for 6 days with gentle shaking at 37°C. Supernatant was collected by centrifuging cultures at 2500 × g for 15 min followed by filtration through a 0.2 µm filter. Clarified supernatants of secretory IgG were then incubated with Protein A agarose (Thermo Scientific, cat#20333) followed by washing with IgG binding buffer (Thermo Scientific, cat#21007). Antibodies were eluted with IgG Elution Buffer (Thermo Scientific, cat#21004) into a neutralization buffer containing 1 M Tris-base pH 9.0. Purified antibody was concentrated and buffer exchanged into PBS using an Amicon ultrafiltration unit with a 50 kDa molecular weight cutoff for IgG. His-Fabs were purified with Ni-NTA affinity resin (Takara Bio, cat#635661) followed by SEC on a Superdex 200 16/600 column (Cytiva). Fractions containing His-Fabs were then pooled and concentrated using an Amicon ultrafiltration unit with a 10 kDa cutoff.

Bio-layer interferometry
Bio-layer interferometry (BLI) assays were performed on the Octet Red instrument (ForteBio) at room temperature with shaking at 500 rpm. For binding analyses, anti-human IgG Fc capture sensors (ForteBio, cat#18–5060) were loaded in kinetics buffer (PBS with 0.01% bovine serum albumin, 0.02% Tween 20, and 0.005% NaN3, pH 7.4) containing 40 µg/mL mAb for 150 s. After loading, the baseline signal was recorded for 60 s in kinetics buffer. The sensors were then immersed in kinetics buffer containing 30 µM purified HMPV preF for a 300 s association step, followed by immersion in kinetics buffer for a dissociation phase of 300 s. For competitive binding assays, anti-penta His capture sensors were loaded in kinetics buffer containing 1 µM His-tagged GCN4-stabilized HMPV preF for 300 s. After loading, the baseline signal was recorded for 30 s in kinetics buffer. The sensors were then immersed for 300 s in kinetics buffer containing 40 µg/mL of the first antibody, followed by immersion for another 300 s in kinetics buffer containing 40 µg/mL of the second antibody. Percent competition was determined by dividing the maximum increase in signal of the second antibody in the presence of the first antibody by the maximum signal of the second antibody alone.

ELISA
Nunc MaxiSorp 96-well plates (Thermo Fisher, cat#442404) were coated with 100 ng of HMPV preF for 90 min at 4°C. Wells were washed three times with 1× DPBS and then incubated with 1× DPBS containing 1% bovine serum albumin (Sigma-Aldrich, cat#A2153) for 1 h at room temperature. Antigen-coated plates were incubated with serial dilutions of mAb for 90 min at 4°C. Wells were washed three times with 1× DPBS followed by incubation for 1 h with horseradish peroxidase-conjugated goat anti-human total Ig at a dilution of 1:6000 (Invitrogen, cat#31412). Wells were then washed four times with 1× DPBS followed by a 5–15 min incubation with TMB substrate (SeraCare, cat#5120–0053). Absorbance was measured at 405 nm using a Softmax Pro plate reader (Molecular Devices).

Neutralization assays
For neutralization screening of culture supernatants, Vero cells were seeded in 96-well flat-bottom plates and cultured for 48 h. After 13 days of culture, 40 µL of B cell culture supernatant was mixed with 25 µL of sucrose-purified GFP-HMPV A2 diluted to 2000 plaque-forming units (pfu)/mL for 1 h at 37°C. Vero cells were then incubated with 50 µL of the supernatant/virus mixture for 1 h at 37°C to allow viral adsorption. Next, each well was overlaid with 100 µL DMEM containing 0.8% methylcellulose and 1.2% of 0.05% trypsin. Fluorescent plaques were counted at 5 days post-infection using a Typhoon imager.

Neutralizing titers of monoclonal antibodies were determined by a 50% plaque reduction neutralization test (PRNT50). Vero cells were seeded in 24-well plates and cultured for 48 h. Monoclonal antibodies were serially diluted 1:4 in 120 µL DMEM and mixed with 120 µL of sucrose-purified HMPV diluted to 2000 pfu/mL for 1 h at 37°C. Vero cells were incubated with 100 µL of the antibody/virus mixture for 1 h at 37°C to allow viral adsorption. Each well was then overlaid with 500 µL DMEM containing 0.8% methylcellulose. Fluorescent plaques were counted at 5 days post-infection using a Typhoon imager. PRNT50 titers were calculated by linear regression analysis.

CryoEM complex and grid preparation
The complex of DS-CavEs2 P185A monomer bound to 4F11 and MxR Fabs was made by incubating DS-CavEs2 P185A trimer with a 1:3.2 molar ratio of trimer:MxR Fab for 5 min at room temperature with shaking, then adding a 1:1.2 molar ratio of trimer:4F11 Fab and incubating for 10 min with shaking at room temperature. The complex was then purified using a Superdex 200 10/300 column (Cytiva) to remove excess Fab, and fractions containing DS-CavEs2 P185A monomer bound to MxR and 4F11 Fabs were collected. The complex of DS-CavEs2 P185A monomer bound to 4F11 and MxR Fabs was frozen on UltrAuFoil 1.2/1.3 Au 300 mesh grids (Ted Pella, cat#688-300-AU-50) using a Vitrobot Mk. IV (Thermo Fisher) at 4°C and 100% humidity. Dodecyl-β-D-maltoside (DDM) was added to the sample before freezing at a final concentration of 0.1× CMC, and the grids were glow-discharged using a plasma cleaner. The final grids used for collection were frozen with 3 µL of complex at 3.5 mg/mL, a 5 s wait time, a 10 s blot time, and a blot force of 1. The grid of the complex of MPV-2c trimer and 4F11 Fab was prepared similarly, except the final grids used for collection were frozen with 3 µL of complex at 2 mg/mL, a 30 s wait time, a 6 s blot time, and a blot force of 2.

Data collection, processing, and model refinement
Data for the complex of DS-CavEs2 P185A monomer bound to 4F11 and MxR Fabs were collected on a Glacios (Thermo Fisher) operating at 200 kV with a Gatan K3 DED. Data were collected using SerialEM at 92,000× magnification (1.122 Å/pix) and 60 e/Å2 exposure dose to get 1048 micrographs. Data was processed using CryoSPARC v4.3.1 after being motion corrected with WARP86. After importing the motion corrected micrographs, patch CTF estimation was performed. Micrographs were then curated to include <7 Å CTF fit and to remove micrographs from thick ice. Blob picker was used to select 1,332,453 particles that were then extracted at 128 px box size before undergoing multiple rounds of 2D classification. The 101,166 particles in the final set of good 2D classes were re-extracted at 256 px box size. A single ab-initio class was generated. This map was further refined using homogeneous refinement and non-uniform refinement with C1 symmetry. A custom mask was then used to crop out the CH1 domain of the 4F11 Fab to produce a map of 4.03 Å resolution. Model refinement was performed in the Phenix software suite, Coot, and the ISOLDE plugin of ChimeraX. The cryoEM map was deposited to the EMDB. No model was built to fit this cryoEM map. All figures were produced in ChimeraX.

Data for the complex of MPV-2c trimer and 4F11 Fab was collected on a Glacios (Thermo Fisher Scientific) operating at 200 kV with a Gatan K3 DED. Data were collected using SerialEM at 92,000× magnification (1.122 Å/pix) and 60 e/Å2 exposure dose to get 3327 micrographs. Data was processed using CryoSPARC v4.3.1 after being motion corrected with WARP86. After importing the motion corrected micrographs, patch CTF estimation was performed. Micrographs were then curated to include <7 Å CTF fit and to remove micrographs from thick ice. Blob picker was used to select 3,524,733 particles that were then extracted at 180 px box size before undergoing multiple rounds of 2D classification. The 276,088 particles in the final set of good 2D classes were re-extracted at 360 px box size. A single ab-initio class was generated. This map was further refined using homogeneous refinement and non-uniform refinement with C1 symmetry. A custom mask was then used to crop out the CH1 domain of the 4F11 Fab to produce a map of 4.13 Å resolution. Model refinement was performed in the Phenix software suite, Coot, and the ISOLDE plugin of ChimeraX. The cryoEM map and model were deposited to the PDB and EMDB. All figures were produced in ChimeraX.

Escape mutation studies
Vero cells were seeded in 24-well flat-bottom plates and cultured for 48 h. Antibody was serially diluted 1:2 starting at 30 µg/mL in 500 µL of DMEM (Gibco, cat#11965092) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin (Gibco, cat#15140122), and 1.2% of 0.05% trypsin (Gibco, cat#25300054). HMPV was diluted to 1 × 106 pfu/mL, and 500 µL was mixed with each antibody dilution and incubated at 37°C for 1 h. Virus/antibody mixtures were added to Vero cells, and viral replication was monitored by GFP expression. After 7 days, supernatants were collected and incubated with fresh antibody at 37°C for 1 h, then used to inoculate new Vero cells in 24-well plates. After 3 passages for MxR and 5 passages for 4F11, wells containing the highest antibody concentration with visible viral replication were selected for expansion. From each selected well, 200 µL of supernatant was incubated with 2 mL of DMEM containing the same antibody concentration present in that well. After 1 h at 37°C, the mixture was used to infect Vero cells in T-25 flasks. After 5–7 days in culture, virus was harvested for sequencing.

Plaque purification
Vero cells were plated in 24-well plates and inoculated with serial 1:10 dilutions of virus, overlaid with methylcellulose containing 1.2% trypsin (Gibco, cat#25300054), and cultured for 5–7 days. Individual plaques were identified with fluorescent microscopy and picked with a P200 pipette tip. The aspirate containing the plaque was transferred onto Vero cells in 96-well plates containing 0.234 µg/mL of 4F11. After 7 days, supernatant was transferred to Vero cells in a T25 flask containing 0.234 µg/mL of 4F11. Purified virus was re-sequenced by Sanger sequencing.

Metagenomic next-generation sequencing and data interpretation
RNA was extracted from a 500 µL volume of cell culture supernatant using the Quick-RNA Viral Kit (Zymo Research, cat#R1034). Extracted RNA was treated with Turbo DNA-free kit (Invitrogen, cat#AM1907) to remove genomic DNA. First-strand cDNA synthesis was performed using random hexamers (Invitrogen, cat#N8080127) and SuperScript IV enzyme (Invitrogen, cat#18090200), and second-strand synthesis was performed using Sequenase Version 2.0 (Applied Biosystems, cat#70775Z1000UN). The cDNA was purified using 1.0× AMPure XP beads (Beckman Coulter, cat#A63881) and quantified on a Qubit 3.0 (LifeTech). Purified cDNA was sent to Azenta Life Sciences, where it underwent tagmentation and library preparation, followed by metagenomic Next-Generation Sequencing as 2× 150-bp runs on a NovaSeq X Plus (Illumina).

A Snakemake (v8.14) pipeline (available at https://github.com/jbloomlab/Virus-Antibody-Passaging/tree/main) was used to process sequencing reads and identify viral mutations that emerged during passaging87. The pipeline first preprocesses the unaligned reads with fastp (v0.23) using default settings to automatically identify and trim adapter sequences and filter low-quality bases88. The processed reads from the sequenced viral stock were then used to assemble a reference genome by aligning to the HMPV CAN97-83 sequence (AY297749) with BWA mem (v0.7), followed by generating a consensus sequence from the most frequent base at each position using iVar (v1.4.3)89,90. The remaining processed sequencing data—comprising two replicates each of 4F11-passaged virus and MxR-passaged virus, plus an untreated control—were aligned to the stock consensus using BWA mem. Following alignment, variants were identified using iVar with a minimum base quality threshold of 20. To remove potential false positives, heuristic filters were applied such that final reported variants must exceed 3% frequency, occur at sites covered by more than 200 reads, and be assigned a statistically significant p-value of <0.05 by iVar.

Animals and viral challenge
All procedures were reviewed and approved by the Fred Hutch Institutional Animal Care and Use Committee (PROTO202300013) and conducted in accordance with institutional and National Institutes of Health guidelines. Golden Syrian hamsters (Mesocricetus auratus) were infected intranasally with 100 µL of 105 pfu HMPV. Sample sizes (n=38) with at least 5 animals per group were consistent with previously published experiments testing the efficacy of RSV mAbs in the cotton rat model60,91–93. Only male animals (4–8 weeks of age) were assessed in this work, because no association between sex and clinical outcomes has been observed in adults with RSV or HMPV94,95. Monoclonal antibody was administered intramuscularly at 2.5 or 1.25 mg/kg in 50 µL PBS, 24 h after infection. Nasal turbinates and lungs were removed for viral titration by plaque assay 4 days post-infection and clarified by centrifugation in DMEM. Confluent Vero cell monolayers were inoculated in duplicate with diluted homogenates in 24-well plates. After incubating for 1 h at 37°C, wells were overlaid with 0.8% methylcellulose (made with 1.2% of 0.05% trypsin). After 5 days, plaques were counted using the Typhoon imager to determine titers as pfu per gram of tissue.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 10. Pairwise statistical comparisons were performed using Mann–Whitney two-tailed testing. p<0.05 was considered statistically significant. Data points from individual samples are displayed.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript is exceptionally well written and presents significant findings on a novel human monoclonal antibody, 4F11, targeting hMPV. The authors describe 4F11 as binding to site 0 at the apex of the hMPV F protein, with structural characterization revealing a unique angle of Fab engagement that spans across protomers and strongly interacts with the N-linked glycan at position 172. These features clearly distinguish 4F11 from previously characterized site 0 antibodies, SAN32-2 and ADI-61026. Furthermore, the authors raise an important concern regarding the A185P mutation, which may affect vaccine immunogenicity. This insight has meaningful implications for rational vaccine design and underscores the importance of accounting for such mutations in future immunogen development.\nOverall, the manuscript makes a valuable contribution to the field and provides novel insights that could inform both therapeutic antibody development and vaccine strategies against hMPV.\nThere are three major concerns related to the structural and biophysical characterization of Fab 4F11 in complex with hMPV F.\n• Model building could be largely improved given all the refinement tools available today (Coot, Phenix and Isolde). In supplemental table 1, the clashscore ideally should be below 5 and Ramachandran favored regions should be easily refined to be greater than 95%. In addition, rotamer outliers can be lower than 1%.\n• We recommend adding a supplemental figure showing the map-to-model fit at the interface. This would help assess sidechain placement and contacts and provide strong support for the interpretation shown in Figure 2f.\n• In supplemental figure 4, authors may include both experimental binding curves and fitted curves. What fitting model was used? Without all this information, kinetics data shown in figure 2e might not be accurate.\nIn addition, there are a few minor comments and would appreciate the authors’ thoughts on these points.\n• The authors raised a concern about A185P mutation potentially affecting vaccine immunogenicity. Do you have any supporting evidence showing that 4F11 type of antibodies is immunodominant? Or has this type of antibodies turned out to be rare?\n• In the protection study shown in Figure 4, the authors used DPBS as a control and tested two doses of 4F11 in a therapeutic setting, showing reduced virus loads in both the upper and lower respiratory tracts. While the results are encouraging, including a well-known monoclonal antibody as a comparator (e.g., MxR, MPE8, etc.) would make the findings stronger. This would help show how 4F11 performs relative to existing antibodies.\n• Based on the MS, 4F11 appears to be designed for therapeutic use, given its high potency, broad cross-neutralizing activity, and resistance to escape mutations. However, it still faces a fundamental challenge common to therapeutic settings: once infection is established, much of the disease severity is driven by the host immune response rather than ongoing viral replication. We would appreciate hearing the authors’ perspective on this issue. Specifically, how do you envision overcoming this limitation to achieve meaningful therapeutic benefit in humans?", "reviewer_2": "Harris et al. discovered a potent neutralizing antibody 4F11 against human metapneumovirus (hMPV). The approach is to use single B cell sorting to isolate naturally occurring human monoclonal antibodies from human donors who have been previously infected naturally. The bait and switch strategy of binding to hMPV B2 preF antigen and screening for neutralization against hMPV A2 appears robust in this study for discovery of cross-neutralizing Abs, although the conservation of F antigen between the two subtypes are high and it is expected that anti-F antibody should cover both subtypes. The authors characterized 4F11 using several biochemical and structural analysis, including cryoEM. A novel binding mode for the antibody to interact with hMPV F antigen site 0 was identified and the binding is also glycan dependent. Interesting, 4F11 binds to hMPV preF trimer at a 1:1 ratio rather than 3:1, and appears to disrupt the trimer formation in some in vitro conditions. The authors further characterized the potency of the antibody using in vitro neutralization assays and resistance selection, as well as in vivo hamster challenge model, demonstrating its potential application for hMPV therapy.\nThe manuscript is overall well written. Antibodies against RSV F has been extensively studied, and recently a few publications also described profiling of antibody response against hMPV. However, 4F11 appears to be a novel class of site 0 antibody against hMPV which has not been identified previously, making this manuscript a valuable addition for the field to further understand immune response against hMPV.\nMajor Comments:\nThe authors should consider further discuss potential (unique) mechanism of this antibody to neutralize hMPV. It is possible that the antibody disassociates hMPV prefusion F and/or prevent the conformational change of prefusion to postfusion conformation. The author should discussion another hMPV antibody described in the literature M8C10 (Structural characterization of M8C10, a neutralizing antibody targeting a highly conserved prefusion-specific epitope on the metapneumovirus fusion trimerization interface - PubMed) as it also binds the trimer interface although the epitope appears to be different than 4F11. There are a few trimer disassociating antibodies described in the literature against other viruses as well (such as flu) and can be included in discussion as a general theme.\nThe authors suggest that the primary goal is to develop an antibody for therapeutic indication against hMPV. In that case, the role of Fc should be discussed and evaluated in in vitro (such as Fc mediated assays) and/or in vivo studies. The authors performed in vivo experiments to demonstrate the protection efficacy of these antibodies as a therapy. While the data did suggest that these antibodies could offer protection, the lack of benchmark antibodies in the in vivo study prevents comparison of these antibodies to others previously described in the field.\nSpecific Comments:\nP1 line 21 and 31: should not use the word “broad” or “broadly” here and throughout. hMPV F is highly conserved between the two subtypes, it is expected anti-F antibodies cross-react to both subtypes and different strains. In fact, this is true for most previously described hMPV F antibodies.\nP1 line 40-43: should indicate disease burden is also high in older adults. Also, should clarify the approved RSV vaccines are for older adults, while there are two approved RSV monoclonal antibodies indicated for infants.\nP2 line 40: “subtypes” instead of “strains”.\nP2 line 45: I am not convinced the construct (GCN4) used in Wen et al. is stabilized in prefusion conformation. Authors should discuss rationale for this antigen choice in the B cell sorting experiment.\nP3 line 10-11: surprised to see only 6 out of 1140 clones neutralize hMPV A2. How many of them neutralize B strain? How many of them bind preF from A strain? Authors should discuss whether the majority of the B cells binds preF but couldn’t neutralize hMPV.\nP5 line 38: is there a structural explanation as to how N466K could compensate K179E?\nP6 line 17 and 21: I believe the correct figure cited should be Fig 4, not Fig 5.\nP6 line 24-27: the dose comparison with the cited previous studies is apple to orange, as they were for prophylaxis. Especially the last sentence on the clinical dose of palivizumab and nirsevimab is not relevant here.\nP6 line 36: please clarify the the “1:1” stoichiometry is Ab to preF trimer.\nP21 line 16: “WT indicates the same WT hMPV A2 expressing a GFP reporter” – it is confusing that “WT” actually refers to a GFP reporter virus, suggest to rephrase.\n(Remarks on code availability)\nI can't access the website provided: https://github.com/jbloomlab/Virus-Antibody-Passaging/tree/main", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The use of 1:1 stoichiometry vs 3:1 stoichiometry terminology used throughout the paper is not correct. hMPV F is a homotrimer, so the majority of antibodies discovered previously are actually 1:1 (1 Fab per 1 F monomer), and it is 3:1 in this case.\nIs it possible that two or three Fabs could bind? Provide a size exclusion curve comparing this complex to that of one where other previously discovered Fabs binds in a 1:1 ratio.\nGiven the very high sequence conservation between hMPV A and B genotypes, the bait and switch methodology appears to be overstated. Based on the hundreds of antibodies previously isolated for hMPV F, it appears most neutralize both genotypes anyways.\nIs the original isotype of the sequenced B cells that produced the monoclonal antibodies known?\nPage 4 line 1: The hMPV F protein is known to dissociate into monomers easily, it is not appropriate to say that 4F11 “caused” the trimer to dissociate. However, please provide the data for this in the supplemental section.\nThe resolution of the cryoEM structure is low, which limits the significance of the work. Perhaps trying to complex with MPE8 and 4F11 would help improve the resolution, since MPE8 binds across protomers. Alternatively, complexing 4F11 with DsCavEs2-IPDS could work since this construct has interprotomer disulfide bonds.\nA map to model image of the whole complex and interaction site should be provided given the low resolution.\nDoes 4F11 bind other viral glycoproteins similar to antibodies like 2G12, given its interaction with glycan residues? At a minimum, provide binding data with pre-fusion RSV F or some other negative control antigen.\nIs the glycan important for binding? N172T is used, but why was threonine chosen and not aspartate?\nPlaque purification of viral clones is mentioned in the results, but the methods don’t mention picking individual plaques, how was this done?\nThere are no cells in the last gated population in Figure 1 b.\nIn supplemental 4, provide the raw data as well as the fit curves on top in the figures."}, "author_response": "We appreciate the generally positive comments from the the Reviewers. We would like to thank the reviewers for their detailed feedback and are grateful for the opportunity to respond. The manuscript has been revised, and the point-by-point response is found below. Reviewer comments are presented in a numbered outline (1, 2, 3), with our responses provided indented below each comment and labeled alphabetically (a, b, c). Page and line numbers refer to the manuscript document in which changes are tracked. Of note, during the initial review period, we realized that the heavy and light chain structures of our 4F11 antibody were flipped. We rebuilt our model of 4F11 with the correct heavy and light chains fitted for the cryo-EM map. We regret not catching this mistake earlier. Importantly, this correction reinforces and does not undermine the overall message of our paper. These changes are also tracked and a new PDB validation report has been provided.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript is exceptionally well written and presents significant findings on a novel human monoclonal antibody, 4F11, targeting hMPV. The authors describe 4F11 as binding to site 0 at the apex of the hMPV F protein, with structural characterization revealing a unique angle of Fab engagement that spans across protomers and strongly interacts with the N-linked glycan at position 172. These features clearly distinguish 4F11 from previously characterized site 0 antibodies, SAN32-2 and ADI-61026. Furthermore, the authors raise an important concern regarding the A185P mutation, which may affect vaccine immunogenicity. This insight has meaningful implications for rational vaccine design and underscores the importance of accounting for such mutations in future immunogen development. Overall, the manuscript makes a valuable contribution to the field and provides novel insights that could inform both therapeutic antibody development and vaccine strategies against hMPV. There are three major concerns related to the structural and biophysical characterization of Fab 4F11 in complex with hMPV F.\n1. Model building could be largely improved given all the refinement tools available today (Coot, Phenix and Isolde). In supplemental table 1, the clashscore ideally should be below 5 and Ramachandran favored regions should be easily refined to be greater than 95%. In addition, rotamer outliers can be lower than 1%.\na. We are grateful to the reviewer for their highly positive feedback. We fixed the model to have a clash score less than 5, Ramachandran favored regions greater than 95%, and rotamer outliers below 1% (Table S1).\n2. We recommend adding a supplemental figure showing the map-to-model fit at the interface. This would help assess sidechain placement and contacts and provide strong support for the interpretation shown in Figure 2f.\na. We have added a new Fig. S4 showing the map-to-model fit at the interface.\n3. In supplemental figure 4, authors may include both experimental binding curves and fitted curves. What fitting model was used? Without all this information, kinetics data shown in figure 2e might not be accurate.\na. We have added the fitted curves to Fig. S8. The use of a 1:1 fitting model is now mentioned in the figure legend.\n4. In addition, there are a few minor comments and would appreciate the authors’ thoughts on these points. The authors raised a concern about A185P mutation potentially affecting vaccine immunogenicity. Do you have any supporting evidence showing that 4F11 type of antibodies is immunodominant? Or has this type of antibodies turned out to be rare?\na. The reviewer brings up an important point about immunodominance and the potential impact of the A185P-stabilizing mutation on vaccine immunogenicity. We have included a more in-depth discussion regarding this point on page 8, lines 33-41: “The glycan shield of HMPV F contributes to the rarity of neutralizing monoclonal antibodies targeting the apex (Rush, et. al., Cell Rep, 2022; Rappazzo et. al., Immunity, 2022; Guo, et. al., Lancet Microbe, 2023), and the A185P mutation is protected by this glycan shield. Since no other neutralizing monoclonal antibody to HMPV has previously been described to bind vertically at the apex, the 4F11 epitope is subdominant. Although we have shown that 4F11-like antibodies can be extremely potent, they may not make a major contribution to vaccine immunogenicity due to their rarity. Further studies comparing vaccine candidates with and without the A185P mutation will be needed to assess potential differences in vaccine immunogenicity. Nevertheless, the introduction of stabilizing mutations within neutralizing epitopes, such as Pro185, could lead to vaccines that miss an opportunity to elicit a subset of potent neutralizing mAbs.”\n5. In the protection study shown in Figure 4, the authors used DPBS as a control and tested two doses of 4F11 in a therapeutic setting, showing reduced virus loads in both the upper and lower respiratory tracts. While the results are encouraging, including a well-known monoclonal antibody as a comparator (e.g., MxR, MPE8, etc.) would make the findings stronger. This would help show how 4F11 performs relative to existing antibodies.\na. We thank the reviewer for this feedback. As suggested, we have now included a new Fig. 4c comparing the in vivo therapeutic efficacy of 4F11 with the cross-neutralizing monoclonal antibody MxR. We have discussed these new results on page 7, lines 8-9: “In contrast to 4F11, the cross-neutralizing mAb MxR failed to block viral replication in the lungs of any animals at a dose of 2.5 mg/kg (Fig. 4c).”\n6. Based on the MS, 4F11 appears to be designed for therapeutic use, given its high potency, broad cross-neutralizing activity, and resistance to escape mutations. However, it still faces a fundamental challenge common to therapeutic settings: once infection is established, much of the disease severity is driven by the host immune response rather than ongoing viral replication. We would appreciate hearing the authors’ perspective on this issue. Specifically, how do you envision overcoming this limitation to achieve meaningful therapeutic benefit in humans?\na. The reviewer raises a good point. We have included a more in-depth discussion regarding this point on page 9, lines 34-46 and page 10, line 1: “Although the therapeutic window for treatment of HMPV infections with monoclonal antibodies is currently undefined, the COVID-19 pandemic provided several examples of monoclonal antibodies that met endpoints as post-exposure prophylaxis or early treatment of symptomatic disease in phase III clinical trials. For instance, sotrovimab, when administered within 5 days of symptom onset, reduced the risk of hospitalization or death among high-risk patients with mild to moderate COVID-19 (NCT04545060) (Gupta, et. al., N Engl J Med, 2021). The cocktail of antibodies casirivimab and imdevimab, when administered within 7 days of symptom onset, also reduced hospitalization or death and led to more rapid symptom resolution and reduction of SARS-CoV-2 viral load among patients with risk factors for severe disease (NCT04425629) (Weinreich, et. al., N Engl J Med, 2021). This cocktail also demonstrated efficacy as post-exposure prophylaxis of symptomatic disease when administered to participants within 4 days after a household contact tested positive for SARS-CoV-2 (NCT04452318) (O’Brien, et. al., N Engl J Med, 2021). These examples provide proof-of-concept that administration of monoclonal antibodies within a few days of exposure or symptom onset can prevent respiratory viral disease. Future clinical trials will be needed to define the therapeutic window for HMPV.”\nReviewer #2 (Remarks to the Author):\nHarris et al. discovered a potent neutralizing antibody 4F11 against human metapneumovirus (hMPV). The approach is to use single B cell sorting to isolate naturally occurring human monoclonal antibodies from human donors who have been previously infected naturally. The bait and switch strategy of binding to hMPV B2 preF antigen and screening for neutralization against hMPV A2 appears robust in this study for discovery of cross-neutralizing Abs, although the conservation of F antigen between the two subtypes are high and it is expected that anti-F antibody should cover both subtypes. The authors characterized 4F11 using several biochemical and structural analysis, including cryoEM. A novel binding mode for the antibody to interact with hMPV F antigen site 0 was identified and the binding is also glycan dependent. Interesting, 4F11 binds to hMPV preF trimer at a 1:1 ratio rather than 3:1, and appears to disrupt the trimer formation in some in vitro conditions. The authors further characterized the potency of the antibody using in vitro neutralization assays and resistance selection, as well as in vivo hamster challenge model, demonstrating its potential application for hMPV therapy. The manuscript is overall well written. Antibodies against RSV F has been extensively studied, and recently a few publications also described profiling of antibody response against hMPV. However, 4F11 appears to be a novel class of site 0 antibody against hMPV which has not been identified previously, making this manuscript a valuable addition for the field to further understand immune response against hMPV.\n1. Major Comments: The authors should consider further discuss potential (unique) mechanism of this antibody to neutralize hMPV. It is possible that the antibody disassociates hMPV prefusion F and/or prevent the conformational change of prefusion to postfusion conformation. The author should discussion another hMPV antibody described in the literature M8C10 (Structural characterization of M8C10, a neutralizing antibody targeting a highly conserved prefusion-specific epitope on the metapneumovirus fusion trimerization interface - PubMed) as it also binds the trimer interface although the epitope appears to be different than 4F11. There are a few trimer disassociating antibodies described in the literature against other viruses as well (such as flu) and can be included in discussion as a general theme.\na. We are grateful to reviewer #2 for their positive comments and helpful feedback. A potential mechanism of neutralization by 4F11 is stabilization of the prefusion conformation to prevent the transition to the postfusion conformation. We have added a new Fig. S9 demonstrating this. We describe this figure on page 4, lines 40-43: “When the F trimer transitions from the prefusion conformation to the postfusion conformation, site Ø undergoes large rearrangements that would cause 4F11 to clash with portions of the postfusion trimer (Fig. S9).” We have also included the following discussion to page 8, lines 3-5: “Since 4F11 binds to site Ø and is prefusion-specific, 4F11 likely neutralizes HMPV by preventing the conformational switch of F from a prefusion to postfusion state, a step essential for viral entry.”\nb. We have also added a new Fig. S5 and S6 comparing the binding of 4F11 versus M8C10 with the HMPV F trimer. We describe these figures on page 4, lines 28-35: “Indeed, when we model the trimer with two or three Fabs of 4F11 bound, the Fabs clash with each other, suggesting that there is structurally only enough room for binding by a single 4F11 Fab at a time (Fig. S5). M8C10 is another mAb that causes trimer dissociation, but it binds an epitope that is buried deep within the trimerization interface. M8C10 interferes with trimer formation likely by clashing with the non-binding protomers of the trimer (Fig. S6). In contrast, 4F11 likely facilitates trimer dissociation when more than one Fab tries to bind the trimer, because the Fabs clash with each other (Fig. S6).” We have also included more in-depth discussion on the potential mechanism of neutralization by 4F11 and how this compares to M8C10 on page 8, lines 7-17: “The HMPV prefusion F-specific monoclonal antibody M8C10 also disrupts trimer formation, similar to 4F11 (Xiao, et.al., J Virol, 2023). However, unlike 4F11 which approaches vertically to bind a quaternary epitope at the apex, the M8C10 epitope is buried deep within the core of the trimer, near antigenic sites II and III, that may only be accessible when the trimer is widely open during the process of “breathing”. Compared to 4F11 (IC range 1.0 to 10.7 50 ng/mL), M8C10 has relatively weaker neutralization potency (IC range 50 371 to 661 ng/mL). Neutralizing antibodies targeting the trimer interface of type 1 fusion proteins, like M8C10 for HMPV and several anti-HA antibodies for influenza, neutralize virus by disrupting the trimer thereby preventing subsequent fusion and viral entry (Bangaru, et. al., Cell, 2019; Turner, et. al., PLoS Biol, 2019; Zost, et. al., J Clin Invest, 2021). Therefore, it is possible that trimer dissociation by 4F11 may be another mechanism that contributes to in vitro neutralization and in vivo efficacy.”\n2. The authors suggest that the primary goal is to develop an antibody for therapeutic indication against hMPV. In that case, the role of Fc should be discussed and evaluated in in vitro (such as Fc mediated assays) and/or in vivo studies. The authors performed in vivo experiments to demonstrate the protection efficacy of these antibodies as a therapy. While the data did suggest that these antibodies could offer protection, the lack of benchmark antibodies in the in vivo study prevents comparison of these antibodies to others previously described in the field.\na. Please see our response above to Reviewer #1, comment #6 regarding the efficacy of monoclonal antibodies as therapy for respiratory viral infections.\nb. We have also included a more in-depth discussion of the role of Fc effector functions in the context of therapy on page 10, lines 1-10: Notably, similar to 4F11, sotrovimab and REGN-COV2 were formulated as IgG1 with preserved Fc effector function. The relative contribution of Fc effector function compared to neutralization for protection against disease in the setting of treatment may be dose-dependent (DiLillo, et. al., Nat Med, 2014; Yamin, et. al., Nature, 2021). For instance, in a hamster challenge model of SARS-CoV-2 infection, a clinically relevant dose of a Fc receptor binding null variant of a potent neutralizing antibody had equal potency to wild-type or Fc receptor binding enhanced variants, indicating that Fc independent neutralization was the primary mode of protection. However, at lower doses, Fc effector functions contributed to decreased viral replication and decreased weight loss. Future studies in hamsters will be needed to define the relative contribution of Fc effector functions to protection at varying doses of 4F11.”\nc. Please see our response above to Reviewer #1, comment #5 regarding the addition of a benchmark antibody to the in vivo experiments.\n3. Specific Comments: P1 line 21 and 31: should not use the word “broad” or “broadly” here and throughout. hMPV F is highly conserved between the two subtypes, it is expected anti-F antibodies cross-react to both subtypes and different strains. In fact, this is true for most previously described hMPV F antibodies.\na. We thank the reviewer for this feedback and have removed the term “broad” and “broadly” as suggested.\n4. P1 line 40-43: should indicate disease burden is also high in older adults. Also, should clarify the approved RSV vaccines are for older adults, while there are two approved RSV monoclonal antibodies indicated for infants.\na. We thank the reviewer for this feedback and included the following information on page 1, lines 42-43: “Disease burden is also high for adults aged 60 years or older (Kulkarni, et. al., Lancet Healthy Longev, 2025; Walsh, et. al., Arch Intern Med, 2008).\nb. We have modified the sentence on page 1, lines 43-45 to clarify that RSV vaccines are approved for older adults: “While several vaccines for older adults to prevent infection caused by respiratory syncytial virus (RSV), a virus related to HMPV, were approved by the Food and Drug Administration in 2023, protective vaccines for HMPV are not yet clinically available.\nc. We have also modified the sentence on page 7, lines 9-12 to clarify that RSV monoclonal antibodies are approved for prophylaxis in infants: “For comparison, RSV antibodies such as palivizumab and nirsevimab are clinically administered for prophylaxis in infants at doses higher than that used in the present study (15 mg/kg and approximately 10-20 mg/kg, respectively) (Boukhvalova, et. al., Antivir Chem Chemother, 2018; Domachowske, et. al., Pediatrics, 2024; Clegg, et. al., J Clin Pharmacol, 2024)\n5. P2 line 40: “subtypes” instead of “strains”.\na. We have made this correction and changed the terminology to “subtypes”.\n6. P2 line 45: I am not convinced the construct (GCN4) used in Wen et al. is stabilized in prefusion conformation. Authors should discuss rationale for this antigen choice in the B cell sorting experiment.\na. We agree with the reviewer that, of all the recombinant versions of HMPV F used in this paper, recombinant HMPV F with GCN4 alone is relatively less stable. However, the original paper describing this construct did observe a mixture of prefusion and postfusion conformations of the GCN4-HMPV F by negative stain electron microscopy (Wen, et. al., Nat Struct Mol Biol, 2012). We used GCN4-HMPV F as our probe for the B cell sorting experiments, because, our initial experiments were performed in 2021, and this was the only recombinant version of HMPV described at the time that demonstrated a prefusion conformation by structural analysis. Later in 2022, DS-CavE2 was reported (Hsieh, et. al., Nat Commun, 2022). It was serendipitous that we performed our B cell sorting experiments with GCN4-HMPV F, even though this was relatively less stable than the newer recombinant versions of HMPV F, because GCN4-HMPV F lacked the proline mutation at amino acid 185. We have clarified this point on page 8, lines 21-31: “We successfully identified 4F11 in our antibody discovery campaign because we had serendipitously used HMPV F without the Pro185 stabilizing mutation. GCN4-stabilized HMPV F was first described in 2012 and newer, more stable constructs of HMPV F have since been reported, including DS-CavES2 in 2022, v3B in 2023, and MPV-2c in 2024 (Wen, et. al., Nat Struct Mol Biol, 2012; Hsieh, et. al., Nat Commun, 2022; Ou, et.al., PLoS Pathog, 2023; Bakkers, et. al., Nat Commun, 2024). We used GCN4-stabilized HMPV as the probe in our B cell sorting experiments, because this was the only recombinant version of HMPV F reported at the time to adopt a prefusion conformation. Structural analysis of GCN4-HMPV F has revealed that a mixture of prefusion and postfusion conformations is present, indicating that GCN4-HMPV F is relatively less stable compared to newer versions of recombinant HMPV F (Wen, et. al., Nat Struct Mol Biol, 2012). However, if newer constructs with the Pro185 mutation had been used to sort B cells, then 4F11-like antibodies would have been missed.”\n7. P3 line 10-11: surprised to see only 6 out of 1140 clones neutralize hMPV A2. How many of them neutralize B strain? How many of them bind preF from A strain? Authors should discuss whether the majority of the B cells binds preF but couldn’t neutralize hMPV.\na. The reviewer raises a good question about the overall frequency of B cells that bind HMPV preF, neutralize HMPV A subtypes, and neutralize HMPV B subtypes. We focused on functionally screening supernatant for neutralization of HMPV A2, since our objective was to identify highly potent antibodies that could neutralize HMPV subtypes A and B. As a result, in our screen, we did not determine the frequency of B cells producing antibodies that could bind preF and that could neutralize HMPV B subtypes. To further contextualize the frequency of neutralizing B cells in our screen with the published literature on the frequency of B cells in the human repertoire that bind HMPV preF and neutralize HMPV, we have added the following discussion to page 7, lines 21-37: “Others have observed binding to HMPV preF by 0.07-3.69% of class-switched B cells (Rush, et. al., Cell Reports, 2022; Rappazo, et. al., Immunity, 2022). The expressed antibodies from 22-69% of these B cells had detectable neutralization of HMPV at concentrations up to 10 µg/mL. The majority of HMPV-neutralizing B cells (75%) were able to produce antibodies that neutralized both subtypes A and B (Rush, et. al., Cell Reports, 2022). In comparison, our study identified a relatively lower frequency (0.5%) of neutralizing B cells among isotype-switched HMPV preF-binding B cells, because our screening strategy was designed to functionally select for B cells that produce highly potent neutralizing antibodies upfront, without the need to sequence, clone, and produce monoclonal antibodies from all HMPV F-binding B cells. This screening strategy leads to detectable IgG in the supernatant of approximately 40-65% of wells with individually sorted B cells (Whaley, et. al., J Immunol Methods, 2020; Boonyaratanakornkit, et. al., Nat Commun, 2023). Of the wells with detectable IgG, approximately 35-100% have antibodies in culture supernatant that bind to the same antigen used as the B cell probe. The median IgG level is approximately 4 ng/mL, although some B cells can produce over 475 ng/mL (Whaley, et. al., J Immunol Methods, 2020). Therefore, this screening strategy favors the selection of neutralizing antibodies with an IC in the ng/mL range, like 4F11, and tends 50 to miss weaker neutralizing antibodies. Furthermore, the recombinant GCN4-HMPV F used as the B cell probe is known to contain a mixture of prefusion and postfusion conformations of HMPV F (Wen, et. al., Nat Struct Mol Biol, 2012). Therefore, HMPV postF-binding B cells were also likely individually sorted into wells and contributed to the relatively lower frequency of neutralizing B cells observed in our screen.”\n8. P5 line 38: is there a structural explanation as to how N466K could compensate K179E?\na. The reviewer raises a good question about the mechanism of epistasis for the N466K mutation. Although we do not currently have a structural explanation, we have added the following discussion to page 9, lines 9-14: “A secondary mutation N466K appears to have an epistatic effect by partially compensating for the fitness cost associated with K179E. These two amino acids, however, are on opposite ends of the trimer and do not interact in either the prefusion or postfusion conformation. Further biophysical and structural characterization of F with K179E alone and in combination with N466K are needed to elucidate the mechanism of attenuation and partial compensation.”\n9. P6 line 17 and 21: I believe the correct figure cited should be Fig 4, not Fig 5.\na. The figure numbers have been corrected.\n10. P6 line 24-27: the dose comparison with the cited previous studies is apple to orange, as they were for prophylaxis. Especially the last sentence on the clinical dose of palivizumab and nirsevimab is not relevant here.\na. We agree with the reviewer that a direct comparison of potency between prophylactic and therapeutic dosing of monoclonal antibodies cannot be made. Our intent was to indicate that the doses used in our in vivo experiments are lower than the doses of other monoclonal antibodies in clinical use, which is an important consideration from the standpoint of toxicity with escalating doses. We have clarified this comparison by deleting the sentence, “In contrast, we found in a previous study that hamsters that received palivizumab at a higher dose of 5 mg/kg as prophylaxis experienced breakthrough lung infection and had no reduction in viral replication in nasal turbinates,” and adding the following sentence to page 7, lines 12-14: “Therefore, the dosage used in our in vivo experiments is lower than the dosages of other monoclonal antibodies in clinical use, which is an important consideration from the standpoint of potential toxicity in humans.”\n11. P6 line 36: please clarify the “1:1” stoichiometry is Ab to preF trimer.\na. We have clarified this point and reworded the phrase to specifically state: “1:1 stoichiometry of 4F11 Fab : preF trimer”.\n12. P21 line 16: “WT indicates the same WT hMPV A2 expressing a GFP reporter” – it is confusing that “WT” actually refers to a GFP reporter virus, suggest to rephrase.\na. We have modified the text in the legend for Fig. 1 to specifically state: “WT-GFP indicates the same HMPV-A2 expressing a GFP reporter used in the neutralization assay shown in (c). This recombinant virus was generated based on the wild-type sequence of CAN97-83.” We have also modified the label in the legend to read, “WT-GFP”.\n13. Reviewer #2 (Remarks on code availability): I can't access the website provided: https://github.com/jbloomlab/Virus-Antibody-Passaging/tree/main\na. The link is now active and accessible.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\n1. The use of 1:1 stoichiometry vs 3:1 stoichiometry terminology used throughout the paper is not correct. hMPV F is a homotrimer, so the majority of antibodies discovered previously are actually 1:1 (1 Fab per 1 F monomer), and it is 3:1 in this case.\na. The reviewer is correct that the majority of antibodies previously discovered bind with 1 Fab per 1 F monomer, or 3 Fabs per 1 preF trimer. In contrast, 4F11 binds with 1 Fab per 1 preF trimer. We have clarified that the stoichiometry is in reference to the preF trimer and now specifically state on page 1, lines 26-28: “4F11 targets an epitope located at the apex of the prefusion F protein (site Ø) with a 1:1 stoichiometry of Fab to trimer, distinct from the 3:1 stoichiometry observed with other HMPV site Ø antibodies.”\n2. Is it possible that two or three Fabs could bind? Provide a size exclusion curve comparing this complex to that of one where other previously discovered Fabs binds in a 1:1 ratio.\na. Based on our structural analysis, this is not possible since a second or third Fab would clash with the first Fab when they try to bind the trimer. We have included a new Fig. S5 showing this clash, and we have added the following description to page 4, lines 28-31: “Indeed, when we model the trimer with two or three Fabs of 4F11 bound, the Fabs clash with each other. This suggests that there is structurally only enough room for binding by one 4F11 Fab at a time to the trimer (Fig. S5).” Furthermore, in our cryo-EM analysis, we did not observe any particles with 2-3 Fabs bound to trimer.\n3. Given the very high sequence conservation between hMPV A and B genotypes, the bait and switch methodology appears to be overstated. Based on the hundreds of antibodies previously isolated for hMPV F, it appears most neutralize both genotypes anyways.\na. The reviewer is correct that most HMPV-neutralizing antibodies neutralize both subtypes A and B of HMPV. However, previous studies of the isotype-switched B cell repertoire have observed that approximately, 25% of HMPV-neutralizing B cells are subtype-specific (Rush, et. al., Cell Reports, 2022). We have clarified this on page 2, lines 39-43: “Although the majority of HMPV-neutralizing B cells (approximately 75%) produce antibodies that neutralize both subtypes A and B (Rush, et. al., Cell Reports, 2022), we leveraged a ‘bait and switch’ strategy to further enrich for B cells that could neutralize both strains. This strategy is based on the rationale that B cells capable of binding to one strain, while neutralizing another strain, are more likely to cross-neutralize both strains.”\n4. Is the original isotype of the sequenced B cells that produced the monoclonal antibodies known?\na. Based on sequencing, the isotype of monoclonal antibodies 4E11 and 4F11 is IgG. Sequencing analysis was unable to determine the isotype of 3B5. We have added this information to page 3, lines 14-16: “Based on sequencing analysis, 4E11 and 4F11 were derived from B cells expressing the IgG isotype. Our sequencing analysis was unable to determine the isotype for 3B5.”\n5. Page 4 line 1: The hMPV F protein is known to dissociate into monomers easily, it is not appropriate to say that 4F11 “caused” the trimer to dissociate. However, please provide the data for this in the supplemental section.\na. We agree with the reviewer that HMPV F is relatively unstable and known to dissociate on its own into monomers over time. However, the mechanism of binding by 4F11 to the trimer also likely facilitates trimer dissociation. Please see our response “b” to Reviewer #2, comment #1 regarding a comparison to other neutralizing antibodies that facilitate trimer dissociation.\nb. We have also included a new Fig. S2 comparing the size exclusion chromatography tracings of the DS-CavEs2 P185A trimer in the presence and absence of 4F11 to show that 4F11 facilitates trimer dissociation in vitro. We discuss this new figure on page 4, lines 5-12: “In contrast to 4F11, MxR binds across protomers and could help stabilize the trimer. The addition of MxR at a 3:1 molar ratio of Fab : DS-CavEs2 P185A trimer led to a peak in the SEC representing intact trimer bound to three Fabs (Fig. S2). To help stabilize the trimer, MxR Fab was precomplexed with DS-CavEs2 P185A trimer before the addition of 4F11 Fab. After addition of 4F11 to this complex, the peak representing the trimer prebound to three MxR Fabs disappeared and a new peak appeared that was shifted to the right and exited the SEC at a fraction corresponding to the size of the F monomer bound to two Fabs (one Fab each of MxR and 4F11) (Fig. S2).”\n6. The resolution of the cryoEM structure is low, which limits the significance of the work. Perhaps trying to complex with MPE8 and 4F11 would help improve the resolution, since MPE8 binds across protomers. Alternatively, complexing 4F11 with DsCavEs2-IPDS could work since this construct has interprotomer disulfide bonds.\na. We are grateful for the reviewer’s feedback. We did attempt to stabilize the trimer by adding the monoclonal antibody MxR, which is a site III-binding monoclonal antibody, similar to MPE8, and that also binds across protomers. However, we still only observed monomers. Please see our response “b” above to comment #5.\nb. Although we did not specifically complex 4F11 with DsCavEs2-IPDS, we did use v3B which is a version of HMPV preF that is stabilized with the same interprotomer disulfide bonds (V84C-A249C) as DsCavEs2-IPDS. We have added this clarification to page 4, lines 17-19: “Recombinant v3B is a prefusion stabilized variant of HMPV F that includes amino acid substitutions V84C-A249C to generate interprotomer disulfide bonds, similar to another variant DSCavEs2-IPDS (Ou, et. al., PLoS Pathog, 2023; Banerjee, et. al., Proc Natl Acad Sci U S A, 2022).”\n7. A map to model image of the whole complex and interaction site should be provided given the low resolution.\na. We have added a new Fig. S4 showing the map-to-model fit at the interface.\n8. Does 4F11 bind other viral glycoproteins similar to antibodies like 2G12, given its interaction with glycan residues? At a minimum, provide binding data with pre-fusion RSV F or some other negative control antigen.\na. Unlike the 2G12 monoclonal antibody which specifically binds only the high mannose patch of HIV gp120 with little to no contact with the polypeptide, 4F11 binds mostly to amino acids at the apex of HMPV preF. Since contact by 4F11 with the glycan at Asn172 contributes only a fraction of the overall buried surface area, we would not expect 4F11 to bind non-specifically to other viral glycoproteins. In addition, 4F11 is not expected to bind another viral glycoprotein like RSV F, because our B cell sorting strategy selected for B cells that could bind HMPV but not RSV F (Fig. 1a, b). To further demonstrate this, we have added a new Fig. S10b and discuss these binding results on page 5, lines 8-12: “Since contact by 4F11 with the glycan at Asn172 contributes only a fraction of the overall buried surface area, we would not expect 4F11 to bind non-specifically to other viral glycoproteins. Indeed, 4F11 does not bind to RSV preF (Fig. S10b), as expected since our B cell sorting strategy already selected for B cells that could bind HMPV but not RSV preF (Fig. 1a, b).”\n9. Is the glycan important for binding? N172T is used, but why was threonine chosen and not aspartate?\na. We removed the glycan three different ways: 1) N172T substitution in the glycan motif; 2) T174A substitution in the glycan motif; and 3) EndoH treatment of recombinant HMPV F after production in GnTI- 293 cells. We have added the results of EndoH treated HMPV produced in GnTI- 293 cells as new Fig. S10a. These 3 lines of evidence, along with the structural data, together indicate that the glycan is important for binding by 4F11.\nb. The reviewer brings up an excellent point about the rationale for mutating the asparagine to threonine. We have added a new Fig. S13 and the following explanation to page 6, lines 31-33: “The N172T substitution was chosen, because we found a single HMPV F sequence with a threonine at position 172 in Nextstrain. All other 998 sequences (99.9%) in Nextstrain had an asparagine.” We have also added a discussion of the potential effect of this mutation on viral escape and fitness to page 9, lines 22-27: “Mutation of the glycosylation motif at Asn172 represents another potential route of escape. However, only 1 out of 999 (0.1%) sequences on Nextstrain have a N172T substitution. This rarity in circulating HMPV strains likely reflects a substantial fitness cost of the mutation. Indeed, site-directed mutagenesis and reverse genetics were used to recover recombinant HMPV lacking glycosylation at position 172, and this virus was profoundly attenuated in vitro and in vivo (Zhang, et. al., J Gen Virol, 2011).”\n10. Plaque purification of viral clones is mentioned in the results, but the methods don’t mention picking individual plaques, how was this done?\na. We have added a description of the plaque purification technique to page 16, lines 37-43: “Vero cells were plated in 24-well plates and inoculated with serial 1:10 dilutions of virus, overlaid with methylcellulose containing 1.2% trypsin (Gibco, cat#25300054), and cultured for 5-7 days. Individual plaques were identified with fluorescent microscopy and picked with a P200 pipette tip. The aspirate containing the plaque was transferred onto Vero cells in 96-well plates containing 0.234 µg/mL of 4F11. After 7 days, supernatant was transferred to Vero cells in a T25 flask containing 0.234 µg/mL of 4F11. Purified virus was re-sequenced by Sanger sequencing.”\n11. There are no cells in the last gated population in Figure 1 b.\na. The last flow plot is derived from cells contained in the flowthrough fraction after magnetic enrichment. We included the flow plot to demonstrate the effectiveness of our magnetic enrichment strategy, which leads to depletion of antigen-specific cells in the flowthrough fraction. Therefore, the lack of cells is expected and further evidence of successful enrichment. We have clarified this in the figure legend by adding the following description: “The flowthrough fraction contains cells that did not bind the magnet and were thus depleted of antigen-specific cells.”\n12. In supplemental 4, provide the raw data as well as the fit curves on top in the figures.\na. Please see our response to reviewer #1, comment #3. We have added the fitted curves to Fig. S8."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you for addressing our three major concerns from the previous review (reviewer #1). However, after careful consideration of the revised manuscript and the authors' responses, we still have reservations about all three major concerns that require further attention.\nFor Major concerns 1 and 2\nWe appreciated authors made efforts to show the model-to-fit interface in FigS4 and figured out the heavy and light chains should flip. However, cryo-EM map seems not meet the high quality for the high-impact factor journal and dataset seems suffers from orientation bias. I will encourage the authors collect another cryo-EM dataset to achieve higher resolution, which enables accurate model building. If glycan N172 plays a key role in contacting with 4F11 Fab, then authors should also show the map and model at this binding interface instead of only showing glycan alone on F.\nFor major concern 3\nWe appreciated authors added fit and experimental curves for BLI. However, KD value of 4F11 Fab bound to monomer in the table seems incorrect if KD is truly derived from kon and koff.", "reviewer_2": "The revised manuscript has addressed my comments.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors addressed all of my concerns."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\n1. Thank you for addressing our three major concerns from the previous review (reviewer #1). However, after careful consideration of the revised manuscript and the authors' responses, we still have reservations about all three major concerns that require further attention.\nFor Major concerns 1 and 2\nWe appreciated authors made efforts to show the model-to-fit interface in FigS4 and figured out the heavy and light chains should flip. However, cryo-EM map seems not meet the high quality for the high-impact factor journal and dataset seems suffers from orientation bias. I will encourage the authors collect another cryo-EM dataset to achieve higher resolution, which enables accurate model building. If glycan N172 plays a key role in contacting with 4F11 Fab, then authors should also show the map and model at this binding interface instead of only showing glycan alone on F.\na. We agree that our cryo-EM data set would ideally have higher resolution. We have made numerous cryo-EM grids with various conditions, including pre-complexing with other monoclonal antibodies, trying different prefusion-stabilized versions of HMPV F, and trying different detergents, but we still have observed an orientation bias. We have submitted the best dataset available to enable modeling the antibody interface. We think that it is reasonable to expect that higher-resolution analysis would overall reveal the same binding mode. We have also modified the following sentence in the discussion on page 9, lines 8-11: “Further biophysical and higher resolution structural characterization of 4F11 in complex with F, F with K179E alone, and F with K179E in combination with N466K are needed to elucidate the mechanisms of neutralization escape, growth attenuation, and partial compensation.” As suggested, we have also now added Supplemental Figure 4c to show the map around the glycan at the binding interface.\n2. For major concern 3\nWe appreciated authors added fit and experimental curves for BLI. However, KD value of 4F11 Fab bound to monomer in the table seems incorrect if KD is truly derived from kon and koff.\na. The reviewer is correct to point out that the K value of 1.01E-07 differs from the D ratio = 0.74E-07 for the monomer in Figure S8. The reason for this slight discrepancy is because the values reported in Figure S8 include the average K , D average k , and average k of each binding curve. Since K is a ratio equal to dis on D , the average K can be calculated either by 1) averaging the K of each D D curve; or 2) taking the ratio . The first approach is an “average of ratios”, which weights each individual ratio ( ) equally. The second approach is a “ratio of averages”, which weights each group (k and k ) equally. dis on Mathematically, the “average of ratios” does not always equal the “ratio of averages”. In general, taking the “average of ratios” is preferred and more robust, because this approach gives each experimental condition (each binding curve) equal weight and, as a result, provides a better representation of the central tendency for affinity across multiple experiments (Liu, et al., Mabs, 2014; Kamat, et al., Anal Biochem, 2017; Lee, et al., J Mol Biol, 2020). In contrast, taking the “ratio of averages” is less preferred, because k and k are derived from fitting dis on kinetic data to non-linear models. The raw data for K , k , and k can be found D dis on in the file “Source data.xlsx”. To avoid confusion, we have clarified in the figure legend for Figure S8 that the values reported are the average K , average k , and average k of each binding curve.\non\nReviewer #2 (Remarks to the Author):\nThe revised manuscript has addressed my comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors addressed all of my concerns."}]

Decision letter