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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the most recent global pandemic that has caused more than a million deaths around the world. The spike glycoprotein (S) drives the entry and fusion of this virus and is the main determinant of cell tropism. To explore S requirements for entry under BSL2 conditions, S has been pseudotyped onto vesicular stomatitis virus (VSV) or retroviral particles with varied success. Several alterations to S were demonstrated to improve pseudoparticle titers, but they have not been systematically compared. In this study, we produced pseudotyped VSV particles with multiple modifications to S, including truncation, mutation, and tagging strategies. The main objective of this study was to determine which modifications of the S protein optimize cell surface expression, incorporation into pseudotyped particles, and pseudoparticle entry. Removal of the last 19 residues of the cytoplasmic tail produced a hyper-fusogenic S, while removal of 21 residues increased S surface production and VSV incorporation. Additionally, we engineered a replication-competent VSV (rVSV) virus to produce the S-D614G variant with a truncated cytoplasmic tail. While the particles can be used to assess S entry requirements, the rVSV∆G/SMet1D614G∆21 virus has a poor specific infectivity (particle to infectious titer ratio).

The pneumoviruses respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two widespread human pathogens that can cause severe disease in the young, the elderly, and the immunocompromised. Despite the discovery of RSV over 60 years ago, and hMPV nearly 20 years ago, there are no approved vaccines for either virus. Antibody-mediated immunity is critical for protection from RSV and hMPV, and, until recently, knowledge of the antibody epitopes on the surface glycoproteins of RSV and hMPV was very limited. However, recent breakthroughs in the recombinant expression and stabilization of pneumovirus fusion proteins have facilitated in-depth characterization of antibody responses and structural epitopes, and have provided an enormous diversity of new monoclonal antibody candidates for therapeutic development. These new data have primarily focused on the RSV F protein, and have led to a wealth of new vaccine candidates in preclinical and clinical trials. In contrast, the major structural antibody epitopes remain unclear for the hMPV F protein. Overall, this review will cover recent advances in characterizing the antigenic sites on the RSV and hMPV F proteins.

13Human metapneumovirus is a leading cause of viral respiratory infection in children, and can 14 cause severe lower respiratory infection in infants, the elderly, and immunocompromised patients. 15However, there remain no licensed vaccines or specific treatments for hMPV infection. Although 16 the hMPV fusion (F) protein is the sole target of neutralizing antibodies, the immunological 17 properties of hMPV F are still poorly understood. To further define the humoral immune response 18 to the hMPV F protein, we isolated two new human monoclonal antibodies (mAbs), MPV458 and 19 MPV465. Both mAbs are neutralizing in vitro and target a unique antigenic site harbored within 20 the trimeric interface of the hMPV F protein. We determined both MPV458 and MPV465 have 21 higher affinity for monomeric hMPV F than trimeric hMPV F. MPV458 was co-crystallized with 22 hMPV F, and the mAb primarily interacts with an alpha helix on the F2 region of the hMPV F 23 protein. Surprisingly, the major epitope for MPV458 lies within the trimeric interface of the hMPV 24 F protein, suggesting significant breathing of the hMPV F protein must occur for hMPV F protein 25 recognition of the novel epitope. In addition, significant glycan interactions were observed with a 26 somatically mutated light chain framework residue. The data presented identifies a novel epitope 27 on the hMPV F protein for structure-based vaccine design, and provides a new mechanism for 28 human antibody neutralization of viral glycoproteins.Human metapneumovirus (hMPV) is a leading cause of viral respiratory infections in children, the 32 majority of which are seropositive for hMPV by five years of age 1 . Although hMPV was discovered 33 in 2001 2 , there are no vaccines or therapeutics approved to prevent or treat viral infection. Similar 34 to other respiratory pathogens, children, the elderly, and the immunocompromised are the major 35 groups for which hMPV infection may require hospitalization [3][4][5][6][7][8][9][10][11] . Several reports have 36 demonstrated hMPV infection can be lethal in both adults and children. In particular, haemopoietic 37 stem cell transplant patients are at high risk of severe hMPV infection [10][11][12][13] , and several outbreaks 38 of hMPV in nursing homes have been reported [14][15][16] . In addition, fatal hMPV has been observed in 39 one child during an outbreak of hMPV in a daycare center. 17 hMPV is also a significant cause of 40 febrile respiratory illness in HIV-infected patients 18 , and has been linked to exacerbations of 41 chronic obstructive pulmonary disease 19 . Co-circulation of hMPV was observed during the SARS 42 outbreak of 2003, suggesting interactions with other circulating respiratory viruses. 20-22 43 44 hMPV circulates as two genotypes, A and B, and based on the sequence variability of the surface 45 proteins, hMPV is further grouped into four subgroups, A1, A2, B1, and B2 23,24 , and two additional 46 subgroups, A2a and A2b, have been proposed 12 . hMPV has three surface glycoproteins, the small 47 hydrophob...

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