The human pathogen Staphylococcus aureus causes a wide range of infections, including skin abscesses and sepsis. There is currently no licensed vaccine to prevent S. aureus infection, and its treatment has become increasingly difficult due to antibiotic resistance. One potential way to inhibit S. aureus pathogenesis is to prevent iron acquisition. The iron-regulated surface determinant (Isd) system has evolved in S. aureus to acquire hemoglobin from the human host as a source of heme-iron. In this study, we investigated the molecular and structural basis for antibody-mediated correlates against a member of the Isd system, IsdB. The association of immunoglobulin heavy chain variable region IGHV1-69 gene-encoded human monoclonal antibodies with the response against S. aureus IsdB is described using structural and functional studies to define the importance of this antibody class. We also determine that somatic hypermutation in the development of these antibodies hinders rather than fine-tunes the immune response to IsdB.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis. IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

word count: 243 19 Text word count: 6,601 20 21 2 ABSTRACT 22Rotavirus is an important cause of diarrheal disease in young mammals. Group A 23 rotavirus (RVA) causes most human rotavirus diarrheal disease and primarily affects 24 infants and young children. Group B rotavirus (RVB) has been associated with sporadic 25 outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode 26 mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 27 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of 28 unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 29 mediates syncytia formation in cultured human cells. Based on sequence alignment, 30 NSP1-1 from groups B, G, and I contain features consistent with fusion-associated 31 small transmembrane (FAST) proteins, which have previously been identified in other 32Reoviridae viruses. Like some other FAST proteins, RVB NSP1-1 is predicted to have 33 an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts 34 NSP1-1-mediated fusion, consistent with a role for this fatty-acid modification in NSP1-1 35 function. NSP1-1 from a human RVB mediates fusion of human cells but not hamster 36 cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance 37 RVA replication in human cells, both in single-cycle infection studies and during a multi-38 cycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. 39These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, 40 immune evasion, and pathogenesis. 41 42

J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, and GlaxoSmithKline, is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols) and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study.Vanderbilt University has applied for patents for some of the antibodies in this paper.

Sosuga virus (SOSV) is a recently discovered paramyxovirus with a single known human case of disease. There has been little laboratory research on SOSV pathogenesis or immunity, and no approved therapeutics or vaccines are available. Here, we report the discovery of human monoclonal antibodies (mAbs) from the circulating memory B cells of the only known human case and survivor of SOSV infection. We isolated six mAbs recognizing the functional attachment protein (HN) and 18 mAbs against the fusion (F) protein. The anti-HN mAbs all target the globular head of the HN protein and can be organized into 4 competition-binding groups that exhibit epitope diversity. The anti-F mAbs can be divided into pre- or post-fusion conformation-specific categories and further into 8 competition-binding groups. Generally, pre-fusion conformation-specific anti-F mAbs showed higher potency in neutralization assays than did mAbs only recognizing the post-fusion conformation of F protein. Most of the anti-HN mAbs were more potently neutralizing than the anti-F mAbs, with mAbs in one of the HN competition-binding groups possessing ultra-potent (<1 ng/mL) half maximal inhibitory (IC50) virus neutralization values. These findings provide insight into the molecular basis for human antibody recognition of paramyxovirus surface proteins and the mechanisms of SOSV neutralization.