Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

A broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus.

BACKGROUND: Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infection, with significant morbidity and mortality. No licensed vaccines or therapeutic agents exist. Monoclonal antibodies (mAbs) are effective at preventing other infectious diseases and could be used against HMPV in high-risk hosts. METHODS: In vitro assays were performed to assess the neutralizing activity and affinity kinetics of human mAb 54G10. A new mouse model was developed to assess prophylactic and therapeutic efficacy in vivo. The epitope of 54G10 was identified by generating mAb-resistant mutants (MARMs). RESULTS: At low concentrations, 54G10 neutralized all 4 subgroups of HMPV in vitro and had subnanomolar affinity for the fusion protein. DBA/2 mice were permissive for all 4 HMPV subgroups, and 54G10 was effective both prophylactically and therapeutically against HMPV in vivo. Sequencing of HMPV MARMs identified the 54G10 epitope, which was similar to an antigenic site on respiratory syncytial virus (RSV). 54G10 also exhibited in vitro neutralizing activity and in vivo protective and therapeutic efficacy against RSV. CONCLUSIONS: Human mAb 54G10 has broad neutralizing activity against HMPV and could have prophylactic and therapeutic utility clinically. The conserved epitope could represent a structural vaccine target for HMPV and RSV.

Lack of prion infectivity in fixed heart tissue from patients with Creutzfeldt-Jakob disease or amyloid heart disease.

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP(Sc)) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP(Sc) in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP(Sc). Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.

Antibodies produced by clonally expanded plasma cells in multiple sclerosis cerebrospinal fluid.

OBJECTIVE: Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B-cell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic. METHODS: We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy- and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections. RESULTS: Whereas humanized control rAbs derived from anti-myelin hybridomas and anti-myelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immunoreactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells. INTERPRETATION: The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.

Identifying key components of the PrPC-PrPSc replicative interface.

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.

Pathologic prion protein infects cells by lipid-raft dependent macropinocytosis.

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP(C)) to the infectious scrapie form (PrP(Sc)). However, the mechanism that exogenous PrP(Sc) infects cells and where pathologic conversion of PrP(C) to the PrP(Sc) form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP(C) to the pathologic PrP(Sc) form in N2a cells exposed to strain RML PrP(Sc) infected brain homogenates, suggesting that a critical determinant of PrP(C) conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP(Sc) infects cells by lipid raft dependent, macropinocytosis.

A recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 mug/ml and bound to hMPV F with an affinity of 9.8 x10(-10) M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

Motif-grafted antibodies containing the replicative interface of cellular PrP are specific for PrPSc.

Prion diseases are closely associated with the conversion of the cellular prion protein (PrPC) to an abnormal conformer (PrPSc) [Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. USA 95, 13363-13383]. Monoclonal antibodies that bind epitopes comprising residues 96-104 and 133-158 of PrPC potently inhibit this process, presumably by preventing heterodimeric association of PrPC and PrPSc, and suggest that these regions of PrPC may be critical components of the PrPC-PrPSc replicative interface. We reasoned that transplanting PrP sequence corresponding to these regions into a suitable carrier molecule, such as an antibody, could impart specific recognition of disease-associated forms of PrP. To test this hypothesis, polypeptides containing PrP sequence between residues 89-112 or 136-158 were used to replace the extended heavy chain complementarity-determining region 3 of an IgG antibody specific for the envelope glycoprotein of HIV-1. Herein the resulting engineered PrP-IgGs are shown to bind specifically to infective fractions of PrP in mouse, human, and hamster prion-infected tissues, but not to PrPC, other cellular components, or the HIV-1 envelope. PrPSc reactivity was abolished when the sequence of the PrP 89-112 and 136-158 grafts was mutated, scrambled, or N-terminally truncated. Our findings suggest that residues within the 89-112 and 136-158 segments of PrPC are key components of one face of the PrPC-PrPSc complex. PrPSc-specific antibodies produced by the approach described may find widespread application in the study of prion biology and replication and in the detection of infectious prions in human and animal materials.

Single-cell repertoire analysis demonstrates that clonal expansion is a prominent feature of the B cell response in multiple sclerosis cerebrospinal fluid.

Single-cell RT-PCR was used to sample CD19(+) B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominantly polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for approximately 70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.