Prevalence and Significance of Substitutions in the Fusion Protein of Respiratory Syncytial Virus Resulting in Neutralization Escape From Antibody MEDI8897.

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.

A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants.

Prevention of respiratory syncytial virus (RSV) illness in all infants is a major public health priority. However, no vaccine is currently available to protect this vulnerable population. Palivizumab, the only approved agent for RSV prophylaxis, is limited to high-risk infants, and the cost associated with the requirement for dosing throughout the RSV season makes its use impractical for all infants. We describe the development of a monoclonal antibody as potential RSV prophylaxis for all infants with a single intramuscular dose. MEDI8897*, a highly potent human antibody, was optimized from antibody D25, which targets the prefusion conformation of the RSV fusion (F) protein. Crystallographic analysis of Fab in complex with RSV F from subtypes A and B reveals that MEDI8897* binds a highly conserved epitope. MEDI8897* neutralizes a diverse panel of RSV A and B strains with >50-fold higher activity than palivizumab. At similar serum concentrations, prophylactic administration of MEDI8897* was ninefold more potent than palivizumab at reducing pulmonary viral loads by >3 logs in cotton rats infected with either RSV A or B subtypes. MEDI8897 was generated by the introduction of triple amino acid substitutions (YTE) into the Fc domain of MEDI8897*, which led to more than threefold increased half-life in cynomolgus monkeys compared to non-YTE antibody. Considering the pharmacokinetics of palivizumab in infants, which necessitates five monthly doses for protection during an RSV season, the high potency and extended half-life of MEDI8897 support its development as a cost-effective option to protect all infants from RSV disease with once-per-RSV-season dosing in the clinic.

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus.

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized "gold standard" panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the "gold standard" diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis.

INTRODUCTION: A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients. METHODS: Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry. RESULTS: Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01). CONCLUSIONS: In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.

Infection and propagation of human rhinovirus C in human airway epithelial cells.

Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.

Prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model.

Human metapneumovirus (HMPV) is a paramyxovirus causing acute respiratory tract infections in humans. The effects of a monoclonal antibody (MAb 338, MedImmune, Inc.) directed against the HMPV fusion protein were assessed in vivo. Different groups of BALB/c mice received an intraperitoneal injection of 25 or 50mg/kg of MAb 338 either 24h before or 48h after viral infection. Lung samples were collected on days 5 and 42 after infection for determination of viral titers and histopathological changes. Pulmonary functions were also evaluated by plethysmography. On day 5 post-infection, lung viral titers were significantly decreased in mice treated with 25 or 50mg/kg before or after viral infection compared to HMPV-infected control mice. Similarly, HMPV copy numbers on day 42 were decreased for all prophylactic and therapeutic interventions. Histopathological changes were also less severe in all treated groups of mice on days 5 and 42 post-infection, correlating with decreased airways obstruction. Finally, on day 42, all treated groups had a significant decrease in airways hyperresponsiveness following treatment with MAb 338. Both prophylactic and, to a lesser extent, therapeutic administration of MAb 338 improved acute and late consequences of HMPV infection in a relevant mouse model.

Respiratory syncytial virus-neutralizing monoclonal antibodies motavizumab and palivizumab inhibit fusion.

Respiratory syncytial virus (RSV) is a major cause of virus-induced respiratory disease and hospitalization in infants. Palivizumab, an RSV-neutralizing monoclonal antibody, is used clinically to prevent serious RSV-related respiratory disease in high-risk infants. Motavizumab, an affinity-optimized version of palivizumab, was developed to improve protection against RSV. These antibodies bind RSV F protein, which plays a role in virus attachment and mediates fusion. Determining how these antibodies neutralize RSV is important to help guide development of new antibody drugs against RSV and, potentially, other viruses. This study aims to uncover the mechanism(s) by which palivizumab and motavizumab neutralize RSV. Assays were developed to test the effects of these antibodies at distinct steps during RSV replication. Pretreatment of virus with palivizumab or motavizumab did not inhibit virus attachment or the ability of F protein to interact with the target cell membrane. However, pretreatment of virus with either of these antibodies resulted in the absence of detectable viral transcription. These results show that palivizumab and motavizumab act at a point after F protein initiates interaction with the cell membrane and before virus transcription. Palivizumab and motavizumab also inhibited F protein-mediated cell-to-cell fusion. Therefore, these results strongly suggest that these antibodies block both cell-to-cell and virus-to-cell fusion, since these processes are likely similar. Finally, palivizumab and motavizumab did not reduce viral budding. Based on models developed from numerous studies of viral fusion proteins, our results indicate that these antibodies may prevent conformational changes in F protein required for the fusion process.

Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

Recombinant respiratory syncytial virus F protein expression is hindered by inefficient nuclear export and mRNA processing.

Studies of the fusion activity of respiratory syncytial virus (RSV) F protein are significantly hindered by low recombinant expression levels. While infection produces F protein levels detectable by western blot, recombinant expression produces undetectable to low levels of F protein. Identifying the obstacles that hinder recombinant F protein expression may lead to improved expression and facilitate the study of F protein function. We hypothesized that nuclear localization and/or inefficient RNA polymerase II-mediated transcription contribute to poor recombinant F protein expression. This study shows a combination of stalled nuclear export, premature polyadenylation, and low mRNA abundance all contribute to low recombinant F protein expression levels. In addition, this study provides an expression optimization strategy that results in greater F protein expression levels than observed by codon-optimization of the F protein gene, which will be useful for future studies of F protein function.

Identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus.

Human metapneumovirus (hMPV) is genetically related to respiratory syncytial virus (RSV); both cause respiratory tract illnesses ranging from a mild cough to bronchiolitis and pneumonia. The F protein-directed monoclonal antibody (mAb) palivizumab has been shown to prevent severe lower respiratory tract RSV infection in animals and humans. We have previously reported on a panel of mAbs against the hMPV F protein that neutralize hMPV in vitro and, in two cases, in vivo. Here we describe the generation of hMPV mAb-resistant mutants (MARMs) to these neutralizing antibodies. Sequencing the F proteins of the hMPV MARMs identified several neutralizing epitopes. Interestingly, some of the epitopes mapped on the hMPV F protein coincide with homologous regions mapped previously on the RSV F protein, including the site against which the broadly protective mAb palivizumab is directed. This suggests that these homologous regions play important, conserved functions in both viruses.

Human metapneumovirus reinfection among children in Thailand determined by ELISA using purified soluble fusion protein.

BACKGROUND: Human metapneumovirus (hMPV) is a newly discovered paramyxovirus that causes acute respiratory illness. Despite apparent near-universal exposure during early childhood, immunity is transient. METHODS: An indirect screening enzyme-linked immunosorbent assay using a recombinant soluble fusion (F) glycoprotein derived from hMPV was used to test for anti-F IgG in 1,380 pairs of acute- and convalescent-stage serum samples collected from children in Kamphaeng Phet, Thailand. RESULTS: Of the 1,380 serum sample pairs tested, 1,376 (99.7%) showed evidence of prior infection with hMPV. Sixty-six paired specimens demonstrated a >or=4-fold rise in titer, for an overall reinfection rate of 4.9%. Two children demonstrated evidence of an initial infection. Forty-eight of the 68 new infections or reinfections occurred in 2000, accounting for 13.2% of all nonflaviviral febrile illnesses in the study population in that year. Of 68 positive cases, 85.3% complained of cough and 66.2% complained of rhinorrhea, compared with 61.4% and 49.0% of negative cases, respectively (P < .01). All positive samples were also tested for an increase in titer of antibodies to respiratory syncytial virus F, and 27% exhibited a >or=4-fold rise. CONCLUSION: These results demonstrate that hMPV reinfections cause illness at a rate equal to that seen for initial infections. hMPV may have a more significant impact in older children than previously realized and may be the cause of significant outbreaks in this population.

The prophylactic administration of a monoclonal antibody against human metapneumovirus attenuates viral disease and airways hyperresponsiveness in mice.

BACKGROUND: Human metapneumovirus (hMPV) is one of the most frequent causes of respiratory tract infections in children. Our objective was to assess the prophylactic benefit of a monoclonal antibody (mAb) against the hMPV fusion protein in a murine model. METHODS: BALB/c mice received one intramuscular injection of either 5 or 10 mg/kg of mAb 338 (MedImmune, Inc.) and were infected intranasally 24 h later with 1x10(8) TCID50 (50% tissue culture infectious dose) of hMPV. On days 5 and 42 post-infection, lung samples were collected for determination of viral titres and for histopathological studies. Pulmonary function was characterized by plethysmography. RESULTS: Mean lung viral titres were significantly lower in mice treated with 5 or 10 mg/kg of mAb 338 compared with infected controls on day 5 (283, 45.6 and 1.49x10(5) TCID50/g, respectively; P<0.05). Similarly, lung viral RNA copies were significantly reduced in treated mice on day 42 (292, 101 and 607 copies per 0.01 g of lungs for mice that received 5 mg/kg, 10 mg/kg or no mAb, respectively; P<0.05). Histopathological changes characterized by important alveolar and interstitial inflammation were less severe in treated mice on days 5 and 42 compared with control. Airways obstruction was also significantly reduced in both treated groups on days 5 and 42, but development of hyperresponsiveness following the acute phase of infection was only significantly reduced in 10 mg/kg treated mice. CONCLUSIONS: Prophylactic administration of mAb 338 attenuates acute and late consequences of hMPV disease in this mouse model.

Immunization of Syrian golden hamsters with F subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains.

Human metapneumovirus (hMPV), a newly discovered paramyxovirus, is associated with acute respiratory-tract illness, primarily in young children, individuals with underlying disease and the elderly. Two genetic lineages of hMPV circulate around the world, and viruses from these two lineages demonstrate antigenic differences. The clinical impact of hMPV warrants the development of vaccines. Recombinant soluble fusion (F) proteins of prototype viruses of the two main lineages of hMPV that can be produced in high yields have been constructed. In this study, the antigenicity, immunogenicity and protective efficacy of these soluble F subunit vaccines were evaluated in Syrian golden hamsters (Mesocricetus auratus). Immunization of hamsters with the soluble F proteins, adjuvanted with Specol or iscom matrix, induced high virus-neutralization titres, with higher titres against the homologous than the heterologous virus. The neutralizing antibodies protected from subsequent infection of the lungs with both homologous and heterologous virus. Upon challenge, viral titres in the nasal turbinates of immunized animals were reduced significantly compared with those of PBS-immunized animals. In conclusion, a soluble F subunit vaccine for hMPV that induces cross-protective immunity for infection of the lower respiratory tract in Syrian golden hamsters has been generated.

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo.

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters.

Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.

Analysis of human and primate CD2 molecules by protein sequence and epitope mapping with anti-human CD2 antibodies.

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.