Nancy Ulbrandt

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

IntroductionA 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.MethodsBlood 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.ResultsLymphocytes 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).ConclusionsIn 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.

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

ABSTRACT 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.

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.

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

ABSTRACT 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.

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.

Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells

ABSTRACT 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.

Evidence for a novel GTPase priming step in the SRP protein targeting pathway.

Protein targeting by the signal recognition particle (SRP) pathway requires the interaction of two homologous GTPases that reciprocally regulate each others GTPase activity, the SRP signal peptide‐ binding subunit (SRP54) and the SRP receptor α‐subunit (SRα). The GTPase domain of both proteins abuts a unique ‘N domain’ that appears to facilitate external ligand binding. To examine the relationship between the unusual regulation and unique architecture of the SRP pathway GTPases, we mutated an invariant glycine in Escherichia coli SRP54 and SRα orthologs (’Ffh‘ and ‘FtsY’, respectively) that resides at the N–GTPase domain interface. A G257A mutation in Ffh produced a lethal phenotype. The mutation did not significantly affect Ffh function, but severely reduced interaction with FtsY. Likewise, mutation of FtsY Gly455 produced growth defects and inhibited interaction with Ffh. The data suggest that Ffh and FtsY interact only in a ‘primed’ conformation which requires interdomain communication. Based on these results, we propose that the distinctive features of the SRP pathway GTPases evolved to ensure that SRP and the SR engage external ligands before interacting with each other.

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.

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.

Evidence for Vaccine Synergy between Borrelia burgdorferi Decorin Binding Protein A and Outer Surface Protein A in the Mouse Model of Lyme Borreliosis

ABSTRACT Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected againstBorrelia burgdorferi challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous B. burgdorferi, B. garinii, andB. afzelii isolates, suggesting that there is synergy between these two immunogens.