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1.  Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis 
Critical Care  2014;18(1):R3.
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.
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.
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).
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.
PMCID: PMC4056005  PMID: 24387680
2.  Infection and Propagation of Human Rhinovirus C in Human Airway Epithelial Cells 
Journal of Virology  2012;86(24):13524-13532.
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.
PMCID: PMC3503113  PMID: 23035218
3.  Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion▿  
Journal of Virology  2010;84(16):8132-8140.
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.
PMCID: PMC2916538  PMID: 20519399
4.  Cell-type specific recognition of human Metapneumoviruses by RIG-I and TLR7 and viral interference of RIG-I ligand recognition by HMPVB1 Phosphoprotein 
Human Metapneumoviruses (HMPV) 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 germline encoded pattern recognition receptors and activation of cytokine and type I interferon genes. Recently, the RNA helicase Retinoic acid inducible gene (RIG-I) has been shown to sense HMPV. In this study, we investigated the ability of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I interferons (IFN). Despite the ability of both HMPV-A1 and 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 (PDC) displayed a unique ability to sense both the A1 and B1 strains and in this case sensing was via Toll-like receptor (TLR)-7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell-type specific manners.
PMCID: PMC2834787  PMID: 20042593
Viral; Signal Transduction; Knockout mouse
5.  Human Metapneumovirus Reinfection among Children in Thailand Determined by an Enzyme-Linked Immunosorbent Assay Using Purified Soluble Fusion Protein 
The Journal of infectious diseases  2008;198(6):836-842.
Human metapneumovirus (hMPV) is a newly discovered paramyxovirus that causes acute respiratory illness. Despite apparent near-universal exposure during early childhood, immunity is transient.
An indirect screening ELISA using a recombinant, soluble, fusion (F) glycoprotein derived from hMPV was used to test for anti-F IgG in 1,380 acute and convalescent sera collected from children in Kamphaeng Phet, Thailand
1,376 (99.7%) tested sera showed evidence of prior infection with hMPV. 67 children demonstrated a four-fold or greater rise in titer for an overall re-infection rate of 4.9%. Two children demonstrated evidence of an initial infection. 49 of the 69 new or re-infections occurred in 2000, accounting for 13.2% of all non-flaviviral febrile illnesses in the study population in that year. Of 69 positive cases, 89.9% reported a respiratory symptom compared to 69.2% of tested negative cases (p<.001). All positive specimens were also tested for an increase in titer to RSV F and 27% exhibited a four-fold or greater rise in titer.
These results demonstrate hMPV reinfection causing illness at rates equal to that seen for initial infections. hMPV may represent a more significant impact in older children than previously realized and may be the cause of significant outbreaks.
PMCID: PMC2648801  PMID: 18680407
Human metapneumovirus; fusion protein; reinfection; Thai children
6.  Identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus 
The Journal of General Virology  2008;89(Pt 12):3113-3118.
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.
PMCID: PMC2885031  PMID: 19008400
7.  Isolation and Characterization of Monoclonal Antibodies Which Neutralize Human Metapneumovirus In Vitro and In Vivo 
Journal of Virology  2006;80(16):7799-7806.
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.
PMCID: PMC1563801  PMID: 16873237
8.  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 
Journal of Virology  2005;79(16):10678-10689.
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.
PMCID: PMC1182652  PMID: 16051860
9.  Conformational Nature of the Borrelia burgdorferi Decorin Binding Protein A Epitopes That Elicit Protective Antibodies 
Infection and Immunity  2001;69(8):4799-4807.
Decorin binding protein A (DbpA) has been shown by several laboratories to be a protective antigen for the prevention of experimental Borrelia burgdorferi infection in the mouse model of Lyme borreliosis. However, different recombinant forms of the antigen having either lipidated amino termini, approximating the natural secretion and posttranslational processing, or nonprocessed cytosolic forms have elicited disparate levels of protection in the mouse model. We have now used the unique functional properties of this molecule to investigate the structural requirements needed to elicit a protective immune response. Genetic and physicochemical alterations to DbpA showed that the ability to bind to the ligand decorin is indicative of a potent immunogen but is not conclusive. By mutating the two carboxy-terminal nonconserved cysteines of DbpA from B. burgdorferi strain N40, we have determined that the stability afforded by the putative disulfide bond is essential for the generation of protective antibodies. This mutated protein was more sensitive to thermal denaturation and proteolysis, suggesting that it is in a less ordered state. Immunization with DbpA that was thermally denatured and functionally inactivated stimulated an immune response that was not protective and lacked bactericidal antibodies. Antibodies against conformationally altered forms of DbpA also failed to kill heterologous B. garinii and B. afzelii strains. Additionally, nonsecreted recombinant forms of DbpAN40 were found to be inferior to secreted lipoprotein DbpAN40 in terms of functional activity and antigenic potency. These data suggest that elicitation of a bactericidal and protective immune response to DbpA requires a properly folded conformation for the production of functional antibodies.
PMCID: PMC98567  PMID: 11447153
10.  Evidence for Vaccine Synergy between Borrelia burgdorferi Decorin Binding Protein A and Outer Surface Protein A in the Mouse Model of Lyme Borreliosis 
Infection and Immunity  2000;68(11):6457-6460.
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 against Borrelia 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, and B. afzelii isolates, suggesting that there is synergy between these two immunogens.
PMCID: PMC97733  PMID: 11035759
11.  The Structure of Multiple Polypeptide Domains Determines the Signal Recognition Particle Targeting Requirement of Escherichia coli Inner Membrane Proteins 
Journal of Bacteriology  1999;181(15):4561-4567.
The signal recognition particle (SRP) targeting pathway is required for the efficient insertion of many polytopic inner membrane proteins (IMPs) into the Escherichia coli inner membrane, but in the absence of SRP protein export proceeds normally. To define the properties of IMPs that impose SRP dependence, we analyzed the targeting requirements of bitopic IMPs that are structurally intermediate between exported proteins and polytopic IMPs. We found that disruption of the SRP pathway inhibited the insertion of only a subset of bitopic IMPs. Studies on a model bitopic AcrB-alkaline phosphatase fusion protein (AcrB 265-AP) showed that the SRP requirement for efficient insertion correlated with the presence of a large periplasmic domain (P1). As previously reported, perturbation of the SRP pathway also affected the insertion of a polytopic AcrB-AP fusion. Even exhaustive SRP depletion, however, failed to block the insertion of any AcrB derivative by more than 50%. Taken together, these data suggest that many proteins that are normally targeted by SRP can utilize alternative targeting pathways and that the structure of both hydrophilic and membrane-spanning domains determines the degree to which the biogenesis of a protein is SRP dependent.
PMCID: PMC103587  PMID: 10419954
12.  DbpA, but Not OspA, Is Expressed by Borrelia burgdorferi during Spirochetemia and Is a Target for Protective Antibodies 
Infection and Immunity  1998;66(11):5379-5387.
DbpA is a target for antibodies that protect mice against infection by cultured Borrelia burgdorferi. Infected mice exhibit early and sustained humoral responses to DbpA and DbpB, suggesting that these proteins are expressed in vivo. Many antigens expressed in mammals by B. burgdorferi are repressed in vitro at lower growth temperatures, and we have now extended these observations to include DbpA and DbpB. To confirm that the protective antigen DbpA is expressed in vivo and to address the question of its accessibility to antibodies during infection, we examined B. burgdorferi in blood samples from mice following cutaneous inoculation. B. burgdorferi was visualized by dark-field microscopy in plasma samples from spirochetemic mice, and an indirect immunofluorescence assay showed that these spirochetes were DbpA positive and OspA negative. We developed an ex vivo borreliacidal assay to show that hyperimmune antiserum against DbpA, but not OspA, killed these plasma-derived spirochetes, demonstrating that DbpA is accessible to antibodies during this phase of infection. Blood transferred from spirochetemic donor mice readily established B. burgdorferi infection in naive recipient mice or mice hyperimmunized with OspA, while mice hyperimmunized with DbpA showed significant protection against challenge with host-adapted spirochetes. Antiserum from persistently infected mice had borreliacidal activity against both cultured and plasma-derived spirochetes, and adsorption of this serum with DbpA substantially depleted this killing activity. Our observations show that immunization with DbpA blocks B. burgdorferi dissemination from the site of cutaneous inoculation and suggest that DbpA antibodies may contribute to control of persistent infection.
PMCID: PMC108673  PMID: 9784547

Results 1-12 (12)