Roadblocks to translational challenges on viral pathogenesis.

Distinct roadblocks prevent translating basic findings in viral pathogenesis into therapies and implementing potential solutions in the clinic. An ongoing partnership between the Volkswagen Foundation and Nature Medicine resulted in an interactive meeting in 2012, as part of the "Herrenhausen Symposia" series. Current challenges for various fields of viral research were recognized and discussed with a goal in mind--to identify solutions and propose an agenda to address the translational barriers. Here, some of the researchers who participated at the meeting provide a concise outlook at the most pressing unmet research and clinical needs, identifying these key obstacles is a necessary step towards the prevention and cure of human viral diseases

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

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

Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance

INVESTIGATIONS OF 3-DEOXY-D-ARABINO - HEPTULOSONATE 7-PHOSPHATE SYNTHASE: I. IMMUNOLOGICAL STUDIES ON THE ISOZYMES FROM ESCHERICHIA COLI. II. PURIFICATION AND MOLECULAR CHARACTERIZATION OF ONE ISOZYME FROM CARROT (DAUCUS CAROTA)

The immunological relationships between the isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) (DAHP synthase) from Escherichia coli were studied by immunodiffusion, immunoinactivation and immunoprecipitation from solution, and competitive enzyme-linked immunosorbent assay. Antibodies directed against the phenylalanine sensitive DAHP synthase inactivated the tyrosine sensitive isozyme in solution. The tyrosine sensitive DAHP synthase reacted with antibodies against the tryptophan sensitive isozyme in solution. No cross reactivity between the three DAHP synthases was detected by immunodiffusion or competitive enzyme-linked immunosorbent assay. It was concluded that there is little immunological relationship between the DAHP synthase isozymes from E. coli. No data was obtained to indicate that there was even a single antigenic site shared by all three enzymes. DAHP synthase activity was detected in extracts of cultured cells and whole root tissue of Daucus carota after partial purification by ammonium sulfate. Unlike the enzyme from E. coli, carrot DAHP synthase was not inhibited by the aromatic amino acids; rather, the enzyme was feedback activated by tryptophan and tyrosine. The physiological significance of feedback activation of DAHP synthase in relation to shikimate pathway regulation was discussed. Chromatography of carrot root extracts on phosphocellulose resolved three DAHP synthase activities. The three isozymes exhibited similar molecular properties. One of the carrot root isozymes, DAHP synthase III, was purified about 10,000-fold. DAHP synthase III sub-units represented the major protein component in the purest enzyme preparations analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme subunits were used to prepare anti-DAHP synthase III antibodies. Native DAHP synthase III is a dimer with an estimated molecular weight of 103,000. The subunit molecular weight was estimated to be 53,000. DAHP synthase III activity in highly purified preparations was stimulated by micromolar concentrations of tryptophan and Mn(\u272+). The isozyme was inhibited by 1,3-propanediol and activated by potassium phosphate. DAHP synthase III was shown to be a hysteretic enzyme. The enzyme responded slowly to the addition of substrates which resulted in a measurable lag in its catalytic activity. Preincubation of DAHP synthase III with tryptophan or Mn(\u272+) reduced the lag in the catalytic activity. The hysteretic behavior of the enzyme was entirely eliminated by preincubation with 1,3-propanediol

Roadblocks to translational challenges on viral pathogenesis.

Distinct roadblocks prevent translating basic findings in viral pathogenesis into therapies and implementing potential solutions in the clinic. An ongoing partnership between the Volkswagen Foundation and Nature Medicine resulted in an interactive meeting in 2012, as part of the "Herrenhausen Symposia" series. Current challenges for various fields of viral research were recognized and discussed with a goal in mind--to identify solutions and propose an agenda to address the translational barriers. Here, some of the researchers who participated at the meeting provide a concise outlook at the most pressing unmet research and clinical needs, identifying these key obstacles is a necessary step towards the prevention and cure of human viral diseases