Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor.

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

Development and characterization of rabbit monoclonal antibodies that recognize human spermine oxidase and application to immunohistochemistry of human cancer tissues.

The enzyme spermine oxidase (SMOX) is involved in polyamine catabolism and converts spermine to spermidine. The enzymatic reaction generates reactive hydrogen peroxide and aldehydes as by-products that can damage DNA and other biomolecules. Increased expression of SMOX is frequently found in lung, prostate, colon, stomach and liver cancer models, and the enzyme also appears to play a role in neuronal dysfunction and vascular retinopathy. Because of growing evidence that links SMOX activity with DNA damage, inflammation, and carcinogenesis, the enzyme has come into view as a potential drug target. A major challenge in cancer research is the lack of characterization of antibodies used for identification of target proteins. To overcome this limitation, we generated a panel of high-affinity rabbit monoclonal antibodies against various SMOX epitopes and selected antibodies for use in immunoblotting, SMOX quantification assays, immunofluorescence microscopy and immunohistochemistry. Immunohistochemistry analysis with the antibody SMAB10 in normal and transformed tissues confirms that SMOX is upregulated in several different cancers. Together, the panel of antibodies generated herein adds to the toolbox of high-quality reagents to study SMOX biology and to facilitate SMOX drug development.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

Transient humoral protection against H5N1 challenge after seasonal influenza vaccination of humans.

Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing 'universal' protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain 'universal' protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines.

Mechanisms of hemagglutinin targeted influenza virus neutralization.

Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing antibodies to critical processes in the viral life cycle. HA-stem binding antibodies can act intracellularly by blocking fusion between the viral and endosomal membranes and extracellularly by preventing the proteolytic activation of HA. HA-head binding antibodies prevent viral attachment and release. These insights into newly identified ways by which the human immune system can interfere with influenza virus infection may aid the development of novel universal vaccines and antivirals.

PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine.

There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to virulent poliovirus strains which can result in Vaccine Associated Paralytic Poliomyelitis (VAPP) in vaccinees. Furthermore, some OPV revertants with high transmissibility can circulate in the population as circulating Vaccine Derived Polioviruses (cVDPVs). IPV does not convey VAPP and cVDPVs but the high costs per dose and insufficient supply have rendered IPV an unfavorable option for low and middle-income countries. Here, we explored whether the human PER.C6(®) cell-line, which has the unique capability to grow at high density in suspension, under serum-free conditions, could be used as a platform for high yield production of poliovirus. PER.C6(®) cells supported replication of all three poliovirus serotypes with virus titers ranging from 9.4 log(10) to 11.1 log(10)TCID(50)/ml irrespective of the volume scale (10 ml in shaker flasks to 2 L in bioreactors). This production yield was 10-30 fold higher than in Vero cell cultures performed here, and even 100-fold higher than what has been reported for Vero cell cultures in literature [38]. In agreement, the D-antigen content per volume PER.C6(®)-derived poliovirus was on average 30-fold higher than Vero-derived poliovirus. Interestingly, PER.C6(®) cells produced on average 2.5-fold more D-antigen units per cell than Vero cells. Based on our findings, we are exploring PER.C6(®) as an interesting platform for large-scale production of poliovirus at low costs, potentially providing the basis for global supply of an affordable IPV.

Various expression-augmenting DNA elements benefit from STAR-Select, a novel high stringency selection system for protein expression.

The creation of highly productive mammalian cell lines often requires the screening of large numbers of clones, and even then expression levels are often low. Previously, we identified DNA elements, anti-repressor or STAR elements, that increase protein expression levels. These positive effects of STAR elements are most apparent when stable clones are established under high selection stringency. We therefore developed a very high selection system, STAR-Select, that allows the formation of few but highly productive clones. Here we compare the influence of STAR and other expression-augmenting DNA elements on protein expression levels in CHO-K1 cells. The comparison is done in the context of the often-used cotransfection selection procedure and in the context of the STAR-Select system. We show that STAR elements, as well as MAR elements induce the highest protein expression levels with both selection systems. Furthermore, in trans cotransfection of multiple copies of STAR and MAR elements also results in higher protein expression levels. However, highest expression levels are achieved with the STAR-Select selection system, when STAR elements or MARs are incorporated in a single construct. Our results also show that the novel STAR-Select selection system, which was developed in the context of STAR elements, is also very beneficial for the use of MAR elements.

Toxoplasmosis, an overview with emphasis on ocular involvement.

Toxoplasmosis is a common parasitic zoonosis and an important cause of abortions, mental retardation, encephalitis, blindness, and death worldwide. Although a large body of literature has emerged on the subject in the past decades, many questions about the pathogenesis and treatment of the disease remain unanswered. This review aims to provide an overview of the current insights regarding the causative parasite and the mechanisms leading to symptomatic infection with emphasis on ocular toxoplasmosis.

Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans.

Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disulfide isomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were already detectable in tears of infants. To determine the epitope containing-regions on PDI, we generated truncated versions of recombinant PDI that differ by 8-10 amino acids in length. By IB, it was found that the sIgA epitopes were confined to conserved regions of PDI, including the functionally essential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of other species, which was confirmed by recognition of human PDI by IgA in tears. In contrast, anti-T. gondii PDI antibodies generated by immunization were not able to cross-react. Binding to the thioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constituting the same domain. By consecutive investigation of the function of the protein targeted by sIgA, the presence of antibody in relation to age and analysis of the epitope constituting regions on PDI we demonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore, analysis of antibody epitopes on an antigen is a useful method to distinguish conventional, affinity-matured antibodies from natural antibodies. The presence at early age and continuity of anti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encoded antibodies in human mucosa outside of the gut. Overall, the PDI-specific antibodies are clearly part of the natural antibody repertoire, suggesting an active role for these antibodies in the innate defense against pathogens.

Protein disulfide isomerase of Toxoplasma gondii is targeted by mucosal IgA antibodies in humans.

Mass spectrometric analysis identified a 49 kDa antigen from Toxoplasma gondii as protein disulfide isomerase (PDI). This antigen is generally recognized by IgA in tears of healthy humans. We determined the complete open reading frame and expressed PDI recombinantly. Recombinant PDI was recognized by IgA in human tears known to contain antibodies specific for the 49 kDa antigen. High expression level and similarity to other protozoan PDIs suggest that T. gondii PDI might be a suitable target for recently described anti-protozoan drugs. PDI-specific antibodies clearly constitute part of the mucosal antibody repertoire possibly involved in defence against parasites.