A broad-spectrum and highly potent human monoclonal antibody cocktail for rabies prophylaxis.

Post-exposure prophylaxis (PEP) is highly effective in preventing disease progression of rabies when used in timely and appropriate manner. The key treatment for PEP is infiltration of rabies immune globulin (RIG) into lesion site after bite exposure, besides wound care and vaccination. Unfortunately, however, RIG is expensive and its supply is limited. Currently, several anti-rabies virus monoclonal antibody (mAb) products are under development as alternatives to RIG, and two recently received regulatory approval in India. In this study, fully human mAbs that recognize different rabies virus glycoprotein conformational antigenic site (II and III) were created from peripheral blood mononuclear cells of heathy vaccinated subjects. These mAbs neutralized a diverse range of lyssavirus types. As at least two anti-rabies virus mAbs are recommended for use in human PEP to ensure broad coverage against diverse lyssaviruses and to minimize possible escape variants, two most potent mAbs, NP-19-9 and 11B6, were selected to be used as cocktail treatment. These two mAbs were broadly reactive to different types of lyssaviruses isolates, and were shown to have no interference with each other. These results suggest that NP-19-9 and 11B6 are potent candidates to be used for PEP, suggesting further studies involving clinical studies in human.

Broader neutralization of CT-P27 against influenza A subtypes by combining two human monoclonal antibodies.

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.

An Anti-Influenza Virus Antibody Inhibits Viral Infection by Reducing Nucleus Entry of Influenza Nucleoprotein.

To date, four main mechanisms mediating inhibition of influenza infection by anti-hemagglutinin antibodies have been reported. Anti-globular-head-domain antibodies block either influenza virus receptor binding to the host cell or progeny virion release from the host cell. Anti-stem region antibodies hinder the membrane fusion process or induce antibody-dependent cytotoxicity to infected cells. In this study we identified a human monoclonal IgG1 antibody (CT302), which does not inhibit both the receptor binding and the membrane fusion process but efficiently reduced the nucleus entry of viral nucleoprotein suggesting a novel inhibition mechanism of viral infection by antibody. This antibody binds to the subtype-H3 hemagglutinin globular head domain of group-2 influenza viruses circulating throughout the population between 1997 and 2007.

A potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza A virus.

Effective annual influenza vaccination requires frequent changes in vaccine composition due to both antigenic shift for different subtype hemagglutinins (HAs) and antigenic drift in a particular HA. Here we present a broadly neutralizing human monoclonal antibody with an unusual binding modality. The antibody, designated CT149, was isolated from convalescent patients infected with pandemic H1N1 in 2009. CT149 is found to neutralize all tested group 2 and some group 1 influenza A viruses by inhibiting low pH-induced, HA-mediated membrane fusion. It promotes killing of infected cells by Fc-mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. X-ray crystallographic data reveal that CT149 binds primarily to the fusion domain in HA2, and the light chain is also largely involved in binding. The epitope recognized by this antibody comprises amino-acid residues from two adjacent protomers of HA. This binding characteristic of CT149 will provide more information to support the design of more potent influenza vaccines.

Phosphorylation of phospholipase C-delta 1 regulates its enzymatic activity.

Phosphorylation of phospholipase C-delta(1) (PLC-delta(1)) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC-delta(1) most potently. It was also demonstrated that PLC-delta(1) directly bound PKC-alpha via its pleckstrin homology (PH) domain. Using deletion mutants of PLC-delta(1) and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC-delta(1). In vitro phosphorylation of PLC-delta(1) by PKC stimulated [(3)H]PtdIns(4,5)P(2) hydrolyzing activity and [(3)H]Ins(1,4,5)P(3)-binding of the PLC-delta(1). On the other hand, endogenous PLC-delta(1) was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we determined that Thr209 of PLC-delta(1) is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC-delta(1) was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC-alpha reduced serine phosphorylation of PLC-delta(1) detected by an anti-phosphoserine antibody and PLC-delta(1)-dependent basal production of inositol phosphates in NIH-3T3 cells, suggesting PKC-alpha activates phosphatase or inactivates another kinase involved in PLC-delta(1) serine phosphorylation to modulate the PLC-delta(1) activity in vivo. Taken together, these results suggest that PLC-delta(1) has multiple phosphorylation sites and phosphorylation status of PLC-delta(1) regulates its activity positively or negatively depends on the phosphorylation sites.

Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation.

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

Weak response of porcine C5a receptor towards human C5a in miniature pig endothelial cells and PMNs.

BACKGROUND: The anaphylatoxin C5a is a potent inflammatory molecule generated during complement activation. Although some reports have implicated C5a in xenograft rejection, to date, the molecular compatibility between human C5a and porcine C5a receptor (C5aR) has been little studied. To examine the need for pC5aR-deficient pig in xenotransplantaion, we aimed to look at the degree of direct interaction between human C5a (recipient side) and porcine endothelial cells (PECs) and porcine polymorphonuclear neutrophils (PMNs) (donor side). METHODS: Following the treatment of human C5a to isolated porcine PMNs, transmigration of PMNs was measured by Transwell system and superoxide generation by cytochrome c reduction assay. Next, the effects of human C5a on several intracellular signaling pathways were further checked; actin cytoskeletal change was observed under a confocal microscope after staining with Alexa Fluor-546-phalloidin, intracellular calcium mobilization was measured by spectrofluorophotometer. The degree of direct effect of human C5a on porcine PMNs was compared with that in human PMNs. Finally, microarray was performed to monitor the effect of human C5a on gene expression of PEC and the expression of several candidate proteins was checked by flow cytometry. RESULTS: We found that human C5a was able to induce chemotaxis, superoxide generation, actin cytoskeletal change, and intracellular calcium mobilization in porcine PMNs. However, higher concentration of human C5a was required to stimulate porcine PMNs in comparison with activating human PMNs. The amino acid sequences of porcine C5aR with those of human C5aR showed a sequence homology of only 67%. To elucidate the effect of human C5a to PECs, microarray analysis following the treatment of PECs with human C5a was performed. These data showed that human C5a did not significantly affect gene transcription patterns in PECs. Additionally, treatment of PECs with human C5a also did not induce protein expression of several cell adhesion molecules, including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, P-selectin, and E-selectin, or secretion of interleukin-8 from PECs. CONCLUSIONS: These results suggest that human C5a may play a minor role on PEC activation possibly due to molecular incompatibility across the species barrier.

Detection of abnormally high amygdalin content in food by an enzyme immunoassay.

Amygdalin is a cyanogenic glycoside compound which is commonly found in the pits of many fruits and raw nuts. Although amygdalin itself is not toxic, it can release cyanide (CN) after hydrolysis when the pits and nuts are crushed, moistened and incubated, possibly within the gastrointestinal tract. CN reversibly inhibits cellular oxidizing enzymes and cyanide poisoning generates a range of clinical symptoms. As some pits and nuts may contain unusually high levels of amygdalin such that there is a sufficient amount to induce critical CN poisoning in humans, the detection of abnormal content of amygdalin in those pits and nuts can be a life-saving measure. Although there are various methods to detect amygdalin in food extracts, an enzyme immunoassay has not been developed for this purpose. In this study we immunized New Zealand White rabbits with an amygdalin-KLH (keyhole limpet hemocyanin) conjugate and succeeded in raising anti-sera reactive to amygdalin, proving that amygdalin can behave as a hapten in rabbits. Using this polyclonal antibody, we developed a competition enzyme immunoassay for determination of amygdalin concentration in aqueous solutions. This technique was able to effectively detect abnormally high amygdalin content in various seeds and nuts. In conclusion, we proved that enzyme immunoassay can be used to determine the amount of amygdalin in food extracts, which will allow automated analysis with high throughput.

Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades.

Generation of a rabbit V(H) domain antibody polyspecific to c-Met and adenoviral knob protein.

Several types of bispecific antibodies with affinity to both adenoviral coat proteins and a targeted antigen have been developed with the aim of providing the specific delivery of adenoviral gene therapy vehicle. From a phage display library of combinatorial dAb2s (each with an anti-adenoviral knob protein V(H) fragment linked with an anti-c-Met V(H)), we serendipitously enriched and isolated a clone, JS5, that has polyspecificity such that it binds both the adenoviral knob protein and c-Met, despite having only one V(H) domain. Our indirect observations suggest that the polyspecificity of JS5 is developed through accumulation of antibody specificity. The method of sequential immunization of a rabbit, first with the adenoviral knob protein and then with target antigens, may provide a method by which monoclonal antibodies with stand-alone polyspecificity may be developed. Such targeted polyspecific antibodies could readily be used for re-directing adenoviral vectors to target cells.

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment.

Recent development of recombinant antibody technology has enabled fusion of recombinant antibody fragment with fluorescent proteins for various applications such as flow cytometry, fluorescence immunoassay, and fluorescent microscopy. In this study, we generated various forms of green fluorescence protein (EGFP)-fused anti-c-Met antibody fragment. Among these fusion proteins, EGFP fusion to the light chain showed high expression in a soluble form of protein in E. coli, and high binding activity to c-Met. A feasibility of the constructs was further examined by replacing the Fab gene by a Fab library of catalytic subunit of protein kinase A (PKA) to construct the Fab library in EGFP fused form. We also constructed the conventional Fab library. After a series of biopanning, we found that the binding capability of EGFP-anti-PKA Fab was comparable with anti-PKA Fab. Sequence analysis of the selected clones showed > or =99% identity in amino acid sequence and shared the same CDR sequence. These results demonstrate that EGFP fusion to the light chain using our vector system does not influence the selection of reactive Fab and that this vector system is useful for EGFP fusion to Fab to develop a one-step detection system.

Increase of intracellular Ca(2+) during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose.

While the molecular mechanisms by which oxidants cause cytotoxicity are still poorly understood, disruption of Ca(2+) homeostasis appears to be one of the critical alterations during the oxidant-induced cytotoxic process. Here, we examined the possibility that oxidative stress may alter the metabolism of cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing second messenger in the heart. Isolated heart perfused by Langendorff technique was subjected to ischemia/reperfusion injury and endogenous cADPR level was determined using a specific radioimmunoassay. Following ischemia/reperfusion injury, a significant increase in intracellular cADPR level was observed. The elevation of cADPR content was closely correlated with the increase in ADP-ribosyl cyclase activity. Inclusion of oxygen free radical scavengers, 2,2,6,6-tetramethyl-1-piperidinyloxy and mannitol, in the reperfusate prevented the ischemia/reperfusion-induced increases in cADPR level and the ADP-ribosyl cyclase activity. Exposure of isolated cardiomyocytes to t-butyl hydroperoxide increased the ADP-ribosyl cyclase activity, cADPR level, and intracellular Ca(2+) concentration ([Ca(2+)](i)) and consequently resulting in cell lethal damage. The oxidant-induced elevation of [Ca(2+)](i) as well as cell lethal damage was blocked by a cADPR antagonist, 8-bromo-cADPR. These results provide evidence for involvement of cADPR and its producing enzyme in alteration of Ca(2+) homeostasis during the ischemia/reperfusion injury of the heart.