Monospecific Antibodies Research Articles

7-P

Aim Solid phase antibody detection assays using single antigens represent a cutting edge technology for screening the sera of transplant patients. Considering the large number of HLA targets, it has been difficult to QC all single allelic HLA entities with well characterized serological reagents. Our aim is to identify, generate, and characterize agreeable sera/antibodies for the quality control of single HLA antigens produced by various manufacturers. Methods Monospecific antibodies are considered the most appropriate means for the quality control of single antigens. A number of human and mouse monoclonal antibodies represent excellent reagents for quality assessment. However, monoclonal antibodies are not available for many HLA. Here, deconvolution of serological specimens, isolating mono-specific anti-HLA antibodies by an absorption-elution technology further assisted in increasing the reagent pool. We then utilize these monospecific antibodies for single HLA antigen QC purposes. Results We characterized HLA monospecific antibodies from dozens of candidate reagents. The purified antibodies were highly specific for specific HLA targets. Initial titration curves indicate an optimal performance range with a specific HLA. Thereafter, each monospecific reagent was screened for its HLA cross-reactive pattern. Crossreactivity coincides with known epitopes and suggests that clinically relevant and irrelevant epitopes are indicated. Conclusions Well-characterized sera/antibodies provide not only a consensus for antibody specificity, helping to standardize and QC existing HLA platforms, but also allow a consensus for the strength (MFI) of the antibody-HLA interaction. The provision of multiple HLA monospecific antibodies that can be harvested from patient sera by individual laboratories will enable multiple HLA single antigen QC. Furthermore, harvest of HLA monospecific sera will provide insights into HLA epitope structures, supporting the efforts to understand crossreactive HLA in the clinical lab.

Development of a caspase-3 antibody as a tool for detecting apoptosis in cells from rainbow trout (Oncorhynchus mykiss)

Background: Apoptosis is an active cell death process mediated by caspases activation, in which different extrinsic or intrinsic signalling pathways result in direct activation of effector caspases. Caspase-3 is considered to be the most important of the executioner caspases, which cause the morphological and biochemical changes detected in apoptotic cells. Different bacterial and virus pathogens have developed different strategies to survive inside the host and overcome natural protections, one of them is inducing apoptotic death in infected cells. We have demonstrated previously that Piscirickettsia salmonis activates this process in monocytes/macrophages from salmonid RTS11 cell line both by morphological and caspase detection assays; nevertheless, recognition of caspase activation by western blot was impossible since most of the commercially available antibodies for mammalian caspases are not cross-reacting. Results: We have generated a monospecific polyclonal antibody directed to an epitope region of salmonid caspase-3; the selected epitope present high homology with caspase-3 from others teleost species and includes the active site of the enzyme. The peptide was designed using bioinformatics tools and was chemically synthesized using the Fmoc strategy, analysed by RP-HPLC, its molecular weight confirmed by mass spectrometry and its structure analyzed by circular dichroism. The synthetic peptide was immunized and antibodies from ascitic fluid were enriched for immunoglobulins using caprylic acid and then purified by activated affinity columns. The anti-peptide activity of purified antibodies was verified by ELISA, and the ability of the anti-peptide to recognize salmonid caspase-3 activation was demonstrated with the molecule in P. salmonis RTS11 infected cells by western blotting, ELISA and immunocytochemistry. Conclusions: This is the first antibody available for a fish caspase, specifically for trout caspase-3, whose applications were validated by different immunological assays.

Anti-peptide antibodies: A tool for detecting IL-8 in salmonids

Background: Interleukin 8 is a chemokine that is produced by several types of cells, like macrophages and has chemotactic activity in particular on neutrophils, playing a key role during the inflammatory process. It has been demonstrated at the molecular level that this molecule is present and conserved in several vertebrate groups, pointing its importance. Analysis of the amino acid sequence of IL-8, projected from cDNA of Salmo salar, presents homology with the sequences of mammals, poultry and lamprey, indicating the presence of a homologous molecule in higher fish. However, there is no information at protein level, which allows characterizing the regulatory role of this molecule during the immune response in fish. Results: In this work, we designed and synthesized an epitope peptide of 10 residues with a purity of 95% and mass of 1158.7 kDa, which showed a random coil structure. From this peptide it was able to generate a polyclonal mono-specific antibody which was capable of detecting the whole molecule of IL-8 in tissue and cellular model of salmonids. Conclusions: The resulting antibody is a versatile tool for detecting IL-8 by different immune techniques such as ELISA, dot blot, western blotting and immunocytofluorescence. Analysis of IL-8 at proteomic level is a useful method for characterizing immune properties of this molecule in fish.

Anti-SAE antibodies in autoimmune myositis: Identification by unlabelled protein immunoprecipitation in an Italian patient cohort

IntroductionMyositis specific autoantibodies (MSAs) are useful in the diagnosis of idiopathic inflammatory myopathies and in the definition of disease subsets. The aim of this study was to set up an unlabelled protein immunoprecipitation technique for MSA identification in the sera of myositis patients, in order to identify and investigate new antibody reactivity, undetectable by currently used methods. MethodsSera of 183 patients with connective tissue diseases (75 adult dermatomyositis, 12 juvenile dermatomyositis, 43 polymyositis, 53 other connective tissue diseases) and 30 healthy controls were screened by an in-house procedure of unlabelled protein immunoprecipitation. In the same sera MSAs and myositis associated antibodies were determined by immunoblotting and immunoprecipitation for RNA. ResultsThe analytical specificity of unlabelled protein immunoprecipitation was demonstrated by testing reference sera with known antibody reactivity.Sera from five patients, affected with dermatomyositis (5/75=7%), immunoprecipitated two proteins of 40 and 90kDa apparent molecular weights respectively, consistent with the subunits of the small ubiquitin like modifier activating enzyme heterodimer (SAE1/SAE2). The identity of putative SAE immunoprecipitated proteins was confirmed by immunoblotting on immunoprecipitates using commercial monospecific antibodies to SAE1 and SAE2. Major clinical features were compared between anti-SAE positive and negative patients. Interestingly, anti-SAE positive patients had mainly skin and muscle manifestations while dysphagia, interstitial lung disease, arthritis and constitutional symptoms were absent. ConclusionsUnlabelled protein immunoprecipitation is a specific analytical approach, appropriate for the identification of the recently described anti-SAE autoantibody. We confirmed the role of anti-SAE antibody as marker of dermatomyositis.

Characterization of lacrimal sac histology: an immunohistochemical study

A prospective observational study in a university hospital setting to study the immunohistochemical (IHC) characteristics of non-neoplastic human lacrimal sac epithelium. Twenty paraffin-embedded specimens of human lacrimal sac were studied using monospecific monoclonal antibodies to 34 beta E12, cell adhesion molecule (CAM 5.2), epithelial membrane antigen (EMA), cytokeratins (CK) 7 and 20, estrogen receptor and progesterone receptor. The distribution and histologic location of IHC staining were examined qualitatively, and the IHC stains scored as positive (+) or negative (-). The haematoxylin-eosin stains were reviewed for tissue morphology. All 20 specimens were positive for 34 beta E12, CAM 5.2, EMA and CK 7 and negative for CK 20, estrogen receptor and progesterone receptor. To our knowledge, this is the first study to characterize the IHC properties of human lacrimal sac epithelium. This epithelium appears to possess consistent IHC properties as it stains for 34 beta E12, CAM 5.2, EMA and CK 7 and this information would be potentially useful in differentiating tumours arising in the region of the lacrimal sac.

Human Monoclonal Antibodies Targeting Nonoverlapping Epitopes on Insulin-like Growth Factor II as a Novel Type of Candidate Cancer Therapeutics

Soluble ligands are important targets for therapy of cancers and other diseases. Therapeutic monoclonal antibodies (mAb) against such ligands block their interactions with corresponding receptors but do not enhance their removal from the circulation and can increase their half-lives because of the long half-lives of the antibodies. We have hypothesized that mAbs targeting two or more nonoverlapping epitopes on the same ligand could form oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (FcγRs) with high avidity leading to their fast and irreversible removal from the circulation. Insulin-like growth factor II (IGF-II) is an example of such ligands and an important target for human cancer therapy. We identified two mAbs, m610.27 and m630.3, which bound to nonoverlapping epitopes on IGF-II with nanomolar affinity, and generated a bispecific antibody, m660. m660 inhibited the interaction of human IGF-II (hIGF-II) with the human breast cancer cell line MCF-7, hIGF-II-mediated IGF receptor type I and insulin receptor phosphorylation, and cell growth. In the presence of hIGF-II, large complexes of m660 were formed that bound to FcγRII-expressing BJAB cells much more efficiently than the monospecific antibody-hIGF-II complexes and were presumably phagocytosed by phorbol 12-myristate 13-acetate-stimulated macrophage-like U937 cells. A mixture of m610.27 and m630.3 exhibited similar properties. To our knowledge, these mAbs are the first reported to target nonoverlapping epitopes on a cancer-related ligand and could represent a novel class of candidate therapeutics against cancers. This approach could also be used to irreversibly eliminate other disease-related soluble ligands.

Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limited antibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccines to attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as a protective antigen. Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC) was raised in rabbits using nitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, was incubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and patho physiolgical responses were recorded. Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on im munoblot and gave protection against fatality (p< 0.01) and lung consolidation (p <0.01). Mouse weight gain showed significant difference (p< 0.05). Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussis vaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol Infect Dis 2012; 2(2): 36-43

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and −20 °C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18 kDa, as observed on SDS–PAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at −20 °C.

Development and ELISA-based detection of anti-M2e IgY antibodies using an encoding plasmid for M2e-Hsp70 C-terminal gene

Background: The use of IgYs in a variety of methods in different areas of research, diagnostics, medical application and biotechnology should be considered widely. Objectives: Development of antibodies against extra cellular domain of influenza M2 (M2e) protein in egg yolk of laying hens. Methods: A Fusion construct harboring C-terminal of bovine heat shock protein 70 (Hsp70) and influenza M2e coding genes was injected to laying hens. Serum and egg yolk antibodies were screened for the presence of anti-M2e antibodies by indirect enzyme-linked immunosorbent assay (ELISA). Results: Anti-M2e antibodies were detected in egg yolks and sera of injected hens from 13 and 7 days post injection (PI), with the peak titer detected on 41 and 35 days PI, respectively. Conclusions: Anti-M2e IgY titers could be an index for expression potential of pcDNA3.1-M2e-HspC-terminal construct in laying hens. This construct could be considered as a promising tool in production of anti-M2e polyclonal, monospecific IgY antibodies. Such anti-M2e antibodies could be exploited for influenza diagnostic and therapeutic measures.

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate &amp;lt;italic&amp;gt;Tetrahymena thermophila&amp;lt;/italic&amp;gt;

In the present study, we purified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is involved in cellular energy production and has important housekeeping functions, from the ciliate Tetrahymena thermophila using a three-step procedure. The enzyme was purified ~68 folds by ammonium sulfate precipitation, followed by two steps of column chromatography (DEAE-cellulose and Mono-S). The purified enzyme is a homotetramer with a molecular weight of ~120 kDa. Isoelectric focusing analysis showed the presence of only one basic GAPDH isoform with an isoelectric point of 8.8. Western blot analysis showed a single 32-kDa band corresponding to the enzyme subunit using a monospecific polyclonal antibody against the T. thermophila GAPDH. The maximum of enzyme activity occurred at pH 8.0 and at 30-35°C. The apparent K(m) values for both NAD(+) and D-glyceraldehyde-3-phosphate were 0.102 ± 0.012 and 0.360 ± 0.018 mM, respectively. The maximal velocity (V(max)) was 39.40 ± 2.95 U/mg. The T. thermophila GAPDH is inhibited by oxidative and nitrosative stress reagents.

Stromal cell-associated expression of kallikrein-related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients

Several members of the human kallikrein-related peptidase family, including KLK6, are up-regulated in ovarian cancer. High KLK6 mRNA or protein expression, measured by quantitative polymerase chain reaction and enzyme-linked immunoassay, respectively, was previously found to be associated with a shortened overall and progression-free survival (OS and PFS, respectively). In the present study, we aimed at analyzing KLK6 protein expression in ovarian cancer tissue by immunohistochemistry. Using a newly developed monospecific polyclonal antibody, KLK6 immunoexpression was initially evaluated in normal tissues. We observed strong staining in the brain and moderate staining in the kidney, liver, and ovary, whereas the pancreas and the skeletal muscle were unreactive, which is in line with previously published results. Next, both tumor cell- and stromal cell-associated KLK6 immunoexpression were analyzed in tumor tissue specimens of 118 ovarian cancer patients. In multivariate Cox regression analysis, only stromal cell-associated expression, besides the established clinical parameters FIGO stage and residual tumor mass, was found to be statistically significant for OS and PFS [high vs. low KLK6 expression; hazard ratio (HR), 1.92; p=0.017; HR, 1.80; p=0.042, respectively]. These results indicate that KLK6 expressed by stromal cells may considerably contribute to the aggressiveness of ovarian cancer.

Abstract 1212: The dual action antibody MEHD7945A targeting EGFR and HER3 enhances chemotherapy induced cytotoxicity in vitro and in vivo

Abstract Dysregulation of the epidermal growth factor receptor family (EGFR, HER2, HER3, HER4) by mutation and/or overexpression plays an important role in tumorigenesis, and targeted agents directed against two members of the HER/ErbB family, epidermal growth factor receptor (EGFR/HER1), and HER2/ErbB2, are used in the treatment of cancer. Extensive crosstalk seen among these receptors implies that blocking signaling of more than one receptor may be more effective in inhibiting tumor growth and circumventing resistance mechanisms than targeting individual receptors. In particular, HER3 is considered a key mediator of resistance to many targeted agents. We generated a “two-in-one” antibody, MEHD7945A, that binds to EGFR and HER3 with high affinity, inhibits receptor function and is more broadly efficacious in various tumor types when compared to monospecific anti-EGFR or anti-HER3 antibodies. Given the ability of HER3 to potently activate the PI3K survival pathway, we investigated if antagonizing ligand-dependent HER signaling with MEHD7945A in the presence of chemotherapy augments cytotoxicity. We calculated combination index values the effects of combining MEHD7945A with commonly used chemotherapeutic agents in NSCLC and colorectal cell lines in vitro and in vivo. The NSCLC lines NCI-H292, NCI-H1666, NCI-H358 and HCC827 were treated with MEHD7945A plus gemcitabine over a wide range of drug concentrations. Cell proliferation data were analyzed using CalcuSyn software and all combination index values were &amp;lt;1, demonstrating that the combination of MEHD7945A and gemcitabine inhibited proliferation synergistically in these cells. To further explore the enhanced cytotoxic effect we evaluated the combination of MEHD7945A and gemcitabine, versus each single agent, in the NCI-H1975 NSCLC xenograft model, and observed enhanced tumor regression. Similar results were observed when MEHD7945A was combined with a range of chemotherapeutic agents, including pemetrexed, docetaxel and irinotecan in H1975, H441 or SW948 xenograft models. In summary, these in vitro and in vivo results demonstrate that MEHD7945A potentiates various cytotoxic agents in a variety of tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1212. doi:1538-7445.AM2012-1212

Development of a new antibody for detecting natural killer enhancing factor (NKEF)‐like protein in infected salmonids

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.

Development of tetravalent IgG1 dual targeting IGF-1R–EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine–glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R–EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R–EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.

Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.

Structure of the Fab-Labeled “Breathing” State of Native Poliovirus

At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and N terminus of VP1 (residues 1 to 53) externalize reversibly. An Fab fragment of a monospecific antibody, which binds to residues 39 to 55 of VP1, was utilized to locate the N termini of VP1 in native (160S) particles in this "breathing" state. Fab and virus were mixed and imaged via cryogenic electron microscopy. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered cell entry intermediate (135S) particle, but the N terminus of VP1 is located near the 2-fold axes, instead of the "propeller tip" as in 135S particles.

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.

Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

The molecular biology of rotaviruses X: intercellular dissemination of rotavirus NSP4 requires glycosylation and is mediated by direct cell-cell contact through cytoplasmic extrusions

The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions, which, through site-directed mutagenesis of the N-linked glycosylation sites near the amino terminus of the protein, were shown to be dependent on its ability to become fully glycosylated. Real-time confocal microscopy was used to follow the appearance of similar cytoplasmic extrusions in virus-infected cells and revealed them to grow at a rate of ~2 microns/min to more than three cell diameters and to have a lifespan of 30-60 minutes. CellTracker dyes were used to label cell populations and facilitate the monitoring of the transfer of cytoplasm from virus-infected to surrounding uninfected cells through cytoplasmic extrusions that broke down to vesicles, seen both on the surface of and within uninfected cells in mixed cell populations. Staining of these tagged cell mixtures with monospecific antibody to NSP4 revealed the presence of the protein on uninfected cells, suggesting that the cytoplasmic extrusions formed by virus-infected cells facilitates the direct cell-cell spread of NSP4. This direct cell-cell transfer of infected cell material triggered by expression of glycosylated NSP4 in virus-infected cells may contribute to viral pathogenesis and facilitate host invasion by rotaviruses.

Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies

Abstract 1114▪▪This icon denotes a clinically relevant abstractAntibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells. Disclosures:No relevant conflicts of interest to declare.