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Background & Aims: Substitution of lysine for glutamic acid at residu 167 in Transmembrane 6 superfamily member 2 (TM6SF2) is associated with fatty liver disease and reduced plasma lipid levels. Tm6sf2-/- mice replicate the human phenotype but were not suitable for detailed mechanistic studies. As an alternative model, we generated Tm6sf2-/- rats to determine the subcellular location and function of TM6SF2. Methods: Two lines of Tm6sf2-/- rats were established using gene editing. Lipids from tissues and from newly secreted very low density lipoproteins (VLDLs) were quantified using enzymatic assays and mass spectrometry. Neutral lipids were visualized in tissue sections using Oil Red O staining. The rate of dietary triglyceride (TG) absorption and hepatic VLDL-TG secretion were compared in Tm6sf2-/- mice and in their wild-type littermates. The intracellular location of TM6SF2 was determined by cell fractionation. Finally, TM6SF2 was immunoprecipitated from liver and enterocytes to identify interacting proteins. Results: Tm6sf2-/- rats had a 6-fold higher mean hepatic TG content (56.1 ± 28.9 9 vs 9.8 ± 3.9 mg/g; P < .0001) and lower plasma cholesterol levels (99.0 ± 10.5 vs 110.6 ± 14.0 mg/dL; P = .0294) than their wild-type littermates. Rates of appearance of dietary and hepatic TG into blood were reduced significantly in Tm6sf2-/- rats (P < .001 and P < .01, respectively). Lipid content of newly secreted VLDLs isolated from perfused livers was reduced by 53% (TG) and 62% (cholesterol) (P = .005 and P = .01, respectively) in Tm6sf2-/- mice. TM6SF2 was present predominantly in the smooth endoplasmic reticulum and endoplasmic reticulum–Golgi intermediate compartments, but not in Golgi. Both apolipoprotein B-48 and acyl-CoA synthetase long chain family member 5 physically interacted with TM6SF2. Conclusions: TM6SF2 acts in the smooth endoplasmic reticulum to promote bulk lipidation of apolipoprotein B–containing lipoproteins, thus preventing fatty liver disease.

Trop-2 is a transmembrane signal transducer that can induce cancer growth. Using antibody targeting and N-terminal Edman degradation, we show here that Trop-2 undergoes cleavage in the first thyroglobulin domain loop of its extracellular region, between residues R87 and T88. Molecular modeling indicated that this cleavage induces a profound rearrangement of the Trop-2 structure, which suggested a deep impact on its biological function. No Trop-2 cleavage was detected in normal human tissues, whereas most tumors showed Trop-2 cleavage, including skin, ovary, colon, and breast cancers. Coimmunoprecipitation and mass spectrometry analysis revealed that ADAM10 physically interacts with Trop-2. Immunofluorescence/confocal time-lapse microscopy revealed that the two molecules broadly colocalize at the cell membrane. We show that ADAM10 inhibitors, siRNAs and shRNAs abolish the processing of Trop-2, which indicates that ADAM10 is an effector protease. Proteolysis of Trop-2 at R87-T88 triggered cancer cell growth both in vitro and in vivo. A corresponding role was shown for metastatic spreading of colon cancer, as the R87A-T88A Trop-2 mutant abolished xenotransplant metastatic dissemination. Activatory proteolysis of Trop-2 was recapitulated in primary human breast cancers. Together with the prognostic impact of Trop-2 and ADAM10 on cancers of the skin, ovary, colon, lung, and pancreas, these data indicate a driving role of this activatory cleavage of Trop-2 on malignant progression of tumors.

Tumor cells often switch from mitochondrial oxidative metabolism to glycolytic metabolism even under aerobic conditions. Tumor cell glycolysis is accompanied by several nonenzymatic activities among which induction of drug resistance has important therapeutic implications. In this article, we review the main aspects of glycolysis-induced drug resistance. We discuss the classes of antitumor drugs that are affected and the components of the glycolytic pathway (transporters, enzymes, metabolites) that are involved in the induction of drug resistance. Glycolysis-associated drug resistance occurs in response to stimuli, either cell-autonomous (e.g., oncoproteins) or deriving from the tumor microenvironment (e.g., hypoxia or pseudohypoxia, mechanical cues, etc.). Several mechanisms mediate the induction of drug resistance in response to glycolytic metabolism: inhibition of apoptosis, induction of epithelial-mesenchymal transition, induction of autophagy, inhibition of drug influx and increase of drug efflux. We suggest that drug resistance in response to glycolysis comes into play in presence of qualitative (e.g., expression of embryonic enzyme isoforms, post-translational enzyme modifications) or quantitative (e.g., overexpression of enzymes or overproduction of metabolites) alterations of glycolytic metabolism. We also discern similarities between changes occurring in tumor cells in response to stimuli inducing glycolysis-associated drug resistance and those occurring in cells of the innate immune system in response to danger signals and that have been referred to as danger-associated metabolic modifications. Eventually, we briefly address that also mitochondrial oxidative metabolism may induce drug resistance and discuss the therapeutic implications deriving from the fact that the main energy-generating metabolic pathways may be both at the origin of antitumor drug resistance.

Background and aims: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4β7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. Methods: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. Results: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. Conclusions: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues. Keywords: Chemokine Receptors; Dopaminergic Regulation; G Protein-Coupled Receptors Heteromers; Gut Tropism; Inflammatory Bowel Diseases; Inflammatory Colitis; T Cell Migration.

Malaria vaccine candidate RTS,S/AS01 is based on the central and C-terminal regions of the circumsporozoite protein (CSP) of P. falciparum. mAb397 was isolated from a volunteer in an RTS,S/AS01 clinical trial, and it protects mice from infection by malaria sporozoites. However, mAb397 originates from the less commonly used VH3-15 germline gene compared to the VH3-30/33 antibodies generally elicited by RTS,S to the central NANP repeat region of CSP. The crystal structure of mAb397 with an NPNA4 peptide shows that the central NPNA forms a type I β-turn and is the main recognition motif. In most anti-NANP antibodies studied to date, a germline-encoded Trp is used to engage the Pro in NPNA β-turns, but here the Trp interacts with the first Asn. This “conserved” Trp, however, can arise from different germline genes and be located in the heavy or the light chain. Variation in the terminal ψ angles of the NPNA β-turns results in different dispositions of the subsequent NPNA and, hence, different stoichiometries and modes of antibody binding to rsCSP. Diverse protective antibodies against NANP repeats are therefore not limited to a single germline gene response or mode of binding., Graphical abstract Image 1, Highlights • mAb397 from an RTS,S trial binds NANP repeats of malaria circumsporozoite protein. • Protective mAb397 is encoded by the VH3-15 gene that is rare for NANP antibodies. • Anti-NANP mAbs bind type I β-turns in three modes using germline-encoded Trp residues. • The Trp of mAb397 interacts with Asn instead of Pro in the NPNA type I β-turn. • Diversity in NANP immune responses can aid in next-generation vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease among children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of 3 anti–heat-labile enterotoxin mAbs (sIgA1, sIgA2, and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37°C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world.

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 μm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.

In 2003 the sequences of mammalian PCSK9 were reported. Radiolabeling pulse-chase analyses demonstrated that PCSK9 was synthesized as a zymogen (proPCSK9) that undergoes autocatalytic cleavage in the endoplasmic reticulum. Distinct from the other proprotein convertases, PCSK9 is secreted as an inactive enzyme in complex with its inhibitory prodomain. Its high mRNA expression in liver hepatocytes and its gene localization on chromosome 1p32, a 3rd locus associated with familial hypercholesterolemia (FH), prompted us to screen a cohort of FH patients with no variations in the LDLR and APOB, two other FH-associated loci, whereupon three patient families expressing the PCSK9 variants S127R or F216L were identified. Although Pcsk9 and Ldlr were downregulated in mice fed a cholesterol-rich diet, PCSK9 overexpression led to the degradation of the low-density lipoprotein receptor (LDLR). This led to the demonstration that gain-of-function and loss-of-function variations in PCSK9 modulate its bioactivity, whereby PCSK9 binds the LDLR in a non-enzymatic fashion to induce its degradation in endosomes/lysosomes. PCSK9 was also shown to play major roles in targeting other receptors for degradation, thereby regulating processes including hypercholesterolemia and associated atherosclerosis, vascular inflammation, viral infections, and immune checkpoint regulation in cancer. Injectable PCSK9 mAb or siRNA are currently used in clinics worldwide to treat hypercholesterolemia and could be combined with current therapies in cancer/metastasis. In this review, we discuss the critical information leading to the discovery of PCSK9 and its functional mechanism in the regulation of LDL cholesterol, as well as its evolving novel functions in both health and disease states.

Funding Information: Funding and additional information—C. G. A. has obtained funding from Maratona da Saúde 2016; CEECIND/00410/2017 financed by FCT (Portugal); ALZ AARG-19–618007 (Alzheimer’s Association); the European Union’s Horizon 2020 research and innovation program under grant agreement No 811087 (Lysocil). C. B. P. was the recipient of an FCT doctoral fellowship (PD/BD/128374/2017). T. B. is the recipient of an FCT doctoral fellowship (SFRH/BD/131513/ 2017). M. A. B. is the recipient of an FCT doctoral fellowship (2020.06758.BD). Funding Information: C. G. A. has obtained funding from Maratona da Sa?de 2016; CEECIND/00410/2017 financed by FCT (Portugal); ALZ AARG-19-618007 (Alzheimer's Association); the European Union's Horizon 2020 research and innovation program under grant agreement No 811087 (Lysocil). C. B. P. was the recipient of an FCT doctoral fellowship (PD/BD/128374/2017). T. B. is the recipient of an FCT doctoral fellowship (SFRH/BD/131513/ 2017). M. A. B. is the recipient of an FCT doctoral fellowship (2020.06758.BD). Funding Information: Acknowledgments—We thank M. Arpin (I Curie) and Z. Lenkei (ESPCI-ParisTech) for the gift of plasmids and cells, respectively. We thank Ana Cláudia Marques for her preliminary observations and lab members for helpful discussions and critical reading of the manuscript. We thank S. Marques (CEDOC Animal Facility), T. Pereira (CEDOC Microscopy Facility), and Ana Oliveira (CEDOC Cell Culture Facility) for their technical assistance. This project has received institutional funding from iNOVA4Health— UID/Multi/ 04462/2019; H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 - GA739572; the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). Publisher Copyright: © 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Genetic studies have identified BIN1 as the second most important risk locus associated with late-onset Alzheimer's disease (LOAD). However, it is unclear how mutation of this locus mechanistically promotes Alzheimer's disease (AD) pathology. Here we show the consequences of two coding variants in BIN1 (rs754834233 and rs138047593), both in terms of intracellular beta-amyloid (iAbeta) accumulation and early endosome enlargement, two interrelated early cytopathological AD phenotypes, supporting their association with LOAD risk. We previously found that Bin1 deficiency potentiates iAbeta production by enabling BACE1 cleavage of the amyloid precursor protein in enlarged early endosomes due to decreased BACE1 recycling. Here, we discovered that the expression of the two LOAD mutant forms of Bin1 does not rescue the iAbeta accumulation and early endosome enlargement induced by Bin1 knockdown and recovered by wild-type Bin1. Moreover, the overexpression of Bin1 mutants, but not wild-type Bin1, increased the iAbeta42 fragment by reducing the recycling of BACE1, which accumulated in early endosomes, recapitulating the phenotype of Bin1 knockdown. We showed that the mutations in Bin1 reduced its interaction with BACE1. The endocytic recycling of transferrin was similarly affected, indicating that Bin1 is a general regulator of endocytic recycling. These data demonstrate that the LOAD-coding variants in Bin1 lead to a loss of function in endocytic recycling, which may be an early causal mechanism of LOAD. publishersversion published

International audience; With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05g/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1g/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98ng/ml when virus output was measured at 24h post-infection, and 7.07ng/ml measured at 48h post-infection. Therapeutic (post-infection) treatment up to 24h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1g/ml and 0.05g/ml concentrations causing greater than 100-fold reduction as measured at 48h, and the 0.05g/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10g/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130g/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (alpha Tm), with a detectably higher affinity (similar to 10 mu M) of HcTnI-C27 than that of HcTnI-C27-H (similar to 15 mu M), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to aTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAMIO underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated Delta cytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from beta-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of beta-catenin signaling, which were further enhanced by the Delta cytoTrop-2 mutant. This Trop-2/E-cadherin/beta-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer. Funding Agencies|grants of Fondazione of the Cassa di Risparmio della Provincia di Chieti; Compagnia di San PaoloCompagnia di San Paolo [2489IT]; Italian Ministry of Development [MI01_00424]; Region Abruzzo (POR FESR) [C78C14000100005]; Oncoxx Biotech (Italian Ministry of University and Research, Smart Cities and Communities) [SCN_00558]; Programma Per Giovani Ricercatori "Rita Levi Montalcini", Italian Ministry of University and Research [PGR12I7N1Z]

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with nonsmall cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and crossphosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF beta-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

Graphical abstract, On March 11, 2020, the World Health Organization (WHO) declared the severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) a global pandemic. As of July 2020, SARS-CoV-2 has infected more than 14 million people and provoked more than 590,000 deaths, worldwide. From the beginning, a variety of pharmacological treatments has been empirically used to cope with the life-threatening complications associated with Corona Virus Disease 2019 (COVID-19). Thus far, only a couple of them and not consistently across reports have been shown to further decrease mortality, respect to what can be achieved with supportive care. In most cases, and due to the urgency imposed by the number and severity of the patients’ clinical conditions, the choice of treatment has been limited to repurposed drugs, approved for other indications, or investigational agents used for other viral infections often rendered available on a compassionate-use basis. The rationale for drug selection was mainly, though not exclusively, based either i) on the activity against other coronaviruses or RNA viruses in order to potentially hamper viral entry and replication in the epithelial cells of the airways, and/or ii) on the ability to modulate the excessive inflammatory reaction deriving from dysregulated host immune responses against the SARS-CoV-2. In several months, an exceptionally large number of clinical trials have been designed to evaluate the safety and efficacy of anti-COVID-19 therapies in different clinical settings (treatment or pre- and post-exposure prophylaxis) and levels of disease severity, but only few of them have been completed so far. This review focuses on the molecular mechanisms of action that have provided the scientific rationale for the empirical use and evaluation in clinical trials of structurally different and often functionally unrelated drugs during the SARS-CoV-2 pandemic.

Highlights • Hepatitis B core antigen (HBcAg) protein with PEDV epitopes can assemble into virus like particles. • Epitope placement within HBcAg can affect the immunogenicity of a vaccine. • The YSNIGVCK antigen from PEDV has a strong correlation with virus neutralization., Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.

Objective Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. Methods A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. Results In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. Conclusion Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology., Highlights • A novel anti-insulin receptor monoclonal antibody (IRAB-A) was identified that has both agonist and sensitizing activities. • IRAB-A increases the receptor's affinity for insulin by binding to an allosteric site and does not compete with insulin. • Mice injected once with IRAB-A show improved glycemia and reduced insulinemia, indicative of enhanced insulin sensitivity. • In diet induced obese mice, the insulin sensitizing effect of IRAB-A appears to depend on the degree of insulin resistance. • Chronic treatment of obese mice showed mixed effects on glucose homeostasis under normal fed or meal challenged conditions.

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process., Highlights • A Microchiroptera-specific monoclonal anti-immunoglobulin antibody was developed. • The mAb, BT1-4F10, binds to lambda immunoglobulin light chains. • All classes of immunoglobulins present in blood and secretions can be visualized. • B lymphocytes can be identified and isolated using flow cytometry. • Bat splenic B cell areas (follicles) are visualized using microscopy.

HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1JR-CSF and HIV-1Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development., Highlights • Only broadly-neutralizing IgG and IgA Abs were protective in two relevant mucosal models of HIV-1 infection. • IgG isotype variants were typically more protective than the IgA isotype variants against vaginal or rectal challenge. • Neutralization rather than IgA-specific functions may afford better protection against mucosal HIV-1 infection. Antibodies are likely to play a key role in preventing HIV-1 infection following mucosal exposure. We used two mucosal models, in conjunction with IgG and IgA versions of the same antibodies, to identify protective antibody properties relevant to vaginal and rectal transmission. Antibodies that can potently neutralize most HIV-1 strains were protective against viral challenge, but those without this capability were non-protective. The IgA antibodies were no more protective than their IgG counterparts even though IgA antibody forms are more abundant in the mucosa. These findings can help prioritize vaccine designs to those that induce potent broadly neutralizing IgG antibodies.

Background: Primary graft dysfunction (PGD) is a known acute lung injury (ALI) and a major cause of fatality post-lung transplantation. Though some long non-coding RNAs (lncRNAs) have been studied in ALI through regulation of microRNAs (miRNAs), their effects on PGD remain undefined. The present study aims to explore the underlying mechanism of lncRNA X-inactive specific transcript (XIST) in PGD after lung transplantation. Methods: Initially, the expression of miR-21, IL-12A and XIST was determined by RT-qPCR and western blot analysis. The dual luciferase reporter assay, RNA pull-down and RIP assay were performed to identify the targeting relationship between miR-21 and IL-12A and the binding relationship between miR-21 and XIST. Loss- and gain-of-function investigations were conducted in rats treated with prolonged cold ischemia and polymorphonuclear neutrophils (PMNs). Findings: miR-21 was decreased, whilst XIST and IL-12A were increased in the bronchoalveolar lavage fluid of PGD patients after lung transplantation. Enhanced miR-21 expression in rats and PMNs resulted in downregulated expression of pro-inflammatory factors and chemokines, and enhanced the apoptosis of PMNs. XIST was found to upregulate IL-12A expression in a miR-21-dependent manner. Additionally, XIST silencing enhanced the apoptosis of PMNs and inhibited the neutrophil extracellular trap (NET) formation through upregulation of miR-21 but downregulation of IL-12A in vivo. Interpretation: In summary, lncRNA XIST upregulates IL-12A by binding to miR-21, thereby inducing NET formation and accelerating PGD after lung transplantation. This suggests that inhibition of XIST and NET may be beneficial for the treatment of PGD. Keywords: Long non-coding RNA, X-inactive specific transcript, Primary graft dysfunction, Interleukin-12A, MicroRNA-21, Neutrophil extracellular trap, Competing endogenous RNA

Background Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. Objective We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. Methods The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. Results Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. Conclusions ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Neural cell adhesion molecules 1 (NCAM1) and 2 (NCAM2) belong to the cell adhesion molecules of the immunoglobulin superfamily and have been shown to regulate formation, maturation, and maintenance of synapses. NCAM1 and NCAM2 undergo proteolysis, but the identity of all the proteases involved and how proteolysis is used to regulate their functions are not known. We report here that NCAM1 and NCAM2 are BACE1 substrates in vivo. NCAM1 and NCAM2 overexpressed in HEK cells were both cleaved by metalloproteinases or BACE1, and NCAM2 was also processed by γ-secretase. We identified the BACE1 cleavage site of NCAM1 (at Glu 671) and NCAM2 (at Glu 663) using mass spectrometry and site-directed mutagenesis. Next, we assessed BACE1-mediated processing of NCAM1 and NCAM2 in the mouse brain during aging. NCAM1 and NCAM2 were cleaved in the olfactory bulb of BACE1+/+ but not BACE1−/− mice at postnatal day 10 (P10), 4 and 12 months of age. In the hippocampus, a BACE1-specific soluble fragment of NCAM1 (sNCAM1β) was only detected at P10. However, we observed an accumulation of full-length NCAM1 in hippocampal synaptosomes in 4-month-old BACE1−/− mice. We also found that polysialylated NCAM1 (PSA-NCAM1) levels were increased in BACE1−/− mice at P10 and demonstrated that BACE1 cleaves both NCAM1 and PSA-NCAM1 in vitro. In contrast, we did not find evidence for BACE1-dependent NCAM2 processing in the hippocampus at any age analyzed. In summary, our data demonstrate that BACE1 differentially processes NCAM1 and NCAM2 depending on the region of brain, subcellular localization, and age in vivo.

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.

Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8+ and CD4+ T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8+ T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4+ T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8+ or CD4+ T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8+ T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections., Highlights • Antigen delivery to DCs via CD40 is more efficient than through nine other receptors at eliciting CD8 T+ cell response. • Antigen delivery via lectins (e.g., LOX-1 and Dectin-1) is more efficient than CD40 at eliciting CD4+ T cell responses. The success of an immunotherapeutic vaccine for cancer is largely dependent on its ability to evoke potent cellular immunity. Although targeting antigens to dendritic cells (DCs) has been known to be an efficient strategy to evoke cellular immunity, which targeted receptors yield the optimal cellular immunity remained elusive. We report that targeting CD40, compared to 9 other DC receptors, results in the greatest levels of CD8+ cytotoxic T cell responses, while targeting lectins results in enhanced CD4+ helper T cell responses. The findings of this study will assist us in the rational design of immunotherapeutic vaccines against cancers.

Highlights • Recombinant TCoV N protein was efficiently produced by a prokaryotic expression system. • An antibody-capture ELISA was established with the recombinant N protein as coating antigen. • Recombinant N protein-based ELISA is more sensitive than IBV-based ELISA for anti-TCoV antibody. • The N protein-based ELISA can be applied for a rapid diagnosis and effective control of outbreaks., Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen–whose native state is susceptible to the formation of a proto-oligomeric intermediate–we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states., We show that a monoclonal antibody can act as a ‘molecular template’ in aberrant protein oligomerization, and the transient intermediate of α1-antitrypsin, a key to the molecular mechanism of disease pathogenesis, expresses a cryptic epitope also present in the oligomer.

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP–mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP–His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP–mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence., Acid ecto-phosphatase activity has been linked to Leishmania donovani virulence. In the present study, we confirm the molecular identity and characterize molecular and functional properties of an enzyme contributing to this activity, the LdMAcP, an L. donovani specific membrane histidine acid phosphatase (HAcP).

Growth hormone (GH) and structurally related cytokines regulate a great number of physiological and pathological processes. They do this by coupling their single transmembrane domain (TMD) receptors to cytoplasmic tyrosine kinases, either as homodimers or heterodimers. Recent studies have revealed that many of these receptors exist as constitutive dimers rather than being dimerized as a consequence of ligand binding, which has necessitated a new paradigm for describing their activation process. In the present study, we describe a model for activation of the tyrosine kinase Janus kinase 2 (JAK2) by the GH receptor homodimer based on biochemical data and molecular dynamics simulations. Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm. This movement slides the pseudokinase inhibitory domain of one JAK kinase away from the kinase domain of the other JAK within the receptor dimer–JAK complex, allowing the two kinase domains to interact and trans-activate. This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation. We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

Graphical abstract, Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β1-adrenoceptor (β1AR-m23 StaR), on β1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ1-AR, but not its human homologue. Specific binding of mAb3 to tβ1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β1-AR conformational states and oligomeric complexes.

Immune tolerance mechanisms supporting normal human pregnancy are exploited by breast cancer and other malignancies. We cloned from human placenta and breast cancer cells the novel human immunomodulator named placenta immunosuppressive ferritin (PLIF). PLIF is composed of a ferritin heavy chain-like domain and a novel cytokine-like domain, named C48. Both intact PLIF and C48 inhibit T cell proliferation. Blocking PLIF by specific antibodies in a tolerant breast cancer model in nude mice resulted in tumor cell apoptosis and rejection. This prompted us to study active immune preventive strategies targeting PLIF activity. Currently, we report on the design and synthesis of the novel C24D polypeptide, which inhibits the binding of PLIF to T cells and therefore inhibits the immune suppressive effect of PLIF. The effect of C24D on the generation of anti-breast cancer cytotoxic T lymphocytes (CTLs) was studied in vitro in cultures of MCF-7 (HLA-A2(+)) or T47D (HLA-A2(-)) breast cancer cells incubated with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. We found that C24D treatment exclusively induced development of CTLs. On reactivation by their specific target cells, the CTLs secreted interferon-γ and induced target apoptosis. Anti-MCF-7 CTLs were cross-cytotoxic to MDA-MB-231 (HLA-A2(+)) triple-negative breast cancer but not to T47D. Moreover, C24D treatment in vivo inhibited the growth of MCF-7 tumors engrafted in immune-compromised nude mice transfused with naïve allogeneic human PBMCs. Our results demonstrate that C24D treatment breakdown breast cancer induced tolerance enabling the initiation of effective anti-tumor immune response.

The discoidin domain receptors, DDR1 and DDR2, are two closely related receptor tyrosine kinases that are activated by triple-helical collagen in a slow and sustained manner. The DDRs have important roles in embryo development and their dysregulation is associated with human diseases, such as fibrosis, arthritis and cancer. The extracellular region of DDRs consists of a collagen-binding discoidin (DS) domain and a DS-like domain. The transmembrane region mediates the ligand-independent dimerisation of DDRs and is connected to the tyrosine kinase domain by an unusually long juxtamembrane domain. The major DDR binding site in fibrillar collagens is a GVMGFO motif (O is hydroxyproline), which is recognised by an amphiphilic trench at the top of the DS domain. How collagen binding leads to DDR activation is not understood. GVMGFO-containing triple-helical peptides activate DDRs with the characteristic slow kinetics, suggesting that the supramolecular structure of collagen is not required. Activation can be blocked allosterically by monoclonal antibodies that bind to the DS-like domain. Thus, collagen most likely causes a conformational change within the DDR dimer, which may lead to the formation of larger DDR clusters. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases., Highlights ► DDR1 and DDR2 are receptor tyrosine kinases that are activated by collagen. ► DDR activation by collagen is slow and sustained. ► The principles of collagen recognition are understood from structural studies. ► The mechanism of transmembrane signalling is currently not understood. ► Possible activation mechanisms are discussed.

Highlights • Evolution of global PEDV strains. • Cross-reactivity between PEDV and other coronaviruses and antigenic variations among different PEDV strains. • Pathologic features of different PEDV strains. • Considerations for vaccine strain selection: PEDV virulence attenuation and in vivo cross-protection among PEDV variants., Emerging and re-emerging coronaviruses cause morbidity and mortality in human and animal populations, resulting in serious public and animal health threats and economic losses. The ongoing outbreak of a highly contagious and deadly porcine epidemic diarrhea virus (PEDV) in Asia, the Americas and Europe is one example. Genomic sequence analyses of PEDV variants have revealed important insights into the evolution of PEDV. However, the antigenic variations among different PEDV strains are less explored, although they may contribute to the failure of PEDV vaccines in Asian countries. In addition, the evolution of PEDV results in variants with distinct genetic features and virulence differences; thus PEDV can serve as a model to explore the molecular mechanisms of coronavirus evolution and pathogenesis. In this article, we review the evolution, antigenic relationships and pathologic features of PEDV strains. This information and review of researches will aid in the development of strategies for control and prevention of PED.

Mitochondrial protein tyrosine phosphorylation is an important mechanism for the modulation of mitochondrial functions. In the present study, we have identified novel substrates of c-Src in mitochondria and investigated their function in the regulation of oxidative phosphorylation. The Src family kinase inhibitor PP2 {amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4d] pyrimidine} exhibits significant reduction of respiration. Similar results were obtained from cells expressing kinase-dead c-Src, which harbours a mitochondrial-targeting sequence. Phosphorylation-site analysis selects c-Src targets, including NDUFV2 (NADH dehydrogenase [ubiquinone] flavoprotein 2) at Tyr193 of respiratory complex I and SDHA (succinate dehydrogenase A) at Tyr215 of complex II. The phosphorylation of these sites by c-Src is supported by an in vivo assay using cells expressing their phosphorylation-defective mutants. Comparison of cells expressing wild-type proteins and their mutants reveals that NDUFV2 phosphorylation is required for NADH dehydrogenase activity, affecting respiration activity and cellular ATP content. SDHA phosphorylation shows no effect on enzyme activity, but perturbed electron transfer, which induces reactive oxygen species. Loss of viability is observed in T98G cells and the primary neurons expressing these mutants. These results suggest that mitochondrial c-Src regulates the oxidative phosphorylation system by phosphorylating respiratory components and that c-Src activity is essential for cell viability.

Pdx1 (pancreatic and duodenal homeobox 1), Ngn3 (neurogenin 3) and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta (β) cells in vivo. We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM (adenoviral Pdx1, Ngn3, MafA construct). When introduced into AR42j-B13 cells, Ad-PNM caused a rapid change to a flattened morphology and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are up-regulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 (insulin 1) and Ins2 (insulin 2) gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID (non-obese diabetic severe combined immunodeficiency) mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7–104%). The working range is 0.15–34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes., Highlights ► The ELISA is based on monoclonal antibodies, highly sensitive and specific, and is the first of its kind. ► Validation shows excellent reproducibility, dilution linearity and recovery. ► We find that mean serum CL-11 concentration in 100 blood donors is 284 ng/ml (95%CI = 269–299 ng/ml). ► The ELISA allows for direct comparison of plasma and serum samples. ► We measure the CL-11 concentration in persons with 3MC syndrome to be less than 2.1 ng/ml.

Cytokines are secreted from macrophages and other cells of the immune system in response to pathogens. Additionally, in autoinflammatory diseases cytokine secretion occurs in the absence of pathogenic stimuli. In the case of TRAPS [TNFR (tumour necrosis factor receptor)-associated periodic syndrome], inflammatory episodes result from mutations in the TNFRSF1A gene that encodes TNFR1. This work remains controversial, however, with at least three distinct separate mechanisms of receptor dysfunction having been proposed. Central to these hypotheses are the NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase) families of transcriptional activators that are able to up-regulate expression of a number of genes, including pro-inflammatory cytokines. The present review examines each proposed mechanism of TNFR1 dysfunction, and addresses how these processes might ultimately impact upon cytokine secretion and disease pathophysiology.

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [3H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes.

The present study examined the overloading of ion-exchange membrane adsorbers, a form of frontal chromatography, as the final purification step in the production of mAbs (monoclonal antibodies) produced from CHO (Chinese-hamster ovary) cells. Preferential binding of impurities over antibody product was exploited using commercially available cation- and anion-exchange membranes. Three different antibody feedstreams previously purified over Protein A and ion-exchange column chromatography were tested. Feedstream conductivity and pH were adjusted to induce product and impurity adsorption. Membranes were then overloaded in a normal flow mode, resulting in retention of impurities and breakthrough of purified antibody. Although some amount of the product also binds to the membranes (usuallyor =30 g mAb/l membrane), yields ofor =99% were achieved by marginalizing the losses, typically by loading more than 3 kg mAb/l membrane. Analyses of the purified pools show consistent removal of impurities despite strong mAb-ligand interactions and high membrane loadings. The clearance of host cell proteins was affected by pH and conductivity, but was unaffected by flow rate, membrane properties or scale. The importance of the present study lies in our demonstration of an alternative use of ion-exchange membranes for fast, effective and high yielding purification of mAbs.

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.

There is great interest in the antibodies-to-DNA transformation, since this change is characteristic of autoimmune diseases and contributes to its pathology. After immunization and fusions, 14 hybridomas bearing DNA-hydrolysis activity against pUC19 plasmid DNA were obtained. Genes coding for V H and V L regions of the 14 monoclonal antibodies (mAbs) were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. V genes of the H chains’ genes expressed four genes of the V H 1/J558 family, three of V H 5/V H 7183, and three of V H 8/V H 3609. The genetic repertoire of these mAbs was examined by determining the nucleotide sequences of their H chain V regions. This V H and V L domain was most similar to an anti-ssDNA (DNA-1) antibody as well as to catalytic autoimmune mAb (m3D8). Computer-generated models of the three-dimensional structures of V H and V L (VHL4) of the IgG4 combinations were used to define the positions occupied by the important sequence motifs at the binding sites. The modeling structure showed that VHL4 binds to oligo (dT3) primarily by sandwiching thymine bases between Tyr L32, Tyr L49 and Tyr H97 side-chains. Superposing VHL4 with anti-nucleic acid m3D8 catAbs revealed a common ssDNA recognition module consisting of His L93, His H35 residues which are critical for DNA-hydrolyzing antibodies. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies’ hydrolyzing activities.

L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/gamma-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominant-negative PS1 or use of a specific gamma-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or gamma-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/gamma-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines.

Leucine-rich alpha-2-glycoprotein-1 (LRG) is a serum glycoprotein of unknown function that has shown promise based on qualitative assessments as a biomarker for certain diseases including microbial infections and cancer. However, the lack of a quantitative assay for LRG has limited its application. Here an indirect enzyme-linked immunosorbent assay (ELISA) for quantifying LRG in human serum is described in which cytochrome c is employed as the capturing ligand and a monoclonal antibody specific for LRG is used to detect the captured glycoprotein. Application of this assay in quantifying LRG in various patients' sera is demonstrated. The concentration of LRG in sera of control subjects as determined by this assay is approximately 50 microg/ml. Consistent with expectations from published reports, LRG was found to be significantly elevated in the sera of some patients with a bacterial infection (toxic shock syndrome, TSS). LRG was only slightly elevated in patients infected with the human immunodeficiency virus as compared to uninfected control subjects, while normal levels of LRG were observed in patients with non-infectious diseases (inflammatory arthritis and neurological disorders, primarily Parkinson's disease). Although LRG and C-reactive protein (CRP) are both produced by the liver and are classified as acute-phase proteins, there was no significant correlation between the levels of LRG and CRP in the sera of the patients. Thus, LRG and CRP measurements are non-redundant and indicate different physiological contexts. The ELISA described in this report should be useful to further assess serum LRG as a biomarker for clinical diagnostics.

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS–CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402–622) and the N4 fragment (aa 220–336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS–CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS–CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS–CoV, these assays will be a useful tool to trace the origin and transmission of SARS–CoV and to minimise the risk of animal-to-human transmission.

Asthma is a common and debilitating disease that has substantially increased in prevalence in Western Societies in the last 2 decades. Respiratory tract infections by respiratory syncytial virus (RSV) and rhinovirus (RV) are widely implicated as common causes of the induction and exacerbation of asthma. These infections in early life are associated with the induction of wheeze that may progress to the development of asthma. Infections may also promote airway inflammation and enhance T helper type 2 lymphocyte (Th2 cell) responses that result in exacerbations of established asthma. The mechanisms of how RSV and RV induce and exacerbate asthma are currently being elucidated by clinical studies, in vitro work with human cells and animal models of disease. This research has led to many potential therapeutic strategies and, although none are yet part of clinical practise, they show much promise for the prevention and treatment of viral disease and subsequent asthma.

The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).

International audience; Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Many factors influence the choice of methods used to develop antibody to infectious agents. In this paper, we review the current status of the main technologies used to produce monoclonal antibodies (mAbs) from the B cells of antigen-sensitized animals. While companies are adopting advanced high-throughput methods, the major technologies used by veterinary and medical research laboratories are classical hybridoma fusion and recombinant library selection techniques. These methods have inherent advantages and limitations but have many common aspects when using immunized rodents. Laboratories with expertise in both methods of antibody development have a distinct advantage in their ability to advance mAb technology. New and re-emerging infectious threats in today’s world emphasize the need for quality immunoreagents and the need to maintain expertise in mAb development. We provide examples of some common applications for mAb reagents used to identify pathogens such as the SARS-coronavirus (SARS-CoV), Bacillus anthracis, and foot-and-mouth disease (FMD) virus. We also outline a framework for investigators to make rational decisions concerning which method to use to develop mAbs based upon characteristics of the pathogen under study and the intended downstream application. Lastly, we provide parameters for the immunisation of mice and a classification system which describes the expected outcome for mAb development strategies when using classes of immunogens to generate mAbs with desired activities.