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The immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells.

The bursa of Fabricius (BF) is the acknowledged central humoural immune organ unique to birds and plays a vital role in B lymphocyte development. In addition, the unique molecular immune features of bursal-derived biological peptides involved in B cell development are rarely reported. In this paper, a novel bursal heptapeptide (BP7) with the sequence GGCDGAA was isolated from the BF and was shown to enhance the monoclonal antibody production of a hybridoma. A mouse immunization experiment showed that mice immunized with an AIV antigen and BP7 produced strong antibody responses and cell-mediated immune responses. Additionally, BP7 stimulated increased mRNA levels of sIgM in immature mouse WEHI-231 B cells. Gene microarray results confirmed that BP7 regulated 2465 differentially expressed genes in BP7-treated WEHI-231 cells and induced 13 signalling pathways and various immune-related functional processes. Furthermore, we found that BP7 stimulated WEHI-231 cell autophagy and AMPK-ULK1 phosphorylation and regulated Bcl-2 protein expression. Finally, chicken immunization showed that BP7 enhanced the potential antibody and cytokine responses to the AIV antigen. These results suggested that BP7 might be an active biological factor that functions as a potential immunopotentiator, which provided some novel insights into the molecular mechanisms of the effects of bursal peptides on immune functions and B cell differentiation.

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Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of from blood in a quantitative qPCR mouse model.

Patients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity.

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A method to induce hen egg lysozyme-specific humoral immune tolerance in mice by pre-exposition with the protein's oligomers.

During treatment with protein therapeutics, such as monoclonal antibodies, the development of anti-drug antibodies is a serious side-effect of modern pharmacology. Anti-drug antibodies are produced as the number and exposure to therapeutic proteins increase. In this context, less immunogenic responses could diminish these noxious effects. Biophysical characterization of antigens, that is size, chemical composition, physical form, and degrability, are known to influence the outcome of immune responses. Here, using chemical modification, we have prepared oligomers of hen egg lysozyme (HEL), 3- to 5-mer, as a typical antigen in immunology and evaluated the efficacy as a tolerogen in HEL-specific antibody responses. Our results clearly demonstrated that pre-exposed the HEL-oligomers into mice effectively suppressed HEL-specific IgG responses regardless of the cross-linking mode. Therefore, the oligomerization is a method to induce tolerogenicity of proteins and may emerge as a promising strategy to control the production of undesirable anti-protein drug antibodies.

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Monoclonal antibody responses after recombinant HA vaccine versus subunit inactivated influenza virus vaccine: a comparative study.

Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok), and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (mAbs) and 38 Flucelvax-induced mAbs for avidity, cross reactivity and any selectivity towards the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of mAbs targeting epitopes near the receptor-binding domain on HA head (HAI+ mAbs) compared to Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive mAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive ASCs showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but may differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head. There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax that use alternate production strategies, can induce protective influenza specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.

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The p21 dependent G2 arrest of the cell cycle in epithelial tubular cells links to the early stage of renal fibrosis.

Renal fibrosis is accompanied by the progression of chronic kidney disease. Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis using a mouse model of unilateral ureter obstruction. We found that in the initial stage of damage, where extracellular matrix was not yet deposited, proximal tubular cells arrested at G2 of the cell cycle. Further analyses indicated that the cyclin-dependent kinase inhibitor p21 is partially involved in the G2 arrest after the damage. A newly produced monoclonal antibody against p21 revealed that levels of p21 were sharply upregulated in response to the damage during the initial stage but dropped toward the later stage. To investigate the requirement of p21 for the progression of renal fibrosis, we constructed the novel p21 deficient mice by i-GONAD method. Compared with wild-type mice, p21 deficient mice showed exacerbation of the fibrosis. Thus we propose that during the initial stage of the renal damage, tubular cells arrest in G2 partially depending on p21, thereby safeguarding kidney functions.

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Respiratory syncytial virus prefusogenic fusion (F) protein nanoparticle vaccine: Structure, antigenic profile, immunogenicity, and protection.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in the very young, elderly, and immunocompromised for which there is no vaccine. The surface exposed RSV fusion (F) glycoprotein is required for membrane fusion and infection and is a desirable vaccine candidate. RSV F glycoprotein structure is dynamic and undergoes significant rearrangements during virus assembly, fusion, and infection. We have previously described an RSV fusion-inactive prefusogenic F with a mutation of one of two furin cleavage sites resulting in the p27 region on the N-terminus of F1 with a truncated fusion peptide covalently linked to F2. A processing intermediate RSV prefusogenic F has been reported in infected cells, purified F, budded virus, and elicited a strong immune response against p27 in RSV infected young children. In this report, we demonstrate that prefusogenic F, when expressed on the cell surface of Sf9 insect and human 293T cells, binds monoclonal antibodies (mAbs) that target prefusion-specific antigenic sites Ø and VIII, and mAbs targeting epitopes common to pre- and postfusion F sites II and IV. Purified prefusogenic F bound prefusion F specific mAbs to antigenic sites Ø and VIII and mAbs targeting pre- and postfusion sites II, IV, and p27. Mice immunized with prefusogenic F antigen produced significantly higher levels of anti-F IgG and RSV neutralizing antibodies than prefusion or postfusion F antigens and induced antibodies competitive with mAbs to sites Ø, VIII, II, and IV. RSV prefusogenic F neutralization antibody responses were enhanced with aluminum phosphate adjuvant and significantly higher than prefusion F. Prefusogenic F vaccine protected cotton rats against upper and lower respiratory tract infection by RSV/A. For the first time, we present the structure, antigenic profile, immunogenicity, and protective efficacy of RSV prefusogenic F nanoparticle vaccine.

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Infliximab-Tumor Necrosis Factor Complexes Elicit Formation of Anti-drug Antibodies.

Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation.

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Generation of a single-chain variable fragment phage display antibody library from naïve mice panned against Fasciola hepatica antigens.

Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites. Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors. In this study, we produced a single-chain variable fragment (scFv) phage display library from naïve mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naïve phage display antibody library to the study of F. hepatica.

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The Alpha Emitter Astatine-211 Targeted to CD38 can Eradicate Multiple Myeloma in a Disseminated Disease Model.

Minimal residual disease (MRD) has become an increasingly prevalent and important entity in multiple myeloma (MM). Despite deepening responses to frontline therapy, roughly 75% of MM patients never become MRD negative to {less than or equal to}10, concerning because MRD negative status predicts significantly longer survival. MM is highly heterogeneous, and MRD persistence may reflect survival of isolated single cells and small clusters of treatment-resistant sub-clones. Virtually all MM clones are exquisitely sensitive to radiation, and the α-emitter astatine‑211 (At) deposits prodigious energy within three cell diameters, ideal for eliminating MRD if effectively targeted. CD38 is a proven MM target, and we conjugated At to an anti-CD38 monoclonal antibody to create an At‑CD38 therapy. When examined in a bulky xenograft model of MM, single-dose At‑CD38 at 15-45 µCi at least doubled median survival of mice relative to untreated controls (p <0.003), but no mice achieved complete remission and all died within 75 days. In contrast, in a disseminated disease model designed to reflect low burden MRD, three studies demonstrated that single-dose At-CD38 at 24-45 µCi produced sustained remission and long-term survival (>150 days) for 50-80% of mice, where all untreated mice died in 20-55 days (p <0.0001). Treatment toxicities were transient and minimal. These data suggest that At-CD38 offers the potential to eliminate residual MM cell clones in low disease burden settings, including MRD. We are optimistic that, in a planned clinical trial, addition of At-CD38 to an autologous stem-cell transplant (ASCT) conditioning regimen may improve ASCT outcomes for MM patients.

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Camelid VHHs Fused to Human Fc Fragments Provide Long Term Protection Against Botulinum Neurotoxin A in Mice.

The bacterium is the causative agent of botulism-a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.

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