Recombinant Fusion Protein Research Articles

Blastic Plasmacytoid Dendritic Cell Neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with an aggressive clinical course and poor prognosis. BPDCN is most often characterized by its presentation with distinct cutaneous lesions. Bone marrow involvement, lymphadenopathy, splenomegaly, and/or cytopenias are also seen to varying degrees. BPDCN presents with diffuse, monomorphous blasts with irregular nuclei, fine chromatin, and scant, agranular cytoplasm. Expression of CD4, CD56, and CD123 is the hallmark of BPDCN. The presence of ≥4 of CD4, CD56, CD123, TCL1, TCF4, and CD303 is necessary for the diagnosis of BPDCN. Prior to December 2018, management of BPDCN revolved around intensive chemotherapy using acute myeloid leukemia or acute lymphoblastic leukemia regimens. However, responses were transient with poor overall survival (OS). Allogeneic stem cell transplantation (alloSCT) is the only potentially curative treatment for BPDCN. Even so, only a minority of patients are candidates for alloSCT given the preponderance of disease in older individuals. For the few fit patients who are candidates for alloSCT, the aim is to achieve complete remission prior to alloSCT. Tagraxofusp (SL-401), a recombinant fusion protein containing interleukin-3 fused to truncated diphtheria toxin, was the first approved CD123-targeted therapy for BPDCN based on a phase I/II clinical trial showing a 90% overall response rate. It was approved by the FDA on December 21, 2018. Capillary leak syndrome is an important adverse effect of tagraxofusp that requires close monitoring. Several clinical trials are underway to study other regimens for the treatment of BPDCN, including IMGN632 (pivekimab sunirine), venetoclax (alone and in combination with hypomethylating agents), CAR-T cells, and bispecific monoclonal antibodies.

Fusion partners matter: Factor VIII light chain binding enables CD16-mediated recombinant Factor VIII-Fc fusion protein natural killer cell activation.

Abstract Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with multiple Fc gamma receptors (FcγR) resulting in the activation or inhibition of a wide variety of FcγR expressing immune cells. We demonstrated that rFVIIIFc activates natural killer (NK) cells via Fc mediated interactions with FcγRIIIA or CD16. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, we assessed cellular activation by measuring inflammatory cytokine secretion (IFNγ by ELISA) or cellular degranulation (flow cytometry-based surface LAMP1/CD107a). In some cases, we introduced specific blocking molecules (anti-FVIII, anti-Fc, or anti-CD16) into our assays to act as inhibitors of NK cell activation. We identified the FVIII light chain as playing a key role in the activation of CD16 +NK cells by rFVIIIFc. Using FVIII domain-specific inhibitors, we demonstrate that blocking C1 or C2 domain-mediated membrane binding potently inhibits rFVIIIFc-mediated CD16 +NK cell activation, while targeting FVIII heavy chain domains does not. Our results suggest FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface and allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. This working model may help explain our previous results where we observed recombinant Factor IX-Fc fusion protein and rFVIIIFc exhibited different CD16-signaling properties despite having identical IgG1 Fc domains.

Plasmacytoid dendritic cell neoplasms.

Plasmacytoid dendritic cells (pDCs) are type I interferon-producing cells that modulate immune responses. There are two types of pDC neoplasms: 1) mature pDC proliferation (MPDCP) associated with myeloid neoplasm and 2) blastic pDC neoplasm (BPDCN). MPDCP is a clonal expansion of mature pDCs that is predominantly associated with chronic myelomonocytic leukemia. In contrast, BPDCN is a clinically aggressive myeloid malignancy involving the skin, bone marrow, lymphatic organs, and central nervous system. There are various types of skin lesions, ranging from solitary brown or violaceous to disseminated cutaneous lesions, which often spread throughout the body. The expression of CD4, CD56, CD123, and pDC markers (TCL-1, TCF4, CD303, and CD304, etc.) are typical immunophenotype of BPDCN. Historically, BPDCN treatment has been based on acute leukemia regimens and allogeneic hematopoietic cell transplantation in selected patients. Recent advances in molecular biology and genetics have led to the development of targeted agents, such as tagraxofusp (a recombinant fusion protein targeting CD123), anti-CD123 CAR-T cells, XmAb14045, and IMGN632. Lastly, this review provides a comprehensive overview of pDC neoplasms.

Vaccination with recombinant Lactococcus lactis expressing HA1-IgY Fc fusion protein provides protective mucosal immunity against H9N2 avian influenza virus in chickens

BackgroundH9N2 virus is mainly transmitted through the respiratory mucosal pathway, so mucosal immunity is considered to play a good role in controlling avian influenza infection. It is commonly accepted that no adequate mucosal immunity is achieved by inactivated vaccines, which was widely used to prevent and control avian influenza virus infection. Thus, an improved vaccine to induce both mucosal immunity and systemic immunity is urgently required to control H9N2 avian influenza outbreaks in poultry farms.MethodsIn this study, we constructed a novel Lactococcus lactis (L. lactis) strain expressing a recombinant fusion protein consisting of the HA1 proteins derived from an endemic H9N2 virus strain and chicken IgY Fc fragment. We evaluated the immunogenicity and protective efficacy of this recombinant L. lactis HA1-Fc strain.ResultsOur data demonstrated that chickens immunized with L. lactis HA1-Fc strain showed significantly increased levels of serum antibodies, mucosal secretory IgA, T cell-mediated immune responses, and lymphocyte proliferation. Furthermore, following challenge with H9N2 avian influenza virus, chickens immunized with L. lactis HA1-Fc strain showed reduced the weight loss, relieved clinical symptoms, and decreased the viral titers and the pathological damage in the lung. Moreover, oropharyngeal and cloacal shedding of the H9N2 influenza virus was detected in chicken immunized with L. lactis HA1-Fc after infection, the results showed the titer was low and reduced quickly to reach undetectable levels at 7 days after infection.ConclusionOur data showed that the recombinant L. lactis HA1-Fc strain could induce protective mucosal and systemic immunity, and this study provides a theoretical basis for improving immune responses to prevent and control H9N2 virus infection.

Evaluation of the Diagnostic Efficacy of EC-Test for Latent Tuberculosis Infection in Ambulatory People Living with HIV.

Latent tuberculosis infection (LTBI) co-infected with human immunodeficiency virus (HIV) is more likely to develop into active tuberculosis (ATB), recombinant Mycobacterium tuberculosis fusion protein (ESAT6/CFP10, EC) Test is a latest developed method for LTBI. Compared with the interferon γ release test assays (IGRAs), the diagnostic performance of EC-Test to LTBI screening in HIV needs to be evaluated. A population-based multicenter prospective study was conducted in Guangxi Province, China. The baseline data was collected and LTBI were measured by QuantiFERON-TB Gold In-Tube (QFT-GIT), EC-Test and T-cell spot of the TB assay (T-SPOT.TB). A total of 1478 patients were enrolled. when taking T-SPOT.TB as reference, the value of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and consistency that EC-Test to diagnosis LTBI in HIV is 40.42%, 97.98%, 85.26%, 85.04% and 85.06% respectively; when taking QFT-GIT as reference, the value is 36.00%, 92.57%, 55.10%, 85.09% and 81.13%, respectively. When the CD4+ <200 /μl, the accuracy of EC-Test to T-SPOT.TB and QFT-GIT were 87.12% and 88.89%, respectively; when the 200 ≤ CD4+ ≤ 500 /μl, the accuracy of EC-Test were 86.20% and 83.18%, respectively; when the CD4+ >500/μl, the accuracy of EC-Test were 84.29% and 77.94%, respectively. The incidence of adverse reactions in EC-Test is 34.23% and the serious adverse reactions is 1.15%. EC-Test has good consistency compared with IGRAs in detecting LTBI in HIV no matter in different immunosuppression status or different regions, and the safety of EC-Test is also well, suitable for LTBI screening in HIV in high prevalence settings.

Plant-derived PAP proteins fused to immunoglobulin A and M Fc domains induce anti-prostate cancer immune response in mice

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacterium-meditated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system.

Synthesis of plant-based, self-adjuvanted, dual antigen specific to Mycobacterium tuberculosis as a novel tuberculosis subunit vaccine that elicits immunogenicity in rabbit.

The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy. A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.

Abstract CT258: A phase 1/2 study of JK08, an IL-15 antibody fusion protein targeting CTLA-4, in patients with advanced solid tumors

Abstract Background: CTLA-4 is a transmembrane receptor that functions as an immune checkpoint and fundamental negative regulator of T cell activation. CTLA-4 is highly expressed on most regulatory T cells (Tregs) but only upregulated in effector T cells following activation. Emerging evidence suggests anti-tumor activity of antibodies targeting CTLA-4 may be modulated by the local presence of NK cells. Interleukin 15 (IL-15) is a pleiotropic cytokine important in both innate and adaptive immunity. The IL-15/IL-15Rα complex can stimulate adjacent cells through the IL-2Rβ/γ complex. This receptor complex is present on many hematopoietic cells; however, IL-2Rβ expression is highest on NK cells and CD8+ T cells. Trans-presentation of IL-15 is believed to be the optimal mode of action for IL-15-induced proliferation and activation of NK cells and CD8+ T cells, two essential mediators of anti-tumor responses. JK08 is a recombinant fusion protein consisting of two functional elements - a fully human monoclonal antibody directed against CTLA-4 and a protein complex consisting of human IL-15 and the Sushi domain of human IL-15Rα. JK08 is intended to widen the therapeutic window for IL-15 cytokine-mediated cancer therapy and CTLA-4-targeted antibody-mediated cancer therapy. This widening of the therapeutic window can be achieved by local activation and expansion of NK cells at sites of Tregs, and by IL-15 enhancement of the activity and potency of the proximal CTLA-4 antibody, mirroring the endogenous trans-presentation orientation. Preclinical studies demonstrate JK08 can elicit ADCC-mediated killing of CTLA-4-expressing cells and interact with IL-2Rβ to promote robust T cell proliferation independent of IL-15Rα expression, suggesting that JK08 could effectively activate IL-2Rβ/γC-expressing cells preferentially at sites of Tregs, and achieve enhanced anti-tumor responses including through ADCC-mediated depletion of T regulatory cells. In vivo studies show JK08 induces robust NK and CD8+ T cell expansion in cynomolgus monkeys and anti-tumor activity in syngeneic murine models. In summary, JK08 treatment may lead to clinical activity in multiple cancer indications by counteracting the contribution of Tregs to cancer progression. Methods: The Phase 1/2 study of JK08 will enroll patients with advanced relapsed/refractory solid tumors. The study will employ an accelerated 3+3 escalation design to explore the safety, PK, immuno-regulatory activity, and preliminary anti-tumor activity of JK08. Patients will receive treatment with JK08 subcutaneously once weekly until confirmed disease progression or intolerable toxicity. Four tumor specific expansion cohorts will be initiated once dose and schedule are established from dose escalation and include melanoma, breast cancer, colorectal cancer, and a basket of advanced solid tumors. Response will be assessed every 9 weeks per RECIST v1.1. Citation Format: Nuria Kotecki, Sylvie Rottey, Elena Garralda, Maria de de Miguel, Omar Saavedra, Valentina Boni, Judit W. Johnson, Jonathan P. McNally, Mohit Mathew, Adam Hughes, Sam Murphy, Naimish Pandya. A phase 1/2 study of JK08, an IL-15 antibody fusion protein targeting CTLA-4, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT258.

Expression of a Siglec-Fc Protein and Its Characterization.

The emerging importance of the Siglec-sialic acid axis in human disease, especially cancer, has necessitated the identification of ligands for Siglecs. Recombinant Siglec-Fc fusion proteins have been widely used as ligand detectors, and also as sialic acid-targeted antibody-like proteins for cancer treatment. However, the heterogenetic properties of the Siglec-Fc fusion proteins prepared from various expression systems have not been fully elucidated. In this study, we selected HEK293 and CHO cells for producing Siglec9-Fc and further evaluated the properties of the products. The protein yield in CHO (8.23 mg/L) was slightly higher than that in HEK293 (7.46 mg/L). The Siglec9-Fc possesses five N-glycosylation sites and one of them is located in its Fc domain, which is important for the quality control of protein production and also the immunogenicity of Siglec-Fc. Our glycol-analysis confirmed that the recombinant protein from HEK293 received more fucosylation, while CHO showed more sialylation. Both products revealed a high dimerization ratio and sialic acid binding activity, which was confirmed by the staining of cancer cell lines and bladder cancer tissue. Finally, our Siglec9-Fc product was used to analyze the potential ligands on cancer cell lines.

Abstract 2938: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy

Abstract The overexpression of human epidermal growth factor receptor 2 (HER2) has been identified in a variety of solid tumors including breast cancer (BC), gastric cancer and is associated with tumor recurrence, brain metastasis, and an overall poor prognosis. As such, HER2 has become a major target in the research and development of anticancer drugs. CD47, an integrin-associated protein, is expressed in many human cancer cells, especially upregulated preferentially in HER2-expressing cells. Co-expression of HER2 and CD47 is more frequently detected in recurrent BC patients with poor prognosis when comparing with the counterpart primary tumors. IMM2902 is a first-in-class, clinical-stage, novel recombinant bispecific mAb-Trap fusion protein targeting both CD47 and HER2 simultaneously. The binding activities of IMM2902, measured as EC50, to total CD47+ and HER2+ in BT474, NCI-N87 and SKOV-3 cancer cell lines are 16, 11 and 5 nM respectively. The affinity of IMM2902 to HER2 is about 15 times higher than that of CD47. IMM2902 not only prevents the engagement of CD47 with SIRPα and hence blocks “don’t eat me” signal of CD47/SIRPα, but also induces strong antibody-dependent cell-mediated cytotoxicity (ADCC, EC50 = 0.0045 - 0.0544 nM) and antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.05 - 0.13 nM) activity with specifically engineered IgG1 Fc fragment. Most importantly, IMM2902 induces accelerated HER2 degradation in tumor tissues. Despite its high affinity to CD47, IMM2902 does not binds to CD47 on erythrocyte membrane, therefore, unlike conventional CD47 mAb, IMM2902 demonstrated a minimal impact on red blood cells (RBC). In preclinical research, IMM2902 has demonstrated a much stronger antitumor activity with excellent tolerability in a mouse model of human breast cancer (dose range 3.5 - 10 mg/kg) and a mouse model of human gastric cancer (dose range 1 -18 mg/kg) than that of trastuzumab (targeting HER2 only) alone, IMM01 (a SIRPα-Fc fusion protein) alone, and the combination of trastuzumab and IMM01. In HER2-low SUN-1 xenograft models, IMM2902 has showed a strong tumor growth inhibition at doses between 2 to 18 mg/kg. Intriguingly, IMM2902 also has exhibited potent tumor killing activity against breast cancer resistant to trastuzumab (HCC-1954 and patient-derived xenografts herceptin-resistant model) at doses between 3.5 to 10 mg/kg. Given the strong preclinical antitumor activity as well as the favorable safety profile, IMM2902 may serve as a potent immunotherapy for HER2-expressing cancers via dual blockade of CD47 and HER2. A Phase 1 clinical trial exploring safety, tolerability, and preliminary efficacy of IMM2902 in patients with HER2-expressing advanced solid tumors is currently ongoing in both China (CXSL2101035) and USA (NCT05076591). Citation Format: Binglei Zhang, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Frank X. Gan, Wenzhi Tian, Fan Zhang, Yongping Song. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2938.

Abstract 704: MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models

Abstract Aims: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can suppress MDSC expansion and activate tumor immunity in part through agonism of the CXCR4 receptor. The aim of this study is to investigate whether a novel TFF2 - albumin fusion peptide (TFF2-MSA) can improve survival in anti-PD-1 treated syngeneic colorectal cancer (CRC) mouse models. Methods: Two syngeneic colon carcinoma mouse models were developed using cell lines grafted subcutaneously into mice. MC38 CRC cells were engrafted into C57BL/6 mice while CT26.wt CRC cells were implanted into BALB/C mice We generated a recombinant fusion protein, designated TFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing dose frequency. Mice subsequently received either TFF2-MSA or anti-PD-1 antibody (clone 29F.1A12) or both, and tumor volume, and survival were measured. At the endpoint, flow cytometry was performed to examine treatment-induced effects on immune profiles. Results: In the MC38 model, administration of TFF2-MSA suppressed tumor growth (TGI 38%), the combination of TFF2-MSA and anti-PD-1 had an additive effect and suppressed tumor growth dramatically (TGI 74%). The combination also exhibited a survival rate of 90% after 50 days, while vehicle and single TFF2-MSA therapy were 30% and 65%, respectively. The percentage of exhausted CD8+ T cells was markedly reduced in the draining lymph node by the combination treatment, as measured by flow cytometry using antibodies against LAG3, TIM3 and PD-1. In the CT26.wt model, administration of TFF2-MSA alone exhibited little effect, but the combination of anti-PD-1 and TFF2-MSA showed a profound effect. In the CT26.wt model, administration of TFF2-MSA suppressed tumor growth (TGI 17%), anti-PD-1 alone (TGI 43%) and the combination of TFF2-MSA and anti-PD-1 (TGI 54%). Conclusion: Targeting MDSCs using TFF2-MSA fusion protein synergizes well with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of colorectal cancer. In a separate abstract, additive effects between TFF2-MSA and anti-PD-1 antibody were also demonstrated in a separate ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) gastric cancer model, suggesting combination therapy may also be applicable to gastric cancer. Citation Format: Bruce L. Daugherty. MDSC-targeted TFF2-MSA suppresses tumor growth and increases survival in anti-PD-1 treated MC38 and CT26.wt murine colorectal cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 704.

The Effect Of Hepatitis E Virus Open Reading Frame 3 Protein on The Expression Levels of IFN-β,IL6,TNF-α and RANTES in SW480 Colon Cancer Cells

The Hepatitis E virus (HEV) could induce chronic hepatitis and liver failure with high mortality through unclear mechanisms. The purpose of this study was to determine the effect of (recombinant fusion protein, ORF3 of genotype 1 HEV) on the expression levels of cytokine or chemokine genes in colon cancer cells (SW480) using a quantitative assay of “SYBR green" real-time PCR. An ORF3 protein expression system (Orf3-GFP plasmid) was synthesized in the company of GenScript (USA) and used as an expression vector, SW480 cells were infected by (Orf3-GFP plasmid) or GFP- control vector. Result of this study was approved after assessing the expression level of the ORF3 gene in ORF3 expressing cells and in control groups using ORF3 specific primer to ensure that the transfection was performed successfully, The expression levels of three genes (IFN-β, IL-6, and TNF-α) were estimated in ORF3-expressing cells using specific primers for all genes. In addition, the role of HEV Orf3 in upregulating RANTES expression levels was revealed. In conclusion: In SW480 cells, this study found that the ORF3 protein of genotype 1 HEV is capable of regulating the expression of multiple immune response genes including Interferon- β (IFN-β), Interleukin-6 (IL-6) Tumor necrosis factor-α (TNF-α) and regulated on activation, normal T cell expressed and secreted (RANTES) which may enhance viral replication and pathogenicity.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1β secretion and their contribution to the overall immune responses remains elusive. To investigate the mechanisms underlying the production of IL-1β from rFlaA:Betv1 stimulated macrophages. Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1β to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1β secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1β-expression in THP-1 macrophages. Finally, lack of IL-1β positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1β, IL-6, and TNF-α from peritoneal macrophages. The mechanisms contributing to rFlaA:Betv1-induced IL-1β secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Antibody response to Newcastle disease virus recombinant fusion protein in post-vaccinated laying hens

This research was aimed to analyze antibody response in laying hens post vaccinated by Newcastle Disease Virus (NDV) recombinat Fusion (F) protein which has been succesfully expressed from the F gene of local isolates of NDV from Kulon Progo strain (0663/04/2013), Yogyakarta, Indonesia. The F gene cloned into expression vector plasmid pBT7-N-His. Two types of NDV recombinant vaccine, a concentrated and pure F recombinant protein were used for vaccination. A concentrated recombinant F protein was collected from the centrifugal ultrafiltration process and a pure recombinant F protein was collected from the electroeluted process. Recombinant F protein of NDV was successfully expressed, purified, and visualized by Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining with a molecular weight of 28 kDa. All two types of recombinant F vaccines and a commercial live vaccine as a positive control were injected two times at 14 and 18th weeks old laying hens to analyze the antibody response in serum. In comparison with a commercial live NDV vaccine, indirect Enzyme-linked Immunosorbent Assay (ELISA) revealed that antibody responses were high in both recombinant F protein vaccinated groups. In conclusion, the recombinant F protein has the potential to be developed as a recombinant vaccine candidate to obtain a higher antibody response in laying hens compared to commercially available live NDV vaccines.

RMBP-NAP Suppresses OXA-induced Allergic Dermatitis by Regulating the Th1/Th2 Balance.

Allergic dermatitis (AD) is an inflammatory skin disease that arises from abnormal T lymphocyte activation. A recombinant fusion protein comprising Helicobacter pylori neutrophil-activating protein and maltose binding protein, rMBP-NAP, has been documented as a novel immunomodulatory TLR agonist. To explore the effect of the rMBP-NAP on the OXA-induced AD in a mouse model and clarify the possible action mechanism. The AD animal model was induced by repeated administration of oxazolone (OXA) in BALB/c mice. H&E staining was used to analyze the ear epidermis thickness and the number of infiltrating inflammatory cells. TB staining was used to detect mast cell infiltration in the ear tissue. ELISA was used to analyze the secretion of cytokines IL-4 and IFN-γ in peripheral blood. qRT-PCR was used to determine the expression levels of IL-4, IFN-γ, and IL-13 in ear tissue. OXA induced the establishment of an AD model. After the rMBP-NAP treatment, the thickness of the ear tissue and the number of mast cells infiltrated in AD mice reduced, and the serum and ear tissue levels of IL-4 and IFN-γ increased, but the ratio of IFN-γ (rMBP-NAP group)/IL-4 (rMBP-NAP group) was greater than the ratio of IFN-γ (sensitized group)/IL-4 (sensitized group). The rMBP-NAP improved the disease symptoms including skin lesions in AD, alleviated the inflammation in ear tissue, and restored the Th1/2 balance by inducing a shift from the Th2 to the Th1 response. The results of our work support the use of rMBP-NAP as an immunomodulator for AD treatment in future investigations.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

Efficacy of tumor necrosis factor inhibitor combined with intra-articular injection of triamcinolone acetonide in the treatment of refractory rheumatoid arthritis synovitis: a retrospective study.

To investigate whether there is a difference in the efficacy of intra-articular injection of tumor necrosis factor (TNF) inhibitor and triamcinolone acetonide (HA) in rheumatoid arthritis (RA) patients with recurrent synovitis after the first intra-articular injection of HA. RA patients who relapsed 12weeks after the first HA treatment were enrolled in this study. After joint cavity extraction, recombinant human TNF receptor-antibody fusion protein (TNFR:FC) (25mg or 12.5mg) or HA (1ml or 0.5ml) was injected then. The changes in the visual analog scale (VAS), joint swelling index, and joint tenderness index before and 12weeks after reinjection were compared and analyzed. The changes in synovial thickness, synovial blood flow, and fluid dark zone depth before and after reinjection were observed by ultrasound. Forty-two RA patients were enrolled, including 11 males and 31 females, with an average age of 46.79 ± 12.61years and an average disease duration of 7.76 ± 5.44years. After 12weeks of intra-articular injection of HA or TNFR:FC, the VAS scores were significantly lower than those before treatment (P < 0.01). After 12weeks of injection, the scores of the joint swelling index and tenderness index in both groups were significantly decreased compared with those before treatment. There was no significant difference in synovial thickness under ultrasound in the HA group before and after injection, while the synovial thickness in the TNFR:FC group was significantly improved after 12weeks (P < 0.01). After 12weeks of injection, the grade of synovial blood flow signal in both groups decreased significantly compared with that before treatment, especially in the TNFR:FC group. After 12weeks of injection, the depth of the liquid dark area under ultrasound decreased significantly in the HA group and TNFR:FC group compared with that before treatment (P < 0.01). Intra-articular injection of a TNF inhibitor is an effective method for the treatment of recurrent synovitis after conventional hormone therapy. Compared with HA treatment, it can reduce synovial thickness. Key Points • Intra-articular injection of a TNF inhibitor is an effective method for the treatment of recurrent synovitis after conventional hormone therapy. • Compared with HA treatment, intra-articular injection of biological agents combined with glucocorticoids can not only relieve joint pain but also significantly inhibit joint swelling. • Compared with HA treatment, intra-articular injection of biological agents combined with glucocorticoids cannot only improve synovial inflammation but also inhibit synovial proliferation. • For the treatment of refractory RA synovitis, biological agents combined with glucocorticoid injection are an effective and safe option.

A possible role of the ecdysone receptor in modulating gonad-inhibiting hormone gene expression in the black tiger prawn, Penaeus monodon

Gonad-inhibiting hormone (GIH) is a neuronal eyestalk peptide that inhibits ovarian maturation in crustaceans. In this study, the aim was to investigate the transcriptional regulation of the GIH gene in the black tiger prawn (Penaeus monodon). An analysis of a 1158 bp DNA fragment 5′ upstream of the GIH gene revealed a putative half-site of the hormone response element (HRE) at −561 to −566 relative to the predicted transcription start site with an upstream imperfect palindromic sequence separated by one nucleotide. Deletion of the HRE from the GIH 5′ fragment increased the promoter activity in Sf9 cells, whereas the addition of 20-hydroxyecdysone (20E) reduced the promoter activity by approximately 60–70%. A candidate HRE-binding nuclear receptor, an ecdysone receptor (EcR) that serves to bind 20E in crustaceans, was therefore functionally characterized. EcR was expressed ubiquitously in female prawns, with the highest expression levels occurring in eyestalks, suggesting that EcR possibly stimulates vitellogenesis by binding to the HRE in the GIH 5′ regulatory sequence, thereby inhibiting GIH expression. This was verified by specific binding between a recombinant EcR-GST fusion protein and the HRE in the GIH 5′ DNA fragment. In addition, in vivo knockdown of EcR resulted in a 3-fold elevation of GIH expression levels in the eyestalks of adult female prawns. Taken together, the results obtained in this study indicate that EcR acts as an inhibitory factor in the regulation of the GIH gene.

Advancing Luciferase-Based Antibody Immunoassays to Next-Generation Mix and Read Testing

Antibody measurements play a central role in the diagnosis of many autoimmune and infectious diseases. One antibody detection technology, Luciferase Immunoprecipitation Systems (LIPS), utilizes genetically encoded recombinant luciferase antigen fusion proteins in an immunoglobulin capture format to generate robust antibody measurement with high diagnostic sensitivity and specificity. The LIPS technology has been highly useful in detecting antibodies for research diagnostics and the discovery of new autoantigens. The methodology of the assay requires immunoglobulin binding reagents such as protein A/G beads and washing steps to process the immune complex before antibody levels are measured by light production with a luminometer. Recently, simplified mix and read immunoassays based on split components of the nanoluciferase enzyme in a complementation format have been developed for antibody measurements without requiring immunoglobulin-capturing beads or washing steps. The mix and read immunoassays utilize two or three nanoluciferase fragments which when reconstituted via antigen-specific antibody binding generate a functional enzyme. At present, these split luciferase tests have been developed mainly for detecting SARS-CoV-2 antibodies. Here, we describe the traditional LIPS technology and compare it to the new split luciferase methodologies focusing on their technical features, strengths, limitations, and future opportunities for diagnostic research, and clinical applications.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.