Liming Xu

Synergistic effects of interleukin-1β and interleukin-17A antibodies on collagen-induced arthritis mouse model.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Both interleukin-1β (IL-1β) and Interleukin-17 (IL-17) are important proinflammatory cytokines involved in the pathogenesis of RA. We investigated whether combination therapy with IL-1β and IL-17A antibodies would generate the potential for synergistic effects on a collagen-induced arthritis (CIA) mouse model. Mice with CIA were subcutaneously injected with humanized IL-1β antibody, IL-17A antibody, or combination treatment. The effects of treatment were determined by arthritis severity score, histological damage and bone destruction, autoreactive humoral and cellular immune responses and cytokine production. Treatment with IL-1β antibody or IL-17A antibody alone resulted in beneficial effects on clinical and histological parameters of CIA mice. Compared with the single antibody treatments, the combination therapy resulted in a more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-17A, IFN-γ, RANKL and MMP-3 in inflammatory tissue. In conclusion, combination treatment with humanized IL-1β and IL-17A antibodies demonstrates synergistic beneficial effects for preventing joint inflammation and cartilage destruction and bone damage in CIA mice model. These studies also provide evidence that combination with IL-1β and IL-17A antibodies may lead to a new combinatorial therapy for RA patients.

Therapeutic efficacy of three bispecific antibodies on collagen-induced arthritis mouse model.

Interleukin-1β (IL-1β) and interleukin-17A (IL-17A) are inducible factors and important cytokines in the pathogenesis of rheumatoid arthritis (RA). In the present study, three bispecific and neutralizing antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1β and hIL-17A were constructed, their therapeutic efficacy was compared on collagen induced arthritis (CIA) model mice. In vitro assays demonstrated that the three antibodies could simultaneously bind to target both hIL-1β and hIL-17A. Mice with CIA were subcutaneously administered with one of three antibodies every two days for 29 days, we noticed that, compared with the BsAB-2 and BsAB-3, BsAB-1 antibody therapy resulted in more significant effect on alleviating the severity of arthritis by preventing bone damage and cartilage destruction and substantially decreasing production of CII-specific antibodies. In addition, BsAB-1 antibody was more potent in the inhibition of mRNA expression of IL-2, IL-1β, IL-17A, TNF-α and MMP-3 in the spleen of CIA mice compared to the other two. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 for the treatment of RA model mice, and may be chosen as an ideal candidate for further development of therapeutic drugs for treatment of RA.

scFv antibodies against infectious bursal disease virus isolated from a combinatorial antibody library by flow cytometry.

Infectious bursal disease is an economically important disease that affects chickens worldwide. Here, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry against VP2 protein through a bacteria display technology, resulting in the enrichment of scFv. Three scFv clones with different fluorescence intensity were obtained by random colony pick up. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis. The potential use of the selected IBDV-specific scFv antibodies was demonstrated by the successful application of the isolated antibodies in western blotting assay and ELISA.

Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry

The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

A effective DNA vaccine against diverse genotype J infectious hematopoietic necrosis virus strains prevalent in China

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In this study, a DNA vaccine, designated pIHNch-G, was constructed with the glycoprotein (G) gene of a Chinese IHNV isolate SD-12 (also called Sn1203) of genotype J. The minimal dose of vaccine required, the expression of the Mx-1 gene in the muscle (vaccine delivery site) and anterior kidney, and the titers of the neutralizing antibodies produced were used to evaluate the vaccine efficacy. To assess the potential utility of the vaccine in controlling IHNV throughout China, the cross protective efficacy of the vaccine was determined by challenging fish with a broad range of IHNV strains from different geographic locations in China. A single 100ng dose of the vaccine conferred almost full protection to rainbow trout fry (3g) against waterborne or intraperitoneal injection challenge with IHNV strain SD-12 as early as 4days post-vaccination (d.p.v.), and significant protection was still observed at 180d.p.v. Intragenogroup challenges showed that the DNA vaccine provided similar protection to the fish against all the Chinese IHNV isolates tested, suggesting that the vaccine can be widely used in China. Mx-1 gene expression was significantly upregulated in the muscle tissue (vaccine delivery site) and anterior kidney in the vaccinated rainbow trout at both 4 and 7d.p.v. Similar levels of neutralizing antibodies were determined with each of the Chinese IHNV strains at 60 and 180d.p.v. This DNA vaccine should play an important role in the control of IHN in China.

Preliminary study of an oral vaccine against infectious hematopoietic necrosis virus using improved yeast surface display technology

&NA; Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Because oral vaccines induce more efficient mucosal immunity than parenteral immunization, an oral vaccine was developed with an improved yeast cell surface display technology to induce an immune response to IHNV. The oral yeast vaccine, designated EBY100/pYD1‐bi‐G, was delivered orally to rainbow trout (Oncorhynchus mykiss) on days 1 and 32, and the nonspecific and specific immune responses were measured 50 days after the first vaccination. In the hindgut, spleen, and head kidney, the expression of IFN‐1 and Mx‐1 was significantly upregulated after oral vaccination with EBY100/pYD1‐bi‐G, and the highest expression of IFN‐1 and Mx‐1 was observed in the spleen (7.5‐fold higher than the control group) and head kidney (3.9‐fold higher than the control group), respectively. Several markers of the adaptive immune response (IgM, IgT, CD4, and CD8) were also significantly upregulated, and the highest expression of these markers was observed in the hindgut, suggesting that the mucosal immune response was successfully induced by oral vaccination with EBY100/pYD1‐bi‐G. Sera from the orally vaccinated rainbow trout showed higher anti‐IHNV neutralizing antibody titers (antibody titer 81 ± 4) than the control sera (antibody titer 7 ± 3), and the relative percentage survival after IHNV challenge was 45.8% compared with 2% in the control group. Although the protection afforded by this orally delivered vaccine was lower than that of a DNA vaccine (83%–98%), it is a promising candidate vaccine with which to protect larval fish against IHNV, which are most susceptible to the virus and difficult to inject with a DNA vaccine. HighlightsPreliminary study of an oral vaccine against infectious hematopoietic necrosis virus.The oral vaccine was designed using improved yeast surface display technology.The yeast oral vaccine induced an efficient mucosal immune response.It can be used as primer or booster vaccination before or after DNA vaccination.

Bi-specific antibodies with high antigen-binding affinity identified by flow cytometry.

Using conventional approaches, the antigen-binding affinity of a novel format of bi-specific antibody (BsAb) cannot be determined until purified BsAb is obtained. Here, we show that new lipoprotein A (NlpA)-based bacteria display technology, combined with flow cytometry (FCM), can be used to detect antigen-binding affinity of BsAbs, in the absence of expression and purification work. Two formats of BsAb, scFv2-CH/CL and Diabody-CH/CL, specific for human interleukin 1β (hIL-1β) and human interleukin 17A (hIL-17A), were constructed and displayed in Escherichia coli using NlpA-based bacteria display technology. Conversion of these cells to spheroplasts, and their incubation with fluorescently conjugated antigens resulted in the selective labeling of spheroplasts expressing BsAb; enabling their antigen-binding affinity to be analyzed with FCM. The association and dissociation of BsAbs for binding to hIL-1β and hIL-17A were analyzed using FCM-based assays. The results showed that antigen-binding affinity of Diabody-CH/CL was significantly higher than that of scFv2-CH/CL. To confirm these results of FCM-based assays, BsAbs were expressed, purified and subjected to relative affinity measurements, in vitro and in vivo bioactivity analysis. The results showed that Diabody-CH/CL had greater relative affinities for both antigens, resulting in better blocking bioactivities on cellular level and effects on alleviating joint inflammation, and cartilage destruction and bone damage in collagen induced arthritis (CIA) mice model. These results indicate that BsAbs with good antigen-binding affinity can be identified by FCM-based assays without expression and purification work, and the indentified BsAb can serve as a lead compound for further drug development.

Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens of salmon and trout. An active bivalent DNA vaccine was constructed with the glycoprotein gene of Chinese IHNV isolate Sn1203 and VP2–VP3 gene of Chinese IPNV isolate ChRtm213. Rainbow trout (5 g) were vaccinated by intramuscular injection with 1.0 µg of the bivalent DNA vaccine and then challenged with an intraperitoneal injection of IHNV, IPNV, or both, at 30 and 60 days post-vaccination (d.p.v.). High protection rates against IHNV were observed, with 6% and 10% cumulative mortality, respectively, compared with 90–94% in the mock-vaccinated groups. IPNV loads (531-fold and 135-fold, respectively) were significantly reduced in the anterior kidneys of the vaccinated trout. Significant protection against co-infection with IHNV and IPNV was observed, with cumulative mortality rates of 6.67% and 3.33%, respectively, compared with 50.0% and 43.3%, respectively, in the mock-vaccinated groups. No detectable infective IHNV or IPNV was recovered from vaccinated trout co-infected with IHNV and IPNV. The bivalent DNA vaccine increased the expression of Mx-1 and IFN-γ at 4, 7, and 15 d.p.v, and IgM at 21 d.p.v., and induced high titres (≥160) of IHNV and IPNV neutralizing antibodies at 30 and 60 d.p.v.

The kinetics and protection of the antiviral state induced by recombinant iIFN1a in rainbow trout against infectious hematopoietic necrosis virus

The iIFN1a (intracellular IFN-a1), that is one of the IFN-a1 variants, was shown to be functional intracellularly and act as a novel defense against an infectious hematopoietic necrosis virus (IHNV). To determine its antiviral properties, a recombinant iIFN1a was generated in Escherichia coli. Its antiviral activity against IHNV was 1.69×10(7)U/mg in CHSE-214 cells. Additionally, iIFN1a was capable of inducing comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver tissues at an early time point (6h), that was followed by a rapid decline 24h after induction. The recombinant protein also elicited protection against IHNV in vivo. At 6 and 24h after induction there was 100% protection against the virus, however, at 48 and 72h the protection decreased to 57 and 40%, respectively. The in vivo protection kinetics correlated with the kinetics of gene expression. The results of this study provide details of the antiviral state that was induced by iIFN1a in vivo for the first time. Additionally, this information will facilitate the development of this recombinant protein as a potential anti-viral treatment and/or adjuvant.

A Neutralization scFv Antibody against IL-1β Isolated from a NIPA-based Bacterial Display Library

OBJECTIVE RA is one of autoimmune diseases, has drawn great attention of the world. Currently, the anti- IL-1β monoclonal antibody Canakinumab (ACZ885) for treatment of RA has entered into clinical trials. However, Full length antibody has large molecular weight, and is difficult to penetrate the tissue or the nidus. In contrast, scFv has low molecular weight and strong penetration ability, and is favorable to increase the drug concentration in the indus, hence improving the efficacy of the drug. The aim of this study is to obtain a neutralizing scFv antibody from a combinatorial scFv library against hIL-1β by the modified NLPA-based bacterial display system, for further development of the small molecule antibody drug for treatment of RA. METHODS The modified NIPA-based bacterial display system was used to construct the combinatorial scFv library derived from the spleen cDNA of immunized mice with hIL-1β. FACS was used to screen hIL- 1β-binding clones with FITC-labeled hIL-1β protein. Three clones were randomly selected from the third round of screening, and their nucleotide sequences were aligned with mouse immunoglobulin genes. The single chain antibody genes of the hIL-1β-binding clones were subcloned into the prokaryotic expression vector pET-27b for expression. The molecular mass of the purified anti-hIL-1β single chain antibody was about 28ku. The hIL-1β-binding ability of antibody were examined by ELISA and Western blot assays. Ability of the scFv antibody to neutralize hIL-1β was evaluated by the MTT test. CONCLUSIONS In this study, it is the first time to use the NIPA-based bacteria display system to construct and screen the combinatorial scFv library. Three scFvs against hIL-1β were obtained from the scFv library of the immunized mice. Prokaryotically expressed and purified scFvs demonstrate binding ability with hIL-1β. Among the three clones. The MTT test suggests that scFv-20 is a neutralization antibody against hIL-1β. The study provides a lead candidate for further development of small molecule therapeutic antibodies for treatment of RA.