Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

CTLA-4 Blockade of Natural Killer Cells Increases Cytotoxicity against Acute Lymphoid Leukaemia Cells Neda.

OBJECTIVE: There is interest in using cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy to treat blood cancers. Unfortunately, patients with acute lymphoblastic leukaemia (ALL) frequently exhibit resistance to treatment and natural killer (NK) cell exhaustion. This study aims to increase the cytotoxic potency of natural killer cells by using CTLA-4 to block the Nalm-6 leukaemia cell line. MATERIALS AND METHODS: In this experimental study, NK cells were purified from the peripheral blood mononuclear cells (PBMCs) of 10 healthy people and assessed by flow cytometry for purity and viability. The purified cells were activated overnight at 37°C and 5% CO2 with interleukin-15 (IL-15, 10 ng/ml) followed by evaluation of expressions of CTLA-4, activating and inhibitory receptors, and the release of interferon gamma (IFN-γ) and granzyme B (GZM B). CTLA-4 expression on NK cells from recurrent ALL patients was also evaluated. Finally, the cytotoxic activity of NK cells was assessed after the CTLA-4 blockade. RESULTS: The purity of the isolated cells was 96.58 ± 2.57%. Isolated NK cells activated with IL-15 resulted in significantly higher CTLA-4 expression (8.75%, P<0.05). Similarly, CTLA-4 expression on the surface of NK cells from patients with ALL was higher (7.46%) compared to healthy individuals (1.46%, P<0.05). IL-15 reduced NKG2A expression (P<0.01), and increased expressions of NKP30 (P<0.05) and NKP46 (P<0.01). The activated NK cells released more IFN-γ (P<0.5) and GZM B (P<0.01) compared to unactivated NK cells. Blockade of CTLA-4 enhanced the NK cell killing potential against Nalm-6 cells (56.3%, P<0.05); however, IFN-γ and GZM B levels were not statistically different between the blocked and non-blocked groups. CONCLUSION: Our findings suggest that CTLA-4 blockage of Nalm-6 cells causes an increase in antitumour activity of NK cells against these cells. Our study also provides evidence for the potential of cancer immunotherapy treatment using blocking anti-CTLA-4 mAbs.

TAX and HBZ: hFc Ɣ 1 proteins as targets for passive immunotherapy.

Objectives: Human T leukemia virus type one (HTLV-1) causes two life-threatening diseases in around five percent of infected subjects, a T cell malignancy and a neurodegenerative disease. TAX and HBZ are the main virulence agents implicated in the manifestation of HTLV-1-associated diseases. Therefore, this study aims to produce these HTLV-1 factors as recombinant Fc fusion proteins to study the structures, their immunogenic properties as vaccines, and their capability to produce specific neutralization antibodies. Materials and Methods: TAX and HBZ sequences were chosen from the NCBI-nucleotide database, then designed as human Fc chimers and cloned into Pichia pastoris. Produced proteins were purified by HiTrap affinity chromatography and subcutaneously injected into rabbits. Rabbit Abs were purified by batch chromatography, and their neutralization activities for the HTLV-1-infected MT-2 cell line were assessed. Furthermore, the protective abilities of recombinant proteins were evaluated in Tax or HBZ immunized rabbits by MT-2 cell line inoculation and measurement of HTLV-1-proviral load. Results: Specific Abs against Tax and HBZ can eliminate 2 million MT-2 cells in 1/1000 dilution in vitro. In challenging assays, the immunization of the animals using Tax or HBZ had no protective activity as HTLV-1 PVL was still positive. Conclusion: The result suggests that recombinant TAX and HBZ: hFcγ1 proteins can produce a proper humoral immune response. Therefore, they could be considered a passive immunotherapy source for HTLV-1-associated diseases, while total TAX and HBZ proteins are unsuitable as HTLV-1 vaccine candidates.

Hydrocortisone Long-term Treatment Effect on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stromal/Stem Cells.

Cortisol is secreted in prolonged stress and has therapeutic effects in inflammatory diseases. Considering the immunomodulatory effects of mesenchymal stem cells, here we investigated the effect of hydrocortisone (HC) long-term treatment on immunomodulatory properties of human adipose-derived mesenchymal stromal/stem cells (ASCs). Isolated ASCs from healthy subjects were treated with different HC concentrations for 14 days. The effect of HC-treated ASCs on the proliferative response of peripheral blood mononuclear cells (PBMCs) was evaluated in ASCs/2-way mixed leukocyte reaction coculture using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. HC-treated ASCs were further divided into interferon gamma (IFN-γ) stimulated and unstimulated groups. Transforming growth factor beta 1 (TGF-ß1) and interleukin (IL)-6 levels were measured in culture supernatants by enzyme-linked immunosorbent assay. Relative expression of cyclooxygenase-2 (COX-2), hepatocyte growth factor, indoleamine dioxygenase, and programmed death-ligand 1 genes was assessed by real-time PCR. Levels of TGF-ß1 and COX-2 expression were elevated in unstimulated ASCs, while exposure to high concentration of HC significantly increased TGF-ß1 levels and reduced COX-2 expression. Unstimulated HC-5-µM-treated ASCs increased PBMC proliferation ratio on day 2 of coculture compared to the control group (P = 0.05). In IFN-γ stimulated condition, pretreatment with HC-5 µM resulted in a significantly increased IL-6 and significantly decreased COX-2 expression compared to the HC untreated control group. In conclusion, our results showed various alterations of ASC immunomodulatory related features as a result of long-term exposure of different concentrations of HC. It seems that HC at low concentration pushed the balance toward extended immune response in ASCs, while this observation wasn't persistent in ASCs treated with higher concentrations of HC.

Up-regulation of CD200/CD200R1 Immunomodulatory Axis of Allogenic Peripheral Blood Mononuclear Cells in a Co-culture with Adipose-derived Mesenchymal Stem Cells.

Co-inhibitory molecules modulate immune responses. Immunomodulatory properties of mesenchymal stem cells (MSCs) turn them into ideal candidates for cell therapy. This study was designed to investigate the immunomodulatory effect of adipose-derived stem cells (ASCs) on inflammatory environment of a co-culture of allogenic peripheral blood mononuclear cells (PBMCs) in a two-way mixed leukocyte reaction (twMLR) setting. ASCs were co-cultured with allogenic PBMCs in twMLR setting for four days. The proliferation of peripheral blood mononuclear cells (PBMCs), levels of interleukin (IL)-10, and expression of interferon-gamma (IFN-γ), B7-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death-ligand 1 (PD-L1), +, and CD200R1 genes, as well as cell surface expression of CD200 and CD200R1, were measured in twMLR as control, and co-culture groups on days 0, 2 and 4 of the experiment. The proliferation of PBMCs was suppressed on days 2 and 4 of co-culture. The expression of CD200 (p=0.014), CD200R1, CTLA-4, and PD1 genes increased on days 2 and 4 of the co-culture compared to twMLR. CD200 expressing PBMCs decreased by 1.75% on day 2 of the co-culture but increased by 6.23% on day 4 of the co-culture (p=0.013) compared to the same days of twMLR. IL-10 levels increased in the co-culture supernatants on days 2 and 4 compared to twMLR (p<0.05). Our results showed that ASCs upregulate the CD200/CD200R1 axis more than PD-1/PD-L1 and CTLA-4/B7-1 pathways in the twMLR. Also, elevated expression of CD200R1 in the final day of co-culture was similar to PD-1 expression pattern. This finding suggests a role for the CD200/CD200R1 axis in later modulation of the immune response.

Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction.

The immunomodulatory properties of type 2 diabetic patients' adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peripheral blood mononuclear cells (PBMCs) proliferation ratio, protein levels of IFN-γ and IL-10, mRNA expression of COX-2, TNF-α, TGF-ß1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days (p < 0.001). IFN-γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND-ASCs co-culture of the same days (p < 0.05). The expression of TNF-α and IL-6 on days 2 and 4 of D-ASCs co-culture were higher than ND-ASCs co-culture of the same days (p < 0.001). TGF-ß1 on day 4 of ND-ASCs co-culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expression of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition.

Mycobacterium tuberculosis Ag85b:hfcγ1 recombinant fusion protein as a selective receptor-dependent delivery system for antigen presentation.

Introducing an effective vaccine for tuberculosis (TB) is an urgent need. Mycobacterium tuberculosis (Mtb) Ag85 complex is suggested for making protective immunodominant antigens for design and development of novel TB vaccine. In the present study, a pDR2EF1-Fcγ1 vector has been used to make Ag85B:hFcγ1 recombinant fusion protein. Briefly, specific XbaI and NotI site incorporated primers were used to amplify Mtb-fbpB gene by PCR, TA-cloned and amplified in E.coli DH5α. The resulting vector then subcloned into the pDR2EF1.Fcγ1 vector and transferred to Chinese hamster ovary (CHO) cell line. DNA sequencing was performed to confirm that Ag85B:hFcγ1 construct is precise and in-frame. Then, Ag85B:hFcγ1 protein was produced by CHO expression system and recombinant protein was purified using HiTrap rProtein A Sepharose Fast Flow column. The presence of recombinant fusion protein confirmed by immunofluorescence (IFA) and Western blotting (WB). This fusion protein containing Fc fragment of human IgG1, apart from stability and adjuvanticity potential, could bind to FcRγI (CD64) on the surface of antigen-presenting cells (APCs) and induce cross-presentation in favour of host immune response and can be used as a potential candidate along with other subunit vaccines against Mtb.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Medium-dose estrogen ameliorates experimental autoimmune encephalomyelitis in ovariectomized mice.

Estrogen is a neuro-protective hormone in various central nervous system (CNS) disorders. The present study evaluated the role of estrogen during experimental autoimmune encephalomyelitis (EAE) at doses selected to mimic any suppressive potential from the hormone during pregnancy. Here, mice were ovariectomized and then 2 weeks later treated with MOG antigen to induce EAE. Concurrently, mice then received (subcutaneously) an implanted pellet to deliver varying estrogen amounts over a 21-day period. Clinical scores and other parameters were monitored daily for the 21 days. At the end of the period, brain/spinal cord histology was performed to measure lymphocyte infiltration; T-cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed using real-time PCR; T-cell differentiation was evaluated via flow cytometry. The results demonstrated that estrogen inhibited development of EAE. Histological studies revealed limited leukocyte infiltration into the CNS. High and medium dose of estrogen increased TH2 and Treg cell production of interleukin (IL)-4, IL-10, and transforming growth factor (TGF)-ß, but concurrently resulted in a significant reduction in production of interferon (IFN)-γ, IL-17, and IL-6. Flow cytometry revealed there were also significant decreases in the percentages of TH1 and TH17 cells, as well as significant increase in percentages of Treg and TH2 cells in the spleen and lymph nodes. Real-time PCR results indicated that high- and medium-dose estrogen treatments reduced T-bet and ROR-γt factor expression, but enhanced Foxp3 and GATA3 expression. Collectively, these results demonstrated that a medium dose of estrogen - similar to a pregnancy level of estrogen - could potentially reduce the incidence and severity of autoimmune EAE and possibly other autoimmune pathologies.

Structural properties of a viral orthologue of cellular CD200 protein: KSHV vOX2.

Kaposi׳s sarcoma-associated herpesvirus (KSHV) vOX2 is a cell surface glycoprotein expressed during viral lytic replication to suppress host inflammatory reactions. Here we have characterised vOX2 with biochemical, biophysical and bioinformatics tools and as a result propose a 3-dimensional model for vOX2 based on structural and functional homology with the PD-L1 protein. To validate this model, vOX2 was characterised by analytical ultracentrifugation (AUC) and circular dichroism spectroscopy (CD). The results identified the potential glycosylation sites and revealed that vOX2 is predominantly a beta-folded molecule with an RGD adhesion motif exposed on the C-terminal domain. The protein exists in monomer-dimer equilibrium similar to its IgV-type folded homologues, with 30-36% glycosylation and the molecular weight of the extracellular fragment of molecule is 32.0-33.6 kDa, much less than 50 kDa. Thus, the structural similarity to PD-L1 verifies its immunomodulatory potential and the RGD motif suggests an adhesive capacity.

Pregnancy level of estrogen attenuates experimental autoimmune encephalomyelitis in both ovariectomized and pregnant C57BL/6 mice through expansion of Treg and Th2 cells.

Pregnancy suppressive effect on autoimmune diseases including Multiple Sclerosis and Rheumatoid Arthritis may result from high levels of sex steroids such as estrogen and estriol. This study was designed to reveal the molecular and cellular mechanisms underlying the effect of estrogen on MS alleviation. Female C57BL/6 mice were immunized with MOG35-55. Clinical scores and other relevant parameters were monitored daily. Brain and spinal cord histology was performed to measure lymphocyte infiltration and central nervous system demyelination. Th1/Th2/Th17 and Treg cell profiles were determined through ELISA, flow cytometry, and real-time PCR. Transcription factor expression levels in the CNS were assessed by real-time PCR and T cell differentiation was explored through flow cytometry examination. Pregnancy and pregnancy level of estrogen alleviated clinical manifestations in EAE induced mice, reduced CNS demyelination and cell infiltration, suppressed spleen T cell proliferation, enhanced production of anti-inflammatory cytokines in splenocytes and increased the percentage of Th2 and Treg cells. Furthermore, the results of real-time PCR for transcription factors and related cytokines of Th1/Th2/Th17 and Treg cells in CNS showed reduced expression levels of Th1 and Th17 transcription factors, including T-bet and ROR-γt, and decreased Th1 and Th17 cytokines including IFN-γ, TNF-α, IL-17 and IL-23. These results are the first to indicate that pregnancy and pregnancy level of estrogen ameliorate the EAE condition by favoring Treg and Th2 differentiation through induced expression of Foxp3 and GATA3 in the CNS. Moreover, pregnancy and pregnancy level of estrogen decreased mRNA levels of T-bet and ROR-γt in the CNS.

Candida albicans structural and secreted proteins modulate CD4/CD8 ratio in tumor infiltrating lymphocytes of spontaneous adenocarcinoma bearing mice.

BACKGROUND: Candida albicans is one of the most important opportunistic pathogens that suppress immunologic mechanisms of the host. It is speculated that structural and secretory proteins of C. albicans have immunomodulatory effects in cancer. OBJECTIVE: To evaluate the effects of C. albicans structural and secreted proteins on intratumoral CD4/CD8 ratio as well as the survival rate in BALB/c tumor model. METHODS: Structural and secretory proteins from C. albicans were isolated and examined for their effects on tumor growth and survival of adenocarcinoma bearing mice. RESULTS: The results indicated that in mice treated with C. albicans structural protein, the survival rate significantly decreased compared with the control groups. Also, mice treated with secretory proteins showed a decrease in survival rate but it was not statistically significant (p>0.05). Investigating the frequency of tumor infiltrated CD4+ and CD8+ T lymphocytes indicated that the percentages of tumor infiltrated CD4+ T lymphocytes in response to structural and secreted proteins were higher compared to the control groups. CONCLUSION: Our study suggests that C. albicans structural and secreted proteins modulate intratumor T lymphocyte infiltration.