Incomplete Freund's Adjuvant Research Articles

Synthetic Peptides as Immunogen in the Production of IgY Anti-C-Myc Antibody in Local Chicken (Gallus gallus)

Experiments conducted to determine the effect of doses of immunogen on the levels of IgY c-Myc antibody in chickens. To generate antibody, we used a synthetic peptide as an immunogen, which is developed from the epitope of the c-Myc protein. Hens were immunized a week after their first eggs were produced. Antigen diluted in double distilled water and emulsified with Freund 's Complete Adjuvant. The suspension was injected into the area of the chicken breast subcutaneously with a dose of 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, or 0.16 mg per hen. Booster injection performed on days 10, 20, and 30 with the same volume of emulsion, but using Incomplete Freund 's Adjuvant and the amount of antigen was half of the amount of the first Immunization. Extraction of IgY from eggs was carried out using kits from Gallus Immunotech. The level of IgY was quantified using a spectrophotometer where the absorbance was read at 280 nm. The results showed that after the first immunization IgY content of the egg were reached the level of >5 mg/ml yolk when the dose of immunogen was above 0.1 mg/hen. The level was higher in eggs produced after the third booster ( >8 mg/ml yolk), but it is lower in all groups treated with immunogen above 0.1 mg/hen. We concluded, immunogen dosage of 0.1 mg/hen was optimum in the production of chicken IgY anti-c-Myc.

IL-17A Can Promote Propionibacterium acnes-Induced Sarcoidosis-Like Granulomatosis in Mice.

The etiology of sarcoidosis is unknown. In this study, Propionibacterium acnes (PA) was used to induce sarcoidosis-like granulomatous inflammation in a mouse model. Wild-Type (WT) C57BL/6 mice were divided into three groups: (1) WT-PA group; (2) WT-PA + Incomplete Freund's Adjuvant (IFA) group; and (3) WT-PBS group. Loose granuloma formation was observed in the lungs on day 56 in the WT-PA and WT-PA + IFA groups. The proportions of peripheral Th17 cells in the WT-PA (p = 0.0004) and WT-PA + IFA groups (p = 0.0005) were significantly higher than that in the WT-PBS group. The proportions of peripheral Treg cells in the WT-PA (p < 0.0001) and WT-PA + IFA groups (p < 0.0001) were lower than that in the WT-PBS group. Then, to explore the mechanism of IL-17, Wild-Type (WT) C57BL/6 mice were divided into three groups: (1) WT-PBS group (2) WT-PA group; (3) WT-PA + mouse IL-17A neutralizing antibody (IL-17Ab) group. IL-17A gene knockout mice (KO) were divided into two groups: (1) KO -PA group; (2) KO-PBS group. The KO-PA and WT-PA + IL-17Ab groups showed reduced inflammation and no loose granuloma formation on day 56. As compared to the WT-PA group, the ratio of peripheral Th17 in the KO-PA (p < 0.0001) and WT-PA + IL-17Ab groups (p < 0.0001) decreased, while the ratio of peripheral Treg in the KO-PA (p < 0.0001) and WT-PA + IL-17Ab (p = 0.0069) groups increased on day 56. Hence, PA can be used to establish a mouse model of sarcoidosis-like granuloma. IL-17A plays an important role in experimental sarcoidosis-like granuloma formation.

Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm

Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-notifiable disease of equidae. Despite the importance of B. mallei, little is known about serodiagnosis of glanders. The present study aimed to develop an immunoblotting assay based on whole-cell proteome of B. mallei to enable accurate serodiagnosis of glanders. Three farm horses were subcutaneously immunized with a crude suspension (106 cfu/ml) of heat-inactivated B. mallei formulated with incomplete Freund's adjuvant (IFA) to achieve a hyperimmune sera panel. The immunization was done for 1, 14 and 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The hyperimmunity of sera was confirmed by CFT. B. mallei whole-cell proteome was prepared through sonication and the protein content was visualized by SDS-PAGE and quantified by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative horse sera validated the test. A ladder pattern of the B. mallei immunoreactive antigens was seen within the region of 20-90 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders. The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries.

Immunoexpression of canonical Wnt and NF-κB signaling pathways in the temporomandibular joint of arthritic rats

To investigate the participation of canonical Wnt and NF-κB signaling pathways in an experimental model of chronic arthritis induced by methylated bovine serum albumin (mBSA) in rat temporomandibular joint (TMJ). Wistar rats were sensitized by mBSA+Complete Freund Adjuvant (CFA)/Incomplete Freund Adjuvant (IFA) on the first 14days (1×/week). Subsequently, they received 1, 2 or 3 mBSA or saline solution injections into the TMJ (1×/week). Hypernociceptive threshold was assessed during the whole experimental period. 24h after the mBSA injections, the TMJs were removed for histopathological and immunohistochemical analyses for TNF-α, IL-1β, NF-κB, RANKL, Wnt-10b, β-catenin and DKK1. The nociceptive threshold was significantly reduced after mBSA injections. An inflammatory infiltrate and thickening of the synovial membrane were observed only after mBSA booster injections. Immunolabeling of TNF-α, IL-1β and Wnt-10b was increased in the synovial membrane in arthritic groups. The immunoexpression of nuclear β-catenin was significantly higher only in the group that received 2 booster TMJ injections. However, NF-κB, RANKL and DKK1 immunoexpression were increased only in animals with 3 mBSA intra-articular injections. Our results suggest that canonical Wnt and NF-κB signaling pathways participate in the hypernociception and inflammatory response in TMJ synovial membrane during the development of rheumatoid arthritis in rats.

11C]PIB PET imaging can detect white and grey matter demyelination in a non-human primate model of progressive multiple sclerosis

BackgroundMultiple sclerosis (MS) is a demyelinating and inflammatory disease of the central nervous system. Its diagnosis is clinical, often confirmed by magnetic resonance imaging. This image modality, however, is not ideal for discrimination of demyelination in grey and white matter regions from inflammatory lesions. Positron Emission Tomography (PET), using specific radiopharmaceuticals, can be a tool to differentiate between these processes. The radiopharmaceutical [11C]PIB is widely used for detection of β-amyloid plaques, but has also been suggested for the analysis of myelin content due to its consistent uptake in white matter. The aim of this study was to evaluate [11C]PIB PET imaging as a tool for detecting demyelinated regions in white and grey matter of non-human primate model of progressive MS. MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in marmosets by injection of recombinant human myelin oligodendrocyte glycoprotein (rhMOG) emulsified in either Incomplete Freund's Adjuvant (IFA) or Complete Freund's Adjuvant (CFA). [11C]PIB PET images were acquired prior to immunization (baseline) and after symptoms were present (end of experiment). Brain tissue was isolated for histochemical analysis. ResultsAll rhMOG/IFA-treated and rhMOG/CFA-treated animals showed clinical signs of EAE. The rhMOG/CFA group presented a significant [11C]PIB uptake reduction only in the left motor cortex (9%, P = 0.011). For the rhMOG/IFA group, significant decrease in [11C]PIB uptake was observed in the whole brain (15%, P = 0.015), in the right hemisphere of body of corpus callosum (34%, P = 0.02), splenium of corpus callosum (38%, P = 0.004), hippocampus (19%, P = 0.036), optic tract (13%, P = 0.025), thalamus (14%, P = 0.041), Globus pallidus (23%, P = 0.017), head of caudate nucleus (25%, P = 0.045), tail of caudate nucleus (29%, P = 0.003), putamen (28%, P = 0.047) and left hemisphere of body of corpus callosum (14%, P = 0.037) and head of caudate nucleus (23%, P = 0.023). [11C]PIB uptake significantly correlated with luxol fast blue histology (myelin marker), both in the rhMOG/IFA (r2= 0.32, P < 0.0001) and the rhMOG/CFA group (r2= 0.46, P < 0.0001). Conclusion[11C]PIB PET imaging is an efficient tool for detecting demyelination in grey and white matter, in a non-human primate model of progressive MS.

The effect of crude alcoholic extract of Withania somnifera leaves in experimentally induced arthritis in mice

The alcoholic extract 70% of the leaves of Withania Somnifera was dried and percentage of extract 8.3 were used .Incomplete freunds adjuvant was used to induce arthritis in mice 0.1ml of this adjuvant was injected in the right tarsal joint. The mice were divided into four groups, the first group were treated with alcoholic extract 500mg/kg body weight (T1), the second group was treated with diclofenac sodium 3mg/kg body weight (T2) and third group was treated with distilled water (T3), the fourth group used as a control group (T4) non induced non treated.Vernier calipers was used for measuring the diameter of the joint. The mice in T1group showed a significant decrease in the diameter of tarsal joint of right leg as compared with mice in T2, T3 and T4 groups. The hematological results of mice in T1 group showed significant increase as compared with T4 group. While inT2 and T3 groups showed a significant decrease as compared with T4 group .The histological results showed that the joint of mice in T1 group showed no inflammatory response in the membrane of joint. The joint of mice in T2 group showed inflammatory cell infiltration while the joint of mice in T3 group showed fibrous connective tissue proliferation with mononuclear cell infiltration in the membrane of the joint .

A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model

Engagement of programmed death 1 receptor (PD-1) and its ligand PD-L1/2 induces a signal transduction pathway that inhibits the activity of tumor-infiltrating cytotoxic T lymphocytes and promotes tumor growth and metastasis. Antibodies blocking PD-1 or PD-L1 can restore antitumor T cell responses and cause long-term remission in a subset of cancer patients with advanced or refractory tumors. In this study, we asked whether PD-L1 vaccination could confer tumor control in mouse tumor models. To address the central tolerance toward self-molecules, we fused the extracellular domain of PD-L1 (PD-L1E) to the C-terminal of the translocation domain of diphtheria toxin (DTT). DTT is able to elicit CD4+ T cell responses required for inducing robust immune responses against self-molecules. The fusion molecule is called DPDL1E. When formulated with incomplete Freund’s adjuvant (IFA), DPDL1E elicited robust immune responses biased toward the Th1 type and inhibited tumor growth in both preventive and therapeutic mouse tumor models. We further showed that the anti-DPDL1E sera blocked PD-L1 binding to PD-1 in vitro. The DPDL1E vaccination increased the levels of tumor-infiltrating T lymphocytes (TILs) and reduced the levels of myeloid-derived suppressor cells (MDSCs) as well as exhausted LAG3+PD-1+ CD8+ T cells. All of these data suggest that DPDL1E vaccination reverses the suppressive phenotype of the tumor microenvironment and that it is a promising strategy for cancer therapy.

A multipeptide vaccine plus toll-like receptor agonists LPS or polyICLC in combination with incomplete Freund\u2019s adjuvant in melanoma patients

BackgroundCancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund’s adjuvant (IFA) would reduce magnitude and persistence of immune responses.Patients and methodsParticipants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS).ResultsFifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6.ConclusionsLPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA.Trial registrationThe clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012.

Tumor growth inhibition by mSTEAP peptide nanovaccine inducing augmented CD8+ T cell immune responses.

Poly(lactic-co-glycolic) acid (PLGA) has been successfully used in drug delivery and biomaterial applications, but very little attention has been directed towards the potential in vivo effects of peptide-loaded PLGA nanoparticles (NPs), specifically the potency of intravenous (IV) STEAP peptide-loaded PLGA-NP (nanovaccine) dosing and whether STEAP-specific CD8+ T cells directly play a key role in tumor inhibition. To address these concerns, syngeneic prostate cancer mouse models were established and treated with either mSTEAP peptide emulsified in incomplete Freund's adjuvant (IFA) via subcutaneous (SC) injection or mSTEAP peptide nanovaccine containing the same amount of peptide via IV or SC injection. Meanwhile, mice were treated with either CD8b mAb followed by nanovaccine treatment, free mSTEAP peptide, or empty PLGA-NPs. Immune responses in these mice were examined using cytotoxicity assays at 14days after treatment. Tumor size and survival in various treatment groups were measured and monitored. The results demonstrated that mSTEAP peptide nanovaccine resulted in tumor inhibition by eliciting a significantly stronger CD8+ T cell immune response when compared with the controls. Moreover, the survival periods of mice treated with mSTEAP nanovaccine were significantly longer than those of mice treated with mSTEAP peptide emulsified in IFA or the treatment controls. Additionally, it was observed that the peptide nanovaccine was mainly distributed in the mouse liver and lungs after IV injection. These findings suggest that the peptide nanovaccine is a promising immunotherapeutic approach and offers a new opportunity for prostate cancer therapies.

Stimulation of Nucleotide Oligomerization Domain and Toll-Like Receptors 2 to Enhance the Effect of Bacillus Calmette Guerin Immunization for Prevention of Mycobacterium Tuberculosis Infection: Protocol for a Series of Preclinical Randomized Controlled Trials.

BackgroundBacillus calmette guerin (BCG) immunization has been associated with a reduction in Mycobacterium tuberculosis (MTB) infection. BCG immunization has been shown to enhance innate immunity. This effect of BCG can be explained by an enhancing effect on innate immunity.ObjectiveThis study aimed to test the following hypotheses: (1) BCG immunization can prevent infection with MTB, (2) prevention of infection occurs via stimulation of NOD2 (nucleotide oligomerization domain) and toll-like receptors 2 (TLR2), and (3) the effect of BCG immunization on prevention of infection with MTB can be enhanced by giving stimulators of NOD2 and TLR2.MethodsTo detect the influence of immunization on infection rates, the ultralow dose (ULD) infection model is used. The infection rate of mice vaccinated with BCG and exposed after 6 weeks to ULD of MTB and unvaccinated mice are compared via cultures of lung homogenates and interferon (IFN) gamma release assay. If a reduced infection rate by BCG immunization is confirmed, the experiment is repeated by giving BCG combined simultaneously or in time sequence with the enhancers of innate immunity murabutide or beta-glycan. The influence of murabutide or beta-glycan alone on infection rates is investigated. To quantify the contribution of innate immunity levels of tumor necrosis factor, IFN gamma expression, histone H3 K4me3 trimethylation, and concentrations of monocytes with features of activation of innate immunity as defined by the Ly6Chigh as well as CD11b positive phenotype in immunized versus unimmunized infected and uninfected mice in the various immunization protocols is compared. The experiments will be repeated with prior application of the inhibitors of epigenetic programming of innate immunity histone methyltransferase inhibitor 5’-deoxy-5’-methylthio-adenosine and histone acetyl transferase inhibitor epigallocatechin-3-gallate. The influence of BCG on innate immunity is further corroborated by a prospective observational study in human infants.ResultsInvestigations of derivatives of muramyl dipeptide (MDP) to enhance early immunity in the C57BL/6 mouse strain (mice aged 7 weeks) by another group used 300 micrograms per mouse of oil-associated 6-0-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (mycol-MDP) 50/50 mixed with Freund’s incomplete adjuvant. Comparison of colony-forming unit (CFU) count in the lungs 3 weeks after aerosol challenge with Mycobacterium bovis of groups (n=5) between groups receiving mycol-MDP in oil emulsion (see above) versus controls (n=5) showed a significantly lower CFU count of 94.5 x106 (SD 22.0) in cases versus controls with 204.0 X 106 (SD 77.6). It is important to note that after elimination of T-cells in this model, a reduction of CFU in lungs of mice treated with mycol-MDP persisted albeit without statistical significance, which was possibly related to the small number of animals used.ConclusionsDemonstration of a reduction of MTB infection by enhancement of innate immunity could show a new approach to improving vaccine efficacy against this pathogen.International Registered Report Identifier (IRRID)PRR1-10.2196/13045

The Effects of Red Betel (Piper crocatum) Leaf Extract to Decreasing Profile Population of CD4+ and CD8+ Cells in Rheumatoid Arthritis-Induced Mice

Rheumatoid Arthritis (RA) is one of the autoimmune diseases resulted by oxidative stress. Red betel leaves (Piper crocatum) with a high content of flavonoid are expected to function as antioxidant and to reduce inflammation. This research aimed to identify the effects of red betel leaf extract on the amount of the CD4+ and CD8+ cells. 24 male Swiss mice, aged eight 8 weeks, with the weight of 27±3 g. 20 mice induced-RA were made by injecting 0.01 mL Complete Freund’s Adjuvant (CFA) by intraperitoneal; after seven days, they were then boostered by injecting 0.03 mL of Incomplete Freund’s Adjuvant (IFA) into the forelimbs. The animals were divided into six groups: normal, RA (C-), RA+aspirin (C+), RA+red betel leaf extract 100 mg/kg BW (T1), RA+red betel leaf extract 200 mg/kg BW (T2), and RA+leaf extract red betel nut 400 mg/kg BW (T3). The animals were given treatment for 21 days. At the end of the treatment, the mice were sacrificed, and the removed lymphocyte cells were isolated to measure the number of the CD4+ and CD8+ using Flow-cytometry. The results of this research showed that red betel leaf extract was significantly different to decreasing the amount of the CD4+ cells, but it was not significantly different to reducing the amount of CD8+ cells. The effective dosage in this research was 200 mg/kg BW.

Safety and biocompatibility of injectable vaccine adjuvants composed of thermogelling block copolymer gels

Injectable thermogelling polymers have been recently investigated as novel adjuvants and delivery systems for next generation vaccines. As research into natural and synthetic biocompatible polymers progresses, the safety and biocompatibility of these compounds is of paramount importance. We have developed cationic pentablock copolymer (PBC) vaccine adjuvants based on Pluronic F127, a thermogelling triblock copolymer that has been approved by the FDA for multiple applications, and methacrylated poly(diethyl amino)ethyl methacrylate outer blocks. These novel materials have been demonstrated to effectively create an antigen depot, minimally impact antigen stability, and enhance the immune response to antigens (i.e., adjuvanticity) in mice. In this work, we investigated the safety and biocompatibility of the parent triblock Pluronic gels and the cationic PBC gels in mice. Histological analysis showed no injection site reactions and no damage to the liver or kidneys was observed upon administering the block copolymer formulations. However, the subcutaneous injection of a thermogelling Pluronic solution induced increased levels of lipids in the blood, with no further deleterious effects observed from the addition of the cationic outer blocks. This hyperlipidemia resolved within 30 days after the administration of the Pluronic formulation. To mitigate this adverse effect, the vaccine adjuvant formulations were modified by adding poly(vinyl alcohol), which allowed gelation, while reducing the amount of Pluronic in the formulation. This modified formulation abrogated the observed hyperlipidemia and no adverse effects were observed in the serum through biomarker analysis or at the injection site (i.e., inflammation) in comparison to the responses induced by administration of saline or incomplete Freund's adjuvant. These studies provide a foundation to developing these gels as adjuvants for next generation vaccines. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1754-1762, 2019.

Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate.

Aim:Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate.Materials and Methods:Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund’s complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund’s incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method.Results:Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster.Conclusion:The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Nanoparticle or conventional adjuvants: which one improves immune response against Brucellosis?

Objective(s):Brucellosis is a common infectious disease among animals and humans. While subunit vaccines could be used as an efficient strategy against pathogens, they usually seem to be less immunogenic than live or killed vaccines. However, the use of a suitable adjuvant accompanied by subunit vaccines can be a good alternative to enhance the immune response.Materials and Methods:To find a proper adjuvant against Brucellosis, the immune response of induced mice by Aluminum Hydroxide (AH), Incomplete Freund (IFA), and Chitosan Nanoparticle (CS) adjuvants in individuals and in combination with CS were assessed.Results:Immunization with CS stimulated higher interferon gamma (IFN-γ) immunity, while there were no significant differences between rOMP25 (IFA), rOMP25 (AH), rOMP25 (AH-CS) and rOMP25 (IFA-CS) recombinant proteins. Tumor necrosis factor alpha (TNF-α) analysis revealed there were no significant differencesbetween immunized groups and the positive control group, except for the treatment formulated in single IFA. Furthermore, unlike IFN-γ, there was a reverse interleukin-4 (IL-4) immune response trend for treatments, as rOMP25 (CS) displayed the lowest response. rOMP25 (CS) induced higher titer of total antibody than the other ones. Although the recombinant proteins emulsified in different adjuvants induced similar titer of IgG1 antibody, the ones that were formulated in CS, IFA and IFA-CS showed a higher titer of IgG2a. The cell proliferation assay demonstrating the antigen-specific cell proliferative response could be promoted after immunization with CS. Conclusion:CS whether single or in combination with IF adjuvants has potential to improve Th1-Th2 responses.

Synthetic Peptides as Immunogen in the Production of IgY Anti-C-Myc Antibody in Local Chicken (Gallus Gallus)

Experiments conducted to determine the effect of doses of immunogen on the levels of IgY c-Myc antibody in chickens. To generate antibody, we used a synthetic peptide as an immunogen, which is developed from the epitope of the c-Myc protein. Hens were immunized a week after their first eggs were produced. Antigen diluted in double distilled water and emulsified with Freund 's Complete Adjuvant. The suspension was injected into the area of the chicken breast subcutaneously with a dose of 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, or 0.16 mg per hen. Booster injection performed on days 10, 20, and 30 with the same volume of emulsion, but using Incomplete Freund 's Adjuvant and the amount of antigen was half of the amount of the first Immunization. Extraction of IgY from eggs was carried out using kits from Gallus Immunotech. The level of IgY was quantified using a spectrophotometer where the absorbance was read at 280 nm. The results showed that after the first immunization IgY content of the egg were reached the level of &gt;5 mg/ml yolk when the dose of immunogen was above 0.1 mg/hen. The level was higher in eggs produced after the third booster ( &gt;8 mg/ml yolk), but it is lower in all groups treated with immunogen above 0.1 mg/hen. We concluded, immunogen dosage of 0.1 mg/hen was optimum in the production of chicken IgY anti-c-Myc.

Prophylactic Autologous Cancer Cells Immunization Inhibits Triple Negative Breast Tumors’ Growth and Metastasis in 4T1 Tumor-Bearing BALB/c Mice

Triple negative breast cancer (TNBC), a type of breast cancer, is still incurable and 4T1 tumor is an equal experimental model of TNBC which is highly metastatic and invasive. In the present study, we assessed the prophylactic cancer vaccine effects on survival and metastasis in 4T1 tumor bearing mice. Female BALB/c mice were divided in three groups and subcutaneously (s.c) injected with PBS incomplete Freund’s adjuvant (IFA) and a mix of the homogenized 4T1 cancer cells emulsified with IFA, respectively. Also, in the third group, the last immunization was contained just the homogenized cancer cells. Tumor induction was performed with 4T1 cells injection at the 4th mammary fat pad of each mouse. Tumor diameter and the number of macroscopic metastatic nodules on the lungs’ surface were investigated two weeks after the last immunization. We found that the immunization of mice with homogenized 4T1 cancer cells and IFA significantly inhibited 4T1 tumors’ growth. Also, metastatic colonies in the lungs significantly decreased in mice which immunized with 4T1 cancer cells and IFA. Overall, the effective induction of immune response by the vaccine used in our study seems to have prevented cancer cells migration and metastasis to other organs. However, precise immunological studies are required to determine the mechanism of such a vaccine.

Detection of specific immune response in sole (Solea senegalensis) against Photobacterium damselae subsp. piscicida antigens

The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response.Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis.Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida, and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule.Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related.

Antigen-specific CD4+ and CD8+ T-cell responses in PBMC from pony mares immunized with either native or recombinant zona pellucida vaccines

Few studies have investigated the cell mediated immune response during zona pellucida-based immunocontraception, despite hypothesized cytotoxic T-cell involvement in ovarian dysfunction associated with these vaccines. This study aimed to investigate antigen-specific anamnestic responses of helper (CD4+) and cytotoxic (CD8+) T-lymphocytes in peripheral blood mononuclear cells (PBMC) isolated from pony mares before and after their treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 antigens (reZP) or adjuvanted saline. Mares were randomly assigned to pZP, reZP and control groups (n = 7 per group). Treatments consisted of a primary vaccination or saline (V1; Day 0) incorporating Freund's modified complete adjuvant, followed by a single booster (V2; Day 35) incorporating Freund's incomplete adjuvant. Peripheral blood mononuclear cells were isolated and cryopreserved immediately prior to V1 (Day 0) and five weeks post V2 (Day 70). Relative proliferation of T-lymphocytes in response to pZP antigen was assessed using carboxyfluorescein diacetate succinimidyl ester dilution with immunophenotyping, analysed via flow cytometry. Significant pZP-specific CD4+ and CD8+ T-lymphocyte responses were detected in PBMC isolated from mares treated with either pZP or reZP, in comparison to pre-treatment samples. In the pZP group, but not the reZP group, CD8+ T-cell proliferation showed significant negative correlations to circulating progesterone, oestradiol and anti-Müllerian hormone levels. Results suggest that antigen-specific CD8+ T-cells may play a role in ovarian suppression observed during pZP immunocontraception in this species.

Anti-arthritic effect of pentosan polysulfate in rats with collagen-induced arthritis

Pentosan polysulfate (PPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of PPS on arthritic profiles based on clinical score, ankle size, histological changes, and activity of inflammatory mediators using collagen-induced arthritic rat are reported. Model of arthritis was developed in Sprague Dawley rats by intradermal injection of bovine type II collagen emulsified with incomplete Freund's adjuvant. The rats were randomly divided into four groups: normal control, arthritic control, arthritic rats treated with PPS (at dose level 20 μg/g) and arthritic rats treated with meloxicam (2 μg/g). The treatment was continued daily until the day 30. Arthritic biomarkers (cartilage oligomeric matrix protein and tartrate-resistant acid phosphatase 5b) in synovial fluid, expression of inflammatory mediators (interleukin-1β, and tumor necrosis factor-α) and osteoclast marker genes (cathepsin K, tartrate-resistant acid phosphatase) in synovial membrane were measured. Daily administration of PPS to the arthritic rats significantly decreased the severity of arthritis by effectively suppressing the symptoms of arthritis and improving the functional recovery based on clinical score and histopathological evidence. Intriguingly, identical downregulation pattern of arthritis profiles, biological markers as well as relative mRNA levels of osteoclast markers and cytokines were monitored in arthritic rats treated with PPS. In conclusion, PPS exerted protective effects against collagen-induced arthritis in rats. The results suggest that PPS acts as an anti-inflammatory and anti-arthritic agent in decreasing the arthritic effects in collagen-induced arthritic rats.

In Vivo Enrichment of Genetically Manipulated Platelets for Murine Haemophilia B Gene Therapy

Hemophilia B (HB) is an X chromosome-linked bleeding disorder resulting from a deficiency of factor FIX (FIX). Our previous studies have demonstrated that platelet-targeted FIX gene therapy can provide sustained therapeutic levels of FIX expression in platelets without inhibitory antibody (inhibitor) development, indicating platelet gene therapy could be a promising strategy for the treatment of HB patients. In this study, we aimed to enhance platelet-FIX expression in HB mice with MGMT P140K-mediated in vivo selection of hematopoietic stem cells (HSCs) under a nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT LV), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the drug-resistance MGMT P140K gene. Platelet-FIX expression was introduced in nonmyeloablative preconditioned HB mice after 2bF9/MGMT LV-mediated HSC transduction and transplantation. After 8 weeks of bone marrow reconstitution, recipients were administered with O6-benzylguanine/1, 3-bis-2-chloroethyl-1- nitrosourea (BG/BCNU) once every 4 weeks for a total of 3 treatments. Animals were analyzed starting at 4 weeks after transplantation by PCR, quantitative real-time PCR (qPCR), FIX assays, inhibitor assays, and tail bleeding test.PCR results showed that 2bF9 proviral DNA was detected in all recipients that received 2bF9/MGMT LV-transduced HSCs. Quantitative PCR results confirmed that 2bF9/MGMT LV-transduced cells were effectively enriched after each round of BG/BCNU selective treatment in vivo. The average copy number of the 2bF9 cassette per cell after three BG/BCNU treatments was 0.50 ± 0.04, which was significantly higher than pre-BG/BCNU treatment (0.13 ± 0.01). There was an approximately 2.9-fold higher FIX antigen (FIX:Ag) (4.11 ± 0.76 vs. 1.43 ± 0.06 mU/108 platelets) and a 3.7-fold FIX activity (FIX:C) (2.56 ± 0.50 vs. 0.69 ± 0.04 mU/108 platelets) level, respectively, post-treatment compared to pre-treatment. There was a small amount of FIX:Ag detected in the 2bF9/MGMT LV-transduced recipient plasma. When we normalized FIX:Ag levels to total whole blood FIX content, the results demonstrated that 96.32 ± 0.32% of whole blood FIX in the BG/BCNU treated animals was stored in platelets. To assess whether the bleeding phenotype was rescued in HB mice after receiving 2bF9/MGMT LV-transduced HSCs, we used a 6-hour tail bleeding test. All 2bF9/MGMT LV-transduced recipients stopped bleeding within 6 hours with a clotting time of 2.6 ± 0.5 hours and a remaining hemoglobin level of 65.1 ± 4.9%, which were not significantly different from those of the wild-type control group (1.7 ± 0.9 hours and 70.6 ± 13.9%). In contrast, none of the FIXnull mice stopped bleeding within 6 hours with a remaining hemoglobin level of 38.8 ± 6.7%.Notably, none of the recipients developed anti-FIX inhibitory antibodies as measured by a modified Bethesda assay. To investigate whether immune tolerance was induced in 2bF9/MGMT LV-transduced recipients, all the recipients were challenged with 200U/kg recombinant human FIX (rhFIX) in the presence of Incomplete Freund's adjuvant (IFA) at 9 months after transplantation. Only one out of four recipients developed a low titer of inhibitors (1.3 BU/ml). In contrast, all of the HB controls developed inhibitors ranging from 17-120 BU/ml after the same challenge (n = 4). When we used the ELISA assay for the quantification of total anti-FIX IgG antibodies, low titer antibodies were found in the recipients after they were challenged with rhFIX plus IFA. However, the antibody levels in the HB control mice under the same conditioning were 213-fold higher than those developed in 2bF9 MGMT LV-transduced recipients.In conclusion, our data demonstrated that using the MGMT-mediated drug-selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune suppression in HB mice. Our studies suggest that platelet-targeted FIX gene therapy together with in vivo enrichment of engineered cells may provide a promising strategy for the treatment of HB patients. DisclosuresNo relevant conflicts of interest to declare.