Development and evaluation of cell membrane-based biomimetic nanoparticles loaded by Clostridium perfringens epsilon toxin: a novel vaccine delivery platform for Clostridial-associated diseases.

As Clostridium perfringens (C. perfringens) epsilon toxin (ETX) ranks as the third most potent clostridial toxin after botulinum and tetanus toxins, vaccination is necessary for creatures that can be affected by it to be safe from the effects of this toxin. Nowadays, nanostructures are good choices for carriers for biological environments. We aimed to synthesize biomimetic biodegradable nanodevices to enhance the efficiency of the ETX vaccine. For this purpose, poly(lactic-co-glycolic acid) (PLGA) copolymer loaded with purified epsilon protoxin (proETX) to create nanoparticles called nanotoxins (NTs) and then coated by RBC membrane-derived vesicles (RVs) to form epsilon nanotoxoids (RV-NTs). The resulting RV-NTs shaped smooth spherical surfaces with double-layer core/shell structure with an average particle size of 105.9 ± 35.1 nm and encapsulation efficiency of 97.5% ± 0.13%. Compared with NTs, the RV-NTs were more stable for 15 consecutive days. In addition, although both structures showed a long-term cumulative release, the release rates from RV-NTs were slower than NTs during 144 hours. According to the results of cell viability, ETX loading in PLGA and entrapment in the RBC membrane decreased the toxicity of the toxin. The presence of PLGA enhances the uptake of proETX, and the synthesized structures showed no significant lesion after injection. These results demonstrate that NTs and RV-NTs could serve as an effective vaccine platform to deliver ETX for future in vivo assays.

Evaluating the Immunogenicity of recombinant VP1 protein from the foot-and-mouth disease virus encapsulated in nanoliposome in guinea pig animal model.

Foot-and-mouth disease (FMD) is considered as contagious in livestock, which is caused by the Picornavridae virus family known as FMD virus (FMDV). In the present study, the VP1 gene from FMDV (O strain) was expressed and purified. In addition, nanoliposomes were utilized as an adjuvant. After formulating the nanoliposomes with DMPC, DMPG, and cholesterol, the recombinant VP1 protein was encapsulated in the nanoliposomes. Further, the intended nanoliposomes in the sizes of 400 and 200 nm, nanoliposome-inactivated FMDV, Freund's adjuvant-inactivated FMDV, and Freund's adjuvant-recombinant VP1 mixtures, and PBS buffer (negative control) were injected to six groups of five guinea pigs. Furthermore, the guinea pig serums were analyzed through using ELISA and serum neutralization tests after four boosting vaccinations. Based on the results, the immunogenicity of the 200 nm-nanoliposomes encapsulating recombinant VP1 protein was more than that of 400 nm-ones so that 200 nm-nanoliposomes could trigger the immune response against FMDV in guinea pigs.

The role of MPL and imiquimod adjuvants in enhancement of immune response and protection in BALB/c mice immunized with soluble Leishmania antigen (SLA) encapsulated in nanoliposome.

Adjuvants play an essential role in the induction of immunity against leishmaniasis. In this study, monophosphoryl lipid A (MPL) and imiquimod (IMQ) were used as TLR ligands adjuvants to enhance immunogenicity and rate of protection against leishmaniasis. Nanoliposomes containing soluble Leishmania antigens (SLA) and adjuvants were consisted of DSPC, DSPG and Chol prepared by using lipid film method followed by bath sonication. The size of nanoliposomes was around 95 nm and their zeta potential was negative. BALB/c mice were immunized by liposomal formulations of lip/SLA, lip/MPL/SLA, lip/IMQ/SLA, lip/MPL/IMQ/SLA, lip/SLA + lip/IMQ, lip/SLA + lip/MPL, lip/SLA + lip/MPL/IMQ and five controls of SLA, lip/MPL, lip/IMQ, lip/MPL/IMQ and buffer by subcutaneously (SC) injections, three times in 2 weeks intervals. The synergic effect of two adjuvants when they are used in one formulation showed significantly (p < .001) smaller footpad swelling and the lowest parasite burden in lymph node and foot after the challenge. IgG2a in these groups showed the higher titre compared to control groups, which is compatible with the high IFN-γ production and lowest IL-4. Taken together the results indicated that co-delivery of MPL and IMQ adjuvants and antigen in nanoliposome carrier could be an appropriate delivery system to induce cellular immunity pathway against leishmaniasis.

Comparison of Gold Nanoparticle Conjugated Secondary Antibody with Non-Gold Secondary Antibody in an ELISA Kit Model.

In this study, gold nanoparticles (AuNPs) were used as carriers of the signaling anti-chicken antibody peroxidase in comparison with anti-chicken antibody peroxidase without gold nanoparticle in a commercial avian influenza kit. AuNPs enhanced the absorbance and shortened the assay time. AuNPs act as a carrier of many enzymes and multiply the effect of enzyme when reacting with substrate. They amplify optical signal, while keeping low background signals.

Evaluation of immunogenicity of purified cell wall-associated 34 kDa antigen of Mycobacterium avium subsp. paratuberculosis infection.

The 34 kDa cell wall protein of Mycobacterium avium subsp. paratuberculosis (MAP) has been suggested as a major species-specific immunodominant antigen in Johne's disease. However to date, there has not been a purified 34 kDa protein isolated from bacterial lysates used in immunogenicity analysis. Therefore we attempted to assess the immunogenicity properties of the purified cell wall 34 kDa protein for the first time, and compare the results with previous studies. We used an ELISA test for evaluation of the immunogenicity of this 34 kDa antigen against MAP infection. All serum samples from cattle confirmed to be infected with MAP were positive and those from healthy cattle were negative with the present antigen in ELISA tests. The sensitivity and specificity of 34 kDa antigen were then evaluated in comparison with a standard commercial kit and whole cell wall extracts. The results indicated that the pure 34 kDa antigen specific to MAP with high specificity and sensitivity has a strong potential for use in serodiagnosis assays and screening of Johne's disease.

Differentiating pestes des petits ruminants and rinderpest viruses by a novel monoclonal antibody.

Peste des petits ruminant (PPR) is an acute, febrile, viral disease of small ruminants with great economic importance. PPR and rinderpest (RP) viruses are antigenically related and need to be differentiated serologically. The use of monoclonal antibodies (MAbs) in ELISA for specific diagnostics and separation of PPR and RPV is important. For this purpose, six Balb/c mice were immunized with inactivated antigen from the Nijeria strain. Fusion cloning was performed 3 months later by directly using cloning plates, selecting the hybridoma colonies at an early stage with an inverted microscope, and transferring the colonies into 96-well plates with a micropipette. From 300 wells, nearly 56 hybridoma clones were found, from which, after testing in ELISA, 11 with higher titer were selected. Among these, only two clones were placed for limiting dilution (1H1, 6A12). Only one clone (6A12L1F12) had no cross-reactivity with RP, reacted with the N protein, and was of IgG2 isotype.

Psyllium decreased serum glucose and glycosylated hemoglobin significantly in diabetic outpatients.

Psyllium is a bulk-forming laxative and is high in both fiber and mucilage. The beneficial effect of dietary fiber in the management of type II diabetes, has not been totally demonstrated. The purpose of this study was to determine the plasma-lowering effects of 5.1g b.i.d. of psyllium husk fiber, as an adjunct to dietary and drug therapy on lipid and glucose levels, in patients with type II diabetes. Patients were randomly selected from an outpatient clinic of primary care to participate in a double-blind placebo-controlled study in which Plantago ovata Forsk., or placebo was given in combination with their anti-diabetic drugs. Forty-nine subjects were included in the study that were given diet counseling before the study and then followed for 8 weeks in the treatment period. Fasting plasma glucose (FBS) was measured every 2 weeks, and total plasma cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglyceride (TG), and insulin levels were measured every 4 weeks. Glycosylated hemoglobin (HbA1c) was also measured at the beginning and ending of the study. The test products (psyllium or placebo) were supplied to subjects in identically labeled foil packets containing a 5.1g dose of product, to consume two doses per day, half an hour before breakfast and dinner. Both products were well tolerated, with no serious adverse events related to treatment was reported in either. Better gastric tolerance to metformin was recorded in the psyllium group. FBS, and HbA1c, showed a significant reduction (p<0.05), whereas HDL-C increased significantly (p<0.05) following psyllium treatment. LDL/HDL ratio was significantly decreased (p<0.05). Our results show that 5.1g b.i.d. of psyllium for persons with type II diabetes is safe, well tolerated, and improves glycemic control.