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Background: Necrotic enteritis (NE) is an economically important disease, caused by Clostridium perfringens type G strains, and is one of the major targets of antibiotics used in poultry feed. Aims: This study aimed to genotypically characterize virulent strains of C. perfringens isolated from healthy and diseased birds in Iran. Methods: Eleven isolates were derived from necrotic enteritis cases, and 27 were from healthy chickens. Isolations were performed using blood agar. To assess whether zmp is generally associated with avian NE, 38 C. perfringens isolates were screened using PCR and western blotting. The involvement of these toxins as virulence factors was investigated using cytotoxicity assays. Results: All isolates carried the phospholipase c (plc) gene regardless of their origin and virulence. The zinc metallopeptidase (zmp) gene was found in the isolates collected from birds affected by necrotic enteritis. Furthermore, Necrotic enteritis like B (NetB) was only found in 36.36% of the isolates derived from necrotic enteritisinfected birds. Western blot analysis further confirmed the expression of Alpha toxin, NetB, and Zmp in different isolates. Incubation of Leghorn Male Hepatoma (LMH) cells with crude C. perfringens toxins indicated that the supernatants of all bacterial strains were toxic toward LMH cells at different dilutions. In addition, crude toxins of the Cp28 strain expressing Alpha toxin, Zmp, and NetB showed an approximately 50% cytotoxic dose (CD50) at a 1:34 dilution. Strain Cp119.2, which produces both ZMP and the Alpha toxin, and strain Cp48, which only produces the Alpha toxin, showed CD50 at 1:23 and 1:4 dilutions, respectively. Conclusion: It seems that both NetB and Zmp play major roles in the cytotoxicity and pathogenicity of this organism. [ABSTRACT FROM AUTHOR]

Therapeutic human immunodeficiency virus (HIV) vaccines can boost the anti-HIV host immunity to control viral replication and eliminate viral reservoirs in the absence of anti-retroviral therapy. In this study, two computationally designed multiepitope Gag-Pol-Env-Nef-Rev and Hsp70-Gag-Pol-Env-Nef-Rev constructs harboring immunogenic and highly conserved HIV T cell epitopes were generated in E. coli as polypeptide vaccine candidates. Furthermore, the multiepitope gag-pol-env-nef-rev and hsp70-gag-pol-env-nef-rev DNA vaccine constructs were prepared and complexed with MPG cell-penetrating peptide. The immunogenicity of the multiepitope constructs were evaluated using the homologous and heterologous prime/boost strategies in mice. Moreover, the secretion of IFN-γ was assessed in infected lymphocytes in vitro. Our data showed that the homologous polypeptide regimens could significantly induce a mixture of IgG1 and IgG2a antibody responses, activate T cells to secret IFN-γ, IL-5, IL-10, and generate Granzyme B. Moreover, IFN-γ secretion was significantly enhanced in single-cycle replicable (SCR) HIV-1 virions-infected splenocytes in these groups compared to uninfected splenocytes. The linkage of heat shock protein 70 (Hsp70) epitopes to Gag-Pol-Env-Nef-Rev polypeptide in the homologous regimen increased significantly cytokines and Granzyme B levels, and IFN-γ secretion in virions-infected splenocytes. Briefly, both designed constructs in the homologous regimens can be used as a promising vaccine candidate against HIV infection., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Cholera, a life-threatening disease caused by the Gram-negative bacterium Vibrio cholera, remains a concern in developing countries. The present study investigated the immunogenicity and protective immunity of outer membrane vesicles (OMVs) and combination of OMV and killed whole cells (WC) of a local strain isolated from the last outbreak in Iran in addition to reference and local strains of V. cholerae El Tor O1 in comparison to Dukoral vaccine in mice model. The protein content, morphology, and size of extracted OMVs were evaluated by electrophoresis and microscopic analyses, respectively. The serum titers of total immunoglobulin G (IgG), IgG1, IgG2a, and immunoglobulin A (IgA) in addition to secretory IgA and total IgG in different mice groups were determined by enzyme-linked immunosorbent assay (ELISA). In addition, fluid accumulation (FA) assay regarding the resistance to live strain of V. cholerae in ligated ileal loops was carried out to determine immunogenicity by OMV or combination of OMV and WC in comparison to that reported for Dukoral vaccine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified OMVs indicated protein profiles within the range of 34-52 kDa. Furthermore, transmission electron microscopy demonstrated the spherical shaped vesicles of 50-200 nm. The results of ELISA showed significant titers of systemic and mucosal immune anti-OMV IgGs in immunized BALB/c mice with different vaccine regimens. Additionally, a notable increase in the FA ratio was demonstrated in this study. The obtained results of the present study revealed that the WC-OMV combination of local strain can induce a high level of antibody response indicating more protection than OMV or WC separately. Moreover, it can be considered an effective immunogen against V. cholerae., (Copyright © 2021, Author(s). Published by Kowsar.)

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

Background: Quantitation of Helicobacter pylori (Hp) in the gastric tissue is essential for assessment of vaccination/therapeutic regimens., Materials & Results: Here, the inhibitory effect of mouse gastric DNA (MgDNA) on amplification of Hp genomic DNA (HpDNA) was evaluated by spiking HpDNA with serial dilutions of MgDNA, which yielded concentrations of >10 ng/µl and >0.63-10 ng/µl of MgDNA, as inhibition and interference zones, respectively. Mice were then inoculated with varying doses of Hp and assessed at the inhibition-free concentration of 0.63 ng/µl. The average Hp copy numbers per microgram of gastric tissue discriminated mice having received high vs. low dose inoculums (p < 0.001). Secondly, Hp copy numbers were quantitated in immunized mice, which demonstrated significantly lower numbers, in reference to controls (p = 0.006)., Conclusion: Our method, bypassing the inhibition and/or interference imposed by MgDNA, was able to quantitate gastric tissue-colonizing Hp, segregating mice inoculated with low vs. high doses of Hp, as well as those immunized from controls.

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.