Immunomodulatory in vitro effects of Trichinella cystatin-like protein on mouse splenocytes.

Trichinella parasites have developed specific mechanisms allowing successful completion of their life cycle. These mechanisms are in a great part involved in immunomodulation and studying them may provide a valuable insight into the functioning of the immune system. Trichinella products may be also used as potential therapeutic agents to treat immune diseases. This study investigates the immunomodulatory potential of recombinant multi cystatin-like protein (CLP) derived from T. britovi to determine whether CLP has anti-inflammatory properties in vitro. CLP is a highly antigenic glycoprotein present in Trichinella excetory-secretory (ES) products. AlphaFold structure prediction confirms that it consists of three type-two cystatin-like domains. Mouse splenocytes were stimulated in vitro with lipopolysaccharide (LPS) and co-stimulated with recombinant CLP. The culture supernatants were collected and tested for secreted cytokine levels using ELISA. CLP was found to reduce LPS-induced secretion of inflammatory cytokines TNFα and IL-6. On the contrary, in some experimental groups, co-stimulation with CLP resulted in increased secretion of the regulatory cytokine IL-10. The obtained results indicate that CLP has anti-inflammatory properties and future research on its function is advisable, specifically in the context of the therapy of inflammatory disorders.

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast.

Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

DNA vaccines against influenza.

Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

DNA probe modified with 3-iron bis(dicarbollide) for electrochemical determination of DNA sequence of Avian Influenza Virus H5N1.

In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region.