Andrey Mazeika

Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes

BackgroundThere is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.ResultsThe cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.ConclusionsWe propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

On the possibility of lipid-induced regulation of conformation and immunogenicity of influenza a virus H1/N1 hemagglutinin as antigen of TI-complexes

The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.

The effects of triterpene glycosides and phospholipids from marine invertebrates in the composition of tubular immunostimulating complexes on the immunogenicity of human serum albumin

To study the adjuvant activity and to optimize tubular immunostimulating complexes (TI complexes) with respect to the soluble antigen, an investigation was performed on cucumariosides and their fractions, i.e., individual triterpene glycosides that were obtained from the holothurian Cucumaria japonica, as well as on phospholipids from the sea urchin Strongylocentrotus intermedius and the starfish Distolasterias nippon included in the composition of TI complexes in order to assess their effects on the immunogenicity of human serum albumin (HSA). Generally, TI complexes showed a moderate adjuvant activity with respect to HSA; this activity can be adjusted by substitution of a cucumarioside and/or a polar lipid in the composition of the TI complex. TI complexes based on monogalactosyldyacylglycerol (MGDG) from the sea alga Ulva lactuca and cucumariosides A2-2 and A4-4 maximally stimulated anti-HSA antibody production in mice. Substitution of MGDG for phosphatidylcholine (PC) from the starfish D. nippon with an increased (compared with other investigated phospholipids) ratio of n-3/n-6 polyunsaturated fatty acids did not result in a change in the immunostimulating activity of the TI complex. However, the use of other phospholipids led to a decrease in the adjuvant activity of the TI complex, compared with that of the TI complex based on PC from D. nippon and cucumarioside A2-2. The greatest fluctuations in the contents of cytokines IL-6, IL-12, IFN-γ and GM-CSF depending on the composition of the TI complexes indicate the possibility of regulation of the T-cell immune response. The values of hematological parameters were normal.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis

BACKGROUND Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Nanoparticulate Tubular Immunostimulating Complexes: Novel Formulation of Effective Adjuvants and Antigen Delivery Systems

New generation vaccines, based on isolated antigens, are safer than traditional ones, comprising the whole pathogen. However, major part of purified antigens has weak immunogenicity. Therefore, elaboration of new adjuvants, more effective and safe, is an urgent problem of vaccinology. Tubular immunostimulating complexes (TI-complexes) are a new type of nanoparticulate antigen delivery systems with adjuvant activity. TI-complexes consist of cholesterol and compounds isolated from marine hydrobionts: cucumarioside A2-2 (CDA) from Cucumaria japonica and monogalactosyldiacylglycerol (MGDG) from marine algae or seagrass. These components were selected due to immunomodulatory and other biological activities. Glycolipid MGDG from marine macrophytes comprises a high level of polyunsaturated fatty acids (PUFAs), which demonstrate immunomodulatory properties. CDA is a well-characterized individual compound capable of forming stable complex with cholesterol. Such complexes do not possess hemolytic activity. Ultralow doses of cucumariosides stimulate cell as well as humoral immunity. Therefore, TI-complexes comprising biologically active components turned out to be more effective than the strongest adjuvants: immunostimulating complexes (ISCOMs) and complete Freunds adjuvant. In the present review, we discuss results published in series of our articles on elaboration, qualitative and quantitative composition, ultrastructure, and immunostimulating activity of TI-complexes. The review allows immersion in the history of creating TI-complexes.

The complexation between holothurian triterpene glycosides and Chl as the basis for lipid-saponin carriers of subunit protein antigens

The ability of holothurian triterpene glycosides (cucumarioside A2-2 from Cucumaria japonica, cucumarioside G1 from C. fraudatrix, frondoside A from C. frondosa, and holotoxin A1 from Apostichopus japonicus) to form supramolecular lipid-saponin complexes was studied. TEM demonstrated that all the studied compounds form supramolecular cholesterol-saponin complexes (nanoparticles) in aqueous medium. The complexes formed by cucumarioside A2-2, holotoxin A1, and frondoside A had a tubular structure and fundamentally differed in the structure from the particles produced by cucumarioside G1. The morphology of the nanoparticles formed by cucumarioside A2-2, holotoxin A1, and cucumarioside G1 changed depending on the fraction of cholesterol in the lipid-saponin system; however, this pattern was not observed for frondoside A. At the same molar fraction of cholesterol in the lipid-saponin system, cucumarioside A2-2 formed the particles with the most pronounced tubular structure; the cholesterol-saponin complexes of holotoxin A1 had a less pronounced tubular structure, whereas the structure of frondoside A particles was extremely heterogeneous. Comparative analysis of the morphology of the described supramolecular complexes and specific structural features of the glycosides demonstrated that the structure of the corresponding nanoparticles depended on the degree of branching of the carbohydrate moiety in the glycoside molecule and the complexation with cholesterol was determined by the specific features of aglycone structure. Thus, the feasibility of producing new generation antigen carriers using the complexes in question was proved.

Immunogenicity and Protective Activity of a Chimeric Protein Based on the Domain III of the Tick-Borne Encephalitis Virus E Protein and the OmpF Porin of Yersinia pseudotuberculosis Incorporated into the TI-Complex

Tick-borne encephalitis (TBE) is a widespread, dangerous infection. Unfortunately, all attempts to create safe anti-TBE subunit vaccines are still unsuccessful due to their low immunogenicity. The goal of the present work was to investigate the immunogenicity of a recombinant chimeric protein created by the fusion of the EIII protein, comprising domain III and a stem region of the tick-borne encephalitis virus (TBEV) E protein, and the OmpF porin of Yersinia pseudotuberculosis (OmpF-EIII). Adjuvanted antigen delivery systems, the tubular immunostimulating complexes (TI-complexes) based on the monogalactosyldiacylglycerol from different marine macrophytes, were used to enhance the immunogenicity of OmpF-EIII. Also, the chimeric protein incorporated into the most effective TI-complex was used to study its protective activity. The content of anti-OmpF-EIII antibodies was estimated in mice blood serum by enzyme-linked immunosorbent assay (ELISA). To study protective activity, previously immunized mice were infected with TBEV strain Dal’negorsk (GenBank ID: FJ402886). The animal survival was monitored daily for 21 days. OmpF-EIII incorporated into the TI-complexes induced about a 30–60- and 5–10-fold increase in the production of anti-OmpF-EIII and anti-EIII antibodies, respectively, in comparison with the effect of an individual OmpF-EIII. The most effective vaccine construction provided 60% protection. Despite the dramatic effect on the specific antibody titer, the studied TI-complex did not provide a statistically significant increase in the protection of OmpF-EIII protein. However, our results provide the basis of the future search for approaches to design and optimize the anti-TBEV vaccine based on the OmpF-EIII protein.

Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes

Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection.