CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVß3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process.

Oral Immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10.

Two lines of transgenic carrot plants producing Mycobacterium tuberculosis proteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.002% of TSP for CFP10. As has been shown, oral immunization of mice induces both the cell-mediated and humoral immunities. These data suggest that the proteins in question are appropriate as a candidate edible vaccine against tuberculosis.

A synergistic effect of a combined bivalent DNA-protein anti-HIV-1 vaccine containing multiple T- and B-cell epitopes of HIV-1 proteins.

Immunogenic properties of the combined vaccine CombiHIVvac, comprising polyepitope HIV-1 immunogens, one being the artificial polyepitope protein TBI, containing the T- and B-cell epitopes from Env and Gag proteins, and the DNA vaccine construct pcDNA-TCI coding for the artificial protein TCI, carrying over 80 T-cell epitopes (both CD4+ CTL and CD8+ Th) from Env, Gag, Pol, and Nef proteins, are studied in this work. The data reported demonstrate clearly that a combination of two B- and T-cell immunogens (TBI and TCI) in one construct results in a synergistic increase in the antibody response to both TBI protein and the proteins from HIV-1 lysate. The level of antibodies induced by immunization with the constructs containing either immunogen alone (TBI protein or the plasmid pcDNA-TCI) was significantly lower as compared to that induced by the combined vaccine. The analysis performed suggests that the presence of CD4+ T-helper epitopes, which can be presented by MHC class II, in the protein TCI may be the main reason underlying the increased synthesis of antibodies to TBI protein due to a CD4-mediated stimulation of B-cell proliferation and differentiation.

Combined virus-like particle-based polyepitope DNA/protein HIV-1 vaccine design, immunogenicity and toxicity studies.

We have previously described designing of polyepitope immunogens TBI and TCI, to stimulate the humoral and cellular immune responses to HIV-1. Here, immunogens TBI and TCI were used to create new vaccine construct named CombiHIVvac (Combined HIV-1 vaccine). CombiHIVvac is a virus-like particles (VLP) containing the DNA vaccine pcDNA-TCI as a core encapsulated within a spermidine-polyglucin-TBI conjugate. The immunogenic and toxic properties of the candidate vaccine CombiHIVvac have been studied. CombiHIVvac induces a strong humoral and CTL responses in mice; the antibodies are highly specific and are able to neutralize HIV-1 in vitro. Preclinical study demonstrated that CombiHIVvac does not cause long-term changes in physiological, biochemical and morphological parameters in immunized animals and thus can be recommended for clinical trials.

Designing and engineering of DNA-vaccine construction encoding multiple CTL-epitopes of major HIV-1 antigens.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8(+) CTL) and T-helper lymphocytes (CD4(+) Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database. The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

Comparative analysis using a mouse model of the immunogenicity of artificial VLP and attenuated Salmonella strain carrying a DNA-vaccine encoding HIV-1 polyepitope CTL-immunogen.

Two systems have been examined for delivery of DNA-vaccine encoding a HIV-1 polyepitope CTL-immunogen (TCI). One is intended for i.m. injection and is in the form of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate. The other is intended for mucosal immunization and is based on attenuated Salmonella typhimurium strain 7207, which can deliver pcDNA-TCI directly into professional antigen-presenting cells (APC). After immunization, the artificial VLP and recombinant Salmonella induced an enhanced HIV specific serum antibody, proliferative and CTL responses compared to those induced by naked pcDNA-TCI. The most significant responses were produced when pcDNA-TCI was delivered by Salmonella.