Assessment of Carrot Callus as Biofactories of an Atherosclerosis Oral Vaccine Prototype

Atherosclerosis is a pathology leading to cardiovascular diseases with high epidemiologic impact; thus, new therapies are required to fight this global health issue. Immunotherapy is a feasible approach to treat atherosclerosis and given that genetically engineered plants are attractive hosts for vaccine development; we previously proved that the plant cell is able to synthesize a chimeric protein called CTB:p210:CETPe, which is composed of the cholera toxin B subunit (CTB) as immunogenic carrier and target epitopes from the cholesteryl ester transfer protein (CETP_461–476) and apolipoprotein B100 (p210). Since CTB:p210:CETPe was expressed in tobacco at sufficient levels to evoke humoral responses in mice, its expression in carrot was explored in the present study looking to develop a vaccine in a safe host amenable for oral delivery; avoiding the purification requirement. Carrot cell lines expressing CTB:p210:CETPe were developed, showing accumulation levels up to 6.1 µg/g dry weight. An immunoblot analysis revealed that the carrot-made protein is antigenic and an oral mice immunization scheme led to evidence on the immunogenic activity of this protein; revealing its capability of inducing serum IgG responses against p210 and CETP epitopes. This study represents a step forward in the development of an attractive oral low-cost vaccine to treat atherosclerosis.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies

Key message

An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse.

Abstract

Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60 µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80 days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.

     

Circulating Autoantibodies against the Apolipoprotein B-100 Peptides p45 and p210 in Relation to the Occurrence of Carotid Plaques in 64-Year-Old Women

Objectives

Immune responses against oxidized low density lipoprotein (LDL) play a key role in atherosclerosis. Previous studies have indicated inverse associations between autoantibodies to epitopes in oxidized LDL and cardiovascular disease. In this study we investigated the associations between autoantibodies against the apolipoprotein B-100 (apoB-100) peptides p45 and p210 and occurrence of carotid plaques.

Design

The study cohort consisted of a population-based sample of 64-year-old women with varying degrees of glucose tolerance (n=594). To identify and record the occurrence of carotid atherosclerotic plaques ultrasonography was used. Measurements of plasma IgM and IgG autoantibodies against the native and malondialdehyde (MDA)-modified apoB-100 peptides p45 and p210 were performed by ELISA.

Results

Women with carotid plaques were found to have lower levels of IgM MDA-p210 autoantibodies compared to plaque-free women. The number of carotid plaques in each subject and the total carotid plaque area correlated inversely with IgM MDA-p210 levels (r=-0.11, P=0.009 and r=-0.11, P=0.013, respectively). Furthermore, levels of IgM MDA-p210 above the lowest tertile were associated with an odds ratio of 0.55 (95% CI 0.38-0.79, P=0.001) for occurrence of carotid plaques, independently of other risk markers and statin treatment. Associations between apo-B100 peptide autoantibodies and cardiovascular risk factors were generally weak but subjects with impaired glucose tolerance had higher levels of IgM against MDA-p210.

Conclusion

The present study demonstrates that high levels of IgM against MDA-p210 are associated with less severe carotid disease in women. These findings provide additional support for a role of immune responses against oxidized LDL in cardiovascular disease.     

Expression of cholera toxin B subunit oligomers in transgenic potato plants

A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (Mr 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (Mr 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Comparison of Strategies for the Production of FMDV Empty Capsids Using the Baculovirus Vector System

Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C). Expression levels and assembly into empty capsids were analyzed in insect cells and larvae. We observed that the use of the single-promoter vector allowed higher levels of expression both in insect cells and larvae. Recombinant capsid proteins produced by both vectors were recognized by monoclonal antibodies (mAbs) directed against conformational epitopes of FMDV A/Arg/01 and proved to self-assemble into empty capsids (75S) and pentamers (12S) when analyzed by sucrose gradient centrifugation.     

Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated Nicotiana benthamiana leaves

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636–789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2?days post infiltration (dpi), reached 0.04?% of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB–S1D fusion protein were confirmed by Western blot and G_M1-ELISA. The highest expression level of CTB–S1D fusion protein was 0.07?% of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB–S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB–S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB–S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.     

Development of barcode and proteome profiling of glioblastoma

High grade glioma (glioblastoma) is the most common brain tumor. Its malignancy makes it the fourth biggest cause of cancer death. In our experiments, we used several glioblastoma cell lines to obtain proteomics information specific for this disease. 2DE separation with following imaging, immunochemistry, spot picking, and mass-spectrometry allowed us to detecting more than 600 protein spots and identifying more than 130 of them. Proteome profiles in normal and glioblastoma cell lines are very similar but levels of several proteins have prominent differences between norm and cancer. Among these proteins are alpha-enolase (ENOA_HUMAN), pyruvate kinase M1/M2 (KPYM_HUMAN), cofilin 1 (COF1_HUMAN), translationally-controlled tumor protein TCTP_HUMAN, annexin 1 (ANXA1_HUMAN), PCNA (PCNA_HUMAN), p53 (TP53_HUMAN) and others. Most interesting results were obtained about protein p53. Its level was dramatically up-regulated and enriched by multiple additional isoforms in all glioblastoma cell lines. An immunological analysis (Western blot) of three hub-proteins (p53, 14-3-3, PCNA) allowed us to creating the minimal barcode of glioblastoma cell lines. These preliminary data point to this barcode as a promising diagnostic tool for testing of the biological fluids from patients.     

A comparative proteomic analysis of parthenogenetic lines and amphigenetic lines of silkworm

Parthenogenetic strains of silkworm serve as an effective system for sex-control in silkworms. To determine the molecular mechanism of silkworm parthenogenesis, protein profiles from newly hatched silkworm of a parthenogenetic lines with high pigmentation rate and hatching rate were compared with amphigenetic lines using proteomics approach, including by two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS), and bioinformatics analysis. Several proteins were expressed differentially between the parthenogenetic and amphigenetic lines, and seven of nine interesting proteins were identified successfully using MALDI-TOF/TOF MS analysis. The identified proteins were muscular protein-20, odorant binding protein-LOC100301497, glutathione S-transferase delta, translationally controlled tumor protein homolog, cuticular protein RR-1 motif 19, beta-actin, actins, and muscle-type A₁ actins. These proteins may be associated with the regulation of growth, development, and reproductive processes of silkworm parthenogenetic lines.     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

Apoptotic potential role of Agave palmeri and Tulbaghia violacea extracts in cervical cancer cells

Cervical cancer, a gynaecological malignant disorder, is a common cause of death in females in Sub-Saharan Africa, striking nearly half a million of lives each year worldwide. Currently, more than 50 % of all modern drugs in clinical use are of natural products, many of which have an ability to control cancer cells (Madhuri and Pandey, Curr Sci 96:779–783, 2009; Richter, Traditional medicines and traditional healers in South Africa, 2003). In South Africa, plants used to treat cancer are rare even though majority of our population continue to put their trust in traditional medicine. In this study we aimed to screen Agave palmeri (AG) and Tulbaghia violacea (TV) for potential role in inducing cell death in cervical cancer cell lines HeLa and ME-180, and in normal human fibroblast cell line KMST-6 cell lines. To achieve this, AG and TV crude extracts were utilized to screen for apoptosis induction, inhibition of cell proliferation followed by elucidation of the role of Bax, Bcl-2, p53, Rb, RBBP and Mdm2 genes in cervical cancer. In brief, plant leaves and roots were collected, crushed and methanolic extracts obtained. Different concentrations of the stock extracts were used to treat cancer cells and measure cell death using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and flow cytometry. Western blot was applied to measure gene expression at protein level using RBBP6, p53, Mdm2, Rb, Bax, Bcl-2 and β-actin mouse monoclonal primary antibodies (IgG) and goat anti mouse coupled with horseradish peroxidase secondary antibody from Santa Cruz Biotechnology and real time-PCR was used for mRNA expression level. Plant extracts of AG and TV were time (24 h) and dose (50, 100, 150 μg/ml) dependent in their induction of cell death with an IC₅₀ ~ 150 μg/ml. A further mixed respond by several genes was observed following treatment with the two plant extracts where RBBP6 was seen to be spliced in cancer cells while Bax was induced and Bcl-2 was inhibited with the levels of p53 remaining the same. The two plant extracts do induce cell death, in a p53 independent manner.     

Qualitative differences of pumpkin (Cucurbita pepo L.) proteins influenced by Ca2+ nutrition

The effect of Ca-deficiency on changes in the protein content of cotyledons and roots of germinating pumpkin (Cucurbita pepo L.) plants was studied by means of immunoelectrophoresis. Antiserum against seed proteins gave 11 clear precipitation zones in homological reactions and 7 zones with proteins from cotyledons of 10 day-old plants. Several less mobile zones in the area of start appeared in variant “?Ca”. Using antiserum against root proteins of 10 day-old control plants, 8 precipitation zones in the homological reaction and 6 zones (two cathodic zones are missing) in the heterological reaction were obtained. The patterns of proteins from the root and those arising from the protein of seed are both changed under Ca-deficiency.     

The localization of auxin transporters PIN3 and LAX3 during lateral root development in Arabidopsis thaliana

Fluorophore tagged proteins are used in Arabidopsis thaliana to understand their functional role in plant development. This requires the analysis of their spatial localization in planta. However, the localization analysis is often perturbed by a significant overlap of the fluorophores used to label proteins of interest and the optical filtering methods available on the confocal microscope. This problem can be addressed by the use of spectral imaging with linear unmixing the image data. We applied this method to help us identify double transgenic A. thaliana lines which expressed two fluorescently tagged auxin transporter proteins: the auxin efflux protein PIN-FORMED-3 (PIN3), tagged with green fluorescent protein (GFP), and the auxin influx protein LIKE-AUX1-3 (LAX3), tagged with yellow fluorescent protein (YFP). This method allows the reliable separation of overlapping GFP and YFP fluorescence signals and subsequent localization analysis highlighting the potential benefit of this methodology in studies of lateral root development.     

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg?·?g^?1 leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.     

The expression of ABCG1 transporter gene in peripheral blood mononuclear cells of patients with atherosclerosis

The antiatherogenic role of high-density lipoproteins (HDL) was demonstrated by numerous experimental, clinical and epidemiological studies. The mechanism underlying the antiatherogenic potential of HDL is based on their involvement in reverse cholesterol transport (RCT) from peripheral tissues into the liver. Transmembrane transporter ABCG1 is a key RCT protein. Its function is to remove cholesterol from cells and transfer it to HDL. The role of ABCG1 transporter in the development of atherosclerosis in humans remains unexplored. The goal of our study was to investigate the expression of ABCG1 gene in patients with atherosclerosis. Real-time PCR was applied to study ABCG1 mRNA content in leukocytes, monocytes, and macrophages activated with macrophage colony-stimulating factor (M-CSF) from patients with atherosclerosis and healthy people. The amount of ABCG1 protein in monocytes and macrophages of patients and healthy donors was assayed by immunoblotting. It was found that the level of ABCG1 mRNA (p < 0.001) and ABCG1 protein (p < 0.05) was lower in macrophages of patients with atherosclerosis. The level of ABCG1 mRNA in monocytes of patients with artery occlusion was lower than in patients with features of lesser stenosis and the control group (p < 0.05). No correlation was found between ABCG1 gene expression and total and HDL cholesterol levels in the blood plasma. It can be concluded that reduced ABCG1 gene expression in monocytes and macrophages may be critical for the atherosclerosis progression.