Publications by authors named "Marina Clemente"

23 Publications

  • Page 1 of 1

Heat Shock Proteins 90 kDa: Immunomodulators and Adjuvants in Vaccine Design Against Infectious Diseases.

Front Bioeng Biotechnol 2020 20;8:622186. Epub 2021 Jan 20.

Unidad Biotecnológica 6-UB6, Laboratorio de Molecular Farming y Vacunas, INTECH, UNSAM-CONICET, Chascomús, Argentina.

Heat shock proteins 90 kDa (Hsp90s) were originally identified as stress-responsive proteins and described to participate in several homeostatic processes. Additionally, extracellular Hsp90s have the ability to bind to surface receptors and activate cellular functions related to immune response (cytokine secretion, cell maturation, and antigen presentation), making them very attractive to be studied as immunomodulators. In this context, Hsp90s are proposed as new adjuvants in the design of novel vaccine formulations that require the induction of a cell-mediated immune response to prevent infectious diseases. In this review, we summarized the adjuvant properties of Hsp90s when they are either alone, complexed, or fused to a peptide to add light to the knowledge of Hsp90s as carriers and adjuvants in the design of vaccines against infectious diseases. Besides, we also discuss the mechanisms by which Hsp90s activate and modulate professional antigen-presenting cells.
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http://dx.doi.org/10.3389/fbioe.2020.622186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855457PMC
January 2021

Use of Veterinary Vaccines for Livestock as a Strategy to Control Foodborne Parasitic Diseases.

Front Cell Infect Microbiol 2020 26;10:288. Epub 2020 Jun 26.

Laboratorio de Molecular Farming y Vacunas, Unidad Biotecnológica 6-UB6, INTECH, UNSAM-CONICET, Chascomús, Argentina.

Foodborne diseases (FBDs) are a major concern worldwide since they are associated with high mortality and morbidity in the human population. Among the causative agents of FBDs, spp., and are listed in the top global risk ranking of foodborne parasites. One common feature between them is that they affect domestic livestock, encompassing an enormous risk to global food production and human health from farm to fork, infecting animals, and people either directly or indirectly. Several approaches have been employed to control FBDs caused by parasites, including veterinary vaccines for livestock. Veterinary vaccines against foodborne parasites not only improve the animal health by controlling animal infections but also contribute to increase public health by controlling an important source of FBDs. In the present review, we discuss the advances in the development of veterinary vaccines for domestic livestock as a strategy to control foodborne parasitic diseases.
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http://dx.doi.org/10.3389/fcimb.2020.00288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332557PMC
June 2021

Close relationship between the state of the oxygen evolving complex and rice cold stress tolerance.

Plant Sci 2020 Jul 9;296:110488. Epub 2020 Apr 9.

Laboratorio de Fisiología de Estrés Abiótico en Plantas, Unidad de Biotecnología 1, INTECH-CONICET-UNSAM, Chascomús, Argentina. Electronic address:

The results of the present work suggested a relationship between the growth stability and functional/structural parameters associated to the primary photochemistry and oxygen evolving complex (OEC) in tolerant rice plants under suboptimal low temperatures (SLT) stress. This was concluded from the absence of changes in net photosynthetic rate and in fraction of reaction centers to reduce quinone A, and very small changes in P680 efficiency to trap and donate electrons to quinone A and in fraction of active OEC in tolerant plants under cold stress but not in sensitive plants. The SLT stress also induced OEC activity limitations in both genotypes, but in a greater extent in sensitive plants. However, an assay using an artificial electron donor to replace OEC indicated that the P680 capacity to accept electrons was not altered in both genotypes under SLT stress from the beginning of the stress treatment, suggesting that the OEC structure stability is related to rice SLT tolerance to sustain the photosynthesis. This hypothesis was also supported by the fact that tolerant plants but not sensitive plants did not alter the gene expression and protein content of PsbP under SLT stress, an OEC subunit with a role in stabilizing of OEC structure.
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http://dx.doi.org/10.1016/j.plantsci.2020.110488DOI Listing
July 2020

The potential of a DIVA-like recombinant vaccine composed by rNcSAG1 and rAtHsp81.2 against vertical transmission in a mouse model of congenital neosporosis.

Acta Trop 2019 Oct 16;198:105094. Epub 2019 Jul 16.

Unidad de Biotecnología 6-UB6, INTECH, CONICET-UNSAM, Chascomús, Argentina. Electronic address:

Neospora caninum is the etiological agent of neosporosis, a worldwide infectious disease recognized as the major cause of abortions and reproductive failures in livestock, responsible for significant economic losses in cattle industries. Currently, there are not cost-effective control options for this pathology, and the development of a vaccine involving new and integrated approaches is highly recommended. In this study, we evaluated the immunogenic and protective efficacy, as well as the potential DIVA (Differentiation of Infected from Vaccinated Animals) character of a recombinant subunit vaccine composed by the major surface antigen from N. caninum (NcSAG1) and the carrier/adjuvant heat shock protein 81.2 from Arabidopsis thaliana (AtHsp81.2) in a mouse model of congenital neosporosis. BALB/c female mice were intraperitoneal (i.p.) immunized with a mixture of equimolar quantities of rNcSAG1 and rAtHSP81.2 or each protein alone (rNcSAG1 or rAtHsp81.2). The vaccine containing a mixture of rNcSAG1 and rAtHsp81.2 significantly enhanced the production of specific anti-rNcSAG1 total IgG (tIgG), IgG1 and IgG2a antibodies in immunized mice when compared to control groups (non-vaccinated and rAtHsp81.2 immunized mice) as well as to the group of mice immunized only with the antigen (rNcSAG1). In addition, partial protection against vertical transmission and improvement of the offspring survival time was observed in this group. On the other hand, rAtHsp81.2 induced the production of specific anti-rAtHsp81.2 tIgG, allowing us to differentiate vaccinated from infected mice. Despite further experiments have to be made in cattle to test the capability of this vaccine formulation to differentiate vaccinated from infected animals in the field, our results suggest that the formulation composed by rNcSAG1 and rAtHsp81.2 could serve as a basis for the development of a new vaccine approach against bovine neosporosis.
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http://dx.doi.org/10.1016/j.actatropica.2019.105094DOI Listing
October 2019

Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein.

Plant Sci 2019 Jul 13;284:117-126. Epub 2019 Apr 13.

Laboratorio de Biotecnología Vegetal, IIB-INTECH, CONICET-UNSAM, Chascomús, Provincia de Buenos Aires, Argentina. Electronic address:

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.
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http://dx.doi.org/10.1016/j.plantsci.2019.04.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785835PMC
July 2019

Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide.

Parasit Vectors 2019 Mar 25;12(1):140. Epub 2019 Mar 25.

Unidad de Biotecnología 6-UB6, IIB-INTECH, CONICET-UNSAM, Intendente Marino Km 8.2, B7130IWA, Chascomús, Buenos Aires Province, Argentina.

Background: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1) in a T. gondii infection murine model.

Methods: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1 fusion protein (NbHsp90.3-SAG1 group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA.

Results: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1 group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection.

Conclusions: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1 exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.
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http://dx.doi.org/10.1186/s13071-019-3362-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434815PMC
March 2019

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming.

Int J Mol Sci 2019 Mar 17;20(6). Epub 2019 Mar 17.

Instituto Tecnológico Chascomús (INTECH), UNSAM-CONICET, Chascomús, Provincia de Buenos Aires B7130, Argentina.

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.
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http://dx.doi.org/10.3390/ijms20061345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471620PMC
March 2019

Promising Plant-Derived Adjuvants in the Development of Coccidial Vaccines.

Front Vet Sci 2019 12;6:20. Epub 2019 Feb 12.

Unidad de Biotecnología 6-UB6, Instituto Tecnológico Chascomús (INTECh), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-Universidad Nacional de General San Martín (UNSAM), Chascomús, Argentina.

Coccidial parasites cause medical and veterinary diseases worldwide, frequently leading to severe illness and important economic losses. At present, drugs, chemotherapeutics and prophylactic vaccines are still missing for most of the coccidial infections. Moreover, the development and administration of drugs and chemotherapeutics against these diseases would not be adequate in livestock, since they may generate unacceptable residues in milk and meat that would avoid their commercialization. In this scenario, prophylactic vaccines emerge as the most suitable approach. Subunit vaccines have proven to be biologically safe and economically viable, allowing researchers to choose among the best antigens against each pathogen. However, they are generally poorly immunogenic and require the addition of adjuvant compounds to the vaccine formulation. During the last decades, research involving plant immunomodulatory compounds has become an important field of study based on their potential pharmaceutical applications. Some plant molecules such as saponins, polysaccharides, lectins and heat shock proteins are being explored as candidates for adjuvant/carriers formulations. Moreover, plant-derived immune stimulatory compounds open the possibility to attain the main goal in adjuvant research: a safe and non-toxic adjuvant capable of strongly boosting and directing immune responses that could be incorporated into different vaccine formulations, including mucosal vaccines. Here, we review the immunomodulatory properties of several plant molecules and discuss their application and future perspective as adjuvants in the development of vaccines against coccidial infections.
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http://dx.doi.org/10.3389/fvets.2019.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379251PMC
February 2019

Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

Biochimie 2016 Apr 4;123:85-94. Epub 2016 Feb 4.

Unidad de Biotecnología 6-UB6, IIB-INTECH, CONICET-UNSAM, Chascomús, Argentina. Electronic address:

Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.
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http://dx.doi.org/10.1016/j.biochi.2016.02.002DOI Listing
April 2016

The fusion of Toxoplasma gondii SAG1 vaccine candidate to Leishmania infantum heat shock protein 83-kDa improves expression levels in tobacco chloroplasts.

Biotechnol J 2015 May 22;10(5):748-59. Epub 2015 Apr 22.

Laboratorio de Biotecnología Vegetal, IIB-INTECH, CONICET-UNSAM, Chascomús, Provincia de Buenos Aires, Argentina.

Chloroplast transformation technology has emerged as an alternative platform offering many advantages over nuclear transformation. SAG1 is the main surface antigen of the intracellular parasite Toxoplasma gondii and a promising candidate to produce an anti-T. gondii vaccine. The aim of this study was to investigate the expression of SAG1 using chloroplast transformation technology in tobacco plants. In order to improve expression in transplastomic plants, we also expressed the 90-kDa heat shock protein of Leishmania infantum (LiHsp83) as a carrier for the SAG1 antigen. SAG1 protein accumulation in transplastomic plants was approximately 0.1-0.2 μg per gram of fresh weight (FW). Fusion of SAG1 to LiHsp83 significantly increased the level of SAG1 accumulation in tobacco chloroplasts (by up to 500-fold). We also evaluated the functionality of the chLiHsp83-SAG1. Three human seropositive samples reacted with SAG1 expressed in transplastomic chLiHsp83-SAG1 plants. Oral immunization with chLiHsp83-SAG1 elicited a significant reduction of the cyst burden that correlated with an increase of SAG1-specific antibodies. We propose the fusion of foreign proteins to LiHsp83 as a novel strategy to increase the expression level of the recombinant proteins using chloroplast transformation technology, thus addressing one of the current challenges for this approach in antigen protein production.
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http://dx.doi.org/10.1002/biot.201400742DOI Listing
May 2015

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen.

Vaccine 2013 Dec 10;31(49):5872-8. Epub 2013 Oct 10.

Laboratorio de Biotecnología Vegetal, IIB-INTECH, CONICET-UNSAM, Chascomús, Provincia de Buenos Aires, Argentina.

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP+rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP+rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8(+) T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.
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http://dx.doi.org/10.1016/j.vaccine.2013.09.047DOI Listing
December 2013

A chloroplast-derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice.

Plant Biotechnol J 2012 Dec 1;10(9):1136-44. Epub 2012 Oct 1.

Laboratorio de Biotecnología Vegetal, IIB-INTECH, CONICET-UNSAM, Chascomús, Argentina.

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti-Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34-kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN-γ, IL-4 and IL-10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4-specific serum antibodies and secretion of IFN-γ, IL-4 and IL-10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.
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http://dx.doi.org/10.1111/pbi.12001DOI Listing
December 2012

Overview of plant-made vaccine antigens against malaria.

J Biomed Biotechnol 2012 15;2012:206918. Epub 2012 Jul 15.

Laboratorio de Biotecnología Vegetal, Instituto Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús, Camino de Circunvalación km 8.2, 7130 Buenos Aires, Argentina.

This paper is an overview of vaccine antigens against malaria produced in plants. Plant-based expression systems represent an interesting production platform due to their reduced manufacturing costs and high scalability. At present, different Plasmodium antigens and expression strategies have been optimized in plants. Furthermore, malaria antigens are one of the few examples of eukaryotic proteins with vaccine value expressed in plants, making plant-derived malaria antigens an interesting model to analyze. Up to now, malaria antigen expression in plants has allowed the complete synthesis of these vaccine antigens, which have been able to induce an active immune response in mice. Therefore, plant production platforms offer wonderful prospects for improving the access to malaria vaccines.
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http://dx.doi.org/10.1155/2012/206918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403509PMC
November 2012

Plant Hsp90 proteins interact with B-cells and stimulate their proliferation.

PLoS One 2011 20;6(6):e21231. Epub 2011 Jun 20.

Laboratorio de Biotecnología Vegetal, IIB-INTECH, CONICET-UNSAM, Chascomús, Provincia de Buenos Aires, Argentina.

Background: The molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro.

Methodology: Plant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction.

Conclusions: Our results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021231PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3118808PMC
October 2011

Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.

BMC Biotechnol 2010 Jul 15;10:52. Epub 2010 Jul 15.

IIB-INTECH, Camino de Circunvalación km 6, Provincia de Buenos Aires, Argentina.

Background: Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.

Results: Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.

Conclusion: Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.
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http://dx.doi.org/10.1186/1472-6750-10-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920232PMC
July 2010

Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans.

Exp Parasitol 2010 Oct 28;126(2):263-6. Epub 2010 Apr 28.

Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnologico Chascomús (IIB-INTECH), UNSAM/CONICET, Chascomús 7130, Argentina.

Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools.
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http://dx.doi.org/10.1016/j.exppara.2010.04.013DOI Listing
October 2010

Efficient expression of a Toxoplasma gondii dense granule Gra4 antigen in tobacco leaves.

Exp Parasitol 2008 Sep 10;120(1):118-22. Epub 2008 Jun 10.

IIB-INTECH, Camino Circunvalación Laguna km. 6, Chascomús, prov. de Bs. As, 7130, Argentina.

A His-tagged truncated version of Toxoplasma gondii dense granule 4 protein (Gra4(163-345)) was transiently expressed in tobacco leaves. Two genetic constructions were used to accomplish this goal. In one of them, based in a Potato virus X (PVX) amplicon, the sequence encoding His-Gra4(163-345) was placed under control of an additional PVX coat protein subgenomic promoter. In the other, the same sequence was fused to an apoplastic transport signal and placed under the direction of the cauliflower mosaic virus 35S promoter. His-Gra4(163-345) accumulation in agroinfiltrated tobacco leaves was estimated by Western blot analysis using mouse anti-Gra4 antibody and a seropositive human serum. Here, we demonstrated the feasibility of producing a Gra4 antigen using transient expression methods in plants.
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http://dx.doi.org/10.1016/j.exppara.2008.06.002DOI Listing
September 2008

Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe-S proteins and induces oxidative stress.

Plant J 2006 Dec 8;48(6):873-82. Epub 2006 Nov 8.

Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH) CONICET/UNSAM, Camino Circunvalación Km 6, 7130 Chascomús, Argentina.

Frataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO(2) assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.
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http://dx.doi.org/10.1111/j.1365-313X.2006.02923.xDOI Listing
December 2006

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH).

Protein Expr Purif 2007 Feb 22;51(2):157-61. Epub 2006 Jun 22.

IIB-INTECH, UNSAM-CONICET, CC 164 (7130) Chascomús, Argentina.

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.
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http://dx.doi.org/10.1016/j.pep.2006.06.007DOI Listing
February 2007

Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity.

Mol Biotechnol 2005 May;30(1):41-50

IIB-INTECH, Camino de Circunvalación Laguna Km. 6, B7130IIWA, Chascomús, Prov. Buenos Aires, Argentina.

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 microg and 6 microg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.
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http://dx.doi.org/10.1385/MB:30:1:041DOI Listing
May 2005

Structure analysis of two Toxoplasma gondii and Neospora caninum satellite DNA families and evolution of their common monomeric sequence.

J Mol Evol 2004 May;58(5):557-67

INGEBI, Ciudad de Buenos Aires, Argentina.

A family of repetitive DNA elements of approximately 350 bp-Sat350-that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1). an arrangement of AB1CB2 350-bp repeats and (2). an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database (http://ToxoDB.org ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.
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http://dx.doi.org/10.1007/s00239-003-2578-3DOI Listing
May 2004

Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis.

Diagn Microbiol Infect Dis 2003 Dec;47(4):609-13

Departamento de Parasitología, ANLIS Dr. Carlos G. Malbran, Av. Velez Sarsfield 563, 1281-Ciudad Autónoma de, Buenos Aires, Argentina.

The value of T. gondii recombinant antigens rRop2, rGra4, rGra7 and rSAG1m (mature version) or rSAG1ct (C-terminal version) in differentiating recently acquired from chronic infections was determined by IgG-ELISA. The general highest sensitivity was observed with rRop2 whereas rSAG1m was not recognized by any of the serum samples, suggesting an incorrect folding. rGra4 and rGra7 showed significant higher sensitivity and absorbance values with serum samples from recently infected individuals compared to those with chronic infection. In contrast, rRop2 and rSAG1ct did not show differences in the reactivity pattern between both groups of serum samples.
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http://dx.doi.org/10.1016/s0732-8893(03)00156-1DOI Listing
December 2003
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