Molecular Farming and Vaccines Laboratory

Molecular Farming refers to the production of recombinant proteins in plants (including pharmaceutical products, industrial proteins and other secondary metabolites). Between 1986 and 1989, the first pharmaceutic product, the human growth hormone and the first recombinant antibody were expressed in transgenic plants. However, in 1997, the chicken egg avidin protein was commercialized as the first recombinant protein produced in plants. These finding demonstrated that plants can be employed as platforms to produce large-scale recombinant proteins. Over the years it was demonstrated that plants have the capability to express functionally active proteins from mammals and other eukaryotic organisms with therapeutic activity like human sera, growth factors, vaccines, hormones, cytokines, enzymes and antibodies. This is possible due to the ability of plants to realize the post-translational modifications required for the correct folding of the proteins in order to keep their functionality and integrity. Thus, there is a great interest to use plants as bio-factories to produce drugs, antigens, nutritional supplements, biopolymers and biofuels.

One of the most important challenges for the science community for using plants as a commercial productive platform is to improve the yields of recombinant proteins expressed within them. The degradation of recombinant proteins by proteases due to imperfect synthesis or deficient folding can strongly affect the protein accumulation levels. Therefore, the recombinant protein stability is considered the most important factor that limits the yields in molecular farming.

The 90-kDa heat shock proteins are soluble proteins expressed in all organisms. The expression levels of Hsp90s are high, reaching between 1 and 2% of the total soluble proteins within the cell. The main role of Hsp90s is related to the correct folding during the synthesis of certain proteins, as well as to the re-folding of denatured or partially denatured proteins. In this sense, we propose Hsp90s as carriers of recombinantproteins produced in plants to guarantee their high expression, their correct folding and to avoid the formation of insoluble aggregates. In our lab., we are developing a platform based on the use of tobacco and lettuce plants to produce vaccine antigens fused to Hsp90 as a strategy to improve the protein of interest expression in plants.

Another alternative to diminish the degradation of recombinant proteins expressed in plants by proteases is to co-express the recombinant protein with protease inhibitors. In addition, it was observed that the over-expression of protease inhibitors may contribute to the resistance to certain phytopathogens. Up to now, different serine protease inhibitors have been identified in plants. Some of them were related to physiological processes such as defense against pathogens. Kazal-type serine protease inhibitors belong to the family of serine protease inhibitors. This kind of inhibitors are present in many organisms included plants and play a regulatory role that involve serine proteases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. Although Kazal-type serine protease inhibitors have been identified in plants, little is known about their role. In our lab., we are exploring the different biotechnological applications of these family of inhibitors from plants and parasites.

In recent times, it was recognized the importance of Hsp90s role in the modulation of the immune response against infections and tumors. In addition, several studies showed that Hsp90s fused to antigenic peptides or proteins increase their humoral and cellular immune response demonstrating their activity as adjuvants. Under the hypothesis that these adjuvant properties observed in different Hsp90s are also conserved in plant Hsp90,  we are carrying out the characterization of the immunomodulatory properties of different plant Hsp90 isoforms using Toxoplasma gondii and Neospora caninum models. Likewise, we are carrying out comparative studies among different Hsp90s isoforms to determine their respective roles on the modulation of the immune response and their potential use as vaccine adjuvants. In addition, we are exploring the use of plant Hsp90 in DIVA vaccines. The use of Hsp90 as a positive mark may be incorporated into vaccine formulations, especially in those that require vaccination monitoring.

Mendoza Morales LF, Lagorio V, Corigliano MG, Sánchez-López E, Ramos Duarte VA, Legarralde A, Ganuza A, Clemente M, Sander VA. Dysfunction, oxidative stress markers, and cytokine expression in the placentae of mice experimentally infected with Neospora caninum. Parasitology Research. En prensa. https://doi.org/10.1007/s00436-023-07995-0

Contreras SM, Zambrano Siri RT, Rivera EM, Cristaldi C, Kamenetzky L, Kim K, Clemente M, Ocampo J, Vanagas L, Angel SO. Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres. Epigenomes 6, 29. 2022. https://doi.org/10.3390/epigenomes6030029

Mendoza-Morales LF, Lagorio V, Corigliano MG, Sanchez-López E, Ramos-Duarte VA, Clemente M, Sander V. Neosporosis in sheep: A systematic review and meta-analysis of global seroprevalence and related risk factors. Acta Tropica 233, 106569. 2022. https://doi.org/10.1016/j.actatropica.2022.106569

Rivera EM, Moscatelli G, Ballering G, Ganuza A, Alonso AM, Moroni S, Clemente M, Altcheh J, Angel SO. Evaluation of Toxoplasma gondii recombinant antigens for early diagnosis of congenital toxoplasmosis. Diagnostic Microbiology and Infectious Disease 102, 115608. 2022. https://doi.org/10.1016/j.diagmicrobio.2021.115608

Corigliano MG, Sander VA, Sanchez López EF, Ramos Duarte VA, Mendoza Morales LF, Angel SO, Clemente M. Heat Shock Proteins 90 kDa: Immunomodulators and adjuvants in vaccine design against infectious diseases. Frontiers in Bioengineering and Biotechnology. En prensa. https://doi.org/10.3389/fbioe.2020.622186

Sánchez-López EF, Corigliano MG, Oliferuk S, Ramos-Duarte VA, Rivera M, Mendoza-Morales LF, Angel SO, Sander VA, Clemente M. Oral immunization with a plant HSP90-SAG1 fusion protein produced in Tobacco elicits strong immune responses and reduces cyst Number and clinical signs of Toxoplasmosis in mice. Frontiers in Plant Science 12, 726910.  2021. https://doi.org/10.3389/fpls.2021.726910

Sander VA, Sánchez López EF, Mendoza Morales L, Ramos Duarte VA, Corigliano MG, Clemente M.Use of Veterinary Vaccines for Livestock as a Strategy to Control Foodborne Parasitic Diseases.  Front Cell Infect Microbiol. 2020 Jun 26;10:288. doi: 10.3389/fcimb.2020.00288. eCollection 2020

Vilas JM, Corigliano MG, Clemente M, Maiale S, Rodriguez AA. Close relationship between the state of the oxygen evolving complex and rice cold stress tolerance. Plant Sci 296, 110488. 2020. https://doi.org/10.1016/j.plantsci.2020.110488

Bengoa-Luoni SA, Corigliano MG, Sánchez-López E, Albarracín RM, Legarralde A, Ganuza A, Clemente M, Sander VA. 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. 198: 105094. 2019. https://doi.org/10.1016/j.actatropica.2019.105094

Clemente M, Corigliano MG, Pariani SA, Sánchez-López EF, Sander VA, Ramos-Duarte VA. Plant serine protease inhibitors: Biotechnology application in agriculture and molecular farming. Int J Mol Sci.; 20: e1345. 2019. https://doi.org/10.3390/ijms20061345

Corigliano MG, Albarracín RM, Vilas JM, Sanches López EF, Bengoa Luoni SA, Deng B, Farran I, veramendi J, Maiale SJ, Sander VA, Clemente M.  Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein. Plant Sci. 284: 117-116. 2019. https://doi.org/10.1016/j.plantsci.2019.04.011

Sánchez-López EF, Corigliano MG, Albarracín RM, Sander VA, Legarralde A, Bengoa-Luoni SA, Clemente M. 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. 12: 140. 2019. https://doi.org/10.1186/s13071-019-3362-6

Sander VA, Corigliano MG, Clemente M. Promising plant-derived adjuvants in the development of coccidial vaccines. Front Vet Sci. 6: 20. 2019. https://doi.org/10.33 89/fvets.2019.00020