AGBIOTECH PROJECTS · Molecular Pharming). Agriculture CIGB 42: For the control of in-fectious...
Transcript of AGBIOTECH PROJECTS · Molecular Pharming). Agriculture CIGB 42: For the control of in-fectious...
AGBIOTECHPROJECTS
PORTFOLIO 2018
AGBIOTECHPROJECTS
PORTFOLIO 2018
AGBIOTECHPROJECTS
PORTFOLIO 2018
Group of the Biotechnological and Pharmaceutical Industries of Cuba.
The pharmaceutical and biotechnology sectors in Cuba have a worldwide reputation thanks to their high standards of innovation and quality. BioCubaFarma, the business group that represents the sector, is made up of 34 companies, which in turn have 61 produc-tion lines and more than 20,000 employees. BioCubaFarma has a consolidated international presence, with exports to more than 40 countries.
The main products in its portfolio are biopharmaceuticals for the prevention and treatment of di_erent diseases, such as cancer, in-fectious diseases, cardiovascular diseases, diabetes, as well as diag-nostic reagents and medical equipment. In addition, it has a wide portfolio of projects in di_erent stages of development, both for human health, as well as animal and agricultural use. The high sci-entific level of human resources in the Cuban biopharmaceutical industry guarantees the quality and competitiveness of its projects.
The Mariel Special Development Zone (ZEDM) is contributing sig-nificantly to the growth of the sector through its regulatory frame-work, favorable for those international companies wishing to in-vest in research-production facilities with high added value. The Portfolio of Opportunities for Investment offers several options to be inserted as an investor in the companies of the new biopharma-ceutical hub of that Special Zone.
CONTACTS
Dr. Normando E. Iznaga Escobar, PhDdirector of commercial policy, business development and internationational collaboration. [email protected]+53 7 643 8513 / +53 5 280 7579
Norkis Arteaga Moralesbusiness development manager, [email protected]+53 7 274 5113 / +53 5 280 6036
PROJECT AREA RESEARCH DEVELOPMENT-PILOT SCALE FIELD TESTS REGISTER
GAVAC: Immonogen for biologi-
cal control of bovine cattle ticks.
Veterinary
HeberNem: Ecological bionemato-
cide and growh promother in plant.
Agriculture
PorVac: Subunit vaccine against
classical swine fever virus (PPC).
Veterinary
KestoZyme: Biocatalizer for the
production of fructooligosacári-
dos (FOS) from sacarose.
Industrial
Enzyme
Highly thermostable biocata-
lysts for the enzymatic inversion
of sucrose and molasses.
Industrial
Enzyme
ACUABIO V: Nutritional suplement
and growth and immune system
promoter in aquatic organism.
Aquaculture
Soybean transgenic plants ex-
pressing nmdef02 defensin to pro-
tect against fungi diseases.
Agriculture
CIGB -552vet: Biological citostatic for
the treatment of cancer in pet animals.
Veterinary
ACUABIO 4: Nutritional suple-
ment for aquatic organism.
Aquaculture
SalVac: Vaccine candidate
against sea lice in salmonids.
Aquaculture
VACCINE P0: Vaccine candidate against
garrapatas based on protein P0.
Veterinary
CunVac: Recombinant vaccine against
hemorrhagic disease of the rabbit.
Veterinary
Plants as bioteactors (Plant
Molecular Pharming).
Agriculture
CIGB 42: For the control of in-
fectious diseases in plants.
Agriculture
Controling geminivirus diseases. Agriculture
Vaccine for biologic immunocastration. Veterinary
Antimicrobial peptides. Veterinary
Index
VETERINARY
GAVAC: Immonogen for biological control of bovine cattle ticks.
3
PorVac: Subunit vaccine against classical swine fever virus (PPC).
6
CIGB -552vet: Biological citostatic for the treatment of cancer in pet animals.
9
CunVac: Recombinant vaccine against hemorrhagic disease of the rabbit.
12
VACCINE P0: Vaccine candidate against garrapatas based on protein P0.
16
Antimicrobial peptides. 19
Vaccine for biologic immunocastration. 22
AGRICULTURE
HeberNem: Ecological bionematocide and growh promother in plant.
27
Soybean transgenic plants expressing nmdef02 defensin to protect against fungi diseases.
32
Plants as bioteactors (Plant Molecular Pharming). 35
CIGB 42: For the control of infectious diseases in plants. 38
Controling geminivirus diseases. 42
AQUACULTURE
ACUABIO 4: Nutritional suplement for aquatic organism. 47
ACUABIO V: Nutritional suplement and growth and immune system promoter in aquatic organism.
50
SalVac: Vaccine candidate against sea lice in salmonids. 52
INDUSTRIAL ENZYME
KestoZyme: Biocatalizer for the production of fructooligosacáridos (FOS) from sacarose.
57
Highly thermostable biocatalysts for the enzymatic inversion of sucrose and molasses.
60
VETERINARY
www.biocubafarma.cu 3
GOAL
The use of this immunogen for the purpose of incorporating it into integrated control strate-gies to reduce dependence on costly and environmentally erro-neous chemical control of ticks affecting cattle.
DESCRIPTION
When GAVAC is administered to the bovine, the Bm86 antigen it contains is processed by the animal’s immune system gen-erating an immune response measured by antibodies. These antibodies pass to the tick when it suctions its blood and causes irreversible lesions that destroy its intestinal wall and cause dam-ages that are manifested in the repletion, the oviposition and the viability of the larvae.
These damages have a cumu-lative effect on the generations of ticks, causing the gradual re-duction of the population in the grasses and in the animals.
PATENT STATUS
The product in this moments not have patent; the technology is protected by the Know How of the whole productive process.
GAVAC: Immonogen for biological control of bovine cattle ticks.AREA: VETERINARY
PROJECT STATUS
Product in commercial phase.
TYPE OF COLLABORATION REQUESTED
Counterpart interested in effica-cy trials with different tick species for the registration and com-mercialization of this product in different markets of interest.
COMPETITIVE ADVANTAGES - MILESTONES
Only commercially available tick-borne vaccine. More than 4 million head of cattle have been immunized with GAVAC, its main advantages have been: Great reduction of the inci-dence of hemo-parasitic dis-eases, More than 80% reduc-tion in chemical treatments for tick control, Drastic reduction of tick control costs, complete-ly ecological product.
DESCRIPTION
Tick-borne diseases constitute important constraints on the livestock industry, in particu-lar for the production of cattle in tropical and sub-tropical re-gions of the world. Tick-borne protozoan diseases (babesioses
and theilerioses) and ehrlichial diseases (cowdriosis and ana-plasmosis) are main health and management problems, which prevent upgrading of livestock in many developing countries. Integrated control strategies have been advocated based on host resistance to ticks and the diseases they transmit, strategic tick control taking into account the seasonal dynamics of tick infestation, availability of vac-cines against tick-borne diseas-es, and cost/benefit analysis of chemical acaricide application.
The group at CIGB developed a vaccine against tick based on the recombinant expres-sion of Bm86 antigen from Rhipicephalus Boophilus mi-croplus. This vaccine, togeth-er with appropriate tick control integral strategies has shown to be effective in field appli-cations in Cuba and in several Latin American countries, ex-pressed in less amount of de-sease transmise by ticks and reductions in the use of chem-ical product. (Figures 1A, B and C) Therefore the group has a vast experience in the cloning,
Agbiotech Proyects. Portfolio 2018.4
expression, and testing of potential vaccine an-tigens against ticks.
Active immunization of bovine cattle against the tick of the genus Rhipicephalus (Boophilus) sp. From a month of birth.
More than 4 million cattle have been immunized with GAVAC under a Pest Management Program for controlling ticks showing:
Advantages
∞ Large reduction in the incidence of he-mo-parasitic diseases.
∞ More than 80% reduction in Chemical treat-ments for tick control.
∞ A drastic reduction in expenses for tick control.
∞ Environmental friendly product.
∞ USDA has already tested GAVAC, show-ing more than 95% of efficacy against R. B. annulatus.
The Venezuela program report and effective re-sults in the controlling the ticks and reduction of chemical treatments (Figure 2) with:
∞ Large scale field trial in Cuba wherein 509 758 cattle were vaccinated across the country.
∞ Reduction in frequency of acaricidal treatments: from 7-14 days to 115-1095 days (1095 days at certain locations).
∞ Reduction in chemical consumption: from 371 t annual average to 15 t.
∞ Reduction in incidence of death and sick by he-moparasites of 98%. Enzootic stability.
∞ Estimated reduction in about 15 Millions USD per year in tick controls.
∞ More than 2 million of cattle have received the vaccination with Gavac under the Integrated Management Program in Venezuela with an im-portant reduction in the use of acaricides and in the expenses for the tick control.
FIGURE 1. A: Average period between chemical treatments, B: Number of animals sick with babesiosis and Anaplamoses,
C: Number of death by Babesiosis or Anaplasmoses deseases.
FIGURE 2: Venezuela results in the integrate Gavac programs.
The National program in the Republic Bolivariana of Venezuela included more than 2 million of bovines spread over 18 states.
www.biocubafarma.cu 5
PUBLICATIONS
1. Valle MR., Rubiera R., Penichet M., Montesinos R.,
Cremata J., Falacon V., Sánchez G., Bringas R., Cordovez
C., Valdez M., Lleonard R., Herrera L. and De la Fuente J.
High level expression of a protective BM86 antigen from
the cattle tick Boophilos microplus in yeast Pichia pas-
toris. Recombinant and Synthetic Vaccine, 1994, Narosa
Publishing House, New Delhi, India.
2. Manuel Rodriguez, Roger Rubiera, Manuel Penichet,
Raquel Montesinos, Jose Cremata, Viviana Falcon,
Giselle Sanchez, Ricardo Bringas, Carlos Cordoves,
Mario Valdes, Ricardo Lleonart, Luis Herrera, Jose de
la Fuente II. High level expression of the B. microplus
Bm86 antigen in the yeast Pichia pastoris forming
highly immunogenic particles for cattle. Journal of
Biotechnology 1994, 33: 135-146.
3. Machado H., Santizo C., A rapid visual immunoassay for
the quantification of bovine antibodies against recombi-
nant Boophilo microplus BM86 antigen. Recombinant
Vaccine for the Control of Cattle Tick, 1995.
4. Mario Canales, Antonio Enriquez, Eduardo Ramos,
Deborah Cabrera, Hubert Dandie, Alejandro Soto,
Viviana Falcon, Manuel Rodriguez and Jose de la Fuente.
Large- scale production in Pichia pastoris of the recom-
binant vaccine Gavac@ against cattle tick. Vaccine, 1997,
Vol. 15, No. 4, pp. 414-422.
5. Manuel Rodrlguez, Jose de la Fuente, Carlos Montero,
Miguel Redondo, Luis Mendez, Emerio Serrano, Mario
Valdes, Antonio Enrique Mauriz, Eduardo Ramos, Mario
Canales y Jorge Lodos. ESTRATEGIA PARA EL CONTROL
DE Boophilus microplus MEDIANTE LA INMUNlZACIÓN
CON UNA VACUNA RECOMBINANTE QUE CONTIENE
EL ANTiGENO Bm86. Biotecnología Aplicada 1997, Vol.
14, No.2.
6. Labarta V., Rodriguez M., Penichet M., Lleonard R.,
Luaces L., De la Fuente J. Simulation of control strate-
gies for the cattle tick Boophilus microplus employing
vaccination with a recombinant Bm86 antigen prepara-
tion. Veterinary Parasitology 1996, 63: 131-160.
7. Jose de la Fuente, Manuel Rodriguez, Miguel Redondo,
Carlos Montero, Jose Carlos Garcia-Garcia, Luis
Mendezt, Emerio Serranot, Mario Valdest, Antonio
Enriquez, Mario Canales, Eduardo Ramos, Oscar Boue,
Hector Machado, Ricardo Lleonart, C.A. de Armas, Sara
Rey, J.L.Rodriguez, Mirna Artiles and Libertad Garcia.
Field studies and cost-effectiveness analysis of vaccina-
tion with GavacTM against the cattle tick Boophilus mi-
croplus, Vaccine, 1998, Vol. 16, No.4, pp. 366-373.
8. Mario Canales, Jose A. Buxad, Lazaro Heynngnezz,
and Antonio Enriquez. Mechanical disruption of Pichia
pastoris yeast to recover the recombinant glycopro-
tein BM86. Enzyme and Microbial Technology 1998,
23:58- 63.
9. Lodos J., Ochagavia ME., Rodriguez M., De la Fuente J.
Simulations study of the effects of acaricide an vacci-
nation on Boophilos cattle tick populations. Preventive
Veterinary Medicine 1999, 38: 47-63.
10. Fragoso, P. Hoshman Radt, M. Ortiz, M. Rodriguez, M.
Redondo, L. Herrera and J. de la Fuente. Protection
against Boophilus annulatus infestations in cattle vacci-
nated with the B. microplus Bm86-containing Vaccine
Gavac. Vaccine 1998, Volume 16 Number 20.
11. Oscar Bouea, Karina Sanchez, Grisel Tamayo, Laura
Hernandez, Edel Reytor and Antonio Enriquez. Single-
step purification of recombinant Bm86 protein produc-
es in Pichia pastoris by salting-out and by acid precipita-
tion of contaminants. Biotechnology Techniques, 1997,
Vol11, No 8, pp. 561-565.
12. Boue, M. Redondo, C. Montero, M. Rodrigue and J. de
la Fuente. REPRODUCTIVE AND SAFETY ASSESSMENT
OF VACCINATION WITH GAVACTM AGAINST THE
CATTLE TICK (Boophilus microplus). Theriogenology
1999, 51: 1547-1554.
13. O. BOUE, O. FARNOS, A. GONZALEZ, R. FERNANDEZ,
J.A. ACOSTA, R. VALDES, L.J. GONZALEZ, Y. GUANCHE,
G. IZQUIERDO, M. SUAREZ, I. DOMINGUEZ, H.
MACHADO, M. RODRIGUEZ and R. LLEONART.
Production and biochemical characterization of the
recombinant Boophilus microplus Bm95antigen from
Pichia pastoris. Experimental and Applied Acarology
2004, 32: 119–128.
14. Manuel Rodríguez Valle, Carlos Montero, Héctor
Machado and Marisdania Joglar. The evaluation of yeast
derivatives as adjuvants for the immune response to the
Bm86 antigen in cattle. BMC Biotechnology 2001, 1:2.
15. Jose C. Garcia-Garcia, Carlos Montero, Manuel
Rodriguez, Alejandro Sotot, Miguel Redondo, Mario
Valdes, Luis Mkndez and José de la Fuente. Effect of
particulation on the immunogenic and protective prop-
erties of the recombinant Bm86 antigen expressed in
Pichia pastors. Vaccine, 1998, Vol. 16, No. 4, pp. 374-380.
Agbiotech Proyects. Portfolio 2018.6
GOAL
Development of a subunit vac-cine against classical swine fever virus as part of a comprehensive approach to the control and eradication of this disease.
DESCRIPTION
Classical swine fever is a highly contagious disease, classified in the list of notifiable diseases of the International Office of Epizootics (OIE).
It is a viral infection that affects domestic and wild pigs. It is con-sidered as one of the most dev-astating diseases for the swine industry worldwide, from the economic and health point of view.
In the CIGB, a candidate vaccine against this virus has been de-veloped, based on the expres-sion in mammalian cells of a chimeric antigen comprising by E2 protein of classical swine fe-ver virus. The antigen was fused to the porcine CD154 molecu-lar adjuvant. This vaccine can stimulate cellular and humoral responses, very similar to those generated with the inactivated virus vaccine; we obtain early protection against the virus, only 7 days after the first dose ap-plied. Porvac also cuts the hor-izontal and vertical transmission of the virus, and can considerate
PorVac: Subunit vaccine against classical swine fever virus (PPC).AREA: VETERINARY
marker vaccine, that allows dif-ferentiating the vaccinated pigs from the infested ones. Our vac-cine can be used in programs of control and eradication of this disease in zones of endemism.
PATENT STATUS
CHIMERIC VACCINE ANTIGENS AGAINST CLASSIC SWINE FEVER VIRUS. Antígenos vacu-nales quiméricos contra el virus de la peste porcina clásica (PPC) Patente PCT/CU2007/000008, WO07/098717.
PROJECT STATUS
For register in Cuba.
TYPE OF COLLABORATION REQUESTED
Corporate association for licens-ing and marketing.
COMPETITIVE ADVANTAGES - MILESTONES
It is a subunit vaccine based on the E2 protein of the virus to which the CD154 pig molecule was fused, this fusion protein is produced recombinantly in cells and mammals. It presents intel-lectual property and potential for use in government programs for the control and eradication of classical swine fever within a territory, prevents transmission from mother to offspring and
allows differentiation between vaccinated and infested pigs.
DESCRIPTION
Classical swine fever virus (CS-FV) elicits high mortality in in-fected herds during recurrent outbreaks. E2-viral envelope gly-coprotein is responsible for the induction of neutralizing antibod-ies, which become this molecule in a prospective candidate to de-velop vaccines.
In order to obtain a high immu-nogenic vaccine, viral E2 glyco-protein was fused to the swine CD154 as a molecular adjuvant. This candidate vaccine has been produced by genetic engineer-ing in CHO-DG44, CHO-K1 and HEK 293 cells. The protective ca-pacity of the chimeric E2-CD154 glycoprotein against a virulent strain of CSFV was demonstrated in a challenge experiment using a biphasic immunization sched-ule with doses of 50 and 25 μg/pigs. The immunized animals de-veloped neutralizing antibodies that were protective when the animals were faced to a chal-lenge with 105 LD50 of the homologous CSFV ‘‘Margarita’’ strain administered by intramus-cular injection. Consequently, no clinical signs of the disease were detected in the vaccinated pigs
www.biocubafarma.cu 7
(Figure 1). The E2-CD154 immu-nized animals were also able to overcome the challenge with a high virulent CSF virus strain performed 7 days after a unique dose of the vaccine without clini-cal manifestations of the disease. Specific anti-CSFV neutralizing
antibodies and IFN- were in-duced 8 days after challenge equivalent to 14 days post-vac-cination. This is the first subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. In addition, this vaccine is also
able to inhibit horizontal and ver-tical transmission of the virus. These results demonstrate that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.
FIGURE 1. Vaccine Protect in challenge pigs.
FIGURE 2. DIVA diagnostic system.
In parallel, a DIVA diagnostic system has been developed (Figure 2) which allows distinguishing vaccinated animals from infected animals. Both tools will allow establishing a program to assist in the eradication of Classical Swine Fever in coun-tries where this disease is endemic.
The main achievements of this project are:
∞ Cloning and characterization of CSFV E2 gly-coprotein fused to swine CD154 molecule.
∞ Expression of chimeric E2-CD154 protein in mammalian cell cultures (CHO-DG44, CHO-K1 and HEK 293).
∞ Demonstration of protective capacity of the E2-CD154 glycoprotein in an experiment us-ing a biphasic immunization schedule and a challenge with 105 LD50 of the high virulent CSFV ‘‘Margarita’’.
∞ Demonstration of protective capacity against 105 LD50 of the high virulent homologous CSFV ‘‘Margarita’’ after 7 days post vaccina-tion with E2-CD154 glycoprotein using a sin-gle immunization.
∞ Demonstration of trans placental transmis-sion inhibition of CSFV.
∞ Vaccination with this subunit marker vaccine E2-CD154 allows distinguishing vaccinated animals from infected animals.
Agbiotech Proyects. Portfolio 2018.8
PUBLICATIONS
1. Marisela Suárez, Yusmel Sordo, Yanet Prieto, María P.
Rodríguez, Lídice Méndez, Elsa M. Rodríguez, Alina
Rodríguez-Mallon, Elianet Lorenzo, Elaine Santana,
Nemecio González, Paula Naranjo, María Teresa Frías,
Yamila Carpio, Mario Pablo Estrada. A single dose of
the novel chimeric subunit vaccine E2-CD154 confers
early full protection against classical swine fever virus.
Vaccine 2017.
2. Prieto Y, Mendez L, Rodríguez E, Carpio Y, Rodríguez A,
Venereo A, Lorenzo E, Rodríguez MP, Suárez M, Sordo
Y, Joglar M, Santana E, Oramas N, González E, Pons T
and Estrada MP. Generation and efficacy assessment
of a chimeric antigen E2-CD154 as a marker Classical
Swine Fever Virus subunit vaccine produced in HEK
293 mammalian cells. (articule in writting).
3. Llilianne Ganges, Sara Muñoz-González, Yusmel
Sordo, Marta Pérez-Simó, Marisela Suarez, Albert
Canturri, Maria Pilar Rodriguez Molto, Maria Teresa
Frías-Lepoureau, Mariano Domingo, Mario Pablo
Estrada. Efficacy of E2 glycoprotein fused to porcine
CD154 as a novel chimeric subunit vaccine to pre-
vent classical swine fever virus vertical transmission
in pregnant sows. Veterinary Microbiology 2017.
www.biocubafarma.cu 9
GOAL
To conclude the development, license the patent and assume registration and commercializa-tion of the product in diverse markets of interest, this is a new drug directed to molecular tar-gets useful in the treatment of cancer and its metastases.
DESCRIPTION
CIGB-552 is a second-genera-tion peptide that increases the levels of COMMD1 and caus-es selective degradation of the RelA subunit (p65) of the NFkB transcription factor. CIGB-552 induces apoptosis and blocks cell cycle progression in sever-al tumor cell lines without caus-ing negative effects on normal cells. It has been shown to elic-it a potent antitumor effect in various tumor models in im-munocompetent animals and a broad therapeutic window in xenograft models of human co-lon cancer lines in nude mice. In addition, its antimetastatic ef-fect has been demonstrated in the model of lung metastasis of Lewis carcinoma. Its subcutane-ous administration is effective, and its biodistribution guaran-tees a direct apoptotic effect on the tumor. Its mechanism of
CIGB -552VET: Biological citostatic for the treatment of cancer in pet animals.AREA: VETERINARY
action presupposes a new ther-apy directed to the regulation of the antiapoptotic activity of the factor NFkB in cancer cells that would provide greater selectivity and specificity. This new peptide represents a potential therapy for various types of cancer that affect companion animals as has been demonstrated in the tests performed on dogs such as sarcomas, lymphomas and others.
PATENT STATUS
1. Immunomodulatory and antitumor peptides, PCT/CU2007/000006 Otorgada en Europa, China, México, India, Australia, Rusia y EUA.
2. Cell penetrating peptides and their use fused to biomole-cules with therapeutic action, PCT/CU2008/000006. Otor-gada en EUA, Canadá, Méxi-co, China, Corea y Sudáfrica.
3. CIGB-552 second-genera-tion peptide and COMMD1 protein as a new molecular target for cancer treatment. PCT/CU2011/000003. Otor-gada en EUA, México, China, Sudáfrica, Australia, Japón, y Ucrania.
PROJECT STATUS
Extensive laboratory data, con-ducted two clinical trials in dogs with spontaneous tumors.
TYPE OF COLLABORATION REQUESTED
Corporate association for licens-ing, co-development, registra-tion and marketing.
COMPETITIVE ADVANTAGES - MILESTONES
Product “First in its class”. There are no drugs in the market whose mechanism is based on the inhibition of the transcription factor NF-kB by levels recovered from the COMMD1 protein in cancer cells. Biological proper-ties of as cell penetrator, cyto-toxic and immunomodulatory. It does not produce immunosup-pression and produces few ad-verse effects.
It can be combined with ex-isting therapies. The mecha-nism of action demonstrates that the peptide has a great po-tential for application in various solid tumors, including colorec-tal, breast, lung and pancreatic cancer, in addition to those in-flammatory diseases at risk of developing cancer.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
Agbiotech Proyects. Portfolio 2018.10
CIGB-552 is a novel synthet-ic peptide derived from the an-timicrobial peptide LALF 32-51 (Limulus sp) which has been shown to be a potential candi-date for the anticancer thera-py and one of its useful property is the cell-penetrating capacity. COMMD1 is a newly recognized pleotropic protein that plays an
The mechanism of action of CIGB-552 assumes a new targeted anticancer therapy to regulate on-cogenic and inflammatory activity of NFkB in can-cer cells, providing greater selectivity and specificity.
important role in inflammation, hypoxic response and cell surviv-al. The recovery of COMMD1 pro-tein by CIGB-552 inhibits NF-kB, a key factor involved in the survival, proliferation and dissemination of tumor cells (2). In addition, CIGB- 552 inhibits the transcriptional activity of NFkB by TNF and IL-1ß in human colon cancer cells. The
This novel peptide presupposes a potential ap-plicability in solid tumors and inflammation asso-ciated- cancer including colorectal, breast, lung, melanoma, lymphomas and others.
peptide CIGB-552 demonstrates potent in vivo efficacy against hu-man colon and lung cancer with a considerable therapeutic win-dow in murine xenograft models (3) (Figure 1). Besides, safety and tolerability of the therapy with the peptide CIGB-552 in a model of spontaneous tumor in dog was demonstrated.
FIGURE 1. In vivo efficacy on a xenograft mouse model: colorectal (HT-29 human xenograft). Development by
Experimental Pharmacology Oncology-Berlin.
FIGURE 2. Propuse Mechanism: We have demonstrated
that CIGB-552 mediated-estabilization of COMMD1 pro-
motes RelA (p65) ubiquitination and subsequent repres-
sion of the NFkB activity, an event linked to the apoptosis
of cancer cells.
www.biocubafarma.cu 11
Phase I clinical trial in Dogs
TABLE 1. Responding population of dogs treated with CIGB-552vet. Abbreviations: SCC, squamous cell carcinoma;
TNM, tumor-node-metastasis; CR, comple esponse; PD, progressive disease; PR, partial response; SD, stable disease.
FIGURE 3. Design of study. CIGB-552 was evaluat-
ed at three dosage levels: 0.075, 0.15 and 0.3 mg/kg, s.c.
Schedule: Three injections per week every other day for
2 weeks. Clinical assessments were done on days 0 , 7, 15
and 30 of the study.
BREED DOSE OF CIGB-552 mg/Kg TUMOR HISTOLOGY TNM STAGE 1 RESPONSE
RESPONSE DURATION
(DAYS)
1. Boxer 0.075 Cutaneous lymphoma Stage IV PD -
2. Pekinese 0.075 Soft tissue sarcoma T2bN0M0 Stage III PD -
3. Mexican hairless terrier 0.075 Cutaneous lymphoma T1N0M1 CR 250
4. Teckel 0.075 Orbital melanoma Stage III SD 60
5. Cross-breed 0.15 Cutaneous lymphoma Stage III SD 60
6. Bichon habanero 0.15 Oral carcinoma (SCC) T3bN0M0 Stage III PD -
7. Labradors 0.3 Spleen lymphoma Stage I PR 120
8. Cross-breed 0.3 Cutaneous mastocytoma Stage III PR 60
PUBLICATIONS
1. Maribel G. Vallespí, Julio R. Fernandez, Isis Torrens, Isbel
Garcia, Hilda Garay, Osmani Mendoza, Milaid Granadillo,
Viviana Falcon, Boris Acevedo, Raimundo Ubieta, Gerar-
do E. Guillen and Osvaldo Reyes. Identification of a nov-
el antitumor peptide based on the screening of an Ala
library derived from the LALF 32–51 region. J Pept Sci.
2010, Jan;16(1):40-7.
2. Julio R Fernández Massó, Brizaida Oliva Arguellez, Yelaine
Tejeda, Soledad Astrada, Hilda Garay, Osvaldo Reyes,
Livan Delgado-Roche, Mariela Bollati-Fogolín and Maribel
G Vallespí. The Antitumor Peptide CIGB-552 increas-
es COMMD1 and Inhibits Human Lung Cancer Cells
Growth. Journal of Amino Acids, 2013, Article ID 251398.
3. Maribel G. Vallespí, Gilmara Pimentel, Ania Cabrales-Rico,
Julio Garza, Brizaida Oliva Arguellez, Osmani Mendo-
za, Yolanda Gomez, Tais Basaco, Iraida Sánchez, Carlos
Calderón, Juan C. Rodriguez, Maria Rivera Markelova, Idu-
na Fichtner, Soledad Astrada, Mariela Bollati-Fogolín, Hilda
E. Garay and Osvaldo Reyes Acosta. Antitumor efficacy,
pharmacokinetic and biodistribution studies of the antican-
cer peptide CIGB-552 in mouse models. Journal of Pep-
tide Science. 2014, Volume 20, Issue 11, pages 850–859.
4. Teresa Núñez de Villavicencio-Díaz, Yassel Ramos
Gómez, Brizaida Oliva Argüelles, Julio R. Fernández
Mass, Arielis Rodríguez-Ulloa, Yiliam Cruz García, Luis
Javier González, Vladimir Besada Pérez, Inés Tiscornia,
Victoria, S., Mariela Bollati-Fogolín and Maribel G Vallespí.
Comparative proteomics analysis of the antitumor effect
of CIGB-552 peptide in HT-29 colon adenocarcinoma
cells. Journal of Proteomics 2015, 126: 163–171.
5. Arielis Rodríguez-Ulloa, Jeovanis Gil, Yassel Ramos,
Lilian Hernández-Álvarez, Lisandra Flores, Brizaida Oliva,
Dayana García, Aniel Sánchez-Puente, Alexis Musacchio-
Lasa, Jorge Fernández-de-Cossio, Gabriel Padrón,
Luis J. González López, Vladimir Besada, and Maribel
G Vallespí. Proteomic Study to Survey the CIGB-552
Antitumor Effect. BioMed Research International 2015,
Article ID 124082, 18 pages.
Agbiotech Proyects. Portfolio 2018.12
GOAL
Development and evaluation of vaccine preparation, based on the VP60 protein of the viral capsid, against the devastating rabid hemorrhagic disease.
DESCRIPTION
Rabbit haemorrhagic disease is caused by the highly conta-gious RHDV virus. The incuba-tion period is approximately 24 to 48 hours. Young adult rab-bits and adult rabbits usually die suddenly within 6 to 24 hours after the onset of fever and show few clinical symptoms. It has a high mortality that oscil-lates between 70-100%.
This pandemic emerged in the People’s Republic of China in 1984. Since then cases have been observed in countries in Asia, Europe and Latin America.
The first report of RHD in the Western Hemisphere was re-ceived in 1988 in domestic rab-bits in Mexico City, since that time there have been repeat-ed outbreaks in several coun-tries of the continent. In Cuba there have been outbreaks of the disease every four years or so being the last in 2014.
CunVac: recombinant vaccine against hemorrhagic disease of the rabbit.AREA: VETERINARY
VP60 is the major structur-al protein of the RHDV virus and has been shown to be highly immunogenic and ca-pable of conferring full pro-tection against lethal doses of the RHD-producing virus when delivered to inoculated or oral rabbits.
In our group, expression of the VP60 protein was obtained in the methylotrophic yeast Pich-ia pastoris. This protein is ob-tained forming particles similar to the virus, which confer high immunogenicity. In rabbit im-munization experiments with a biphasic scheme, high titers of antibodies have been ob-tained, capable of inhibiting the haemagglutination of different viral strains of RHDV and RH-DVa, from 15 days after immu-nization and maintained up to more than 365 days post-im-munization. These antibody ti-ters have been protective in rabbits immunized and chal-lenged with 100 LD50 of a Eu-ropean reference viral strain.
PATENT STATUS
No patent. Specific Know how.
PROJECT STATUS
Proof of concept completed. In the technological development stage for its production and en-try into clinical trials for Registry.
TYPE OF COLLABORATION REQUESTED
Corporate association for licensing and marketing.
COMPETITIVE ADVANTAGES - MILESTONES
The vaccines that are commer-cialized are conventional. This has the disadvantage of manip-ulating and multiplying the virus and the possibility of introduc-ing other viruses like myxomato-sis. In addition, the infection of healthy rabbits to be used in the production of these vaccines is being ethically questioned.
Our preparation is a subunit vac-cine so it is safe, there is no viral reversion, and is obtained in the yeast Pichia pastoris, forming vi-rus-like particles, which guaran-tees an adequate and effective im-mune response in the rabbits that protects them against the Chal-lenge with virulent RHDV strains.
Immunization with this type of vaccine is advisable.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 13
FIGURE 1. Expression of the recombinant VP60 in P. pastoris PVP12 strain studied by Western blot using a hyper immune
anti-RHDV serum. Lane 1 corresponds to VP60 obtained insoluble associated to the cell debris fraction of PVP11 strain,
studied under reducing conditions. Lanes 2, 3 and 4 correspond to cell lysates, disruption pellet and disruption super-
natant of PVP12-induced cells, respectively, analyzed under reducing conditions. The protein showed the expected size
of about 60 kDa. Lane 5 corresponds to PVP12 disruption supernatant run under non-reducing conditions, in which no
band could be detected. MW and numbers in the left indicate the migration of standard molecular weight markers.
FIGURE 2. Analysis by sec-HPLC in a
TSK G5000 PW column of VP60 from
P. pastoris PVP12 and PVP11 strains.
(A) Raw disruption supernatant from
PVP12 strain, in which VP60 was de-
tected by mAb 1H8 with a retention
time (RT) of approximately 33min.
The inner box shows VP60 detec-
tion in the first of the maximums
collected. c-indicates raw disruption
supernatant from wild-type yeast. (B)
Purified VP60 protein. (C) The par-
ticulate form of HBsAg expressed in
P. pastoris MP36-transformed cells
(molecular mass of about 2×106 Da,
RT = 32min) was analyzed under the
same conditions to confirm the mul-
timeric structure of VP60. (D) VP60
solubilized from the disruption pellet
of P. pastoris PVP11 strain. As deter-
mined by immunodot, this protein
eluted with a RT = 31min. Monomeric
VP60was detected in the same run
with a RT of approximately 53 min.
A
B
C
D
Agbiotech Proyects. Portfolio 2018.14
FIGURE 3. Detection by sandwich ELISA (A and B) and immunodot (C) of RHDV conformational epitopes in the recombi-
nant VP60 variants produced in P. pastoris. Equivalent quantities of the proteins were analyzed using the monoclonal an-
tibodies 1H8 (A and C), 6H6 (B and C) or 6G2 (C), directed against the reference RHDV Bs.89 strain. As capture antibody,
a polyclonal anti-RHDV serum was used. ELISA determinations were performed in duplicate and standard deviation bars
are indicated. The sample numbers in each panel indicate the following:
1. RHDV (Bs.89 strain).
2. Raw disruption supernatant from P. pastoris PVP12 strain.
3. Same sample as 2, subjected to filtration through a 300,000 Da cut-off membrane.
4. VP60 expressed by the baculovirus/Sf9 system.
5. Cell lysates from MP36 P. pastoris strain. No significant differences were found in the absorbance values among sam-
ples 1 to 4 in ELISA. Sample
6 was included only in the immunodot and corresponds to insoluble disruption pellet from PVP11 strain.
FIGURE 4. Analysis by electron microscopy of recombinant PVP12 yeast cells. (A) The study showed high-density struc-
tures resembling multimers or particles of VP60 with a diameter of approximately 30 nm. These structures, signalled with
arrows, were relatively uniform in size and shape and were observed only in PVP12 cells. (B) The latter structures were
not detected in cells from P. pastoris wild-type MP36 strain. Panel (C) shows the formation of protein aggregates in VP60
solubilized from the cell debris fraction of PVP11 disrupted cells.
A
A CB
C
B
www.biocubafarma.cu 15
FIGURE 5. Mean antibody titers to RHDV in rabbits immunized with the recombinant variants of VP60. Titers were deter-
mined using a competition ELISA and sera from days 0, 60 and 365 of the experiment. Titers were expressed as the re-
ciprocal of the highest dilution in which the absorbance value decreased by more than 25% with respect to the equivalent
dilution in the placebo group. Positive standard deviations bars are indicated in the figure. Statistical significant differences
(p < 0.05) were found at days 60 and 365 between rabbits orally immunized with VP60 or injected with inactivated RHDV
and the rest of immunized groups. Animals from the placebo group remained seronegative. An analysis of variance and
a Bonferroni’s Multiple Comparison test were used.
PUBLICATIONS
1. Omar Farnós, Oscar Boué, Francisco Parra, José Manuel
Martín-Alonso, Odaysa Valdés, Marisdania Joglar, Leonor
Navea, Paula Naranjo, Ricardo Lleonart. High-level ex-
pression and immunogenic properties of the recombi-
nant rabbit hemorrhagic disease virus VP60 capsid pro-
tein obtained in Pichia pastoris. Journal of Biotechnology
2005, 117: 215–224.
2. Omar Farnós, Manuel Rodríguez, Maylin Chiong,
Francisco Parra, Oscar Boué, Norailys Lorenzo, Manuel
Colás, Ricardo Lleonart. The recombinant rabbit hemor-
rhagic disease virus VP60 protein obtained from Pichia
pastoris induces a strong humoral and cell-mediated
immune response following intranasal immunization in
mice. Veterinary Microbiology 2006, 114: 187–195.
3. O. Farnós; D. Rodríguez; O. Valdés, M. Chiong, F. Parra,
J. R. Toledo, E. Fernández, R. Lleonart and M. Suárez.
Molecular and antigenic characterization of rabbit hem-
orrhagic disease virus isolated in Cuba indicates a distinct
antigenic subtype. Arch Virol. 2007, 152: 1215–1221.
4. Omar Farnós, Erlinda Fernández, Maylin Chiong,
Francisco Parra, Marisdania Joglar, Lídice Méndez,
Elsa Rodríguez, Galina Moya, Dalia Rodríguez, Ricardo
Lleonart, Ernesto M. González, Alena Alonso, Pastor
Alfonso, Marisela Suárez, María P. Rodríguez, Jorge R.
Toledo. Biochemical and structural characterization of
RHDV capsid protein variants produced in Pichia pasto-
ris: Advantages for immunization strategies and vaccine
implementation. Antiviral Research 2009, 81: 25–36.
5. Erlinda Fernándeza, Jorge R. Toledo, Maylin Chiong,
Francisco Parra, Elsa Rodríguez, Carlos Montero,
Lídice Méndez, Lorenzo Capucci, Omar Farnós. Single
doseadenovirusvectoredvaccineinducesapotentand
long- lasting immune response against rabbit hemor-
rhagic disease virus after parenteral or mucosal adminis-
tration. Veterinary Immunology and Immunopathology
2011, 142: 179– 188.
6. Erlinda Fernández, Jorge Roberto Toledo, Lídice Méndez,
Nemecio González, Francisco Parra, José M. Martín-
Alonso, Miladys Limonta, Kosara Sánchez, Ania Cabrales,
Mario P. Estrada, Alina Rodríguez-Mallon, Omar Farnós.
Conformational and thermal stability improvements for the
large-scale production of yeast-derived rabbit hemorrhagic
disease virus- like particles as multipurpose vaccine. PLoS
ONE 2013, 8(2): e56417. doi:10.1371/journal.pone.0056417.
Agbiotech Proyects. Portfolio 2018.16
GOAL
Development and evaluation of vaccine preparation against ticks, based on a peptide of the ribosomal protein P0 of Rhipicephalus genus ticks.
DESCRIPTION
Ectoparasites are vectors for transmission of infectious agents causing diseases. The use of chemical pesticides is the tradi-tional method for tick control. The intensive use of these acar-icides contaminates food, envi-ronment, and develops resistant ticks. The vaccination is consid-ered an alternative for tick infes-tation control. Although new tick proteins have been proposed as potential protective molecules, only a limited number of them have been evaluated as recombi-nant antigens in vaccination trials.
Ribosomal protein P0 of Rhipicephalus genus ticks is a promising vaccine candidate. A synthetic peptide of 20 ami-no acids of an immunogenic region of the protein that is not conserved with respect to their
P0 Vaccine: vaccine candidate against ticks based in the P0 antigen.AREA: VETERINARY
hosts, showed an efficacy of 90 % and 89% as a vaccine against infestation of Rhipicephalus san-guineus in experiments in rabbits and dogs, respectively, causing a drastic decrease in the viability of the ticks. The same peptide used to immunize cattle showed an efficacy of 96 % leading to a sig-nificant decrease in recovery and the weight of ticks Rhipicephalus Boophilus microplus and a signif-icant reduction in the weight of egg masses and the hatch per-cent. These results suggest the promising possibilities of ribo-somal protein P0 peptide for the effective control of ectoparasites.
PATENT STATUS
Patent (WO2012041260A1), granted in Cuba, published by the PCT) and granted in USA, China, Chile, Europe, Australia, South Africa and Rusia.and in progress in several coun-tries where it was presented as Canada, Costa Rica, Colombia, Brazil, Dominican Rep., Perú,, Mexico and Argentina.
PROJECT STATUS
Research and proof of concept ongoing. In process to obtain an immunogenic and effective vari-ant of the candidate for the pro-duction process.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing, co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
Gavac is the only registered trademark vaccine against ticks produced by recombinant DNA techniques.
This vaccine was more ef-fective than GAVAC against Rhipicephalus sanguineus and Rhipicephalus microplus ticks.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 17
Ribosomal protein P0 of Rhipicephalus genus ticks is a promising vaccine candidate. A synthetic peptide of 20 ami-no acids of an immunogenic region of the protein that is not conserved with respect to their hosts, showed an efficacy of 90 % and 89% as a vaccine against infestation of Rhipicephalus san-guineus in experiments in rabbits and dogs, respectively, caus-ing a drastic decrease in the viability of the ticks. The same peptide used to immunize cat-tle showed an efficacy of 96 % leading to a significant decrease in recovery and the weight of ticks Rhipicephalus Boophilus microplus and a significant re-duction in the weight of egg masses and the hatch percent. These results suggest the prom-ising possibilities of ribosomal protein P0 peptide for the effec-tive control of ectoparasites. At the moment, research and proof of concept of peptide fused to carrier proteins using recombi-nant DNA techniques are ongo-ing. The objective is to obtain an immunogenic and effective vari-ant of the candidate feasible for the production process.
FIGURE 1. In red, the sequence of P0 peptide used as immunogen against
ticks.
Experiments of immunization and challenge against ticks using different mammalian hosts.
FIGURE 2. In dog, rabbit or cows,
the vaccine carry out induce a spe-
cific immunological response against
P0 antigen. The ticks collected in the
challenge experiments have smallest
size in the vaccine group compare
with placebo; the amount of eggs
was also less in this group.
Agbiotech Proyects. Portfolio 2018.18
PUBLICATIONS
1. Rodríguez-Mallon A, Fernández E, Encinosa PE,
Bello Y, Méndez-Pérez L, Cepero L, et al. A novel
tick antigen shows high vaccine efficacy against the
dog tick, Rhipicephalus sanguineus. Vaccine. 2012,
30:1782-9.
2. Rodríguez-Mallon A, Encinosa PE, Méndez-Pérez
L, Bello Y, Rodríguez Fernández R, Garay H, et al.
High efficacy of a 20 amino acid peptide of the
acidic ribosomal protein P0 against the cattle tick,
Rhipicephalus microplus. Ticks Tick Borne Dis.
2015, Jun;6(4):530-7.
www.biocubafarma.cu 19
GOAL
Obtain a new product with Intel-lectual Property, which could be used as a new generation anti-microbial or molecular adjuvant.
DESCRIPTION
This is an applied research proj-ect, aimed at obtaining a new product with Intellectual Prop-erty. The candidates obtained in this project could be used main-ly as antimicrobial of new gener-ation for the prevention and / or treatment of infectious diseases. They could also be used as mo-lecular adjuvants.
Acuabio 6, a new antimicrobial candidate and immunostimulato-ry potential, comprises three an-timicrobial peptides: Oreochro-micin-1, Oreochromicin-2 and Oreochromicin-3, which are re-lated to dicentracin, moronecidin and pleurocidin-like peptides, re-spectively. A patent including anti-microbial spectrum and adjuvant properties was presented and published between 2011- 2013 (1).
Antimicrobial peptides.AREA: VETERINARY
PATENT STATUS
The antimicrobial spectrum of these molecules and their property as molecular adju-vants are included in a patent filed and published between 2011 and 2013 (Amino Acid Se-quences for Pathogen Control, PCT / CU2012 / 000005, WO 2013/044890 A1). Granted in Cuba, Europe, United States, Japan, Australia and China. At national level in other countries.
PROJECT STATUS
In the research phase.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for li-censing, co-development and marketing.
COMPETITIVE ADVANTAGES - MILESTONES
Peptides have a potency of ef-fect on bacteria and fungi, in some cases superior to the anti-biotics and antifungal tested.
DESCRIPTION
For Antimicrobial Treatment.
Cloning and characterization of Oreochromicins regarding its antimicrobial and hemolytic ac-tivities were accomplished (2).
In vitro antimicrobial assays carried out with Oreochromi-cins indicated that these peptides displayed a broad-spectrum anti-microbial activity against Gram- negative and Gram-positive bac-teria, and fungi (1) (Table 1).
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
Agbiotech Proyects. Portfolio 2018.20
MICROORGANISMS OREOCHROMICIN 1 OREOCHROMICIN 2 OREOCHROMICIN 3
Staphylococcus aurus (Gram +) MIC = 5 μM MIC = 5 μM >106 μM
Bacillus subtilis (Gram +) MIC = 3 μM MIC = 1.7 μM MIC = 106 μM
Pseudomonas aeroginosa (Gram -) MIC = 35 μM MIC = 6.67 μM >160 μM
Escherichia coli (Gram -) MIC = 6.7 μM MIC = 5 μM >160 μM
Aeromonas hydrophila (Gram -) >160 μM MIC = 160 μM >160 μM
Edwardsiella tarda (Gram -) MIC = 160 μM MIC = 20 μM MIC = 160 μM
Vibrio sp. (Gram -) ND ND MIC = 106 μM
Candida albicans (Fungi) MIC = 20 μM MIC = 26.7 μM MIC = 40 μM
Phytophthora parasitica (Oomycete) MIC = 2.5 μM MIC = 2.5 μM MIC = 5 μM
TABLE 1. Antimicrobial spectrum of piscidin-like peptides Oreochromicin I, II and III.
FIGURE 1. Hemolytic activity of the three piscidin-like peptides against human (A) and fish (B) erythrocytes.
Piscidin-like peptides were al-so evaluated for hemolytic activ-ity in human and fish red blood cells. Oreoch-1 and Oreoch-3 were not hemolytic for human red blood cells at concentrations below 25 μM. Oreoch-2 was he-
For adjuvant applications
The ability of Oreochromicins to en-hance adaptive immune respons-es in mice and tilapia has been demonstrated (3). When co-ad-ministrated with ovalbumin in mice, Oreochromicin-1 induced a TH1 humoral immune response. Oreo-chromicin-2 and 3 induce a TH1 cellular immune response charac-
molytic in human erythrocytes reaching 50% of hemolysis at 19 μM (Fig. 1A). In addition, hemo-lytic activity in fish red blood cells was observed for Oreoch-1 and Oreoch-2 above 16 μM and 7 μM, respectively, in a dose-dependent
terized by the induction of interfer-on- in a dose depend manner.
Additionally, co- administration of Oreochromicin-1 with the sea lice my32 from Lepeophtheirus sal-monis antigen (my32-Ls) increas-es the humoral immune response in mice and tilapia. Moreover, the administration of different combi-nations of these Oreochromicins
manner. Oreoch-1 and Oreoch-2 reached 23% and 57% of hemo-lysis, respectively, at the highest concentration analyzed (Fig. 1B). In contrast, Oreoch-3 was not hemolytic for fish erythrocytes at concentrations below 25 μM.
with the sea lice antigen my32-Ls in mice also showed to exert im-mune-stimulating humoral and cellular effects.
Oreochromicins are able to boost TH1 cellular and/or hu-moral immune responses in mammals and fish, suggesting their use as TH1 molecular adju-vants to subunit antigens.
A B
www.biocubafarma.cu 21
PUBLICATIONS
1. Jannel Acosta, Vivian Montero, Yamila Carpio, Janet
Velázquez, Hilda Elisa Garay, Osvaldo Reyes, Ania
Cabrales, Yordanka Masforrol, Antonio Morales, Mario
Pablo Estrada. Cloning and functional characteriza-
tion of three novel antimicrobial peptides from tilapia
(Oreochromis niloticus). Aquaculture 2013, 372-375:
9–18.
2. Jannel Acosta, Yamila Carpio, Iris Valdés, Janet Velázquez,
Yasser Zamora, Reynold Morales, Antonio Morales, Elsa
Rodríguez, Mario P. Estrada. Co-administration of tilapia
alpha-helical antimicrobial peptides with subunit antigens
boost immunogenicity in mice and tilapia (Oreochromis
niloticus). Vaccine 2014, 32: 223– 229.
Agbiotech Proyects. Portfolio 2018.22
GOAL
Developing a vaccine based on GnRH hormone peptides can create an immunological cas-tration in animals.
DESCRIPTION
Castration in affective animals is used not only as a way to control fertility but to elimi-nate behaviors associated with sex such as aggressiveness, territoriality, response to es-trus, avoiding escaping from homes after the female in zeal, etc. There is a growing interest in eliminating traditional cas-tration methods in addition to chemical sterilants in affective animals because they cause pain, stress, complications, and death.
The proposed vaccine uses different mutated GnRH ana-log peptides and fused to a tet-anus toxoid fragment, which gives rise to a peptide of about 30 amino acids. The formula-tion employed in the vaccine candidates tested includes wa-ter-oil oily adjuvants Monta-nide type, in the presence or otherwise of VSSP (very small size proteoliposomes).
In rats, dogs and prepubertal pigs of both sexes, rapid and
Vaccine for biological immunocastration.AREA: VETERINARY
significant ablation of andro-gens and estrogens occurred, as well as vigorous immuno-castration and decreased size of the organs that make up the reproductive system in males.
PATENT STATUS
US6132720. Vaccine for revers-ible immunocastration in mam-mals, presentada por Bringas y col, publicada en 1998.
WO 2010037352 A1. Pharma-ceutical compositions utilizing combinations of variants of the gonadotropin- liberating hor-mone (GnRH) as immunogen.
Basulto y col., publicada en el 2010.
PROJECT STATUS
In transit to the development phase.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for field trials, licensing, co-de-velopment, registration and marketing.
COMPETITIVE ADVANTAGES - MILESTONES
Several years of study at labora-tory scale and under controlled
conditions that guarantee the proof of concept.
DESCRIPTION
Gonadotropins releasing hor-mone (GnRH-Gonodatro-pin-Releasing Hormone), also known as Ia Releasing Hor-mone Luteinizing Hormone (LHRH-Luteinizing Hormone Releasing Hormone) is a hy-pothalamic decapeptide which acts on the anterior pituitary gland, causing the release of FSH (follicle- stimulating hor-mone) and LH (luteinising hor-mone) towards the blood, which in turn stimulate the syn-thesis of testicular steroids and thus the development of male gonads in young animals and adolescent boys. In the female this hormone stimulates the de-velopment of ovarian follicles and the synthesis of ovarian ste-roids and ovulation.
The proposed vaccine con-templates the chemical binding by synthesis, the conjuga-tion or cloning as fusion pro-tein GnRH or its mimetics with carrier molecules. The carri-er molecules can be complete proteins or fragments or epi-topes, says Tetanus Toxoid (TT),
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 23
conjugate to Montanide and VSSP adjuvants. The immuniza-tion whit this vaccine, exhibit a close relationship between the titers of anti-GnRH generated, the reduction of the levels of sex hormones (androgens and estrogens) to castration levels, and these in turn, with the ef-fect produced over the target organs. These results, in addi-tion to presupposing clear ef-fects with the use of the type oil adjuvants Montanide allows an additional advantage, the use of much less potent adju-vants such as aluminum salts and other adjuvants not con-sidered “strong” but more safe in the process of preparing vaccines. These aspects offer a considerable advantage to the mixture of the amino and car-boxyl terminal variants over any of the GnRH immunogens.
The use of GnRH and its ana-logues for reversible immunolog-ical castration in mammals offers great possibilities to achieve suc-cessful fertility control in domes-tic animals. The goal of this work was to evaluate the biological ef-fect of GnRHm1-TT, a synthetic vaccine formulation as an immu-nogen, on the reproductive or-gans and semen quality of adult male Beagle dogs. Eight dogs of 16-17 years of age and 9-13 kg, were used. The sample was dis-tributed at random in two groups of four animals each. One group was placebo, and the other re-ceived two doses of 1 mg of the formulation with an eight-week interval and equivalent to 0.8 mg of GnRHm1 per animal. Respec-tively, complete and incomplete Freund adjuvants were used on the first and second immuniza-tions. The experiment lasted 16
weeks. In the doses and scheme used, peptide GnRHm1-TT in-duced structural and functional changes in testicles and epididy-mides, probably due to the neu-tralizing capacity of anti GnRH an-tibodies on endogenous GnRH. At the end of the experiment it was concluded that the ejaculates from dogs corresponded to one animal ready for reproduction, another not ready, and the oth-er two uncertain. In the three last cases it was caused by low con-centration of viable spermatozoa and high percentage of patho-logical spermatozoa; however, all placebo animals were up for re-production. The results achieved allow us to suggest the possibility of trying other schemes and dos-es with this peptide to achieve ef-fective inhibition of the reproduc-tive capacity of dogs and other adult mammal species.
FIGURE 1. Immunogenecity profile of
male rats immunized with GnRHm1-
TT vaccine candidate. B) Fortnightly
scheme. Rats were immunized sub-
cutaneously. After immunization, an-
imals were bled and serum tested by
indirect ELISA for anti-GnRH antibody
seroconversión. It correlated with the
androgen withdrawal observed in ma-
jor vaccine responders.
FIGURE 2. Testorene levels in rats
immunized with GnRHm1 vaccine. B)
Fortnightly scheme. The variation of
testorene level in serum is expressed
as the percent of testosterone relative
to the basal testorene levels.
Agbiotech Proyects. Portfolio 2018.24
FIGURE 3. Effects of vaccination with
the GnRHm1-TT vaccine candidate in
rat’s prostate weight. Prostate weights
were standarized against each animal
body weight (g) at different doses.
PUBLICATIONS
1. Roberto Basulto, César Milanes, Alain Rojas, Franklin
Fuentes, Nelson Izquierdo, José A Bertot, Héctor
Hernández, Delmiro Sánchez, Lesvia Calzada, Jesús
Junco. Efectos de la inmunización contra GnRH so-
bre la estructura y función testicular en perros adultos.
Biotecnología Aplicada 2003, 20:20-24.
2. Franklin Fuentes, Jesús Junco, Lesvia Calzada, Yovisleydis
Lopez, Eulogio Pimentel, Roberto Basulto Baker, Osvaldo
Reyes Acosta, Hilda E Garay-Pérez, Gerardo Guillén-
Nieto. Effect of a GnRH vaccine formulation on testos-
terone concentrations and reproduction in adult male
rats. Biotecnología Aplicada 2014, 31:222-227.
This vaccine represents a promising for immunocastration animal in order to avoid quirugic methods.
AGRICULTURE
www.biocubafarma.cu 27
GOAL
To implement a previous evalu-ation and business joint ventures for the commercial exploitation of the bionematicide HeberNem.
DESCRIPTION
At present, biological products for the control of pest and diseas-es are highly demanded world-wide. Their designs are oriented towards a friendly environmental nature compatible with organic agriculture and capable to strong-ly reduce or substitute the use of chemical pesticides. HeberNem is a biological nematicide, envi-ronmentally friendly, which has proven to be an efficient alterna-tive, supplementing the action of chemical nematicides, under a system of integrated pest man-agement. The product has been successfully tested in green hous-es dedicated to vegetable produc-tion in semi-protected crops and plantations of banana and guava. A nationwide mass marketing of bionematocide HeberNem is cur-rently carried out by Permissions Nr. 001/2007 and 050/08 of National Pesticides Registry. A new registry of a more efficient solid formulation for greenhous-es and semiprotected crops was
HeberNem: Ecological bionematocide and growh promother in plant.AREA: AGRICULTURE
recently obtained: Volume 6, Folio 0581, Permit 02616.
More than 18 years of toxicolog-ical and ecotoxicological studies (25 studies) have supported the product biosafety and bione-maticide activity against several genera of plant parasitic nema-todes. The product has proven to be environmentally friendly. Its mechanism of action has been elucidated. There are two formu-lations already registered: a liquid formulation (HeberNem-L) and solid (HeberNem-S).
PATENT STATUS
“Pesticides and antiparasitic com-positions”. Cuba: CU2001-0004: PCT/CU01/00014 Granted in: Cuba, Europe, China, Vietnam, Israel, Mexico, Panama, United States, South Africa, Peru, Austra-lia, Colombia, Russia, India, Can-ada, Brazil, Iran and Nigeria. Filed in Venezuela and Paraguay.
“Biofertilizer Composition”. CU 2007-0092:PCT/CU 2008/000002 Granted in: Cuba, South Africa, Mexico, Panama, Canada and China. Requested in: Europe, USA, Canada, Brazil, Costa Rica, China, Argentina, Venezuela, Peru, Chile.
PROJECT STATUS
National Marketing and product effectiveness trials abroad.
TYPE OF COLLABORATION REQUESTED
Development and Evaluation Agreements to perform clinical trials to validate the effective-ness of Hebernem as biopesti-cide and biofertilizer. Identify-ing appropriate partners to ex-tend its use and application.The results will lead to subsequent commercial agreements for specific territories and during a period of time.
COMPETITIVE ADVANTAGES - MILESTONES
More than 18 years of toxi-cological and ecotoxicolog-ical studies (25 studies) have supported the product bio-safety and bionematicide ac-tivity against several genera of plant parasitic nematodes. The product has proven to be environmentally friendly. Its mechanism of action has been elucidated. There are two formulations already reg-istered: a liquid formulation (HeberNem-L) and solid (He-berNem-S).
Agbiotech Proyects. Portfolio 2018.28
FIGURE 2. HeberNem powder formulation in 250 g bags.
FIGURE 1. Tomatoes plantation: Without HeberNem (left side) and with HeberNem (right side).
www.biocubafarma.cu 29
FIGURE 3. Example of application of HeberNem in banana plantations.
Agbiotech Proyects. Portfolio 2018.30
PUBLICATIONS
1. Mena J, de la Riva G, Vázquez RP, Fernández M,
Coego A, García M, Pimentel E, López A, García R,
Zaldúa Z, Mencho JD. Nematicidic agent and meth-
od for the bio-control of nematodes. International
Application Published Under the Patent Cooperation
Treaty (PCT 0397), 1996, WO 96/04749.
2. Mena J, Vázquez RP, León L, Ramírez Y, Hernández
AT, Pimentel E. Efectividad de Corynebacterium pau-
rometabolum en el control de Meloidogyne incogni-
ta. Nematropica 2001, 31:144-145.
3. Mena J, Pimentel E, Hernández A T, Veloz L, Vázquez
RP, León L, Mencho JD, Ramírez Y, Pujol M, Borroto, C.
Mechanism of action of Corynebacterium paurome-
tabolum strain C-924 on nematodes. Nematology
2002, 4: 287.
4. Jesús Mena Campos, Eulogio Pimentel Vázquez,
Liuven Veloz González, Armando Tomás Hernández,
Licette León, Yamilka Ramírez, Ileana Sánchez, Juan
Diego Mencho, Alina López, Merardo Pujol, Carlos
Borroto, Eralio Ramos, José Manuel Alvarez, Marieta
Marín, Gilda Jiménez, Graciela García, Víctor M
Pico, Manuel Expósito, Yaiza Coca, Miguel Gómez,
Amparo Olazabal, Abel Hernández,Viviana Falcón,
María C De la Rosa, Ivón Menéndez, Manuel Raíces.
Aislamiento y determinación de cepas bacterianas
con actividad nematicida. Mecanismo de acción de
C. 148 149 paurometabolum C-924 sobre nem-
atodos. Biotecnología Aplicada 2003, Vol.20, No.4.
5. Emilio Fernández, Jesús Mena, Julián González and
María Elena Márquez. Biological control of nema-
todes in banana. Turner D.W. and F.E. Rosales (eds).
Banana Root System: towards a better understanding
for its productive management: Proceedings of an in-
ternational symposium / Sistema Radical del Banano:
hacia un mejor conocimiento para su manejo pro-
ductivo: Memorias de un simposio internacional.
INIBAP, Montpellier, France. 2005, p 193-200.Mena
J, Pimentel E, Borroto C, Smith E, Mesa L, León L,
Hernández M, Ramírez Y, Sosa A, García M, Raíces
M, Hernández AT, Wong I, Basulto R, Oliva O, Herrera
L. Extensión Nacional del bionematicida HeberNem,
para el control de fitonematodos en los cultivos prote-
gidos. MINAG. Instituto de Investigaciones Hortícolas
“Liliana Dimitrova”. Investigaciones Científicas al
servicio de la Agricultura tropical.-La Habana 2006.
Liliana ISBN 959- 7111-39-X.
6. Armando Hernández, Fréderic Weekers, Jesús Mena,
Carlos Borroto, and Philippe Thonart. Freeze-drying
of the biocontrol agent Tsukamurella paurometabo-
la C-924. Predicted stability of formulated powders.
GEN PUBLISHING INC., A MARY ANN LIEBERT INC.
COMPANY • VOL. 2 NO. 3 • FALL 2006 INDUSTRIAL
BIOTECHNOLOGY.
7. Mena J, Pimentel E, Hernández AT, León L, Ramírez
Y, Wong I, Marín M, Mencho JD, Hernández M, del
Castillo A, Sánchez I, Expósito M, Jiménez G, Fleitas
M, García G, González N, Zamora J, Zalazar E,
Olivera V, Rodríguez G, Álvarez B, Dandie H, Sánchez
MC, Pimentel R, Pérez C, Compte O, Sardiñas, M,
Martínez L, Salinas D, García C, Borroto C. Uso de
bionematicida HeberNem en los cultivos protegidos.
Resúmenes Taller Latinoamericano Biocontrol de fi-
topatógenos con Trichoderma y otros antagonistas.
Fitosanidad 2006, 10: 168.
8. Mena J. Manual de Aplicación del Bionematicida
HeberNem. 1er Seminario Técnico sobre Sanidad
Vegetal en Cultivos Protegidos, La Habana 2007:
http://revistas. mes.edu.cu/eduniv/evento/fitosani-
dad/Manual-Aplicacion- Bionematicida-HeberNem.
pdf.
9. Mena J, Pimentel E, Marín M, Hernández AT,
Sánchez I, Ramírez Y, González S, García M, Borroto
C. Composición biofertilizante. Oficina Cubana
de la Propiedad Intelectual, OCPI Nro. solicitud:
2007-0092.
10. Mena J, Pimentel E, Borroto C, Smith E, Mesa L, León
L, Hernández M, Ramírez Y, Sosa A, García M, Raíces
M, Hernández ET, Wong I, Basulto R, Oliva O, Herrera
L. Empleo del bionematicida HeberNem como sus-
tituto de productos químicos en los cultivos protegi-
dos. CITRIFRUT, 2007, ISBN 978-959-296-001-5.
www.biocubafarma.cu 31
11. Nemecio González, Idania Wong, Eulogio Pimentel,
Jesús Zamora, Eladio Salazar, Jesús Mena. EXPRESIÓN
DE PROTEASAS EXTRACELULARES EN EL CULTIVO
DE ALTA DENSIDAD DEL MICROORGANISMO CON
ACTIVIDADBIONEMATICIDA Tsukamurella pau-
rometabola, C924. TECNOLOGÍA QUÍMICA 2009,
Vol. XXIX, No. 2.
12. Armando Hernández, Jesús Mena, Nemecio
González, Jesús Zamora, Eulogio Pimentel, Eladio
Salazar, María del C Sánchez, Carlos Borroto, Alain
Moreira, Lourdes Mesa, Eduardo Smith, Viviana
Falcón, Ivon Menéndez, Rafael Pimentel, Roger
Ríos, Idania Wong. Anhydrobiotic cells of the nema-
tocidic agent Tsukamurella paurometabola C-924.
Biotecnología Aplicada 2009; Vol.26, No.4.
13. Marieta Marín Bruzos, Jesús Mena Campos, Pavel
Chaveli Chávez, Rolando Morán Valdivia y Eulogio
Pimentel Vázquez. Interaction among Tsukamurella
paurometabola C-924 and Rhizobium legumino-
sarum biovar phaseoli CFH in bean plants. Acta
Agronómica. 2013, 62 (1), p 52-58.
14. Marieta Marín, Idania Wong, Graciela GarcíaI, R.
Morán, Rosa Basulto, E. Pimentel, J. Mena. Actividad
antagónica in vitro de Tsukamurella paurometabola
C-924 frente a fitopatógenos. Rev. Protección Veg.
2013, Vol. 28 No. 2:132-137.
15. Marusia Stefanova, Maria Elena Díaz de Villegas, Jesús
Mena Campos. Capítulo 8: Control biológico de en-
fermedades de plantas en Cuba. Control Biológico
de Enfermedades de Plantas en América Latina y el
Caribe. 2014, p 201-218.
16. Marieta Marín, Jesus Mena, Idania Wong, Rolando
Morán, Ramón Franco, Marcia Rojas, Pavel Chavelis,
Graciela García, Rosa Basulto, Armando Hernández,
Eulogio Pimentel, Alain Moreira, Sonia González.
Characterization of the vegetal growth promot-
ing capacity of Tsukamurella paurometabola C-924
and the main mechanisms involved. Biotecnología
Aplicada 2014, 31:168-171.
Agbiotech Proyects. Portfolio 2018.32
GOAL
To evaluate resistance to the fungus Phakopsora pachyrhizi in soybean plants transformed with the defensin gene nmdef02.
DESCRIPTION
Plant diseases caused by fun-gi and oomycetes have an important impact for crop pro-duction in Cuba and world-wide. Every year huge amounts of fungicides for control are ap-plied, with the effect thereof on the environment and human health. Find alternatives with biotechnological approaches for disease control is the big-gest challenge for researchers related with this topic. Plant de-fensins are small, cysteine-rich peptides, which inhibit the growth of a wide range of mi-croorganisms and are part of the defense mechanisms of plants against diseases.
The application of the plant de-fensin as alternative to reduce crop losses due to the attack of pathogen constitutes an ad-vantage with regard to the ap-plication of chemical fungicides. First: plant defensin are derived from seeds, roots and tubers, for
Obtaining soybean plants bearing the defensin gene nmdef02 and its evaluation against the hongo Phakopsora pachyrhiziAREA: AGRICULTURE
what they constitute nature sub-stances that are not toxic to the host plant and neither to people that consume the products from these plants. Second: as oth-er protein, the defensin quickly degrades like native substanc-es not leaving any residual after their effectiveness expires. This defensin was isolated from a plant and develops a strong anti- microbe activity in vitro and ex vitro against important bacteria, fungus and oomycetes patho-gens. Herewith, the constitutive expression of this defensin in transgenic plants increased the resistance to many plant diseas-es with high economic impact.
PATENT STATUS
“Nucleic and amino acid se-quences for the control of patho-gen agents”. CU2009-000003/WO 2009/117975 A1. Granted in: Cuba, United States, Europ, China, Japan, Vietnam, Israel, Mexico, South Africa, Australia, Colombia, Russia, India, Canada, Brazil, Iran, Nigeria.
Publication: NmDef02, a novel antimicrobial gene isolated from Nicotiana megalosiphon confers
high-level pathogen resistance under greenhouse and field conditions. Plant Biotechnology Journal, 8: 678-690, 2010.
PROJECT STATUS
In field test.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing , co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
More than 10 years of efficacy studies in experimental areas of several crops against different important from the economic point of view plant pathogens.
DESCRIPTION
This is an applied research proj-ect aimed to develop molecular strategies to inhibit fungi diseas-es like asian rust in the commer-cial soybean genotypes come together with it resistance to herbicide glyphosate.
The widespread and sustainable exploitation of crops around the world is continuously
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 33
challenged to pest control (Delgado, 2015) and particular-ly those come from fungi. The scale-up of crops is a factor that triggers the emergence of diseases that affect its yielding; such is the damage of Asian rust Phakopsora pachyrhizi as-sociated with the extending of soybean around the world. Soybean rust is a severe foli-ar disease of soybean that oc-curs throughout most soybean producing regions of the world. Soybean rust causes significant yield loss to soybean crops in Asia, Africa, Australia, and near-ly all tropical countries in the
Several Incasoy36 and DT84 transgenic lines nmdfe02- and cp4epsps- expressing genes were obtained and challenged in two indepen-dent field trials. It demonstrated that some of these lines were able to protect against not only for asian rust but also to the most important fungi of soybean under field condition. (Figure 2).
Eastern where soybeans are grown have reported its occur-rence. Rust is considered to be a major threat to soybean pro-duction in the Western hemi-sphere. In Brazil, this disease was estimated to cost growers ap-proximately $1.2 billion (USD) in 2003 alone. If P. pachyrhizi be-comes established in the con-tinental US, serious yield losses are likely to occur (Aragao, 2011; Enriquez et al., 2011). There are other fungal diseases associat-ed with the cultivation of soy-bean that are of great interest because of their high frequency of incidence on the crop.
To solve the problem of dis-eases in cultivable plants, sev-eral interesting strategies have been approached (Borras et al., 2009; Portieles et al., 2010; Enriquez et al., 2011).
Thus, one of the research-es carried out in the laborato-ry was to express the defensin NmDef02 in the Cuban soy-bean genotype in order to protect it against Asian rust produced by P. pachyrhizi, we followed for plant regeneration the procedure applicate by Cuban soybean cv Incasoy36, (Figure 1).
FIGURE 1. Procedure for plant regeneration in Cuban soybean cv Incasoy36.
FIGURE 2. Transgenic defensin-expressing soybean cv
DT84 were tested for fungus in an in vitro growth assay.
Agbiotech Proyects. Portfolio 2018.34
In experiments with this transgenic events in the field trial comparing with the not transgenic soybean plant was demonstrated the protections of the transformed plants against challenged with P. pachyrhizi, Figure 3.
FIGURE 3. Procedure for plant regeneration in Cuban soybean cv Incasoy36.
PUBLICATIONS
1. R Portieles, C Ayra, E González, A Gallo, R Rodríguez,
O Chacón, Y López, M Rodríguez, J Castillo, M Pujol,
G Enriquez, C Borroto, L Trujillo, B.P.H.J. Thomma, O
Borrás- Hidalgo. NmDef02, a novel antimicrobial pro-
tein isolated from Nicotiana megalosiphon confers
high-level pathogen resistance under greenhouse and
field conditions. Plant Biotechnol. J. 2010, 8: 678-690.
2. G Enriquez et al, Biotechnology for the genetic improve-
ments of Cuban soybean varieties. In book of Abstracts
of Biotechnology Havana’2011 “Ag-Biotech: contribut-
ing to face global challenges”, 2011, Havana, Cuba.
3. F Aragao, Development of the imidazolinone tolerant
soybean by Embrapa/BASF and future perspectives. In
book of Abstracts of Biotechnology Havana’2011 “Ag-
Biotech: contributing to face global challenges”, 2011,
Havana, Cuba, pp. 157.
4. C Delgado, G Enriquez, R Ortiz, O Cespedes, N Soto, Y
Hernandez, M Pujol. Glyphosate resistance trait into soy-
bean Cuban varieties: agronomical assessment of trans-
genic lines until F6 generation. International Journal of
Agronomy and Agricultural Research. 2015, 7, (4): 75-85.
5. Soto, N., Delgado, C., Hernández, Y. et al. Efficient par-
ticle bombardment-mediated transformation of Cuban
soybean (INCASOY-36) using glyphosate as a selec-
tive agent. Plant Cell Tissue and Organ Culture 2017,
128:187–196
www.biocubafarma.cu 35
GOAL
Development of expression strategies of recombinant pro-teins of pharmaceutical and in-dustrial interest in plant cells.
DESCRIPTION
For more than 20 years plants have been described as an al-ternative host of great interest for the production of molecules for pharmaceutical, industrial and veterinary use through ge-netic engineering. In the CIGB, an initial project was developed based on the expression of the monoclonal antibody CB.Hep1 in transgenic plants of the ge-nus Nicotiana. After several years of research and devel-opment, the plant-produced recombinant antibody (PHB-1) in CIGB was approved by the Center for State Control of Medicines, Equipment and Medical Devices (CECMED), a regulatory body of the Ministry of Health (MINSAP), to be used in the industrial production of the Heberbiovac vaccine.
However, the increasing re-quirements of the CB.Hep1 antibody, as well as the poten-tially large volumes of other an-tibodies whose expression in plants is currently investigated
Plants as bioteactors (Plant Molecular Pharming).AREA: AGRICULTURE
in CIGB, indicate the need to develop a system that pro-motes high levels of expres-sion and production in plants.
In this sense, in this project new protein candidates are evaluated as they are produced in this system and new produc-tive platforms are investigated, such as accumulation at seed levels and rapid production in transient expression systems in Nicotiana benthamiana.
PATENT STATUS
Specific know how.
PROJECT STATUS
In Developments.
TYPE OF COLLABORATION REQUESTED
Counterpart interested in ex-ploiting our technology.
COMPETITIVE ADVANTAGES - MILESTONES
There is more than 20 years of experience in the expression of recombinant proteins in plants at CIGB, at all stages of a pro-duction process, from research, development, large-scale pro-duction as well as registration strategy. CIGB is a center for
the generation of new proteins of pharmaceutical-veterinary interest, plants are an econom-ical alternative for the produc-tion of drugs and there is an increasing amount of technol-ogy available for use as host of recombinant proteins.
DESCRIPTION
The use of plants as heterolo-gous hosts is one of the most promising technologies for manufacturing valuable recom-binant proteins. The goal of this project is to use plant as host sys-tems for the production of anti-gens, antibodies and industrial proteins to be used in therapy, vaccine production and diag-nostic. Our experience includes the production and registration of monoclonal antibodies and human or animal antigens. We have explored several plant ex-pression systems including:
1. Stable expression in tobacco leaves and seeds.
2. Transient expression in Ni-cotiana benthamiana using non-viral vectors.
3. Development of viral repli-cons to transient and stable proteins expression.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
Agbiotech Proyects. Portfolio 2018.36
FIGURE 1. Antitumour effect of
monoclonal antibody produced in
tobacco leaf PhR3 and nimotuzum-
ab injected 24 h after A431 human
tumour cell implantation in nude
mice. Nimotuzumab (hR3) or PhR3
was injected at (A) 0.1, (B) 0.5 and (C)
1 mg/animal, for 10 days, 24 h after
s.c. inoculation of A431 tumour cells
to nude mice. Phosphate–buffered
saline (PBS) was used as negative
control. Both antibodies exhibited
a strong negative effect on tumour
growth kinetics with respect to PBS.
No statistical differences in effect
were found between the two anti-
bodies on day 36 (D) of the exper-
iment. Points/bars in graphics are
volume mean values and standard
deviations. Plant Biotechnol J. 2013
Jan;11(1):53-65.
TABLE 1. Comparative analysis of the production of monoclonal antibody PHB-01 in tobacco leaves vs. seeds.
(a) Data according the Monoclonal Antibody Production Department at CIGB.
(b) Dataaccordingtoourexperienceandassumptions.
(c) Production batches from 840 plants.
(d) Time in a greenhouse after 6 weeks in seedbed.
(e) Ratio biomass weight / extraction buffer volume: 1/1 for leaves, 1/10 for seeds.
(f) Assuming that the purification process recovers 50% of the starting amount of PHB-01.
(g) Assuming that each plant produces 8 g of seeds.
A
C
B
D
TOBACCO LEAVES (a) TOBACCO SEEDS (b)
Biomass production batch (c) 400-600 kg leaves 6.5-7.0 kg seed (g)
Time to harvest (d) 6-8 weeks 18-20 weeks
Expression level 30 mg PHB-01/kg leaves 6.5 g PHB-01/kg seeds
Purification batch (L) 400 L (1/1, w/v) (e) 70 L (1/10, w/v) (e)
Process yield (f) 9 g 24 g
www.biocubafarma.cu 37
PUBLICATIONS
1. Nadia Ramírez. Expression and long-term stability of a
recombinant single-chain Fv antibody fragment in trans-
genic Nicotiana tabacum seeds. BIOTECHNOLOGY
LETTERS 2001, 23: 47-49.
2. Nadia Ramírez et al. Expresión de fragmentos de an-
ticuerpos activos en plantas transgénicas de tabaco.
BIOTECNOLOGÍA APLICADA 2001, 18: 101-102.
3. Nadia Ramírez et al. Expression and characteriza-
tion of an anti-Hepatitis B surface antigen glycosylat-
ed mouse antibody in transgenic tobacco plants, and
its use in the immunopurification of its target antigen.
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
2003, 38 (3): 223-30.
4. Rodolfo Valdés et al. Hepatitis B surface antigen immuno-
purification using a plant-derived specific antibody pro-
duced in large scale. BIOCHEMICAL AND BIOPHYSICAL
RESEARCH COMMUNICATIONS 2003, 310: 742-747.
5. Meilyn Rodríguez et al. Transient expression of a full-size
antibody specific for Hepatitis B surface antigen in plant
cell suspension cultures. BIOTECNOLOGÍA APLICADA
2003, 20: 152-154.
6. Alejandro Fuentes et al. Development of a highly effi-
cient system for assessing recombinant gene expres-
sion in plant cell suspensions via Agrobacterium tume-
faciens transformation. BIOTECHNOLOGY APPLIED
BIOCHEMISTRY 2004, 39: 355–361.
7. Meilyn Rodríguez et al. Transient expression in to-
bacco leaves of an aglycosylated recombinant anti-
body against the Epidermal Growth Factor receptor.
BIOTECHNOLOGY AND BIOENGINEERING 2005, 89
(2): 188-194.
8. Merardo Pujol et al. An integral approach towards a prac-
tical application for a plant-made monoclonal antibody
in vaccine purification. VACCINE 2005, 23: 1833-1837.
9. Merardo Pujol et al. Fighting cancer with plant-expressed
pharmaceuticals. TRENDS IN BIOTECHNOLOGY 2007,
25 (10): 455-459.
10. Alina López et al. Expression of the hepatitis A virus
empty capsids in suspension cells and transgenic to-
bacco plants (Nicotiana tabacum L.). BIOTECNOLOGÍA
APLICADA 2008, 25 (42-46).
11. Lincidio Pérez et al. Anti-carcinoembryonic antigen di-
abody expressed in transiently transformed tobacco
leaves. MINERVA BIOTECNOLOGICA 2009, 21: 197-200.
12. Ada Triguero et al. Differential Nglycosylation of a mono-
clonal antibody expressed in tobacco leaves with and
without. PLANT BIOTECHNOLOGY JOURNAL. 2011,
Volume 9, Issue 9, pages 1120–1130.
13. Meilyn Rodríguez et al. Comparative in vitro and experi-
mental in vivo studies of the anti–epidermal growth fac-
tor receptor antibody nimotuzumab and its aglycosylat-
ed form produced in transgenic tobacco plants. PLANT
BIOTECHNOLOGY JOURNAL, 2013, Vol. 11, pp. 53–65.
14. Meilyn Rodríguez et al. Transgenic plants of Nicotiana
tabacum L, express aglycosylated monoclonal antibody
with antitumor activity. BIOTECNOLOGÍA APLICADA,
2013, Vol. 30, pp 157-161.
15. Abel Hernández et al. High-level production and aggre-
gation of hepatitis B surface antigen in transgenic tobac-
co seeds. BIOTECNOLOGÍA APLICADA 2013, 30(2).
16. Abel Hernández et al. Tobacco seeds as efficient pro-
duction platform for a biologically active anti-HBsAg
monoclonal antibody. Transgenic Research. Transgenic
Res. 2015, Oct;24(5):897-909.
Agbiotech Proyects. Portfolio 2018.38
GOAL
Develop a formulated product suitable for the treatment of infec-tious diseases in plants, especially for Huanglongbing (HLB) of citrus.
DESCRIPTION
The “Huanglongbing” (HLB) caused by the bacterium Can-didatus ‘Liberibacter asiaticus’ is the most destructive disease of citrus worldwide by the severi-ty of symptoms, the rapidity with which it spreads and affects all commercial citrus species. The economic impact for the pres-ence of HLB in citrus - produc-ing countries has increased year by year, with losses estimat-ed in about 30 to100%, due to reduced yields and fruit quali-ty. Until now, there is no region in the world where the HLB is adequately controlled and the disease does not exist, which contributes to increase its sever-ity and incidence. CIGB is devel-oping a product that contributes to solve the problem mentioned before, because offers an effi-cient alternative to activate the natural defense of citrus plants against the HLB. The applications of the product allow a significant
CIGB-42: For the control of infectious diseases in plants.AREA: AGRICULTURE
reduction of the bacteria as caus-al agent of the disease.
PATENT STATUS
1. A method for the protec-tion of plants against diseas-es. “CU2011-000049 / WO 2012/053994 A2. Awarded in Cuba, United States, Eu-rope, China, Vietnam, Israel, Mexico, South Africa, Austra-lia, India, Canada, Brazil.
2. Method for the treatment of plant diseases. PCT / CU2016 / 050001. WO 2017/020874 A2. Published February 9, 2017.
PROJECT STATUS
Research and Formulation development.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing , co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
More than 7 years of studies both at laboratory and plot scale
have supported the product ac-tivity against citrus HLB.
DESCRIPTION
The project start with the objec-tive to treatment of Huanglong-bing (HLB) desease which con-stitutes the most destructive dis-ease of citrus worldwide, yet no established efficient manage-ment measures exist for it.
Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, devel-opment and stress tolerance. In the experiments make as a pos-sible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’.
In the results of this experi-ment the bacterial titers in plants were reduced after treatment with epibrassinolide under both greenhouse and field condi-tions, demonstrate that epibrass-inolide may provide a useful tool for the management of HLB and the concept was demontrate. (Figure 1)
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 39
A
C
B
FIGURE 1. Induction of response of citrus plants to ‘Ca. L. asiaticus’ bacterium by eBL under greenhouse conditions.
(A) Quantification of ‘Ca. L. asiaticus’ in HLB-affected citrus plants treated or untreated with eBL every 15 days for 12
months. Each point represents mean values with standard error (10 replicates per treatment). Significant difference
among means was determined by two-way ANOVA least significant difference mean separation at *P < 0.05; **P
<0.01; NS, not significant, F = 3.762. Data are representative of three independent experiments. Phenotype of HLB-
affected citrus plants treated (B) and untreated (C) with eBL after 12 months.
FIGURE 2. Induction of new de-
fense related genes determined by
SuperSAGE combined with next
generation sequencing. Number of
genes in treated and non-treated
samples with eBL belonging to the
different categories.
We observe that some known defense genes were induced in leaves by epibrassinolide.
In order to identificate the posible activate genes with this treatments we used the SuperSAGE tech-nology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. (Figure 2)
Agbiotech Proyects. Portfolio 2018.40
thora infestans, and in some cases the protection was high-er than for plants treated with standard fungicides. However, the induction of resistance by eBL in citrus already infected with HLB disease has not pre-viously been documented.
The results suggest that eBL treatment dynamically in-creased the transcripts of an array of defense genes in the citrus plant against the ‘Ca. L. asiaticus’ bacterium. Sev-eral defense response genes are highly activated after eBL treatment. These findings sug-
gest an ability of the host to effectively enhance defense resulting in the reduction of the bacterial titers in the plant.
The application of eBL holds promise to improve the in-nate immune response to this pathogen and change the en-vironment to reduce the mul-tiplication and spread of ‘Ca. L. asiaticus’. Finally, the influence of eBL application on sever-al parameters such as disease symptomatology, citrus phys-iology, and yields should be studied to better understand the complexity of citrus HLB.
TABLE 1. Summary of the SuperSAGE.
FIGURE 3. Select candidate for treatments.
To obtain gene function cate-gories of the differentially ex-pressed UniTags, gene ontol-ogy (GO) annotation of the nucleotide sequences was performed by BLASTX against the nonredundant GenBank and UniProtKB/TrEMBL pro-tein databases. For this anal-ysis, we used UniTags that showed a maximum of two mismatches (24/26) with en-tries in the GenBank nucleo-tide database. Of the differ-entially expressed UniTags, a total of 643 from the treated library and 259 from the un-treated library had FC values ≥ 2.5 (Table 1). The largest set of these genes was assigned to oxidation/ reduction, while genes involved in cellular re-sponse to deprivation con-stituted the smallest group. Genes involved in defense re-sponse to bacteria formed the eighth biggest group.
Brassinosteroids induce dis-ease resistance in tobacco, rice, cucumber, potato, toma-to and barley plants against bacteria, fungi, oomycetes and viruses. For example, treat-ment of potato plants under field conditions with 24-epi-brassinolide decreased the level of infection by Phytoph-
LIBRARY UNTREATED TREATED TOTAL
Sequenced tags 180,002 601,748 781,750
Number of unique transcripts (UniTags) 47,747 65,536 113,283
Differential expressed “Tags” 2531 10505 13036
Function according to Gene Ontology 259 643 902
www.biocubafarma.cu 41
PUBLICATIONS
1. Canales E et al. ‘Candidatus Liberibacter asiaticus’,
Causal Agent of Citrus Huanglongbing, is Reduced by
Treatment with Brassinosteroids. Plos One, 2016, 11:
e0146223. doi:10.1371/journal.pone.0146223.
2. Silva Y et al. Expression of a microbial serine proteinase
inhibitor gene enhances the tobacco defense against
oomycete pathogens. Physiological and Molecular Plant
Pathology, 2013, 84: 99-106.
3. Moran Y et al. Identification and molecular characteriza-
tion of Nocardia sp. as a new causal agent of tobacco
false broomrape. Journal of Phytopathology, 2013, 161:
86-91.
Agbiotech Proyects. Portfolio 2018.42
GOAL
Develop molecular strategies that allow the inhibition of dis-eases by geminiviruses in plants.
DESCRIPTION
Geminiviruses are important plant pathogens that affect crop yields and fiber and pose a serious threat to food security around the world. In Cuba, beans and toma-toes are the most affected and significant yield losses in these crops cause Cuban researchers to develop ways to overcome the threat of geminiviruses. Follow-ing this objective, our research project focuses on modern mo-lecular tools to identify local geminiviruses and characterize the plant-virus interaction. In fu-ture, the development of resis-tant plants is the main objective. The small size of the geminivirus genome, the mechanisms it us-es to replicate and predispose the host, also supports our approach to validate geminiviral replicons for recombinant gene expression and silencing.
Control of geminivirus diseases in plants.AREA: AGRICULTURE
PATENT STATUS
Specific know how.
PROJECT STATUS
Research and development of the formulation. Biological assays.
TYPE OF COLLABORATION REQUESTED
Corporate association for li-censing, co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
Several years of study at labora-tory scale and under controlled conditions that guarantee the proof of concept. New strate-gies were developed that stim-ulate defense mechanisms in plants.
DESCRIPTION
This is an applied research proj-ect aimed to develop molecular strategies to inhibit geminivirus disease in plants.
Two transgenic tomato lines designed to generate siRNAs cognate to a 728 nt of Toma-to yellow leaf curl virus (TYL-CV) C1 gene fragment (Figure 1a) from an intron- contain-ing hairpin (HP) RNA transgene construct (Figure 1b) showed high resistance against TYLCV in greenhouse and in field (Fig-ure 2). Our findings demon-strate stable expression of highly abundant antiviral siR-NAs from the transgene in plant lines for at least six gen-erations under both the con-trolled greenhouse conditions and the natural hot summer conditions in the open field (Figure 3). The robust expres-sion of transgenic siRNAs, which constituted 6-8% of total sRNA population in the trans-genic plants, does not appear to exhaust the tomato RNA si-lencing machinery. Indeed, the transcriptome profiling did not reveal any changes in expres-sion of the tomato genes that mediate RNA silencing.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 43
FIGURE 1. (A) Representation of TYLCV
genome; (B) transgene cassette.
FIGURE 2. Transgenic healthy toma-
to inoculated with TYLCV in green-
house (A) and in field (B), compared
to inoculated non-transgenic symp-
tomatic plants.
FIGURE 3. Graphic representation
of transgen (blue) and viral (red) RNAi
content in tomato plants cultivated in
greenhouse and in field. C1 and C2 –
non-transgenic non-inoculated plants;
C1+ and C2+ -non-transgenic inocu-
lated plants; T1,T2-transgenic non-in-
oculated plants; T1-, T2-, T1+, T2+-
transgenic inoculated plants.
A
B
A B
Agbiotech Proyects. Portfolio 2018.44
PUBLICATIONS
1. Ramos, P. L., Guerra, O., Dorestes, V., Ramírez, N., Rivera-
Bustamante, R. y Oramas, P. Detection of TYLCV in Cuba.
Plant Disease. 1996, 80(10):1208.
2. Ramos, P. L., Guerra, O., Peral, R., Oramas, P., Guevara-
González, R. G. y Rivera-Bustamante, R. F. Taino Tomato
Mottle Virus, a new bipartite geminivirus from Cuba.
Plant Disease. 1997, 81(9):1095.
3. Echemendía A. L., Ramos P.L., Peral R., Fuentes A.,
González G., Sanpedro J. y Morales. Cuban Isolate of
Bean golden yellow mosaic virus is a member of the
Mesoamerican BGMYV Group. F. Plant Disease 2001,
85(9).1030.
4. Domínguez M., P. L. Ramos, A. L. Echemendía, J. Crespo,
R. Peral, M. Pujol, V. Andino y C. Borroto. Molecular char-
acterization of Tobacco leaf rugose virus, a new bego-
movirus infecting tobacco in Cuba. Plant Disease 2002,
86(6). 1050.
5. Echemendía A. L., P. L. Ramos, R. Peral, A. Fuentes, M.
Pujol, González G. First report of Dicliptera yellow mottle
virus (DiYMoV) infecting Dicliptera vahliana in Cuba. Plant
Pathology. 2003,52. 787.
6. Echemendía A. L., P. L. Ramos, L. Díaz, R. Peral, A.
Fuentes, M. Pujol, González G. First report of Sida golden
yellow vein virus infecting Sida species in Cuba. 2004.
Plant Pathology. 53(2). 234.
7. Dagmara Plana, Alejandro Fuentes, Marta Alvarez, Regla
M. Lara, Félix Alvarez and Merardo Pujol. A new approach
for in vitro regeneration of tomato plants devoid of ex-
ogenous plant growth hormones. Biotechnol. J. 2006,1:
1153–1157.
8. Fuentes A., P.L. Ramos, E. Fiallo, D. Callard, Y. Sánchez,
R. Peral, R. Rodríguez y M. Pujol. Intron-hairpin RNA
derived from replicase associated protein C1 gene
confers immunity to Tomato yellow leaf curl virus
infection in transgenic tomato plants.. Transgenic
Research. 2006, 15. 291-304.
9. Alejandro D. Fuentes, Pedro L. Ramos, Yadira
Sánchez, Danay Callard, Aleines Ferreira, Kenia Tiel,
Karen Cobas, Raisa Rodríguez, Carlos Borroto, Vivian
Doreste and Merardo Pujol. A transformation proce-
dure for recalcitrant tomato by addressing transgenic
plant-recovery limiting factors.. Biotechnol. J. 2008,
3: 1088–1093.
10. Alejandro D Fuentes, Pedro L Ramos, Ana I Fernández,
Kenia Tiel, Danay Callard, Yadira Sánchez, Merardo
Pujol. Transgenic tobacco cells: a permissive system
for the assessment of resistance strategies against
Tomato yellow leaf curl virus. Biotecnología Aplicada.
2009, 26.127-132.
11. Alejandro Fuentes, Natacha Carlos, Yoslaine Ruíz,
Danay Callard, Yadira Sánchez, María Elena Ochagavía,
Jonathan Seguin, Nachelli Malpica-Lopez, Thomas
Hohn, Maria Rita Lecca, Rosabel Pérez, Vivian Doreste,
Hubert Rehrauer, Laurent Farinelli, Merardo Pujol,
Mikhail Pooggin. Field trial and molecular characteri-
zation of RNAi-transgenic tomato plants that exhib-
it resistance to tomato yellow leaf curl gemini-
virus. Molecular Plant Microbe Interaction. 2016, 29.
197-209.
AQUACULTURE
Agbiotech Proyects. Portfolio 2018.46
www.biocubafarma.cu 47
GOAL
Share with the counterpart the development, registration and commercialization of the final product. Aquabio 4 Stimulates growth and immunity in fish and crustaceans.
DESCRIPTION
The Center of Genetic Engineering and Biotechnology (CIGB) has discovered and de-velops a formulation (Acuabio 4), capable to improve growth, survival, innate immune param-eters, appetite and resistance to infection by pathogens in aquat-ic organisms whether as fish feed additive or as part of an an-tiviral compound or a combined veterinary therapy.
The results have been already tested in Cuba at laboratory lev-el. This product is a pituitary ade-nylate cyclase activation peptide that stimulates the growth hor-mone release. Non transgene-sis mediated technology. When tested in catfish, tilapia, carp and shrimps, Acuabio 4 generated:
∞ Short term growth
∞ Survival increase
∞ Appetite stimulation
Acuabio 4: a potent immune stimulator and nutritional supplement for aquatic organisms.AREA: AQUACULTURE
∞ Immune system stimula-tion (innate and adaptive im-mune system)
∞ Resistance to infection by pathogens
Evaluation in vivo against vi-rus (IPNV, el VHSV, ISAV, WSSV, YHV, TSV, IHNNV and MBV) af-fecting salmonids, shrimps and bivalves demonstrated a syner-gic response when combined with antiviral compounds, com-pared with animals treated with the antiviral or the peptide alone, regarding viral clearance and survival increase.
PATENT STATUS
1. WO 2007059714 A1. Neu-ropeptides for the culture of aquatic organisms.
2. WO/2012/072055 A1. Use of pacap for treating viral infec-tions in aquatic organisms.
3. WO 2013029570 A1. Use of the pacap as a molecular adjuvant for vaccines.
PROJECT STATUS
In an advanced state of develop-ment. So far two formulations have been developed: a solid
formulation for use in immer-sion baths and an oily formu-lation to be incorporated as an additive to the feed.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing, co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
Acuabio 4 has demonstrated the ability to improve the innate and adaptive immune system, with a survival increase; to significantly stimulate the food intake, com-pared with non-treated animals and to reduce the development cycle of fish, with a better weight gaining in time.
DESCRIPTION
We have cloned and charac-terized the PRP/PACAP cDNA from the commercially import-ant North African catfish Clar-ias gariepinus. The sequence obtained agrees with the high-er conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
Agbiotech Proyects. Portfolio 2018.48
fish PACAP in mammalian cells and bacteria and al-so demonstrated that the growth rate of fish is en-hanced by both PRP and PACAP recombinant pep-tides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreo-chromis niloticus), and carp (Cyprinus carpio) sup-port the finding that PACAP plays a primordial role in growth control in teleost fish. (Figure 1).
We also evaluated the effect of PACAP adminis-tered by bath immersion, on important parameters of innate immunity and antioxidant defenses in Af-rican catfish (C. gariepinus) fry. We have shown, for the first time, that administration of recom-binant C. gariepinus PACAP not only promotes growth but also increases lysozyme, nitric oxide synthase-derived metabolites and antioxidant de-fenses in treated fry.
In addition, the effects of recombinant Clarias gariepinus PACAP administration by intraperitone-al injection on growth performance and feeding behavior in juvenile fish were studied. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stim-ulated growth in juvenile tilapia Oreochromis niloticus. We have observed that the growth-pro-moting effect of PACAP in juvenile tilapia was cor-related with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth
control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia.
C. gariepinus PACAP administration by intraper-itoneal injection is also on important immunep-arameters in juvenile fish. We observed that a single injection of the recombinant peptide was able to increase the nitric oxide synthase-derived metabolites (NOS) and totalimmunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia O. niloticus respectively, after 24h of its administration. In addition, our re-sults showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juve-nile fish. (Table 1).
Finally, the PACAP mRNA expression and PACAP im-munoreactivity detected in peripheral blood leuco-cytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish.
FIGURE 1. The growth rate of fish is enhanced by both
PRP and PACAP administrations.
TABLE 1. Innate immune Parameters and weight in tilapia larvae treated with Acuabio V after 30 days of culture.
* Statistical differences t student p< 0.05
ANTIOXIDANT DEFENSES
Parameters PACAP
Catalase +
SOD -
GSH +
INNATE IMMUNITY
Parameters PACAP
Lisozyme +
Lectins +
NO +
A A
Later on, we have studied both the expression of the two PACAP transcriptional variants (PRP/PA-CAP and PACAP) together with their receptors in diverse lymphoid organs of the rainbow trout (Oncorhynchus mykiss). Our results demonstrate
www.biocubafarma.cu 49
for the first time in fish the presence of both tran-scripts in spleen, in which immunohistochemistry confirmed the production of PACAP by lympho-cyte-like cells. In contrast, PACAP but not PRP/PA-CAP mRNA was detected in gills. Additionally, we observed a differential expression pattern of the PAC1, the PACAP specific receptor, with respect to VPAC1 and VPAC2 in lymphoid organs of fish.
All receptors were detected in brain, intestine and spleen. By contrast, PAC1 and VPAC1 receptors but not VPAC2 were found in peripheral blood and in RTS11 rainbow trout monocyte/macrophage cells. Besides, in gills and skin, PAC1 and VPAC2 but not VPAC1 were observed, whereas in head kidney, the PAC1 receptor was the only one detected.
The expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was also eval-uated in laboratory- reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV- Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 e biot. 2) were used in infection challeng-es. Kidney and spleen, the teleost main lympho-poietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective patho-gen burden, assessed targeting the VHSV-glyco-protein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic in-fections in brown trout. These gene expression re-sults provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.
In general, our finding added PACAP and its recep-tors to the list of neuroendocrine molecules pres-ent in the fish immune system, suggesting a direct autocrine/paracrine mechanism of PACAP action to mediate immune function in fish.
PUBLICATIONS
1. Lugo JM, Rodríguez A, Helguera Y, Morales R, González
O, Acosta J, Besada V, Sánchez A and Estrada MP.
Recombinant novel pituitary adenilate cyclase activat-
ing polypeptide (PACAP) from African catfish (Clarias
gariepinus) authenticates its biological function as a
growth promoting factor in lower vertebrates. Journal of
Endocrinology, 2008, 197: 583-597.
2. Carpio Y, Lugo JM*, León K, Morales R and Estrada MP.
*Both authors contribute equally to this work. Novel
function of recombinant pituitary adenylate cyclase-ac-
tivating polypeptide as stimulator of innate immunity
in African catfish (Clarias gariepinus). Fish and Shellfish
Immunology, 2008, 25:439-45.
3. Estrada MP*, Lugo JM*, Carpio Y, Morales R et al. * Both
authors contribute equally to this work. Growth stimu-
lating neuropeptides and the innate immune system in
aquatic organisms. Biotecnología Aplicada, 2008, oct.-
dic., vol.25, no.4, p.362-366. ISSN 1027-2852.
4. Lugo JM, Carpio Y, Oliva A, Morales A and Estrada
MP. Pituitary adenylate cyclase-activating polypeptide
(PACAP): A regulator of the innate and acquired immune
functions in juvenile fish Fish and Shellfish Immunology
2010, Sep;29(3):513-20.
5. Lugo JM, Oliva A, Morales A, Reyes O, Garay HE, Herrera F,
Cabrales R, Perez E and Estrada MP. The biological role of
pituitary adenylate cyclase-activating polypeptide (PACAP)
in growth and feeding behavior in juvenile fish. Journal of
Peptide Science, 2010, Sep 16. DOI 10.1002/psc.1275.
6. Lugo JM, Tafalla C, Leceta J, Gomariz R and Estrada MP.
Differential expression pattern of pituitary adenylate cy-
clase- activating polypeptide (PACAP) alternative splic-
ing variants and its receptors in the immune system of
rainbow trout (Oncorhynchus mykiss) Fish & Shellfish
Immunology 2011, 30: 734-738.
7. Lugo JM, Carpio Y, Morales R, Rodríguez-Ramos T, Ramos
L, Estrada MP. First report of the pituitary adenylate cyclase
activating polypeptide (PACAP) in crustaceans: Conser-
vation of its functions as growth promoting factor and
immunomodulator in the white shrimp Litopenaeus van-
namei.Fish Shellfish Immunol. 2013, Dec;35(6):1788-96.
8. Bartolomeo Gorgoglione*, Yamila Carpio*, Christopher
J. Secombes, Nick G.H. Taylor, Juana María Lugo, Mario
Pablo Estrada. (*) Both authors contribute equally to this
work. Viral and bacterial septicaemic infections modu-
late the expression of PACAP splicing variants and VIP/
PACAP receptors in brown trout immune organs. Fish &
Shellfish Immunology. 2015, 47 923-932.
Agbiotech Proyects. Portfolio 2018.50
GOAL
To assess in important species of aquatic organisms o (eg, shrimp, salmon, tilapia, carp, bivalves and other) a potent stimulator of growth in the early life stages.
DESCRIPTION
The Center for Genetic Engi-neering and Biotechnology has developed a formulation able to improve the survival, growth and homogeneity in sizes of aquat-ic species grown in aquaculture. It has been evaluated in Cuba at laboratory and pilot scales.
The product is based on Releasing Peptide Growth Hormone (GRHP-6), a secret-agogue of growth hormone.
During its assessments on fish and shrimp Acuabio V showed:
∞ Increase in fish and shrimp’s larvae surviving during farm-ing (10-20% in larvae stage and ponds culture).
∞ Increase in standard size and weight of whole fish and shrimps population during fish/shrimp farming.
Acuabio V: Nutritional supplement, in aquatic species for aquaculture improve-ments of growth and immune system.AREA: AQUACULTURE
∞ Increase immune in-nate response and antioxi-dant defense in the species evaluated.
∞ Increase pathogens resis-tance in animals treated compared with controls one.
PATENT STATUS
“Analogue compound to pep-tide secretagogues of the grouth horme and prepara-tions containing them”: PCT/ CU2007/000007. Date of Inter-national Application: February 28th, 2007. Granted in Australia, Europe, USA, Singapore, Japan, China, India, Mexico, South Ko-rea, Canada, Cuba, Russia and South Africa.Filed in: Brazil, Chile and Malaysia.
Method of stimulating growth and resistance to disease of aquatic organisms. No. In-ternational Application: WO 03/080102 A1. Granted in Aus-tralia, Europe, USA, Singapore, Japan, China, India, Mexico, South Korea, Canada, Cuba, Rus-sia, South Africa. Chile, Malaysia.
PROJECT STATUS
In assessment a formulation to be applied by immersion baths.
In Development oil formulation to be added to the aquaculture feed.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing, co-development and commercialization.
Identification of appropriate partners to:
∞ Register CIGB 777 formu-lation to be applied by im-mersion baths in regional aquaculture sectors linked to production.
∞ Jointly develop the oily for-mulation of CIGB 777 to be added to the aquaculture feed.
COMPETITIVE ADVANTAGES - MILESTONES
It promotes growth, survival, innate immune responses and antioxi-dant defense of economic impor-tance´s aquaculture species.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 51
CONTROL ACUABIO V
Weight (g) 0.188 ± 0.06* 0.231 ± 0.09*
Protein concentration (mg/g of tissue) 14.47 ± 5.92 9.53 ± 1.75
Antiprotease activity (anti-tripsin) 13.19 ± 2.31 23.11 ± 3.98*
Lectins (titre) 6.00 ± 2.19 14.67 ± 9.35*
SOD activity (U/mL•min)** 1.06 ± 0.09* 7.54 ± 1.36*
BSA
4 baths
FIGURE 1. 33 days of culture in ponds shrimp samples
after four treatments with the Acuabio V. Control group
represents larvae treated with BSA.
TABLE 1. Innate immune Parameters and weight in tilapia larvae treated with Acuabio V after 30 days of culture.
* Statistical differences t student p< 0.05
PUBLICATIONS
1. Rebeca Martinez, Yamila Carpio, Antonio Morales,
Juana María Lugo, Fidel Herrera, Claudina Zaldívar,
Olimpia Carrillo, Amílcar Arenal, Eulogio Pimentel,
Mario Pablo Estrada. Oral administration of the
growth hormone secretagogue-6 (GHRP-6) enhances
growth and non-specific immune responses in tilapia
(Oreochromis sp.). Aquaculture 2016, 452: 304–310
http://dx.doi. org/10.1016/j.aquaculture.2015.11.014.
2. Martinez R., Ubieta K, Herrera , F, Forellat, A , Morales
R, de la Nuez A, Rodriguez, R, Reyes,O, Oliva , A,
Estrada, MP. A novel GH secretagogue, A233, ex-
hibits enhanced growth activity and innate im-
mune system stimulation in teleosts fish. Journal
of Endocrinology 2012, 214: 409- 419, 2012. DOI:
10.1530/JOE-11-0373.
Agbiotech Proyects. Portfolio 2018.52
GOAL
To develop and evaluate the ef-ficacy of vaccine against sea lice in salmonids aquacultures in other countries.
DESCRIPTION
The sea lice are ectoparasites that live on another organism (salmonides), from which they obtain part or all the nutritive substances that they needs to live, without giving any com-pensation to the host in return.
The fixation system of these ectoparasites generates in sal-monids wounds in the skin that finally provoke stress and de-crease of the appetite, making them susceptible to other bac-terial and viral infestations. To fight it there have been applied chemical treatments, which, in addition to the high economic involved cost, have poisonous effects on the marine life and the human health.
The CIGB has a vast expe-rience in the development, production and commercial-ization of vaccines against vet-erinarian ectoparasites and it is provided with a vaccine can-didate against sea lice that af-fects salmonids.
SalVac: vaccine candidate against sea lice in salmonids.AREA: AQUACULTURE
PROJECT STATUS
Challenge experiments success-fully concluded. Development phase.
∞ 2016-2017: Fields trial.
∞ 2017: Conclude development phase.
∞ 2018: Register.
∞ 2019: Commercialization.
TYPE OF COLLABORATION REQUESTED
Corporate partnership for out-li-censing, co-development and commercialization.
COMPETITIVE ADVANTAGES - MILESTONES
There are no registered vaccines against sea lice.
Immunization in aquatic stations in Chile. Challenge of immu-nized salmonids against Caligus R. successfully concluded.
DESCRIPTION
Infestations with sea lice (Copep-oda, Caligidae) have grown to be-come the greatest challenge in salmon aquaculture. The salmon louse Lepeophtheirus salmonis is responsible for the main disease outbreaks on salmon farms in the northern hemisphere. In the southern hemisphere, Caligus rogercresseyi is the most im-portant species affecting Chile’s salmon industry. This is an ap-plied research project, aimed to obtain a new product with Intel-lectual Property. In this con-text, we have been developing candidate vaccines based on akirin-2 (designated as my32 for C. rogercresseyi) and ribosom-al protein P0 from L. salmonis and C. rogercresseyi. These can-didate vaccines have been pro-duced by genetic engineering in E. coli. The final goal is to reduce sea lice infestations in vaccinat-ed salmon.
PATENT STATUS
1. Nucleid acid and aminoacids sequences and a vaccine against ectoparasites infestations in fish. Yamila Carpio, Ma-rio Pablo Estrada. Patent PCT/CU2008/000003.
2. Vaccine composition for controlling ectoparasite infestations US 9085634 B2. PCT/CU2011/000005. Alina Rodriguez Mal-lon, Erlinda Fernández, Mario Pablo Estrada, Yamila Carpio.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 53
The main achievements of this project are:
∞ Cloning and characterization of akirin-2 (my32) and ribosomal protein P0 from L. salmonis and C. rogercresseyi.
∞ Recombinant production of different chime-ric antigens based on these two antigens in E. coli.
FIGURE 1. Localization of my32 by immunohistochemistry.
(A) Hematoxilin-eosin staining (magnification 4X). (B, C,
G, H) Immunohistochemistry developed after incubation
with the substrate 3’, 3’-diaminobenzidine tetrahydro-
chloride (10X) (B, C) Ovaries (ov). (G,H) Mature oocytes
(mo) and oviducts (o) in the genital segment. (B, G) re-
acted with pre-immune rabbit serum. (C, H) reacted with
rabbit my32 antisera. (D, E, F) Immunoflourescence. (D)
Reacted with pre- immune rabbit serum (4X) (E, F) reacted
with rabbit my32 antisera. (E) mature female (4X), cuticle
(c), (F) Ovary (40X).
SAMPLING DAY POST- CHALLENGE
SAMPLE SIZE
EXPERIMENTAL GROUPS
AVERAGE NUMBER OF PARASITES PER FISH (c)
NUMBER OF FISH PER GROUP (d)
INHIBITION OF INFESTATION (e)
STATISTICAL ANALYSIS
10 5Placebo (a) 13 ± 3 25 -
p<0.01f
Vaccine (b) 23 ± 5 25 -
24 20Placebo (a) 37 ± 10 20 -
p<0.0001f
Vaccine (b) 16 ± 7 20 57 %
TABLE 1. Results of vaccination with recombinant my32 in Salmo salar and challenge with Caligus rogercresseyi.
(a) Placebo: Salmo salar injected with PBS formulated in oil adjuvant Montanide 888 Vg (Seppic).
(b) Vaccine group: Salmo salar injected with purified my32 formulated in oil adjuvant Montanide 888 Vg (Seppic)
(c) Average number of parasites per fish ± standard deviation
(d) Total number of fish at the time of sampling
(e) Percent of inhibition of infestation calculated as (1-T/C)*100. (T: parasite number per fish in vaccinated group, C:
parasite number per fish in placebo group)
(f) Statistical significant differences were determined using a two-tailed unpaired Student’s t-test
∞ Demonstration of immunogenicity using tila-pia as a fish model.
∞ Successful vaccination-challenge experiment in Salmo salar employing my32-Cr and P0-my32-Ls in combination with an immunostim-ulant of the mucosal immunity.
∞ Assessment of different adjuvant efficacy.
Agbiotech Proyects. Portfolio 2018.54
PUBLICATIONS
1. Carpio, Y, Basabe, L, Acosta, J, Rodríguez, A, et al. Novel
gene isolated from Caligus rogercresseyi: a promis-
ing target for vaccine development against sea lice.
Vaccine. 2011, 29(15):2810-20.
2. Yamila Carpio, Claudia García, Tirso Pons, Denise
Haussmann, Tania Rodríguez-Ramos, Liliana Basabe,
Jannel Acosta and Mario Pablo Estrada. Akirins in
sea lice: First steps towards a deeper understanding.
Experimental Parasitology.. 2013, 135(2):188-199.
3. Liliana Basabe, Yamila Carpio, Diamilé Gonzalez,
Antonio Morales, Mario P Estrada. Yield improvement of
the sea lice MY32/Cr novel antigen production and IgM
immune response characterization in Oreochromis ni-
loticus as a model.. Biotecnología Aplicada.. 2014, Vol.
31 No. 1, 28-32.
4. Carlos Pérez, Nemecio González, Eladio Salazar,
Eulogio Pimentel, Yamila Carpio, Miladys Limonta.
Design and Optimization of a Purification Process for
MY32/Ls Protein Solubilizing Inclusion Bodies for a
New Vaccine against Sea Lice. Bioprocessing Journal.
2015, Volume 14/Issue 1. ISSN 1538-8786.
5. Jannel Acosta, Yamila Carpio, Iris Valdés, Janet
Velázquez, Yasser Zamora, Reynold Morales, Antonio
Morales, Elsa Rodríguez, Mario P. Estrada. Co- admin-
istration of tilapia alpha-helical antimicrobial peptides
with subunit antigens boost immunogenicity in mice
and tilapia (Oreochromis niloticus). Vaccine. 2014,
32:223– 229
INDUSTRIAL ENZYME
Agbiotech Proyects. Portfolio 2018.56
www.biocubafarma.cu 57
GOAL
Commercial Exploitation of the enzyme Fructosyltransferase (KestoZyme) for the produc-tion of high quality FOS from sucrose.
DESCRIPTION
Fructooligosaccharides (FOS) are natural nondigestible sugars, consisting of 2-4 units of fructose attached to a terminal glucose. Given its activity as soluble fiber and its demonstrated prebiotic ef-fect in humans and animals, FOS maintains an increasing demand in the functional foods market. At present, commercial production of FOS from sucrose is carried out using biocatalysts based on fungal cells immobilized on cal-cium alginate spheres. CIGB has developed a new biocatalyst for the production of FOS, based on a fructosyltransferase of vegeta-ble origin produced at high levels in the yeast Pichia pastoris. The recombinant enzyme (1-SSTrec) is formulated as a lyophilized powder of specific high activity (> 10,000 U / g), is fully soluble in water and can be operated at temperatures of 40 ° C. It is sta-ble at room temperature (25-30 ° C) for one year. The composition (w / w) of the reaction products are: F (1%), G (21%), GF (20%), GF2
KestoZyme: Biocatalizer for the production of fructooligosacáridos (FOS) from sacarose.AREA: INDUSTRIAL ENZYME
(55%), GF3 (3%). The enzyme can be operated without reuse in a batch reactor or with continuous reuse in a membrane bioreactor.
PATENT STATUS
The technology for the pro-duction and operation of the Fructosyltransferase enzyme (1-SSTrec) is backed by the pat-ent “Method of obtaining 1-kes-tose”. No. of application in Cuba: CU2012-0138. International Application No.PCT / CU2013 / 000005. International Publication No. WO 2014/044230.
PROJECT STATUS
In Commercial step.
TYPE OF COLLABORATION REQUESTED
License of our technology for the production of FOS by the use of the enzyme Fructosyltransferase (KestoZyme).
COMPETITIVE ADVANTAGES - MILESTONES
No recombinant fructosyltrans-ferases are available for the commercial production of FOS.
The enzyme 1-SSTrec obtained in the CIGB is recovered from the culture supernatant with
purity above 90% and has com-petitive advantages over the cur-rent biocatalysts based on cells / fungi enzymes, in terms of:
∞ High specific activity (> 10,000 U / g)
∞ High performance of total FOS (55-60%, w / w) in re-action products.
∞ High quality in the final composition of FOS (90% 1-kestose and 10% nystose).
∞ No hydrolysis activity of su-crose substrate.
Simple and flexible operation to produce small and large scale FOS.
DESCRIPTION
FOS intake has been proven to promote the selective prolifera-tion of intestinal Bifidobacteria and Lactobacilli with import-ant benefits to health, includ-ing: competitive exclusion of intestinal pathogens, reduction of serum cholesterol, increase of calcium and magnesium absorption, prevention of co-lon cancer, and production of B-vitamins. The trisaccharide 1-kestose (GF2) is commercial-ly more attractive FOS due to
Agbiotech Proyects. Portfolio 2018.58
its superiority in terms of bi-fidogenic activity and more sweetening power, in compar-ison with nystose (GF2) and fructosylnystose GF3), so it is more appropriate for use as a low-caloric sweetener for dia-betics. Current systems for FOS production from sucrose are based on fungal cells entrapped in calcium alginate beads.
CIGB has developed a nov-el biocatalyst for the produc-tion of FOS, based on a plant Fructosyltransferase produced at high levels in the yeast Pichia pastoris (KestoZyme). The re-combinant enzyme (1-SSTrec) is formulated as a freeze-dried powder with high specific ac-tivity (>10,000 U/g), is total-ly soluble in water and can be
operated at temperature of 40ºC. This enzyme is stable at room temperature (25-30ºC) during one year. The average product composition (w/w) is as follows: F(1%), G(21%), GF(20%), GF2(55%), GF3(3%). The soluble enzyme is operated, without re-use, in a batch stirred tank reac-tor or, with continuous reuse, in a membrane bioreactor.
TABLE 1. Comparison between Immobilized fungus enzyme and KestoZyme.
BIOCATALYST COMPANY FOS CONTENT (DRY MATTER)
COMPOSITION AT HIGHEST GF2
COMPOSITION AT HIGHEST FOS
1-SSTrec
KestoZyme
HeberBiotec 55-60% GF2 ~ 91%
GF3 ~ 9%
GF4 ~ 0%
GF2 ~ 86%
GF3 ~ 14%
GF4 ~ 0%
Aspergillus niger
Immobilized
Beghin Meiji,
Meiji Seika
55-60% GF2 ~ 76%
GF3 ~ 22%
GF4 ~ 2%
GF2 ~ 37%
GF3 ~ 53%
GF4 ~ 10%
www.biocubafarma.cu 59
PUBLICATIONS
1. E.R. Pérez, L.E. Trujillo, J.G. Arrieta, H. Pérez, M.A.
Brizuela, G. Trujillo, L. Hernández. A pH shift-based
procedure to screen fructooligosaccharides ferment-
ing yeast and bacterial strains. Biotecnologia Aplicada
2010, 27:216-220.
2. C. Menéndez, D. Martínez, L.E. Trujillo, Y. Mazola, E.
González, E. Pérez, L. Hernández. Constitutive high
level expression of a codon-optimized –fructosidase
gene from the hyperthermophile Thermotoga ma-
ritima in Pichia pastoris. Applied Microbiology and
Biotechnology 2012, 97:1201-1212.
3. D. Martínez, B. Cutiño-Avila, E.R. Pérez, C. Menéndez,L.
Hernández, A. del Monte-Martínez. A thermostable
exo--fructosidase immobilized through rational de-
sign. Food Chemistry 2014, 145:826-831.
4. D. Martínez, C. Menéndez, F.M. Echemendia, E.R.
Pérez, L.E. Trujillo, A. Sobrino, R. Ramírez, Y. Quintero,
L. Hernández. Complete sucrose hydrolysis by heat-
killed recombinant Pichia pastoris cells entrapped in
calcium alginate. Microbial Cell 2014, Factories doi:
10.1186/1475- 2859-13-87.
5. C Menéndez, D Martínez, LE Trujillo, R Ramírez, A
Sobrino, BV Cutiño-Ávila, L Basabe, A del Monte-
Martínez, ER Pérez, L Hernández. Development of sol-
uble and immobilized biocatalysts based on a
recombinant thermostable -fructosidase enabling
complete sucrose inversion at pasteurization tempera-
tures. Biotecnologia Aplicada 2014, 31:249-253.
6. D Martínez, C Menéndez, FM Echemendia, L
Hernández, A Sobrino, LE Trujillo, I Rodríguez, ER Pérez.
Kinetics of sucrose hydrolysis by immobilized recom-
binant Pichia pastoris cells in a batch reactor. Journal
of Microbial & Biochemical Technology, 2015, 7(5):294-
298 doi:10.4172/1948-5948.1000228.
Agbiotech Proyects. Portfolio 2018.60
GOAL
Commercial exploitation of ther-mostable biocatalysts for the en-zymatic inversion of sugar and honey.
DESCRIPTION
The hydrolysis of sucrose pro-duces a mixture of glucose and fructose in equal propor-tion (known commercially as Inverted Sugar or Inverted Syrup). The use of Inverted Syrup pres-ents advantages over its precur-sor, sucrose, in terms of higher total dry matter, higher solubili-ty, higher retention of moisture, greater sweetening power and faster action as an energy food. The enzymatic hydrolysis of su-crose, unlike the convention-al inversion with acid, does not use chemical substances nor generate toxic byproducts, and is therefore the most recom-mended process for the pro-duction of Inverted Sugar, with a destination in the food industry.
CIGB has developed two bio-catalysts for total or partial in-version of sucrose:
1. The first biocatalyst is based on the immobilization of
Highly thermoestable biocatalysts for the production of sirope invested from sacarose.AREA: INDUSTRIAL ENZYMES
Pichia pastoris cells contain-ing high levels of a thermo-stable invertase (TmINVrec) of bacterial origin in peri-plasm. A heat treatment elim-inates the viability of the cells prior to immobilization in cal-cium alginate. This biocata-lyst can be stably operated at 60-70 ° C to reverse concen-trated solutions (55-60 ° Bx) of sucrose or honey, either in repeated batch cycles or in continuous flow columns.
2. The second biocatalyst con-sists of the recombinant en-zyme (TmINVrec) recovered from the culture supernatant and formulated as a soluble high specific activity powder (> 15,000 U / g). The enzy-matic activity is stable after one year of storage at room temperature (25-30 ° C). In both cases, the hydrolysis reaction is completed in a short period of time and re-sults in syrup directly fit for human consumption.
PATENT STATUS
Specific Know–how.
PROJECT STATUS
The following technologies have been developed:
∞ Pilot scale production and operation of the immobilized cell biocatalyst.
∞ Production and pilot scale operation of the free enzyme biocatalyst.
TYPE OF COLLABORATION REQUESTED
Strategic alliance and associa-tion for the production and / or commercialization of thermo-stable biocatalysts for the enzy-matic inversion of sucrose.
COMPETITIVE ADVANTAGES - MILESTONES
The biocatalysts developed in the CIGB are more competi-tive than the current enzymat-ic systems of sugar inversion, in that:
∞ Its high thermal stability al-lows to operate in continu-ous reuse cycles.
∞ Total sucrose inversion is achieved in short reactions.
∞ Operating costs are low.
HOST INSTITUTION: Centro de Ingeniería Genética y Biotecnología (CIGB).
E-mail: [email protected] / Web site: http://gndp.cigb.edu.cu
www.biocubafarma.cu 61
The table summarizes the pro-ductivity in each of the con-centrations tested with two biocatalyst charges given by
The productivity (P) represents the amount of hydrolyzed sucrose (500 g/L, 600 g/L, 700 g/L) per amount of biocatalyst obtained from 1 L of culture in the reaction time (g/L/h). Calculations were performed for more than 98% hydrolysis or at 10 h reaction. *
PUBLICATIONS
1. C. Menéndez, D. Martínez, L.E. Trujillo, Y. Mazola, E.
González, E. Pérez, L. Hernández. Constitutive high
level expression of a codon-optimized ß–fructosi-
dase gene from the hyperthermophile Thermotoga
maritima in Pichia pastoris. Applied Microbiology and
Biotechnology 2013, 97:1201-1212.
2. D. Martínez, C. Menéndez, F.M. Echemendia, E.R.
Pérez, L.E. Trujillo, A. Sobrino, R. Ramírez, Y. Quintero,
L. Hernández. Complete sucrose hydrolysis by heat-
killed recombinant Pichia pastoris cells entrapped in
calcium alginate. Microbial Cell Factories 2014, doi:
10.1186/1475- 2859-13-87.
3. C Menéndez, D Martínez, LE Trujillo, R Ramírez, A
Sobrino, BV Cutiño-Ávila, L Basabe, A del Monte-
Martínez, ER Pérez, L Hernández. Development of
soluble and immobilized biocatalysts based on a re-
combinant thermostable ß-fructosidase enabling com-
plete sucrose inversion at pasteurization temperatures.
Biotecnologia Aplicada 2014, 31:249-253.
TABLE 1. Productivity of biocatalysts at different initial concentrations of sucrose
the biocatalyst (g) mass ra-tios: reaction volume (mL) or U/g sucrose, according to the amount of biocatalyst obtained
from 1 L of culture. Under all tested conditions more than 50% hydrolysis of the sucrose was achieved.
BIOCATALYSTS BIOCATALYST /L*BIOCATALYST
CHARGES CHARGES RELACIÓN
500 G/L 600 G/L 700 G/L
PpABfrA 725 g1:10 445,6 348,3 316,5
1:5 226,3 226,8 215,4
BfrA libre 97379,8 U4 U/g 2233,5 2160,0 2178,8
8 U/g 2066,0 2000,0 1531,3
Quantity of biocatalyst obtained by L of culture. The stability of the biocatalysts under reactive con-ditions showed average life times of 360 and 168 h for PpABfrA and free BfrA, respectively. The bio-catalysts developed in the CIGB are more competitive than the
current enzymatic systems of sugar inversion (invertase from Saccharomyces cerevisiae), in that: Its high thermal stability al-lows to operate in continuous reuse cycles, total sucrose inver-sion is achieved in short reactions and operating costs are low.
4. D Martínez, C Menéndez, FM Echemendia, L Hernández,
A Sobrino, LE Trujillo, I Rodríguez, ER Pérez. Kinetics of
sucrose hydrolysis by immobilized recombinant
Pichia pastoris cells in a batch reactor. Journal of
Microbial & Biochemical Technology, 2015, 7(5):294-
298 doi:10.4172/1948-5948.1000228.
5. D Martínez, C Menéndez, L Hernández, A Sobrino, LE
Trujillo, I Rodríguez, ER Pérez. Scaling-up batch condi-
tions for efficient sucrose hydrolysis catalysed by im-
mobilized recombinant Pichia pastoris cells in a stirrer
tank reactor. Electronic Journal of Biotechnology 2017,
25:39-42) doi:10.1016/j.ejbt.2016.11.003.