Papers by Jorge R. Toledo
The mammary gland: bioreactor for the production of recombinant proteins. Biological systems for ... more The mammary gland: bioreactor for the production of recombinant proteins. Biological systems for the expression of recombinant proteins have evolved from microorganisms, such as bacteria and yeast, to animal and plant cells. This evolution has been driven mainly by the specific posttranslational modifications required for many recombinant proteins to display full biological activity. In this review we discuss the main
... Biotecnología Animal. Centro de Ingeniería Genética y Biotecnología Ave. 31, e/ 158 y 190, CP... more ... Biotecnología Animal. Centro de Ingeniería Genética y Biotecnología Ave. 31, e/ 158 y 190, CP 10 600, Apartado Postal 6162, Playa, Ciudad de La Habana, Cuba E-mail:[email protected] ABSTRACT Biological systems ...
Page 1. Biotecnología Aplicada 2005; Vol.22, No.1 62 ✍Corresponding author ...
Veterinary Immunology and Immunopathology, 2011
Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disea... more Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 108 gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 109 GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.
Applied Biochemistry and Biotechnology, 2009
HIV-1-derived lentiviral vectors (LvV) are within the most attractive gene delivery vehicles in t... more HIV-1-derived lentiviral vectors (LvV) are within the most attractive gene delivery vehicles in the context of both dividing and quiescent cells. LvV is currently produced by the conventional calcium phosphate precipitation method. Nevertheless, this procedure is highly susceptible to variations in pH and impurities, which lead to inconsistencies in LvV production. Here, we present a simple and robust procedure for LvV production using branched 25 kDa polyethylenimine, with a transfection efficiency of over 90% and viral titer yields of about 1 × 107 infective lentiviral particles per milliliter. The procedure outlined is simple, consistent, and as inexpensive as the CaPO4-based method.
Florida Entomologist, 2014
The efficacy of Heterorhabditis bacteriophora (Poinar) infective juveniles (IJs) was evaluated ag... more The efficacy of Heterorhabditis bacteriophora (Poinar) infective juveniles (IJs) was evaluated against third instar Anastrepha ludens (Loew) (Diptera: Tephritidae) under laboratory conditions in a sandy clay soil at various levels of soil moisture. Three experiments were performed in which the efficacy of the IJs against A. ludens was estimated, i.e., (a) at 6 different levels of soil moisture, (b) in soil that was allowed to lose moisture over a 15 day period, and (c) in soil with an initial moisture content of 16% and in which moisture loss was partially mitigated by adding water at 5-day intervals. In the first experiment, the greatest A. ludens mortality (80%) was observed in soil with 18% moisture (-63.1 bars), although this was not significantly greater than A. ludens mortality at 21% moisture (-20.4 bars). At 24% soil moisture (-7.70 bars), percentage of mortality of A. ludens declined to about 50%. Likewise insect mortality was substantially lower at soil moisture levels of 15% (-240.1 bars) and 12% (-1,232 bars) and very much lower (about 16%) at 9% soil moisture (-10,147 bars). In the second experiment, as soil moisture declined from 16% to less than 10% over a 15 day period, the infectivity of IJs, as indicated by A. ludens larval mortality, progressively declined from more than 55% to less than 10%. In the third experiment, in which moisture loss was partially mitigated by adding water at 5-day intervals, the decline in infectivity of IJs was gradual up to 21 days, but decreased thereafter. We conclude that soil moisture levels must be carefully considered when applying H. bacteriophora IJs to control A. ludens under field conditions, because soil moisture has a marked effect on the efficacy of IJs for the biological control of this pest.
Journal of Proteome Research, 2009
Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target t... more Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man 8 GlcNAc 2 isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.
Archives of Biochemistry and Biophysics, 2007
We have established a continuous, non-transformed cell line from primary cultures from Capra hirc... more We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N′-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.
Veterinary Immunology and Immunopathology, 2010
Vaccine, 2010
Subunit vaccines are a suitable alternative for the control of classical swine fever. However, su... more Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, type I interferon is used as an immunostimulating molecule in order to increase the immunogenicity of a vaccine candidate based on the E2-CSFV antigen produced in goat milk. Pigs vaccinated with E2-CSFV antigen co-formulated with recombinant human alpha interferon were protected against clinical signs and viremia as early as 7 days post-vaccination. It was also demonstrated that interferon stimulates a response of specific anti-CSFV neutralizing antibodies. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after vaccination. These results suggest that the E2-CSFV antigen combined with type I interferons could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.
Journal of Biotechnology, 2004
The production of large quantities of complex proteins with biopharmaceutical purposes is the mai... more The production of large quantities of complex proteins with biopharmaceutical purposes is the main drawback for their more extensive use. Here we demonstrated that a direct instillation of a recombinant adenoviral vector containing an expression cassette for the human growth hormone gene into the mammary gland of mice and goats allowed for the efficient secretion of human growth hormone in the milk. Through this approach we were able to express human growth hormone at maximal levels of 2.8 mg/ml in the milk of mice and up to 0.3 mg/ml in goat milk. We found that the expression levels were closely dependent on both the degree of differentiation of the secretory epithelium and on the adenoviral dose used. Here we demonstrated that the direct transduction of mammary epithelial cells by means of a recombinant adenovirus could be a suitable alternative to transgenic technology for the production of recombinant proteins of biopharmaceutical interest.
Archives of virology, 2012
BoHV-5 was detected in one of several extended semen samples from a healthy donor bull during rou... more BoHV-5 was detected in one of several extended semen samples from a healthy donor bull during routine virus screening. This was achieved by polymerase chain reaction assay (PCR) and virus isolation, with primary identification by the fluorescent antibody test. The isolated virus, B4180, was characterized by sequencing a cloned fragment of the gC gene and by restriction enzyme analysis (REA). The nucleotide sequence shared 99 % similarity with published sequences of BoHV-5, and the REA showed that the isolate was of the BoHV-5a subtype. This study provides the first evidence of intermittent BoHV-5 shedding in bull semen as well as information about its geographic distribution.
Vaccine, 2008
E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever v... more E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3′ terminus. The recombinant antigen was produced at a range of 120–150 μg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 μg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 105 PLD50 of homologous CSFV “Margarita” strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.
Journal of Economic Entomology, 2009
The demographic changes in Anastrepha obliqua (Macquart) (Diptera: Tephritidae) throughout labora... more The demographic changes in Anastrepha obliqua (Macquart) (Diptera: Tephritidae) throughout laboratory colonization were characterized over the course of 11 generations. Four significant changes were determined. The first change was a reduction in the preoviposition period from the G(p) to G(1). The second was that wild female flies had difficulty ovipositing in an artificial substrate, yielding the lowest fecundity rates observed throughout the experiment. The third significant change was a decrease in longevity and life expectancy from G(p) to G(1), which then continued to decrease with successive generations. This resulted in a lab strain with high fecundity limited to a short period of oviposition. The last significant change was a reduction in larval and pupal weight. In addition, larval recovery decreased from G(p) to G(1) but displayed rapid recovery over the course of generations. There was no change in adult emergences for all generations, and flight ability increased with successive generations. These changes were correlated with demographic parameters, indicating that the increased investment in early age reproduction incurs costs such as a reduction in life expectancy or fecundity later in life. This trend was also correlated with an increase in early fecundity and reduction in the oviposition period.
Journal of Economic Entomology, 2004
Mangoes infested with third instar larvae were irradiated using Co-60 gamma rays and a dose inter... more Mangoes infested with third instar larvae were irradiated using Co-60 gamma rays and a dose interval of 2-250 Gy to assess the irradiation dose required to prevent adult emergence of the Mexican fruit fly (Anastrepha ludens), the West Indies fruit fly (A. obliqua), the sapote fruit fly (A. serpentina), and the Mediterranean fruit fly (Ceratitis capitata). Doses of 76.9, 87.3, 91.4 and 112.7 Gy, were estimated to inhibit 99.9968% (probit 9) of adult emergence forA. obliqua, A. serpentina, A. ludens, and C. capitata, respectively. Using mangoes infested with a total of 100,000 larvae of each species, the results obtained in the laboratory were confirmed using a dose of 100 Gy for the Anastrepha species and 150 Gy for C. capitata. No adult emergence was observed for any of the four species compared with approximately 80% emergence in the controls. A dose of 150 Gy is recommended as a generic quarantine treatment against potential infestation of these species in exported mangoes. A minor decrease in the ascorbic acid content was the only adverse effects observed in irradiated mangoes.
Journal of Economic Entomology, 2004
The effects of irradiation doses increasing from 0 to 100 Gy (1 Gy is energy absorbed in J kg(-1)... more The effects of irradiation doses increasing from 0 to 100 Gy (1 Gy is energy absorbed in J kg(-1) of irradiated material) on fertility, flight ability, survival, and sterile male mating performance were evaluated for mass-reared Anastrepha obliqua (Macquart). High sterility values (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 98.2%) for irradiated males were obtained for doses as low as 25 Gy. Egg hatch was inhibited for irradiated males crossed with irradiated females at a low dose of 20 Gy. However, we estimated that to achieve 99.9% sterility (standard goal of many sterile insect technique programs), irradiation doses had to be increased to a dose between 50 and 75 Gy. At doses of 25 Gy and greater, we observed a decreasing trend in adult flight ability and an increasing trend in adult mortality. Such differences were greater for pupae irradiated at a young age compared those irradiated 24 h before emergence. Our single most relevant finding was that sterility induction (i.e., oviposition of nonfertilized eggs) was two times greater for males irradiated at low doses (40 Gy) than for males irradiated at high doses (80 Gy) when used at a 3:1:1 sterilized male to fertile male to fertile female ratio. Males irradiated at high doses may have been outcompeted by unirradiated males when courting unirradiated females. Implications of our findings for sterile insect technique programs are discussed.
Journal of Chemical Ecology, 2006
Nonirradiated males and females of Anastrepha obliqua (Macquart) were attracted to and landed mor... more Nonirradiated males and females of Anastrepha obliqua (Macquart) were attracted to and landed more frequently on ripe fruits of Spondias mombin L. than on artificial fruit in wind tunnel bioassays. Porapak Q volatile extracts of S. mombin were also attractive and elicited landing on artificial fruit for both sexes. Combined gas chromatographic-electroantennographic detection (GC-EAD) analysis of volatile extracts showed that nine volatile compounds elicited repeatable antennal responses from females and males. The EAD-active compounds were identified by GC-mass spectrometry (MS) as follows: ethyl butyrate, isopropyl butyrate, hexan-1-ol, propyl butyrate, isobutyl butyrate, ethyl hexanoate, isopentyl butyrate, ethyl benzoate, and ethyl octanoate. In wind tunnel bioassays, males and females were attracted and landed more frequently on lures containing the nine-component blend of synthetic compounds than on unscented controls. Field cage bioassays showed that multilure traps baited with the nine-synthetic blend captured significantly more A. obliqua than traps baited with hydrolyzed protein or water.
Journal of Biotechnology, 2006
The high degree of structural conservation of erythropoietin between species, make it, especially... more The high degree of structural conservation of erythropoietin between species, make it, especially, difficult to produce this protein growth factor in the milk of transgenic animals. Here, we show that through the direct transduction of the mammary epithelium, it is possible to produce high levels of recombinant human erythropoietin in the milk of non-transgenic goats without causing harm to the animals. The efficiency of viral transduction was improved through a temporal disruption of tight-junctions with EGTA allowing for the expression of human erythropoietin at levels of up to 2 g/L in milk. The human erythropoietin was purified from the milk using a multi-step protocol involving milk clarification, two precipitation steps and two affinity chromatographies, with a yield of about 70% and purity over 98%. However, the human erythropoietin expressed in milk was underglycosylated, which seems to be the main cause for its low in vivo hematopoietic activity. Nonetheless, these results demonstrate that through the direct transduction of the mammary epithelium it is possible to produce potentially toxic proteins in milk, at levels high enough for their purification and biological characterization.
Journal of Biotechnology, 2008
Classical swine fever virus is the etiological agent of the most economically important highly co... more Classical swine fever virus is the etiological agent of the most economically important highly contagious disease of swine worldwide. E2 is the major envelope glycoprotein present as a homodimer on the outer surface of the virus and represents an important target for the induction of neutralizing immune response against the viral infection. The E2 extracellular domain was expressed in the milk of adenoviral transduced goats at the highest level about 1.2g/L. The recombinant glycoprotein was purified from clarified serum milk by a single metal chelate affinity chromatography step, as a homodimer of approximately 100kDa and purity over 98%. Glycosylation analysis showed the presence of oligomannoside, hybrid and complex type N-glycans, attached to the recombinant E2. The capacity of goat milk-derived E2 antigen to protect pigs from both classical swine fever clinical signs and viral infection was assessed in a vaccination and challenge trial. The immunized pigs became protected after challenge with 10(5) LD(50) of a highly pathogenic CSFV strain. In the context of veterinary vaccines, this expression system has the advantages that the recombinant antigen could be harvested in about 48h after adenoviral transduction with expression levels in the range of g/L. This approach may turn into a scalable expression system for the assessment and production of veterinary vaccines.
Archives of Virology, 2007
Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throu... more Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004–2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.
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Papers by Jorge R. Toledo