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Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: Preliminary study

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Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: Preliminary study
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  © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim1049Biotechnol. J. 2012, 7, 1049–1053DOI 10.1002/biot.201100455www.biotechnology-journal.com 1 Introduction The mammary gland is considered to be an effi-cient bioreactor for producing recombinant proteinfor biopharmaceutical or veterinary applications[1]. Direct transduction of the mammary gland hasbeen considered a faster, simpler and more eco-nomical alternative for expressing heterologousgenes [2]. Adenoviral vectors (AdVs) have becomea powerful tool for gene delivery and production of foreign proteins. They have been used for severalapplications, such as gene therapy, the productionof recombinant proteins and vaccination [3, 4]. Asthese vectors have a favorable biosafety profile,they are the most frequently used vectors for clin-ical assays in humans (www.wiley.co.uk/genmed/clinical).The methodology for in vivo adenoviral trans-duction of the mammary gland was initially estab-lished for the production of human growth hor-mone (hGH) [5]. Since then, several valuables pro-teins have been produced in the milk of mice, rab-bits and goats [6–8]. Although regulatory approvalof these products requires an accurate safety pro-file [9, 10], only one study on the persistence of AdVs after mammary gland transduction has beenperformed [11].We have performed a preliminary biosafety evaluation based on the persistence of viable ade-noviral particles in the milk, serum, saliva, urineand feces after adenoviral transduction of goatmammary glands, and measurements of anti-ade-noviral antibodies. We also assayed adenoviral in- Rapid Communication Biosafety evaluation of recombinant protein production in goatmammary gland using adenoviral vectors: Preliminary study Elaine Santana Rodríguez 1, *, Alaín González Pose 1, *, María Pilar Rodríguez Moltó 1 , Angela Sosa Espinoza 1 ,Pastor Alfonso Zamora 2 and Marisela Suárez Pedroso 1 1 Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba 2 Animal Health and Production Branch, National Center for Animal and Plant Health (CENSA), San José de Las Lajas,Mayabeque, CubaThe production of recombinant proteins in the milk of non-transgenic goats can be achieved bytransducing the mammary gland with recombinant adenoviral vectors. However, this process in-volves several regulatory issues. The current study evaluates the biosafety of this production sys-tem. We present a preliminary biosafety profile based on detection of adenoviral particles in dif-ferent body fluids and the antibody response after adenoviral transduction of the goat mammarygland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenovi-ral particles were detected in the milk until day 4 after transduction, they were absent in serum,saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus in-activation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant pro-teins using adenoviral vectors. Keywords: Adenoviral transduction · Biosafety · Goat mammary gland · Mammalian biotechnology · Viral inactivation Correspondence: Elaine Santana Rodríguez, MSc, Department of AnimalBiotechnology, Center for Genetic Engineering and Biotechnology, P. O. Box 6162, Havana 10600, Cuba E-mail: elaine.santana@cigb.edu.cu Abbreviations: AdV , adenoviral vector; BPL , β -propiolactone; MB , methyl-ene blue; rhEPO , recombinant human erythropoietinReceived22OCT 2011Revised10FEB 2012Accepted08MAR 2012Acceptedarticle online12MAR 2012 Supporting information available online * These authors contributed equally to this work.  BiotechnologyJournal Biotechnol. J. 2012, 7, 1049–10531050© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim activation using methylene blue (MB) and β -propi-olactone (BPL). 2 Materials and methods Details of the recombinant adenovirus, the trans-duction of goat mammary glands in vivo, samplecollection and processing, AdV detection and am-plification, recombinant human erythropoietin(rhEPO) detection and immunodetection of E2 pro-tein are given in the Supporting information online. 2.1 Antibody detection in sera and milk samples Antibodies against adenovirus in sera and milk were detected by indirect ELISA. MaxiSorp plates(NUNC, NY, USA) were coated with 1 µ g protein/ well using the recombinant AdV AdCA/CD40L in0.05 M sodium carbonate buffer pH 9.6 for 3 h at37°C. Plates were washed in PBS plus 0.05% Tween20 (Merck, Hohenbrunn, Germany) and blocked with 1% BSA (Sigma) for 1 h at 37 ° C. After washing,serial dilutions of serum and milk were added. Theplates were washed and anti-goat IgG mAb conju-gated to peroxidase (ICN, NY, USA) was added. Af-ter 1 h at 37°C, plates were washed and visualizedusing 0.04 M 3,3’,5,5’-tetramethylbenzidine dihy-drochloride (Sigma). Negative milk and sera sam-ples were taken as background. Antibody titer wasconsidered positive when the highest dilution of the sample gave OD 450  values that were two timeshigher than the background. 2.2 Inactivation experiments One of our future goals is to test the biological ac-tivity of the protein of interest after the adenoviralinactivation treatment. As the biological activity of the EPO protein produced in goat’s milk was low [12], we looked at the E2 protein. This protein hasbeen produced at high levels in goat’s milk and isbiologically active. Its activity was demonstrated by using the E2 protein as a vaccine candidate againstthe classical swine fever virus [8]. Fresh milk con-taining E2 protein and AdE2 was used for the inac-tivation experiments. Each sample contained 5 mLmilk and 1 × 10 8 GTU/mL AdE2. BPL (Sigma) wasadded to samples at different concentration (0.1,0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1%). The pH was adjusted to 7–8 with 0.3 M HEPES (Sigma). Allsamples were stirred at either room temperature or4°C overnight. Samples were stored at –20 ° C untiltitration.MB (Sigma) was added at different con-centration (1.25, 3.1, 6.25, 12.5, 25, 50, 100, 200 or400 µM). Illuminations were done at room temper-ature for 1 h using a slide projector with a 360 WApollo, Orizon EYB 71 lamp as light source. Thedose rate of white-light irradiation was more thanof 6000 lux. Negative controls were goat’s milk con-taining E2 protein and AdE2 without MB or BPL. 2.3 Statistical analysis All statistical procedures were performed using thestatistical software GraphPad Prism v.4.02 (Graph-Pad, San Diego, CA, USA). rhEPO concentration,adenoviral titer in milk and adenoviral inactivationlevels with MB were compared by ANOVA andNewman-Keuls post-test. A Kruskal-Wallis testand a Dunn post-test were performed to comparethe milk volumes. The antibody titers against theAdV AdrhEPO and adenoviral inactivation levels with BPL were compared at each time point usingthe unpaired t -test with Welch’s correction. 3 Results and discussion 3.1 In vivo production of the rhEPO The production level of rhEPO in the milk of goatstransduced with AdrhEPO was 1.8 g/L on day 2 af-ter transduction (Supporting information, Fig. S1A).The rate of production dropped with time; by day 9the rhEPO concentration was 0.032 g/L, and norhEPO was detected on days 10 and 11. The aver-age rhEPO production was 0.6 g/L. During the first3 days, the rhEPO concentration decrease rapidly in a significant way (  p <0.001). There were no sig-nificant differences in rhEPO concentration ondays 4 and 5, but the drop at day 6 was significantcompared with the previous days (compared todays 2 and 3  p <0.001, to days 4 and 5  p <0.01). A rapid decrease of rhEPO concentration was ob-served on days 7–9, which was significantly lowerthan preceding measurements (  p <0.001). Such adrop in production has also been observed previ-ously [13]. The volumes of milk produced 10 daysafter lactation induction and transduction rangedfrom 290 to 425 mL per udder half per day (Sup-porting information, Fig. S1B). The lowest volume was observed at day 5 (  p <0.01 compared with days2, 7 and 11,  p <0.05 compared with days 8 and 10).There were no signs of lesions in the goat’s udderafter adenoviral inoculation and no protein was de-tected in the negative control. 3.2 Presence of viral particles in secretion samples Infective adenoviral particles were detected in milkup to day 4 after transduction; particles were de-  © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim1051 tected through GFP production in a HEK-293 cellline (Fig. 1A). Titers declined significantly frommore than 4log 10 on day 2 to around 0.3log 10 onday 4 (  p <0.001). No virus was detected in the milkfrom day 5 to day 21 after transduction. No aden-oviral particles were detected in samples of serum,saliva, urine and feces despite the titration afterthree adenoviral amplification rounds. The fact thatthe infection of the goats’ udders with the recombi-nant AdV is confined to the mammary glands pro-tects the animal health by avoiding adenoviralspread through other tissues. 3.3 Detection of anti-adenovirus antibodies in sera and milk Using an indirect ELISA, samples of serum andmilk were shown to contain anti-adenovirus anti-bodies (Fig. 1B). The humoral response againstadenovirus was substantial in all animals. Antibody titers began to rise by day 4 in both fluids, which co-incided with the decrease of viral particles in milk.These titers reached their maximum on day 8 inmilk and day 12 in serum; these levels were main-tained until the last day tested. Titers in milk weresignificantly higher than in serum from day 6 to thelast day tested (comparison between milk andserum,  p <0.001 on days 6, 8, 10, 12, 14, 18 and 20,  p <0.01 on day 16). OD values from negative con-trols indicated the absence of antibodies. Similarresults were described by Fan et al. [11]. They ob-served low expression levels of the recombinantprotein lysostaphin due to the potent immune re-sponse raised against AdVs and the protein itself.These results are important from a biosafety pointof view, because they could indicate that recombi-nant adenoviral particles are not released into theenvironment. 3.4 Viral inactivation Milk containing adenoviral particles carrying theE2 nucleotide sequence was treated with severalconcentrations of MB. The result indicated a de-crease in viral activity as MB concentrations in-creased (Fig. 2A). The viral charge in milk was sig-nificantly diminished at all MB concentrations ap-plied compared to the negative control. An MB con-centration of 1.25 µM was able to significantly reduce the initial viral charge from 7.7log 10 to6.6log 10 (  p <0.001). Although this effect was not ob-served using 3.1 µM MB, 6.25 and 12.5 µM MB did Biotechnol. J. 2012, 7, 1049–1053www.biotechnology-journal.com Figure 1. Immune response induced by AdV transduc-tion. ( A ) Adenoviral particle detected in the milk of goats transduced with AdrhEPO during active lacta-tion. Titration was performed in HEK-293 cells by ob-servation of green fluorescent protein expression un-der a fluorescence microscope. Arithmetic means arethe result of one experiment with six replicates perday. Antibody titers are expressed as the log 10 value of the reciprocal of the highest dilution of milk. SD val-ues are shown. Titers were compared by ANOVA us-ing the Newman-Keuls post-hoc test. ( B ) Detection of anti-adenovirus antibodies in serum and milk of goatstransduced with AdrhEPO. Indirect ELISA was per-formed by coating with 1 µg protein/well of the recom-binant AdV AdCA/CD40L. Anti-adenovirus antibodieswere detected with an anti-goat IgG mAb conjugatedto peroxidase. Arithmetic means are the result of threeexperiments with three replicates per goat. SD valuesare shown. Statistics were performed by comparingthe titers of milk and serum for each time point. Datawere compared using the unpaired t -test with Welch’scorrection. NC: negative control. ** p <0.01;*** p <0.001.  BiotechnologyJournal Biotechnol. J. 2012, 7, 1049–10531052© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim cause a significant drop in the viral charge to5.6log 10 and 4.6log 10 , respectively (  p < 0.001). In-creasing the MB concentration to 25 µM did notproduce in any further significant drop in viralcharge. However, unexpectedly, increasing the MBconcentration from 25 to 50 µM resulted in anabrupt drop in adenoviral titer. Thus, a concentra-tion of 50 µM was able to inactivate the adenoviralparticles; this was also seen with higher concentra-tions of MB. This abrupt drop in the adenoviral titerobserved between treatment with 25 and 50 µM MBindicates that concentrations between these valuescould also totally inactivate AdE2. Here total aden-oviral inactivation using MB required a concentra-tion 18-fold higher and a longer time than thatfound by Schagen et al. [14]. The fact that aden-oviruses are non-enveloped viruses and the ex-treme complexity of milk composition may pro-foundly influence the results [15, 16].Virus particles in the milk could also be inacti- vated using BPL at different temperatures and con-centrations (Fig. 3A). No adenoviral proliferation was detected in HEK-293 cells after treatment with0.2% or higher concentrations of BPL at 4 ° C. At thistemperature, 0.1% and 0.18% BPL also produced asignificant reduction in the viral charge. However,at room temperature, 0.3% BPL was needed to elim-inate adenoviral proliferation. Thus, higher con- Figure 2. Methylene blue (MB) adenoviral inactivation in milkusing the AdV AdE2 and E2 protein. ( A ) Adenoviral titers aftertreatment with different concentrations of MB. ( B ) Western blotof E2 protein after treatment with 200 µM MB. Lane 1, negativecontrol; lane 2, milk sample treated with MB; lane 3, positivecontrol; lane 4, molecular weight marker (prestained standardBio-Rad). Arithmetic means of adenoviral titers are the result of six experiments and are expressed as the log 10 value of thereciprocal of the highest dilution of milk. SD values are shown.Titers were compared by ANOVA using the Newman-Keulspost-hoc test. *** p <0.001. Figure 3.  β -propiolactone (BPL) adenoviral inactivation inmilk using the AdE2 and E2 protein. ( A ) Adenoviral titersafter treatment with different concentrations of BPL. ( B ) Western blot of E2 protein after treatment with 0.5%BPL at 4°C. Lane 1, negative control; lane 2, milk sampletreated with BPL; lane 3, positive control; lane 4, molecularweight marker (prestained standard Bio-Rad). ( C ) Westernblot of E2 protein after treatment with 0.5% BPL at roomtemperature (RT). Lane 1, negative control; lane 2, milksample treated with BPL; lane 3, positive control; lane 4,molecular weight marker (prestained standard Bio-Rad).Arithmetic means of adenoviral titers are the result of sixexperiments and are expressed as the log 10 value of the reciprocal of the highest dilution of milk. SD values areshown. Statistics were performed by comparing the titersat different temperatures for each time point. Adenoviraltiters were compared by the unpaired t -test with Welch’scorrection. ** p <0.01.  © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim1053 [4]Modric, T., Mergia, A., The use of viral vectors in introducinggenes into agricultural animal species. Anim. Biotechnol.2009,  20 , 216–230.[5]Sánchez, O., Toledo, J. R., Rodríguez, M. P., Castro, F. O., Ade-noviral vector mediates high expression levels of humangrowth hormone in the milk of mice and goats.  J. Biotechnol. 2004, 114 , 89–97.[6]Toledo, J. R., Sánchez, O., Montesino, R., Fernandez, Y. et al.,New procedure for production of biopharmaceutical pro-teins in the milk of non-transgenic animals.  Biotecnología Aplicada 2005,  22 , 62–67.[7]Han, Z. S., Li, Q. W., Zhang, Z. Y., Yu, Y. S. et al., Adenoviral vector mediates high expression levels of human lactofer-rin in the milk of rabbits.  J. Microbiol. Biotechnol. 2008, 18 ,153–159.[8]Toledo, J. R., Sánchez, O., Montesino, R., Farnos, O. et al.,Highly protective E2-CSFV vaccine candidate produced inthe mammary gland of adenoviral transduced goats.  J. Biotechnol. 2008, 133 , 370–376.[9]Fallaux, F. J., van der Eb, A. J., Hoeben, R. C., Who’s afraid of replication-competent adenoviruses? Gene Ther. 1999  ,6 ,709–712.[10]Dingermann, T., Recombinant therapeutic proteins: Pro-duction platforms and challenges.  Biotechnol. J. 2008,  3 ,90–97.[11]Fan, W., Plaut, K., Bramley, A. J., Barlow, J. W. et al., Persis-tency of adenoviral-mediated lysostaphin expression ingoat mammary glands.  J. Dairy Sci. 2004  , 87  , 602–608.[12]Toledo, J. R., Sánchez, O., Montesino, R., García, G. et al., Highexpression level of recombinant human erythropoietin inthe milk of non-transgenic goats.  J. Biotechnol. 2006, 123 ,225–235.[13]Yang, Y., Nunes, A., Berencsi, K., Furth, E. et al., Adenoviralmediated gene transfer into primary human and mousemammary epithelial cells in vitro and in vivo. Cancer Lett. 1995, 98 , 9–17.[14]Schagen, F. H. E., Moor, A. C. E., Cheong, S. C., Cramer, S. J. etal., Photodynamic treatment of adenoviral vectors with vis-ible light: An easy and convenient method for viral inacti- vation. Gene Ther. 1999, 6 , 873–881.[15]Washburn, K. E, Streeter, R. N, Saliki, J. T, Lehenbauer, T. W.,The use of phenothiazine dyes to inactivate bovine viral di-arrhea virus in goat colostrum. Can. J. Vet. Res. 2004, 68 ,105–111.[16]Wainwright, M., Photoinactivations of viruses.  Photochem. Photobiol. Sci. 2004,  3 , 406–411.[17]LoGrippo, G. A., Investigations of the use of beta-propiolac-tone in virus inactivation.  Ann. N. Y. Acad. Sci. 1960, 83 ,578–594.[18]Anderson, R. D., Haskell, R. E., Xia H., Roessler, B. J., David-son, B. L., A simple method for the rapid generation of re-combinant adenovirus vectors. Gene Ther. 2000, 7  , 1034–1038.[19]Morral, N., O’Neal, W., Rice, K., Leland, M. et al., Adminis-tration of helper-dependent adenoviral vectors and se-quential delivery of different vector serotype for long-termliver-directed gene transfer in baboons.  Proc. Natl. Acad. Sci.USA 1999, 96 , 12816–12821.[20]Kim, I. H., Jozkowicz, A., Piedra, P. A., Oka, K., Chan, L., Life-time correction of genetic deficiency in mice with a singleinjection of helper-dependent adenoviral vector.  Proc. Natl. Acad. Sci. USA 2001, 98, 13282–13287. Biotechnol. J. 2012, 7, 1049–1053www.biotechnology-journal.com centrations of BPLwere required for viral inactiva-tion at room temperature than at 4°C. This is con-sistent with the increased stability of BPL at 4°C[17].Using western blot analysis, the immunologicalidentity of E2 protein appeared not to be affectedby either of these adenoviral inactivation methods(Figs. 2B, 3B and 3C). A band of approximately 110 kDa was detected in milk samples treated withMB or BPL, corresponding to the molecular massobserved for the E2 protein purified from the milk, which was used as positive control. No signs of degradation of the E2 protein treated with the in-activating agents was observed in the western blotanalysis. 4 Concluding remarks This study demonstrates that recombinant AdVscan be used to produce high levels of recombinantprotein in the milk of non-transgenic goats with ahigh biosafety profile. Further studies have to beperformed to gain a complete environmental as-sessment of the system. It may be possible to in-crease the efficiency and biosafety using a differ-ent complementing cell line or AdV [18–20]. TheGTU titration method and the amplification pro -cess used here exhibited a very high detection pro-file under our test conditions, and could representexcellent methods for detecting small quantities of adenoviral particles in goat fluids; this would helpin avoiding the escape of virus to the environment.However, other methods with similar or higher detection levels are needed to corroborate these results. Although MB and BPL were shown to besuccessful for viral inactivation, the safety and bio-logical activity of final product still have to bedemonstrated. We thank Dr. Jannel Acosta Alba for the reviewing of manuscript.The authors declare no conflict of interest. 5 References [1]Castro, F. O., Toledo, J. R., Sánchez, O., Rodríguez, L., All roadslead to milk: Transgenic and non-transgenic approaches forexpression of recombinant proteins in the mammary gland.  Acta Sci. Vet. 2010,  38 , s615–s626.[2]Niemann, H., Kues, W. A., Transgenic farm animals: An up-date.  Reprod. Fert .  Dev. 2007, 19 , 762–770.[3]Ferreira, T. B., Alves, P. M., Aunins, J. G., Carrondo, M. J. T., Useof adenoviral vectors as veterinary vaccines. Gene Ther. 2005, 12 , S73–S83.
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