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      Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production


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          Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (h IGH), kappa-chain (h IGK), and lambda-chain (h IGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, b IGHM and b IGHML1, were homozygously inactivated (b IGHM −/− , b IGHML1 −/− ; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the h IGK and h IGL genomic loci on the HAC and from the endogenous bovine kappa-chain (b IGK) and lambda-chain (b IGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire b IGL joining (J) and constant (C) gene cluster (b IGLJ1-IGLC1 to b IGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (b IGHM −/− , b IGHML1 −/− and b IGL −/− ; TKO) Tc cattle. We further demonstrate that b IGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

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          Experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a Taiwan hospital

          Abstract Objectives: To describe the immunological responses and clinical outcome of coronavirus (SARS) infected healthcare workers (HCW) who had been administered with convalescent plasma as a treatment. Methods: Convalescent plasma (500 mL) was obtained from each of three SARS patients and transfused into the three infected HCW. Donors were blood type O and seronegative for hepatitis B and C, HIV, syphilis and human T-cell lymphotropic virus types I and II (HTLV-I and -II). Serum antibody (IgG) titre was >640. Apharesis was performed with a CS 3000 plus cell separator followed by the forming of the convalescent phase plasma. As part of the routine check with donated plasma, the convalescent plasma was confirmed free of residual SARS-CoV by RT–PCR. Serial serum samples obtained from the recipients of the convalescent plasma were collected to undertake real-time quantitative RT–PCR for SARS-CoV for direct measurement of viral concentration. Specific immunoglobulin IgM and IgG concentrations were titrated using an antigen microarray developed in-house. Results: Viral load dropped from 495 × 103, 76 × 103 or 650 × 103 copies/mL to zero or 1 copy/mL one day after transfusion. Anti-SARS-CoV IgM and IgG also increased in a time-dependent manner following transfusion. All three patients survived. One HCW became pregnant subsequently, delivering 13 months after discharge. Positive anti-SARS-CoV IgG was detected in the newborn. Passive transfer of anti-SARS-CoV antibody from the mother was considered as a possibility. Conclusions: All infected HCW whose condition had progressed severely and who had failed to respond to the available treatment, survived after transfusion with convalescent plasma.
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            Antigen-specific human polyclonal antibodies from hyperimmunized cattle.

            Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and kappa-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human kappa-light chain (up to approximately 650 microg/ml, fully human) or with bovine kappa- or lambda-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.
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              Purification of intravenous immunoglobulin G from human plasma--aspects of yield and virus safety.

              Plasma-derived intravenous immunoglobulin (IVIG) preparations have been successfully applied for the prophylactic prevention of infectious diseases in immunodeficient patients. In addition to its replacement therapy of primary and secondary antibody deficiencies, IVIG has found increased use in autoimmune and inflammatory diseases. IVIG has become the major plasma product on the global blood product market. The world wide consumption nearly tripled between 1992 and 2003, from 19.4 to 52.6 tons. Classical manufacturing processes of IVIG, but also new strategies for purification are discussed with respect to practicability and yield. Ethanol fractionation is still the basis for most IVIG processes, although isolation and purification of immunoglobulin G (IgG) by chromatography has gained ground. The efficiency of virus inactivation methods and virus removal techniques in terms of logarithmic reduction factors are analyzed, but also the IgG losses are taken into consideration. Some of these methods also have the ability to separate prions. High pathogen safety and high yields have become the dominant goals of the plasma fractionation industry.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                6 March 2014
                : 9
                : 3
                : e90383
                [1 ]Sanford Applied Biosciences L.L.C., Sioux Falls, South Dakota, United States of America
                [2 ]Kyowa Hakko Kirin, Co., Ltd., Chiyoda-ku, Tokyo, Japan
                [3 ]Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, Utah, United States of America
                [4 ]Trans Ova Genetics, Sioux Center, Iowa, United States of America
                [5 ]Hematech, Inc., Sioux Falls, South Dakota, United States of America
                Michigan State University, United States of America
                Author notes

                Competing Interests: The authors have the following interests: The work contained in the manuscript resulted from paid employment of each of the authors and was funded by Hematech, Inc., a former company which no longer exists. The work contained in the manuscript now belongs to Sanford Applied Biosciences, a non-profit company owned by Sanford Health of Sioux Falls, South Dakota. Co-authors Hiroaki Matsushita, Hua Wu, Jin-an Jiao and Eddie J Sullivan declare their paid employment by Sanford Applied Biosciences. Co-author Poothappillai Kasinathan is employed by Trans Ova Genetics. Sanford Applied Biosciences is a paying customer of Trans Ova Genetics. Co-authors Akiko Sano and Yoshimi Kuroiwa are employed by Kyowa Hakko Kirin, Co., Ltd. The authors also have the following patent pending related to the work (“Complex Chromosome Engineering for Production of Human Antibodies in Transgenic Animals”, PCT No.: US2013053618). The authors have developed cattle for the production of antibodies, and have trademarked Tc (Transchromosomal) Bovine, a genetically engineered cattle, which would be considered a product of the company. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. There are no further patents, products in development, or marketed products to declare.

                Conceived and designed the experiments: HM AS ZW YK PK ES JJ HW. Performed the experiments: HM AS. Analyzed the data: HM AS HW YK JJ ES ZW. Contributed reagents/materials/analysis tools: ES YK. Wrote the paper: HM ZW YK ES HW.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                : 18 November 2013
                : 28 January 2014
                Page count
                Pages: 14
                This work was financed by internal funding of Hematech, Inc., which no longer exists and no longer has financial interest in the work. Hematech has transferred all of its business to Sanford Applied Biosciences, LLC. No current external funding sources for this study. Financial interest in this work is currently owned by Sanford Applied Biosciences, who did have a role in the decision to publish as well as the preparation of the manuscript. While it is considered beneficial to the interests of Sanford Applied Biosciences to publish this work, this consideration played no role in the study design, data collection and analysis and preparation of the manuscript. This is evidenced by the inclusion of now independent authors with no affiliation to Sanford Applied Biosciences.
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                Biological Systems Engineering
                Biomedical Engineering
                Genetic Engineering
                Genetically Modified Organisms
                Drug Discovery
                Organismal Cloning
                Gene expression
                DNA modification
                Animal Genetics
                Gene Function
                Immune Cells
                Antibody-Producing Cells
                B Cells
                Genetics of the Immune System
                Immune Response



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