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J Virol, May 1998, p. 3762-3772, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Transgenic Mice Secreting Coronavirus Neutralizing Antibodies into the Milk

Isabel Sola,1 Joaquín Castilla,1 Belén Pintado,2 José M. Sánchez-Morgado,1 C. Bruce A. Whitelaw,3 A. John Clark,3 and Luis Enjuanes1,*

Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid,1 and Departamento de Reproducción Animal, Instituto Nacional de Investigaciones Agrarias (INIA), 28040 Madrid,2 Spain, and Division of Molecular Biology, Roslin Institute, Midlothian EH25 9PS, United Kingdom3

Received 27 October 1997/Accepted 20 January 1998

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 104 by radioimmunoassay (RIA) and neutralized virus infectivity up to 104-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 106 as determined by RIA, neutralized virus infectivity by 106-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta -lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta -lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta -lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Department of Molecular and Cell Biology, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone and Fax: 341-585-4555. E-mail: L.Enjuanes{at}cnb.uam.es.

J Virol, May 1998, p. 3762-3772, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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