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Clinical and Vaccine Immunology, June 2007, p. 685-692, Vol. 14, No. 6
1071-412X/07/$08.00+0     doi:10.1128/CVI.00028-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fruit-Specific Expression of the Human Immunodeficiency Virus Type 1 Tat Gene in Tomato Plants and Its Immunogenic Potential in Mice{triangledown}

Yuri Jorge Peña Ramírez,2,{dagger},# Ennio Tasciotti,1,# Abel Gutierrez-Ortega,2,{ddagger} Alberto J. Donayre Torres,2 María Teresa Olivera Flores,3 Mauro Giacca,1 and Miguel Ángel Gómez Lim2*

Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy,1 Departamento de Ingeniería Genética, Cinvestav, Campus Guanajuato, Irapuato, Km 9.6 Libramiento norte, Apartado Postal 629, Irapuato, Gto., México 36500,2 Facultad de Química, Conjunto E, Laboratorio 116, Paseo de la Investigación Científica, Universidad Nacional Autónoma de México, DF México, México 045103

Received 10 January 2007/ Returned for modification 7 March 2007/ Accepted 2 April 2007

The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 µg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.

* Corresponding author. Mailing address: Centro de Investigación y de Estudios Avanzados del I.P.N. Departamento de Ingeniería Genética de Plantas, Km 9.6 del Libramiento Norte de la carretera Irapuato-León, Apdo. Postal 629, 36500 Irapuato, Gto., México. Phone: (52) 462 623 9679. Fax: (52) 462 458 49. E-mail: mgomez{at}ira.cinvestav.mx

{triangledown} Published ahead of print on 25 April 2007.

{dagger} Present address: Instituto Tecnológico Superior de Acayucan, Carretera Costera del Golfo Km 216.4, Acayucan, Ver., México 96100.

# These authors contributed equally to the paper.

{ddagger} Present address: Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C. Av. Normalistas #800, Colinas de la Normal C.P. 44270, Guadalajara, Jalisco, México.

Clinical and Vaccine Immunology, June 2007, p. 685-692, Vol. 14, No. 6
1071-412X/07/$08.00+0     doi:10.1128/CVI.00028-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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