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J. Biol. Chem., Vol. 278, Issue 39, 37926-37936, September 26, 2003

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HIV-1 p55Gag Encoded in the Lysosome-associated Membrane Protein-1 as a DNA Plasmid Vaccine Chimera Is Highly Expressed, Traffics to the Major Histocompatibility Class II Compartment, and Elicits Enhanced Immune Responses^*

Ernesto T. A. Marques, Jr., Priya Chikhlikar, Luciana Barros de Arruda , Ihid C. Leao , Yang Lu, Justin Wong, Juei-Suei Chen, Barry Byrne ¶ and J. Thomas August ||

From the Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4⁺ responses, a 4- to 5-fold increase in CD8⁺ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.

Received for publication, April 1, 2003 , and in revised form, May 27, 2003.

^* This work was supported in part by NIAID, National Institutes of Health Grants R37-AI41908 and R21-AI44317 (to J. T. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by Coordenação de Aperfeicoamento de Pessoal de Nível Superior (CAPES), Brazil.

Supported by World Health Organization Grant TDR 960158.

^¶ Present address: University of Florida, 1600 S. West Archer Rd., Gainesville, FL 32610.

^|| To whom correspondence should be addressed: Dept. of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 N. Wolfe St., Biophysics Rm. 309, Baltimore, MD 21205. Tel.: 410-955-8484; Fax: 410-955-1894; E-mail: taugust{at}jhmi.edu.

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