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Multimeric Soluble CD40 Ligand and GITR Ligand as Adjuvants for Human Immunodeficiency Virus DNA Vaccines

Geoffrey W. Stone,¹ Suzanne Barzee,^1, Victoria Snarsky,^1, Kristin Kee,^1, Celsa A. Spina,^2,3 Xiao-Fang Yu,⁴ and Richard S. Kornbluth^1*

Department of Medicine,¹ Department of Pathology, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0679,² VA San Diego Healthcare System—151, 3350 La Jolla Village Drive, San Diego, California 92161,³ The John Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Suite E513, Baltimore, Maryland 21205⁴

Received 20 September 2005/ Accepted 26 November 2005

For use in humans, human immunodeficiency virus (HIV) DNA vaccines may need to include immunostimulatory adjuvant molecules. CD40 ligand (CD40L), a member of the tumor necrosis factor (TNF) superfamily (TNFSF), is one candidate adjuvant, but it has been difficult to use because it is normally expressed as a trimeric membrane molecule. Soluble trimeric forms of CD40L have been produced, but in vitro data indicate that multimeric, many-trimer forms of soluble CD40L are more active. This multimerization requirement was evaluated in mice using plasmids that encoded either 1-trimer, 2-trimer, or 4-trimer soluble forms of CD40L. Fusion with the body of Acrp30 was used to produce the 2-trimer form, and fusion with the body of surfactant protein D was used to produce the 4-trimer form. Using plasmids for secreted HIV-1 antigens Gag and Env, soluble CD40L was active as an adjuvant in direct proportion to the valence of the trimers (1 < 2 < 4). These CD40L-augmented DNA vaccines elicited strong CD8⁺ T-cell responses but did not elicit significant CD4⁺ T-cell or antibody responses. To test the applicability of the multimeric fusion protein approach to other TNFSFs, a 4-trimer construct for the ligand of glucocorticoid-induced TNF family-related receptor (GITR) was also prepared. Multimeric soluble GITR ligand (GITRL) augmented the CD8⁺ T-cell, CD4⁺ T-cell, and antibody responses to DNA vaccination. In summary, multimeric CD40L and GITRL are new adjuvants for DNA vaccines. Plasmids for expressing multimeric TNFSF fusion proteins permit the rapid testing of TNFSF molecules in vivo.

S.B. and V.S. contributed equally to this paper.

Present address: Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892.

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