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

Abstract

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.

CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in vitro expansion of viral specific CD8+ T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

“…In our current study, we found no discernible signs of adverse effects of CD40L-expressing canarypox vector when used in mice. In contrast to the work by Stone et al [31] who demonstrated that multimeric secreted CD40L was more potent than membrane expressed CD40L in a DNA vaccine, we found no differences in adjuvant activity when comparing both forms in the context of canarypox virus vectors. These differences highlight the complexity of transferring similar immune enhancement strategies between different vaccine vectors.…”

“…Surprisingly, our vaccination strategy had no effect on humoral immunity indicating that expressing the TNFSF molecule CD40L in the context of a canarypox virus based vector does not affect B cell responses. This is consistent with a previous report showing a lack of effect of CD40L DNA vector in humoral immunity [31].…”

“…Recent advances in immunology have shown that several types of molecules may be used as novel adjuvants to enhance immunogenicity of vaccines, such as cytokines, chemokines, Toll-like receptor ligands, and some costimulatory molecules [44][45][46][47]. Among these molecules, CD40L is attractive since it is one of the main mechanisms by which CD4 + T cells provide help to CTL and B cells [16,18,19,31]. The CD4 + T-cell-replacement activity of CD40L in our DC co-culture experiments is especially intriguing in the setting of development of a therapeutic vaccine for HIV-1.…”

“…A previous study reported that soluble multimeric form of CD40L, SP-D-CD40L, was superior to membrane CD40L in augmenting immunogenicity elicited by HIV-1 DNA vaccine [31]. We thus constructed ALVAC vectors expressing both membrane CD40L (vCPmCD40L) and soluble multimeric CD40L (vCPmSP-D-CD40L).…”

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

“…Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity. [6][7][8] The structural hallmark of the ligands of the TNF family is the carboxy-terminal 'TNF homology domain', which is part of both the transmembrane and soluble forms of TNF ligands. [1][2] The TNF homology domains (THDs) of the various TNF ligands are composed of a framework of aromatic and hydrophobic residues that adopt an almost identical tertiary fold and cause self association into trimers.…”