A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells

Abstract

BackgroundOne reason why subunit protein and DNA vaccines are often less immunogenic than live-attenuated and whole-inactivated virus vaccines is that they lack the co-stimulatory signals provided by various components of the more complex vaccines. The HIV-1 envelope glycoprotein complex (Env) is no exception to this rule. Other factors that limit the induction of neutralizing antibodies against HIV-1 lie in the structure and instability of Env. We have previously stabilized soluble trimeric mimics of Env by introducing a disulfide bond between gp120 and gp41 and adding a trimer stabilizing mutation in gp41 (SOSIP.R6 gp140).ResultsWe further stabilized the SOSIP.R6 gp140 using a GCN4-based isoleucine zipper motif, creating SOSIP.R6-IZ gp140. In order to target SOSIP.R6-IZ to immune cells, including dendritic cells, while at the same time activating these cells, we fused SOSIP.R6-IZ to the active domain of CD40 ligand (CD40L), which may serve as a 'cis-adjuvant'. The Env component of the SOSIP.R6-IZ-CD40L fusion construct bound to CD4 and neutralizing antibodies, while the CD40L moiety interacted with CD40. Furthermore, the chimeric molecule was able to signal efficiently through CD40 and induce maturation of human dendritic cells. Dendritic cells secreted IL-6, IL-10 and IL-12 in response to stimulation by SOSIP.R6-IZ-CD40L and were able to activate naïve T cells.ConclusionsChimeric HIV-1 gp140 - CD40L trimers can target and activate dendritic cells. Targeting and activating immune cells using CD40L and other 'cis-adjuvants' may improve subunit protein vaccine immunogenicity for HIV-1 and other infectious diseases.

Targeting HIV-1 Envelope Glycoprotein Trimers to B Cells by Using APRIL Improves Antibody Responses

“…Note that the internal D7324 epitope in the C5 domain of SOSIP constructs is mutated by the introduction of the cysteine at residue 501 that forms the intramolecular disulfide bond with gp41 and that it is also occluded by the proximity of the gp41 subunit; hence, unmodified SOSIP gp140s do not react efficiently with D7324 (6,83). Because of the addition of C-terminal sequences to SOSIP gp140, SOSIP.R6-IZ-D7324 trimers are poorly cleaved (63).…”

“…We introduced (i) a disulfide bond between residues 501 in gp120 and 605 in gp41 (A501C, T605C) (6); (ii) a trimer-stabilizing substitution in gp41 (I559P) (83); and (iii) a sequence-enhanced site for furin cleavage (RRRRRR) (7). We further modified the JR-FL SOSIP.R6 gp140 construct to include a C-terminal GCN4-based trimerization domain (isoleucine zipper [IZ]) (30,63). We have shown that this domain further improves trimer stability.…”

“…The addition of costimulatory molecules can augment the induction of immune responses to poor antigens; covalently linking the costimulatory molecule to the antigen enhances the immunostimulatory effect, because the antigen and "cis-adjuvant" preferentially need to contact the same immune cells, generally antigenpresenting cells (19,27,31,41,42,53,63,95,104,105). The addition of costimulatory molecules also provides an opportunity to skew the immune response in the appropriate and desired direction.…”

“…We have shown that DCs can be activated by fusing trimeric Env to the active domain of CD40 ligand (CD40L; TNFSF5/CD154) (63). CD40L, a tumor necrosis factor (TNF) superfamily (TNFSF) member, is an important immunostimulatory molecule mainly expressed by activated T-helper cells.…”

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

“…7, D and F). Thus, CPP or another molecule such as the CD40 ligand (61) can be oriented at the quasi-2-fold axes for targeting of the nanoparticle to antigen presenting cells such as the dendritic cells.…”