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

Melchers M, Matthews K, de Vries RP, Eggink D, van Montfort T, Bontjer I, van de Sandt C, David K, Berkhout B, Moore JP, Sanders RW - Retrovirology (2011)

Bottom Line: 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.Chimeric 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam, Netherlands.

ABSTRACT

Background: One 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).

Results: We 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.

Conclusions: Chimeric 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.

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Improved trimerization of JRFL-SOSIP.R6 gp140 by addition of an heterologous trimerization domain. A. Schematic of the SOSIP.R6.IZ design. The clade B JR-FL gp140 (amino acids 31-681) contains several modifications that have been previously described (see Materials and Methods). Trimer formation was further enhanced by insertion of a GCN4-based isoleucine zipper (IZ) to the C-terminus of SOSIP.R6. B. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6 and SOSIP.R6-IZ proteins secreted from transiently transfected 293T cells. Note that no exogenous furin was added in these experiments, therefore the proteins are predominantly (> 90%) uncleaved. C. Gel filtration analysis of SOSIP.R6 and SOSIP.R6-IZ proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6 or SOSIP.R6-IZ proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.
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Figure 2: Improved trimerization of JRFL-SOSIP.R6 gp140 by addition of an heterologous trimerization domain. A. Schematic of the SOSIP.R6.IZ design. The clade B JR-FL gp140 (amino acids 31-681) contains several modifications that have been previously described (see Materials and Methods). Trimer formation was further enhanced by insertion of a GCN4-based isoleucine zipper (IZ) to the C-terminus of SOSIP.R6. B. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6 and SOSIP.R6-IZ proteins secreted from transiently transfected 293T cells. Note that no exogenous furin was added in these experiments, therefore the proteins are predominantly (> 90%) uncleaved. C. Gel filtration analysis of SOSIP.R6 and SOSIP.R6-IZ proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6 or SOSIP.R6-IZ proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.

Mentions: We have previously described modifications that improve the stability of soluble, cleaved gp140 trimers, based on the R5 subtype B isolate JR-FL [11]. The amino-acid sequence of gp120 and the gp41 ectodomain was modified as follows (Figure 2A). We introduced: (i) a disulfide bond between residues 501 in gp120 and 605 in gp41 (A501C, T605C; [11]); (ii) a trimer-stabilizing substitution in gp41 (I559P; [12]); (iii) a sequence-enhanced site for furin cleavage (RRRRRR; [45]). Despite these modifications, the resulting JR-FL SOSIP.R6 gp140 protein (hereafter called SOSIP.R6) is expressed as heterogeneous oligomers, with monomers, dimers and tetramers present as well as the desired trimers (Figure 2B).


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

Melchers M, Matthews K, de Vries RP, Eggink D, van Montfort T, Bontjer I, van de Sandt C, David K, Berkhout B, Moore JP, Sanders RW - Retrovirology (2011)

Improved trimerization of JRFL-SOSIP.R6 gp140 by addition of an heterologous trimerization domain. A. Schematic of the SOSIP.R6.IZ design. The clade B JR-FL gp140 (amino acids 31-681) contains several modifications that have been previously described (see Materials and Methods). Trimer formation was further enhanced by insertion of a GCN4-based isoleucine zipper (IZ) to the C-terminus of SOSIP.R6. B. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6 and SOSIP.R6-IZ proteins secreted from transiently transfected 293T cells. Note that no exogenous furin was added in these experiments, therefore the proteins are predominantly (> 90%) uncleaved. C. Gel filtration analysis of SOSIP.R6 and SOSIP.R6-IZ proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6 or SOSIP.R6-IZ proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141652&req=5

Figure 2: Improved trimerization of JRFL-SOSIP.R6 gp140 by addition of an heterologous trimerization domain. A. Schematic of the SOSIP.R6.IZ design. The clade B JR-FL gp140 (amino acids 31-681) contains several modifications that have been previously described (see Materials and Methods). Trimer formation was further enhanced by insertion of a GCN4-based isoleucine zipper (IZ) to the C-terminus of SOSIP.R6. B. Reducing SDS-PAGE and Blue Native-PAGE analysis of SOSIP.R6 and SOSIP.R6-IZ proteins secreted from transiently transfected 293T cells. Note that no exogenous furin was added in these experiments, therefore the proteins are predominantly (> 90%) uncleaved. C. Gel filtration analysis of SOSIP.R6 and SOSIP.R6-IZ proteins. Concentrated culture supernatants, derived from transiently transfected 293T cells, containing the SOSIP.R6 or SOSIP.R6-IZ proteins were fractionated on a Superose-6 column, followed by analysis by SDS-PAGE and western blot. The elution of standard proteins is indicated.
Mentions: We have previously described modifications that improve the stability of soluble, cleaved gp140 trimers, based on the R5 subtype B isolate JR-FL [11]. The amino-acid sequence of gp120 and the gp41 ectodomain was modified as follows (Figure 2A). We introduced: (i) a disulfide bond between residues 501 in gp120 and 605 in gp41 (A501C, T605C; [11]); (ii) a trimer-stabilizing substitution in gp41 (I559P; [12]); (iii) a sequence-enhanced site for furin cleavage (RRRRRR; [45]). Despite these modifications, the resulting JR-FL SOSIP.R6 gp140 protein (hereafter called SOSIP.R6) is expressed as heterogeneous oligomers, with monomers, dimers and tetramers present as well as the desired trimers (Figure 2B).

Bottom Line: 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.Chimeric 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam, Netherlands.

ABSTRACT

Background: One 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).

Results: We 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.

Conclusions: Chimeric 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.

Show MeSH
Related in: MedlinePlus