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Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs.

Feng H, Zhang H, Deng J, Wang L, He Y, Wang S, Seyedtabaei R, Wang Q, Liu L, Galipeau J, Compans RW, Wang BZ - Sci Rep (2015)

Bottom Line: Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines.A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function.Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory Vaccine Center, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
HIV vaccines should elicit immune responses at both the mucosal portals of entry to block transmission and systemic compartments to clear disseminated viruses. Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines. A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function. Herein a membrane-anchored form of GIFT4 was constructed by fusing a glycolipid (GPI)-anchoring sequence and incorporated into Env-enriched HIV virus-like particles (VLPs) as a molecular adjuvant. Guinea pigs were immunized with the resulting HIV VLPs through an intramuscular priming-intranasal boosting immunization route. The GIFT4-containing VLPs induced higher levels of systemic antibody responses with significantly increased binding avidity and improved neutralizing breadth and potency to a panel of selected strains, as well as higher levels of IgG and IgA at several mucosal sites. Thus, the novel GPI-GIFT4-containging VLPs have the potential to be developed into a prophylactic HIV vaccine. Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

No MeSH data available.


Related in: MedlinePlus

Characterization of VLPs.a, Western blotting analysis of the protein components of VLPs. VLP samples containing 1 μg of total protein were loaded for SDS-PAGE followed by western blotting. Protein bands were probed with: a.1, anti-HIV Gag antibody; a.2, anti-gp120 polyclonal antibodies; a.3, anti-GM-CSF antibody. Lane 1, Gag only VLPs; Lane 2, sVLPs; Lane 3, GIFT4/Gag VLPs; lane 4, cVLPs; M, Molecular weight (kDs). b, FACS analysis of GIFT4 GPI anchoring into VLPs using anti-GM-CSF antibody. VLP samples (1 ml at 1 mg/ml) were used for antibody staining as described for cell surface staining in Figure 1. Blue, sVLPs; Green, cVLPs; Red, cVLPs post-PIPLC treatment. c, proliferation of guinea pig splenocytes in vitro stimulated by VLPs or GIFT4. Spleen cells isolated from guinea pig were cultured in vitro for 2 days in the presence of 1 μg/ml of various HIV VLPs or the soluble GIFT4 (50 ng/ml) and photographed under an EVOS microscope. C.1 to c.5, cells treated with PBS, soluble GIFT4, sVLPs, GIFT4/Gag VLPs, or cVLPs, respectively. Scale bar, 1000 μm.
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f2: Characterization of VLPs.a, Western blotting analysis of the protein components of VLPs. VLP samples containing 1 μg of total protein were loaded for SDS-PAGE followed by western blotting. Protein bands were probed with: a.1, anti-HIV Gag antibody; a.2, anti-gp120 polyclonal antibodies; a.3, anti-GM-CSF antibody. Lane 1, Gag only VLPs; Lane 2, sVLPs; Lane 3, GIFT4/Gag VLPs; lane 4, cVLPs; M, Molecular weight (kDs). b, FACS analysis of GIFT4 GPI anchoring into VLPs using anti-GM-CSF antibody. VLP samples (1 ml at 1 mg/ml) were used for antibody staining as described for cell surface staining in Figure 1. Blue, sVLPs; Green, cVLPs; Red, cVLPs post-PIPLC treatment. c, proliferation of guinea pig splenocytes in vitro stimulated by VLPs or GIFT4. Spleen cells isolated from guinea pig were cultured in vitro for 2 days in the presence of 1 μg/ml of various HIV VLPs or the soluble GIFT4 (50 ng/ml) and photographed under an EVOS microscope. C.1 to c.5, cells treated with PBS, soluble GIFT4, sVLPs, GIFT4/Gag VLPs, or cVLPs, respectively. Scale bar, 1000 μm.

Mentions: VLPs were produced using the rBV expression system in insect cells. The protein composition of the resulting VLPs was characterized by western blot using antibodies specific to Gag, Env or GM-CSF. As shown in Fig. 2a (a.2), the Env incorporated into standard Env/Gag VLPs (sVLPs, lane 2) or cVLPs (lane 4) was observed to have a molecular mass of about 120 kDa19. A band migrating at the expected size of GPI-GIFT4 was seen in cVLP and GIFT4/Gag VLPs (lanes 3 and 4 in a.3), demonstrating the incorporation of GPI-GIFT4 into these VLPs. The results shown in lane 4 of both a.2 and a.3 in Fig. 2a further indicate that membrane-anchored GIFT4 and Env can be co-incorporated into HIV cVLPs. To verify that the integration of GIFT4 into VLPs is through GPI anchoring on the membrane surface, FACS assay was carried out. GIFT4 was detected by the enhanced fluorescent intensity in cVLPs (green curve in Fig. 2b) but not sVLPs (blue curve in Fig. 2b) after anti-GM-CSF antibody staining. Further, the GIFT4 signal from cVLPs was completely eliminated by treatment with PIPLC, a phosphatidylinositol-specific phospholipase which releases GPI-anchored molecules from membranes22, as shown in Fig. 2b (red curve). Together, these data demonstrated that GIFT4 can be incorporated into VLPs, or co-incorporated into cVLPs, through GPI anchoring.


Incorporation of a GPI-anchored engineered cytokine as a molecular adjuvant enhances the immunogenicity of HIV VLPs.

Feng H, Zhang H, Deng J, Wang L, He Y, Wang S, Seyedtabaei R, Wang Q, Liu L, Galipeau J, Compans RW, Wang BZ - Sci Rep (2015)

Characterization of VLPs.a, Western blotting analysis of the protein components of VLPs. VLP samples containing 1 μg of total protein were loaded for SDS-PAGE followed by western blotting. Protein bands were probed with: a.1, anti-HIV Gag antibody; a.2, anti-gp120 polyclonal antibodies; a.3, anti-GM-CSF antibody. Lane 1, Gag only VLPs; Lane 2, sVLPs; Lane 3, GIFT4/Gag VLPs; lane 4, cVLPs; M, Molecular weight (kDs). b, FACS analysis of GIFT4 GPI anchoring into VLPs using anti-GM-CSF antibody. VLP samples (1 ml at 1 mg/ml) were used for antibody staining as described for cell surface staining in Figure 1. Blue, sVLPs; Green, cVLPs; Red, cVLPs post-PIPLC treatment. c, proliferation of guinea pig splenocytes in vitro stimulated by VLPs or GIFT4. Spleen cells isolated from guinea pig were cultured in vitro for 2 days in the presence of 1 μg/ml of various HIV VLPs or the soluble GIFT4 (50 ng/ml) and photographed under an EVOS microscope. C.1 to c.5, cells treated with PBS, soluble GIFT4, sVLPs, GIFT4/Gag VLPs, or cVLPs, respectively. Scale bar, 1000 μm.
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Related In: Results  -  Collection

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f2: Characterization of VLPs.a, Western blotting analysis of the protein components of VLPs. VLP samples containing 1 μg of total protein were loaded for SDS-PAGE followed by western blotting. Protein bands were probed with: a.1, anti-HIV Gag antibody; a.2, anti-gp120 polyclonal antibodies; a.3, anti-GM-CSF antibody. Lane 1, Gag only VLPs; Lane 2, sVLPs; Lane 3, GIFT4/Gag VLPs; lane 4, cVLPs; M, Molecular weight (kDs). b, FACS analysis of GIFT4 GPI anchoring into VLPs using anti-GM-CSF antibody. VLP samples (1 ml at 1 mg/ml) were used for antibody staining as described for cell surface staining in Figure 1. Blue, sVLPs; Green, cVLPs; Red, cVLPs post-PIPLC treatment. c, proliferation of guinea pig splenocytes in vitro stimulated by VLPs or GIFT4. Spleen cells isolated from guinea pig were cultured in vitro for 2 days in the presence of 1 μg/ml of various HIV VLPs or the soluble GIFT4 (50 ng/ml) and photographed under an EVOS microscope. C.1 to c.5, cells treated with PBS, soluble GIFT4, sVLPs, GIFT4/Gag VLPs, or cVLPs, respectively. Scale bar, 1000 μm.
Mentions: VLPs were produced using the rBV expression system in insect cells. The protein composition of the resulting VLPs was characterized by western blot using antibodies specific to Gag, Env or GM-CSF. As shown in Fig. 2a (a.2), the Env incorporated into standard Env/Gag VLPs (sVLPs, lane 2) or cVLPs (lane 4) was observed to have a molecular mass of about 120 kDa19. A band migrating at the expected size of GPI-GIFT4 was seen in cVLP and GIFT4/Gag VLPs (lanes 3 and 4 in a.3), demonstrating the incorporation of GPI-GIFT4 into these VLPs. The results shown in lane 4 of both a.2 and a.3 in Fig. 2a further indicate that membrane-anchored GIFT4 and Env can be co-incorporated into HIV cVLPs. To verify that the integration of GIFT4 into VLPs is through GPI anchoring on the membrane surface, FACS assay was carried out. GIFT4 was detected by the enhanced fluorescent intensity in cVLPs (green curve in Fig. 2b) but not sVLPs (blue curve in Fig. 2b) after anti-GM-CSF antibody staining. Further, the GIFT4 signal from cVLPs was completely eliminated by treatment with PIPLC, a phosphatidylinositol-specific phospholipase which releases GPI-anchored molecules from membranes22, as shown in Fig. 2b (red curve). Together, these data demonstrated that GIFT4 can be incorporated into VLPs, or co-incorporated into cVLPs, through GPI anchoring.

Bottom Line: Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines.A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function.Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory Vaccine Center, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
HIV vaccines should elicit immune responses at both the mucosal portals of entry to block transmission and systemic compartments to clear disseminated viruses. Co-delivery of mucosal adjuvants has been shown to be essential to induce effective mucosal immunity by non-replicating vaccines. A novel cytokine, GIFT4, engineered by fusing GM-CSF and interleukin-4, was previously found to simulate B cell proliferation and effector function. Herein a membrane-anchored form of GIFT4 was constructed by fusing a glycolipid (GPI)-anchoring sequence and incorporated into Env-enriched HIV virus-like particles (VLPs) as a molecular adjuvant. Guinea pigs were immunized with the resulting HIV VLPs through an intramuscular priming-intranasal boosting immunization route. The GIFT4-containing VLPs induced higher levels of systemic antibody responses with significantly increased binding avidity and improved neutralizing breadth and potency to a panel of selected strains, as well as higher levels of IgG and IgA at several mucosal sites. Thus, the novel GPI-GIFT4-containging VLPs have the potential to be developed into a prophylactic HIV vaccine. Incorporation of GPI-anchored GIFT4 into VLPs as a molecular adjuvant represents a novel approach to increase their immunogenicity.

No MeSH data available.


Related in: MedlinePlus