Abstract
Background:Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist.Methods:We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested.Results:Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions:Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.
Highlights
Liposomes are highly useful carriers for delivering drugs or antigens
Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes
The desired protein is produced in frame with the C-terminus of murine CD14 containing an omega domain for GPI attachment, which results in an extractable membrane association of the recombinant protein
Summary
Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. External layer modifications include immunoliposomes targeting specific cells using antibodies [4,5,6] and vaccinal liposomal particles [7]. The association of a GPI anchor in co-incubation leads to the correct orientation on the outer layer of liposomes resulting in enhanced interactions with B cells for experimental vaccines [19]. We created three different proteins in fusion with the C-terminus of murine CD14 containing an omega domain that post-translationally receives a GPI anchor. These proteins were produced in CHO cells for different applications, as suggested before by other groups [16]. We tested the capacity of our immunoliposomes loaded with anti-CD4 antibodies to bind to CD4+ cells ex vivo
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