Visualization of CombiHIVvac Vaccine Particles Using Electron Microscopy

Acandidate vaccine CombiHIVvac is developed; presently the clinical phase I trial has been completed successfully. CombiHIVvac combines the conserved polyepitope immunogens approaches in a novel self-adjuvanted microparticle concept. The artificial TBI (T cell and B cell immunogen) polypeptide used in the vaccine comprises epitopes from Env and Gag. The polypeptide is conjugated to dextran and mixed with DNA, which leads to formation of microparticles presenting TBI on the surface and containing the DNA inside. The DNA (pcDNA-TCI) enclosed in the microparticles codes for the TCI (T cell immunogen) polypeptide, which contains CD8 and CD4 epitopes from Env, Gag, Pol, and Nef conserved among HIV subtypes A, B, and C. The proposed technique enables the vaccine components to combine into particles on the principle of self-assembly (Fig. 1A). The conjugate molecule consists of 1 dextran molecule, 1 protein TBI molecule, and 10–15 spermidine molecules. Positively charged spermidine provides binding of the conjugate dextran/protein TBI with negatively charged DNAvaccine promoting formation of particles on the self-assembly principle. According to our estimation, the plasmid pcDNA-TCI (6,583 bp) is able to present about 100–110 dextran (60 kDa) molecules on its surfaces. For vaccine assembly, pcDNA-TCI was added to conjugate in the proportion of 1 DNA molecule:120 conjugate molecules. To prove that CombiHIVvac has actually the form of particles, we used gel filtration chromatography and atomic force microscopy. During sepharose CL-2B gel filtration, the vaccine was eluted in the volume, corresponding to 12–14 MDa size material. Experimental visualization of a theoretically predicted formation of artificial microparticles was performed with transmission electron microscopy with negative staining (1%