*2.1. Study Vaccine*

A candidate DNA-vaccine "DNA-4" has been developed at The Biomedical Center (St. Petersburg, Russia) in collaboration with the Research Institute of Ultra Pure Biologicals (St. Petersburg, Russia). The vaccine contains four plasmid DNA encoding consensus sequences of *nef*, *gag*, *rt*, or *gp140* HIV-1 FSU subtype A genes [2]. Amino acid sequences of viral proteins were modified to increase their expression level and optimize their immunological properties. Nucleotide sequences were designed to replace most wild-type codons with codons from highly expressed human genes. In reverse transcriptase (RT), N-terminal methionine and hystidines were introduced to replace catalytic aspartic acids residues 110, 185, and 186 within the active site of RT. In Nef, glycine residues 2 and 3 were deleted to remove the myristylation site. In gp140, the signal peptide was replaced with the signal sequence of human tissue plasminogen activator to increase its transport and secretion; the transmembrane and cytoplasmic regions of gp160 (amino acids 676–860) were removed to obtain a soluble form of the HIV-1 envelope glycoprotein, region 500–534 containing the cleavage site and fusion peptide domain was removed to prevent the proteolytic processing of the envelope, to stabilize the protein by linking it covalently to the gp41 extracellular domain, and to reduce toxicity; and region 589–618 containing the sequence between the heptad repeats was removed to stabilize the formation of trimers and eliminate formation of the hairpin intermediate [2].

Each gene was inserted into the vector pBMC that had been created at The Biomedical center. Inserted genes were expressed in eukaryotic cells under the control of the cytomegalovirus promoter and the bovine growth hormone polyadenylation signal [2].

DNA-4 was manufactured by the production facility of the Research Institute of Ultra Pure Biologicals (St. Petersburg, Russia) in accordance with the existing Russian federal regulations. The plasmids were equally formulated in 0.5 mL of sterile saline solution with overall plasmid concentration of 0.25 mg/mL. No adjuvants were added to the vaccine. Placebo vials contained 0.5 mL of saline solution without plasmids.
