*2.2. Phase II Clinical Trial Design*

Phase II clinical trial was a multicenter, double-blind, placebo-controlled study. It was conducted to assess the safety of two "DNA-4" doses (0.5 mg and 0.25 mg) in patients with HIV-1 receiving ART by the analysis of frequency and severity of adverse events.

The study was conducted in 7 Centers for the Prevention and Control of AIDS and Infectious Diseases situated in different Russian cities: Moscow region, Kazan, Tolyatti, Volgograd, Lipetsk, Kaluga, Izhevsk.

During screening (visit 1) the following data were obtained: medical history, assessment of weight and height, electrocardiography, chest X-ray (both direct and lateral projection), laboratory tests of blood and urine were performed, viral load, levels of CD4 and CD8 T cells. For women pregnancy tests were performed. Patients eligible for inclusion were included in the study. The inclusion and exclusion criteria used in the study are listed in Appendix A. All trial participants were randomized into three equal groups and vaccinated four times with corresponding dose (0.5 mg or 0.25 mg or placebo) on days 1, 7, 11, and 15 with a 22-week follow-up period. Vaccine doses were selected based on the results of the phase I clinical trials of DNA-4 vaccine [3]. The highest dose of 1.0 mg/mL was excluded from this study since it did not show the enhancement of the immunogenicity.

Randomization was performed centrally by an unblinded study monitor according to the randomization list and stratum. At screening, each subject was allocated an individual registration. Investigator completed the Inclusion form including following information: screening date, site number, subject number, subject initials, date of birth, and basic ART. At Randomization visit the eligible patients were randomized to one of three treatment groups with the ratio 1:1:1. Trial participants were stratified by basic ART. Investigator indicated basic ART for each subject during randomization: 2NRTI + NNRTI or 2NRTI + PI. Patients with different basic ART were allocated equally to one of three treatment groups.

A dose of the studied vaccine was blinded by using two types of packages for each patient (box A and box B). Each package contained 4 ampoules with the DNA-4 vaccine with a dosage of 0.25 mg or with placebo.

Patients from 0.25 mg DNA-4 group were immunized with one ampoule from box A with DNA-4 vaccine of 0.25 mg intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with placebo intramuscularly to the deltoid muscle of the left shoulder.

Patients from 0.5 mg DNA-4 group were immunized with one ampoule from box A with DNA-4 vaccine of 0.25 mg intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with DNA-4 vaccine of 0.25 mg intramuscularly to the deltoid muscle of the left shoulder.

Patients from the placebo group were immunized with one ampoule from box A with placebo intramuscularly strictly to the deltoid muscle of the right shoulder and one ampoule from box B with placebo intramuscularly to the deltoid muscle of the left shoulder.

The candidate vaccine was administered intramuscularly in 1 mL of sterile saline solution in the deltoid muscle of each shoulder. Figure 1 shows the clinical trial design.

**Figure 1.** Trial scheme. Arrows show days of immunization.

Safety and tolerability were evaluated by the frequency and severity of adverse events (AE) according to subjective complaints from the patient's diary, vital signs, physical examination, laboratory tests and development of local reactions. The severity of AE was assigned in accordance with the DAIDS scale, Version 1.0, December 2004. Each adverse event was graded using a 4-grade scale: 1—mild, 2—moderate, 3—severe, 4—potentially life threatening.

Association of AE with vaccine administration was determined as associated, possibly associated, unlikely associated, or not associated. AE associated with the vaccine injection should meet the following criteria: occurs in a short time after injection, accompanies a known response to the use of the vaccine, terminates after cessation of the vaccination, re-occurs after the resumption of the vaccination.

The viral load was assessed at screening and at visits 2 and 6–11 by real-time PCR analysis. "AmpliSense HIV-Monitor-M-FL" kit (Russia) were used to detect transient viral increases above 50 copies/mL (the sensitivity of the kit was 20 copies/mL). The magnitude of viral blips, the number of viral increases as well as the number of patients with viral increases were compared between vaccinated groups and placebo group.

The quantity and ratio of CD4 and CD8 T cells were measured by flow cytometry analysis.

The viral load and CD4 and CD8 T cell levels at visit 2 were the baselines for assessing the dynamics of the viral load.
