*3.3. CD4 and CD8 T Cells Measurement*

The number of CD4 and CD8 T cells were measured by flow cytometry at visits 2, 6, 7, 9, and 11. At visit 2 blood donation was performed before the first vaccination. Results at visit 2 present the data on the CD4 and CD8 level at the time of study entry. The results are shown in Tables 4 and 5.

There was a weak trend to increase in the absolute number of CD4 T cells in the group receiving 0.25 mg of the studied vaccine, but the differences with the placebo group were not statistically significant.


**Table 4.** Data on CD4 T cells number in trial participants at different visits (cells <sup>×</sup> <sup>10</sup>9/L).

**Table 5.** Data on CD8 T cells number in trial participants at different visits (cells <sup>×</sup> <sup>10</sup>9/L).


#### **4. Discussion**

Different therapeutic vaccine strategies including tools based on DNA, viral vectors such as modified vaccinia Ankara (MVA) and vesicular stomatitis virus (VSV), RNA, peptide, or protein, Lentiviral vector and dendritic cell have been used in numerous clinical trials. Despite the major advances in our immunological understanding of HIV-1 specific T cell responses and HIV-1 reservoir, we have not been able to achieve a cure and none of these vaccines have proven to be effective [5]. Combination strategies are now being considered as the most promising approach for therapeutic HIV vaccine development. Interleukins, immune checkpoint inhibitors and Treg modulation were suggested as candidates for effective vaccine, but failed to yield any significant clinical benefit [5]. At the CROI 2017 conference data on clinical trial BCN02 were presented. It was the combined use of therapeutic vaccination with a vaccine based on the MVA vector (MVA.HIVconsv vaccine) and Romidepsin, specific drugs that can reactivate latent virus from the reservoir (Kick and kill strategies) followed by ART treatment interruption. At the time of the report 11 patients had interrupted treatment, 7 of them had to resume ART within the first 4 weeks while 4 participants (36%) remained off ART after 7, 12, 14,

and 22 weeks, respectively. The authors suggested that therapeutic vaccination targeting conserved regions of HIV-1 combined with HIV latency reactivation strategies may facilitate clearance of the viral reservoir in early-treated individuals [6]. The DNA vaccine, since it induces HIV specific cytotoxic T cells, in case of latent viral reservoirs destruction may be an ideal strategy for HIV eradication.

In our previous studies we have developed a candidate DNA vaccine against HIV-1 consisting of four plasmids encoding four HIV-1 subtype A genes: *gag*, *env*, *rt*, and *nef* [2]. The preclinical studies and phase I clinical trial of the vaccine were conducted [2,3]. The phase I trial was conducted to access safety, tolerability and immunogenicity of the DNA-4 HIV vaccine in healthy HIV-1-negative adult volunteers. We found that our DNA vaccine was safe and well-tolerated at three used doses (0.25 mg, 0.5 mg, and 1.0 mg). Altogether, T-cell immune responses were elicited in all participants. We observed the increase in lymphocyte proliferation after fourth immunization that can show the advantage of fourfold against triple immunization. The frequency of detection positive cytokine responses decreases with increasing the vaccine dose. The humoral responses were induced in 5 people (24%). We did not observe any correlation between the antibody production and the DNA-4 vaccine doses. We also found the important correlation with our results obtained for the HIV specific immune responses in exposed seronegative individuals, i.e., TNFa production in immunized group [3].

This study was conducted as a multicenter, double-blind, placebo-controlled study of safety and dose selection of a candidate HIV vaccine for HIV-infected people receiving ART. It can be concluded that the DNA-4 candidate vaccine at doses of 0.25 mg and 0.5 mg was safe and well-tolerated by HIV-infected individuals receiving ART. In vaccinated groups, three spontaneous increases of viral load with largest amplitude were detected.

The proportion of trial participants who demonstrated adverse events associated or possibly associated with the vaccine administration was 7.4% higher in the vaccinated group than in the placebo group. The frequency of local reactions in group immunized with 0.25 mg of the vaccine and the placebo group was similar, and in the group immunized with 0.5 mg no local reactions were revealed. This is in contrast with other AE, which were highest in the group receiving 0.25 mg of the vaccine.

Immunogenicity of the DNA-4 vaccine was performed in Phase I clinical trial using IFNγ-ELISpot, intracellular cytokine staining (ICS) of IFNγ, TNFα and IL-2, lymphocyte proliferation assay (LPA) and ELISA [3]. For specific T cell stimulation, a panel of 451 overlapping peptides spanning HIV-1 subtype A-Eastern European (EE) Env, Gag, RT, and Nef proteins was used. Peptides were synthesized at the Research Institute of Ultra Pure Biologicals (St. Petersburg, Russia). HIV-specific cellular immune responses were detected in 21/21 (100%) trial participants: 9 patients were IFNγ-ELISpot reactive, 18 patients expressed cytokines to specific antigen stimulation, and 12 patients had positive lymphocyte proliferation. Using ICS we detected the increased TNFα expression by CD4 T cells in response to the specific peptide stimulation in 3/21 trial participants [3]. The humoral response was induced in 5 people (24%). The titer of HIV-specific antibodies did not exceed 1/100.

For complete eradication of the HIV infection the destruction of latent viral reservoirs is necessary, and this cannot be achieved by modern ART. The only example of HIV cure is the so-called "Berlin patient" who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) from a donor carrying homozygous mutation in the HIV coreceptor CCR5 [7,8]. Recently information about HIV-1 remission maintained over a further 18 months after a similar procedure has been published [9]. However, this procedure is very expensive, high-risk and cannot be widely used.

One of the approaches used for eliminating viral reservoirs is reactivation of latent proviral genomes during ART treatment by histone deacetylase inhibitors and some cytokines [10,11].

Another way is enhancement of cellular immunity in HIV-infected individuals using therapeutic vaccines capable of inducing functional CD8<sup>+</sup> T cells specific for HIV-1 epitopes [12–16]. The next generation of therapeutic vaccines will also be combined with reservoir activating agents [17]. DNA vaccines, in case of provirus activation, may be an ideal drug for viral reservoirs eradication.

Proviral genome reactivation may be caused by TNFα expression. TNFα activates transcription factor NFkB and HIV transcription [12,18]. DNA-4 vaccination induced increased TNFα expression in some individuals, as shown in a phase I clinical trial by intracellular cytokine staining followed by flow cytometry [3]. The expression of TNFα was also demonstrated by us in a cohort of exposed, seronegative individuals [4]. That is why we hypothesized that therapeutic "DNA-4" vaccine immunization may activate latent provirus and destroy at least some virus reservoirs. In order to measure that, we assessed the frequency and magnitude of transient viral load increases above 50 copies/mL (blips). Such spontaneous viral load increases occurring during ART treatment may be associated with latent viral reservoirs activation.

To investigate the possible effects of DNA vaccination on viral reservoirs we analyzed the magnitude and frequency of the blips in the placebo and immunized groups (Table 3). Neither relative frequency of the blips nor relative numbers of patients with blips differ between groups. But the amplitudes of blips in patients 21 and 37 immunized with 0.25 mg of the candidate DNA vaccine were much higher than that in placebo group—2800 and 18,000 copies/mL, respectively. In participant #21 an increase of the viral load was detected from the 6th to the 10th visit with the dynamics of increasing, peak and decreasing of the viral load, while most of the other blips were detected only during a single visit. The third largest blip, 709 copies/mL, was found in trial participant 43 vaccinated with 0.5 mg of DNA vaccine.

The number of patients is small. However, the largest increases were registered in double-blinded vaccinated groups. The results suggest that the lower DNA concentration (0.25 mg) is more active than 0.5 mg. This is in correspondence with more AE in the group vaccinated with 0.25 mg.

These results may have several explanations. The participation of therapeutic DNA-4 vaccination during ART in destruction of latent viral reservoirs in some patients due to the reactivation of a latent provirus by TNFα is possible but is not proved. The destruction of latent cells containing viral RNA can be the source of the viral blips."Repliclones", populations of replicating cells with HIV's genome nested inside them can also produce new virions [19].

The studied vaccine contains Nef protein which has been shown to have an ability to induce viral reactivation. It was demonstrated that exogenous Nef activated virus production in latent cell lines and in peripheral blood mononuclear cells isolated from asymptomatic HIV-infected individuals [20]. Early production of Nef during viral reactivation might enhance latent T cell activation.

Nef increases the production of exosomes containing activated ADAM17 (a disintegrin and metalloprotease domain 17), an enzyme that converts pro-TNF-α into its active form. The uptake of ADAM17-containing exosomes by target cells can induce the release of TNF-α, which subsequently binds to TNF receptor type 1 and activates NF-κB and c-Jun N-terminal kinase (JNK) pathways [20].

On the other hand, Nef is able to selectively downregulate surface CD4 and HLA-I molecules that may lead to evade immune surveillance by reactivated cells. Moreover, Nef can counteract multiple apoptotic pathways and promote cell survival could further hinder the clearance of reactivating reservoirs [20]. So, Nef protein can has dual effect on latent viral reservoirs reactivation.
