**3. CombiHIVvac: a combined vaccine containing two immunogens in a single construct. Preclinical and clinical trials**

Since an effective immunoprophylactic vaccine against HIV-infection must induce specific humoral and T-cell immune responses [2, 3, 12], we constructed CombiHIVvac vaccine comprising both above mentioned immunogens, i.e. TBI and TCI [21].

CombiHIVvac was constructed in the form of micelle-like particles based on the original technique combining two different immunogens in a single construct, i.e. polyepitope TBI protein and DNA-vaccine pcDNA-TCI encoding polyepitope protein TCI [19, 21] (**Figure 3**).

TBI protein is conjugated to dextran and mixed with DNA, which leads to formation of microparticles presenting TBI on the surface and containing the DNA inside. Positively charged spermidine provides the binding of the conjugate dextran/protein TBI with negatively charged DNA-vaccine promoting formation of particles on the self-assembly principle (50–250 nm in diameter) [22].

We have previously shown that by combining two immunogens (TBI and TCI) in one construct significant enhancement HIV-specific B cell response was observed [23]. In our opinion, the formation of such particles plays a critical role in the registered effect. CombiHIVvac particles enable more effective absorption by antigen-presenting cells (APCs) compared to individual immunogens. Since TBI protein is fixed on the particle surface and is represented in multiple copies, this provides multiple enhancement of vaccine antigenicity. Besides, pcDNA-TCI enclosed in the vaccine structure is more protected against degradation by DNase I than free pcDNA-TCI, as it was previously demonstrated, resulting in prolongation of DNA-vaccine presence in an organism. Finally, the presence of CD4+ T-helper epitopes in the protein TCI may be the main reason underlying the increased synthesis of antibodies to TBI protein due to a CD4-mediated stimulation of B-cell proliferation and differentiation.

To carry out CombiHIVvac preclinical and clinical trials, we produced experimental series of vaccine of the standard quality according to WHO recommendations. Preclinical studies indicating the safety of the vaccine in tests with animals have been performed, namely, the acute and chronic toxicity has been studied in mice and guinea pigs and the absence of deviations in the vital organs of animals, as well as no changes in hematological and morphological parameters and no immunotoxicity and allergenic activity, have been shown for both single and tenfold administration of vaccine. Specific activity was evaluated based on the parameters of humoral and cellular immunity in BALB/c mice after their twofold immunization. The CombiHIVvac vaccine has been shown to induce formation of HIV-specific antibodies and CTLs [19, 21, 24, 25]. The vaccine did not cause any pyrogenic reaction in rabbits and did not affect the central nervous system and the detoxification liver function in mice. The duration of vaccine persistence in the organisms of laboratory animals has also been estimated and it has been shown that such vaccine component as the plasmid DNA completely eliminated from the organs and tissues of mice for 2 months after vaccination [21]. Thus, preclinical studies showed that CombiHIVvac is safe in animal trials.

Phase I clinical trials were carried out in healthy volunteers to study reactogenicity, safety, and immune activity of CombiHIVvac. The results of clinical trials published in [26]

In the following, recombinant protein TBI and plasmid pcDNA-TCI were used for the devel-

**Figure 2.** Design of the CTL immunogen, a candidate for the in HIV-1 vaccine: a general schematic. The bar patterns indicate the polyepitope CTL immunogen and the origin of the sequences. The positions of individual epitopes and their MHC restrictions (HLA-A, B, Cw – human; H-2a, b, d, f, k, p, u, q – mouse; Mamu-A\*01 – *Macaca mulatta*) are depicted

Since an effective immunoprophylactic vaccine against HIV-infection must induce specific humoral and T-cell immune responses [2, 3, 12], we constructed CombiHIVvac vaccine com-

CombiHIVvac was constructed in the form of micelle-like particles based on the original technique combining two different immunogens in a single construct, i.e. polyepitope TBI protein

TBI protein is conjugated to dextran and mixed with DNA, which leads to formation of microparticles presenting TBI on the surface and containing the DNA inside. Positively charged spermidine provides the binding of the conjugate dextran/protein TBI with negatively charged DNA-vaccine promoting formation of particles on the self-assembly principle (50–250 nm in

We have previously shown that by combining two immunogens (TBI and TCI) in one construct significant enhancement HIV-specific B cell response was observed [23]. In our opinion, the formation of such particles plays a critical role in the registered effect. CombiHIVvac particles enable more effective absorption by antigen-presenting cells (APCs) compared to individual immunogens. Since TBI protein is fixed on the particle surface and is represented in multiple copies, this

and DNA-vaccine pcDNA-TCI encoding polyepitope protein TCI [19, 21] (**Figure 3**).

**3. CombiHIVvac: a combined vaccine containing two immunogens** 

**in a single construct. Preclinical and clinical trials**

prising both above mentioned immunogens, i.e. TBI and TCI [21].

opment of CombiHIVvac vaccine [21].

208 Advances in HIV and AIDS Control

as lines below the CTL immunogen. Th stands for helper epitopes.

diameter) [22].

**Figure 3.** TEM images of CombiHIVvac microparticles with different magnification. A – scale bar 1000 nm, the insert in the left upper corner is a scheme of a CombiHIVvac particle (1 – pcDNA-TCI, 2 – spermidine/dextran, 3 – TBI).

demonstrated that CombiHIVvac is well-tolerated and safe. Neither single nor twofold (with 28-days interval) intramuscular vaccine administration induced significant changes in biochemical and physiological indicators as compared to the baseline values. Local reactions to vaccine administration were absent. There were no pathological changes in volunteers for all observation time. The mean values of the examined biochemical and clinical parameters were within the physiological ranges or near their limits. We failed to detect any regularity in changes of indicators depending on the time from the date of immunization. All studied indicators of immune status came back to the baseline level registered before the vaccination [26].

of virus lysate were sorbed. Using immunoblot analysis, we demonstrated the presence of antibodies to HIV-1 proteins p17, p24, p55, p68, and gp120, i.e. to those proteins which epitopes compose B-cell vaccine component – TBI protein. The response rates differed among volunteers within the same group. Furthermore, during 1 year after the second immunization

Artificial Epitope-Based Immunogens in HIV-Vaccine Design

http://dx.doi.org/10.5772/intechopen.77031

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The results of the study of T-cell response via the IFN-γ ELISpot in repeatedly vaccinated volunteers (**Table 1**) show the HIV-specific response of T-lymphocytes in all volunteers (100%) on the 14th day after the first vaccination, remaining sufficiently strong for 6 months after the second vaccination. Using MHC pentamers in a complex with Env peptide (KLTPLCVTL, gp120 aa 120–128) of HIV-1, it was demonstrated that KLTPLCVTL CD8 T lymphocytes occur

Thus, the performed clinical trials showed that the CombiHIVvac vaccine is well tolerated and safe (does not induce any significant changes in biochemical and physiological parameters in comparison to the background values), characterized by low reactogenicity (local reactions to the vaccine are absent) and most importantly capable of inducing the specific humoral and

Based on the obtained results, the Ministry of Health and Social Development of the Russian

Preclinical and clinical trials of CombiHIVvac demonstrated that a combination of two completely artificial polyepitope T- and B-cell antigens is capable of inducing HIV-specific CTLs and antibodies in laboratory animals and human. Furthermore, TCI protein expressed in cells as part of pcDNA-TCI plasmid fulfills a double function: (1) induces specific CD8+ CTL responses and (2) acts as an adjuvant synergistically effecting on synthesis of antibodies to TBI protein with virus-neutralizing activity at least to two HIV-1 subtypes (A and B) [23, 26]. The obtained results imply that CombiHIVvac is actually an original platform for the development and further improvement of combined DNA-protein HIV-vaccines using a broad range of conservative T- and B-cell epitopes based on virus antigens. Providing that TBI and TCI immunogens in CombiHIVvac composition were developed more than 15 year ago concurrently with clinical trials of CombiHIVvac, we carried out works on enhancement of immunogenic and protective properties of artificial polyepitope antigens utilizing new data on the

we registered antibodies at least to one of those proteins in 100% of volunteers.

in all volunteers (100%) up to the sixth month after the second vaccination (**Table 1**).

Federation has recommended the vaccine for advanced (Phase II) clinical trials.

**4. Possible development of CombiHIVvac vaccine platform**

structural-functional organization and immunology of HIV-1.

**4.1. B-cell epitopes to HIV-1 generating broadly neutralizing antibodies (bNAbs)**

years dozens of B-cell HIV epitopes recognized by bNAbs have been detected [27].

At present when developing efficient B-cell immunogens, researchers mainly rely on epitopes recognized by antibodies neutralizing a broad spectrum of HIV-1 strains (bNAbs). In recent

It was shown that many of these antibodies can prevent infection, and some can suppress active infection in hu-mice or macaques [28–32]. Recently results of Phase I clinical trials of

cellular immunity.

When carrying out Phase I clinical trials, we assessed CombiHIVvac specific activity in addition to its safety. The obtained results revealed that CombiHIVvac induces both humoral and cell HIV-specific immune responses. It was was confirmed by several methods including immunoblotting, ELISA, env-pseudotyped virus neutralization assay, IFN-γ ELISpot and peptide-МНС-pentamers.

The specific immune response was detected via ELISA 14 days after the first immunization; the second immunization led to enhance the immune response. The maximum immune response is observed by ELISA on the 14th day after the second vaccine administration. Up to the end of observation time (1 year) we detected antibodies in 29% of volunteers.


To confirm antibody production capable of recognizing native HIV-1 during CombiHIVvac administration, we used a kit "New Lav blot" (Bio Rad) on which strips of separate proteins

a The neutralization of virus was evaluated as IC50 value obtained by neutralizing the clones of pseudoviruses of subtypes A (SP-2010 and SP-392) and B (SF162 and PVO4) with blood sera of volunteers vaccinated with CombiHIVvac. The reaction was considered as positive if the neutralization titer was greater than or equal to 1: 100. The neutralizing activities of sera on the 14th and 28th days after immunization were lower than 1: 100.

b The ELISpot responses were considered as positive if the number of IFN-γ-producing cells in the vaccinated volunteers was two times larger than the control value.

c The results of determination of HIV-specific CD8+ T-lymphocytes in HLA-A\*0201-positive volunteers repeatedly vaccinated with CombiHIVvac obtained with the use of MHC pentamers in a complex with Env peptide (KLTPLCVTL) of HIV-1 are given.

**Table 1.** Evaluation of the response of HIV-specific T-lymphocytes and the activity of virus-neutralizing antibodies in repeatedly vaccinated volunteers.

of virus lysate were sorbed. Using immunoblot analysis, we demonstrated the presence of antibodies to HIV-1 proteins p17, p24, p55, p68, and gp120, i.e. to those proteins which epitopes compose B-cell vaccine component – TBI protein. The response rates differed among volunteers within the same group. Furthermore, during 1 year after the second immunization we registered antibodies at least to one of those proteins in 100% of volunteers.

demonstrated that CombiHIVvac is well-tolerated and safe. Neither single nor twofold (with 28-days interval) intramuscular vaccine administration induced significant changes in biochemical and physiological indicators as compared to the baseline values. Local reactions to vaccine administration were absent. There were no pathological changes in volunteers for all observation time. The mean values of the examined biochemical and clinical parameters were within the physiological ranges or near their limits. We failed to detect any regularity in changes of indicators depending on the time from the date of immunization. All studied indicators of immune status came back to the baseline level registered before

When carrying out Phase I clinical trials, we assessed CombiHIVvac specific activity in addition to its safety. The obtained results revealed that CombiHIVvac induces both humoral and cell HIV-specific immune responses. It was was confirmed by several methods including immunoblotting, ELISA, env-pseudotyped virus neutralization assay, IFN-γ ELISpot and

The specific immune response was detected via ELISA 14 days after the first immunization; the second immunization led to enhance the immune response. The maximum immune response is observed by ELISA on the 14th day after the second vaccine administration. Up to

To confirm antibody production capable of recognizing native HIV-1 during CombiHIVvac administration, we used a kit "New Lav blot" (Bio Rad) on which strips of separate proteins

**Time after the second vaccination, days**

**14 28 14 28 90 180 270 360**

B/PVO4 — — 36 36 29 29 15 0 A/392 — — 86 86 86 71 57 0 A/SP2010 — — 79 79 79 64 7 0

the end of observation time (1 year) we detected antibodies in 29% of volunteers.

**Analysis Percentage of vaccinated volunteers with positive responses Time after**

**days**

activities of sera on the 14th and 28th days after immunization were lower than 1: 100.

**the first vaccination,** 

Neutralization of virusa B/SF162 — — 71 71 71 64 57 0

IFN-γ ELISpot<sup>b</sup> 71 79 100 93 86 79 50 43 MHC pentamersc 100 100 100 100 80 100 80 60

The neutralization of virus was evaluated as IC50 value obtained by neutralizing the clones of pseudoviruses of subtypes A (SP-2010 and SP-392) and B (SF162 and PVO4) with blood sera of volunteers vaccinated with CombiHIVvac. The reaction was considered as positive if the neutralization titer was greater than or equal to 1: 100. The neutralizing

The ELISpot responses were considered as positive if the number of IFN-γ-producing cells in the vaccinated volunteers

The results of determination of HIV-specific CD8+ T-lymphocytes in HLA-A\*0201-positive volunteers repeatedly vaccinated with CombiHIVvac obtained with the use of MHC pentamers in a complex with Env peptide (KLTPLCVTL)

**Table 1.** Evaluation of the response of HIV-specific T-lymphocytes and the activity of virus-neutralizing antibodies in

the vaccination [26].

210 Advances in HIV and AIDS Control

peptide-МНС-pentamers.

a

b

c

of HIV-1 are given.

was two times larger than the control value.

repeatedly vaccinated volunteers.

The results of the study of T-cell response via the IFN-γ ELISpot in repeatedly vaccinated volunteers (**Table 1**) show the HIV-specific response of T-lymphocytes in all volunteers (100%) on the 14th day after the first vaccination, remaining sufficiently strong for 6 months after the second vaccination. Using MHC pentamers in a complex with Env peptide (KLTPLCVTL, gp120 aa 120–128) of HIV-1, it was demonstrated that KLTPLCVTL CD8 T lymphocytes occur in all volunteers (100%) up to the sixth month after the second vaccination (**Table 1**).

Thus, the performed clinical trials showed that the CombiHIVvac vaccine is well tolerated and safe (does not induce any significant changes in biochemical and physiological parameters in comparison to the background values), characterized by low reactogenicity (local reactions to the vaccine are absent) and most importantly capable of inducing the specific humoral and cellular immunity.

Based on the obtained results, the Ministry of Health and Social Development of the Russian Federation has recommended the vaccine for advanced (Phase II) clinical trials.
