**2. Artificial TBI and TCI immunogens**

the vaccine strategy is to mimic the infectious process and natural immunity against a particular pathogen. However, for HIV-1 and for many other chronic viral, bacterial, fungal and parasitic infections, and cancer natural immunity is insufficient for protection [1, 2]. Novel and effective approaches in polyepitope HIV-vaccine development are needed today [3, 4].

Problems that retard development of HIV-1 vaccine are well-known. Firstly, HIV-1 has a high rate of escape mutations with the result that the virus can change the antigenic structure quicker than the immune system is switched to new antigenic variants. Secondly, it is still unclear which type of immune response is more significant when preventing infection: induction of HIV neutralizing antibodies (in systemic vs. mucosal compartments), CD4+ T-helper cells, cytotoxic CD8+ T-cells (both potent high avidity CD8+ T-effector/memory responses and central memory responses), innate immunity, or all factors together. However, recent publications demonstrate that a humoral response to vaccine may be critical to prevent acquisition of HIV, while CD8+ T-cells may be required to control viral replication in vaccinated individual. Thirdly, virus proteins include regions with pathogenic properties due to molecular mimicry of physiologically significant functions or induction of autoimmune responses that might contribute to immunodeficiency. Finally, when studying HIV-infection, experimental models

Well-known HIV-1 vaccine design strategies are based on the use of different forms of viral antigen including inactivated virus, modified or attenuated virus, native and genetically engineered proteins, and peptides [5]. The first generation candidate vaccines (such as AIDSVAX B/B and AIDSVAX B/E) were constructed for inducing humoral immunity, to elicit virusneutralizing antibodies. Development of such vaccines was based on the use of full-length proteins of HIV envelope or their fragments [8]. The second generation vaccines (e.g. Merck Ad5 gag/pol/nef of B subtype) were aimed to mediate protection by inducing HIV-specific cytotoxic T-lymphocytes (CTLs) capable to recognize and eliminate HIV-infected cells [9]. Many candidates were tested in human or animals; however, none of them has demonstrated

The first promising and statistically significant results were obtained in clinical trials of RV144 vaccine stimulating both humoral and cellular immunity. It is a combination of two previously developed vaccines ALVAC-HIV (Sanofi Pasteur) and AIDSVAX B/E gp120 (VAXGEN) [11]. Despite rather low protective efficacy (31.2%), RV144 clinical trials made it possible to draw several weighty conclusions, i.e. (1) HIV-1 vaccine is not a myth but a reality; (2) efficient vaccine should induce both humoral and cell immune responses against HIV-1, and (3) new

One of them includes construction of completely artificial polyepitope (mosaic) anti-HIV-1 immunogens comprising a broad range of protective T- and B-cell epitopes based on the main viral antigens capable of inducing production of neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helper (CD4+ Th) T-lymphocytes. This approach seems to be rather promising when developing new generation HIV-vaccines. In theory, it makes it possible to overcome HIV-1 antigenic variability, focus immune responses on protective epitopes and allows to exclude undesirable determinants from a vaccine compound capable of inducing autoantibodies or antibodies increasing virus infectivity [3, 4, 13]. This paper discusses our

experience in designing artificial polyepitope antigens – HIV-1 candidate vaccines.

are very limited [1, 5–7].

206 Advances in HIV and AIDS Control

efficacy in phase II-III trials [10].

approaches are needed to increase vaccine efficacy [2, 3, 12].

The first immunogen designed in our project, short for T- and B-cell epitopes containing Immunogen (TBI), was constructed with the use of conservative epitopes from Env and Gag HIV-1 based on a well-known protein space motif, i.e. four helix bundle (**Figure 1**). When designing immunogen, four Th-cell epitopes (amphipathic α-helix) and five B-cell epitopes (regions with flexible hydrophilic loops) were used as blocks [14, 15]. The rationale for TBI design was that combining T- and B-cell epitopes in one construct will stimulate both proper B-cell and T-cell responses and the necessary interplay between B- and T-cells. Recombinant protein TBI has a CD spectra similar to ones in α-helical proteins and was able to form crystals - that was demonstrated for artificial protein with a predicted tertiary structure for the first time [16]. Based on its ability to crystallize we assumed that TBI protein structure is similar to that of the natural proteins.

Mice and Macaque rhesus immunized with TBI formed both cell and humoral responses to HIV-1. TBI-induced antibodies showed virus-neutralizing activity to HIV-1 [17].

The second artificial polyepitope immunogen we developed was TCI (short for T-Cell Immunogen) aimed at stimulation of T-cell immunity [18]. When constructing immunogen, we selected highly conservative T-cell epitopes among three main HIV-1 subtypes (A, B, and C) (**Figure 2**). TCI comprises more than 80 T-cell epitopes (both CD8+ CTL and CD4+ Th) from Env, Gag, Pol, and Nef proteins [18]. We analyzed CTL-epitopes that were together restricted with 10 different optimally selected alleles of human MHC class I. As known, it is sufficient to cover genetic diversity of MHC class I antigens in population from almost all geographic regions. Since antigen processing and presentation through MHC class I pathway were found to be the most efficient for proteins synthesized inside the cell, the target vaccine construct was designed in the form of DNA-vaccine via cloning a gene encoding TCI protein into vector plasmid pcDNA3.1 [18].

The obtained DNA-vaccine pcDNA-TCI was used for genetic immunization; we showed that the vaccine is capable of inducing both specific T-cell responses and specific antibodies in immunized BALB/c mice [18–20].

**Figure 1.** A model of TBI protein tertiary structure. T-cell epitopes are located in the region of α-helices, B-cell epitopes are located at loop sites and N- and C-terminuses.

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

Artificial Epitope-Based Immunogens in HIV-Vaccine Design

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

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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

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]

**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).

B-cell proliferation and differentiation.

showed that CombiHIVvac is safe in animal trials.

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

In the following, recombinant protein TBI and plasmid pcDNA-TCI were used for the development of CombiHIVvac vaccine [21].
