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

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 structural-functional organization and immunology of HIV-1.

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

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

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 mAbs VRC01 were published [33]. It is shown that they are safe and well tolerated after multiple intravenous or subcutaneous administrations in humans, in addition VRC01 from participants' sera were found to avidly capture HIV virions and to mediate antibody-dependent cellular phagocytosis [33].

Exceptional features of bNAbs inspire many researches to develop immunogen capable of their producing (induction). One of the evolving research areas focusing on the design of such immunogens is based on the development of HIV-1 envelope (Env) trimers [6, 34–36]. Despite substantial progress in this area, (a number of questions must be addressed). Firstly, although trimers are rather stable in solution, they produce conformational conditions that fail to provide binding and induction of bNAbs. Secondly, trimers expose undesired immunodominant non-protective HIV epitopes that could prevent adaptive immune response from recognizing neutralizing epitopes, block protective immunity and/or induce increased HIVinfection [4, 36].

An alternative approach to solving this problem includes constructing completely artificial polyepitope anti-HIV-1 immunogens comprising a set of protective epitopes assembled in a single mosaic (polyepitope) construct. Unfortunately, the most bNAbs recognize conformational epitopes and considerably more rarely linear epitopes [37–39]. Furthermore, conformational B-cell epitopes are frequently formed in HIV by lipids and glycans or their combinations [37–40]. It complicates the design of immunogens capable of inducing sufficient B-cell response. Phage peptide libraries offer the unique possibility to obtain mimics of such epitopes [41–46].

Using phage peptide library we can select peptides mimicking epitopes recognized by bnAbs, that make it possible to construct mosaic immunogen on their base to simultaneously induce several neutralizing antibodies [43, 47–52]. **Figure 4** depicts general working scheme.

HIV-1 subtypes A, B, and AG [20, 47, 48, 53]. Consequently, we succeeded to demonstrate immunologic imitation of conformational antigenic determinants, i.e. HIV-1 epitopes, by linear peptides. Obtained peptide-mimics are material that can serve as a basis for the development of immunoprophylactic HIV-1 vaccine. Besides, peptides can be used when designing

assay of immune serum, selection of the most promising mimotope; (F) artificial immunogen design.

**Figure 4.** Phage display applications for artificial immunogen design. (A) Randomized peptide library are used to map the residues forming the epitope(s) recognized by monoclonal antibodies immobilized on a solid support; (B) amplification of selected phages; (C) ELISA assay or western blot to determine specificity of selected phages binding; (D) phage particles are used to presentation isolated peptides-mimotopes to the immune system; (E) virus-neutralization

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The progress in identification of T-cell epitopes as well as understanding mechanisms of processing and presentation of antigens through MHC class I and II pathway make it possible to

It is known that CTL recognizes viral protein-antigens synthesized inside the cell not as fulllength molecules but as short peptides (8–10 amino acid residues) in complex with MHC class I molecules. These short antigenic epitopes emerge from endogenously synthesized proteins due to proteasome-mediated processing and then are transported to the lumen of endoplasmic reticulum (ER) using transport proteins TAP (transporter associated with antigen processing) where they bind to emerging MHC class I molecules [55, 56]. Since antigen must be synthesized in a cell to induce response of CTL, target T-cell vaccine should be designed as DNA-vaccine because it is the most natural way of presenting CTL-epitopes to

As opposed to stimulation of CTL, when inducing CD4+ T-lymphocyte-helpers response, antigen should be presented to these cells in a complex with MHC class II molecules. Usually

diagnostic systems for the detection of antibodies to HIV-1.

rational design artificial polyepitope vaccines [13, 54].

CD8+ T-lymphocytes through MHC class I pathway [57].

**4.2. Design of polyepitope T-cell antigens**

In our study we used a number of bNAbs against HIV-1, i.e. 2G12, 2F5, IgG1b12, Z13е1, VRC-01, VRC-03, and 697-30D to obtain peptide-mimics. The last five bNAbs were kindly furnished upon NIH AIDS Reagent Program, USA. Each monoclonal antibody was used to perform biopanning using phage peptide libraries (New England Biolabs, USA) [20, 47, 48, 53].

After biopanning of phage libraries using monoclonal antibody 2G12 (recognizes conformational epitope) and 2F5 (recognizes linear epitope), we isolated peptide-mimics that have another amino acid sequences compared to natural epitopes, but able to elicit antibodies in laboratory animals capable to compete with initial bNAbs and neutralizing the virus.

As a result we obtained a collection of phagotops carrying on their surface peptide-mimics of epitopes recognized by above mentioned bNAbs. Specific activity of selected peptides was studied both free and in the compound of phage particles. We carried out chemical synthesis of 134 free peptides. Evaluation of their capacity to compete with HIV-1 epitope for binding to monoclonal antibodies VRC-01, VRC-03, and IgG1b12 was carried out using pseudovirus particles in virus-neutralization assay. To study peptides immunogenicity in the compound of phage particles, the latter were produced in preparative amount using bacterial cells. We used the obtained samples to immunize laboratory animals from which we sampled sera to study their virus-neutralizing activity. It was shown that sera of rabbits immunized with a mix of bacteriophages are able to neutralize pseudotyped viruses obtained on the base of

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**Figure 4.** Phage display applications for artificial immunogen design. (A) Randomized peptide library are used to map the residues forming the epitope(s) recognized by monoclonal antibodies immobilized on a solid support; (B) amplification of selected phages; (C) ELISA assay or western blot to determine specificity of selected phages binding; (D) phage particles are used to presentation isolated peptides-mimotopes to the immune system; (E) virus-neutralization assay of immune serum, selection of the most promising mimotope; (F) artificial immunogen design.

HIV-1 subtypes A, B, and AG [20, 47, 48, 53]. Consequently, we succeeded to demonstrate immunologic imitation of conformational antigenic determinants, i.e. HIV-1 epitopes, by linear peptides. Obtained peptide-mimics are material that can serve as a basis for the development of immunoprophylactic HIV-1 vaccine. Besides, peptides can be used when designing diagnostic systems for the detection of antibodies to HIV-1.

#### **4.2. Design of polyepitope T-cell antigens**

mAbs VRC01 were published [33]. It is shown that they are safe and well tolerated after multiple intravenous or subcutaneous administrations in humans, in addition VRC01 from participants' sera were found to avidly capture HIV virions and to mediate antibody-dependent

Exceptional features of bNAbs inspire many researches to develop immunogen capable of their producing (induction). One of the evolving research areas focusing on the design of such immunogens is based on the development of HIV-1 envelope (Env) trimers [6, 34–36]. Despite substantial progress in this area, (a number of questions must be addressed). Firstly, although trimers are rather stable in solution, they produce conformational conditions that fail to provide binding and induction of bNAbs. Secondly, trimers expose undesired immunodominant non-protective HIV epitopes that could prevent adaptive immune response from recognizing neutralizing epitopes, block protective immunity and/or induce increased HIV-

An alternative approach to solving this problem includes constructing completely artificial polyepitope anti-HIV-1 immunogens comprising a set of protective epitopes assembled in a single mosaic (polyepitope) construct. Unfortunately, the most bNAbs recognize conformational epitopes and considerably more rarely linear epitopes [37–39]. Furthermore, conformational B-cell epitopes are frequently formed in HIV by lipids and glycans or their combinations [37–40]. It complicates the design of immunogens capable of inducing sufficient B-cell response. Phage peptide libraries offer the unique possibility to obtain mimics of such

Using phage peptide library we can select peptides mimicking epitopes recognized by bnAbs, that make it possible to construct mosaic immunogen on their base to simultaneously induce

In our study we used a number of bNAbs against HIV-1, i.e. 2G12, 2F5, IgG1b12, Z13е1, VRC-01, VRC-03, and 697-30D to obtain peptide-mimics. The last five bNAbs were kindly furnished upon NIH AIDS Reagent Program, USA. Each monoclonal antibody was used to perform biopanning using phage peptide libraries (New England Biolabs, USA) [20, 47, 48, 53]. After biopanning of phage libraries using monoclonal antibody 2G12 (recognizes conformational epitope) and 2F5 (recognizes linear epitope), we isolated peptide-mimics that have another amino acid sequences compared to natural epitopes, but able to elicit antibodies in

several neutralizing antibodies [43, 47–52]. **Figure 4** depicts general working scheme.

laboratory animals capable to compete with initial bNAbs and neutralizing the virus.

As a result we obtained a collection of phagotops carrying on their surface peptide-mimics of epitopes recognized by above mentioned bNAbs. Specific activity of selected peptides was studied both free and in the compound of phage particles. We carried out chemical synthesis of 134 free peptides. Evaluation of their capacity to compete with HIV-1 epitope for binding to monoclonal antibodies VRC-01, VRC-03, and IgG1b12 was carried out using pseudovirus particles in virus-neutralization assay. To study peptides immunogenicity in the compound of phage particles, the latter were produced in preparative amount using bacterial cells. We used the obtained samples to immunize laboratory animals from which we sampled sera to study their virus-neutralizing activity. It was shown that sera of rabbits immunized with a mix of bacteriophages are able to neutralize pseudotyped viruses obtained on the base of

cellular phagocytosis [33].

212 Advances in HIV and AIDS Control

infection [4, 36].

epitopes [41–46].

The progress in identification of T-cell epitopes as well as understanding mechanisms of processing and presentation of antigens through MHC class I and II pathway make it possible to rational design artificial polyepitope vaccines [13, 54].

It is known that CTL recognizes viral protein-antigens synthesized inside the cell not as fulllength molecules but as short peptides (8–10 amino acid residues) in complex with MHC class I molecules. These short antigenic epitopes emerge from endogenously synthesized proteins due to proteasome-mediated processing and then are transported to the lumen of endoplasmic reticulum (ER) using transport proteins TAP (transporter associated with antigen processing) where they bind to emerging MHC class I molecules [55, 56]. Since antigen must be synthesized in a cell to induce response of CTL, target T-cell vaccine should be designed as DNA-vaccine because it is the most natural way of presenting CTL-epitopes to CD8+ T-lymphocytes through MHC class I pathway [57].

As opposed to stimulation of CTL, when inducing CD4+ T-lymphocyte-helpers response, antigen should be presented to these cells in a complex with MHC class II molecules. Usually processing and presentation of antigen take place for extracellular antigens which are delivered in cells via endocytosis and phagocytosis. In this case antigen processing occurs in lysosome.

The obtained results became the basis for the development of original software TEpredict and PolyCTLDesigner that we consider as a universal platform for rational design of polyepitope immunogens – candidate DNA vaccines for induction of T-cell immunity both against infec-

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PolyCTLDesigner enables the user to select a minimal set of epitopes with known or predicted specificity to different allelic variants of MHC class I molecules. This set covers selected repertoire of HLA alleles with the given degree of redundancy. After that PolyCTLDesigner uses the model by Peters et al. [73] to predict binding affinity to TAP for the selected set of known or predicted epitopes. According to this model, the main contribution into peptide binding to TAP is provided by the first three N-terminal amino acid peptide residues and the last C-terminal residue. Considering that epitope C-terminus must be unchanged since C-terminus should contain the site of proteasomal cleavage [74], only N-terminus of antigenic peptide can be extended (if necessary) for optimization of interaction with heterodimer

Then PolyCTLDesigner analyzes all possible matching of the selected peptides and detects an optimal spacer sequence for each pair providing adequate cleavage of epitopes with release of C-terminus of proximal peptide. To predict proteasomal and/or immunoproteasomal cleav-

When analyzing epitopes matching, PolyCTLDesigner creates a directed graph with nodes corresponding to epitopes and edges corresponding to acceptable matching. Each edge has relevant weight vector characterized by the efficiency of proteasomal cleavage, spacer length

**Figure 5.** PolyCTLDesigner operation algorithm. I – Selection of minimal set of CD8+ T cell epitopes with the known or predicted specificity towards various allelic variants of MHC class I molecules; II – Prediction of binding affinity of peptides to TAP and, if necessary, addition of N-terminal flanking residues to optimize this binding; III – Prediction of optimal spacer sequences for each pair of peptides; IV – Creating weighted graph, where the nodes are the target epitopes, and the edges are possible variants of their association. Each edge is a weight vector whose attributes are: efficiency of proteasomal cleavage, length of the spacer, and number of predicted non-target epitopes at junction; V – Designing polyepitope immunogen sequence. Resulting sequence is defined as the longest simple path in the graph that

age, PolyCTLDesigner uses models developed by Toes et al. [75].

has the lowest weight; Prot1, Prot2, Prot3 – proteasomal cleavage sites.

tious and oncological diseases [71, 72] (**Figure 5**).

TAP1/TAP2.

Thus, when designing polyepitope T-cell immunogens capable of inducing high levels of CD4+ and CD8+ T-lymphocyte responses to all epitopes in its compound, one should provide efficient proteasome- and/or lysosome-mediated processing of expression product of target gene through MHC class I and II pathway. For the purpose the following strategies are appropriate:


To evaluate which of these strategies provide a rational approach to constructing T-cell antigens, we designed a set of polyepitope constructs covering a range of possible structural variants.

To assess the influence of ubiquitin and spacer sequences flanking epitopes on immunogenicity of the polyepitope construct, we designed a set of polyepitope immunogens considering different strategies of processing and presentation of the target antigens. The designed constructs comprised similar set from 10 HLA-A2-restricted CTL-epitopes of the main HIV-1 antigens Env, Gag, Pol, Nef, and Vpr, but differed in a number of structural properties, namely (i) the presence or absence of spacers; (ii) the structure of spacer sequences, and (iii) the presence of N- or C-terminal sequence of ubiquitin. Genes encoding the designed antigens were cloned into plasmid vector and vaccinia virus.

Immunogenicity of the designed immunogens were evaluated after 3-fold prime-boost immunization of HLA-A2 transgenic mice with the obtained recombinant plasmids and recombinant vaccinia virus (rVV). It was demonstrated that the vaccine construct inducing the majority of complexes [peptide/MHC class I] *in vitro* was also the most immunogenic during animal vaccination. This construct comprises N-terminal ubiquitin to target the polyepitope on proteasome. Besides, in the compound of this construct epitopes are divided by spacer sequences comprising sites of proteasomal cleavage of the polyepitope and motifs for TAP-dependent transport of the released peptides into ER where they bind to MHC class I molecules [54].

The obtained results became the basis for the development of original software TEpredict and PolyCTLDesigner that we consider as a universal platform for rational design of polyepitope immunogens – candidate DNA vaccines for induction of T-cell immunity both against infectious and oncological diseases [71, 72] (**Figure 5**).

processing and presentation of antigen take place for extracellular antigens which are delivered in cells via endocytosis and phagocytosis. In this case antigen processing occurs in

Thus, when designing polyepitope T-cell immunogens capable of inducing high levels of CD4+ and CD8+ T-lymphocyte responses to all epitopes in its compound, one should provide efficient proteasome- and/or lysosome-mediated processing of expression product of target gene through MHC class I and II pathway. For the purpose the following strategies are appropriate:

**1.** To design poly-CTL-epitope construct one may use spacer sequences dividing epitopes that comprise sites of proteasomal cleavage [58–60] and/or motif for binding to TAP [61–63] to provide polyepitope processing and transport of released peptides (epitopes)

**2.** To induce T helper lymphocytes response fragments with T-helper epitopes can be combined with the use of motif [KR][KR] which is a cleavage site for a number of lysosomal

**3.** To target polyepitope immunogen into proteasome and presentation of CTL-epitopes to CD8+ T-lymphocytes through MHC class I pathway, researchers typically use genetic at-

**4.** To degrade polyepitope immunogen and present released Th-epitopes to CD4+ T-lymphocytes through MHC class II pathway, researchers typically use a genetic attachment of the sequence of LAMP-1 protein tyrosine motif (Lysosomal-associated membrane protein 1) to its C-terminus to direct the polyepitope immunogen from the secretory pathway to the

To evaluate which of these strategies provide a rational approach to constructing T-cell antigens, we designed a set of polyepitope constructs covering a range of possible structural variants.

To assess the influence of ubiquitin and spacer sequences flanking epitopes on immunogenicity of the polyepitope construct, we designed a set of polyepitope immunogens considering different strategies of processing and presentation of the target antigens. The designed constructs comprised similar set from 10 HLA-A2-restricted CTL-epitopes of the main HIV-1 antigens Env, Gag, Pol, Nef, and Vpr, but differed in a number of structural properties, namely (i) the presence or absence of spacers; (ii) the structure of spacer sequences, and (iii) the presence of N- or C-terminal sequence of ubiquitin. Genes encoding the designed antigens were

Immunogenicity of the designed immunogens were evaluated after 3-fold prime-boost immunization of HLA-A2 transgenic mice with the obtained recombinant plasmids and recombinant vaccinia virus (rVV). It was demonstrated that the vaccine construct inducing the majority of complexes [peptide/MHC class I] *in vitro* was also the most immunogenic during animal vaccination. This construct comprises N-terminal ubiquitin to target the polyepitope on proteasome. Besides, in the compound of this construct epitopes are divided by spacer sequences comprising sites of proteasomal cleavage of the polyepitope and motifs for TAP-dependent transport of the released peptides into ER where they bind to MHC class I

cathepsins participating in antigen processing [64, 65].

tachment of ubiquitin sequence to its N- or C-termini [66].

lysosome.

214 Advances in HIV and AIDS Control

into ER.

lysosome [67–70].

molecules [54].

cloned into plasmid vector and vaccinia virus.

PolyCTLDesigner enables the user to select a minimal set of epitopes with known or predicted specificity to different allelic variants of MHC class I molecules. This set covers selected repertoire of HLA alleles with the given degree of redundancy. After that PolyCTLDesigner uses the model by Peters et al. [73] to predict binding affinity to TAP for the selected set of known or predicted epitopes. According to this model, the main contribution into peptide binding to TAP is provided by the first three N-terminal amino acid peptide residues and the last C-terminal residue. Considering that epitope C-terminus must be unchanged since C-terminus should contain the site of proteasomal cleavage [74], only N-terminus of antigenic peptide can be extended (if necessary) for optimization of interaction with heterodimer TAP1/TAP2.

Then PolyCTLDesigner analyzes all possible matching of the selected peptides and detects an optimal spacer sequence for each pair providing adequate cleavage of epitopes with release of C-terminus of proximal peptide. To predict proteasomal and/or immunoproteasomal cleavage, PolyCTLDesigner uses models developed by Toes et al. [75].

When analyzing epitopes matching, PolyCTLDesigner creates a directed graph with nodes corresponding to epitopes and edges corresponding to acceptable matching. Each edge has relevant weight vector characterized by the efficiency of proteasomal cleavage, spacer length

**Figure 5.** PolyCTLDesigner operation algorithm. I – Selection of minimal set of CD8+ T cell epitopes with the known or predicted specificity towards various allelic variants of MHC class I molecules; II – Prediction of binding affinity of peptides to TAP and, if necessary, addition of N-terminal flanking residues to optimize this binding; III – Prediction of optimal spacer sequences for each pair of peptides; IV – Creating weighted graph, where the nodes are the target epitopes, and the edges are possible variants of their association. Each edge is a weight vector whose attributes are: efficiency of proteasomal cleavage, length of the spacer, and number of predicted non-target epitopes at junction; V – Designing polyepitope immunogen sequence. Resulting sequence is defined as the longest simple path in the graph that has the lowest weight; Prot1, Prot2, Prot3 – proteasomal cleavage sites.

and number of predicted non-target epitopes at the junction. Finally, the software designs an optimal polyepitope immunogen sequence that is calculated as a complete simple way in the constructed graph with the least length (weight).

N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. N-terminal signal peptides are believed to provide delivery of immunogen in ER, while LAMP-1 protein motif directs immunogen from the secretory pathway to the lysosome and presents epitopes released after the cleavage to CD4+ T-lymphocytes through MHC class II pathway. The sequence polyE of TCI-N3 comprises N-terminal ubiquitin for its delivery into proteasome and presentation of epitopes released after the cleavage to CD8+ T-lymphocytes through MHC class I pathway.

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Immunogenicity of the obtained DNA-vaccine constructs was studied in BALB/c mice according to capacity of CD4+ and CD8+ T-cells to produce IL-2 and IFNγ in ELISpot. The obtained results revealed that DNA-vaccine constructs encoding TCI-N2 and TCI-N3 immunogens induce responses of HIV-specific CD4+ and CD8+ T-lymphocytes that are significantly higher than that of the negative control the group of animals immunized with vector plasmid pcDNA3.1 as well as of group of mice that received a construct encoding core immunogen TCI-N1 with no additional signal sequences. At the same time DNA-vaccine construct encoding TCI-N3 immunogen comprising N-terminal ubiquitin induces the highest statistically significant level (Р ≤ 0.05) of

Thus, the obtained results point to a regular correlation between the structure of polyepitope

• it is possible to significantly increase the immunogenic potential of the target polyepitope vaccine via optimization of the immunogen structure using the spacer sequences comprising motifs for binding to TAP and the sites of proteasomal and lysosomal cleavage flanking

• ubiquitin-dependent targeting of polyepitope at proteasome is the most efficient strategy to induce specific T-cell immune response as compared to LAMP-dependent targeting at lysosome.

Our findings support the concept of vaccine rational design based on existing knowledge on

We did not set ourselves the task of covering all challenges facing designers of HIV-1 vaccine. The paper presents our experience on designing artificial polyepitope HIV-1 immunogens constructed using a broad spectrum of conservative T- and B-cell epitopes. This approach is believed to be promising for the design of new generation HIV-vaccines. In theory, it makes it possible to overcome HIV-1 antigenic variability, focuses immune responses on protective determinants, and allows to exclude from vaccine composition undesired determinants capable of inducing autoantibodies or antibodies increasing virus infectivity. The results demonstrate that completely artificial molecules designed with the use of bioinformatic and combinatorial biology methods are able to induce production of broad-spectrum neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helper (CD4+ Th)

It is our belief that the proposed approach can play an important and positive role in the

mechanism of presentation of T-cell antigens through MHC class I and II pathway.

CD4+ and CD8+ T-lymphocytes as compared with two other immunogens.

CTL- and Th-epitopes in the compound of the polyepitope construct;

construct and its antigenic and immunogenic properties:

T-lymphocytes in laboratory animals and human.

development of HIV-1 vaccine.

**5. Conclusions**

Besides, PolyCTLDesigner makes it possible to construct a sequence of the epitope fragment comprising T-helper epitopes. In the compound of the selected antigens software predicts peptide fragments with the length of 20–40 amino acid residues with the majority of overlapping T-helper epitopes restricted by the widest possible repertoire of HLA class II allomorphs. Then five C- and N-terminal amino acid residues from the initial antigen sequence are added to each of the selected fragments since it was shown that they can play significant role in binding to T-cell receptors of CD4+ T-lymphocytes [76, 77]. Fragments with T-helper epitopes are combined using [KR][KR] motif which is a cleavage site for a number of lysosomal cathepsins involved in antigen processing.

More detailed information on PolyCTLDesigner software is available at http://tepredict. sourceforge.net/PolyCTLDesigner.html.

We used the developed software when designing new polyepitope constructs – candidate DNA-vaccines against HIV-1. Particularly, when evaluating the influence of proteasomedependent and lysosome-dependent degradation of polyepitopes on immunogenicity of the target polyepitope construct, we designed three polyepitope HIV-1 immunogens, i.e. TСI-N1, TСI-N2, and TСI-N3 using cytotoxic and helper T-cell epitopes of HIV-1 [78].

All three polyepitope immunogens are based on the same core sequence of polyE, while differences between immunogens lie in the use of different terminal signal sequences (**Figure 6**). Immunogen TCI-N1 comprises only the core sequence polyE. Sequence polyE of TCI-N2 immunogen includes

**Figure 6.** Design of T-cell polyepitope immunogens. polyE – common for all antigens sequence polyE designed using cytotoxic and helper T-cell epitopes of HIV-1; ER-signal – N-terminal signal peptide (in our case MRYMILGLLALAAVCSAA – the signal sequence of the adenovirus protein E3/gp19K); LAMP1 – C-terminal tyrosine-based motif of LAMP-1 glycoprotein (RKRSHAGYQTI); Ub – N-terminal ubiquitin with substitution of the C-terminal Gly to Val to prevent liberation of Ub cleavage by Ub hydrolases.

N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. N-terminal signal peptides are believed to provide delivery of immunogen in ER, while LAMP-1 protein motif directs immunogen from the secretory pathway to the lysosome and presents epitopes released after the cleavage to CD4+ T-lymphocytes through MHC class II pathway. The sequence polyE of TCI-N3 comprises N-terminal ubiquitin for its delivery into proteasome and presentation of epitopes released after the cleavage to CD8+ T-lymphocytes through MHC class I pathway.

Immunogenicity of the obtained DNA-vaccine constructs was studied in BALB/c mice according to capacity of CD4+ and CD8+ T-cells to produce IL-2 and IFNγ in ELISpot. The obtained results revealed that DNA-vaccine constructs encoding TCI-N2 and TCI-N3 immunogens induce responses of HIV-specific CD4+ and CD8+ T-lymphocytes that are significantly higher than that of the negative control the group of animals immunized with vector plasmid pcDNA3.1 as well as of group of mice that received a construct encoding core immunogen TCI-N1 with no additional signal sequences. At the same time DNA-vaccine construct encoding TCI-N3 immunogen comprising N-terminal ubiquitin induces the highest statistically significant level (Р ≤ 0.05) of CD4+ and CD8+ T-lymphocytes as compared with two other immunogens.

Thus, the obtained results point to a regular correlation between the structure of polyepitope construct and its antigenic and immunogenic properties:


Our findings support the concept of vaccine rational design based on existing knowledge on mechanism of presentation of T-cell antigens through MHC class I and II pathway.
