Larisa I. Karpenko

Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble

Summary. Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high β-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing.

Novel approaches in polyepitope T-cell vaccine development against HIV-1

RV144 clinical trial was modestly effective in preventing HIV infection. New alternative approaches are needed to design improved HIV-1 vaccines and their delivery strategies. One of these approaches is construction of synthetic polyepitope HIV-1 immunogen using protective T- and B-cell epitopes that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helpers (CD4+ Th) T-lymphocytes. This approach seems to be promising for designing of new generation of vaccines against HIV-1, enables in theory to cope with HIV-1 antigenic variability, focuses immune responses on protective determinants and enables to exclude from the vaccine compound that can induce autoantibodies or antibodies enhancing HIV-1 infectivity. Herein, the authors will focus on construction and rational design of polyepitope T-cell HIV-1 immunogens and their delivery, including: advantages and disadvantages of existing T-cell epitope prediction methods; features of organization of polyepitope immunogens, which can generate high-level CD8+ and CD4+ T-lymphocyte responses; the strategies to optimize efficient processing, presentation and immunogenicity of polyepitope constructs; original software to design polyepitope immunogens; and delivery vectors as well as mucosal strategies of vaccination. This new knowledge may bring us a one step closer to developing an effective T-cell vaccine against HIV-1, other chronic viral infections and cancer.

Construction of artificial virus-like particles exposing HIV epitopes, and the study of their immunogenic properties

One of the major problems in the development of successful recombinant vaccines against human immunodeficiency virus (HIV) is that of correct identification of a safe and effective vaccine delivery system with which to induce protective immunity using soluble protein antigens. An original method for constructing artificial immunogens in the form of spherical particles with yeast dsRNA in the center and hybrid proteins exposing epitopes of an infectious agent on the surface is reported. The dsRNA and the proteins were linked with spermidine-polyglucin-glutathione conjugates. Particles exposing HIV-1 epitopes were constructed, and their immunogenicity tested.

Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.

Attenuated Salmonella enteritidis E23 as a vehicle for the rectal delivery of DNA vaccine coding for HIV-1 polyepitope CTL immunogen

This study is focusing on elucidation of the capacity of attenuated Salmonella enteritidis E23 (cya, crp) to serve as a vehicle for the rectal delivery of the DNA vaccine. Earlier for creation HIV‐1 candidate DNA vaccine we have designed the polyepitope protein TCI (T‐cell immunogen), which comprises over 80 CTL epitopes from subtype A, B and C HIV‐1 proteins. The gene coding for TCI protein was used to construct the eukaryotic expression plasmid pcDNA‐TCI. The attenuated S. enteritidis E23 was transformed by electroporation with recombinant plasmid pcDNA‐TCI and the expression of the TCI gene was determined in vitro and in vivo. BALB/c mice were rectally immunized with S. enteritidis E23/pcDNA‐TCI (108 cfu) twice at 4 week interval. Bacteria were not pathogenic for mice and spontaneously eliminated from mice spleen and liver to 60 days post the immunization. Detectable antibodies were generated in 2 weeks after immunization and their level increased after second immunization. The results of INF‐γ ELISpot show that mice immunized with S. enteritidis E23/pcDNA‐TCI elicited HIV‐specific cellular immune response. This study demonstrates that attenuated S. enteritidis E23 is an effective live vector for rectal delivery of the DNA vaccine pcDNA‐TCI to generate humoral and T‐cellular responses against HIV‐1.

Localization of the amino terminus of the hepatitis B virus core antigen within the core particle

The position of the amino terminus of the hepatitis B virus core antigen (HBcAg) within the core particle was studied. For this purpose, three recombinant analogs of HBcAg were designed. One analog, HBcAgR, was identical in amino acid sequences to the core polypeptide of the hepatitis B virus; the second, HBeAgR, differed from the authentic protein in deletion of 39 carboxy-terminal amino acids. The amino acid sequences of the third polypeptide, HBe delta N and of HBeAgR were similar, HBe delta N differed from HBeAgR only in replacement of 3 N-terminal amino acids by 16 amino acids of beta-galactosidase. The HBcAg analogs were compared with respect to their reaction with monoclonal antibody (mAb E1A7) to the amino-terminal linear epitope of hepatitis B virus e antigen. Although able to assemble into virus-like particles, the three analogs of HBcAg, reacted differently with mAb E1A7. It was demonstrated that mAb E1A7 reacted with both native and denatured HBeAgR. HBe delta N was not recognized by mAb E1A7. In contrast, HBcAgR reacted with mAb E1A7 only when denatured. Native HBcAgR did not react with mAb E1A7 when assembled into particles. Thus evidence was obtained that the amino terminus of HBcAg is not exposed on the particle surface.

Peptide-MHC multimer-based monitoring of CD8 T-cells in HIV-1 infection and AIDS vaccine development

The use of MHC multimers allows precise and direct detecting and analyzing of antigen-specific T-cell populations and provides new opportunities to characterize T-cell responses in humans and animals. MHC-multimers enable us to enumerate specific T-cells targeting to viral, tumor and vaccine antigens with exceptional sensitivity and specificity. In the field of HIV/SIV immunology, this technique provides valuable information about the frequencies of HIV- and SIV-specific CD8+ cytotoxic T lymphocytes (CTLs) in different tissues and sites of infection, AIDS progression, and pathogenesis. Peptide-MHC multimer technology remains a very sensitive tool in detecting virus-specific T -cells for evaluation of the immunogenicity of vaccines against HIV-1 in preclinical trials. Moreover, it helps to understand how immune responses are formed following vaccination in the dynamics from priming point until T-cell memory is matured. Here we review a diversity of peptide-MHC class I multimer applications for fundamental immunological studies in different aspects of HIV/SIV infection and vaccine development.

P19-04. A synergistic effect of a combined bivalent DNA-protein anti-HIV-1 vaccine containing multiple T- and B-cell epitopes of HIV-1 proteins

Immunogenic properties of the combined vaccine CombiHIVvac, comprising polyepitope HIV-1 immunogens, one being the artificial polyepitope protein TBI, containing the T- and B-cell epitopes from Env and Gag proteins, and the DNA vaccine construct pcDNA-TCI coding for the artificial protein TCI, carrying over 80 T-cell epitopes (both CD4+ CTL and CD8+ Th) from Env, Gag, Pol, and Nef proteins, are studied in this work. The data reported demonstrate clearly that a combination of two B- and T-cell immunogens (TBI and TCI) in one construct results in a synergistic increase in the antibody response to both TBI protein and the proteins from HIV-1 lysate. The level of antibodies induced by immunization with the constructs containing either immunogen alone (TBI protein or the plasmid pcDNA-TCI) was significantly lower as compared to that induced by the combined vaccine. The analysis performed suggests that the presence of CD4+ T-helper epitopes, which can be presented by MHC class II, 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.

Artificial polyepitope HIV-1 immunogen containing mimotope of 2F5 epitope

Constructing a vaccine against HIV-1, able to induce production of broadly neutralizing antibodies, is crucial. We report here the selection and characterization of RDWSFDRWSLSEFWL peptide mimotope that binds specifically to bNAbs 2F5. The peptide mimotope was selected from 15-mer phage-displayed peptide library by using Mab 2F5 as the selecting agent. The most abundant RDWSFDRWSLSEFWL peptide was inserted into a carrier, an artificial polyepitope immunogen - TBI (T- and B-cell immunogen). TBI-2F5 polyepitope immunogen that includes the mimotope of 2F5 epitope was constructed. It was shown that sera of mice immunized with TBI-2F5 protein recognized TBI protein as well as RDWSFDRWSLSEFWL peptide. The capacity of sera of immunized mice to neutralize HIV-1 was demonstrated using subtype B env-pseudoviruses of HIV-1 QH0692.42 and PVO.4. Based on these results, we conclude that peptide mimotope of 2F5 epitope RDWSFDRWSLSEFWL can be an essential component for a successful HIV-vaccine.