1. SYNTHETIC POLY-CTL EPITOPE DNA VACCINE AGAINST HIV-1 Bazhan S.I, Belavin P.A., SereginS.V., Danilyuk N.K., Babkina I.K., Karpenko L.I., Nekrasova N.A., Lebedev L.R., Ignatyev G.M., Agafonov A.P., Ilyichev A.A. SRC VB Vector; Koltsovo, Novosibirsk Region, 630559, Russia State Research Center of Virology and Biotechnology "Vector", Novosibirsk region, Koltsovo, 630559, Russia *E. mail: bazhan@vecror.nsc.ru 1. Introduction One of the promising approaches to the development of a new generation of effective and safe vaccines is based on identification of T- and B-cell epitopes in virus proteins for use as a basis for synthetic polyepitope vaccines. To provide protection effectively, HIV vaccine should be able to induce both humoral and cell immune responses. Although new promising approaches to the induction of neutralizing antibodies are still being developed [Binley et al., 2000], the mainstream is the induction of cell immunity [Thomson et al., 1995, 1996; Hanke et al., 1998a, b, c]. And that is for a reason, because convincing evidence exists that the responses of cytotoxic T-lymphocytes (CD8 + CTL) associated with HIV infection are important mediators of antiviral immunity and, therefore, induction of HIV-specific CTLs could be an important component of an effective vaccine against HIV-1 [Thomson et al., 1995, 1996; Hanke et al., 1998a, b, c]. This work describes the engineering of a synthetic poly-CTL-epitope T-cell immunogen (TCI), which is considered to be a candidate for use as an effective and safe DNA vaccine against HIV-1. 4. Immunochemical study of the TCI gene product In order to ensure that the target gene does express HIV-1 epitopes, the TCI gene was cloned into expression vectors pGEX-2T and pET-32a in E. coli cells. The immunogenic properties of the synthesized TCI protein associated both with GST and TRN-buffer proteins were examined by ELISA using a panel of HIV-1-positive human sera (10 samples) and a panel of HIV-1-negative human sera (10 blood samples). Both chimeric proteins were absolutely detectable on the positive serum panel; however they had no binding activity to any of the negative sera. Therefore, the TCI protein has HIV-1 antigen activity indeed. The antigenic properties of the TCI protein were also examined by immunoblotting using МABs 29F2 and 30A6, which bind to the EPFRDYVDRFYKTLR epitope of HIV-1 р24 and TCI. Acknowledgements This work was supported grants from Russia’s State Scientific and Technical Programs “New Generation Vaccines and Future Medical Diagnostics” and “First Priorities in Science and Technology” subprogram “Protection against Pathogens”. 2. Design of a synthetic immunogen containing multiple CTL epitopes of the major HIV-1 antigens The amino acid sequences of the CTL epitopes implicated in the induction of HIV-1- specific CTL responses in infected individuals are present in the Los Alamos HIV Molecular Immunology Database. In order to be chosen for the CTL-immunogen design, the epitopes were supposed to meet the following eligibility criteria: 1) They induce both CD8 + CTL and CD4 + Th and represent three major HIV-1 subtypes—А, В, and С—occurring in Russia, Western Europe and the USA. 2) They belong to the major viral proteins-antigens: Env, Gag, Pol, and Nef. 3) All the CD8 + CTL-epitopes are restricted by ten different optimally selected HLA class I molecules. As is known, this is sufficient for covering the genetic diversity of МНС class I antigens in whatever geographical region [Hanke 1998a; Lalvani et al., 1994; Sidney et al., 1996]. 4) They may not induce autoimmune antibodies. To meet this criterion, the HIV-1 protein regions that have local homology with human proteins had been identified and the corresponding epitopes excluded from consideration [Maksiutov et al., 2002]. A general schematic of the design of the TCI immunogen is presented in Fig. 1. As can be seen, the target protein is an arrangement of p17, p24, gp120, gp41, Pol, and Nef protein fragments depicted in Fig. 1. The TCI protein is 392 amino acids in length and contains more than eighty CD8 + CTL and CD4 + Th epitopes restricted by HLA class I and II alleles. To be able to study CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, which were restricted by mouse and Macaque rhesus МНС class I molecules, were included in the target immunogen (Fig. 1).. 3. Assembly of a synthetic gene The primary structure of the TCI gene and the corresponding amino acid sequence is presented in Fig. 2. The 1176 bp gene was synthesized by assembling the target gene from three synthetic DNA fragments. Fragments I, II, and III were synthesized by combining a matrix and a matrix-free technique using PCR. A general schematic of the process is presented in Fig. 3. The authenticity of the synthesized TCI gene was confirmed by sequencing. After that the target gene was cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. 5. Comparative analysis of the immunogenicity of DNA vaccine constructs encoding TCI immunogen In this work we carried out comparative estimating immunogenicity of three artificial DNA vaccine constructs obtained on the basis of plasmid pcDNA−TCI, which was engineering using vector pcDNA3.1 (Invitrogen, USA). To compare immunogenicity of DNA vaccine constructs carrying plasmid pcDNA−TCI, mice immunization was realized using “naked” DNA, virus-liked particle (VLP/ pcDNA−TCI, Fig. 4) and attenuated recombinant strain S. typhimurium 7207/pcDNA- TCI. Control animals were immunized with vector plasmid pcDNA3.1 and S. typhimurium strain SL7207 transformed by vector plasmid pcDNA3.1. While plasmid DNA and VLP were administered by intramuscular injection, immunization with Salmonella was performed by the mucosal route (per rectum). The following parameters of the immune response were measured:  CTL response, which was assessed by an ELISPOT assay (Fig. 5);  antibody titers, which were assessed by ELISA using a number of antigens, including the TCI protein, a mixture of recombinant proteins, Gag and Env, and inactivated HIV-1 lysate (Fig. 6);  proliferative response of lymphocytes, which was assessed by blast transformation (Fig. 7). Based on analysis of the immunogenicity of the constructs, the following conclusions were made: 1) All obtained DNA vaccine constructs containing plasmid pcDNA-TCI can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in immunized animals. The most significant responses were produced when pcDNA-TCI was delivered by Salmonella. 2) The use of a full-length TCI protein and separate epitopes (peptides N15 and N16) for in vitro re-stimulation of splenocytes indicate that DNA-immunization incorporates all the stages required for delivery of the target immunogen to the immune system:  expression of the TCI gene,  processing of the target protein, and  presentation of the resulting peptides (determinants) and MHC class I molecules to CD8 + lymphocytes (CTL); 3) The highest titer was the same following immunization with the vaccine construct containing 20  g of plasmid pcDNA-TCI within VLP-(pcDNA-TCI) as with 100  g of naked pcDNA-TCI. Additionally, a more prolonged response was produced by the plasmid within VLP than naked plasmid DNA. 4) All vaccine constructs induce production of specific antibodies, which bind to both the TCI protein and native (HIV-1 lysate) and recombinant (Env and Gag) HIV-1 proteins. This implies that the artificial TCI protein being synthesized in vivo behaves as a HIV- specific immunogen and contains HIV B cell epitopes accessible by the immune system. Additional features were revealed after DNA immunization with naked plasmid pcDNA−TCI. Firstly, a dose-dependent effect is observed after immunization with pcDNA-TCI. An earlier, stronger and more prolonged T and B cell responses are induced by plasmid DNA in a dose of 100 than 20  g. Secondly, re-immunization of animals, which were boosted by plasmid pcDNA-TCI in a dose of 50  g, enhances T and B cell responses. Fig. 2. Nucleotide and amino acid sequences of the TCI gene and TCI- encoded protein The 1176 bp gene encoding TCI immunogen (392 a.o.) was synthesized by assembling the target gene from three synthetic DNA fragments (blocks I, II, III). Fig. 3. The Scheme of Synthesis and Assembly of Gene Encoding CTL Immunogen Fragments (blocks I, II, and III) were synthesized by combining a matrix and a matrix-free technique using PCR Electron microscopy of artificial virus-like particles. Magnification, 70,000. Fig. 4. Diagram of Virus-Like Particles To improve immunogenicity of developing vaccines, original vehicles have been developed in Vector, for targeted delivery of protein and genetic material to target cells for providing of induction of a specific immune response (Sizov et al., 2000; Lebedev et al., 2000). These delivery vehicles are in the form of virus-like particles (VLPs), their structure being as follows (Fig. 4):  internal part (2) is represented by plasmid DNA vaccine construct;  envelope is made of a polyglucine - spermidine conjugate (1 and 3); A B C Fig. 5. IFNγ-ELISPOT responses in groups (n=4) of BALB/c mice immunized with naked pcDNA-TCI, dose 100  g (A); VLP/pcDNA-TCI, dose pcDNA-TCI 20  g (B); and recombinant S. typhimurium SL7207/pcDNA-TCI (C). For all animals splenosytes were separately restimulated in vitro with recombinant TCI (black bars), mixture of peptides N15 and N16 (gray bars) and EHEC polypeptide as a negative control (white bars). The results are expressed as the mean numbers of IFNγ secreting cells (spots) per 106 splenocytes. Error bars represent the standard errors of means. 5000 A B C Fig. 6. Serum IgG titer in groups (n=4) of mice immunized with naked pcDNA-TCI, dose 100  g (white bars); VLP, dose pcDNA-TCI 20  g (gray bars); and recombinant S. typhimurium SL7207/pcDNA-TCI (black bars). Titer of antibodies against recombinant TCI protein (A); mixture of the recombinant Gag and Env proteins (B); and inactivated HIV-1 lysate (C). A B C Fig. 7. Proliferative responses in groups (n=4) of BALB/c mice immunized with naked pcDNA-TCI, dose 100  g (A); VLP, dose pcDNA-TCI 20  g (B); and recombinant S. typhimurium SL7207/pcDNA-TCI (C). Splenosytes form 4 animals were pooled and restimulated in vitro with recombinant TCI (black bars), mixture of peptides N15 and N16 (gray bars) and EHEC polypeptide as a negative control (white bars). The results are expressed as the index of splenocyte proliferation. The bar patterns indicate the schematic representation of the polyepitopes CTL immunogen and 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 CTL immunogen. Th - helper epitopes. Fig. 1. H-2a,b,f Mamu-A*01(SHIV) H-2d,p,u,q H-2k Fig. 1. Design of the CTL immunogen, a candidate for use as an anti-HIV-1 vaccine