Creation of superagonist epitope sequences in the hepatitis B envelope protein using mutagenesis and DNA vaccination.

DNA vaccination is a simple and efficient method for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we have examined the effect of the mutations of each of the 12 amino acids of the HBsAg Ld-restricted CTL epitope on the ability of the modified proteins to induce CTLs after DNA-based immunization. Replacement of glutamine or serine by alanine codons in the whole envelope gene created a protein that induced higher CTL activity against cells bearing the wildtype peptide-MHC complex than against the wildtype sequence itself. These results represent the first example of immunogenic mutant sequences (superagonists) that induce higher CTL activity against the wildtype CTL epitope than does the wildtype protein. Because the entire mutant protein is being expressed from the modified plasmid, any of the various steps in epitope processing could be affected by the mutations and lead to increased class I immunogenicity of the peptide sequence.

Bioinformatics tools for identifying class I-restricted epitopes.

The lack of simple methods to identify relevant T-cell epitopes, the high mutation rate of many pathogens, and restriction of T-cell response to epitopes due to human lymphocyte antigen (HLA) polymorphism have significantly hindered the development of cytotoxic T-lymphocyte (CTL) epitope-based or "epitope-driven" vaccines. Previously, CTL epitopes were mapped using large arrays of overlapping synthetic peptides. The large number of protein sequences available for mapping is now making this method prohibitively expensive and time-consuming. Bioinformatics tools such as EpiMatrix and Conservatrix, which search for unique or multi-HLA-restricted (promiscuous) T-cell epitopes and identify epitopes that are conserved across variant strains of the same pathogen, accelerate epitope mapping. These tools offer a significant advantage over other methods of epitope selection because high-throughput screening can be performed in silico, followed by confirmatory studies in vitro. CTL epitopes discovered using these tools might be used to develop novel vaccines and therapeutics for the prevention and treatment of infectious diseases such as human immunodeficiency virus, hepatitis C, tuberculosis, and some cancers.

Immuno-informatics: Mining genomes for vaccine components.

The complete genome sequences of more than 60 microbes have been completed in the past decade. Concurrently, a series of new informatics tools, designed to harness this new wealth of information, have been developed. Some of these new tools allow researchers to select regions of microbial genomes that trigger immune responses. These regions, termed epitopes, are ideal components of vaccines. When the new tools are used to search for epitopes, this search is usually coupled with in vitro screening methods; an approach that has been termed computational immunology or immuno-informatics. Researchers are now implementing these combined methods to scan genomic sequences for vaccine components. They are thereby expanding the number of different proteins that can be screened for vaccine development, while narrowing this search to those regions of the proteins that are extremely likely to induce an immune response. As the tools improve, it may soon be feasible to skip over many of the in vitro screening steps, moving directly from genome sequence to vaccine design. The present article reviews the work of several groups engaged in the development of immuno-informatics tools and illustrates the application of these tools to the process of vaccine discovery.

Role of transfection in the priming of cytotoxic T-cells by DNA-mediated immunization.

DNA vaccination results in remarkably strong, broad-based immune responses to the encoded proteins and it is a simple and effective method of inducing cytotoxic T-lymphocyte (CTL) responses. Bone marrow-derived cells can take up and present exogenous antigenic protein liberated by transfected fibroblasts or myoblasts after the injection of such cells. In addition, dendritic cells can carry the injected plasmid DNA, supporting the hypothesis that dendritic cells can be directly transfected. It is, however, unclear from the current data what proportion of the cytotoxic immune response is initiated by the transfer of protein compared to that resulting from direct transfection of professional antigen presenting cells. This question is addressed here by using a matched series of plasmid DNA vectors expressing the wild-type or several mutant forms of HBsAg that are secretion-defective or severely truncated. The data indicate that neither HBsAg particle formation nor its secretion or liberation plays a significant role in the development of the cytotoxic immune response. The results argue that direct transfection of bone marrow-derived cells is the major, and possibly the only, mechanism used for priming of naive CTL precursors directed against the HBsAg.

Mapping cross-clade HIV-1 vaccine epitopes using a bioinformatics approach.

UNLABELLED: The genomic variability of HIV viruses circulating in different regions of the world has impeded the development of a globally relevant HIV vaccine. Broadly conserved HIV-1 cytotoxic T cell (CTL) epitopes were identified by screening protein sequences in the Los Alamos National Laboratory (LANL) HIV sequence database with a sequence parsing and matching algorithm (Conservatrix). Putative HIV-1 CTL epitopes were selected from this list using the epitope prediction tool EpiMatrix. METHODS: One hundred peptides representing putative HLA A*0201, HLA A*1101, HLA A*0301, and HLA B*07 ligands conserved in many isolates of HIV-1 were synthesized. Seventy-five HLA A*0201, HLA A*1101 and HLA B*07 peptides were incubated with transport associated protein (TAP)-deficient T2 cells transfected with the gene for the corresponding human HLA molecule (HLA A*0201, HLA A*1101, and HLA B*07). Binding and stabilization of peptide-HLA complexes on the surface of the T2 cells was measured by FACS. T cell responses to the entire set of 100 peptides (HLA A*0201, HLA A*1101, HLA A*0301, and HLA B*07) were measured in ELIspot assays using PBMC from healthy HIV-1 infected subjects who possessed a matching HLA allele. RESULTS: Fifty-seven (76%) of the 75 peptides tested in binding studies, including all (three of three) of the control (published) ligands bound to the T2 cells expressing the corresponding MHC molecule. Forty-three of the 100 peptides (43%) including all (four of four) of the control (published) epitopes tested in ELIspot assays stimulated gamma-interferon release. Thirty-one of these 43 epitopes are novel, highly conserved HIV-1 epitopes. EpiMatrix predicted and assays confirmed MHC-restriction by more than one HLA allele for nine of the 43 novel epitopes; of these epitopes five were recognized in the context of MHC "supertypes" and four were promiscuous epitopes. CONCLUSION: Epitopes identified using this approach were conserved in a broad range of HIV-1 sequences derived from isolates obtained in Latin America, Africa, Asia, the Pacific Islands, Europe and the US. The successful identification of cross-clade epitopes by this bioinformatics approach may accelerate the development of a globally relevant HIV-1 vaccine.