From genome to vaccine: in silico predictions, ex vivo verification.

Bioinformatics tools enable researchers to move rapidly from genome sequence to vaccine design. EpiMer and EpiMatrix are computer-driven pattern-matching algorithms that identify T cell epitopes. Conservatrix, BlastiMer, and Patent-Blast permit the analysis of protein sequences for highly conserved regions, for homology with other known proteins, and for homology with previously patented epitopes, respectively. Two applications of these tools to epitope-driven vaccine design are described in this review. Using Conservatrix and EpiMatrix, we analyzed more than 10000 HIV-1 sequences and identified peptides that were potentially immunostimulatory and highly conserved across HIV-1 clades. MHC binding assays and CTL assays have been carried out: 50 (69%) of the 72 candidate epitopes bound in assays with cell lines expressing the corresponding MHC molecule; 15 of the 24 B7 peptides (63%) stimulated gamma-interferon release in ELISpot assays. These results lend support to the bioinformatics approach to selecting novel, conserved, HIV-1 CTL epitopes. EpiMatrix was also applied to the entire 'proteome' derived from two Mycobacterium tuberculosis (Mtb) genomes. Using EpiMatrix, BlastiMer, and Patent-Blast, we narrowed the list of putative Mtb epitopes to be tested in vitro from 1600000 to 3000, a 99.8% reduction. The pace of vaccine design will accelerate when these and other bioinformatics tools are systematically applied to whole genomes and used in combination with in vitro methods for screening and confirming epitopes.

Prediction of well-conserved HIV-1 ligands using a matrix-based algorithm, EpiMatrix.

This preliminary study was undertaken to identify new human leucocyte antigens (HLA) ligands from human immunodeficiency virus type 1 (HIV-1) which are highly conserved across HIV-1 clades and which may serve to induce cross-reactive cytotoxic T lymphocytes (CTLs). EpiMatrix was used to predict putative ligands from HIV-1 for HLA-A2 and HLA-B27. Twenty-six peptides that were both likely to bind and also highly conserved across HIV-1 strains in the Los Alamos HIV sequence database were selected for binding assays using the T2 stabilization assay. Two peptides that were also highly likely to bind (for A2 and B27, as determined by EpiMatrix) and well conserved across HIV-1 strains, and had previously been described to bind in the published literature, were also selected to serve as positive controls for the assays. Ten new major histocompatibility complex (MHC) ligands were identified among the 26 study peptides. The control peptides bound, as expected. These data confirm that EpiMatrix can be used to screen HIV-1 protein sequences for highly conserved regions that are likely to bind to MHC and may prove to be highly conserved HIV-1 CTL epitopes.

Co-infection with Mycobacterium tuberculosis and HIV in high risk clinical care setting in Rhode Island.

We assessed the extent to which co-infection with HIV and Mycobacterium tuberculosis (Mtb) was diagnosed at several high risk clinical care sites from 1992 to 1994 to determine whether surveillance for co-infection was performed. Information on PPD skin testing, HIV status, and HIV risk exposures was extracted from records at HIV clinics in Rhode Island and a large database (HIV sites) and from records at the state TB clinic and the Rhode Island Health Department (TB sites). At the HIV sites, 34 of 1,408 HIV infected subjects were newly diagnosed with Mtb infection in the study period. At the TB sites, 16 of 1,389 subjects with newly diagnosed Mtb infection or disease were identified as HIV infected. Eighty per cent of the records reviewed for this study were incomplete. Hispanic subjects were at higher risk of being identified as co-infected at the HIV sites. At the TB sites, US-born subjects were at higher risk of being identified as co-infected Recommendations for high risk individuals include yearly tuberculosis skin testing. Adherence to these guidelines in selected high-risk clinical care sites in Rhode Island was substandard during the study period; the importance of Mtb screening was demonstrated in this study. Identification of groups that are at higher risk of having HIV and Mtb co-infection identified may enable health care providers to improve testing and prevention of tuberculosis at high risk clinical care settings.

An interactive Web site providing major histocompatibility ligand predictions: application to HIV research.

EpiMatrix/HIV, a tool that is currently available on the World Wide Web, enables researchers to screen HIV proteins for potential MHC ligands. We have performed a comparison of EpiMatrix predictions to 158 published allotype-specific HLA-associated peptides (MHC ligands) derived from 133 proteins. The top 10 ranked EpiMatrix predictions for each of the 158 HLA allotype-protein pairs were selected for comparison with these published ligands. EpiMatrix correctly identified 134 of 158 published ligands (85%). The algorithm is now available for use by the HIV research community at the URL http:/(/)www.EpiMatrix.com/HIV.

Prediction of HIV peptide epitopes by a novel algorithm.

Identification of promiscuous or multideterminant T cell epitopes is essential for HIV vaccine development, however, current methods for T cell epitope identification are both cost intensive and labor intensive. We have developed a computer-driven algorithm, named EpiMer, which searches protein amino acid sequences for putative MHC class I- and/or class II-restricted T cell epitopes. This algorithm identifies peptides that contain multiple MHC-binding motifs from protein sequences. To evaluate the predictive power of EpiMer, the amino acid sequences of the HIV-1 proteins nef, gp160, gag p55, and tat were searched for regions of MHC-binding motif clustering. We assessed the algorithm's predictive power by comparing the EpiMer-predicted peptide epitopes to T cell epitopes that have been published in the literature. The EpiMer method of T cell epitope identification was compared to the standard method of synthesizing short, overlapping peptides and testing them for immunogenicity (overlapping peptide method), and to an alternate algorithm that has been used to identify putative T cell epitopes from primary structure (AMPHI). For the four HIV-1 proteins analyzed, the in vitro testing of EpiMer peptides for immunogenicity would have required the synthesis of fewer total peptides than either AMPHI or the overlapping peptide method. The EpiMer algorithm proved to be more efficient and more sensitive per amino acid than both the overlapping peptide method and AMPHI. The EpiMer predictions for these four HIV proteins are described. Since EpiMer-predicted peptides have the potential to bind to multiple MHC alleles, they are strong candidates for inclusion in a synthetic HIV vaccine.