Evaluation of Approaches to Identify the Targets of Cellular Immunity on a Proteome-Wide Scale

Background: Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT) strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products. Principal Findings: We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems. Conclusions: This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re-emerging diseases that pose a threat to public health

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments.

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases

Kartierung der zytotoxischen T-Zell-Antwort gegen zwei humane Herpesviren

Das Humane Cytomegalievirus (HCMV) und das Epstein-Barr-Virus sind weitverbreitet in der Bevölkerung. Beide können schwere Erkrankungen auslösen, die vor allem in immunsupprimierten Infizierten lebensgefährlich werden können. Neben Standardtherapien werden seit geraumer Zeit T-Zell-basierte Immuntherapien wie der adoptive T-Zell-Transfer erfolgreich zur Behandlung eingesetzt. Voraussetzung dafür ist die möglichst umfangreiche Kenntnis der Virus-spezifischen T-Zell-Epitope. Die T-Zell-Epitope beider Viren waren bereits Gegenstand zahlreicher Studien. Dennoch weist die Epitopkartierung in beiden Fällen gewisse Lücken auf. Bei EBV gibt es eine eklatante Lücke bei HLA-A*01-restringierten Epitopen. Während für alle anderen häufigen HLA-Allotypen dominante Epitope bekannt und charakterisiert sind, ist kein einziges HLA-A*01-restringiertes Epitop beschrieben. Aufgrund der Größe von HCMV haben sich Studien bezüglich seiner Epitope dagegen meist auf bestimmte Antigene konzentriert, was ebenfalls eine umfassende Kartierung verhinderte. Ziel dieser Arbeit war es deshalb, neue HCMV-spezifische und HLA-A*01-restringierte, EBV-spezifische T-Zell-Epitope zu identifizieren und zu charakterisieren. Im Falle von EBV wurde hierfür eine Epitopvorhersage durchgeführt, welche die potenziell HLA-A*01-bindenen Peptide im gesamten EBV-Proteom identifizieren sollte. Aus dieser Liste wurden 171 Peptide mittels IFNγ ELISpot Assays auf ihre Immunogenität hin untersucht und nachfolgend die HLA-Restriktion stark immunogener Peptide mittels Durchflusszytometrie verifiziert. Von den untersuchten Peptiden waren 29 immunogen; sieben davon waren dominante Epitope. Für alle dominanten Epitope konnte die HLA-A*01-Restriktion bestätigt und reaktive CD8+ T-Zellen in gesunden Spendern identifiziert werden. Im Gegensatz dazu wurden im zweiten Projekt potenzielle HCMV-Epitope als HLA-Liganden aus mit Virusmutanten infizierten Zelllinien isoliert und danach mittels Massenspektrometrie sequenziert. Von den 181 Liganden konnten 50 (30%) als Epitope charakterisiert werden. Für alle 13 dominanten Epitope konnte nachfolgend die vorhergesagte HLA-Restriktion bestätigt werden. Die Infektion von zwei weiteren Zelllinien erlaubte die Isolation hunderter neuer Peptide, die einer Immunogenitätstestung unterzogen werden können. Da die HLA-Liganden aus über 100 verschiedenen Proteinen stammen, eignet sich diese Methode hervorragend für eine umfangreiche Analyse der Epitope über das gesamte Proteom hinweg. Für beide Viren konnten damit neue, stark immunogene Epitope identifiziert werden, die in Zukunft für Antigen-spezifische Immuntherapien Verwendung finden können.Both human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are widespread in the population. They can cause severe morbidity and mortality, especially in immunocompromised hosts. Apart from the standard treatment with antiviral drugs, T cell-based immune therapies such as adoptive T cell transfer have been successfully used in recent years. A prerequisite for this treatment is the comprehensive knowledge about virus-specific T cell epitopes. The T cell epitopes of both viruses have already been the subject of numerous studies. Nevertheless, the epitope mapping in both cases shows certain gaps. In EBV there is a striking gap in HLA-A*01-restricted epitopes. While dominant epitopes are known and characterized for all other common HLAs, not a single useful HLA-A*01-restricted epitope has been described. In contrast, due to the size of HCMV, studies on its epitopes have mostly focused on specific antigens, which also prevented comprehensive mapping. The aim of this work was therefore the identification and characterization of novel HCMV-specific and HLA-A*01-restricted, EBV-specific T cell epitopes. To this end, epitope prediction was performed to identify HLA-A*01-binding peptides throughout the entire EBV proteome. From this list of peptides, 171 peptides were evaluated for immunogenicity using IFNγ ELISpot assays. Subsequently, the HLA restriction of strongly immunogenic peptides was verified via flow cytometry. Of the tested peptides, 29 were immunogenic; seven of them were dominant epitopes. For all dominant epitopes the HLA-A*01 restriction was confirmed and reactive CD8+ T cells in healthy donors were identified. In the second project, candidate HCMV epitopes were isolated as HLA ligands from cell lines infected with HCMV mutants and then sequenced by mass spectrometry. Of the 181 ligands, 50 (30%) could be characterized as epitopes. For all 13 dominant epitopes the predicted HLA restriction could be confirmed. The infection of two further cell lines allowed the isolation of hundreds of new peptides, which can be subjected to immunogenicity testing. Since the HLA ligands originate from more than 100 different proteins, this method is ideally suited for an unbiased, comprehensive analysis of the epitopes throughout the entire proteome. In summary, novel, strongly immunogenic epitopes were identified for both viruses, which could be employed for vaccination and antigen-specific immunotherapy in the future

Demonstration of new subtypes of adenovirus 7 in South Africa, and probing oesophageal carcinoma cell lines for evidence of adenovirus or of other oncogenic viruses

This study was carried out in 2 parts: 1. Genome analysis of human adenovirus species 7; 2. Search for a possible viral aetiology in oesophageal carcinoma. Sixteen laboratory isolates of adenovirus species 7, isolated in South Africa between 1975 and 1986, were characterized by restriction endonuclease analysis of their DNA genomes. Virus was propagated in human embryo fibroblast cells; genomic DNA, extracted and purified from cellular DNA extracts, was analyzed using 9 different restriction enzymes. Results of this study have demonstrated 2 new genome types of adenovirus 7c which have not previously been identified. The 2 novel strains, designated as genome types Ad7c1 and Ad7c2, were shown to differ from prototype Ad7 c according to restriction profiles with EcoRI; 2 new EcoRI sites were demonstrated in Ad7c1 and 1 in Ad7c2. The restriction sites were mapped on the viral genomes (at 3.68kb and 5.32kb from the left terminus) by double enzyme digestions, cloning of viral DNA, and nucleic acid hybridization using a cloned Ad7 probe. Strains resembling the prototype Ad7c and Ad7p (Gomen) genome types were also identified in the 1985 and 1986 Ad7 isolates. In order to investigate the possible role of a viral co-factor in the aetiology of oesophageal carcinoma, 18 probes, derived from potentially oncogenic viruses, were used to screen 3 human oesophageal carcinoma cell lines for the possible presence of integrated viral DNA. One of these, an Ad7 recombinant plasmid probe, was developed by cloning DNA from the transforming region of the Ad7cl strain into the plasmid vector pUC19. Cellular DNA, extracted from the 3 tumor lines HCU18, HCU33 and HCU39, was tested by means of both DNA dot hybridization and Southern blot hybridization for the presence of Epstein-Barr virus, human papillomavirus (types 1, 5, 6, 8, 11, 16, 18), human adenovirus (strains 5, 7, 12, 31) and human T-lymphotropic virus type I DNA. Both assays were demonstrated to be sensitive enough to detect 1 copy of viral DNA per cell. No hybridization between HPV, EBV, HTLV-I or adenovirus DNA probes, and the cellular DNA was detected. These findings indicate that the stable integration of these tumor viruses in host chromosomes did not play a role in the maintenance of the malignant phenotype of the 3 extensively passaged cell lines. Cells of the 3 oesophageal tumor lines were further examined by transmission electron microscopy, but the presence of virus particles in these cells was not observed

The role of specific MHV-68 genes in persistent infection in the lung and virus pathogenesis

The gammaherpesvirus subfamily has long been the study of intensive investigation owing to the association between infection and development of lymphoproliferative disease. Well-known members ofthe Gammaherpesvirinae include Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV). Common properties of gammaherpesviruses include a narrow host range of infection and limited productive growth in vitro, and these factors make the study of acute infection problematic. Murine gammaherpesvirus-68 (MHV-68) is able to undergo lytic replication in a range of cell types in vitro and can infect inbred strains of mice. These properties make MHV-68 an excellent model for the study of gammaherpesvirus pathogenesis.Herpesviruses have been indicated in development of diseases in the lung, including pneumonia and idiopathic pulmonary fibrosis. MHV-68 allows investigation of gammaherpesvirus infection of and persistence in the lung - following intranasal inoculation the virus establishes a life-long infection in this organ, with virus persisting in epithelial cells and/or B cells. Identification of key viral genes required for persistence may allow for development of vaccination and/or treatment strategies. Using real-time PCR the long-term viral load in the lungs was reduced following the deletion of key genes from the viral genome. Genes identified are the thymidine kinase gene, previously shown to play a role during acute infection of the lung and ORF73, a homologue of the KSHV LANA-1 gene. Initial data also suggests that the ORF72 and Mil genes, both involved in reactivation from latency, may play a role in maintaining viral load at late time points post-infection.In vivo investigation of the Ml gene of MHV-68 has demonstrated a potential role in control of viral reactivation from latency in the spleen. A novel MHV-68 mutant, MIA, lacking 1171 bp of the Ml ORF, was used to study the role of Ml in pathogenesis. Initial data suggests that in vivo infection with MIA results in increased viral titres during acute infection of the lung, indicating a potential role in control of initial infection. The major role of Ml appears to be during acute phase latency in the spleen, with the MIA virus failing to drive splenomegaly and establishing latency at lower levels. Despite the presence of fewer latently infected splenocytes, MIA reactivates at significantly higher levels, indicating that a function of Ml is to control viral reactivation from latency.A viral mutant (M4Inl) was created that carries a stop codon inserted at genome co¬ ordinate 8386 in the region between the M3 and M4 genes. The mutation is thought to be in an untranscribed region of the genome, potentially in the promoter region of the M3 or M4 genes. Studies demonstrated that the virus is attenuated following infection of both wild-type and IFNyR " mice with respect to lung pathology scores. The lethality of M4Inl in juvenile IFNyR." mice is reduced compared with wild-type MHV-68 infection. Despite the location of the mutation within potential promoter regions, M4Inl transcribes both M3 and M4 at wild type levels in vitro, and in vivo in the spleen. This evidence suggests an apparently untranscribed region of the MHV-68 genome is able to influence pathogenesis in the lung independent of the neighbouring genes

Ribosome Profiling of EBV-infected cells

The Epstein-Barr-Virus is an oncogenic herpesvirus that establishes a lifelong infection in humans. It is ubiquitous in the population and is responsible for the development of multiple diseases including cancer. Analysis of the EBV DNA and RNA sequences has predicted that the viral genome encodes approximately 100 protein-coding genes. However, the existence of many putative proteins has not been confirmed by biochemical methods yet. Furthermore, the identification of EBV open reading frames (ORFs) is difficult as viral genes are encoded on both strands of the double-stranded DNA genome and often overlap. Moreover, EBV encodes different types of non-coding RNAs. In order to identify the full scope of EBV’s coding capacity, ribosome profiling of replicating and non-replicating EBV strains was performed. Ribosome profiling combines classical ribosome footprinting experiments with current deep sequencing technology to map translating ribosomes on mRNA at single nucleotide resolution. This approach confirmed the majority of previously identified ORFs and has enabled the identification of 28 novel small open reading frames and of 8 alternative translation initiation sites. 25 of the 28 small ORFs were localized in the 5’leaders of several mRNA transcripts and are classified as upstream open reading frames (uORFs). Several of these uORFs were found to repress the translation of the downstream encoded main ORF. In summary, ribosome profiling of EBV-infected cells has allowed a comprehensive identification and annotation of the EBV ORFs and has revealed a novel mode of viral gene expression regulation at the translational level