Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation

Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics

RNA metabolism is the primary target of formamide in vivo

The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes

A Wide Extent of Inter-Strain Diversity in Virulent and Vaccine Strains of Alphaherpesviruses

Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity, which likely seeds future strain evolution

Analysis and clinical correlation of genetic variation in cytomegalovirus

Background <br/> Cytomegalovirus (CMV) displays genetic polymorphisms in multiple genes, which may result in important virulence differences. Glycoprotein N (gN) and immediate early 1 (IE1) are key viral genes and immune targets. We aimed to characterize the molecular epidemiology of gN and IE1 genotypes in organ transplant patients with CMV disease in the context of clinical and virologic endpoints. <br/> Methods <br/> A total of 240 patients with CMV disease had genotyping analysis by nested polymerase chain reaction assays and sequencing using blood samples obtained at disease onset. Results were correlated with viral clearance kinetics and recurrence. <br/> Results <br/> Complex patterns of gN and IE1 genotypes were present with no clear genetic linkages. No single genotype of IE1 or gN was associated with poorer outcome. For example, different gN or IE1 genotypes had comparable baseline viral load, clearance half-lives, time to clearance, and rates of virologic recurrence. Mixed infection was present at IE1 in 15.8% and gN in 21.9%, but analysis of a single gene was insufficient to detect all mixed infections. Infections caused by multiple strains, as opposed to single strains, were associated with higher baseline viral loads (P = 0.011), delayed viral clearance (P = 0.033), and higher rates of virologic recurrence (P = 0.008). <br/> Conclusions <br/> Genetic diversity in CMV is complex. Specific gN or IE subtypes do not seem to affect in vivo viral virulence patterns in single-strain infections. Mixed infections demonstrate associations with virologic outcomes that single-strain infections do not

Associations among Epstein-Barr virus subtypes, human leukocyte antigen class I alleles, and the development of posttransplantation lymphoproliferative disorder in bone marrow transplant recipients

The association between Epstein-Barr virus subtype, human leukocyte antigen class I alleles, and the development of posttransplantation lymphoproliferative disorder was examined in a group of 25 bone marrow transplant recipients. A highly statistically significant correlation was observed between the human leukocyte antigen B51 allele and development of posttransplantation lymphoproliferative disorder (P=.0016). This study provides, to our knowledge, the first evidence that the human leukocyte antigen B51 allele might predispose bone marrow transplant recipients to Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder