Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

BACKGROUND: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+) and CD8(+) T cells, with the CD8(+) T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell immune responses. HIV-1-specific CD4(+) T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+) T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+) T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env. CONCLUSIONS/SIGNIFICANCE: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines

Cyclin-Dependent Kinase Activity Controls the Onset of the HCMV Lytic Cycle

The onset of human cytomegalovirus (HCMV) lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE) gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK) inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2) cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state

Systems Analysis of MVA-C Induced Immune Response Reveals Its Significance as a Vaccine Candidate against HIV/AIDS of Clade C

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world

Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe