Dan Frampton

RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

Emergence of a novel subclade of influenza A(H3N2) virus in London, December 2016 to January 2017

We report the molecular investigations of a large influenza A(H3N2) outbreak, in a season characterised by sharp increase in influenza admissions since December 2016. Analysis of haemagglutinin (HA) sequences demonstrated co-circulation of multiple clades (3C.3a, 3C.2a and 3C.2a1). Most variants fell into a novel subclade (proposed as 3C.2a2); they possessed four unique amino acid substitutions in the HA protein and loss of a potential glycosylation site. These changes potentially modify the H3N2 strain antigenicity.

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

Using nearly full-genome HIV sequence data improves phylogeny reconstruction in a simulated epidemic

HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to determine what gene(s) provide(s) the best approximation to the real phylogeny by analysing a simulated epidemic (created as part of the PANGEA_HIV project) with a known transmission tree. We sub-sampled a simulated dataset of 4662 sequences into different combinations of genes (gag-pol-env, gag-pol, gag, pol, env and partial pol) and sampling depths (100%, 60%, 20% and 5%), generating 100 replicates for each case. We built maximum-likelihood trees for each combination using RAxML (GTR + Γ), and compared their topologies to the corresponding true tree’s using CompareTree. The accuracy of the trees was significantly proportional to the length of the sequences used, with the gag-pol-env datasets showing the best performance and gag and partial pol sequences showing the worst. The lowest sampling depths (20% and 5%) greatly reduced the accuracy of tree reconstruction and showed high variability among replicates, especially when using the shortest gene datasets. In conclusion, using longer sequences derived from nearly whole genomes will improve the reliability of phylogenetic reconstruction. With low sample coverage, results can be highly variable, particularly when based on short sequences.

Proteins accessible to immune surveillance show significant T-cell epitope depletion: Implications for vaccine design

T cell activation is the final step in a complex pathway through which pathogen-derived peptide fragments can elicit an immune response. For it to occur, peptides must form stable complexes with Major Histocompatibility Complex (MHC) molecules and be presented on the cell surface. Computational predictors of MHC binding are often used within in silico vaccine design pathways. We have previously shown that, paradoxically, most bacterial proteins known experimentally to elicit an immune response in disease models are depleted in peptides predicted to bind to human MHC alleles. The results presented here, derived using software proven through benchmarking to be the most accurate currently available, show that vaccine antigens contain fewer predicted MHC-binding peptides than control bacterial proteins from almost all subcellular locations with the exception of cell wall and some cytoplasmic proteins. This effect is too large to be explained from the undoubted lack of precision of the software or from the amino acid composition of the antigens. Instead, we propose that pathogens have evolved under the influence of the host immune system so that surface proteins are depleted in potential MHC-binding peptides, and suggest that identification of a protein likely to contain a single immuno-dominant epitope is likely to be a productive strategy for vaccine design.

Molecular Signatures of Regression of the Canine Transmissible Venereal Tumor

Summary The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified CCL5 as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

Host Genetic Variants and Gene Expression Patterns Associated with Epstein-Barr Virus Copy Number in Lymphoblastoid Cell Lines

Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs.

Pre/pro-B cells generate macrophage populations during homeostasis and inflammation

Significance In this report we provide evidence of a source of macrophage (Mφ) populations that are derived from unique biphenotypic early pro-B cells with non-rearranged B-cell receptors. These early precursors give rise to either tissue resident- or monocyte-derived Mφs during homeostasis and inflammatory responses, thereby demonstrating functional plasticity depending on the environmental cues in adult mice. We suggest that these findings significantly advance and expand our understanding of Mφ biology and hematopoiesis, the plasticity of hematopoietic precursors, and the heterogeneity of Mφ subsets. Most tissue-resident macrophages (Mφs) are believed to be derived prenatally and are assumed to maintain themselves throughout life by self-proliferation. However, in adult mice we identified a progenitor within bone marrow, early pro-B cell/fraction B, that differentiates into tissue Mφs. These Mφ precursors have non-rearranged B-cell receptor genes and coexpress myeloid (GR1, CD11b, and CD16/32) and lymphoid (B220 and CD19) lineage markers. During steady state, these precursors exit bone marrow, losing Gr1, and enter the systemic circulation, seeding the gastrointestinal system as well as pleural and peritoneal cavities but not the brain. While in these tissues, they acquire a transcriptome identical to embryonically derived tissue-resident Mφs. Similarly, these Mφ precursors also enter sites of inflammation, gaining CD115, F4/80, and CD16/32, and become indistinguishable from blood monocyte-derived Mφs. Thus, we have identified a population of cells within the bone marrow early pro-B cell compartment that possess functional plasticity to differentiate into either tissue-resident or inflammatory Mφs, depending on microenvironmental signals. We propose that these precursors represent an additional source of Mφ populations in adult mice during steady state and inflammation.

Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells

Early on in human immunodeficiency virus (HIV) type 1 infection, gut T-helper (Th) 17 cells are massively depleted leading eventually to compromised intestinal barrier function and excessive immune activation. In contrast, the functional Th17 cell compartment of the gut is well-maintained in nonpathogenic simian immunodeficiency virus infection as well as HIV-1 long-term nonprogressors. Here, we show that dendritic cells (DCs) loaded with HIV-1 bearing high surface complement levels after incubation in plasma from HIV-infected individuals secreted significantly higher concentrations of Th17-polarizing cytokines than DCs exposed to nonopsonized HIV-1. The enhanced Th17-polarizing capacity of in vitro-generated and BDCA-1(+) DCs directly isolated from blood was linked to activation of ERK. In addition, C3a produced from DCs exposed to complement-opsonized HIV was associated with the higher Th17 polarization. Our in vitro and ex vivo data, therefore, indicate that complement opsonization of HIV-1 strengthens DC-mediated antiviral immune functions by simultaneously triggering Th17 expansion and intrinsic C3 formation via DC activation.

Use of Whole-Genome Sequencing in the Investigation of a Nosocomial Influenza Virus Outbreak

Abstract Traditional epidemiological investigation of nosocomial transmission of influenza involves the identification of patients who have the same influenza virus type and who have overlapped in time and place. This method may misidentify transmission where it has not occurred or miss transmission when it has. We used influenza virus whole-genome sequencing (WGS) to investigate an outbreak of influenza A virus infection in a hematology/oncology ward and identified 2 separate introductions, one of which resulted in 5 additional infections and 79 bed-days lost. Results from WGS are becoming rapidly available and may supplement traditional infection control procedures in the investigation and management of nosocomial outbreaks.