Gut bacteria characteristic of the infant microbiota down-regulate inflammatory transcriptional responses in HT-29 cells.

Immuno-modulatory effects of infant gut bacteria were tested on poly(I:C) stimulated HT-29 intestinal epithelial cells. Blautia producta, Bacteroides vulgatus, Bacteroides fragilis and Bacteroides thetaiotaomicron decreased transcription of poly(I:C)-induced inflammatory genes. Modulation of basal level and poly(I:C)-induced IL-8 secretion varied between bacterial species, and between heat treated and non-heat treated bacterial cells.

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

BACKGROUND: Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. RESULTS: The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. CONCLUSIONS: In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

Hyperproliferation, cancer, and inflammation in mice expressing a Δ133p53-like isoform.

The p53 protein is a pivotal tumor suppressor that is frequently mutated in many human cancers, although precisely how p53 prevents tumors is still unclear. To add to its complexity, several isoforms of human p53 have now been reported. The Δ133p53 isoform is generated from an alternative transcription initiation site in intron 4 of the p53 gene (Tp53) and lacks the N-terminus. Elevated levels of Δ133p53 have been observed in a variety of tumors. To explore the functions of Δ133p53, we created a mouse expressing an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53. Δ122p53 mice show decreased survival and a different and more aggressive tumor spectrum compared with p53 null mice, implying that Δ122p53 is a dominant oncogene. Consistent with this, Δ122p53 also confers a marked proliferative advantage on cells and reduced apoptosis. In addition to tumor development, Δ122p53 mice show a profound proinflammatory phenotype having increased serum concentrations of interleukin-6 and other proinflammatory cytokines and lymphocyte aggregates in the lung and liver as well as other pathologies. Based on these observations, we propose that human Δ133p53 also functions to promote cell proliferation and inflammation, one or both of which contribute to tumor development.

Cross-presentation of epitopes on virus-like particles via the MHC I receptor recycling pathway.

Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.

The chemokine-binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes.

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine-binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV-CBP could disable these gradients using mouse models. Previously we established that ORFV-CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell-based chemotaxis assays, ORFV-CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood-derived DC to lipopolysaccharide-induced sites of skin inflammation and inhibited the migration of ex vivo CpG-activated DC to inguinal lymph nodes. Finally we showed that ORFV-CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.

Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata.

BACKGROUND: Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. RESULTS: Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. CONCLUSIONS: This analysis provides evidence that two distinct secretome genes families have evolved under contrasting selective pressures. The data supports current hypotheses regarding the biological role of TashAT family proteins in the management of host cell phenotype that may have evolved to allow adaptation of T. annulata to a specific host cell lineage. We provide new evidence of extensive allelic diversity in representative members of the enigmatic SVSP gene family, which supports a putative role for the encoded products in subversion of the host immune response.

Gut commensal Lactobacillus reuteri 100-23 stimulates an immunoregulatory response.

Lactobacillus reuteri 100-23 is a bacterial commensal of the gastrointestinal tract of mice. Previous studies have shown that colonization of the murine gut by this strain stimulates small-bowel enterocytes to produce proinflammatory cytokines. This is associated with a mild, transitory inflammatory response 6 days after inoculation of formerly Lactobacillus-free animals. The inflammation subsides by 21 days after colonization, although lactobacilli continue to be present in the bowel. To determine the immunological mechanisms that underpin tolerance to bowel commensals, we investigated cytokine responses of dendritic cells and T cells after exposure to cells of L. reuteri 100-23. Interleukin-10 (IL-10), IL-2 and transforming growth factor-beta (TGF-beta) concentrations in supernatants of cultured immune cells, as well as the results of proliferative assays of mesenteric lymph node (MLN) cells and quantification of Foxp3-positive cells in MLN and spleen, indicated that L. reuteri 100-23 stimulated the development of an increased number of regulatory T cells.

Population genomics of louping ill virus provide new insights into the evolution of tick-borne flaviviruses.

The emergence and spread of tick-borne arboviruses pose an increased challenge to human and animal health. In Europe this is demonstrated by the increasingly wide distribution of tick-borne encephalitis virus (TBEV, Flavivirus, Flaviviridae), which has recently been found in the United Kingdom (UK). However, much less is known about other tick-borne flaviviruses (TBFV), such as the closely related louping ill virus (LIV), an animal pathogen which is endemic to the UK and Ireland, but which has been detected in other parts of Europe including Scandinavia and Russia. The emergence and potential spatial overlap of these viruses necessitates improved understanding of LIV genomic diversity, geographic spread and evolutionary history. We sequenced a virus archive composed of 22 LIV isolates which had been sampled throughout the UK over a period of over 80 years. Combining this dataset with published virus sequences, we detected no sign of recombination and found low diversity and limited evidence for positive selection in the LIV genome. Phylogenetic analysis provided evidence of geographic clustering as well as long-distance movement, including movement events that appear recent. However, despite genomic data and an 80-year time span, we found that the data contained insufficient temporal signal to reliably estimate a molecular clock rate for LIV. Additional analyses revealed that this also applied to TBEV, albeit to a lesser extent, pointing to a general problem with phylogenetic dating for TBFV. The 22 LIV genomes generated during this study provide a more reliable LIV phylogeny, improving our knowledge of the evolution of tick-borne flaviviruses. Our inability to estimate a molecular clock rate for both LIV and TBEV suggests that temporal calibration of tick-borne flavivirus evolution should be interpreted with caution and highlight a unique aspect of these viruses which may be explained by their reliance on tick vectors.

Functionalisation of Virus-Like Particles Enhances Antitumour Immune Responses.

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

The Δ133p53ß isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53ß isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53ß expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53ß as a highly accurate prognostic factor for aggressive prostate cancer.

∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling.

∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.

Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes.

The results of adoptive T-cell therapies (ACTs) are very encouraging and show clinical evidence that ACT can provide a cure for patients with metastatic disease. However, various response rates and long-term cancer remission have been observed in different ACT trials. The types of T cells, prior treatment with chemotherapy and co-administration of other immune-target therapies have been found to influence the efficacy of ACT. In this study, we investigate the ability of ACT using CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) to reject the growth of established B16-ovalbumin (OVA) melanoma. CD8+ CTLs were found to be the main effector T cells that mediated tumour regression. However, low tumour-free survival rates were observed in ACT with CD8+ CTLs only. Co-transferring CD4+ Th1 cells and CD8+ CTLs has been observed to induce a synergistic antitumour response, resulting in complete regression in 80% of the tumour-bearing mice. We also examined a prior Dacarbazine (DTIC) and after virus-like particle (VLP)-OVA vaccine treatment to enhance ACT, but no therapeutic benefit was observed during primary B16-OVA tumour growth. Nevertheless, the ACT-mediated antitumour response was able to generate memory responses to both B16-OVA and B16-gp33 tumours. VLP-OVA vaccination following ACT enhances the memory responses to tumours that express a heterogenic population of both B16-OVA and B16-gp33 cells; however, it abolished the memory response to tumours consisting of only gp33-expressing cells. These findings provide important information for designing therapeutic treatments for patients with metastatic disease and cancer relapse to achieve durable cancer remission.

Combining dendritic cells and B cells for presentation of oxidised tumour antigens to CD8+ T cells.

The dendritic cell (DC) is the foremost antigen-presenting cell (APC) for ex vivo expansion of tumour-specific patient T cells. Despite marked responses in some patients following reinfusion of DC-activated autologous or HLA-matched donor T cells, overall response rates remain modest in solid tumours. Furthermore, most studies aim to generate immune responses against defined tumour-associated antigens (TAA), however, meta-analysis reveals that those approaches have less clinical success than those using whole tumour cells or their components. Tumour lysate (TL) is used as a source of tumour antigen in clinical trials and potentially represents the full range of TAAs in an undefined state. Little is known about how different APCs cooperate to present TL antigens. We examined the effect of oxidised whole-cell lysate (ox-L) versus soluble fraction freeze-thaw lysate (s-L) on bone marrow-derived DCs and macrophages, and magnetic bead-isolated splenic B cells. The APCs were used individually, or in combination, to prime T cells. CD8+ T cells produced interferon (IFN)-γ in response to both s-L and ox-L, but only proliferated in response to ox-L. IFN-γ production and proliferation was enhanced by priming with the DC+B cell combination. Compared to DC alone, a trend toward greater interleukin (IL)-12 production was observed when DC+B cell were loaded with s-L and ox-L antigens. CD8+ T-cell specific lysis in vivo was greatest in ox-L-primed groups and DC+B cell priming significantly increased in vivo cytotoxicity compared to DC alone. These improved T-cell responses with two APCs and stressed cell lysate has implications for APC-based adoptive cell therapies.

Tumor protein 53 mutations are enriched in diffuse large B-cell lymphoma with irregular CD19 marker expression.

Accumulating evidence suggests tumor protein 53 (p53) promotes correct cellular differentiation. Thus, mutant TP53 may be more frequent in tumors with irregular differentiation. This study investigated whether TP53 mutations were more frequent in diffuse large B cell lymphoma (DLBCL) that lacked the B cell lineage marker CD19. Sixteen CD19 negative and 78 CD19 positive DLBCL were sequenced for TP53 mutations. Twenty nine tumors had TP53 mutations and were associated with poorer survival. Mutant TP53 was more frequent in CD19 negative lymphomas (81% versus 21%, p < 0.0001). Analysis of other B cell markers revealed a lack of paired box 5 (PAX5) in CD19 positive lymphomas with mutant TP53 (50%), which was more frequent compared to tumors with wild-type TP53 (15%, p = 0.002). In summary, DLBCL lacking CD19 or PAX5 expression were more likely to have mutant TP53, suggesting irregular B cell marker phenotypes are associated with TP53 mutation.

Conditions for the generation of cytotoxic CD4(+) Th cells that enhance CD8(+) CTL-mediated tumor regression.

Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8(+) cytotoxic T lymphocytes (CTL), others have shown that the presence of tumor-reactive CD4(+) T helper (Th) cells can greatly enhance the anti-tumor activity of CD8(+) CTL. However, difficulties in obtaining adequate numbers of CD4(+) Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study aims to optimize the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4(+) Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4(+) Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4(+) Th1-like cells with CD8(+) CTL significantly enhanced tumor regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4(+) Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8(+) CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4(+) Th1-like cells, with potential applications to cancer treatment involving ACT.

Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12.

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

Mannosylation of virus-like particles enhances internalization by antigen presenting cells.

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.

Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus.

Noroviruses are an emerging threat to public health, causing large health and economic costs, including at least 200,000 deaths annually. The inability to replicate in cell culture or small animal models has limited the understanding of the interaction between human noroviruses and their hosts. However, an alternative strategy to gain insights into norovirus pathogenesis is to study murine norovirus (MNV-1) that replicates in cultured macrophages. While the innate immune response is central to the resolution of norovirus disease, the adaptive immune response is required for viral clearance. The specific responses of macrophages and dendritic cells to infection drive the adaptive immune response, with chemokines playing an important role. In this study, we have conducted microarray analysis of RAW264.7 macrophages infected with MNV-1 and examined the changes in chemokine transcriptional expression during infection. While the majority of chemokines showed no change, there was specific up-regulation in chemokines reflective of a bias toward a Th1 response, specifically CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10 and CXCL11. These changes in gene expression were reflected in protein levels as determined by ELISA assay. This virus-induced chemokine response will affect the resolution of infection and may limit the humoral response to norovirus infection.

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression.

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.

An enhanced heterologous virus-like particle for human papillomavirus type 16 tumour immunotherapy.

Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48-57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.