Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles.

UNLABELLED: At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. IMPORTANCE: From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV.

Comparative preclinical evaluation of AS01 versus other Adjuvant Systems in a candidate herpes zoster glycoprotein E subunit vaccine.

The candidate vaccine HZ/su is being developed to prevent herpes-zoster disease (HZ). HZ occurrence is attributed to declines in varicella-zoster virus (VZV) specific T-cell immunity. HZ/su contains VZV antigen, gE, and Adjuvant System AS01B (liposome-based formulation of MPL and QS-21). In clinical trials, AS01B enhances CD4+ T-cell responses to gE. In clinical trials of other vaccines, Adjuvant Systems AS03 and AS04 also enhance antigen-specific CD4+ T-cell responses. Hence the purpose of this study was to evaluate gE formulated with AS01B, AS01E (50% less MPL and QS-21 than AS01B), AS03 or AS04 in C57BL6 mice primed with live-attenuated VZV. Four-weeks post-vaccination, the gE-specific CD4+ T-cell response to gE/AS01B was 5.4, 2.8 and 2.2-fold greater than those to gE/AS03, gE/AS04 and gE/AS03, respectively (p<0.001). Therefore in the VZV-primed mouse model, CD4+ T-cell responses to gE appeared most enhanced by AS01B, and adds further support for the use of AS01B in the HZ/su formulation.

Antibody avidity measurements in recipients of Cervarix vaccine following a two-dose schedule or a three-dose schedule.

The HPV-16/18 vaccine (Cervarix) is a prophylactic vaccine for the prevention of cervical cancer and contains recombinant virus-like particles (VLPs) assembled from the L1 major capsid proteins of human papillomavirus (HPV) strains 16 and 18. Although a correlate of protection has yet to be identified, HPV-specific antibodies are thought to prevent virus infection of the genital mucosa. Therefore, antigen-specific antibodies as assessed by ELISA or pseudovirion-based neutralisation assay are frequently measured in clinical trials to substantiate the immune responses induced by the vaccine. Measuring antigen-antibody binding avidities, which reflects the degree of affinity maturation in the B-cells, is another valuable method to assess the quality of the antibody responses. Here we describe the antigen-specific antibody avidities in samples taken from a clinical trial examining the feasibility of adopting a two-dose (Months 0 and 6) schedule for 9-14 year olds instead of the three-dose schedule (Months 0, 1 and 6). Antibody avidity (i.e. avidity index [AI]) was determined in the ELISA by the ratio of antibody concentrations in serum samples treated or not with the chaotropic agent NaSCN. Importantly, in the comparison between the groups of two-dose and three-dose recipients, no differences in AIs were observed at Months 7, 24 and 48. The results suggest that from Month 7 to 48, the quality of the antibody response in terms of avidity was similar in the two-dose recipients to that in the three-dose recipients. Hence these results support the adoption of a two-dose schedule in 9-14 year-old girls.

Acetylation at lysine 346 controls the transforming activity of the HTLV-1 Tax oncoprotein in the Rat-1 fibroblast model.

BACKGROUND: Transformation by the Tax oncoprotein of the human T cell leukemia virus type 1 (HTLV-1) is governed by actions on cellular regulatory signals, including modulation of specific cellular gene expression via activation of signaling pathways, acceleration of cell cycle progression via stimulation of cyclin-dependent kinase activity leading to retinoblastoma protein (pRb) hyperphosphorylation and perturbation of survival signals. These actions control early steps in T cell transformation and development of Adult T cell leukemia (ATL), an aggressive malignancy of HTLV-1 infected T lymphocytes. Post-translational modifications of Tax by phosphorylation, ubiquitination, sumoylation and acetylation have been implicated in Tax-mediated activation of the NF-κB pathway, a key function associated with Tax transforming potential. RESULTS: In this study, we demonstrate that acetylation at lysine K(346) in the carboxy-terminal domain of Tax is modulated in the Tax nuclear bodies by the acetyltransferase p300 and the deacetylases HDAC5/7 and controls phosphorylation of the tumor suppressor pRb by Tax-cyclin D3-CDK4-p21(CIP) complexes. This property correlates with the inability of the acetylation deficient K(346)R mutant, but not the acetylation mimetic K(346)Q mutant, to promote anchorage-independent growth of Rat-1 fibroblasts. By contrast, acetylation at lysine K(346) had no effects on the ability of Tax carboxy-terminal PDZ-binding domain to interact with the tumor suppressor hDLG. CONCLUSIONS: The identification of the acetyltransferase p300 and the deacetylase HDAC7 as enzymes modulating Tax acetylation points to new therapeutic targets for the treatment of HTLV-1 infected patients at risk of developing ATL.

An eYFP reporter gene for the yeast two-hybrid system.

The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.

Host-pathogen interactome mapping for HTLV-1 and -2 retroviruses.

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression. RESULTS: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway. CONCLUSIONS: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection.

How the DNA damage response determines the fate of HTLV-1 Tax-expressing cells.

How the Human T lymphotropic virus type 1 (HTLV-1) Tax protein stimulates proliferation while triggering cell cycle arrest and senescence remains puzzling. There is also a debate about the ability of Tax to activate or inhibit the DNA damage response. Here, we comment on these different activities and propose a conceptual rationale for the apparently conflicting observations.

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program.

The Tax oncoprotein encoded by the human T-cell leukemia virus type 1 plays a pivotal role in viral persistence and pathogenesis. Human T-cell leukemia virus type 1-infected cells proliferate faster than normal lymphocytes, expand through mitotic division, and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax modulates the spatiotemporal program of origin activation and fires supplementary origins at the onset of S phase. Thereby, Tax increases the DNA replication rate, accelerates S phase progression, but also generates a replicative stress characterized by the presence of genomic lesions. Mechanistically, Tax favors p300 recruitment and histone hyperacetylation at late replication domains, advancing their replication timing in early S phase.

Safety of long-term treatment of HAM/TSP patients with valproic acid.

HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease of the central nervous system induced by human T-lymphotropic virus type 1. As a potential therapeutic approach, we previously suggested reducing the proviral load by modulating lysine deacetylase activity using valproic acid (VPA) and exposing virus-positive cells to the host immune response. We conducted a single-center, 2-year, open-label trial, with 19 HAM/TSP volunteers treated with oral VPA. Proviral load, CD38/HLA-DR expression, and CD8(+) lysis efficiency were not significantly affected by VPA. Mean scores of HAM/TSP disability did not differ between baseline and final visit. Walking Time Test increased significantly (> 20%) in 3 patients and was in keeping with minor VPA side effects (drowsiness and tremor). Walking Time Test improved rapidly after VPA discontinuation. We conclude that long-term treatment with VPA is safe in HAM/TSP.

Preventive and therapeutic strategies for bovine leukemia virus: lessons for HTLV.

Bovine leukemia virus (BLV) is a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1). BLV is a major animal health problem worldwide causing important economic losses. A series of attempts were developed to reduce prevalence, chiefly by eradication of infected cattle, segregation of BLV-free animals and vaccination. Although having been instrumental in regions such as the EU, these strategies were unsuccessful elsewhere mainly due to economic costs, management restrictions and lack of an efficient vaccine. This review, which summarizes the different attempts previously developed to decrease seroprevalence of BLV, may be informative for management of HTLV-1 infection. We also propose a new approach based on competitive infection with virus deletants aiming at reducing proviral loads.

Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads.

BACKGROUND: Bovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process. RESULTS: We demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads. CONCLUSION: Together, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.

Protein-protein interactions and gene expression regulation in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 (HTLV-1) propagates in CD4 or CD8 T cells and, after extended latency periods of 30-50 years, causes a rapidly fatal leukemia called adult T-cell leukemia/lymphoma (ATL). Infection with HTLV-1 is also associated with a degenerative neuromuscular disease referred to as tropical spastic paraparesis or HTLV-1-associated myelopathy. HTLV genome, in addition to the structural proteins and retroviral enzymes, codes for a region at its 3' end originally designated pX. The products of this region (Tax, Rex, p12I, p13II, p30II and HBZ) play important roles in deregulation of cellular functions by either directly disrupting cellular factors or altering transcription of viral and cellular genes. Here, we will review current knowledge of protein-protein interactions that regulate transcriptional functions of proteins encoded by the pX region.

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli.

BACKGROUND: Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude. RESULTS: The rpoS level has been monitored by the aim of a transcriptional reporter gene based on the synthesis of the green fluorescent protein (GFP). It has been observed that the level of GFP increases during the transition from batch to fed-batch phase. After this initial increase, the GFP content of the cell drops, primarily due to the dilution by cell division. However, a significant drop of the GFP content has been observed if using a partitioned bioreactor, for which the mixing conditions are very bad, leading to the exposure of the cells to cyclic and stochastic extracellular fluctuations. If considering the flow cytometric profile of the cell to cell GFP content, this drop has to be attributed to the appearance of segregation at the level of the GFP content among the microbial population. CONCLUSION: The generation of extracellular perturbations (in the present case, at the level of the sugar concentration and the dissolved oxygen level) has led to a drop at the level of the rpoS expression level. This drop has to be attributed to a segregation phenomenon in microbial population, with a major sub-population exhibiting a low expression level and a minor sub-population keeping its initial elevated expression level. The intensity of the segregation, as well as its time of appearance during the culture can be related to the bioreactor mixing efficiency.

Valproate synergizes with purine nucleoside analogues to induce apoptosis of B-chronic lymphocytic leukaemia cells.

Resistance to chemotherapy and drug toxicity are two major concerns of chronic lymphocytic leukaemia (B-CLL) treatment by purine nucleoside analogues (PNA, i.e. fludarabine and cladribine). We hypothesized that targeting epigenetic changes might address these issues and evaluated the effect of the histone deacetylase inhibitor valproate (VPA) at a clinically relevant concentration. VPA acted in a highly synergistic/additive manner with fludarabine and cladribine to induce apoptosis of B-CLL cells. Importantly, VPA also restored sensitivity to fludarabine in B cells from poor prognosis CLL patients who became resistant to chemotherapy. Mechanism of apoptosis induced by VPA alone or combined with fludarabine or to cladribine was caspase-dependent and involved the extrinsic pathway. VPA, but neither fludarabine nor cladribine, enhanced the production of reactive oxygen species (ROS) and inhibition of ROS with N-acetylcysteine decreases apoptosis of CLL cells. VPA stimulates hyperphosphorylation of p42/p44 ERK, cytochrome c release and overexpression of Bax and Fas. Together, our data indicate that VPA may ameliorate the outcome of PNA-based therapeutic protocols and provide a potential alternative treatment in both the relapsed and front-line resistant patients and in patients with high risk features.

The HTLV-1 Tax interactome.

The Tax1 oncoprotein encoded by Human T-lymphotropic virus type I is a major determinant of viral persistence and pathogenesis. Tax1 affects a wide variety of cellular signalling pathways leading to transcriptional activation, proliferation and ultimately transformation. To carry out these functions, Tax1 interacts with and modulates activity of a number of cellular proteins. In this review, we summarize the present knowledge of the Tax1 interactome and propose a rationale for the broad range of cellular proteins identified so far.

Vaccination of calves using the BRSV nucleocapsid protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects the lungs against BRSV replication and pathology.

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and exacerbation of subsequent RSV disease have occurred. In order to circumvent these problems, calves were vaccinated with the nucleocapsid protein, known to be a major target of CD8(+) T cells in cattle. This was performed according to a DNA prime-protein boost strategy. The results showed that DNA vaccination primed a specific T-cell-mediated response, as indicated by both a lymphoproliferative response and IFN-gamma production. These responses were enhanced after protein boost. After challenge, mock-vaccinated calves displayed gross pneumonic lesions and viral replication in the lungs. In contrast, calves vaccinated by successive administrations of plasmid DNA and protein exhibited protection against the development of pneumonic lesions and the viral replication in the BAL fluids and the lungs. The protection correlated to the cell-mediated immunity and not to the antibody response.

Emphasis on cell turnover in two hosts infected by bovine leukemia virus: a rationale for host susceptibility to disease.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects and induces accumulation of B-lymphocytes in the peripheral blood and lymphoid tissues of cattle, leading to leukemia/lymphoma. BLV can also be experimentally transmitted to sheep, in which disease appears earlier and at higher frequencies. Abnormal accumulation of leukemic B-lymphocytes results from an alteration of different parameters that include cell proliferation and death as well as migration to lymphoid tissues. Interestingly, B lymphocyte turnover is increased in BLV-infected sheep but reduced in cattle, revealing a potential relationship between cell kinetics and disease progression.

Even attenuated bovine leukemia virus proviruses can be pathogenic in sheep.

Based on a reverse genetics approach, we previously reported that bovine leukemia virus (BLV) mutants harboring deletions in the accessory R3 and G4 genes persist at very low proviral loads and are unable to induce leukemia or lymphoma in sheep, indicating that these R3 and G4 gene sequences are required for pathogenesis. We now show that lymphoma can occur, albeit infrequently (1 case of 20) and after extended periods of latency (7 years). Direct sequencing and reinfection experiments demonstrated that lymphomagenesis was not due to the reversion of the mutant to the wild type. Similar observations with another type of attenuated mutant impaired in the transmembrane protein (TM) YXXL signaling motifs were made. We conclude that the R3 and G4 genes and the TM YXXL motifs are not strictly required for pathogenesis but that their integrity contributes to disease frequency and latency.

Development of a 1-step enzyme-linked immunosorbent assay for the rapid diagnosis of bovine respiratory syncytial virus in postmortem specimens.

Bovine respiratory syncytial virus (BRSV) is associated with severe respiratory disease in cattle. BRSV infection frequently leads to the death of young infected animals. The presence of BRSV in postmortem specimens is routinely detected using indirect immunofluorescence (IIF). However, this technique requires special equipment and considerable expertise. The present paper describes the development of a 1-step ELISA for rapid (1.5 hours) detection of BRSV antigen in organ homogenates. The performance of the new 1-step ELISA was evaluated using bovine postmortem specimens (n = 108) in comparison with 3 other BRSV diagnostic techniques: indirect immunofluorescence, the Clearview respiratory syncytial virus (RSV) test, and real-time reverse transcriptase polymerase chain reaction (RT-PCR). The relative sensitivity, specificity, and the kappa coefficient of 1-step ELISA, the Clearview RSV electroimmunoassay (EIA), and IIF were calculated, using real-time RT-PCR as the reference test. The new 1-step ELISA was the most sensitive and specific of the 3 tests. Thus, the new 1-step ELISA is a reliable test for detecting BRSV antigen in organ homogenates.

DNA immunization with plasmids encoding fusion and nucleocapsid proteins of bovine respiratory syncytial virus induces a strong cell-mediated immunity and protects calves against challenge.

Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication, clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected following challenge. These results indicate that DNA vaccination could be a promising alternative to the classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants.