Andres Männik - Academia.edu (original) (raw)

Papers by Andres Männik

Research paper thumbnail of Immunogenic hotspots in the spacer domain of ADAMTS13 in immune‐mediated thrombotic thrombocytopenic purpura

Journal of Thrombosis and Haemostasis

Research paper thumbnail of Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP

Blood Advances

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated ... more Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency of ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats member 13). In healthy individuals, ADAMTS13 has a folded conformation where the central spacer domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G's (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in iTTP patients are directed against the different ADAMTS13 domains but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13 thereby opening ADAMTS13. Therefore, we purified anti-CS and anti-CUB autoantibodies from 13 acute iTTP patients by affinity chromatography. The successfully purified anti-CS (10/13 patients) and a...

Research paper thumbnail of Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura

Blood Advances

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13)... more Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3:...

Research paper thumbnail of N-glycan–mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP

Blood

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by th... more Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to red...

Research paper thumbnail of Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura

Haematologica

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) common... more Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated ‘MDTCS’ variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aro...

Research paper thumbnail of Generation and validation of small ADAMTS13 fragments for epitope mapping of anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura

Research and Practice in Thrombosis and Haemostasis

Research paper thumbnail of Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription

Journal of Virology

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that... more Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the pres...

Research paper thumbnail of The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing

PLOS ONE

Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great... more Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great need for animal models that accurately mimic PV infection in humans for testing therapeutic strategies against human papillomaviruses (HPVs). In this study, we present data that demonstrate that in terms of gene expression during initial viral DNA amplification, Macaca fascicularis PV (MfPV) types 5 and 8 appear to be similar to mucosal oncogenic HPVs, while MfPV1 (isolated from skin) resembles most high-risk cutaneous beta HPVs (HPV5). Similarities were also observed in replication properties during the initial amplification phase of the MfPV genomes. We demonstrate that high-risk mucosal HPV-specific inhibitors target the transient replication of the MfPV8 genomes, which indicates that similar pathways are used by the high-risk HPVs and MfPVs during their genome replication. Taking all into account, we propose that Macaca fascicularis may serve as a highly relevant model for preclinical tests designed to evaluate therapeutic strategies against HPV-associated lesions.

Research paper thumbnail of DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

Scientific reports, Jan 20, 2018

There are currently no licensed therapeutic treatment or preventive vaccines against Ebolavirus d... more There are currently no licensed therapeutic treatment or preventive vaccines against Ebolavirus disease, and the 2013-2016 West African outbreak of Ebolavirus disease spread rapidly and resulted in almost 30,000 cases and more than 11,000 deaths. However, the devastating outbreak has spurred the development of novel Ebolavirus vaccines. Here, we demonstrate that alphavirus-based DNA-launched self-replicating RNA replicon vaccines (DREP) encoding either the glycoprotein (GP) gene or co-expressing the GP and VP40 genes of Sudan or Zaire Ebolavirus are immunogenic in mice inducing both binding and neutralizing antibodies as well as CD8 T cell responses. In addition, antibodies were cross-reactive against another Ebolavirus, although the specificity was higher for the vaccination antigen. DREP vaccines were more immunogenic than recombinant MVA vaccines expressing the same Ebolavirus antigens. However, a DREP prime followed by an MVA boost immunization regimen improved vaccine immunogen...

Research paper thumbnail of Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

PLOS Pathogens, 2017

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well... more Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting highthroughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

Research paper thumbnail of HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

BMC Biotechnology, 2016

Background: The production of recombinant monoclonal antibodies in mammalian cell culture is of h... more Background: The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories. Results: HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. Conclusions: HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.

Research paper thumbnail of Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency

PLOS ONE, 2015

Stable maintenance replication is characteristic of the latency phase of HPV infection, during wh... more Stable maintenance replication is characteristic of the latency phase of HPV infection, during which the viral genomes are actively maintained as extrachromosomal genetic elements in infected proliferating basal keratinocytes. Active replication in the S-phase and segregation of the genome into daughter cells in mitosis are required for stable maintenance replication. Most of our knowledge about papillomavirus genome segregation has come from studies of bovine papillomavirus type 1 (BPV-1), which have demonstrated that the E2 protein cooperates with cellular trans-factors and that E2 binding sites act as cis-regulatory elements in the viral genome that are essential for the segregation process. However, the genomic organization of the regulatory region in HPVs, and the properties of the viral proteins are different from those of their BPV-1 counterparts. We have designed a segregation assay for HPV-18 and used it to demonstrate that the E2 protein performs segregation in combination with at least two E2 binding sites. The cooperative binding of the E2 protein to two E2 binding sites is a major determinant of HPV-18 genome segregation, as demonstrated by the change in spacing between adjacent binding sites #1 and #2 in the HPV-18 Upstream Regulatory Region (URR). Duplication or triplication of the natural 4 bp 5'-CGGG-3' spacer between the E2 binding sites increased the cooperative binding of the E2 molecules as well as E2-dependent segregation. Removal of any spacing between these sites eliminated cooperative binding of the E2 protein and disabled segregation of the URR and HPV-18 genome. Transfer of these configurations of the E2 binding sites into viral genomes confirmed the role of the E2 protein and binding sites #1 and #2 in the segregation process. Additional analysis demonstrated that these sites also play an important role in the transcriptional regulation of viral gene expression from different HPV-18 promoters.

Research paper thumbnail of DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent

Methods in Molecular Biology, 2015

The efficient transfection of cloned genes into cells has a critical role in nucleic acid-based t... more The efficient transfection of cloned genes into cells has a critical role in nucleic acid-based therapeutic applications, molecular and cell biology studies, and the production of recombinant proteins in cultured cells. Using a stearylated cell-penetrating peptide (CPP) NickFect51, we have generated an effective, universal, and convenient method for the delivery of DNA vectors into animal cells derived from different origins (mammalian, avian, insect). The CPP-mediated transfection described in detail herein is efficient for many regular cell lines commonly used for research purposes and it is especially suitable for transfection of protein production cell lines adapted for growth in chemically defined serum-free medium.

Research paper thumbnail of The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

Virology journal, Jan 14, 2015

Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is ... more Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains. U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods. The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related l...

Research paper thumbnail of Predominance of a rare type of HIV-1 in Estonia

Journal of acquired immune deficiency syndromes (1999), Jan 15, 2005

An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in ... more An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in Estonia. The objective of this study was to further investigate the molecular epidemiology and genetic structure of HIV-1 in Estonia. Most of the investigated individuals became infected after August 2000 when HIV-1 started to spread rapidly among Estonian intravenous drug users (IDUs). Two viral DNA regions, gag/pol and gp41, were sequenced and subtyped from peripheral blood mononuclear cells or plasma from 141 individuals. Phylogenetic analysis in the gp41 region revealed that the most frequent type of the virus among IDUs was a circulating recombinant form, CRF06_cpx, whereas a few samples showed highest sequence similarity to a subtype A strain circulating in Ukraine and Russia. Likewise, in the gag/pol region, most of the samples were classified as CRF06_cpx, with a few classified as subtype A. In this region, however, 16% of the sequences turned out to be mosaic unique recombinant ...

Research paper thumbnail of Humoral and cellular immune responses to HIV-1 Nef in mice DNA-immunised with non-replicating or self-replicating expression vectors

Vaccine, 1999

Objective: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and ... more Objective: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an eective immunogen in humoral and cellular immune responses. We have used two dierent self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their eciency in raising humoral and cellular immune responses. Design and methods: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51 Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. Results: Ecient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-speci®c cytotoxic T lymphocyte (CTL) responses within four weeks. Conclusions: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Research paper thumbnail of RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication

PLoS Pathogens, 2013

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associat... more Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 59-triphosphate (59-ppp) RNA and mediate IFN production. Cytosolic 59-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-b independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 59-ppp dsRNA and induces IFN-b through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-b induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-b production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

Research paper thumbnail of Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals

Plasmid, 2003

The objective of our study was to analyze the efficiency and the properties of the inheritance of... more The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals. Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system. We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector. The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid. This method resulted in successful production of transgenic animals. Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50%. The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.

Research paper thumbnail of Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division

Journal of Virology, 2010

Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and K... more Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.

Research paper thumbnail of Induction of the Bovine Papillomavirus Origin "Onion Skin"-Type DNA Replication at High E1 Protein Concentrations In Vivo

Journal of Virology, 2002

We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origi... more We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially “onion skin”-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA...

Research paper thumbnail of Immunogenic hotspots in the spacer domain of ADAMTS13 in immune‐mediated thrombotic thrombocytopenic purpura

Journal of Thrombosis and Haemostasis

Research paper thumbnail of Anti-cysteine/spacer antibodies that open ADAMTS13 are a common feature in iTTP

Blood Advances

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated ... more Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by an autoantibody-mediated deficiency of ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats member 13). In healthy individuals, ADAMTS13 has a folded conformation where the central spacer domain interacts with the C-terminal CUB domains. We recently showed that ADAMTS13 adopts an open conformation in iTTP and that patient immunoglobulin G's (IgGs) can open ADAMTS13. Anti-ADAMTS13 autoantibodies in iTTP patients are directed against the different ADAMTS13 domains but almost all patients have autoantibodies binding to the cysteine/spacer (CS) domains. In this study, we investigated whether the autoantibodies against the CS and CUB domains can disrupt the S-CUB interaction of folded ADAMTS13 thereby opening ADAMTS13. Therefore, we purified anti-CS and anti-CUB autoantibodies from 13 acute iTTP patients by affinity chromatography. The successfully purified anti-CS (10/13 patients) and a...

Research paper thumbnail of Anti-ADAMTS13 autoantibody profiling in patients with immune-mediated thrombotic thrombocytopenic purpura

Blood Advances

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13)... more Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3:...

Research paper thumbnail of N-glycan–mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP

Blood

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by th... more Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to red...

Research paper thumbnail of Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura

Haematologica

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) common... more Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated ‘MDTCS’ variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aro...

Research paper thumbnail of Generation and validation of small ADAMTS13 fragments for epitope mapping of anti‐ADAMTS13 autoantibodies in immune‐mediated thrombotic thrombocytopenic purpura

Research and Practice in Thrombosis and Haemostasis

Research paper thumbnail of Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription

Journal of Virology

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that... more Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the pres...

Research paper thumbnail of The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing

PLOS ONE

Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great... more Due to the extreme tissue and species restriction of the papillomaviruses (PVs), there is a great need for animal models that accurately mimic PV infection in humans for testing therapeutic strategies against human papillomaviruses (HPVs). In this study, we present data that demonstrate that in terms of gene expression during initial viral DNA amplification, Macaca fascicularis PV (MfPV) types 5 and 8 appear to be similar to mucosal oncogenic HPVs, while MfPV1 (isolated from skin) resembles most high-risk cutaneous beta HPVs (HPV5). Similarities were also observed in replication properties during the initial amplification phase of the MfPV genomes. We demonstrate that high-risk mucosal HPV-specific inhibitors target the transient replication of the MfPV8 genomes, which indicates that similar pathways are used by the high-risk HPVs and MfPVs during their genome replication. Taking all into account, we propose that Macaca fascicularis may serve as a highly relevant model for preclinical tests designed to evaluate therapeutic strategies against HPV-associated lesions.

Research paper thumbnail of DNA-launched RNA replicon vaccines induce potent anti-Ebolavirus immune responses that can be further improved by a recombinant MVA boost

Scientific reports, Jan 20, 2018

There are currently no licensed therapeutic treatment or preventive vaccines against Ebolavirus d... more There are currently no licensed therapeutic treatment or preventive vaccines against Ebolavirus disease, and the 2013-2016 West African outbreak of Ebolavirus disease spread rapidly and resulted in almost 30,000 cases and more than 11,000 deaths. However, the devastating outbreak has spurred the development of novel Ebolavirus vaccines. Here, we demonstrate that alphavirus-based DNA-launched self-replicating RNA replicon vaccines (DREP) encoding either the glycoprotein (GP) gene or co-expressing the GP and VP40 genes of Sudan or Zaire Ebolavirus are immunogenic in mice inducing both binding and neutralizing antibodies as well as CD8 T cell responses. In addition, antibodies were cross-reactive against another Ebolavirus, although the specificity was higher for the vaccination antigen. DREP vaccines were more immunogenic than recombinant MVA vaccines expressing the same Ebolavirus antigens. However, a DREP prime followed by an MVA boost immunization regimen improved vaccine immunogen...

Research paper thumbnail of Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

PLOS Pathogens, 2017

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well... more Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting highthroughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

Research paper thumbnail of HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

BMC Biotechnology, 2016

Background: The production of recombinant monoclonal antibodies in mammalian cell culture is of h... more Background: The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories. Results: HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. Conclusions: HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.

Research paper thumbnail of Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency

PLOS ONE, 2015

Stable maintenance replication is characteristic of the latency phase of HPV infection, during wh... more Stable maintenance replication is characteristic of the latency phase of HPV infection, during which the viral genomes are actively maintained as extrachromosomal genetic elements in infected proliferating basal keratinocytes. Active replication in the S-phase and segregation of the genome into daughter cells in mitosis are required for stable maintenance replication. Most of our knowledge about papillomavirus genome segregation has come from studies of bovine papillomavirus type 1 (BPV-1), which have demonstrated that the E2 protein cooperates with cellular trans-factors and that E2 binding sites act as cis-regulatory elements in the viral genome that are essential for the segregation process. However, the genomic organization of the regulatory region in HPVs, and the properties of the viral proteins are different from those of their BPV-1 counterparts. We have designed a segregation assay for HPV-18 and used it to demonstrate that the E2 protein performs segregation in combination with at least two E2 binding sites. The cooperative binding of the E2 protein to two E2 binding sites is a major determinant of HPV-18 genome segregation, as demonstrated by the change in spacing between adjacent binding sites #1 and #2 in the HPV-18 Upstream Regulatory Region (URR). Duplication or triplication of the natural 4 bp 5'-CGGG-3' spacer between the E2 binding sites increased the cooperative binding of the E2 molecules as well as E2-dependent segregation. Removal of any spacing between these sites eliminated cooperative binding of the E2 protein and disabled segregation of the URR and HPV-18 genome. Transfer of these configurations of the E2 binding sites into viral genomes confirmed the role of the E2 protein and binding sites #1 and #2 in the segregation process. Additional analysis demonstrated that these sites also play an important role in the transcriptional regulation of viral gene expression from different HPV-18 promoters.

Research paper thumbnail of DNA Transfer into Animal Cells Using Stearylated CPP Based Transfection Reagent

Methods in Molecular Biology, 2015

The efficient transfection of cloned genes into cells has a critical role in nucleic acid-based t... more The efficient transfection of cloned genes into cells has a critical role in nucleic acid-based therapeutic applications, molecular and cell biology studies, and the production of recombinant proteins in cultured cells. Using a stearylated cell-penetrating peptide (CPP) NickFect51, we have generated an effective, universal, and convenient method for the delivery of DNA vectors into animal cells derived from different origins (mammalian, avian, insect). The CPP-mediated transfection described in detail herein is efficient for many regular cell lines commonly used for research purposes and it is especially suitable for transfection of protein production cell lines adapted for growth in chemically defined serum-free medium.

Research paper thumbnail of The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

Virology journal, Jan 14, 2015

Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is ... more Although prophylactic vaccines have been developed against HPV6, HPV11, HPV16 and HPV18 there is the clear unmet medical need in order to justify the development of drugs targeting human papillomavirus replication. The native host cells of HPVs are human primary keratinocytes which can be cultivated in raft cultures. However, this method is difficult to use in high-throughput screening assays and the need for a cost-effective cellular system for screening potential anti-HPV drug candidates during all stages of HPV genome replication remains. U2OS cells were transfected with HPV11 wt or E8- minicircle genomes and their gene expression was studied via 3' RACE, 5' RACE or via real time PCR methods. The DNA replication of these genomes was detected by Southern blot methods. The analysis of HPV11 transcripts in U2OS cells showed that the patterns of promoter use, splice sites and polyadenylation cleavage sites are identical to those previously characterized in human HPV-related l...

Research paper thumbnail of Predominance of a rare type of HIV-1 in Estonia

Journal of acquired immune deficiency syndromes (1999), Jan 15, 2005

An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in ... more An earlier study has indicated that a complex recombinant HIV-1 strain dominates the epidemic in Estonia. The objective of this study was to further investigate the molecular epidemiology and genetic structure of HIV-1 in Estonia. Most of the investigated individuals became infected after August 2000 when HIV-1 started to spread rapidly among Estonian intravenous drug users (IDUs). Two viral DNA regions, gag/pol and gp41, were sequenced and subtyped from peripheral blood mononuclear cells or plasma from 141 individuals. Phylogenetic analysis in the gp41 region revealed that the most frequent type of the virus among IDUs was a circulating recombinant form, CRF06_cpx, whereas a few samples showed highest sequence similarity to a subtype A strain circulating in Ukraine and Russia. Likewise, in the gag/pol region, most of the samples were classified as CRF06_cpx, with a few classified as subtype A. In this region, however, 16% of the sequences turned out to be mosaic unique recombinant ...

Research paper thumbnail of Humoral and cellular immune responses to HIV-1 Nef in mice DNA-immunised with non-replicating or self-replicating expression vectors

Vaccine, 1999

Objective: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and ... more Objective: HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an eective immunogen in humoral and cellular immune responses. We have used two dierent self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their eciency in raising humoral and cellular immune responses. Design and methods: The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51 Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. Results: Ecient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-speci®c cytotoxic T lymphocyte (CTL) responses within four weeks. Conclusions: We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Research paper thumbnail of RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication

PLoS Pathogens, 2013

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associat... more Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 59-triphosphate (59-ppp) RNA and mediate IFN production. Cytosolic 59-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-b independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 59-ppp dsRNA and induces IFN-b through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-b induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-b production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

Research paper thumbnail of Effective generation of transgenic mice by Bovine papillomavirus type 1 based self-replicating plasmid that is maintained as extrachromosomal genetic element in three generations of animals

Plasmid, 2003

The objective of our study was to analyze the efficiency and the properties of the inheritance of... more The objective of our study was to analyze the efficiency and the properties of the inheritance of the Bovine papillomavirus type 1 (BPV1) replicator-based plasmid used as vector system for generation of transgenic animals. Previously, we have characterized a series of self-replicating plasmid vectors containing all viral factors necessary and sufficient for stable extrachromosomal replication of the BPV1 genome in the tissue culture system. We also demonstrated that the designed replicating vector system has a considerable benefit in the transgene expression, if compared to the regular expression vector. The vector, which showed the highest stability and maintenance function in the tissue culture was chosen for generation of the transgenic mice by pronuclear injections of the circular supercoiled plasmid. This method resulted in successful production of transgenic animals. Transmission efficiency of the vectors into the F(1) generation of animals varied between 0 and 48%, whereas transmission into the F(2) generation was uniformly near 50%. The maintenance of the vector-plasmids in the F(2) generation of transgenic animals as extrachromosomal genetic element was demonstrated by rescue of the plasmid into the Escherichia coli.

Research paper thumbnail of Effective Formation of the Segregation-Competent Complex Determines Successful Partitioning of the Bovine Papillomavirus Genome during Cell Division

Journal of Virology, 2010

Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and K... more Effective segregation of the bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated human herpesvirus type 8 (KSHV) genomes into daughter cells is mediated by a single viral protein that tethers viral genomes to host mitotic chromosomes. The linker proteins that mediate BPV1, EBV, and KSHV segregation are E2, LANA1, and EBNA1, respectively. The N-terminal transactivation domain of BPV1 E2 is responsible for chromatin attachment and subsequent viral genome segregation. Because E2 transcriptional activation and chromatin attachment functions are not mutually exclusive, we aimed to determine the requirement of these activities during segregation by analyzing chimeric E2 proteins. This approach allowed us to separate the two activities. Our data showed that attachment of the segregation protein to chromatin is not sufficient for proper segregation. Rather, formation of a segregation-competent complex which carries multiple copies of the segregation protein is required. Complementation studies of E2 functional domains indicated that chromatin attachment and transactivation functions must act in concert to ensure proper plasmid segregation. These data indicate that there are specific interactions between linker molecules and transcription factors/complexes that greatly increase segregation-competent complex formation. We also showed, using hybrid E2 molecules, that restored segregation function does not involve interactions with Brd4.

Research paper thumbnail of Induction of the Bovine Papillomavirus Origin "Onion Skin"-Type DNA Replication at High E1 Protein Concentrations In Vivo

Journal of Virology, 2002

We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origi... more We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially “onion skin”-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA...