| 1. |
- Alim, Abdul, 1983-, et al.
(författare)
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Tick-borne encephalitis virus protein expression to develop novel subunit vaccines and diagnostic tools
- 2023
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Konferensbidrag (refereegranskat)abstract
- Tick-borne encephalitis virus (TBEV) and Langat virus (LGTV) are both members of Flavivirus genus within the Flaviviridae family. TBEV is the main pathogenic arbovirus circulating in Europe, Russia, and China. Flaviviruses are characterized by a positive sense single-stranded RNA genome and an enveloped icosahedral virion structure. Previously, it has been observed that flavivirus envelope (E) protein and non-structural protein 1 (NS1) both play a critical role in the pathology of flavivirus. Therefore, in this study, we aim to investigate flavivirus E and NS1 protein as a good target for the development of a subunit vaccine with further potential as a putative diagnostic tool to distinguish between TBEV infected from TBEV vaccinated individuals. Thus, we have generated 4 different successful constructs with TBEV (E and NS1) and LGTV (E and NS1) in the pET SUMO vector. Restriction digestion and sequencing analysis confirmed successful clones of interest and their right orientation. Next, the right clones were transformed in BL21 (DE3) one shoot chemically competent E. coli and induce the expression with 0.5 mM IPTG in culture medium following 0-4h, and 24h incubation period. Next, bacterial cell pellets were collected and used for SDSPAGE/Western blot analysis. We used the champion™ pET SUMO expression system which may produce high levels of soluble protein in bacteria. It employs a small ubiquitin-related modifier (SUMO) fusion, belonging to the growing family of ubiquitin-related proteins, to enhance the solubility of expressed fusion proteins. We have stained with 6x-His tag antibody of interest (mouse monoclonal) for targeting both TBEV- E/NS1 and LGTV-E/NS1 proteins. Among them, the expression of TBEV-NS1 and LGTV-E proteins was verified and confirmed. Several attempts have also been made to obtain the TBEV-E and LGTV-NS1 protein in E. coli cells; however, these require further optimization with a suitable time and dose of IPTG induction. We have used the BL21(DE3) expression system, which could maximize the expression of soluble protein. After successful expression, the 13-kd SUMO moiety will be cleaved by the highly specific and active SUMO (ULP1) protease at the carboxyl terminal, producing a native protein. Furthermore, a protein purification assay (e-g., NI-NTA column/ÄKTA Protein Purification Systems) will be developed to obtain native recombinant protein. The purified proteins will be studied in combination with suitable adjuvants as putative TBE subunit vaccines. They will also be characterized with the potential to develop new tools for TBE diagnostics.
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| 2. |
- Lindquist, Richard, 1985-, et al.
(författare)
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The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection
- 2020
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Ingår i: Journal of Neuroinflammation. - : BioMed Central. - 1742-2094. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection.METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated.RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains.CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.
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| 3. |
- Asghar, Naveed, 1983-, et al.
(författare)
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Design, rescue, and characterization of Langat virus infectious clone by next generation sequencing
- 2023
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. TBE virus (TBEV) infections cause mild flu-like symptoms that may lead to severe neurological disorders. The incidence of TBE cases in Sweden has increase remarkably over the last decades. There is no specific antiviral treatment available against TBEV and vaccination remains the best protective measure. The currently available TBE vaccines require repeated injections for long-term immunity and vaccine failure can occur in some patients due to poor immunogenicity in the elderly. Live attenuated viral vaccines are known to provide long-term immunity with fewer doses whereas the commercial TBE vaccines are based on single surface protein of the virus. We aim to develop a modified live attenuated TBE vaccine based on Langat virus (LGTV). LGTV is a naturally attenuated strain of TBEV. We aim to weaken the virus further by introducing modifications within LGTV genome. In this study we have successfully designed and rescued infectious clones of LGTV using RNA and DNA based strategies. We passaged these infections clones in cell culture and performed next generation sequencing to study similarity of rescued viruses to the parental LGTV sequence and their stability in cell culture. Note: Visit my poster to discuss the results of next generation sequencing analysis and rescue strategies for LGTV infectious clones.
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| 4. |
- Asghar, Naveed, 1983-, et al.
(författare)
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DNA launched suicidal flaviviruses as therapeutic vaccine candidates
- 2018
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Konferensbidrag (refereegranskat)abstract
- Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. The relativelyhigh cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect interventions with high efficiency. We aim to develop therapeutic vaccine candidates against HBV, HCV and HDV using DNA based subgenomic flavivirus replicons as a delivery system. Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), or Kunjinvirus (KUNV) replicon with firefly luciferase geneas a reporter were expressed and characterized in cell culture studies. WNV and KUNV replicons showed significantly higher replication compared to their respective negative controls with unfunctional viral RNA dependent RNA polymerase. KUNV and WNV replicons were chosen for cloning the HCV or HB/DV vaccine candidate gene by replacing luciferasegene. Owing to the self-replicating trait of the flavivirus subgenomic replicons, Western blotting demonstrated that the antigen expression by KUNV and WNV replicons was several folds higher than the positive control. These results suggest that DNA based KUNV and WNV replicons may function as carriers for the hepatitis vaccine candidate genes, and these replicons are currently used for in vivostudies in animal models.
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| 5. |
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| 6. |
- Asghar, Naveed, 1983-, et al.
(författare)
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Immunogenicity of DNA launched suicidal flavivirus replicons for protective vaccination against hepatitis viruses
- 2019
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Konferensbidrag (refereegranskat)abstract
- Chronic liver disease, resulting from Hepatitis B virus (HBV), Hepatitis D virus (HDV), or Hepatitis C virus (HCV) infections, contributes to a major health burden worldwide. Chronic infections with the hepatitis C virus (HCV) can be effectively cured by antivirals. However, as cured patients can be re-infected they lack protective immune responses. In addition, the relativelyhigh cost of the HCV treatment brings concerns about the accessibility, especially in the developing countries. Hence, there exists a need for cost effect vaccines with high efficiency to control and possibly eradicate Hepatitis viruses globally. The vaccine should induce either, or both, neutralizing antibodies and protective T cell responses. We therefore have developed DNA based flavivirus replicons as a potent delivery system that effectively prime HCV-specific T cell responses. We generated suicidal subgenomic DNA replicons of Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), and Kunjinvirus (KUNV) expressing either a fusion protein between the HCV NS3/4A and a stork hepatitis B virus core or a vaccine candidate gene of HB/DV. Transfection experiments showed that the antigen expression by KUNV and WNV replicons was several folds higher than the antigen expression by standard DNA plasmid with CMV promoter. The immunogenicity of three suicidal flaviviral DNA replicons expressing HCV NS3/4A was tested in mice and compared to HCV NS3/4A expression by the standard DNA plasmid. The KUNV-HCV replicon was the best replicon-based immunogen with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, dextramer staining, and polyfunctionality. Importantly, a mutant KUNV-HCV immunogen lacking replication failed to induce immune responses. Thus, the newly developed KUNV-based suicidal DNA launched replicon vaccine for HCV is a highly attractive candidate as a prophylactic vaccine against chronic hepatitis C. In addition, we are currently testing the immunogenicity of KUNV-HB/DV replicon in mice.
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| 7. |
- Asghar, Naveed, 1983-, et al.
(författare)
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Local landscape effects on population dynamics of Ixodes ricinus
- 2016
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Ingår i: Geospatial health. - : Page Press Publications. - 1827-1987 .- 1970-7096. ; 11:3, s. 283-289
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Tidskriftsartikel (refereegranskat)abstract
- Ixodes ricinus, a common tick in Europe, transmits severe tickborne pathogens (TBPs). In Sweden, both prevalence and incidence of tick-borne infections have increased during the last few decades, and a majority of the cases is reported from the area around Stockholm. Among ticks, transmission of TBPs involves co-feeding of susceptible larvae or nymphs with infected ticks on the same host. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBPs. We have studied the association between local landscape characteristics and population dynamics and the probability of co-occurrence of different life cycle stages of I. ricinus at different locations south of Stockholm, Sweden. We found significant spatiotemporal variation in tick activity patterns. Mean tick abundance varied with a tenfold difference among study sites. The probability of co-occurrence of larvae, nymphs and female adults was highest in June and decreased significantly with vegetation height. In addition, the amount of forest habitat and open water in the surrounding landscape of the study sites expressed significant negative effects on tick abundance and co-occurrence, indicating that environmental heterogeneity may increase the likelihood of good rodent habitats, which in turn, are suitable hosts for immature ticks.
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| 8. |
- Asghar, Naveed, 1983-
(författare)
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Ticks and Tick-borne Encephalitis Virus : From Nature to Infection
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Vector-borne diseases are an increasing global threat to humans due to climate changes, elevating the risk of infections transmitted by mosquitos, ticks, and other arthropod vectors. Ixodes ricinus, a common tick in Europe, transmits dangerous tick-borne pathogens to humans. Tick-borne encephalitis (TBE) is a vector-borne disease caused by TBE virus (TBEV). Climate change has contributed to increased tick abundance and incidence of tick-borne diseases, and between 10,000 and 15,000 human TBE cases are reported annually in Europe and Asia. TBEV shows a patchy geographical distribution pattern where each patch represents a natural focus. In nature, TBEV is maintained within the tick-rodent enzootic cycle. Co-feeding is the main route for TBEV transmission from infected to uninfected ticks and for maintenance within the natural foci. The increasing number of TBE cases in Scandinavia highlights the importance of characterizing additional TBEV sequences and of identifying novel natural foci, and in this work we sequenced and phylogenetically characterized four TBEV strains: Saringe-2009 (from a blood-fed nymph), JP-296 (from a questing adult male), JP-554 (from a questing adult male), and Mandal-2009 (from a pool of questing nymphs, n = 10). Mandal-2009 represents a TBEV genome from a natural focus in southern Norway. Saringe-2009 is from a natural endemic focus in northern Stockholm, Sweden, and JP-296 and JP-554 originate from a natural focus “Torö” in southern Stockholm. In addition, we have studied the effect of different biotic and abiotic factors on population dynamics of I. ricinus in southern Stockholm and observed significant spatiotemporal variations in tick activity patterns. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBEV among co-feeding ticks. We found that the probability of co-occurrence of larvae, nymphs, and female adults was highest during early summer whereas increasing vegetation height and increasing amounts of forest and open water around the study sites had a significant negative effect on co-occurrence of larvae, nymphs, and female adults.The proximal part of the 3 ́non-coding region (3 ́NCR) of TBEV contains an internal poly(A) tract, and genomic analysis of Saringe-2009 revealed variability in the poly(A) tract indicating the existence of different variants within the TBEV pool of Saringe-2009. Like other RNA viruses, TBEV exists as swarms of unique variants called quasispecies. Because Saringe-2009 came from an engorged nymph that had been feeding on blood for >60 h, we propose that Saringe-2009 represents a putative shift in the TBEV pool when the virus switches from ectothermic/tick to endothermic/mammalian environments. We investigated the role of poly(A) tract variability in replication and virulence of TBEV by generating two infectious clones of the TBEV strain Toro-2003, one with a short/wild-type (A)3C(A)6 poly(A) tract and one with a long (A)3C(A)38 poly(A) tract. The infectious clone with the long poly(A) tract showed poor replication in cell culture but was more virulent in C57BL/6 mice than the wild-type clone. RNA folding predictions of the TBEV genomes suggested that insertion of a long poly(A) tract abolishes a stem loop structure at the beginning of the 3 ́NCR. Next generation sequencing (NGS) analysis of the TBEV genomes after passaging in cell culture and/or mouse brain revealed molecular determinants and quasispecies structure that might contribute to the observed differences in virulence. Our findings suggest that the long poly(A) tract imparts instability to the TBEV genome resulting in higher quasispecies diversity that in turn contributes to TBEV virulence. Phylogenetic analysis of Saringe-2009, JP-296, JP-554, and Mandal-2009 predicted a strong evolutionary relationship among the four strains. They clustered with Toro-2003, the first TBEV strain from Torö, demonstrating a Scandinavian clade. Except for the proximal part of the 3 ́NCR, TBEV is highly conserved in its genomic structure. Genomic analysis revealed that Mandal-2009 contains a truncated 3 ́NCR similar to the highly virulent strain Hypr, whereas JP-296 and JP-554 have a genomic organization identical to Toro-2003, the prototypic TBEV strain from the same natural focus. NGS revealed significantly higher quasispecies diversity for JP-296 and JP-554 compared to Mandal-2009. In addition, single nucleotide polymerphism (SNP) analysis showed that 40% of the SNPs were common between quasispecies populations of JP-296 and JP-554, indicating the persistence and maintenance of TBEV quasispecies within the natural focus.Taken together, these findings indicate the importance of environmental factors for the occurrence pattern of the different life-stages of the tick vector, which are important for the persistence of TBEV in nature. Our findings also show that the selection pressure exerted by specific host also affects the population structure of the TBEV quasispecies. In addition, our results further demonstrate that the evolution of quasispecies has effect on TBEV virulence in mice.
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| 9. |
- Asghar, Naveed, 1983-, et al.
(författare)
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Transient Expression of Flavivirus Structural Proteins in Nicotiana benthamiana
- 2022
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Ingår i: Vaccines. - : MDPI. - 2076-393X. ; 10:10
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Tidskriftsartikel (refereegranskat)abstract
- Flaviviruses are a threat to public health and can cause major disease outbreaks. Tick-borne encephalitis (TBE) is caused by a flavivirus, and it is one of the most important causes of viral encephalitis in Europe and is on the rise in Sweden. As there is no antiviral treatment availa-ble, vaccination remains the best protective measure against TBE. Currently available TBE vaccines are based on formalin-inactivated virus produced in cell culture. These vaccines must be delivered by intramuscular injection, have a burdensome immunization schedule, and may exhibit vaccine failure in certain populations. This project aimed to develop an edible TBE vaccine to trigger a stronger immune response through oral delivery of viral antigens to mucosal surfaces. We demonstrated successful expression and post-translational processing of flavivirus structural pro-teins which then self-assembled to form virus-like particles in Nicotiana benthamiana. We performed oral toxicity tests in mice using various plant species as potential bioreactors and evaluated the immunogenicity of the resulting edible vaccine candidate. Mice immunized with the edible vaccine candidate did not survive challenge with TBE virus. Interestingly, immunization of female mice with a commercial TBE vaccine can protect their offspring against TBE virus infection.
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| 10. |
- Jaafar, Rita, 1995-, et al.
(författare)
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Development of a potential live-attenuated Langat virus as candidate for novel tick-borne encephalitis vaccine
- 2023
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Konferensbidrag (refereegranskat)abstract
- Background. Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. Most infections with the TBE virus (TBEV) are asymptomatic or cause mild flu-like symptoms, but they may induce severe neurological disorders with permanent sequelae. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus. Methods. Infectious clones of Langat virus (LGTV) based on the strain available in our laboratory are created followed by the generation of modified LGTV infectious clone as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models.Preliminary results. We successfully created LGTV infectious clones. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” Langat virus. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Preliminary data on the establishment process of vaccine candidates in vitro as well as cellular and humoral immune response in mice in response to the LGTV infectious clone are presented.Conclusion. Further investigation of modified LGTV clone seems interesting in the development approach of new TBEV vaccine candidate.
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| 11. |
- Jaafar, Rita, 1995-, et al.
(författare)
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Development of Live attenuated Langat virus infectious clone as potential new TBEV vaccine candidate : Study of cellular and humoral immune response in mice
- 2023
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Konferensbidrag (refereegranskat)abstract
- Tick-borne encephalitis (TBE) is one of the most important tick-transmitted diseases in Europe and Asia. The incidence of TBE cases showed a remarkable elevation in recent years probably due to the geographic expansion of TBEV and its vectors, which is concerning in the absence of a specific antiviral treatment. Vaccination remains the best protective measure against TBE. However, currently available vaccines have a burdensome immunization schedule, and poor immunogenicity in the elderly, which may contribute to observed vaccine failures, i.e., TBE occurrence in vaccinated people. One aim within the Developvaccines@oru project is to develop a novel TBE vaccine that could provide improved immunogenicity using fewer doses. Our strategy is to induce an immune response at possible sites of virus infection by a modified live attenuated vaccine based on Langat virus (LGTV). Infectious clones of Langat virus (LGTV IC) based on the strain available in our laboratory are created followed by the generation of modified LGTV IC as potential attenuated virus. Then, we compare them with the ¨original¨ LGTV strain using cell based and animal models. In our laboratory, we f successfully created LGTV IC. In order to establish a baseline for animal experiments with our vaccine candidates, we planned a pilot study using the “original” LGTV and LGTV IC strains. First, we conducted a pre-pilot experiment to optimize the study design and evaluation methods. Our preliminary data shows that intramuscular administration of both strains was well tolerated in mice. In contrast to the original LGTV, LGTV IC was found to cause a transient, but significant reduction in body weight. ELISA results showed that mice antibodies after LGTV IC infection cross reacted with TBEV antigens. The T lymphocytes, isolated from these mice spleens, showed Interferon gamma secretion when stimulated with both LGTV and TBEV peptides. However, this cellular response revealed higher in original LGTV infected mice. Moreover, we confirm that LGTV IC show lower viraemia peak than original LGTV, both occurring at 2 days post infection. Besides, we have successfully rescued modified LGTV infectious clones with individual or combined mutations in genomic regions coding for NS3, NS5 and/or 3’non-coding region of LGTV genome. Further in-vitro and in-vivo investigation of the modified and potentially attenuated LGTV clone, seems interesting in the development approach of new TBEV vaccine candidate.
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| 12. |
- Jaafar, Rita, 1995-, et al.
(författare)
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Pre-Clinical Development of a Novel Tick-Borne Encephalitis Vaccine for Mucosal Immunization
- 2024
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Konferensbidrag (refereegranskat)abstract
- Background. Tick-borne encephalitis (TBE) is a significant disease in Europe and Asia, witha rising incidence due to the spread of the TBE virus (TBEV) and its vectors. Currentvaccines, although essential, have a complex immunization schedule and lower effectiveness in the elderly, leading to occasional vaccine failures. We aim to create a new TBE vaccine that provides better protection with fewer doses.Aim. We aim to develop an effective vaccine that can be delivered via the mucosal route. The current study covers a pilot study that evaluates live-attenuated vaccine using LGTV IC ascandidate for TBE vaccine.Methods. We developed a genetically modified Langat virus (LGTV) so-called LGTV infectious clone (LGTV IC). In-vivo evaluation was done on mice model to assess tolerabilityand immunogenicity of LGTV IC. LGTV IC was delivered to mice via two different routes,mucosal and intramuscular, in two different doses. The study assessed the peak and kinetics ofviremia, as well as the elicited humoral, cellular, and mucosal immune responses.Results. The mucosal administration of LGTV IC intranasally, at 1 x 103 PFU, was well tolerated in mice, as evidenced by the absence of clinical signs post-administration and no observed body weight loss. This immunization regimen consistently elicited anti-TBEV IgG antibodies in serum, irrespective of the dosage. Furthermore, a robust cellular immune response targeting TBEV non-structural protein 3 antigens (NS3), as well as capsid (C) and envelope (E) antigens, was observed. Intriguingly, lower dosage mucosal immunizationproved more effective in eliciting both humoral and cellular immune responses compared tohigher dosage mucosal administration and intramuscular immunization. Evidence of mucosal immunity was detected in select mucosal samples from the low dosage mucosal immunizationgroup.Conclusion and perspective. Administration of LGTV IC via mucosal route seems promising suggesting that LGTV IC can be considered as a candidate for a live TBE vaccine.Ongoing efforts involve further attenuation of LGTV IC, with successful attenuated variants undergoing in-vitro evaluation. Subsequently, these variants will be assessed in vivo as potential live-attenuated vaccine candidates.
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| 13. |
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| 14. |
- Kjær, Lene Jung, et al.
(författare)
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Potential drivers of human tick-borne encephalitis in the Örebro region of Sweden, 2010-2021
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Incidence of tick-borne encephalitis (TBE) has increased during the last years in Scandinavia, but the underlying mechanism is not understood. TBE human case data reported between 2010 and 2021 were aggregated into postal codes within Örebro County, south-central Sweden, along with tick abundance and environmental data to analyse spatial patterns and identify drivers of TBE. We identified a substantial and continuing increase of TBE incidence in Örebro County during the study period. Spatial cluster analyses showed significant hotspots (higher number of cases than expected) in the southern and northern parts of Örebro County, whereas a cold spot (lower number of cases than expected) was found in the central part comprising Örebro municipality. Generalised linear models showed that the risk of acquiring TBE increased by 12.5% and 72.3% for every percent increase in relative humidity and proportion of wetland forest, respectively, whereas the risk decreased by 52.8% for every degree Celsius increase in annual temperature range. However, models had relatively low goodness of fit (R2 < 0.27). Results suggest that TBE in Örebro County is spatially clustered, however variables used in this study, i.e., climatic variables, forest cover, water, tick abundance, sheep as indicator species, alone do not explain this pattern.
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| 15. |
- Koskela, Anita, 1979-, et al.
(författare)
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Comparison of SARS-CoV-2 whole genome sequencing using tiled amplicon enrichment and bait hybridization
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) worldwide pandemic has led to extensive virological monitoring by whole genome sequencing (WGS). Investigating the advantages and limitations of different protocols is key when conducting population-level WGS. SARS-CoV-2 positive samples with Ct values of 14–30 were run using three different protocols: the Twist Bioscience SARS‑CoV‑2 protocol with bait hybridization enrichment sequenced with Illumina, and two tiled amplicon enrichment protocols, ARTIC V3 and Midnight, sequenced with Illumina and Oxford Nanopore Technologies, respectively. Twist resulted in better coverage uniformity and coverage of the entire genome, but has several drawbacks: high human contamination, laborious workflow, high cost, and variation between batches. The ARTIC and Midnight protocol produced an even coverage across samples, and almost all reads were mapped to the SARS-CoV-2 reference. ARTIC and Midnight represent robust, cost-effective, and highly scalable methods that are appropriate in a clinical environment. Lineage designations were uniform across methods, representing the dominant lineages in Sweden during the period of collection. This study provides insights into methodological differences in SARS‑CoV‑2 sequencing and guidance in selecting suitable methods for various purposes.
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| 16. |
- Omazic, Anna, et al.
(författare)
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Dairy milk from cow and goat as a sentinel for tick-borne encephalitis virus surveillance
- 2023
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Ingår i: Comparative Immunology, Microbiology & Infectious Diseases. - : Elsevier. - 0147-9571 .- 1878-1667. ; 95
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Tidskriftsartikel (refereegranskat)abstract
- Tick-borne encephalitis (TBE) is one of the most severe human tick-borne diseases in Europe. It is caused by the tick-borne encephalitis virus (TBEV), which is transmitted to humans mainly via bites of Ixodes ricinus or I. persulcatus ticks. The geographical distribution and abundance of I. ricinus is expanding in Sweden as has the number of reported human TBE cases. In addition to tick bites, alimentary TBEV infection has also been reported after consumption of unpasteurized dairy products. So far, no alimentary TBEV infection has been reported in Sweden, but knowledge about its prevalence in Swedish ruminants is scarce. In the present study, a total of 122 bulk tank milk samples and 304 individual milk samples (including 8 colostrum samples) were collected from dairy farms (n = 102) in Sweden. All samples were analysed for the presence of TBEV antibodies by ELISA test and immunoblotting. Participating farmers received a questionnaire about milk production, pasteurization, tick prophylaxis used on animals, tick-borne diseases, and TBE vaccination status. We detected specific anti-TBEV antibodies, i.e., either positive (>126 Vienna Units per ml, VIEU/ml) or borderline (63-126 VIEU/ml) in bulk tank milk from 20 of the 102 farms. Individual milk samples (including colostrum samples) from these 20 farms were therefore collected for further analysis. Our results revealed important information for detection of emerging TBE risk areas. Factors such as consumption of unpasteurized milk, limited use of tick prophylaxis on animals and a moderate coverage of human TBE vaccination, may be risk factors for alimentary TBEV infection in Sweden.
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| 17. |
- Tran, Pham Tue Hung, 1991-
(författare)
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Characterizing important flavivirus-host interactions : Replication, assembly, restriction factors and vaccine development
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genus Flavivirus (family Flaviviridae) consists of important zoonotic viruses that cause morbidity and mortality worldwide. These viruses are enveloped and have a positive-sense single-stranded RNA genome encoding a polyprotein. Cleavages of the polyprotein by host and viral proteases result in individual viral proteins, including the structural capsid (C), pre-membrane (prM), envelope (E) proteins, and seven nonstructural proteins. Removal of the C-prM-E genes in the flavivirus genome results in replicons that can replicate in transfected cells but do not generate infectious virus particles. The replicon can be co-expressed with the C-prM-E genes in trans, resulting in packaging of the replicon and generation of replicon virus-like particles (RVPs).During cellular infection, various host proteins are employed, supporting multiple stages of the virus life cycle. In this thesis, we identified and characterized functions of the host lunapark protein and two members of the Endosomal Sorting Complexes Required for Transport Machinery – ALIX and CHMP4A. We also revealed how the host proteins were recruited by virus proteins during infection.To counteract the virus infection, virus-infected cells can express antiviral proteins. We demonstrated the antiviral mechanism of interferonstimulated gene (ISG) 15 and the E3 ligase for ISG15 conjugation HERC5, which degrades ALIX and CHMP4A, indirectly targets virus infection. Furthermore, using proteomic screening, we identified tripartite motif-containing proteins (TRIM) – TRIM21 and TRIM14 – as restriction factors to Langat virus and Zika virus.We also established and characterized an RVP production system based on the West Nile virus (WNV) Kunjin strain. The system was used as a vector to express antigens from Ebola virus (EBOV), which can potentially be developed as a vaccine platform against WNV and EBOV.
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| 18. |
- Tran, Pham Tue Hung, 1991-, et al.
(författare)
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Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice
- 2020
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Ingår i: Microorganisms. - Basel, Switzerland : MDPI. - 2076-2607. ; 8:12
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Tidskriftsartikel (refereegranskat)abstract
- Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.
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| 19. |
- Tran, Pham Tue Hung, 1991-, et al.
(författare)
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Identification and characterization of host proteins inducing the endoplasmic reticulum invagination during Flavivirus infection
- 2019
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Ingår i: Positive-Strand RNA Viuses. ; , s. 280-280
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Konferensbidrag (refereegranskat)abstract
- When Flaviviruses infect host cells, they can induce invagination of endoplasmic reticulum (ER) membrane to form vesicle-like compartments. These unique structures are hypothetical to facilitate the viral replication by reducing diffusion of virus replication machinery and viral RNA, providing a scaffold to anchor the replication complex, and protecting viral RNA from host cell intrinsic surveillance. The rearrangements of ER membrane to form these replication compartments (RCs) require modifications in its lipid constituents or binding of proteins to the membrane. Flaviviruses, indeed, use their proteins to generate RCs. It has been implicated that both KUNV and DENV viral NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is recondite whether host proteins can also participate in the formation and maintenance of RCs.In this project, we aimed to identify and characterize of host proteins inducing RC generation during Flavivirus infections. We used A549 as a cell model, and mosquito-borne Zika and Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, ER membranes were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes of infected cells and non-infected cells to identify host candidate proteins that can cause the RC formation. We are attempting to enrich the RC-containing fractions and identifying proteins here, which narrows the list of true candidate proteins. The candidate proteins then will be characterized by using molecular techniques such as gene knock down, overexpression, and microscopy techniques.
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| 20. |
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| 21. |
- Tran, Pham-Tue-Hung, 1991-, et al.
(författare)
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Roles of the Endogenous Lunapark Protein during Flavivirus Replication
- 2021
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Ingår i: Viruses. - Switzerland : MDPI. - 1999-4915. ; 13:7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.
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| 22. |
- Tran, Pham Tue Hung, 1991-, et al.
(författare)
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Screening of host proteins interacting with Kunjin, Langat, Zika replication complex
- 2019
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Ingår i: Positive-Strand Rna Viuses.
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Konferensbidrag (refereegranskat)abstract
- When Flaviviruses infect host cells, they can induce invagination of endoplasmic reticulum (ER) membrane to form vesicle-like compartments. These unique structures are hypothetical to facilitate the viral replication by reducing diffusion of virus replication machinery and viral RNA, providing a scaffold to anchor the replication complex, and protecting viral RNA from host cell intrinsic surveillance. The rearrangements of ER membrane to form these replication compartments (RCs) require modifications in its lipid constituents or binding of proteins to the membrane. Flaviviruses, indeed, use their proteins to generate RCs. It has been implicated that both KUNV and DENV viral NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is recondite whether host proteins can also participate in the formation and maintenance of RCs.In this project, we aimed to identify and characterize of host proteins inducing RC generation during Flavivirus infections. We used A549 as a cell model, and mosquito-borne Zika and Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, ER membranes were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes of infected cells and non-infected cells to identify host candidate proteins that can cause the RC formation. We are attempting to enrich the RC-containing fractions and identifying proteins here, which narrows the list of true candidate proteins. The candidate proteins then will be characterized by using molecular techniques such as gene knock down, overexpression, and microscopy techniques.
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| 23. |
- Tran, Pham Tue Hung, 1991-, et al.
(författare)
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Screening of host proteins interacting with Kunjin, Langat, Zikareplication complex
- 2019
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Ingår i: 16th Smögen Summer Symposium on Virology.
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Konferensbidrag (refereegranskat)abstract
- During infection and eclipse time, Flaviviruses induce invagination of the endoplasmic reticulum (ER) membrane to form compartments, protecting their viral replication complex. The rearrangements of ER membrane require modifications in ER membrane lipid constituents or binding of proteins to bend the membrane. Indeed, it has been implicated that both KUNV and DENV NS1, NS2A, NS4A, NS4B proteins could induce membrane remodelings. However, it is not well known whether host proteins can also participate in the formation and maintenance of these compartments.In this project, we aimed to identify host proteins interacting with Kunjin, Langat, Zika replication complex. These proteins may function for ER invagination during Flavivirus infection. We used human adenocarcinoma epithelial A549 cells as a cell model, mosquito-borne Zika, Kunjin virus, and tick-borne Langat virus as virus models. After virus infections, the ER membranes from infected and non-infected cells were harvested using ultracentrifuge with a sucrose gradient. Proteins from these ERs were identified using mass spectrometry. We compared the differences between the ER proteomes to identify host candidate proteins that can cause the RC formation. To narrows the list of true candidate proteins, we attempted to enrich the RC-containing fractions by doing co-immuno precipitation. We are doing TMT-MS to identify and quantify the host proteins from Co-IP elutions. The functions of these proteins will be characterized by using molecular techniques.
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| 24. |
- Wallenhammar, Amélie, 1986-, et al.
(författare)
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Developing a novel strategy to determine new Tick-borne encephalitis foci
- 2018
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Ingår i: 15th Smögen Summer Symposium on Virology, Smögen, Sweden, August 23-25, 2018..
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Konferensbidrag (refereegranskat)abstract
- TBEV is the most important viral tick-borne zoonosis in Europe, and infection may lead to severeCNS disease, including encephalitis and myelitis. Climate changes have increased the tickdistribution in Sweden, increasing the risk areas of Tick-borne encephalitis (TBE) at severalregions including the Örebro county. The TBE virus (TBEV) is usually transmitted to humans viatick bites, however oral transmission through consumption of non-pasteurized dairy products havealso been described. Both TBEV and antibodies against the viral proteins have been detected inmilk of goats, sheep and cattle. Since the prevalence of TBEV in the tick population is low there is a need for new and robust surveillance techniques identifying novel risk areas of TBEV at earlystages.In this study we have developed a strategy for identifying new TBEV foci. We have collected raw milk and colostrum samples from sheep and goats in the Örebro region. The milk samples were analyzed for the presence of TBEV antibodies by ELISA. In addition, the ELISA results were further verified by an in-house Western Blot assay where milk samples were used asprimary antibody to detect the Envelope-protein of TBEV. This method has so far revealed two novel TBEV foci within the Örebro county. Questing ticks and ticks feeding on sheep have been collected at areas of TBEV positive milk. The ticks are currently being analyzed for TBEV by PCR. The specific Örebro strains will be isolated from TBEV positive ticks and the viral genomeswill be further characterized using established next-generation sequencing technique.
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| 25. |
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