| 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. |
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