| 1. |
- Ahlén, Gustaf
(författare)
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Development of a therapeutic vaccine against the hepatitis C virus
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hepatitis C virus (HCV) infection is a major cause of liver disease and it is estimated that 170 million people worldwide are chronically infected by HCV. There are no protective or curable vaccines available for HCV, however treatment consisting of interferon-α and ribavirin is curative in 45-75% of the chronic infected patients, depending on the viral genotype. The antiviral treatment has the lowest efficacy for patients infected by genotype 1. Today’s treatment regimens are associated with many side effects. Thus, new antiviral treatment regimens and/or vaccines are in urgent need. The majority of infected individuals develop a chronic disease and the reason for the high rate of persistence is in part explained by the highly genetic variability of HCV. Thus, development of a therapeutic vaccine against HCV should therefore be targeted against a region of the HCV genome with a limited genetic variability. We have based our development of a genetic vaccine on the non-structural (NS) 3 and NS4A proteins. The NS3 protein performs essential functions in the viral life cycle including protease and helicase activities. The NS3 co-factor, NS4A, is important for NS3 to stabilize the protein complex and to fully utilize its functions. Previous studies have shown that NS3/4A when delivered as a genetic vaccine induce both humoral and cellular immune responses in mice. We now investigated if we could further enhance the immunogenicity of the NS3/4A DNA vaccine. Codon optimization (co) of the NS3/4A DNA gene resulted in an enhanced immunogenicity explained by the increase of NS3/4A-protein expression. Due to the lack of small animal models to study HCV, we have generated a mouse model with transient expression of HCV proteins in the liver. By using this model we could show that peripherally vaccine-primed T cells could enter the liver, recognize and eradicate NS3/4A expressing hepatocytes, a prerequisite for a functional therapeutic vaccine against HCV. The NS3/4A protein has recently been shown to interfere with the innate immunity through cleavage of Cardif (also known as IPS-1, MAVS, VISA), resulting in suppression of the interferon response within the infected cell. We found that cleavage of Cardif by NS3/4A also occur in murine cells making it possible to study the effect of this interaction also in mouse models with hepatic expressing the NS3/4Aprotein. The effects that NS3/4A exert on the innate immunity do not seem to affect the adaptive immunity, since NS3/4A-protein expression did not prevent clearance of transiently transfected hepatocytes in vivo. This helps to explain why escaping the adaptive immunity through mutations should be beneficial for HCV. To better understand the relationship between immune escape and viral fitness we studied the immunodominant human HLA-A2-restricted epitope at residues 1073-1081 of NS3. Despite that the epitope is immunodominant, only a limited number of mutations occur within this epitope. We now show that the absence of mutations at some of these positions can be explained by a reduced protease activity and viral replication, which reduce the viral fitness. DNA vaccines have been shown to induce promising immune responses in animal models. However, so far DNA vaccines have not been found to prime effective immune responses when tested in primates / humans. The explanation for this is at least partly explained by the poor uptake of the plasmid DNA in humans. We therefore evaluated different delivery methods of our DNA vaccine using transdermal delivery by the gene gun or by intramuscular delivery in combination with in vivo electroporation (EP). These studies revealed that the coNS3/4A vaccine primed the broadest immune responses when delivered with in vivo EP. Importantly, although NS3/4A can block the response to dsRNA, these signal pathways are not activated during DNA immunizations which helps to explain the effectiveness of the DNA-based coNS3/4A vaccine. The coNS3/4A vaccine was evaluated in a toxicological study in rabbits which showed that the vaccine had an acceptable safety profile and biodistribution when administred using in vivo EP. Finally, the current coNS3/4A DNA vaccine delivered using in vivo electroporation was recently approved by the Swedish Medical Products Agency to enter a clinical trial, which will be the first DNA vaccine delivered in combination with in vivo electroporation against an infectious disease in humans.
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| 2. |
- Ahlen, Gustaf, et al.
(författare)
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Limited effect on NS3-NS4A protein cleavage after alanine substitutions within the immunodominant HLA-A2-restricted epitope of the hepatitis C virus genotype 3a non-structural 3/4A protease.
- 2012
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Ingår i: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 93:Pt 8, s. 1680-1686
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Tidskriftsartikel (refereegranskat)abstract
- It has been well established that immunological escape mutations within the hepatitis C virus genotype (gt) 1a non-structural (NS) 3/4A protease is partly prevented by a reduction of the viral protease fitness. Surprisingly little is known whether similar mutations affect proteases from other genotypes. In the present study, we assessed both the human leukocyte antigen (HLA)-A2-restricted cytotoxic T cell response and gt3a NS3/4A protease fitness. Similar to gt1, the 1073-1081 epitope was also immunodominant within the gt3a-specific HLA-A2-restricted cytotoxic T cell response, despite a homology of only 56% between gt1a and gt3a genes. However, unlike the gt1a NS3/4A protease, all residues within the gt3a 1073-1081 epitope could sequentially be replaced by alanine with a, at least in part, retained protease activity.
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| 3. |
- Ahlén, Gustaf, et al.
(författare)
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Mannosylated mucin-type immunoglobulin fusion proteins enhance antigen-specific antibody and T lymphocyte responses
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented.OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays.Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect.Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.
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| 4. |
- Asghar, Naveed, 1983-, et al.
(författare)
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Flavivirus replicon-based novel vaccine candidates against Hepatitis viruses
- 2024
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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, the cured patients can be re-infected as they lack protective immune responses. In addition, the relatively high 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 have developed and utilized flavivirus replicons as delivery system to prime hepatitis-specific T cell responses. We generated subgenomic replicons of Tick-borne encephalitis virus (TBEV), Langat virus (LGTV), West-Nile virus (WNV), and Kunjin virus (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 of control DNA plasmid with CMV promoter. The immunogenicity of these flavivirus replicons was evaluated in mice. The KUNV replicon triggered a potent cellular immune response with respect to priming of HCV NS3/4A-specific T cells as determined by ELISpot, and polyfunctionality. In short, the newly developed KUNV replicon- based vaccine is an attractive candidate to provide protection against hepatitis viruses.
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| 5. |
- 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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| 6. |
- Burm, Rani, et al.
(författare)
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Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections.
- 2023
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Ingår i: Gut. - : BMJ Publishing Group Ltd. - 0017-5749 .- 1468-3288. ; 72:6, s. 1186-1195
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo.DESIGN: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer.RESULTS: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection.CONCLUSION: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection.
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| 7. |
- Chen, Antony, et al.
(författare)
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Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination.
- 2011
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Ingår i: Journal of immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 186:9, s. 5107-18
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Tidskriftsartikel (refereegranskat)abstract
- The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting "healthy" heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2-restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.
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| 8. |
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| 9. |
- Krishnan, Shuba, et al.
(författare)
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Metabolic Perturbation Associated With COVID-19 Disease Severity and SARS-CoV-2 Replication
- 2021
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier BV. - 1535-9476 .- 1535-9484. ; 20
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Tidskriftsartikel (refereegranskat)abstract
- Viruses hijack host metabolic pathways for their replicative advantage. In this study, using patient-derived multiomics data and in vitro infection assays, we aimed to understand the role of key metabolic pathways that can regulate severe acute respiratory syndrome coronavirus-2 reproduction and their association with disease severity. We used multiomics platforms (targeted and untargeted proteomics and untargeted metabolomics) on patient samples and cell-line models along with immune phenotyping of metabolite transporters in patient blood cells to understand viral-induced metabolic modulations. We also modulated key metabolic pathways that were identified using multiomics data to regulate the viral reproduction in vitro. Coronavirus disease 2019 disease severity was characterized by increased plasma glucose and mannose levels. Immune phenotyping identified altered expression patterns of carbohydrate transporter, glucose transporter 1, in CD8+ T cells, intermediate and nonclassical monocytes, and amino acid transporter, xCT, in classical, intermediate, and nonclassical monocytes. In in vitro lung epithelial cell (Calu-3) infection model, we found that glycolysis and glutaminolysis are essential for virus replication, and blocking these metabolic pathways caused significant reduction in virus production. Taken together, we therefore hypothesized that severe acute respiratory syndrome coronavirus-2 utilizes and rewires pathways governing central carbon metabolism leading to the efflux of toxic metabolites and associated with disease severity. Thus, the host metabolic perturbation could be an attractive strategy to limit the viral replication and disease severity.
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| 10. |
- Maravelia, Panagiota, et al.
(författare)
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Blocking entry of hepatitis B and D viruses to hepatocytes as a novel immunotherapy for treating chronic infections
- 2021
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press. - 0022-1899 .- 1537-6613. ; 223:1, s. 128-138
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Chronic hepatitis B and D virus (HBV/HDV) infections can cause cancer. Current HBV therapy using nucleoside analogues (NAs) is life-long and reduces but does not eliminate the risk of cancer. A hallmark of chronic hepatitis B is a dysfunctional HBV-specific T cell response. We therefore designed an immunotherapy driven by naïve healthy T cells specific for the HDV antigen (HDAg) to bypass the need for HBV-specific T cells in order to prime PreS1-specific T cells and PreS1 antibodies blocking HBV entry.METHODS: Ten combinations of PreS1 and/or HDAg sequences were evaluated for induction of PreS1 antibodies and HBV- and HDV- specific T cells in vitro and in vivo. Neutralization of HBV by PreS1-specific murine and rabbit antibodies was evaluated in cell culture, and rabbit anti-PreS1 were tested for neutralization of HBV in mice repopulated with human hepatocytes.RESULTS: The best vaccine candidate induced T cells to PreS1 and HDAg, and PreS1 antibodies blocking HBV entry in vitro. Importantly, adoptive transfer of PreS1 antibodies prevented, or modulated, HBV infection after a subsequent challenge in humanized mice.CONCLUSION: We here describe a novel immunotherapy for chronic HBV/HDV that targets viral entry to complement NAs and coming therapies inhibiting viral maturation.
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| 11. |
- Yan, Jingyi, et al.
(författare)
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Distinct roles of vaccine-induced SARS-CoV-2-specific neutralizing antibodies and T cells in protection and disease
- 2024
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0016 .- 1525-0024. ; 32:2, s. 540-555
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Tidskriftsartikel (refereegranskat)abstract
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (NAbs) lack cross-reactivity between SARS-CoV species and variants and fail to mediate long-term protection against infection. The maintained protection against severe disease and death by vaccination suggests a role for cross-reactive T cells. We generated vaccines containing sequences from the spike or receptor binding domain, the membrane and/or nucleoprotein that induced only T cells, or T cells and NAbs, to understand their individual roles. In three models with homologous or heterologous challenge, high levels of vaccine-induced SARS-CoV-2 NAbs protected against neither infection nor mild histological disease but conferred rapid viral control limiting the histological damage. With no or low levels of NAbs, vaccine-primed T cells, in mice mainly CD8+ T cells, partially controlled viral replication and promoted NAb recall responses. T cells failed to protect against histological damage, presumably because of viral spread and subsequent T cell-mediated killing. Neither vaccine- nor infection-induced NAbs seem to provide long-lasting protective immunity against SARS-CoV-2. Thus, a more realistic approach for universal SARS-CoV-2 vaccines should be to aim for broadly cross-reactive NAbs in combination with long-lasting highly cross-reactive T cells. Long-lived cross-reactive T cells are likely key to prevent severe disease and fatalities during current and future pandemics.
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