Publications by authors named "Jonathan Filskov"

6 Publications

  • Page 1 of 1

HCV p7 as a novel vaccine-target inducing multifunctional CD4 and CD8 T-cells targeting liver cells expressing the viral antigen.

Sci Rep 2019 Oct 1;9(1):14085. Epub 2019 Oct 1.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Despite recent treatment advances for chronic hepatitis C virus (HCV) infection, a vaccine is urgently needed for global control of this important liver pathogen. The lack of robust immunocompetent HCV infection models makes it challenging to identify correlates of protection and test vaccine efficacy. However, vigorous CD4 and CD8 T-cell responses are detected in patients that spontaneously resolve acute infection, whereas dysfunctional T-cell responses are a hallmark of chronic infection. The HCV p7 protein, forming ion-channels essential for viral assembly and release, has not previously been pursued as a vaccine antigen. Herein, we demonstrated that HCV p7 derived from genotype 1a and 1b sequences are highly immunogenic in mice when employed as overlapping peptides formulated as nanoparticles with the cross-priming adjuvant, CAF09. This approach induced multifunctional cytokine producing CD4 and CD8 T-cells targeting regions of p7 that are subject to immune pressure during HCV infection in chimpanzees and humans. Employing a surrogate in vivo challenge model of liver cells co-expressing HCV-p7 and GFP, we found that vaccinated mice cleared transgene expressing cells. This study affirms the potential of a T-cell inducing nanoparticle vaccine platform to target the liver and introduces HCV p7 as a potential target for HCV vaccine explorations.
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http://dx.doi.org/10.1038/s41598-019-50365-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773770PMC
October 2019

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants.

Hepatology 2019 09 5;70(3):771-787. Epub 2019 Jun 5.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance-associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.
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http://dx.doi.org/10.1002/hep.30647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6772116PMC
September 2019

Broadening CD4 and CD8 T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes.

J Virol 2017 07 26;91(14). Epub 2017 Jun 26.

Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.

Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4 and CD8 T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4 T cells, whereas the NS3 pepmix induced a far more vigorous CD4 T cell response and was as potent a CD8 T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ) tumor necrosis factor alpha-positive (TNF-α) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity. With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many chronic infections, including HCV infection, have a remarkable ability to drive initially strong CD4 and CD8 T cell responses against dominant epitopes toward an exhausted, dysfunctional state. Thus, new and innovative vaccine approaches to control HCV should be evaluated. Here, we report on a novel peptide-based nanoparticle vaccine strategy (NS3 pepmix) aimed at generating T cell immunity against potential subdominant T cell epitopes that are not efficiently targeted by vaccination with full-length recombinant protein (rNS3) or infection with HCV. As proof of concept, we found that NS3 pepmix excels in broadening the repertoire of epitope-specific, multifunctional, and cytotoxic CD4 and CD8 T cells compared to vaccination with rNS3, which generated only CD4 T cell responses.
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http://dx.doi.org/10.1128/JVI.00130-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487548PMC
July 2017

Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology.

Infect Immun 2015 Dec 28;83(12):4731-9. Epub 2015 Sep 28.

Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.

When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrient-starved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) or IFN-γ, TNF-α, and interleukin-2 to T cells that only expressed IFN-γ. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission.
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http://dx.doi.org/10.1128/IAI.01055-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645392PMC
December 2015

Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant.

Vaccine 2014 Jun 28;32(31):3927-35. Epub 2014 May 28.

Statens Serum Institut, Department of Infectious Disease Immunology, Artillerivej 5, 2300 Copenhagen S, Denmark. Electronic address:

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.
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http://dx.doi.org/10.1016/j.vaccine.2014.05.050DOI Listing
June 2014
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