This page has only limited features, please log in for full access.
The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.
Alexander Sampson; Jonathan Heeney; Diego Cantoni; Matteo Ferrari; Maria Sans; Charlotte George; Cecilia Di Genova; Martin Mayora Neto; Sebastian Einhauser; Benedikt Asbach; Ralf Wagner; Helen Baxendale; Nigel Temperton; George Carnell. Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses. Viruses 2021, 13, 1579 .
AMA StyleAlexander Sampson, Jonathan Heeney, Diego Cantoni, Matteo Ferrari, Maria Sans, Charlotte George, Cecilia Di Genova, Martin Mayora Neto, Sebastian Einhauser, Benedikt Asbach, Ralf Wagner, Helen Baxendale, Nigel Temperton, George Carnell. Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses. Viruses. 2021; 13 (8):1579.
Chicago/Turabian StyleAlexander Sampson; Jonathan Heeney; Diego Cantoni; Matteo Ferrari; Maria Sans; Charlotte George; Cecilia Di Genova; Martin Mayora Neto; Sebastian Einhauser; Benedikt Asbach; Ralf Wagner; Helen Baxendale; Nigel Temperton; George Carnell. 2021. "Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses." Viruses 13, no. 8: 1579.
Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union’s H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development.
Alexandra Hauser; George Carnell; Kathrin Held; Guidenn Sulbaran; Nadine Tischbierek; Lisa Rogers; Georgios Pollakis; Paul Tonks; Michael Hoelscher; Song Ding; Rogier Sanders; Christof Geldmacher; Quentin Sattentau; Winfried Weissenhorn; Jonathan Heeney; David Peterhoff; Ralf Wagner. Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses. Vaccines 2021, 9, 750 .
AMA StyleAlexandra Hauser, George Carnell, Kathrin Held, Guidenn Sulbaran, Nadine Tischbierek, Lisa Rogers, Georgios Pollakis, Paul Tonks, Michael Hoelscher, Song Ding, Rogier Sanders, Christof Geldmacher, Quentin Sattentau, Winfried Weissenhorn, Jonathan Heeney, David Peterhoff, Ralf Wagner. Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses. Vaccines. 2021; 9 (7):750.
Chicago/Turabian StyleAlexandra Hauser; George Carnell; Kathrin Held; Guidenn Sulbaran; Nadine Tischbierek; Lisa Rogers; Georgios Pollakis; Paul Tonks; Michael Hoelscher; Song Ding; Rogier Sanders; Christof Geldmacher; Quentin Sattentau; Winfried Weissenhorn; Jonathan Heeney; David Peterhoff; Ralf Wagner. 2021. "Stepwise Conformational Stabilization of a HIV-1 Clade C Consensus Envelope Trimer Immunogen Impacts the Profile of Vaccine-Induced Antibody Responses." Vaccines 9, no. 7: 750.
We developed an influenza hemagglutinin (HA) pseudotype library encompassing Influenza A subtypes HA1-18 and Influenza B subtypes (both lineages) to be employed in influenza pseudotype microneutralization (pMN) assays. The pMN is highly sensitive and specific for detecting virus-specific neutralizing antibodies against influenza viruses and can be used to assess antibody functionality in vitro. Here we show the production of these viral HA pseudotypes and their employment as substitutes for wildtype viruses in influenza neutralization assays. We demonstrate their utility in detecting serum responses to vaccination with the ability to evaluate cross-subtype neutralizing responses elicited by specific vaccinating antigens. Our findings may inform further preclinical studies involving immunization dosing regimens in mice and may help in the creation and selection of better antigens for vaccine design. These HA pseudotypes can be harnessed to meet strategic objectives that contribute to the strengthening of global influenza surveillance, expansion of seasonal influenza prevention and control policies, and strengthening pandemic preparedness and response.
Joanne Del Rosario; Kelly da Costa; Benedikt Asbach; Francesca Ferrara; Matteo Ferrari; David Wells; Gurdip Mann; Veronica Ameh; Claude Sabeta; Ashley Banyard; Rebecca Kinsley; Simon Scott; Ralf Wagner; Jonathan Heeney; George Carnell; Nigel Temperton. Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection. Vaccines 2021, 9, 741 .
AMA StyleJoanne Del Rosario, Kelly da Costa, Benedikt Asbach, Francesca Ferrara, Matteo Ferrari, David Wells, Gurdip Mann, Veronica Ameh, Claude Sabeta, Ashley Banyard, Rebecca Kinsley, Simon Scott, Ralf Wagner, Jonathan Heeney, George Carnell, Nigel Temperton. Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection. Vaccines. 2021; 9 (7):741.
Chicago/Turabian StyleJoanne Del Rosario; Kelly da Costa; Benedikt Asbach; Francesca Ferrara; Matteo Ferrari; David Wells; Gurdip Mann; Veronica Ameh; Claude Sabeta; Ashley Banyard; Rebecca Kinsley; Simon Scott; Ralf Wagner; Jonathan Heeney; George Carnell; Nigel Temperton. 2021. "Exploiting Pan Influenza A and Pan Influenza B Pseudotype Libraries for Efficient Vaccine Antigen Selection." Vaccines 9, no. 7: 741.
The delivery of HIV-1 envelope (Env) trimer-based immunogens on the surface of nanoparticles holds promise to promote immunogenicity with the aim of inducing a potent, durable and broad neutralizing antibody (bnAb) response. Towards that goal, we examined the covalent conjugation of Env to 100 nm and 200 nm silica nanoparticles (SiNPs) to optimize conjugation density and attachment stability. Env was redesigned to enable site-specific cysteine-mediated covalent conjugation while maintaining its structural integrity and antigenicity. Env was anchored to different sized SiNPs with a calculated spacing of 15 nm between adjacent trimers. Both particle sizes exhibited high in vitro stability over a seven-day period. After attachment, 100 nm particles showed better colloidal stability compared to 200 nm particles. Importantly, the antigenic profile of Env was not impaired by surface attachment, indicating that the quaternary structure was maintained. In vitro Env uptake by dendritic cells was significantly enhanced when Env was delivered on the surface of nanoparticles compared to soluble Env. Furthermore, multivalent Env displayed efficiently activated B cells even at Env concentrations in the low nanomolar range. In mice, antibody responses to nanoparticle-coupled Env were stronger compared to the free protein and had equivalent effects at lower doses and without adjuvant.
David Peterhoff; Stefanie Thalhauser; Jan Sobczak; Mona Mohsen; Christoph Voigt; Nicole Seifert; Patrick Neckermann; Alexandra Hauser; Song Ding; Quentin Sattentau; Martin Bachmann; Miriam Breunig; Ralf Wagner. Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery. Vaccines 2021, 9, 642 .
AMA StyleDavid Peterhoff, Stefanie Thalhauser, Jan Sobczak, Mona Mohsen, Christoph Voigt, Nicole Seifert, Patrick Neckermann, Alexandra Hauser, Song Ding, Quentin Sattentau, Martin Bachmann, Miriam Breunig, Ralf Wagner. Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery. Vaccines. 2021; 9 (6):642.
Chicago/Turabian StyleDavid Peterhoff; Stefanie Thalhauser; Jan Sobczak; Mona Mohsen; Christoph Voigt; Nicole Seifert; Patrick Neckermann; Alexandra Hauser; Song Ding; Quentin Sattentau; Martin Bachmann; Miriam Breunig; Ralf Wagner. 2021. "Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery." Vaccines 9, no. 6: 642.
SARS-CoV-2 infection fatality ratios (IFR) remain controversially discussed with implications for political measures. The German county of Tirschenreuth suffered a severe SARS-CoV-2 outbreak in spring 2020, with particularly high case fatality ratio (CFR). To estimate seroprevalence, underreported infections, and IFR for the Tirschenreuth population aged ≥14 years in June/July 2020, we conducted a population-based study including home visits for the elderly, and analyzed 4203 participants for SARS-CoV-2 antibodies via three antibody tests. Latent class analysis yielded 8.6% standardized county-wide seroprevalence, a factor of underreported infections of 5.0, and 2.5% overall IFR. Seroprevalence was two-fold higher among medical workers and one third among current smokers with similar proportions of registered infections. While seroprevalence did not show an age-trend, the factor of underreported infections was 12.2 in the young versus 1.7 for ≥85-year-old. Age-specific IFRs were <0.5% below 60 years of age, 1.0% for age 60–69, and 13.2% for age 70+. Senior care homes accounted for 45% of COVID-19-related deaths, reflected by an IFR of 7.5% among individuals aged 70+ and an overall IFR of 1.4% when excluding senior care home residents from our computation. Our data underscore senior care home infections as key determinant of IFR additionally to age, insufficient targeted testing in the young, and the need for further investigations on behavioral or molecular causes of the fewer infections among current smokers.
Ralf Wagner; David Peterhoff; Stephanie Beileke; Felix Günther; Melanie Berr; Sebastian Einhauser; Anja Schütz; Hans Niller; Philipp Steininger; Antje Knöll; Matthias Tenbusch; Clara Maier; Klaus Korn; Klaus Stark; André Gessner; Ralph Burkhardt; Michael Kabesch; Holger Schedl; Helmut Küchenhoff; Annette Pfahlberg; Iris Heid; Olaf Gefeller; Klaus Überla. Estimates and Determinants of SARS-Cov-2 Seroprevalence and Infection Fatality Ratio Using Latent Class Analysis: The Population-Based Tirschenreuth Study in the Hardest-Hit German County in Spring 2020. Viruses 2021, 13, 1118 .
AMA StyleRalf Wagner, David Peterhoff, Stephanie Beileke, Felix Günther, Melanie Berr, Sebastian Einhauser, Anja Schütz, Hans Niller, Philipp Steininger, Antje Knöll, Matthias Tenbusch, Clara Maier, Klaus Korn, Klaus Stark, André Gessner, Ralph Burkhardt, Michael Kabesch, Holger Schedl, Helmut Küchenhoff, Annette Pfahlberg, Iris Heid, Olaf Gefeller, Klaus Überla. Estimates and Determinants of SARS-Cov-2 Seroprevalence and Infection Fatality Ratio Using Latent Class Analysis: The Population-Based Tirschenreuth Study in the Hardest-Hit German County in Spring 2020. Viruses. 2021; 13 (6):1118.
Chicago/Turabian StyleRalf Wagner; David Peterhoff; Stephanie Beileke; Felix Günther; Melanie Berr; Sebastian Einhauser; Anja Schütz; Hans Niller; Philipp Steininger; Antje Knöll; Matthias Tenbusch; Clara Maier; Klaus Korn; Klaus Stark; André Gessner; Ralph Burkhardt; Michael Kabesch; Holger Schedl; Helmut Küchenhoff; Annette Pfahlberg; Iris Heid; Olaf Gefeller; Klaus Überla. 2021. "Estimates and Determinants of SARS-Cov-2 Seroprevalence and Infection Fatality Ratio Using Latent Class Analysis: The Population-Based Tirschenreuth Study in the Hardest-Hit German County in Spring 2020." Viruses 13, no. 6: 1118.
Alternative splicing and the expression of intron-containing mRNAs is one hallmark of HIV gene expression. To facilitate the otherwise hampered nuclear export of non-fully processed mRNAs, HIV encodes the Rev protein, which recognizes its intronic response element and fuels the HIV RNAs into the CRM-1-dependent nuclear protein export pathway. Both alternative splicing and Rev-dependency are regulated by the primary HIV RNA sequence. Here, we show that these processes are extremely sensitive to sequence alterations in the 5’coding region of the HIV genomic RNA. Increasing the GC content by insertion of either GFP or silent mutations activates a cryptic splice donor site in gag, entirely deregulates the viral splicing pattern, and lowers infectivity. Interestingly, an adaptation of the inserted GFP sequence toward an HIV-like nucleotide bias reversed these phenotypes completely. Of note, the adaptation yielded completely different primary sequences although encoding the same amino acids. Thus, the phenotypes solely depend on the nucleotide composition of the two GFP versions. This is a strong indication of an HIV-specific mRNP code in the 5′ gag region wherein the primary RNA sequence bias creates motifs for RNA-binding proteins and controls the fate of the HIV-RNA in terms of viral gene expression and infectivity.
Bastian Grewe; Carolin Vogt; Theresa Horstkötter; Bettina Tippler; Han Xiao; Bianca Müller; Klaus Überla; Ralf Wagner; Benedikt Asbach; Jens Bohne. The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity. Viruses 2021, 13, 997 .
AMA StyleBastian Grewe, Carolin Vogt, Theresa Horstkötter, Bettina Tippler, Han Xiao, Bianca Müller, Klaus Überla, Ralf Wagner, Benedikt Asbach, Jens Bohne. The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity. Viruses. 2021; 13 (6):997.
Chicago/Turabian StyleBastian Grewe; Carolin Vogt; Theresa Horstkötter; Bettina Tippler; Han Xiao; Bianca Müller; Klaus Überla; Ralf Wagner; Benedikt Asbach; Jens Bohne. 2021. "The HIV 5′ Gag Region Displays a Specific Nucleotide Bias Regulating Viral Splicing and Infectivity." Viruses 13, no. 6: 997.
The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.
Duško Lainšček; Tina Fink; Vida Forstnerič; Iva Hafner-Bratkovič; Sara Orehek; Žiga Strmšek; Mateja Manček-Keber; Peter Pečan; Hana Esih; Špela Malenšek; Jana Aupič; Petra Dekleva; Tjaša Plaper; Sara Vidmar; Lucija Kadunc; Mojca Benčina; Neža Omersa; Gregor Anderluh; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Valter Bergant; Alexander Herrmann; Andreas Pichlmair; Roman Jerala. A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response. Vaccines 2021, 9, 431 .
AMA StyleDuško Lainšček, Tina Fink, Vida Forstnerič, Iva Hafner-Bratkovič, Sara Orehek, Žiga Strmšek, Mateja Manček-Keber, Peter Pečan, Hana Esih, Špela Malenšek, Jana Aupič, Petra Dekleva, Tjaša Plaper, Sara Vidmar, Lucija Kadunc, Mojca Benčina, Neža Omersa, Gregor Anderluh, Florence Pojer, Kelvin Lau, David Hacker, Bruno Correia, David Peterhoff, Ralf Wagner, Valter Bergant, Alexander Herrmann, Andreas Pichlmair, Roman Jerala. A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response. Vaccines. 2021; 9 (5):431.
Chicago/Turabian StyleDuško Lainšček; Tina Fink; Vida Forstnerič; Iva Hafner-Bratkovič; Sara Orehek; Žiga Strmšek; Mateja Manček-Keber; Peter Pečan; Hana Esih; Špela Malenšek; Jana Aupič; Petra Dekleva; Tjaša Plaper; Sara Vidmar; Lucija Kadunc; Mojca Benčina; Neža Omersa; Gregor Anderluh; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Valter Bergant; Alexander Herrmann; Andreas Pichlmair; Roman Jerala. 2021. "A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response." Vaccines 9, no. 5: 431.
SARS-CoV-2 infection fatality ratios (IFR) remain controversially discussed with implications for political measures, but the number of registered infections depends on testing strategies and deduced case fatality ratios (CFR) are poor proxies for IFR. The German county of Tirschenreuth suffered a severe SARS-CoV-2 outbreak in spring 2020 with particularly high CFR. To estimate seroprevalence, dark figure, and IFR for the Tirschenreuth population aged ≥14 years in June/July 2020 with misclassification error control, we conducted a population-based study, including home visits for elderly, and analyzed 4203 participants for SARS-CoV-2 antibodies via three antibody tests (64% of our random sample). Latent class analysis yielded 8.6% standardized county-wide seroprevalence, dark figure factor 5.0, and 2.5% overall IFR. Seroprevalence was two-fold higher among medical workers and one third among current smokers with similar proportions of registered infections. While seroprevalence did not show an age-trend, the dark figure was 12.2 in the young versus 1.7 for ≥85-year-old. Age-specific IFRs were <0.5% below 60 years of age, 1.0% for age 60-69, 13.2% for age 70+, confirming a previously reported age-model for IFR. Senior care homes accounted for 45% of COVID-19-related deaths, reflected by an IFR of 7.5% among individuals aged 70+ and an overall IFR of 1.4% when excluding senior care home residents from our computation. Our data underscore senior care home infections as key determinant of IFR additionally to age, insufficient targeted testing in the young, and the need for further investigations on behavioral or molecular causes of the fewer infections among current smokers.
Ralf Wagner; David Peterhoff; Stephanie Beileke; Felix Guenther; Melanie Berr; Sebastian Einhauser; Anja Schütz; Hans Helmut Niller; Philipp Steininger; Antje Knöll; Matthias Tenbusch; Clara Maier; Klaus Korn; Klaus J. Stark; Andre Gessner; Ralph Burkhardt; Michael Kabesch; Holger Schedl; Helmut Küchenhoff; Annette B. Pfahlberg; Iris M. Heid; Olaf Gefeller; Klaus Überla. Estimates and determinants of SARS-CoV-2 seroprevalence and infection fatality ratio using latent class analysis: the population-based Tirschenreuth study in the hardest-hit German county in spring 2020. 2021, 1 .
AMA StyleRalf Wagner, David Peterhoff, Stephanie Beileke, Felix Guenther, Melanie Berr, Sebastian Einhauser, Anja Schütz, Hans Helmut Niller, Philipp Steininger, Antje Knöll, Matthias Tenbusch, Clara Maier, Klaus Korn, Klaus J. Stark, Andre Gessner, Ralph Burkhardt, Michael Kabesch, Holger Schedl, Helmut Küchenhoff, Annette B. Pfahlberg, Iris M. Heid, Olaf Gefeller, Klaus Überla. Estimates and determinants of SARS-CoV-2 seroprevalence and infection fatality ratio using latent class analysis: the population-based Tirschenreuth study in the hardest-hit German county in spring 2020. . 2021; ():1.
Chicago/Turabian StyleRalf Wagner; David Peterhoff; Stephanie Beileke; Felix Guenther; Melanie Berr; Sebastian Einhauser; Anja Schütz; Hans Helmut Niller; Philipp Steininger; Antje Knöll; Matthias Tenbusch; Clara Maier; Klaus Korn; Klaus J. Stark; Andre Gessner; Ralph Burkhardt; Michael Kabesch; Holger Schedl; Helmut Küchenhoff; Annette B. Pfahlberg; Iris M. Heid; Olaf Gefeller; Klaus Überla. 2021. "Estimates and determinants of SARS-CoV-2 seroprevalence and infection fatality ratio using latent class analysis: the population-based Tirschenreuth study in the hardest-hit German county in spring 2020." , no. : 1.
Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.
Eva Wagner-Drouet; Daniel Teschner; Christine Wolschke; Kerstin Schäfer-Eckart; Johannes Gärtner; Stephan Mielke; Martin Schreder; Guido Kobbe; Inken Hilgendorf; Stefan Klein; Mareike Verbeek; Markus Ditschkowski; Martina Koch; Monika Lindemann; Traudel Schmidt; Anne Rascle; Sascha Barabas; Ludwig Deml; Ralf Wagner; Daniel Wolff. Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation. Diagnostics 2021, 11, 312 .
AMA StyleEva Wagner-Drouet, Daniel Teschner, Christine Wolschke, Kerstin Schäfer-Eckart, Johannes Gärtner, Stephan Mielke, Martin Schreder, Guido Kobbe, Inken Hilgendorf, Stefan Klein, Mareike Verbeek, Markus Ditschkowski, Martina Koch, Monika Lindemann, Traudel Schmidt, Anne Rascle, Sascha Barabas, Ludwig Deml, Ralf Wagner, Daniel Wolff. Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation. Diagnostics. 2021; 11 (2):312.
Chicago/Turabian StyleEva Wagner-Drouet; Daniel Teschner; Christine Wolschke; Kerstin Schäfer-Eckart; Johannes Gärtner; Stephan Mielke; Martin Schreder; Guido Kobbe; Inken Hilgendorf; Stefan Klein; Mareike Verbeek; Markus Ditschkowski; Martina Koch; Monika Lindemann; Traudel Schmidt; Anne Rascle; Sascha Barabas; Ludwig Deml; Ralf Wagner; Daniel Wolff. 2021. "Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation." Diagnostics 11, no. 2: 312.
Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and β-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue β-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.
Dusko Lainscek; Tina Fink; Vida Forstneric; Iva Hafner-Bratkovic; Sara Orehek; Ziga Strmsek; Mateja Mancek Keber; Peter Pecan; Hana Esih; Spela Malensek; Jana Aupic; Petra Dekleva; Tjasa Plaper; Sara Vidmar; Lucija Kadunc; Mojca Bencina; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Roman Jerala. Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein. 2020, 1 .
AMA StyleDusko Lainscek, Tina Fink, Vida Forstneric, Iva Hafner-Bratkovic, Sara Orehek, Ziga Strmsek, Mateja Mancek Keber, Peter Pecan, Hana Esih, Spela Malensek, Jana Aupic, Petra Dekleva, Tjasa Plaper, Sara Vidmar, Lucija Kadunc, Mojca Bencina, Florence Pojer, Kelvin Lau, David Hacker, Bruno Correia, David Peterhoff, Ralf Wagner, Roman Jerala. Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein. . 2020; ():1.
Chicago/Turabian StyleDusko Lainscek; Tina Fink; Vida Forstneric; Iva Hafner-Bratkovic; Sara Orehek; Ziga Strmsek; Mateja Mancek Keber; Peter Pecan; Hana Esih; Spela Malensek; Jana Aupic; Petra Dekleva; Tjasa Plaper; Sara Vidmar; Lucija Kadunc; Mojca Bencina; Florence Pojer; Kelvin Lau; David Hacker; Bruno Correia; David Peterhoff; Ralf Wagner; Roman Jerala. 2020. "Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein." , no. : 1.
Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R 2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.
David Peterhoff; Vivian Glück; Matthias Vogel; Philipp Schuster; Anja Schütz; Philip Neubert; Veruschka Albert; Stefanie Frisch; Mara Kiessling; Philip Pervan; Maren Werner; Nicole Ritter; Leon Babl; Maria Deichner; Frank Hanses; Matthias Lubnow; Thomas Müller; Dirk Lunz; Florian Hitzenbichler; Franz Audebert; Viola Hähnel; Robert Offner; Martina Müller; Stephan Schmid; Ralph Burkhardt; Thomas Glück; Michael Koller; Hans Helmut Niller; Bernhard Graf; Bernd Salzberger; Jürgen Wenzel; Jonathan Jantsch; André Gessner; Barbara Schmidt; Ralf Wagner. A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization. Infection 2020, 49, 75 -82.
AMA StyleDavid Peterhoff, Vivian Glück, Matthias Vogel, Philipp Schuster, Anja Schütz, Philip Neubert, Veruschka Albert, Stefanie Frisch, Mara Kiessling, Philip Pervan, Maren Werner, Nicole Ritter, Leon Babl, Maria Deichner, Frank Hanses, Matthias Lubnow, Thomas Müller, Dirk Lunz, Florian Hitzenbichler, Franz Audebert, Viola Hähnel, Robert Offner, Martina Müller, Stephan Schmid, Ralph Burkhardt, Thomas Glück, Michael Koller, Hans Helmut Niller, Bernhard Graf, Bernd Salzberger, Jürgen Wenzel, Jonathan Jantsch, André Gessner, Barbara Schmidt, Ralf Wagner. A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization. Infection. 2020; 49 (1):75-82.
Chicago/Turabian StyleDavid Peterhoff; Vivian Glück; Matthias Vogel; Philipp Schuster; Anja Schütz; Philip Neubert; Veruschka Albert; Stefanie Frisch; Mara Kiessling; Philip Pervan; Maren Werner; Nicole Ritter; Leon Babl; Maria Deichner; Frank Hanses; Matthias Lubnow; Thomas Müller; Dirk Lunz; Florian Hitzenbichler; Franz Audebert; Viola Hähnel; Robert Offner; Martina Müller; Stephan Schmid; Ralph Burkhardt; Thomas Glück; Michael Koller; Hans Helmut Niller; Bernhard Graf; Bernd Salzberger; Jürgen Wenzel; Jonathan Jantsch; André Gessner; Barbara Schmidt; Ralf Wagner. 2020. "A highly specific and sensitive serological assay detects SARS-CoV-2 antibody levels in COVID-19 patients that correlate with neutralization." Infection 49, no. 1: 75-82.
Inducing a long-lasting as well as broad and potent immune response by generating broadly neutralizing antibodies is a major goal and at the same time the main challenge of preventive HIV-1 vaccine design. Immunization with soluble, stabilized and native-like envelope (Env) glycoprotein so far only led to low neutralization breadth and displayed low immunogenicity. A promising approach to generate a potent immune response is the presentation of Env on the surface of nanoparticles. In this review, we will focus on two key processes essential for the induction of immune response that can be addressed by specific features of nanoparticulate carriers: first, the trafficking to and within distinct compartments of the lymph node, and second, the use of multivalent Env display allowing for high avidity interactions. To optimize these pivotal steps critical design criteria should be considered for the presentation of Env on nanoparticles. These include an optimal particle size below 100 nm, distances between two adjacent Env antigens of approximately 10–15 nm, an appropriate orientation of Env, and finally, the stability of both the Env attachment and the nanoparticle platform. Hence, an interdisciplinary approach that combines a suitable delivery system and a straightforward presentation of the Env antigen may have the potential to drive the immune response towards increased breadth and potency.
Stefanie Thalhauser; David Peterhoff; Ralf Wagner; Miriam Breunig. Critical design criteria for engineering a nanoparticulate HIV-1 vaccine. Journal of Controlled Release 2019, 317, 322 -335.
AMA StyleStefanie Thalhauser, David Peterhoff, Ralf Wagner, Miriam Breunig. Critical design criteria for engineering a nanoparticulate HIV-1 vaccine. Journal of Controlled Release. 2019; 317 ():322-335.
Chicago/Turabian StyleStefanie Thalhauser; David Peterhoff; Ralf Wagner; Miriam Breunig. 2019. "Critical design criteria for engineering a nanoparticulate HIV-1 vaccine." Journal of Controlled Release 317, no. : 322-335.
Inducing immune responses protecting from HIV infection or at least controlling replication poses a huge challenge to modern vaccinology. An increasingly discussed strategy to elicit a potent and broad neutralizing antibody response is the immobilization of HIV's trimeric envelope (Env) surface receptor on a nanoparticulate carrier. As a conceptual proof, we attached an Env variant (BG505 SOSIP.664) to highly stable and biocompatible silica nanoparticles (SiNPs) via site-specific covalent conjugation or non-specific adsorption to SiNPs. Firstly, we demonstrated the feasibility of SiNPs as platform for Env presentation by a thorough characterization process during which Env density, attachment stability, and antigenicity were evaluated for both formulations. Binding affinities to selected antibodies were in the low nanomolar range for both formulations confirming that the structural integrity of Env is retained after attachment. Secondly, we explored the recognition of SiNP conjugates by antigen presenting cells. Here, the uptake of Env attached to SiNPs via a site-specific covalent conjugation was 4.5-fold enhanced, while adsorbed Env resulted only in a moderate 1.4-fold increase compared to Env in its soluble form. Thus, we propose SiNPs with site-specifically and covalently conjugated Env preferably in a high density as a promising candidate for further investigations as vaccine platform.
Stefanie Thalhauser; David Peterhoff; Ralf Wagner; Miriam Breunig. Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles. Journal of Pharmaceutical Sciences 2019, 109, 911 -921.
AMA StyleStefanie Thalhauser, David Peterhoff, Ralf Wagner, Miriam Breunig. Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles. Journal of Pharmaceutical Sciences. 2019; 109 (1):911-921.
Chicago/Turabian StyleStefanie Thalhauser; David Peterhoff; Ralf Wagner; Miriam Breunig. 2019. "Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles." Journal of Pharmaceutical Sciences 109, no. 1: 911-921.
The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in nonhuman primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime and poxvirus and protein boost regimen and compared how two poxvirus vectors with various degrees of replication capacity and two different delivery modalities—conventional intramuscular delivery and percutaneous delivery by scarification—impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings.
Benedikt Asbach; Karen V. Kibler; Josef Köstler; Beatriz Perdiguero; Nicole L. Yates; Sherry Stanfield-Oakley; Georgia D. Tomaras; Shing-Fen Kao; Kathryn E. Foulds; Mario Roederer; Michael S. Seaman; David C. Montefiori; Robert Parks; Guido Ferrari; Donald N. Forthal; Sanjay Phogat; James Tartaglia; Susan W. Barnett; Steven G. Self; Raphael Gottardo; Anthony D. Cristillo; Deborah E. Weiss; Lindsey Galmin; Song Ding; Jonathan Heeney; Mariano Esteban; Bertram L. Jacobs; Giuseppe Pantaleo; Ralf Wagner. Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost. Journal of Virology 2019, 93, e01529-18 .
AMA StyleBenedikt Asbach, Karen V. Kibler, Josef Köstler, Beatriz Perdiguero, Nicole L. Yates, Sherry Stanfield-Oakley, Georgia D. Tomaras, Shing-Fen Kao, Kathryn E. Foulds, Mario Roederer, Michael S. Seaman, David C. Montefiori, Robert Parks, Guido Ferrari, Donald N. Forthal, Sanjay Phogat, James Tartaglia, Susan W. Barnett, Steven G. Self, Raphael Gottardo, Anthony D. Cristillo, Deborah E. Weiss, Lindsey Galmin, Song Ding, Jonathan Heeney, Mariano Esteban, Bertram L. Jacobs, Giuseppe Pantaleo, Ralf Wagner. Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost. Journal of Virology. 2019; 93 (3):e01529-18.
Chicago/Turabian StyleBenedikt Asbach; Karen V. Kibler; Josef Köstler; Beatriz Perdiguero; Nicole L. Yates; Sherry Stanfield-Oakley; Georgia D. Tomaras; Shing-Fen Kao; Kathryn E. Foulds; Mario Roederer; Michael S. Seaman; David C. Montefiori; Robert Parks; Guido Ferrari; Donald N. Forthal; Sanjay Phogat; James Tartaglia; Susan W. Barnett; Steven G. Self; Raphael Gottardo; Anthony D. Cristillo; Deborah E. Weiss; Lindsey Galmin; Song Ding; Jonathan Heeney; Mariano Esteban; Bertram L. Jacobs; Giuseppe Pantaleo; Ralf Wagner. 2019. "Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost." Journal of Virology 93, no. 3: e01529-18.
Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.
Karen V. Kibler; Benedikt Asbach; Beatriz Perdiguero; Juan García-Arriaza; Nicole L. Yates; Robert Parks; Sherry Stanfield-Oakley; Guido Ferrari; David C. Montefiori; Georgia D. Tomaras; Mario Roederer; Kathryn E. Foulds; Donald N. Forthal; Michael S. Seaman; Steve Self; Raphael Gottardo; Sanjay Phogat; James Tartaglia; Susan Barnett; Anthony D. Cristillo; Deborah Weiss; Lindsey Galmin; Song Ding; Jonathan Heeney; Mariano Esteban; Ralf Wagner; Giuseppe Pantaleo; Bertram L. Jacobs. Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC. Journal of Virology 2019, 93, 1 .
AMA StyleKaren V. Kibler, Benedikt Asbach, Beatriz Perdiguero, Juan García-Arriaza, Nicole L. Yates, Robert Parks, Sherry Stanfield-Oakley, Guido Ferrari, David C. Montefiori, Georgia D. Tomaras, Mario Roederer, Kathryn E. Foulds, Donald N. Forthal, Michael S. Seaman, Steve Self, Raphael Gottardo, Sanjay Phogat, James Tartaglia, Susan Barnett, Anthony D. Cristillo, Deborah Weiss, Lindsey Galmin, Song Ding, Jonathan Heeney, Mariano Esteban, Ralf Wagner, Giuseppe Pantaleo, Bertram L. Jacobs. Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC. Journal of Virology. 2019; 93 (3):1.
Chicago/Turabian StyleKaren V. Kibler; Benedikt Asbach; Beatriz Perdiguero; Juan García-Arriaza; Nicole L. Yates; Robert Parks; Sherry Stanfield-Oakley; Guido Ferrari; David C. Montefiori; Georgia D. Tomaras; Mario Roederer; Kathryn E. Foulds; Donald N. Forthal; Michael S. Seaman; Steve Self; Raphael Gottardo; Sanjay Phogat; James Tartaglia; Susan Barnett; Anthony D. Cristillo; Deborah Weiss; Lindsey Galmin; Song Ding; Jonathan Heeney; Mariano Esteban; Ralf Wagner; Giuseppe Pantaleo; Bertram L. Jacobs. 2019. "Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC." Journal of Virology 93, no. 3: 1.
Human cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression, and the licensing of a protective HCMV vaccine remains an unmet global need. Here, we designed and validated novel Sendai virus (SeV) vectors delivering the T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a nonproductive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain modified vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to those of the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of alpha interferon and interleukin-6. Finally, in a challenge model with a murine cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens. IMPORTANCE HCMV is a highly prevalent betaherpesvirus that establishes lifelong latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns, causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in posttransplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.
Richard Kiener; Markus Fleischmann; Marian Alexander Wiegand; Niels A. W. Lemmermann; Christiane Schwegler; Christine Kaufmann; Angelique Renzaho; Simone Thomas; Eva Felder; Hans Helmut Niller; Benedikt Asbach; Ralf Wagner. Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors. Journal of Virology 2018, 92, e00569-18 .
AMA StyleRichard Kiener, Markus Fleischmann, Marian Alexander Wiegand, Niels A. W. Lemmermann, Christiane Schwegler, Christine Kaufmann, Angelique Renzaho, Simone Thomas, Eva Felder, Hans Helmut Niller, Benedikt Asbach, Ralf Wagner. Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors. Journal of Virology. 2018; 92 (15):e00569-18.
Chicago/Turabian StyleRichard Kiener; Markus Fleischmann; Marian Alexander Wiegand; Niels A. W. Lemmermann; Christiane Schwegler; Christine Kaufmann; Angelique Renzaho; Simone Thomas; Eva Felder; Hans Helmut Niller; Benedikt Asbach; Ralf Wagner. 2018. "Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors." Journal of Virology 92, no. 15: e00569-18.
The partially protective phenotype observed in HIV-infected long-term-non-progressors is often associated with certain HLA alleles, thus indicating that cytotoxic T lymphocyte (CTL) responses play a crucial role in combating virus replication. However, both the vast variability of HIV and the HLA diversity impose a challenge on elicitation of broad and effective CTL responses. Therefore, we conceived an algorithm for the enrichment of CD8+ T cell epitopes in HIV’s Gag protein, respecting functional preservation to enable cross-presentation. Experimentally identified epitopes were compared to a Gag reference sequence. Amino-acid-substitutions (AAS) were assessed for their impact on Gag’s budding-function using a trained classifier that considers structural models and sequence conservation. Experimental assessment of Gag-variants harboring selected AAS demonstrated an apparent classifier-precision of 100%. Compatible epitopes were assigned an immunological score that incorporates features such as conservation or HLA-association in a user-defined weighted manner. Using a genetic algorithm, the epitopes were incorporated in an iterative manner into novel T-cell-epitope-enriched Gag sequences (TeeGag). Computational evaluation showed that these antigen candidates harbor a higher fraction of epitopes with higher score as compared to natural Gag isolates and other artificial antigen designs. Thus, these designer sequences qualify as next-generation antigen candidates for induction of broader CTL responses.
Benedikt Asbach; Johannes P. Meier; Matthias Pfeifer; Josef Köstler; Ralf Wagner. Computational Design of Epitope-Enriched HIV-1 Gag Antigens with Preserved Structure and Function for Induction of Broad CD8+ T Cell Responses. Scientific Reports 2018, 8, 1 -15.
AMA StyleBenedikt Asbach, Johannes P. Meier, Matthias Pfeifer, Josef Köstler, Ralf Wagner. Computational Design of Epitope-Enriched HIV-1 Gag Antigens with Preserved Structure and Function for Induction of Broad CD8+ T Cell Responses. Scientific Reports. 2018; 8 (1):1-15.
Chicago/Turabian StyleBenedikt Asbach; Johannes P. Meier; Matthias Pfeifer; Josef Köstler; Ralf Wagner. 2018. "Computational Design of Epitope-Enriched HIV-1 Gag Antigens with Preserved Structure and Function for Induction of Broad CD8+ T Cell Responses." Scientific Reports 8, no. 1: 1-15.
Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of uncontrolled CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of a novel IFN-γ ELISpot assay (T-Track® CMV), based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective, longitudinal, observational, multicenter study was conducted in 86 intermediate risk (D-/R+, D+/R+) renal transplant recipients. Patients underwent pre-emptive antiviral therapy. CMV-CMI, CMV viral load and clinical complications (CMV disease, opportunistic infections and graft dysfunction) were monitored over six months post-transplantation. 95% and 88-92% of IFN-γ ELISpot test results were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection. Interestingly, median pp65-specific response was 9-fold higher in patients with self-clearing CMV viral load compared to antivirally-treated patients prior to first detection of viral load (pDiscussion The observation that the proportion of positive test results remains high (88-92%) post-transplantation, under immunosuppressive treatment, demonstrates the sensitivity of the assay and thus its suitability to measure CMV-CMI in immunocompromised patients. Similarly, the detection of reduced CMV-CMI following immunosuppression and of elevated CMV-CMI in association with graft rejection, indicates the ability of the ELISpot assay to monitor patients’ immunosuppressive state. Finally, the increased response to pp65 prior to first detection of viral load in patients with self-limiting viremia suggests that reactivity to pp65 is a potential marker of immunocompetence. Altogether, this novel IFN-γ ELISpot assay (T-Track® CMV) is a highly sensitive immune-monitoring tool, suitable for the follow-up of renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications. This work was supported in part by the Bayerische Forschungsstiftung Grant AZ 924-10 (to LD) and by institutional funds of the University of Regensburg (ReForM C Project 03-082; to BKK, later transferred to BB).
Bernhard Banas; Dominik Steubl; Lutz Renders; Dominik Chittka; Miriam C. Banas; Thomas Wekerle; Martina Koch; Oliver Witzke; Anja Mühlfeld; Claudia Sommerer; Antje Habicht; Christian Hugo; Thomas Huenig; Monika Lindemann; Traudel Schmidt; Anne Rascle; Sascha Barabas; Ludwig Deml; Ralf Wagner; Bernhard K. Kraemer; Bernd Krueger. Clinical Validation of a Novel ELISpot-based in Vitro Diagnostic Assay to Monitor CMV-specific Cell-Mediated Immunity in Kidney Transplant Recipients. Transplantation 2018, 102, S53 .
AMA StyleBernhard Banas, Dominik Steubl, Lutz Renders, Dominik Chittka, Miriam C. Banas, Thomas Wekerle, Martina Koch, Oliver Witzke, Anja Mühlfeld, Claudia Sommerer, Antje Habicht, Christian Hugo, Thomas Huenig, Monika Lindemann, Traudel Schmidt, Anne Rascle, Sascha Barabas, Ludwig Deml, Ralf Wagner, Bernhard K. Kraemer, Bernd Krueger. Clinical Validation of a Novel ELISpot-based in Vitro Diagnostic Assay to Monitor CMV-specific Cell-Mediated Immunity in Kidney Transplant Recipients. Transplantation. 2018; 102 (Supplement):S53.
Chicago/Turabian StyleBernhard Banas; Dominik Steubl; Lutz Renders; Dominik Chittka; Miriam C. Banas; Thomas Wekerle; Martina Koch; Oliver Witzke; Anja Mühlfeld; Claudia Sommerer; Antje Habicht; Christian Hugo; Thomas Huenig; Monika Lindemann; Traudel Schmidt; Anne Rascle; Sascha Barabas; Ludwig Deml; Ralf Wagner; Bernhard K. Kraemer; Bernd Krueger. 2018. "Clinical Validation of a Novel ELISpot-based in Vitro Diagnostic Assay to Monitor CMV-specific Cell-Mediated Immunity in Kidney Transplant Recipients." Transplantation 102, no. Supplement: S53.
In der Vergangenheit wurden verschiedene HIV-Impfstoffkonzepte entwickelt und in Studien getestet. Von einem durchschlagenden Erfolg der Strategien war jedoch kaum die Rede. Wie ist der aktuelle Stand der Entwicklung?
Ralf Wagner; Benedikt Asbach. Dürfen wir noch auf eine HIV-Impfung hoffen? MMW - Fortschritte der Medizin 2018, 160, 24 -27.
AMA StyleRalf Wagner, Benedikt Asbach. Dürfen wir noch auf eine HIV-Impfung hoffen? MMW - Fortschritte der Medizin. 2018; 160 (2):24-27.
Chicago/Turabian StyleRalf Wagner; Benedikt Asbach. 2018. "Dürfen wir noch auf eine HIV-Impfung hoffen?" MMW - Fortschritte der Medizin 160, no. 2: 24-27.
Human Cytomegalovirus (HCMV) remains a major health burden and the development of a vaccine is a global priority. We developed new viral vectors delivering the T cell immunogens IE-1 and pp65 based on Adenovirus 19a/64 (Ad19a/64), a member of subgroup D. In this ex vivo study, the novel vectors were compared side by side to Ad5 or modified Vaccinia Ankara (MVA) strains expressing the same transgenes. We found that unlike Ad5, Ad19a/64 vectors readily transduce a broad panel of immune cells, including monocytes, T cells, NK cells and monocyte-derived dendritic cells (moDCs). Both Ad19a/64- and MVA-transduced moDCs efficiently restimulated IE-1 or pp65-specific T cells but MVA induced a higher amount of cytotoxicity in this cell type. Ad5 and Ad19 induced upregulation of CD86 and HLA-DR in moDCs whereas expression of CD80 and CD83 was largely unaltered. By contrast, MVA transduction led to downregulation of all markers. Taken together, our data demonstrate that Ad19a/64 is a promising vector for the delivery of HCMV immunogens since it transduces dendritic cells with an efficiency that is comparable to MVA, but cytotoxicity and interference with dendritic cell maturation are less pronounced.
Richard Kiener; Markus Fleischmann; Christiane Schwegler; Zsolt Ruzsics; Christian Thirion; Silke Schrödel; Benedikt Asbach; Ralf Wagner. Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo. Scientific Reports 2018, 8, 1 -13.
AMA StyleRichard Kiener, Markus Fleischmann, Christiane Schwegler, Zsolt Ruzsics, Christian Thirion, Silke Schrödel, Benedikt Asbach, Ralf Wagner. Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo. Scientific Reports. 2018; 8 (1):1-13.
Chicago/Turabian StyleRichard Kiener; Markus Fleischmann; Christiane Schwegler; Zsolt Ruzsics; Christian Thirion; Silke Schrödel; Benedikt Asbach; Ralf Wagner. 2018. "Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo." Scientific Reports 8, no. 1: 1-13.